Melanoma is the most aggressive form of skin cancer. Circulating tumor DNA (ctDNA) is a diagnostic and prognostic marker of melanoma. However, whether ctDNA mutations can independently predict survival remains controversial. This meta-analysis assessed the prognostic value of the presence or change in ctDNA mutations in melanoma patients.  We identified studies from the PubMed, EMBASE, Web of Science, and Cochrane databases. We estimated the combined hazard ratios (HRs) for overall survival (OS) and progression-free survival (PFS) using either fixed-effect or random-effect models based on heterogeneity. P < 0.00001; baseline undetectable vs. detectable, pooled HR = 0.19, 95% CI = 0.11–0.36, P < 0.00001; posttreatment detectable vs. undetectable, pooled HR = 2.36, 95% CI = 1.30–4.28, P=0.005). For PFS, baseline detectable ctDNA may be associated with adverse PFS  and baseline high ctDNA and increased ctDNA were significantly associated with adverse PFS . The baseline BRAFV600 ctDNA mutation-positive group was significantly associated with adverse OS compared with the baseline ctDNA-negative group . There were no significant differences in PFS between the baseline BRAFV600 ctDNA mutation-detectable group and the undetectable group .  Sixteen studies including 1,781 patients were included. Both baseline and posttreatment detectable ctDNA were associated with poor OS (baseline detectable vs. undetectable, pooled HR = 1.97, 95% CI = 1.64–2.36, V600 ctDNA mutation was significantly associated with worse prognosis in melanoma patients. The presence or elevation of ctDNA mutation or BRAF Melanoma is the most aggressive form of skin cancer, originating from pigment-producing melanocytes. Though it accounts for only 10% of all skin cancers, it is responsible for more than 80% of skin cancer-related deaths. The development of targeted therapy (MAPK pathway inhibitors)  and immuIn recent years, many studies have focused on the value of circulating tumor DNA (ctDNA) in monitoring disease status and prognosis –12. SomeWe conducted systematic searches of the PubMed, EMBASE, Web of Science, and Cochrane databases for entries up until March, 2020 without language or study period restrictions. The search terms included (“ctDNA” or “circulating tumor DNA” or “circulating DNA” or “free DNA” or “cell free DNA” or “plasma DNA” or “serum DNA”) and (“Melanoma” or “Skin cancer” or “Skin neoplasia” or “skin neoplasm”) and (“prediction” or “outcome” or “predictive” or “prognosis” or “prognostic”). A manual search of related articles and references cited in these articles was performed to identify all available studies.Studies were included in the analysis if they met the following criteria: (1) all patients enrolled in the study were diagnosed with melanoma, (2) ctDNA mutation was assessed using plasma or serum, (3) endpoints included PFS or OS, and sufficient data were presented for determining or calculating the hazard ratio (HR) and 95% confidence interval (95% CI).Studies were excluded if (1) fewer than 15 patients were included in the statistical analysis of prognosis value of ctDNA, (2) samples were not drawn from peripheral blood , and (3) nonresearch publications such as editorials, reviews, and letters, or the publications were duplicated articles.Two investigators independently reviewed all eligible studies and extracted the following information: first author name, publication year, country, number of patients analyzed, tumor stage, sample origin, time of sample collection, clinical therapy, method/platform of ctDNA detection, target genes/variants, cutoff value, number of experimental/control samples, positive ratio, and endpoint, and follow-up duration and confounding factors, if provided. When the article had multiple queues or groups of patients, and an HR for survival curve was provided for each queues/groups, results of all these queues/groups were recorded as independent data. The quality of all included studies was evaluated using the Newcastle–Ottawa Quality Assessment Scale (NOS) . An NOS I2 > 50%), the random effects model was adopted [I2 > 50% and P < 0.05 were considered significant for heterogeneity.Hazard ratios (HRs) and their 95% CIs for PFS and OS were recorded to clarify the prognostic value of ctDNA. For studies in which HRs and 95% CIs were not available, we extracted survival rates from Kaplan–Meier curves by using Engauge Digitizer version 4.1 . To asse adopted . I2 > 50For forest plots with more than 10 included studies or results, we evaluated publication bias using funnel plots for visual inspection and conducted quantitative estimations using Egger's test. Sensitivity analysis was performed by excluding each study in turn to assess the stability of the results. All analyses were carried out using Review Manager version 5.3  and STATA version 12.0 .A total of 2,365 articles were identified after removing duplicates. By reviewing titles and abstracts, 2,305 articles were excluded, of which 2,114 were not related to the disease or subject of our meta-analysis, 86 were reviews or systematic evaluations, and 105 were abstracts, conference papers, or case reports. After detailed reading and evaluation of 60 articles, we excluded eight articles because the number of patients analyzed for prognosis was less than 15. Twelve articles were excluded for lack of prognostic information. Twenty-one articles were excluded because they did not provide sufficient data to extract HRs for PFS or OS. Further three articles with or suspected to have overlapping study populations were excluded. Finally, 16 articles , 22–31 pV600 ctDNA mutation was detected in seven studies [V600 mutation was available. The other studies [The characteristics and quality of studies included in the meta-analysis are described in  studies , 30, 31, studies , althoug studies , 27, 29 P < 0.00001). The I2 statistical heterogeneity was not significant (I2 = 0%). This suggests that baseline detectable ctDNA is associated with adverse OS.A total of 11 studies , 26–30 aP < 0.00001), with no heterogeneity (I2 = 0%)  . This suIn addition, one study  analyzedP=0.005), with no heterogeneity (I2 = 0%)  . One stuP=0.19). The I2 statistical heterogeneity was high (I2 = 55%); thus, a random-effects model was used.A total of 12 studies , 27–30 aP=0.04, I2 statistical heterogeneity was high (I2 = 73%); thus, a random-effects model was used.Three studies , 27, 29 P=0.0003), with no heterogeneity (I2 = 0%) (Two studies (three results) , 16 analI2 = 0%) .In addition, data provided in two studies , 29 not The relationship between posttreatment ctDNA detection and PFS was analyzed in one study , which sP < 0.00001), with no heterogeneity (I2 = 0%) (One study (three results)  indicateI2 = 0%) .V600 mutation detectable ctDNA on PFS and OS, compared with baseline BRAFV600 mutation undetectable ctDNA because of different grouping methods and the limited number of studies. Our results showed that the risk was significantly higher in the baseline BRAFV600 ctDNA mutation-positive group than in the baseline ctDNA-negative group in terms of mortality , with no heterogeneity (I2 = 0%) , with low heterogeneity (I2 = 4%)  . The difI2 = 4%) .A subgroup analysis was performed on the forest plot with the largest number of studies to identify the factors that may influence interstudy heterogeneity, including the effects of baseline detectable ctDNA on PFS and OS, compared with baseline undetectable ctDNA.I2 = 0%). In most subgroups, baseline detectable ctDNA was significantly associated with adverse OS, although there was no significant correlation between baseline detectable ctDNA and OS in the non-ddPCR ctDNA evaluation subgroup and in the subgroup whose data were extracted from the survival curve . For the subgroups that evaluated PFS, heterogeneity was significantly reduced according to whether the target gene included only BRAFV600 or not, and according to whether the data was directly provided by the studies or extracted from the survival curve. As it happened, the two subgroups included the same studies, whose target genes were BRAFV600, and data were extracted from the survival curve, which showed no significant correlation between baseline detectable ctDNA and PFS . Meanwhile, the subgroup whose data was provided directly by the studies suggested that baseline detectable ctDNA was associated with adverse PFS . These results suggest that the sources of heterogeneity may be the method of ctDNA assessment, target genes, and data sources.P=0.005) to 1.61 , and the heterogeneity remained at 0. Two studies [P=0.04) to 0.38  and the heterogeneity was almost unchanged (from I2 = 73% to I2 = 85%). After excluding the other study [P=0.10) and the heterogeneity decreased to 0.Sensitivity analysis based on the change in the combined effect size by excluding the included studies one by one is an important method mainly used to evaluate the robustness and reliability of the combined results of meta-analysis. After sensitivity analysis, the overall effect sizes of the two meta-analyses were significantly modified. Among them, one study  had a si studies , 27 had er study , the oveP=0.306)  .In 2012, melanoma was the 15th most common cancers worldwide . UnfortuV600 mutation is also a prognostic biomarker with similar prognostic value. Patients with detectable BRAFV600 ctDNA at baseline tend to have worse OS compared to patients with undetectable BRAFV600 ctDNA, while baseline detectable BRAFV600 ctDNA may be associated with worse PFS.This meta-analysis included 16 studies including 1,781 melanoma patients for a prognostic analysis. We analyzed differences in OS and PFS at the endpoint of observation for mutations in ctDNA at baseline and after treatment. We also calculated the relevant HRs and 95% CIs. The results revealed that ctDNA mutation was significantly associated with the prognosis of melanoma patients. Specifically, patients with detectable ctDNA mutation tended to have adverse OS compared to patients where ctDNA mutation was not detected, either at baseline or after treatment. Patients with low or undetectable ctDNA mutation at baseline tend to have better PFS compared with patients with high ctDNA mutation, and baseline detectable ctDNA may be associated with adverse PFS. Patients with decreased ctDNA levels tend to have favorable PFS compared with patients with increased ctDNA levels. CtDNA BRAFV600 mutation was significantly associated with worse OS, while patients with BRAFV600 ctDNA tended to have poor PFS, although without statistical significance. However, the subgroups in which the association between multiple gene ctDNA mutations and PFS or OS was analyzed included one study each; therefore, the results of the subgroup analysis were not representative or comparable due to the differences in tumor stage, treatment, and method of ctDNA assessment in the two studies. Melanoma has the highest somatic cell mutation rate [The result of our subgroup analysis stratified by target gene showed that BRAFion rate  and a wiion rate , while tion rate . NRAS muion rate , leadingion rate . The difion rate . TherefoV600 or NRASQ61 mutations in patients with melanoma enhances the effectiveness of ddPCR in a majority of the cases [The results of the subgroup analysis classified by method of ctDNA assessment showed that ddPCR was more effective than other methods. The content of ctDNA in peripheral blood is very small, and the content and nucleic acid polymorphisms in different patients vary greatly . Low-senhe cases .The results of subgroup analysis stratified by sample sources were consistent with the conclusions of Meddeb et al.  in that V600 and other gene mutations has shown significant value in predicting treatment response and outcome in melanoma. In addition to detecting mutated ctDNA, epigenetic changes and loss of heterozygosity (LOH) of ctDNA can also be detected and analyzed. Two studies by Mori et al. [α predicting progression-free and overall survival. Due to the small number of relevant studies, we did not conduct a meta-analysis.There are many biomarkers for the prognosis and prediction of melanoma used clinically. The 8th edition American Joint Committee on Cancer (AJCC) melanoma staging system  determini et al. , 53 demoIn the present meta-analysis, all patients in most of the studies had advanced melanoma, while the patients in one study were at stage II-III. Therefore, our conclusions are applicable to patients with advanced melanoma. Due to the limited number of studies, no subgroup analysis of tumor-node-metastasis (TNM) staging was performed. In addition, several limitations in this study should be addressed. We may not have identified published or unpublished studies with negative results, and some studies had a short follow-up time or small sample sizes; therefore, the results should be interpreted with caution. In addition, we hypothesized that possible sources of bias are as follows, and these factors may also lead to heterogeneity: (1) most studies only used univariate analysis, and the size of effect values may be overestimated compared with multivariate analysis; (2) multivariate analysis was used in some studies to obtain accurate estimates, although the confounders used in multivariate analysis were different, which may affect HR values; (3) in most studies, multiple gene ctDNA mutations were detected, which were not identical and may have introduced bias; (4) not all HRs and 95% CIs included in the meta-analysis were directly collected from the original articles; thus, we extracted survival rates from Kaplan–Meier curves by using Engauge Digitizer version 4.1  to reducV600 ctDNA mutation was significantly associated with worse prognosis in melanoma patients. However, in view of some of the limitations of this study, the results of our meta-analysis need to be verified by further research. With further exploration and understanding of ctDNA biological function and clinical value, as well as the validation of more large-scale, high-quality prospective studies, ctDNA could eventually be applied in clinical practice and may eventually become part of routine melanoma staging.Despite these limitations, our meta-analysis clearly indicates the prognostic value of ctDNA mutations in melanoma; that is, the presence or elevation of ctDNA mutation or BRAF


Chronic undernutrition contributes to the increase in frailty observed among elderly adults, which is a pressing issue in the sector of health care for older people worldwide. Autophagy, an intracellular recycling system, is closely associated with age-related pathologies. Therefore, decreased autophagy in aging could be involved in the disruption of energy homeostasis that occurs during undernutrition; however, the physiological mechanisms underlying this process remain unknown. Here, we showed that 70% daily food restriction (FR) induced fatal hypoglycemia in 23–26-month-old (aged) mice, which exhibited significantly lower hepatic autophagy than 9-week-old (young) mice. The liver expressions of Bcl-2, an autophagy-negative regulator, and Beclin1–Bcl-2 binding, were increased in aged mice compared with young mice. The autophagy inducer Tat-Beclin1 D11, not the mTOR inhibitor rapamycin, decreased the plasma levels of the glucogenic amino acid and restored the blood glucose levels in aged FR mice. Decreased liver gluconeogenesis, body temperature, physical activity, amino acid metabolism, and hepatic mitochondrial dynamics were observed in the aged FR mice. These changes were restored by treatment with hochuekkito that is a herbal formula containing several autophagy-activating ingredients. Our results indicate that Bcl-2 upregulation in the liver during the aging process disturbs autophagy activation, which increases the vulnerability to undernutrition. The promotion of liver autophagy may offer clinical therapeutic benefits to frail elderly patients. Undernutrition leads to frailty, longer hospital stays, frequent readmission, and higher mortality in elderly patients4. Although nutrition support may be helpful for the management of undernourished patients, cyclic enteral and/or total parenteral nutrition are less effective in elderly patients than in younger individuals6.As the world’s population ages, health care for older people has become a pressing issue. In modern society, many factors, including impaired physical function, psychological disorders, and/or limited social interactions, may result in loss of appetite and/or decreased food intake among the elderly. A pooled analysis of data from older people aged 65 years and over in hospitals in 12 countries reported that 39% were at nutritional risk8. Impaired autophagy is closely related to the development of several age-related disorders, including cancer, as well as cardiovascular and neurodegenerative diseases9.Autophagy is an intracellular degradation system that plays a critical role in supplying nutrients during starvation and likewise performs quality control for intracellular organelles such as the mitochondria10; many organisms show signs of decreased capacity for autophagy that correlates with age11, however, the underlying mechanisms are still poorly understood.Aging results in accumulated damage to molecules, cells, and tissues and ultimately leads to organ malfunction12 and is capable of amplified metabolic stress-induced autophagy due to lysosome-enriched hepatocytes13. Liver autophagy contributes to basic hepatic functions, including gluconeogenesis and β‑oxidation, which fluctuate periodically depending on the fasting–feeding cycle. During prolonged fasting, liver autophagy is critical for maintaining systemic energy homeostasis via gluconeogenesis14 and ketogenesis15.The liver is a key metabolic organ that governs energy metabolismWe hypothesized that the undernourished elderly experience disrupted energy homeostasis that might be exacerbated by decreased liver autophagy. To explore this possibility, we subjected young and aged mice to severe food restriction (FR) to generate an undernourished condition; we evaluated the distinct metabolic changes and compared the responses of the two groups. Furthermore, we investigated the mechanism controlling liver autophagy and its relationship to energy metabolism during aging.P = 0.0115) and the age × time interaction (P = 0.0495). None of the young mice experienced hypoglycemia (defined as blood glucose level <50 mg/dL) at any time during the study period, while hypoglycemia-free survival was significantly diminished among the aged mice  and also the age × time interaction (P = 0.0029). Spontaneous activity initially increased in response to FR but ultimately decreased to less than the baseline activity  by day 5 in both the young and aged mice; the overall activity was significantly lower in the aged mice than in the young mice , time (P < 0.001), and the age × time interaction (P < 0.001). Taken together, these results suggest that energy homeostasis was disrupted in the aged mice. However, survivorship bias might occur in these data since the 10-day FR resulted in higher mortality in aged mice. Therefore, we limited the duration of FR and the subsequent experiments to 5 days when the survival rate of the aged mice began to decline.Baseline characteristics of young and aged mice are shown in Supplementary Table 1. There were no significant differences in baseline levels of blood glucose between the two groups. After subjecting the mice to FR, the blood glucose levels were considerably lower than baseline levels on day 5 and had partially recovered in young mice on day 10, but they continued to fall in the aged mice Fig. . Two-wayice Fig.  accompanice Fig. . Transieice Fig. . Althougice Fig. . Two-wayice Fig. , but theice Fig. . Two-wayWe compared the alterations in internal energy sources between the young and aged mice. The body weight of the aged mice was higher than that of the young mice Fig. . The bodSimilarly, FR decreased the liver levels of glucogenic amino acids in the young mice, but not in the aged mice Fig. . MoreoveP = 0.0072), time (P < 0.001), and the drug × time interaction (P = 0.0037). Aged mice on FR with blood glucose levels that were similar to those of young mice on FR before alanine injection  were selected for the alanine tolerance test so that we would be able to perform a rigorous comparison of the capacity for gluconeogenesis. Interestingly, blood glucose levels in the aged FR mice did not increase at all after the alanine injection -1α, glucose 6-phosphatase (G6Pase), or phosphoenolpyruvate carboxykinase (PEPCK) in liver tissues of the young and aged mice Fig. . Expression Fig. ; these rion Fig. . To asseWe examined two major proteolysis pathways in the two mouse cohorts, the ubiquitin–proteasome system and autophagy. Expression of E3 ubiquitin ligases (MuRF1 and Atrogin-1), and autophagy-related protein LC3 in both gastrocnemius and soleus muscles, was increased by 5 day of FR; this increase was significantly attenuated in the aged FR mice compared with the young FR mice Fig. . There wElectron microscopic analysis of liver tissue revealed that the number of autophagic vacuoles within the hepatocytes increased in response to FR in both the young mice but not in the aged mice, and they were significantly lower in the aged FR mice than in the young FR mice Fig. . InteresPparα, encoding the fatty acid oxidation-related gene peroxisome proliferator-activated receptor α (PPARα) and Hmgcs2, encoding the critical enzyme ketogenesis 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMGCS2)  Fig. . To clarS2) Fig. . ExpressS2) Fig. . There wS2) Fig. . ExpressS2) Fig. . An immuS2) Fig. .Fig. 6Ef17 and restore metabolic homeostasis18. The results in Table P < 0.001 vs. vehicle-treated wells). Among 66 chemical ingredients in 10 components of HET tested, 9 ingredients had the capacity to induce autophagy in the in vitro assay system.Next, we investigated the effects of HET on energy homeostasis in aged FR mice, because recent studies have demonstrated the potential of HET to induce autophagyP < 0.001). This was not observed in the aged FR mice who received HET-containing diet, and the blood glucose levels tended to be higher than those measured in the aged FR mice who received control diet  and time (P = 0.0275). The number of mice that exhibited more than 200 counts/60 min of locomotor activity after alanine injection was significantly higher among the HET-treated aged FR mice than in their control counterparts . On day 5 of FR, HET treatment had no significant impact on blood glucose levels and body temperature in aged mice ; furthermore, gluconeogenic substrates, such as lactate, glycerol, and amino acids, are converted to glucose via the process known as gluconeogenesis. In this study, glycogen stores in the liver were depleted both in the young and aged mice at day 5 of FR. Blood βHB increased in the early phase of FR but returned to the basal levels on day 5 in the young mice who had already exhausted their fat stores and had the little additional capacity to produce ketone bodies. In the aged mice, the increase in blood βHB levels was attenuated relative to that detected in the young mice, and weight and proportion of body fat on day 5 of FR were higher in the aged mice than among their younger counterparts. As such, we assumed that the capacity for ketogenesis and/or lipolysis was diminished in the aged FR mice. Liver glycogen shortage during fasting was reported to facilitate lipolysis in adipose tissues23. Since aged mice have a lower baseline of glycogen content, liver glycogen depletion may occur more quickly in them. Thus, the reduction of ketogenesis or lipolysis or both in the aged FR mice may not be related to the liver glycogen levels; further study is needed to clarify this issue. In the young mice subjected to a 5-day FR, muscle weights decreased but blood glucose was maintained >50 mg/dL; these responses were likewise attenuated in the aged FR mice, many of whom became markedly hypoglycemic. These findings indicate that the supply of amino acids due to proteolysis could be insufficient in the aged FR mice. While gluconeogenesis accompanied by accelerated proteolysis was observed among rats fasted for 4 days, plasma levels of amino acids such as alanine decreased with time24. In our study, total and glucogenic amino acids in plasma were decreased in the young FR mice, but were paradoxically increased in their aged counterparts. The relationship between the levels of glucogenic amino acid in the plasma and liver and the comparable expression levels of genes encoding the transporters is similar in the young and aged FR mice. Therefore, amino acids, especially glucogenic amino acids such as alanine, are taken up into the liver in both young and aged mice. On the other hand, alanine is converted into pyruvate in the liver and transported into the mitochondrial matrix via the mitochondrial pyruvate carrier  for gluconeogenesis during fasting. The expression of Mpc2, encoding MPC2, was decreased in the aged FR mice compared to the young FR mice  membrane; autophagy is inhibited by the interaction between Beclin1 and Bcl-2 only when they are located on ER, not on the mitochondria32. Several studies directed at revealing mechanisms of apoptosis reported that expression of Bcl-2 protein on the liver mitochondrial membrane was reduced among aged rats36. As Bcl-2 expression in total liver homogenates was increased in the aged mice cohort in our study, Bcl-2 localization on the ER membrane or in the cytoplasm might increase with aging, although the detailed mechanism remains unclear. Expression of Rubicon, a protein that inhibits autophagy by binding to Beclin1 at the autophagosome maturation step, did not increase in response to aging in the present study. These results suggest that Beclin1-mediated autophagy was inhibited not by Rubicon but by Bcl-2.Bcl-237, QOL and immunological status of elderly patients with weakness38, and nutritional status in elderly patients with chronic obstructive pulmonary disease39. Several studies carried out in vitro have demonstrated the potential of HET to restore metabolic homeostasis18 and promote autophagy17. We confirmed that HET and 9 of its chemical ingredients derived from astragalus root, jujube, ginger, and ginseng induced autophagy in studies featuring reporter cells; these studies suggest that these compounds may contribute to the inductive effects of HET. Among them, 6 components have been identified as capable of inducing autophagy in vitro45. To the best of our knowledge, this is the first study to identify induction of autophagy by 8-gingerol (ginger-derived), oleanonic acid, and betunolic acid (jujube-derived).HET is one of Kampo medicines, which is prescribed for patients after surgery or as treatment for illnesses associated with general fatigue or poor appetite. Clinical studies have reported that HET improves fatigue, lack of energy, and low body temperature in adult patients with appetite loss40 and block the Beclin1–Bcl-2 interaction43 may contribute to its mechanism of action. Therefore, HET could alleviate the disruption of energy homeostasis during the aging process by mainly inducing liver autophagy, but not anti-inflammatory effects. HET may facilitate the recovery from decreased physical strength in older patients with undernutrition.We investigated the impact of HET on energy homeostasis in aged FR mice. Chronic administration of HET facilitated the partial recovery of energy production as indicated by the increased body temperature, locomotor activity, and blood glucose after alanine injection in the aged FR mice treated with HET; these findings were accompanied by increased expression of gluconeogenic enzymes in the liver. Moreover, HET treatment resulted in an increase in plasma levels of various amino acids after alanine injection in the aged FR mice; these findings suggested that HET could restore the impaired hepatic energy metabolism in aged mice. Furthermore, we confirmed that HET was capable of inducing liver autophagy in the aged FR mice as assessed by the increased number of autophagic vacuoles and significantly increased expression of autophagy-related genes. Although the detailed mechanism underlying the responses to HET is unclear, compounds that increase Beclin1 expression46. Further studies will be needed so that we have a clearer understanding of autophagy in the kidney. Moreover, although we demonstrated restoration of energy homeostasis in response to HET in the aged mice, further investigation will be needed to determine if HET has any impact on autophagy and the capacity to generate energy among elderly people.Renal gluconeogenesis is reported to produce 15–25% of the circulating glucose in the postabsorptive state; this fraction increases as starvation progressesIn summary, this study revealed that decreased activation of liver autophagy secondary to increased expression of Bcl-2 and enhancement of the Beclin1–Bcl-2 interaction might lead to the disrupted energy production and contribute to the vulnerability to undernutrition among aged mice. Improvement in liver autophagy is critical for maintaining energy homeostasis in the elderly; this process could be an important therapeutic target for combating frailty and related disorders with poor prognoses.Male C57BL/6J mice  aged 9 weeks (young) and 23–26 months (aged) were used in our experiments. The mice were housed singly in a controlled environment  under a 12-h light/dark cycle with free access to standard powdered chow and water. This study was approved by and was conducted according to the guidelines of the experimental animal ethics committees of Tsumura & Co. , and the animal experiments were conducted in compliance with the ARRIVE guidelines.l-Alanine , chloroquine , and Tat-Beclin1 D11 autophagy-inducing peptide  were dissolved in saline. Rapamycin  was dissolved in saline containing 0.001% dimethyl sulfoxide. The Kampo medicine HET is a dried and powdered hot-water extract of the following ten components: Astragalus Root  4.0 g, Atractylodes Lancea Rhizome (Atractylodis lanceae rhizoma) 4.0 g, Ginseng (Ginseng radix) 4.0 g, Japanese Angelica root (Angelicae radix) 3.0g, Bupleurum Root (Bupleuri radix) 2.0 g, Jujube (Zizyphi fructus) 2.0 g, Citrus Unshiu Peel (Aurantii nobilis pericarpium) 2.0 g, Glycyrrhiza (Glycyrrhizae radix) 1.5 g, Cimicifuga Rhizome (Cimicifugae rhizoma) 1.0 g, and Ginger (Zingiberis rhizoma) 0.5 g. HET was manufactured by Tsumura & Co.  and used by mixing in standard chow.Young and aged mice were placed on a 70% restricted diet, which provided 30% of their daily food intake at 1.1 and 1.3 g/day, respectively, as measured during the acclimatization period. Day 0 was defined as the first day of FR. These mice underwent the 70% FR for 5 or 10 days until the day of the experiment. The mice that were unable to eat within several hours after feeding and displayed noticeable hypothermia were euthanized from an ethical viewpoint.47; hypoglycemia-free survival secondary to FR was assessed. Body temperature was recorded just before feeding with a body temperature maintenance system  connected to a mouse rectal probe . Daily spontaneous activity was assessed with an infrared ray sensor  at times other than during feeding or taking measurements (09:15–11:45).Blood was taken from the tail vein just prior to feeding to determine levels of glucose and ketone body βHB with a OneTouch UltraVue  and FreeStyle Precision Neo , respectively. Hypoglycemia was defined as the blood glucose level below 50 mg/dL; this level of blood glucose is associated with cognitive impairment, coma, seizure, and/or aberrant behavior in both humans and experimental animalsAfter 5-day FR exposure, fluid status and tissue composition of mice were measured by using an ImpediVET (Bio Research Center) under isoflurane anesthesia followed by sacrifice for blood and tissue collection.l-alanine (2 g/kg); blood glucose levels were measured before and 30, 60, 90, and 120 min after the injection. In another set of mice, locomotor activity was evaluated between 60 min and 120 min after alanine injection; plasma amino acid levels were determined at 180 min after the injection. Mice were excluded from further analysis for humane reasons if blood glucose levels were <50 mg/dL prior alanine injection.On day 5, mice were injected via the intraperitoneal route with On day 5 of FR, chloroquine (50 mg/kg) was injected intraperitoneally to the young mice and rapamycin (2 mg/kg) to the aged mice. Blood glucose levels were measured before and 60 min after the injections. On day 5, aged FR mice were intraperitoneally injected with Tat-D11 at 20 mg/kg. Blood was collected from the tail vein of the mice before and 60 min after the Tat-D11 injection to measure glucose and βHB levels. The mice were then sacrificed under isoflurane anesthesia for plasma and liver sample collection.l-alanine on day 5, and the mice were sacrificed under isoflurane anesthesia for blood collection from the abdominal inferior vena cava at 180 min after the injection. The third set of mice was sacrificed under isoflurane anesthesia on day 5 for tissue collection.Three sets of aged mice received HET (1.5%)-containing or control pellet chow and were permitted food ad libitum for 4 weeks, after which they were subjected to a 5-day FR . In the first set of mice, the alanine-tolerance test was performed on day 5 of FR to measure blood glucose levels. In the second set of mice, locomotor activity was assessed from 60 min to 120 min after the injection of Liver tissues were fixed in 2.5% glutaraldehyde for 24 h at 4 °C, rinsed with 0.1 M phosphate buffer (pH = 7.4), postfixed for 1 h in 1% osmium tetroxide, dehydrated in alcohol, and embedded in epoxy resin. Ultrathin sections stained with uranyl acetate and lead citrate were examined under a Hitachi H-7650H electron microscope . Scale bars were added to the microscopy images using PowerPoint .g at 4 °C for 10 min. Protein aliquots  were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a PVDF membrane, and probed with the following primary antibodies: anti-LC3: 1:2500, PM036, MBL International Corporation, Woburn, MA, USA; anti-GAPDH: 1:10,000 and 1:20,000 for western blotting and immunoprecipitation, respectively, 2118, Cell Signaling Technology; anti-Beclin 1: 1:20,000 and 1:50,000 for western blotting and immunoprecipitation, respectively, ab207612 and anti-Bcl-2: 1:1000 and 1:5000 for western blotting and immunoprecipitation, respectively  and secondary antibodies, including anti-rabbit IgG, HRP-linked whole Ab Donkey , or anti-mouse IgG, HRP-linked F(ab’)2 Fragment Sheep . Blots were visualized using the ECL Plus Western Blotting Substrate , scanned by a Typhoon 9410 Imager , and quantified with ImageQuant TL . All blots or gels derive from the same experiment and they were processed in parallel.Liver tissues were homogenized with Cell Lysis Buffer  containing protease inhibitor cocktail . The homogenates were centrifuged twice at 10,000×Immunoprecipitation was performed according to the manufacturer’s instructions accompanying the Dynabeads protein G immunoprecipitation Kit ; western blotting was performed as described above.The total RNA was extracted by using an RNeasy Minikit  and reverse- transcribed by using the TaqMan High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR assays were performed by using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and TaqMan gene-specific primer/probes  on a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific). Expression of mRNA was normalized to that of the 18S rRNA gene.The right lobes of the liver were fixed in 10% buffered formalin and embedded in paraffin for histological examination. Sections (5 µm) were stained with hematoxylin and eosin and examined under a BZ-X800 Microscope . Scale bars were added to the microscopic images using PowerPoint (Microsoft). To improve visibility, contrast enhancement of the microscopy images was performed using PowerPoint (Microsoft).g for 5 min. The glycogen concentration was measured using the EnzyChrom Glycogen Assay Kit .The other liver tissues were homogenized on ice with 25 mM citrate buffer, pH 4.2, supplemented with 2.5 g/L NaF, and the supernatants were recovered by centrifugation at 14,000× The plasma levels of glucose and insulin were measured with the Glucose CII Test Wako Kit (Wako) and the Mouse Insulin ELISA Kit .Amino acids in plasma and liver samples were derivatized by using an AccQ•Tag Ultra Derivatization Kit , and concentrations were determined by an ACQUITY Ultra Performance Liquid Chromatography (UPLC) H-Class system (Waters) equipped with an AccQ•Tag Ultra C18 1.7-μm 2.1 × 100-mm column (Waters) and tunable UV detector (Waters). Amino acid data were processed by using Empower 3 Personal Single System software (Waters). Pathway maps were created by using Visualization and Analysis of Networks software containing Experimental Data V2.6.5 and referenced KEGG pathway databases. The colors in the pathway maps indicated the log2-transformed ratios of each amino acid between groups.Induction of autophagy was assessed with a HEK293 Autophagy LC3 HiBiT Reporter Cell Line and Detection System . Chemical ingredients of HET were purchased or manufactured by Tsumura & Co. . After the cells  were cultured overnight in 96-well microplates, the medium was removed and treated for 24 h with test compounds that were first dissolved in dimethyl sulfoxide  and diluted in a medium containing 0.3% fetal bovine serum. HET was suspended by using an agate mortar. Unless otherwise noted, assays were performed according to the manufacturer’s instructions. Values were reported as percentages calculated from the luminescence intensities of vehicle-treated wells  and Bafilomycin A1-treated wells . Compounds that showed inductive activities >120% at either tested concentration were defined as active ingredients of HET.Aged mice that developed spontaneous tumors were excluded from the experiment because the tumors introduced confounding variables to this protocol.t test was performed for two-group comparisons, and the Tukey–Kramer, Dunnett, or Steel–Dwass tests were used for multiple-group comparisons. The Mann–Whitney test was used when data distributions were not reliably normally distributed. Chi-square test was used for the analysis of the proportion of mice in locomotor activity assessment. Prism 7  and StatLight 2000  were used to perform data analysis. Plasma amino acids were colored according to a color scale, as described in the text, and P values < 0.05 (two-tailed) were considered statistically significant.Hypoglycemia-free survival was analyzed by the log-rank test. Two-way analysis of variance (ANOVA) followed by the Bonferroni or Tukey post-hoc test was used for time-course analyses. Student’s or the Aspin–Welch Further information on research design is available in the Supplementary InformationReporting Summary


Gymnema inodorum leaf extract on Plasmodium berghei-induced alteration of blood coagulation parameters and platelet numbers in mice. Groups of ICR mice were inoculated with 1 × 107 parasitized red blood cells of P. berghei ANKA (PbANKA) and given orally by gavage with 100, 250, and 500 mg/kg of G. inodorum leaf extract (GIE). Chloroquine (10 mg/kg) was used as a positive control. Platelet count and blood coagulation parameters were measured. The results showed that PbANKA induced thrombocytopenia in mice as indicated by markedly decreased platelet count. Decreased platelet count had a negative correlation with the degree of parasitemia with R2 value of 0.6668. Moreover, significantly (p < 0.05) shortened activated partial thromboplastin time was found in PbANKA-infected group, while prothrombin time and thrombin time were still normal. GIE gave significantly (p < 0.05) good results with respect to platelet count, compared with the results obtained from positive and healthy controls. Additionally, GIE reversed the alteration of blood coagulation parameters when compared to untreated mice. The highest efficacy of GIE was observed at a dose of 500 mg/kg. It was concluded that GIE exerted a protective effect on thrombocytopenia and altered blood coagulation parameters induced by PbANKA infection in mice. This plant may be a future candidate for alternative antimalarial development.Malaria remains highly prevalent and one of the major causes of morbidity and mortality in tropical and subtropical regions. Alteration of blood coagulation and platelets has played an important role and attributed to increased morbidity in malaria. Hence, this study was performed to investigate the efficacy of  Plasmodium and transmitted by the female Anopheles mosquito. Five species of malaria parasites that have been well known to cause human malaria include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi [Plasmodium parasite resistance to several available antimalarial drugs have been hindering the control and eradication of malaria [Cinchona ledgeriana and Artemisia annua have been shown the potent antimalarial activity and developed as standard drugs for malaria treatment [Malaria is a parasitic disease caused by protozoa in the genus knowlesi . Globallknowlesi . The eme malaria . The dea malaria –6. Throm malaria , 8. Thes malaria . Researc malaria . For insreatment .Gymnema sylvestre, which belongs to the family of Apocynaceae, is a wild herb found in India, Africa, Australia, and China. It is traditionally used to treat various diseases, including diabetes, malaria, dyspepsia, constipation, jaundice, hemorrhoids, renal and vesicle calculi, cardiopathy, asthma, bronchitis, amenorrhea, and leukoderma [G. sylvestre extract have also been reported, such as phenols, flavonoids, quinones, tannins, saponins, anthraquinones, triterpenoids, and gymnemic acids [Gymnema inodorum (Lour.) Decne, also a member of the genus Gymnema, is popular in Thailand, especially in the northern part. G. inodorum leaf extract has been used as a traditional food and medicine in Asia [G. inodorum leaf extract showed the inhibition of glucose absorption with high antioxidant activity [Gymnema is also traditionally used in wound healing and having a good wound healing property in rat model [G. inodorum leaf extract on blood coagulation disorders in mice has not yet been studied. Hence, this study focused on evaluating the effects of G. inodorum leaf extract on blood coagulation parameters and platelets in Plasmodium berghei-infected mice.ukoderma . Severalukoderma . The impic acids . Gymnema in Asia . It has activity . Gymnemaat model , 17. HowGymnema inodorum were collected from Chiangda organic garden, Chiang Mai, Thailand, in October 2020. Identification and authentication of the collected plant were conducted at the Research Institute for Health Sciences, Chiang Mai University, and the voucher specimen (NRU64/036-001) was deposited.The leaves of G. inodorum were cleaned and dried in a hot air oven at 50°C overnight. The dried plant was ground into a coarse powder with mortar and pestle and subsequently prepared to found powder using an electric blender. 5 g of the plant material was extracted in 100 ml distilled water (DW) at a 60°C incubator with an occasional shaker for 15 min. After centrifugation at 2,500 rpm for 15 min, the supernatant was collected, and the residue was reextracted for a second time by adding another DW. The supernatant was combined and concentrated using lyophilization to obtain the aqueous crude extract of G. inodorum leaves (GIE). Finally, the GIE was stored in a desiccator until use [The fresh leaves of ntil use .ad libitum. All animal experiments with care and handling were conducted according to the NIH guidelines. Ethical approval was obtained from the animal ethics committee, Walailak University (WU-AICUC-63-031).Pathogen-free male ICR mice aged 6-8 weeks, weighing 25-35 g purchased from Nomura Siam International Co., Ltd., were used. Mice were acclimatized for 7 days before being used for the experiments. They were kept at 22-25°C, 12 h light-12 h dark cycle, with a pellet diet and clean drinking water Plasmodium berghei strain ANKA (PbANKA) was obtained from the MR4 . The parasite was maintained by intraperitoneal serial passage of infected blood from mouse to mouse. The infected donor mouse with a rising parasitemia of 20-30% was sacrificed, and the blood was collected by cardiac puncture into a vacuum tube containing heparin as an anticoagulant. The blood was then diluted by phosphate-buffered saline (PBS) based on the parasitemia of the donor mouse and the number of red blood cells (RBC) from the normal mouse (5 × 109 RBC/ml) in such a way that 1 ml of blood contained 5 × 107 parasitized RBC. Naïve mouse was subsequently inoculated intraperitoneally with 0.2 ml of infected blood containing 1 × 107 parasitized RBC of PbANKA.The chloroquine-sensitive Parasitemia was determined by microscopic examination of Giemsa-stained blood smear. Tail blood of each mouse was collected, and a thin smear on microscopic slides was prepared. After being allowed to air-dry, the smeared slides were fixed with absolute methanol and stained with 10% Giemsa at room temperature for 10 min. The stained slides were viewed microscopically using a light microscope with a 100x objective. The parasitemia was estimated by counting the number of parasitized RBC out of RBC in random 5 fields with approximately 200-500 cells. The percentage of parasitemia was calculated using the following formula:3EDTA for coagulation tests and platelet count, respectively. Platelet count was done using an automated blood count . Platelet-poor plasma was prepared from citrate blood by centrifugation at 3000 × g for 15 min and immediately frozen at -80°C until use. Blood coagulation tests, including prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT), were measured with a semiautomated blood coagulation analyzer  using human reagents. The experiments were performed according to the manufacturer's instructions in duplicate.Mouse blood was collected by cardiac puncture into a tube containing 3.2% buffered sodium citrate and K7 parasitized RBC). After three-hour postinfection, the three groups were administered orally by gavage with 100, 250, and 500 mg/kg of GIE by dissolving in 0.2 ml of DW once a day for 4 consecutive days (D0-D3). The other two groups were carried out as negative and positive controls, which were given 0.2 ml of DW and 10 mg/kg of chloroquine (CQ) dissolved in DW, respectively. Additionally, normal mice were also used as healthy controls. On D4, blood was collected by cardiac puncture for measuring platelet count and blood coagulation parameters, including PT, APTT, and TT.To determine the effect of GIE on the alteration of blood coagulation parameters, Peter's 4-day test was used in PbANKA-infected mice . Naïve Ip < 0.05 at the 95% confidence interval.GraphPad Prism version 6.0  was used for analysis in this study. A one-way ANOVA followed by Tukey's posttest was employed to compare the parameters between the control and extract-treated groups at D4 of the study. All results were expressed by the mean ± standard error of mean (SEM), and statistical significance was considered if R2 = 0.6668) between parasitemia and platelet count was also found.As shown in p < 0.001) decrease in platelet count in the untreated group compared to healthy controls. Interestingly, in GIE- (250 and 500 mg/kg) treated mice, platelet count was restored to a normal level compared to healthy controls. There were no statistically significant differences between 250 and 500 mg/kg of GIE- and CQ-treated groups. However, GIE at a dose of 100 mg/kg did not present antithrombocytopenia during PbANKA infection as indicated by a significant (p < 0.01) decrease of platelet count compared to healthy controls.GIE showed antithrombocytopenia induced by PbANKA infection in mice . PbANKA p < 0.05) shortened APTT was observed in untreated and GIE- (at the doses of 100 and 250 mg/kg) treated mice compared to healthy control. However, APTT was normalized compared to healthy controls in 500 mg/kg of GIE- and CQ-treated groups.To evaluate the effect of GIE on blood coagulation parameters during PbANKA infection, PT, APTT, and TT were investigated. As shown in Hematological abnormalities have been observed in patients with malaria as being the most common . Signifiα and interleukin- (IL-) 10 have the greatest impact on the inflammation during malaria infection, followed by thrombocytopenia and blood coagulation disorder [Significant alteration of APTT in PbANKA infection was observed in our study. It could be due to the hepatic involvement associated with malaria infection. Liver damage induced by PbANKA infection in mice has been reported with a strong association to hemostatic changes , 33. Mordisorder –36. The The results obtained from this study revealed that GIE had protective effects against thrombocytopenia during PbANKA infection in mice at the doses of 250 and 500 mg/kg. Moreover, the effect on restored blood coagulation parameters of GIE has also been observed. The significant effects of GIE accompanied by its relative safety may confirm the traditional use of GIE. However, the identification and characterization of active compounds and its mechanisms should be further determined whether these compounds have good activity to be considered good candidates for the development of alternative antimalarial drugs.


Post-trauma patients are at great risk of pulmonary embolism (PE), however, data assessing specific risk factors for post-traumatic PE are scarce. This was a nested case–control study using the Japan Trauma Data Bank between 2004 and 2017. We enrolled patients aged ≥ 16 years, Injury Severity Score ≥ 9, and length of hospital stay ≥ 2 days, with PE and without PE, using propensity score matching. We conducted logistic regression analyses to examine risk factors for PE. We included 719 patients with PE and 3595 patients without PE. Of these patients, 1864 [43.2%] were male, and their median Interquartile Range (IQR) age was 73 [55–84] years. The major mechanism of injury was blunt (4282 [99.3%]). Median [IQR] Injury Severity Score (ISS) was 10 [9–18]. In the multivariate analysis, the variables spinal injury ; long bone open fracture in upper extremity and lower extremity ; central vein catheter ; and any surgery  were independently associated with PE. Spinal injury, long bone open fracture in extremities, central vein catheter placement, and any surgery were risk factors for post-traumatic PE. Prompt initiation of prophylaxis is needed for patients with such trauma. However, patients with lower extremity trauma were unable to receive mechanical prophylaxis. Neither could pharmacologic prophylaxis be applied to patients with high risk of bleeding—especially soon after trauma or surgery. It is thus important to carefully assess risk of PE for each individual patient.Pulmonary embolism (PE) poses a great risk of mortality and morbidity for patients after trauma10 and trauma severity14 are established risk factors. Other, more controversial risk factors of post-traumatic PE are injury sites, blood transfusion, and timing of definitive surgery. Previous studies were not designed to assess specific risk factors for post-traumatic PE, rather investigating the overall outcome of venous thromboembolism (VTE), including deep vein thrombosis (DVT). Few risk assessment studies have focused only on post-traumatic PE. Patients with post-traumatic PE, known as de novo PE, do not often have DVT. The risk of PE and DVT may be different among patients with trauma17.Previous studies evaluated the risk of post-traumatic PE. AgingWe therefore aimed to investigate specific unknown risk factors for acute PE such as injury sites and timing of definitive surgery in trauma patients using a nationwide trauma registry in Japan.18, which includes patient demographics, Abbreviated Injury Scale (AIS) scores, Injury Severity Score (ISS), emergency procedures, in-hospital complications, and clinical outcomes. Data collection was performed as a part of routine clinical patient management.We conducted a nested case–control study between January 2004 and December 2017 utilizing a nationwide trauma registry, the Japan Trauma Data Bank (JTDB) database, established in 2003. In 2017, a total of 264 hospitals, including 95% of all tertiary emergency medical centers in Japan, participated in the database13, which is the same method in previous studies21. We included only patients who survived for more than 2 days after hospital admission in order to exclude the impact of early trauma deaths22. We excluded patients who were pregnant, underwent chronic dialysis20, and received anticoagulation drugs  or general ward. We included only patients who had ISS ≥ 9 because PE is rare among patients with ISS < 924. The database recorded central vein catheter placements only at the emergency department.The primary outcome was occurrence of PE during hospital stay. The diagnosis of PE was based on the reports of the physician in charge. The database has no data on diagnostic measure. We categorized AIS codes representing fractures into open, closed, or unclassifiable, using AIS 90 Update 98 and AIS 2005 Update 2008. We categorized AIS codes such as 752,604.3 (humerus fracture), 752,804.3 (radius fracture), 753,204.3 (ulna fracture), 853,422.3 (tibia fracture), and 852,604.3 (pelvic fracture) in AIS 90 Update 98 as open fractures in this study because almost all were open, although these codes include several other kinds of fractures. We defined blood transfusion as any blood product within the first 24 h—the same method as that used in previous studies25. We employed nearest neighbor matching without replacement in a 1:5 manner. We used a caliper of 0.01 standard deviation of the logit of the PS. We evaluated standardized mean difference of the variables over 0.1 as a meaningful imbalance after PS matching. We calculated descriptive statistics comparing the PE group and the control group, using the Wilcoxon signed-rank test for continuous variables and the chi-square or Fisher exact test for categorical variables, having first evaluated the normality of all continuous variables using the Kolmogorov–Smirnov test. No continuous variables had normal distributions.We included patients from the database without PE as a control, using propensity score (PS) matching to ensure that the PE group and the control group were equally balanced on baseline characteristics and severity of trauma. We used the variables age, gender, vital signs at emergency department , mechanism of injury, transport type, ISS, and admission ward, to calculate PS. We carefully selected these variables based on clinical relevance and previous researchp values were two-sided; we considered p values of < 0.05 statistically significant. We performed statistical analyses using R software (Version 3.6.2)26.We used conditional logistic regression models to identify specific risk factors for post-traumatic PE. In the 1st model, the candidates of risk factors were body regions with AIS ≥ 3. In the 2nd model we included long bone and pelvic fracture instead of AIS ≥ 3 in the upper and lower extremities. In the 3rd model, we categorized long bone and pelvic fracture into open, closed, or unclassifiable fractures. In the final model, we added procedures such as blood transfusion, central vein catheter placement, and any surgery, to the 3rd model. We adjusted all the models for comorbidities . We defined missing values for blood transfusion as no blood transfusion (82 missing among the 4314 patients in this study). We replaced missing values for time of hospital arrival with time when an emergency medical technician had contacted patients at the injury site (67 missing among 1997 patients who received primary bone fixation).The Ethics Committee of the Juntendo University approved this study (IRB No. 19-010). We confirmed that all methods were performed in accordance with the relevant guidelines. We confirmed that the need for informed consent was waived by The Ethics Committee of the Juntendo University. The JTDB administrators also provided permission for use of the data from their database.The Ethics Committee of the Juntendo University, which did not mandate obtaining consent from patients in observational studies using anonymous data, approved this study (IRB No. 19-010).A total of 155,525 patients among the patients who were registered in the JTDB database were eligible for this study after inclusion and exclusion criteria. A total of 772 (0.5%) patients developed post-traumatic PE. After PS matching, the PE group comprised 719 patients and the control group comprised 3595 patients  Table . Such cop = 0.006), central vein catheter placement , and surgery . In both groups, bone fixation was the most frequent surgery (72.5% and 41.4%). Among patients who received bone fixation, a lower proportion of the PE group than that of the control group received bone fixation within 24 h (22.4% vs. 27.5%)  (Table p < 0.001). The PE group had longer hospital stays than the control group (32 [interquartile range (IQR): 19–60] days vs. 23 [IQR: 13–36] days, p < 0.001).The overall in-hospital mortality was higher in the PE group than in the control group  Table . In termTable In the subgroup analysis, we included 1997 patients treated with primary bone fixation. Our non-conditional logistic regression model showed that time to primary bone fixation of 24–120 h—compared to that within 24 h—was associated with PE ; open fracture in lower extremity (OR [95% CI]: 2.11 [1.50–2.96]); central vein catheter placement (OR [95% CI]: 1.91 [1.27–2.86]); and any surgery (OR [95% CI]: 3.53 [2.57–4.85])  led to pulmonary complications such as pneumonia and acute respiratory distress syndrome because post-traumatic and post-surgical inflammation act as “two hit model”In this study, patients with spinal injury, open fracture in the extremities, central vein catheter placement, and any surgery were at high risk of post-traumatic PE. Patients with these characteristics should be probably treated with pharmacological prophylaxis as soon as bleeding risk is adequately controlled.The current study has several strengths. First, our study focused on specific risk factor for post traumatic PE such as open fracture and delayed definitive surgery by using PS matching. By PS matching, our cohort was well balanced in terms of general known risk factors for PE such as age, gender, and severity. Second, our study could have less sampling bias because our study is nationwide cohort study which almost all tertiary emergency centers in Japan participated in. Characteristics of PE in this study could be representative for generalizability of post traumatic PE. Third, we focused on preventable post traumatic PE by excluding the impact of early trauma deaths. There must be no lethal PE occurring just after trauma caused by trauma-induced hypercoagulability in this study.43. Moreover, we performed a sensitivity analysis among ICU patients because prophylaxis has been well adhered to especially in ICU  in this study, which is consistent with previous studies (0.1–2.6%)42. In addition, physicians in Japan have easy access to the use of laboratory data such as D-Dimer, echocardiography, and echocardiography. Moreover, physicians in Japan, compared with those in other countries, have relatively easier access to the use of computed tomography, because there are many computed tomography scanners in Japan45.The current study has several important limitations that warrant discussion. First, neither mechanical nor pharmacological prophylaxis was recorded in this database. It could be potential confounding factors. However, prophylaxis has been well adhered to in Japan since 2004 because of the following reasons: the primary domestic guideline of prevention of VTE was published in 2004; and the payment of management fee to hospitals for the prevention of VTE started in 2004Among general trauma population, long bone open fracture in extremities, spinal injury, central vein catheter placement, and any surgery could be risk factors for post-traumatic PE. Prompt initiation of prophylaxis is needed for patients with such trauma. Among patients with bone fracture, delayed bone fixation also could be a risk factor for PE. Early definitive surgery for bone fracture could reduce risk for PE.Supplementary Tables.


Proton pump inhibitor (PPI) was widely used around the world. Studies suggested conflicting results between PPI treatment and dementia event. This study examined the association between six PPI agents and dementia event by mining the US FDA Adverse Event Reporting System (FAERS) database from 2004 to 2020. We employed proportional reporting ratio (PRR) and information element (IC) methods to detect the signals of dementia relevant to PPI. We also analyzed characteristics of PPI and positive control reports, compared dementia event between long- and short-duration of PPI treatment. Finally, we identified 2396 dementia cases with PPI treatment. We did not detect significant signal between PPI and dementia event: PRR = 0.98, 95%CI 0.94 to 1.02, IC = −0.03, 95%CI − 0.17 to 0.10, even in gastroesophageal reflux disease cases: PRR = 0.65, 95%CI 0.59 to 0.72, IC = −0.62, 95%CI − 0.97 to − 0.27. No significant differences of dementia event were detected between long- and short- duration groups, the OR (95%CI) of the 3 years, 5 years and 10 years comparison were 0.70 (0.48 to 1.02), 0.72 (0.45 to 1.15) and 1.65 (0.75 to 3.63), respectively. Based on the current FAERS data mining, we discovered no association between PPI use and dementia event, even in long-term PPI therapy case. Helicobacter pylori infection, or prevent side effects of glucocorticoids or non-steroidal anti-inflammatory drugs (NSAIDs)1. However, PPI agents were also overused by off label indication, excessive dosage and long-term treatment3.Proton pump inhibitor (PPI) was commonly used worldwide, to treat peptic ulcer disease (PUD), gastroesophageal reflux disease (GERD), 7. The association between PPI therapy and dementia event was a hot issue. PPI agents were reported to increase β-amyloid (Aβ) levels in the mouse brain by affecting the β- and γ-secretase enzymes8, and to lead to vitamin B12 deficiency which was associated with cognitive impairment9. Some studies reported PPI use could increase dementia event16. More recent studies found no significant association between PPI and dementia21. Professor Lai et al. expounded, to test dementia event, the potentially offending agent should be taken for a long time, such as PPI in GERD treatment22. However, the association between long-term PPI use and dementia event was also conflicting25.With the widespread use of PPI agents, numerous studies concerned the safety of PPI treatment26. To the end of 2020, FAERS had gathered more than twelve millions of adverse cases reported by both health professionals and non-health professionals. The FAERS data could be used to detect signals of drug-associated adverse event by data mining methods29. Data mining was shown to be effective in continuous pharmacovigilance monitoring of drug safety issues for old drugs such as PPI30. To the best of our knowledge, there was no research concerning the association between PPI and dementia based on FAERS database. The objective of present study was to detect the association between PPI use and dementia event by comprehensively assessing spontaneous reports submitted to the FAERS database.Adverse event reporting system (AERS) data was an outstanding source for pharmacovigilance analysis and post-marketing drug safety monitoring. The United States Food and Drug Administration AERS (FAERS) is one of the biggest databases open to the publicAfter data cleaning, we retrieved a total of 12,875,561 cases from January 2004 to December 2020, 6,074,285 of which were reported by health professionals. We finally identified 2396 PPI cases, as well as 24,920 anticholinerigc drug (AC) and 9667 benzodiazepine drug (BD) cases as positive controls, with dementia event reported by health professionals  daily dose of each PPI agent was within the range recommended by drug label Table .Table 4DThe current study investigated the association between six PPI agents and dementia event, compared different time interval of PPI treatment and dementia event. The results indicated no association between dementia event and PPI agents, including dexlansoprazole, esomeprazole, lansoprazole, omeprazole, pantoprazole and rabeprazole. To the best of our knowledge, this was the first pharmacovigilance study concerned the association between PPI use and dementia event based on FAERS database.With the widespread use of PPI agents, PPI-associated adverse event had caught health professionals’ attention, as well as the public and the media. In FAERS database, more than half of the PPI adverse event cases were reported by non-health professionals. To reduce the influence of non-health professionals, we only included cases reported by health professionals. However, the proportion of case reported by physician and pharmacist was less than a half for PPI, smaller than AC and BD. Moreover, the number of dementia case treated by PPI was increasing by years, especially from the year of 2018. The suddenly increased case number might be caused by the suddenly increased annual FAERS reports in 2018 and concerns of PPI safety in recent years. The risk of stimulated reporting, both by clinical evidence and media influence, could not be ruled out.8, vitamin B12 deficiency phenomena9 and the widespread PPI use, the association between dementia event and PPI use had become a hot topic. Professor Akter et al. first revealed five different PPI agents had varying degrees of influence on different cognitive domains associated with dementia based on the Cambridge Neuropsychological Test Automated Battery (CANTAB) software test10. Professor Haenisch et al. conducted the first epidemiological investigation, indicated PPI might have an impact on dementia based on the German Study on Aging, Cognition and Dementia in Primary Care Patients (AgeCoDe)11. Then, professor Gomm et al. conducted the first prospective cohort study, revealed regular PPI treatment had a significantly increased risk of dementia using data derived from the largest German statutory health insurer, Allgemeine Ortskrankenkassen (AOK)12, which had been hotly commented.Based on the β-amyloid enhancement31. Professor Taipale et al. finished a nationwide nested case–control study which set a lag window of different duration, found PPI use was not associated with risk of Alzheimer's disease with a 3-year lag window32. Professor Gray et al. reported a prospective cohort study and found no association between PPI exposure and dementia event after a mean follow-up of 7.5 years20. Professor Cooksey et al. conducted a large population-based study based on electronic health-data from the Secure Anonymised Information Linkage (SAIL) Databank from 1999 to 2015, could not confirm an association between PPI use and dementia event17. The current study based the FAERS big data, indicated no significant signal between PPI use and dementia event. Even compared in different time duration, no significant difference of dementia event was found between long- and short-term groups.However, conflicting results had been gradually published. Professor Lochhead et al. conducted a nationwide prospective cohort study and divided PPI users into four groups based on duration of PPI treatment, revealed a modest association between duration of PPI use and cognitive function, however, Lochhead stated that the result could not support PPI use increased dementia event33. The time event comparison only included limited cases with time data reported. Although non-health professionals’ reports excluded, the risk of stimulated reporting could not be eliminated.Our study revealed no association between PPI use and dementia event based on the FAERS real world big data, however, certain limitations existed. FAERS is a spontaneous reporting system, voluntary and opened to health professional as well as the public, so under-reporting, over-reporting or missing data was inevitableIn summary, the current study revealed no association between six PPI agents and dementia event based on the FAERS data mining. Our findings suggested that dementia event might not be considered as a factor in discontinuing PPI treatment.34. FAERS data was processed anonymously, no ethical review was required.We downloaded FAERS data from January 2004 to December 2020 in the FAERS Quarterly Data Extract Files websiteThe FAERS datasets consisted of seven data tables as follow: “DEMO” table for patient demographic and administrative information, “DRUG” table for the drug information, “REAC” table for adverse events information, “OUTC” table for patient outcomes information, “RPSR” table for report sources information, “THER” table for drug therapy start and end dates information and “INDI” table for the indications for drug use. We managed FAERS data in local by Microsoft SQL server 2017 software.We first removed duplicated cases from the original data as the FDA recommended. We removed the same records from “DEMO” table and left one, then deleted the earliest FDA_DT when the CASEIDs were the same and removed the lower PRIMARYID when the CASEID and FDA_DT were the same. We further removed cases listed in the FAERS deleted files. In the current study, we only included cases reported by health professionals, including physicians, pharmacists and other health professionals, for both target drug data and background data.36.In “DRUG” table, drugs could be documented in various forms, such as generic names, brand names, synonymous names or their abbreviations. We used the MedEx software  to standardize different names of the same drug into the “generic name”We tried to identify seven single component PPI agents with the WHO Anatomical Therapeutic Chemical (ATC) code of A02BC from local FAERS database. The seven PPI agents (ATC code) included omeprazole (A02BC01), pantoprazole (A02BC02), lansoprazole (A02BC03), rabeprazole (A02BC04), esomeprazole (A02BC05), dexlansoprazole (A02BC06) and dexrabeprazole (A02BC07). We restricted the drug role as Primary Suspected (PS) drug.37 and 30 BD drugs following three BD data mining studies40, detailed in Supplementary Table We also identified anticholinerigc drugs (AC) and benzodiazepine drugs (BD) as positive controls. We included 56 AC drugs following the Coupland et al. studyAccording to Medical Dictionary for Regularly Activities (MedDRA) and Standardised MedDRA Queries (SMQs) version 23.1. We identified dementia cases in “REAC” table using SMQ  broad searching, including 105 Preferred Terms (PTs). For cases reported more than one PT of the same SMQ, we removed duplicate records and kept one. The PTs details could be found in Supplementary Table We gathered the characteristics of dementia case with PPI, AC and BD, including age and sex, reporter and report country, annual case reported, indications and daily dosage.2 ≥ 441. The IC signal criteria was IC > 0 and the lower limit of 95% CI > 028.We employed both proportional reporting ratio  and information component  to detect signals of dementia event relevant to PPI, as well as AC and BD positive controls. The calculation method of PRR, IC and the 95% confidence interval (95% CI) were shown in Supplementary Table We further calculated signal between PPI use and dementia event in GERD cases who might receive long-term PPI treatment.P value less than 0.05 indicated significant difference.We estimated the time interval from PPI use to adverse event reported in all PPI (PS) cases reported by health professionals in FAERS. We unified the time format as yyyy-mm-dd. The time interval was calculated using event date (EVENT_DT) minus drug start date (START_DT). To make the calculation more accurately, we excluded cases not in the period of 2004–2020, cases without year or month data in either EVENT_DT or START_DT field, and cases with earlier event date than drug start date. For the long- and short-duration comparison, we calculated the odds ratio (OR) using formula in Supplementary Table The statistical analyses were conducted by Microsoft Excel version 2013 , SPSS version 25.0  and GraphPad prism version 8.0.2 .Supplementary Information.


Posttraumatic stress disorder (PTSD) has been shown to be elevated among first responders  compared to the general population. Examining the prevalence of mental health issues in a work force with an elevated occupational risk is fundamental towards ensuring their wellbeing and implementing safeguard measures. The goal of this study is therefore to report the prevalence of depression, anxiety, posttraumatic development, and PTSD in Norwegian ambulance personnel.This study is a cross-sectional, anonymous, web-based survey (Questback®), performed among operative personnel employed in the Emergency Medical Services in the Regional Health Trust of Central Norway between 18. February and 9. April 2021. The study was sent to 1052 eligible participants. Questions reported demographic data, a traumatic events exposure index, Patient Health Questionnaire-9 (Depression), Generalized Anxiety Disorder-7 scale, Posttraumatic symptom scale (PTSD) and Posttraumatic change scale.The response rate in this study was 45.5% (n = 479/1052). The mean age of respondents was 37.1 years (std. 11.1) and 52.8% (n = 253) were male. Of the respondents, 80.6% (n = 386) were married or had a partner, and 91.6% (n = 439) reported having access to a peer support programme, with 34.9% (n = 167) reporting that they had utilized peer support. In this study, 5% (n = 24) showed a prevalence of manifest posttraumatic stress disorder symptoms, while 8.6% (n = 41) reported moderate to severe depression and 2.9% (n = 14) presented moderate to severe symptoms of general anxiety. Of the respondents, 77.2% (n = 370) reported personal growth because of their work experiences.This study indicates that Norwegian ambulance personnel report a prevalence of posttraumatic stress symptoms and depression, which is slightly higher for men, and lower for the female proportion in this study, when compared to an adult Norwegian population. The vast majority of respondents reported personal growth because of their work experience, and both the degree of peer support and having a partner seem to influence levels of posttraumatic stress and -development.The online version contains supplementary material available at 10.1186/s13049-021-00991-2. As part of their regular work load emergency service personnel are exposed to an array of potentially traumatic events, which may influence their physical or psychological wellbeing . Such stStudies indicate that EMS personnel have the highest rate of PTSD among these first responder service branches . This maThe precipitating events and criterion of PTSD are defined by the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) . SpecifiExamining the prevalence of mental health issues in a work force with an elevated occupational risk is fundamental towards ensuring their wellbeing and implementing safeguard measures. The goal of this study is therefore to report the prevalence of depression, anxiety and PTSD in Norwegian ambulance personnel. Due to recent studies showing that traumatic exposure may also impact both negatively and positively on personal development, we also measured the degree of reported posttraumatic growth or deprecation as a consequence of exposure to work-related events .The Regional Health Trust of Central Norway is one of four regional health trusts. It funds and manages 65 EMS stations in a geographical diverse area with mountains, fjords, Atlantic coastline, towns and cities, covering 56,558 square kilometres. In 2019 the region had a population of 731,480, and the EMS performed 94,250 missions, of which 38.1% were emergency red responses.Personnel employed in the Norwegian EMS are primarily emergency medical technicians (EMT). The paramedic title in Norway largely belongs to EMT’s with additional education, but a new Bachelor paramedic program is currently in progress. In addition, qualified nurses also work in EMS. Normally, there is a crew of two EMT’s working on an ambulance. They are dispatched and coordinated by the Emergency Medical dispatch centre, of which there are three in this region. Identical peer-to-peer support programs have been implemented in two of the three health trusts (Nord- and Sør-Trøndelag) involved in the study. This entails an education of selected co-workers in defusing and debriefing techniques based on one-to-one meetings. Criteria for activation are similar to the events described in the Traumatic events exposure index Table  utilizedThis study is a cross-sectional, anonymous, web-based survey (Questback®), performed among operative personnel employed in the EMS in the Regional Health Trust of Central Norway between 18. February and 9. April 2021. The leaders of all the responsible departments and ambulance stations in the Health Trusts were contacted for confirmation to participate, and for updated electronic mail address lists for those eligible to take part in the study.The study was sent to 1052 eligible participants, 558 male and 494 female. Participation was voluntary, and required consent from participants. During the study period, two reminders were sent to non-responders, by the same electronic mail format.Demographical data , cohabitation, peer support access).Traumatic events exposure index, 10 items . The participant reports exposure to defined traumatic events from the working environment during the last 12 months. In addition, the participant is asked to report if the event was perceived as a traumatic experience.PHQ-9  is a tool specific to depression, and scores each of the nine DSM-IV criteria based on the mood modules from the original PRIME-MD  scale. Contains seven items, score for each item 0–3, score range 0–21. Score 5–9 indicates mild anxiety, 10–14 moderate anxiety and 15–21 severe anxiety . In thisCronbach’s α = 0.815 .PTCS  contains 12 items. It investigates three dimensions: self-confidence, interpersonal involvement including social adaptability, and awareness, Developed and validated by the Norwegian Joint Medical Services . Scores Cronbach’s α = 0.885 .PTSS  contains 10 items measuring posttraumatic symptoms during the last 7 days. It consists of a 7-point Likert scale (1–7) with possible total score range 10–70. In this study the total sum score ≥ 35 is considered the cut-off threshold for probable PTSD. It was developed and validated for a Norwegian population .Cronbach’s α = 0.901 .The survey consisted of questions including:All questions were submitted in Norwegian language. The Additional file Data was analyzed using the Statistical Package for the Social Sciences . Statistical data is presented as frequencies, percentages, average with mean and standard deviation (SD), median with interquartile range (IQR), where appropriate. Statistical comparisons between the demographic variables (independent) and dependent test variables  were performed using Pearson’s Chi square test or Fischer’s test, where applicable. Independent Sample t-test was applied to compare means of age. A simple linear regression analysis was performed to estimate strength of association between PHQ-9, GAD-7 and PTSS. Statistical significance was defined by p < 0.05.Internal consistency of the different scales/tests applied in this study was measured utilizing Crombach’s alpha, which is reported in the description of the appropriate scale in this methods section.The study was answered by 500 (47.5%) recipients, including 21 (2%) who declined to take part. Ultimately, 479 (45.5%) consented to, and completed the questionnaire, and are therefore included in the study. The mean age of respondents was 37.1 years (std. 11.1), 52.8% (n = 253) were male (mean age 39.6 (std. 11.2)) and 47.2% (n = 226) were female (mean age 34.4 (std.10.4)). The median number of years of work experience in EMS was 12 (IQR 12–19), and reported education level was as follows: student/trainee 8.4% (n = 40), EMT 46.1% (n = 221), nurse 21.1% (n = 101), paramedic 24.4% (n = 117). Of the respondents, 80.6% (n = 386) were married or had a partner, and 91.6% (n = 439) reported having access to a peer support programme, with 34.9% (n = 167) reporting that they had utilized peer support.The median score in the study population for depression (PHQ-9) was 3 (IQR 2–6), anxiety (GAD-7) was 2 IQR 1–4) and posttraumatic stress (PTSS) was 14 (IQR 11–20). Further results on anxiety, depression and posttraumatic stress symptoms in the study population are reported in Table –4 and poThe threshold for manifest posttraumatic stress disorder symptoms in this self-reporting study was defined as a score ≥ 35 on the PTSS. In this study, n = 24 (5%) attained this score (mean 34 years (std. 1.8))  or GAD-7 (depression) as independent values. The assumption of normality and linearity was not violated in these cases. In both cases we found a positive relationship .The Posttraumatic change scale was applied in this study to determine perceived emotional growth, deprecation or no-growth as a self-reported consequence of work experiences in EMS Table . Mean scOf the 76 respondents who reported deprecation Table , 33  and 8.5% (women), while the current study reported 4.7% men and 5.3% women . Of noteThe relative low prevalence of reported PTSD in this study may have several reasons. First, EMS in Norway has become increasingly professionalized, with focus on selection, training and further education. Almost half of respondents in this study were either qualified nurses or paramedics, and more than 80% were in full time positions. Studies among fire fighters have shown increased PTSD among part-time versus full-time employees . There hA recent Norwegian study of the general population showed a prevalence of self-diagnosed current depression at 8.1%, higher for women (9.8%) than men (6.1%), and another similar Norwegian study reported anxiety at 6.6% , 30. ThiThe implementation of preventative measures in the work environment may improve the mental wellbeing of employees . In thisThis study indicates that Norwegian EMS personnel are regularly exposed to potentially traumatic events. Traditionally, traumatic stress has been understood as relating to life-threatening events and subsequent fear dysregulation, and it may be more intuitive to associate exposure to violent events and threats with increased stress, which we also observed in this survey. However, in many traumatic situations, peritraumatic fear may not be present, and a physical threat may not be the most stressful part of the incident , 37. Accpositive psychological changes experienced as a result of the struggle with highly challenging life circumstances” [Resilience in this context can be described as the ability of an individual to adjust to challenges and influences which potentially may be psychological harmful . That mostances” . This mostances” , 41.This study has several limitations. The response rate was 46%, which means that findings should be interpreted with caution. Missing answers could definitely influence results, and there may be a risk that the most troubled by traumatic experiences did not answer our survey. However, response rates have been described as a typical challenge in studies of related occupational groups . In simiValidated tests were applied to measure posttraumatic stress, posttraumatic growth, depression and anxiety. This study can only give an indication of the prevalence of these serious conditions, and self-reported questionnaires cannot compare to diagnostic interviews in this respect . In addiThis study indicates that Norwegian ambulance personnel report a prevalence of posttraumatic stress symptoms and depression, which is slightly higher for men, and lower for the female proportion in this study, when compared to an adult Norwegian population. We observed an association of increased posttraumatic stress symptoms in workers who were unmarried/had no partner, in those reporting no access to peer support, or who experienced feelings of inadequacy towards patients, including those exposed to ambulance accidents.The vast majority of respondents reported personal growth as a consequence of their work experience, and both the degree of peer support and having a partner seem to influence levels of posttraumatic growth. On an institutional level, we therefore recommend having robust, accessible and widely advertised peer support programs in place, to mitigate the negative effects of an apparently demanding profession, and would advocate for similar studies among other prehospital first responders, for instance the air ambulance or fire department.Additional file 1. English version of inex and scales.Additional file 2. Study invitation and consent to participation.


Deverra tortuosa in streptozotocin-induced diabetic rats were investigated in vivo. A single intraperitoneal injection of 160 mg/kg bodyweights of streptozotocin was used to cause diabetes. Blood glucose levels were tested with an AccuCheck Advantage II glucometer and blood glucose test strips. After diabetes was confirmed, animals were orally treated with the extract, metformin, and insulin according to the experimental design. After extract therapy, histological alterations in the pancreas of diabetic rats were investigated. When compared to a control group, daily oral administration of D. tortuosa extract (300 mg/kg body weight) plus metformin (100 mg/kg) had a positive effect on blood glucose levels as well as showed an increased number of white blood cells (WBCs) and red blood cells (RBCs). The treatment with the extract for two weeks showed a positive impact on pancreatic histopathological changes in the groups with the diabetic rats. Phenolic fraction of the methanol extract was screened by the liquid chromatography-mass spectroscopy (LC-MS) method, which unveiled the existence of flavonoid compounds and phenolics as kaempferol, rutin, isorhamnetin-3-O-rutinoside, caffeic acid, and 4-hydroxybenzoic acid 4-O-glucoside. The results confirmed the use of the plant as an antidiabetic agent; the research recommended further studies on the plant to use the plant as an antidiabetic drug, where the plant extract also showed improvement in blood parameters.Herbal plants represent a new source of hypoglycemic antidiabetic drugs; haematological and histopathological studies of methanol extract of  The use of herbal medicines has increased in the last decade because of their effectiveness, safety, and affordability. WHO recommended the use of traditional plants in the treatment of diabetes. Products isolated from medicinal plants are usually used in the traditional Indian system and have shown clinical antidiabetic activity . One of D. tortuosa is used as “edible food; its aerial parts are used as fuel wood and have many medical uses. Saudi Arabian plant D. tortuosa showed antioxidant, allelopathic, and antifungal activity, used in traditional medicine as a drug for hypertension and against conception [D. tortuosa is a salt-tolerant plant; it is grown in many phytogeographical habitats in Egypt, in desert wadis, sandy, and stony plains. This plant is dispersed in Egypt, Tunisia, Libya, and Saudi Arabia [nception , 4. Azzanception . D. torti Arabia .D. tortuosa  belongs to Apiaceae family, is an Arabic name, and has an aromatic scent; it is characterized by a high concentration of phytochemical compounds, flavonoids, coumarins, furanocoumarin, glucosides, and terpenes [D. tortuosa has anti-Candida activity and antivirulence activity [terpenes , 8. The activity .D. tortuosa as an antidiabetic through studying the effect of plant extract in vivo on albino diabetic rats; histopathology was studied to clarify the role of plant extract on pancreatic tissues; haematological studies were used to show the effect of treatment by herb on blood parameters.The current research was aimed to evaluate the role of plant The fresh plant sample  was collPlant sample was dried at room temperature, the aerial part was grounded for fine powder, methanol extract was prepared by maceration method, and the plant powder (500 g) was soaked in methanol for 24 hours in a shaker at 25°C. Then, the extract was filtered and stored at −4°C for further investigation .Laboratory of Molecular Genetics (Egyptian Association for Sustainable Agri.), Egypt .150–180 gm weighted adult healthy albino Male rats were used for the experimental study. The animals were housed at regular conditions . Water and a normal diet were allowed to all animals . All experimental procedures were in accordance with the guidelines and instructions of Scientific Research Ethics (COSRE) in   Group 1: control group, only saline injection.  Group 2: diabetic group injected with 58 mg/kg STZ i.p.  Group 3: diabetic group injected with metformin with 100 mg/kg body weight for 2 weeks.  Group 4: diabetic group injected with insulin at a dose of 10 U/kg body weight for 2 weeks.  Group 5: diabetic group injected with plant extract at a dose of 300 mg/kg body weight for 2 weeks.Induction of diabetes was done by single intraperitoneal (i.p.) injection of STZ in a dose of 58 mg/kg. Thirty animals were used and divided in to 5 groups :The OECD guideline no. 420 was used to conduct an acute toxicity test on a plant extract (Organization of Economic Co-operation and Development) according to the methods described by Elsharkawy et al. . At the Blood samples were taken from each rat after the treatment and recovery period. The analysis was done for estimation of hemoglobin concentration according to Drabkin and Austin , red bloμm pieces from each block. They were stained with hematoxylin-eosin (HE) staining and prepared for histopathological examination. A light microscope was used to image and examine the relevant regions  [The pancreatic tissue samples were fixed in 10% formalin solution for 48 hours before being rinsed under running water for 8 hours for histopathological examination. During the usual tissue control phase, they were treated with alcohol  and a succession of xylenes before being blocked in paraffin. On the slides, the samples were produced by cutting 4 , Japan) .μg/mL) solution was prepared using high-performance liquid chromatography (HPLC) analytical grade solvent of MeOH, filtered using a membrane disc filter (0.2 μm), and then subjected to LC-ESI-MS analysis. Sample injection volumes (10 μL) were injected into the UPLC instrument equipped with a reverse phase C-18 column . The sample mobile phase was prepared by filtering using a 0.2 μm membrane disc filter and degassed by sonication before injection.The sample  and mass spectrum to the data.A gradient mobile phase included two eluents: eluent A is Ht-test was employed to determine if there were any significant differences between each tested compound with the referenced antifungal drug. The program used was Statistical Package for the Social Sciences (SPSS), version 11.Quantitative data were expressed as the mean ± standard error of means. A paired-sample D. tortuosa in doses up to 2, 000 mg/kg failed to kill albino Male rats within 24 h. The tested extract is considered highly safe, since substances possessing LD50 higher than 50 mg/kg b.wt. are considered nontoxic [Oral administration of alcoholic extract of nontoxic .P < 0.05) in the level of blood insulin (0.33 ng/ml), when compared with the normal control. Similarly, the metformin-treated group showed no noteworthy growth, while insulin diabetic-treated groups recorded a considerable boost (P < 0.05) in blood insulin levels  when compared to the normal group. The treatment with herbal extract showed a mild decrease in blood insulin level (0.93 ng/ml) when compared with the normal group.The impacts of metformin, insulin, and herbal extracts on blood insulin levels in normal and diabetic rats subjected to various treatments for 2 weeks are reported in P < 0.05) in body weight (17%) when compared with the initial weight. Similarly, the metformin and insulin diabetic-treated groups recorded a significant decrease (P < 0.05) in body weight  when compared with the initial weight. The treatment with herbal extracts recorded a significant decrease (P < 0.05) in body weight (7.5%) when compared with the initial weight.The effects of metformin, insulin, and herbal extracts on body weight in normal and diabetic rats subjected to various treatments for two weeks are summarized in P < 0.05) in the RBC count (46.8%), Hb concentration (10.8%), and platelet count (37.4%) when compared with the corresponding control values.The control group showed normal RBC count, Hb concentration, and platelet count. The diabetic group recorded a significant decrease (P < 0.05) in the RBC count , the Hb concentration in metformin and insulin diabetic-treated groups recorded no significant change , and platelet count in metformin diabetic-treated group recorded no significant increase (36.7%), while the insulin diabetic-treated groups recorded significant increase (54.7%) when compared with the corresponding values in the diabetic group. The treatment with herbal extract exhibited a substantial increase (P < 0.05) in the count of RBCs (80.7%); finally, the platelet count showed a significant decrease in the herb by about 76.4%, when compared with the corresponding values in the diabetic-treated group as shown in Similarly, the metformin and insulin diabetic-treated groups recorded a noteworthy upsurge P < 0.0 in the Rrease 36.%, while D. tortuosa on WBC count is illustrated in P < 0.05) in WBC quantities (46.01%) when compared with the corresponding control values. Similarly, the metformin and insulin diabetic-treated groups recorded a substantial increase (P < 0.05) in the count of WBCs  when compared with the corresponding diabetic groups. The treatment with plant extract recorded a substantial increase (P < 0.05) in WBCs (78.7%) when compared with the corresponding diabetic groups.The effect of methanol extract of D. tortuosa, the atrophy of Langerhans islets, degeneration, and apoptosis by B cell decreased, and pancreatic structure was restored in diabetic rats. Additionally, metformin treatment changed the pancreatic structure.Ultraperformance liquid chromatography in combination with mass spectrometry method was deployed to examine the phytochemical components in plant extract, the phenolic fraction of methanol extract was analyzed by UPLC/ESI-MS, phytochemical screening of plant extract revealed the presence of a high amount of flavonoid compounds, and this was confirmed by identification of flavonoid, flavonoid glucoside, and phenolic acid by using positive mode (+) ESI-Ms, and the identification of compounds was determined on the basis of the pattern of mass fragments and ion response. Compounds were identified as listed in The chemical was identified using literature data to compare the molecular ion peak (M-H) and fragmentation patterns derived from MS/MS , 19. FroD. tortuosa is used as a traditional medicinal plant in Egypt and Saudi Arabia. The current study was assumed to examine the antidiabetic activity of methanol extract of the plant. Diabetes is a common disease spread in Arabian countries and the world affecting the major population. It is a metabolic disease, causing complication in many organs such as liver, kidney, and pancreas [pancreas . An expepancreas .The diagnosis of diabetes on male Wister albino rats was confirmed by increased serum glucose level after 28 days of injection of STZ in diabetic rats. The serum glucose level was measured as 541.5 ± 15.39 mg/dL in Group 2, as compared with the nondiabetic rats (control group). It was observed that the glucose levels of Groups 3 and 4 decrease, while the glucose levels of Group 5 were higher than those of Groups 3 and 4 but lower than those of Group 2 (STZ). The serum insulin level of Group 1 was 34.88 ± 0.36 mg/dl. The serum insulin level of the STZ induced rats (Group 2) was lower than that of Group 1 (0.33 ± 0.06 mg/dl), while the herb (Group 5) showed a decrease in serum insulin level (0.93 ± 0.11 mg/dl) more than Group 3 (metformin). This is common and agrees with a pervious study on Apiaceae family used to treat diabetes .Flavonoid compounds have many biological activities; for example, they protect the body against oxidative stress. Flavonoid-rich food reduces the risk of oxidative compounds , 25. IsoNigella sativa extract caused significant increase in hemoglobin and platelet counts. The plant D. tortuosa in this study can be used as an antidiabetic drug and we recommended following the study on this plant.Study of the effect of a phenolic fraction on haematological parameters showed a decrease in RBCs, Hb content, and platelet count in diabetic rats, and this decrease in RBCs and Hb indicated the rats suffer from anemia, as a result of the toxic effect of STZ. Meanwhile the treatment with herbal extract exhibited a significant increase in the RBC count (80.7%). The platelet count showed a significant decrease in the herb-treated group, by approximately 76.4% when compared with the corresponding values in the diabetic-treated group, while there was no significant change in Hb concentration compared with the diabetic group. Current studies came to agree with other previous studies by Helal et al.  and wereD. tortuosa decreases the blood glucose level and improves blood parameters, increasing WBCs and RBCs compared with the diabetic group. The extract also inhibits histopathological changes induced in the pancreas of diabetes group. Phytochemical screening by HPLC/MS of flavonoid fraction revealed the presence of more than 20 peaks; 12 peaks were identified by comparison with literature data. The major flavonoid compounds rutin, kaempferol and isorhamnetin-3-O-rutinoside confirmed the role of the plant as an antidiabetic because these compounds have antioxidant activity and decrease the oxidative stress, which is considered as one of the most important reasons for the pancreatic damage. These obtained results support the use of the plant in antidiabetic traditional preparations after further studies.In the current study, a glucose induction hyperglycemic model was selected to monitor the antihyperglycemic activity of plant extract. The results concluded that methanol extract of