Vitamin B12 affects the dynamics of autonomic nervous system and its deficits has been linked to cardiovascular autonomic neuropathy therefore, vitamin B12 deficiency was hypothesized to be implicated in the development of diabetic gastroparesis. This study was conducted to explore the possible association between vitamin B12 deficiency and gastroparesis in patients with type 2 diabetesA total of 100 T2D patients with diabetes duration > 10 years and 50 healthy controls matched for age and sex were recruited for this study. T2D patients were divided into 2 groups: patients with gastroparesis and patients without gastroparesis. The diagnosis of gastroparesis was based on Gastroparesis Cardinal Symptom Index (GCSI) Score ≥ 1.9 and ultrasonographic findings including gastric emptying ˂ 35.67% and motility index ˂ 5.1. Anthropometric measurements, plasma glucose, glycosylated hemoglobin (HbA1c), lipids profile, vitamin B12 and transabdominal ultrasonography were assessed.The frequency of vitamin B12 deficiency in total patients with T2D was 35% . Vitamin B12 level was negatively correlated with GCSI Score whereas, it was positively correlated with gastric emptying and motility index. Vitamin B12 deficiency was an independent predictor for gastroparesis in patients with T2D; it predicts gastroparesis at a cut off value of 189.5 pmol/L with 69.1% sensitivity and 64.4% specificity, P = 0.002.Beside the known risk factors of diabetic gastroparesis, vitamin B12 deficiency is an independent predictor of diabetic gastroparesis in patients with T2D. Diabetic gastroparesis is a clinical syndrome characterized by delayed gastric emptying in the absence of mechanical obstruction of the stomach . The chaRecently, vitamin B12 levels have been found to be inversely related to glucose intolerance . AdditioThis study comprised 100 adult patients with T2D and 50 age- and sex-matched healthy controls. Patients with T2D were consecutively recruited from Diabetes Outpatient Clinic at Mansoura Specialized Medical Hospital, Mansoura University, Mansoura, Egypt. The inclusion criteria were patients with duration of T2D > 10 years and who had symptoms of gastroparesis [Gastroparesis Cardinal Symptom Index (GCSI) Score ≥ 1.9]. Patients with T2D were submitted for transabdominal ultrasonography accordingly, they divided into 2 groups: patients with gastroparesis (n = 55) and patients without gastroparesis (n = 45). Exclusion criteria were history of digestive tract surgery or prior gastric outlet obstruction, thyroid disease, liver & renal failure, neuropsychiatric disorder, connective tissue disorders, malignancies, pregnancy and participants taking vitamin B12 and alcohol. Drugs that could potentially interfere with gastrointestinal motility such as GLP-1 receptor agonists and the amylin analog, α glucosidase inhibitors, and opioid analgesic were also excluded. Healthy controls were recruited from the same geographic area with the same exclusion criteria.2), and waist circumference (WC) were obtained using standardized techniques. The diagnosis of diabetic gastroparesis was based on the symptom validated questionnaire GCSI Score ≥ 1.9 and ultrasonographic findings including gastric emptying ˂ 35.67% and motility index ˂ 5.1. The cut-off point of gastric emptying and motility index were calculated from our study healthy controls as mean—2SD.All participants were subjected to a thorough medical history and underwent a clinical examination. Anthropometric measurements including height, body weight, body mass index (BMI) , postprandial fullness/early satiety subscale  and bloating subscale (2 symptoms: bloating and stomach distension). All symptoms are rated from 0 to 5 over the prior 2 weeks . GCSI Score was calculated as the average of the 3 symptom subscales . The cliVitamin B12 deficiency was defined as vitamin B12 levels below 125 pmol/L . PeripheFasting plasma glucose (FPG) and 2-h post prandial plasma glucose (PPG) were measured by commercially available kit, Cobas (Integra-400) supplied by Roche Diagnostics . Glycated hemoglobin (HbA1c) was estimated as an index of metabolic control on a DCA 2000 analyzer, fast ion exchange resin , triglycerides (TGs) and high density lipoprotein cholesterol (HDL-C) were measured by commercially available kits (Cobas Integra-400). Low density lipoprotein cholesterol (LDL-C) was calculated according to Friedewald et al. . CompletAfter an overnight fasting, patients sat in a chair, leaned slightly backwards and drank 400 ml meat soup . An ultrasound probe was positioned vertically to permit simultaneous visualization of the gastric antrum, superior mesenteric artery and abdominal aorta for evaluation of the antral contractions Fig This study was a pilot study with an initial sample size of 20 T2D patients who were excluded from the full scale study. The final calculated sample size was 97. Data entry and analysis were done by the SPSS statistical package . The data were expressed as mean ± SD for continuous data, number and percent for categorical data and median (minimum–maximum) for skewed data. Student’s t and Mann–Whitney U tests were used to compare the 2 studied groups for parametric and non-parametric data, respectively. The chi-square and Fischer exact tests were performed to compare 2 or more groups of qualitative variables. The correlations of GCSI Score, gastric emptying and motility index with all other studied variables were analyzed by the Pearson and Spearman correlations analysis. Binary stepwise logistic regression analysis was used to predict the independent variables of binary outcome; the significant predictors in the univariate analysis were entered into the regression model. Receiver operating characteristic (ROC) curve was done to detect the level of vitamin B12 associated with gastroparesis in patients with T2D. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), area under the curve (AUC) and 95% CI were evaluated. P ≤ 0.05 was considered as significant.Patients with T2D had significantly higher BMI, WC, systolic & diastolic blood pressures (SBP & DBP), FPG, 2 h PPG, HbA1c, TC, TGs, LDL-C and lower HDL-C than did healthy controls. Among the studied participants with T2D, the median age of diabetes duration was 17 years, 63% had hypertension, 32% had retinopathy, 24% had diabetic nephropathy and 43% had neuropathy . With regard to anti-diabetic medications, 40% received insulin, 37% received oral anti-diabetic drugs and 23% received combined oral anti-diabetic drugs and insulin. Vitamin B12 was significantly lower in patients with T2D than in healthy controls, the frequency of vitamin B12 deficiency was 35% Table T2D patients with gastroparesis had significantly longer diabetes duration, higher BMI, WC, SBP, DBP, FPG, HbA1c, TC, TGs and LDL-C compared with those without gastroparesis. The frequency of hypertension, proliferative retinopathy, nephropathy and peripheral neuropathy were significantly higher in patients with gastroparesis than in those without gastroparesis. Vitamin B12 level was significantly lower in patients with gastroparesis than in those without gastroparesis. The frequency of vitamin B12 deficiency was 54.5% in patients with gastroparesis and 11.1% in patients without gastroparesis, P = 0.001. The grade of gastroparesis in T2D patients was distributed as 78.2% for grade 1, 21.8% for grade 2 with no reported cases in grade 3. Gastric emptying and motility index were significantly lower in T2D patients with gastroparesis than in those without gastroparesis. With regard to GCSI Score, there was no significant difference between patients with and without gastroparesis, Table GCSI Score was positively correlated with female sex, duration of T2D, BMI, SBP, DBP, retinopathy, nephropathy, neuropathy, FPG, HbA1c and grade of gastroparesis. Gastric emptying and motility index were negatively correlated with female sex, duration of T2D, BMI, SPB, DBP, retinopathy, nephropathy, neuropathy, FPG, HbA1c, TC, TGs and grade of gastroparesis. Table Female sex, duration of T2D, BMI, retinopathy, neuropathy, nephropathy, FPG, HbA1c, TC, TGs and vitamin B12 deficiency were significant positive predictors of gastroparesis in patients with T2D Table In the current study, the diagnosis of diabetic gastroparesis was based on GCSI Score and transabdominal ultrasonography. Although gastric scintigraphy is the gold standard for diagnosis of gastroparesis , 25, ultIn the present study, vitamin B12 level was significantly lower in patients with T2D than in healthy controls moreover, it was significantly lower in T2D patients with gastroparesis than in those without gastroparesis. Of interest, vitamin B12 was negatively correlated with GCSI Score whereas, it was positively correlated with gastric emptying and motility index. Additionally, vitamin B12 deficiency was an independent predictor for gastroparesis in patients with T2D. Vitamin B12 predicts gastroparesis at a cutoff value of 189.5 pmol/L with 69.1% sensitivity, 64.4% specificity, 70.4% PPV and 63% NPV, P = 0.002.In the current study, the frequency of vitamin B12 deficiency in total patients with T2D was 35% (54.5% in patients with gastroparesis and 11.1% in patients without gastroparesis). However, our findings are much higher than estimates from a study conducted by Amjad et al.  where viVitamin B12 deficiency is a major public health problem caused by age, consumption of vegetarian diets, malabsorption and drugs such as chronic use of omeprazole and metformin –38. An aIn our study participants, the independent predictors of gastroparesis other than vitamin B12 deficiency were female sex, duration of diabetes, BMI, diabetic microvasascular complications, FPG, HbA1c, TGs and TC which are consistent with the existing literature –46.In the current study, we did not observe an association between metformin treatment and diabetic gastroparesis. The increased frequency of vitamin B12 deficiency among patients with T2D taking metformin has been previously reported , 48, howFinally, in addition to sex, duration of diabetes, BMI, diabetic microvasascular complications, FPG, HbA1c, TGs and TC, vitamin B12 deficiency was an independent predictor of diabetic gastroparesis. From the previous discussion 2 raising questions arise which needs further studies. First, whether gastroparesis leads to vitamin B12 deficiency therefore, vitamin B12 may be a cause and a consequence of diabetic gastroparesis. Second, whether vitamin B12 supplementation can improve diabetic gastroparesis.Beside the known risk factors of diabetic gastroparesis, vitamin B12 deficiency is an independent predictor of diabetic gastroparesis in patients with T2D.


Introduction: The incidences of diabetes and diabetic retinopathy (DR) in Thai high-risk individuals with prediabetes have not been identified. This study compared diabetes and DR incidences among people at risk with different glycemic levels, using fasting plasma glucose (FPG) and hemoglobin A1C (HbA1c).Materials and methods: A historical cohort study estimating risk of type 2 diabetes and DR was conducted among outpatients, using FPG and HbA1c measurements at recruitment and monitored for ≥5 years. High-risk participants (defined as having metabolic syndrome or atherosclerotic cardiovascular disease) were categorized by glycemic level into 4 groups: 1) impaired fasting glucose (IFG)-/HbA1c- ; 2) IFG+/HbA1c- ; 3) IFG-/HbA1c+ ; and 4) IFG+/HbA1c+ . The incidences of type 2 diabetes mellitus (T2DM) and DR were obtained and estimated using Kaplan-Meier analysis. Cox regression models explored hazard ratios (HRs).Results: We recruited 8,977 people at risk . The baseline cohort consisted of 1) IFG-/HbA1c- ; 2) IFG+/HbA1c- ; 3) IFG-/HbA1c+ ; and 4) IFG+/HbA1c+ . Their 5-year T2DM incidences were 16.0%, 26.4%, 30.8%, and 48.5% (p < 0.001). The median DR follow-up was 7.8 years . The DR incidences were 0.50, 0.63, 1.44, and 2.68/1,000 person-years (p < 0.001) for IFG-/HbA1c-, IFG+/HbA1c-, IFG-/HbA1c+, and IFG+/HbA1c+, respectively. Compared with IFG-/HbA1c-, the multivariable-adjusted HRs (95% CI) for incident diabetes were 1.94 (1.34–2.80), 2.45 (1.83–3.29), and 4.56 (3.39–6.15) for IFG+/HbA1c-, IFG-/HbA1c+, and IFG+/HbA1c+, respectively. As for incident DR, the corresponding HRs were 0.67 (0.08–5.76), 4.74 (1.69–13.31), and 5.46 (1.82–16.39), respectively.Conclusion: The 5-year incidence of T2DM in Thai high-risk participants with prediabetes was very high. The incidences of diabetes and DR significantly increased with higher degrees of dysglycemia. High-risk people with FPG 110–125 mg/dl and HbA1c 6.0%–6.4% were more likely to develop T2DM and DR. Such individuals should receive priority lifestyle and pharmacological management. Prediabetes is a high-risk state for diabetes and the associated macrovascular and microvascular complications. According to meta-analyses, approximately 5%–10% of individuals with prediabetes develop diabetes annually.  MoreoverPrediabetes is a serious healthcare issue affecting one-tenth of the world’s adult population. In 2021, it was estimated that 319 million adults  had IFG, while 541 million adults  had IGT.  The prevNot all forms of prediabetes are the same. Differences in glycemic parameters and their thresholds result in different diabetes and diabetic retinopathy (DR) rates in people with prediabetes. A meta-analysis of prospective studies between 1979 and 2004 showed that 6%–9% of people with IFG progressed to diabetes annually.  SimilarlPrediabetes also impacts the progression of DR. Several studies have elucidated the relationship between the chronic hyperglycemia of prediabetes and diabetes and vascular complications (macrovascular and microvascular damage).  Some stuData for this analysis were derived from the medical records of patients who visited the outpatient clinic of Siriraj Hospital, Bangkok, Thailand. The clinic receives self-referrals and referrals from primary and secondary care services. Generally, clinic attendees have a high risk of diabetes or cardiovascular diseases or already have diabetes, hypertension, obesity, or multiple metabolic risk factors. Attendance at the clinic is usually set for regular intervals. Outpatients receive education, appropriate treatments, prevention strategies, and appropriate medications .n = 47,990). Participants were included if 1) their baseline HbA1c level was less than 6.5% (<48 mmol/mol) and 2) their baseline FPG level was under 7.0 mmol/L. Patients were excluded if they met the following criteria  who regularly visited the clinic for at least 5 years were included. After exclusions, 8,977 participants without diabetes were available for analysis. The study protocol complied with the Declaration of Helsinki and was reviewed by the Siriraj Institutional Review Board (SI956/2021).2, per the Asian-specific BMI cutoff ( • body mass index (BMI) ≥ 23 kg/mI cutoff  • documented hypertension • FPG ≥100 mg/dl or HbA1c ≥ 5.7% • a sex-specific low high-density lipoprotein level  or statin use •hypertriglyceridemia (fasting triglycerides [TG] ≥ 150 mg/dl) or statin use Clinical characteristics, blood pressure (using an automated measurement system), and anthropometric measurements (height and weight) were collected from electronic health records. “High-risk individuals” were defined as having metabolic syndrome or atherosclerotic cardiovascular disease. “Metabolic syndrome” was defined as participants having at least 3 of the following:The blood samples for HbA1c, plasma glucose, and serum lipid measurements were taken in the fasted state. A high-performance liquid chromatography system measured HbA1c with a Tosoh G8 analyzer , using the International Federation of Clinical Chemistry-approved methodology and standardization. FPG was measured by an enzymatic hexokinase method. Standard laboratory procedures measured serum total cholesterol, high-density lipoprotein cholesterol (HDL-c), and TG concentrations. Low-density lipoprotein cholesterol (LDL-c) was calculated using the Friedewald formula.The primary outcomes were the diabetes incidence and the time to the presence of diabetes within 5 years after the baseline. The secondary outcomes were the DR incidence and the time to DR during a follow-up period of up to 9 years from baseline.“Diabetes mellitus” (DM) was defined as when patient history met the criteria for DM. These were a diagnosis of DM documented by a doctor, an FPG of 126 mg/dl or higher, an HbA1c of 6.5% or higher, or oral hypoglycemic-agent use. DR was determined by documentation of the condition by a doctor and International Classification of Diseases (10th Revision) coding. The DR documentation was derived from various sources and reports .• IFG-/HbA1c-: FPG <110.0 mg/dl (6.1 mmol/L), and HbA1c < 6.0% (<42 mol/mol)• IFG+/HbA1c-: FPG 110–125 mg/dl (6.1–6.9 mmol/L), and HbA1c < 6.0%• IFG-/HbA1c+: (HbA1c prediabetes) FPG <110 mg/dl, and HbA1c 6.0%–6.4%• IFG+/HbA1c+: (combined IFG and HbA1c prediabetes) FPG 110–125 mg/dl, and HbA1c 6.0%–6.4%The baseline characteristics of the participants with FPG and HbA1c were compared in these 4 subgroups:2 test . The incidence rates were estimated using Kaplan-Meier analysis. Cox regression models explored the hazard ratios (HRs) of the DM incidence of each group. Adjustments were made for age, alanine aminotransferase, BMI, hypertensive status, mean corpuscular volume (MCV), sex, statin use, TG level, and uric acid. The HR of DR was explored with adjustments for age, BMI, hypertensive status, MCV, sex, and statin use.Continuous, normally distributed variables are summarized as the mean ± standard deviation, while continuous skewed distribution variables are presented as the median with interquartile ranges. Categorical variables are reported as numbers and percentages. Significant variations across the 4 subgroups were identified by ANOVA , the Kruskal–Wallis test , and the ChiIn all, 8,977 participants were enrolled. There were 4221 IFG-/HbA1c-participants (47.0%), 1274 IFG+/HbA1c-participants (14.2%), 2151 IFG-/HbA1c + participants (24.0%), and 1331 IFG+/HbA1c + participants (14.8%). All subjects had comorbidities, metabolic syndrome, or both. These conditions increase the risk of developing diabetes and atherosclerotic cardiovascular diseases. The participants’ diseases were metabolic syndrome (89.9%), dyslipidemia (70.0%), hypertension (69.8%), and atherosclerotic cardiovascular disease (16.9%). The baseline characteristics of the participants are summarized in With increasing degrees of glucose homeostasis in the participants with prediabetes, significant increases were found in age, alkaline phosphatase, BMI, the proportion of people with hypertension, statin use, and TG. However, the participants’ HDL-c levels fell significantly. All changes were especially noticeable among subjects with HbA1c levels between 6.0% and 6.4%.p < 0.001). The IFG+/HbA1c + subgroup had the highest incidence rate (48.5%), followed by the IFG-/HbA1c + subgroup (30.8%), the IFG+/HbA1c-subgroup (26.4%), and the IFG-/HbA1c-subgroup (16.0%). The adjusted multivariable Cox regression model found that subjects in the IFG+/HbA1c+, IFG-/HbA1c+, and IFG+/HbA1c-subgroups had significantly higher risks of 5-year diabetes incidence than subjects in the IFG-/HbA1c-subgroup. The HRs of the first 3 subgroups were 4.56 , 2.45 , and 1.94 , respectively  those with a BMI <23 kg/m2 , 2) those without hypertension , and 3) those who did not use statins  had developed diabetes. The incidence of diabetes varied significantly among the prediabetes subgroups (ectively . Moreovep = 0.001). The adjusted multivariable Cox regression model determined that compared with the IFG-/HbA1c-subgroup, the adjusted HRs were 5.46  for the IFG+/HbA1c + subgroup, 4.73  for IFG-/HbA1c+, and 0.67  for IFG+/HbA1c- . The participants with IFG+/HbA1c + had the highest incidence rate of DR . The other rates were IFG-/HbA1c+, 1.44 per 1,000 person-years; IFG+/HbA1c-, 0.6 per 1,000 person-years; and IFG-/HbA1c-, 0.5 per 1,000 person-years .Previous international studies examining the progression from prediabetes to diabetes found that people with prediabetes had different risks of developing diabetes, depending on their glucose level in the prediabetes range.  This finMost research into the progression from prediabetes to diabetes has examined the relationship using 2-h postload plasma glucose or FPG as prediabetes criteria in general individuals. However, the current work focused on high-risk prediabetes categorized by HbA1c and FPG. HbA1c represents the average blood glucose level during approximately the preceding 3 months  and signOn the other hand, an impairment in FPG indicates a decrease in hepatic insulin sensitivity and a defect in early-phase insulin secretion.  A findinOur model established that hypertension status and statin use significantly increased the risk of diabetes conversion. The expected relationships between either hypertension status or statin use and diabetes incidence were confirmed. In high-risk people with prediabetes, the antihypertensive drug class, blood-pressure-lowering potency of the drug used, and the type of statin may affect these relationships, as revealed by previous meta-analyses.  However,The early stages of retinal complications, renal involvement, and neurological damage were reported in people with prediabetes.  A criticOur investigation demonstrated that an IFG between 110 and 126 mg/dl was not an independent risk factor for progression to DR. A further parallel with this observation was reported by a study assessing the connection between DR prevalence and glycemia in an adult population aged ≥40 years. That work detected a rapid increase in DR prevalence with rising glycemia and found that HbA1c was a better predictive factor than FPG.  FurthermAs for diabetes prevention, dietary control and regular exercise should generally be recommended to individuals with prediabetes. Supplementary intensive lifestyle or pharmacological interventions can increase the effectiveness of basic lifestyle changes and are cost-effective, especially in individuals with IGT.  ResearchOn the other hand, additional lifestyle and pharmacological measures are costly, time-consuming, and require much effort by individuals and healthcare professionals conducting group and monitoring interventions.  MoreoverOur analysis has its strengths. This research is the first to examine the relationships between glycemic range and cardiometabolic risk factors, diabetes, and DR in Thai high-risk individuals with prediabetes. In contrast, the previously mentioned Thai studies only provided information on DM incidence in the low-to moderate-risk population with prediabetes.  In additOur study also has limitations. Perhaps the most fundamental limitation is that our data were derived from a retrospective cohort constructed from databases of healthcare records. The cohort may not represent all possible people with prediabetes in the general population, health records may not have all pertinent risk factors, and diagnoses are likely to have been identified but not subsequently recorded wholly and correctly. Additionally, the DR diagnoses were obtained from various sources and reports. These DR documentation findings depend on the skill and experience of the attending ophthalmologist, and the ophthalmological devices and procedures used. These limitations may have biased the observed associations.However, in our analysis, the exposure to all relevant risk factors was recorded before the occurrence of diabetes and DR. Consequently, the temporal sequence of risk factors and outcomes could be assessed. The diagnoses and definitions of all related risk factors were defined carefully using standard references and all relevant record parameters  coding, and laboratory findings). The diagnoses and risk factors were further reviewed by trained research staff. Last, our model analysis did not include the IGT measurement because, in Thailand, we do not routinely measure IGT in clinical practice. Consequently, there were few data on IGT in our dataset.In conclusion, people with prediabetes are at high risk of developing diabetes in the short term, and the risk of progression depends on the criteria of prediabetes and the number of pathogeneses. Moreover, diabetic retinal complications can be found in people with prediabetes. Using the World Health Organization criteria, this study revealed that people with prediabetes who had an FPG of 110–125 mg/dl or an HbA1c of 6.0%–6.4% were likely to develop type 2 DM and DR. Early intensive intervention and pharmacological treatment should be considered for these high-risk subgroups. Further randomized control trials are needed to confirm the benefits of these interventions in high-risk people with prediabetes, especially those with the combination of FPG and HbA1c prediabetes.


Additionally, it was positively associated with plasma level of IL6 (ρ = 0.17) and IL10 (ρ = 0.14) but not TNF-α. In conclusion, LRG1 levels are increased in obese adolescents and are associated with increased levels of adipogenic markers. These results suggest the usefulness of LRG1 as an early biomarker for obesity and its related pathologies in adolescents.Leucine-rich α-2 glycoprotein1 (LRG1) is a member of the leucine-rich repeat (LRR) family that is implicated in multiple diseases, including cancer, aging, and heart failure, as well as diabetes and obesity. LRG1 plays a key role in diet-induced hepatosteatosis and insulin resistance by mediating the crosstalk between adipocytes and hepatocytes. LRG1 also promotes hepatosteatosis by upregulating de novo lipogenesis in the liver and suppressing fatty acid β-oxidation. In this study, we investigated the association of LRG1 with obesity markers, including leptin and other adipokines in adolescents . BMI-for-age classification based on WHO growth charts was used to define obesity. Plasma LRG1 was measured by ELISA, while other markers were measured by multiplexing assay. Median (IQR) of LRG1 levels was higher in obese  µg/mL) and overweight  µg/mL) adolescents, compared to normal-weight participants  µg/mL). The highest tertile of LRG1 had an OR [95% CI] of 2.55  for obesity. LRG1 was positively correlated to plasma levels of high sensitivity c-reactive protein (HsCRP) (ρ = 0.2), leptin (ρ = 0.2), and chemerin (ρ = 0.24) with  Childhood obesity is one of the most serious health challenges worldwide. The prevalence of childhood obesity in the Arabian Gulf Countries (GCC) is extremely high . This miLeucine-rich α-2 glycoprotein 1 (LRG1) is a member of the highly conserved leucine-rich repeat (LRR) family , which pElevated plasma LRG1 levels were observed in patients with proliferative diabetic retinopathy (PDR) when compared to non-diabetic controls, indicating that it might be involved in the development of pathogenic ocular neovascularization . MoreoveOverall, these previous reports indicate that LRG1 is implicated in the development of diabetes and obesity-related complications in adults. However, the role of LRG1 in these metabolic pathologies in children and adolescents has not been well-established. This study aimed to assess the role of LRG1 as a potential biomarker for childhood obesity. We investigated the association between LRG1 levels and obesity and CVD risk factors. We also aimed to compare LRG1 levels between obese, overweight, and normal-weight adolescents.p = 0.944). The mean (SD) age of the participants was 12.3 (0.8) years, and 197 (45.7%) were males.The demographic characteristics of the study group are summarized in p = 0.007). The median (IQR) level of LRG1 was 27  µg/mL in normal-weight participants compared to 30  µg/mL in overweight and 30  µg/mL in obese (p > 0.05). Multinomial logistic regression showed that the odds of obesity and overweight were significantly associated with LRG1 in univariable analysis  and after adjusting for potential confounders   . This wap < 0.001; p < 0.001; p < 0.001; p < 0.001; p = 0.003; p > 0.05; We found a positive correlation between LRG1 and high sensitivity c-reactive protein (HsCRP) levels  with optimal dilution 1:4000 and HsCRP concentrations were determined using ELISA kit  with optimal dilution 1:1000. All assays were performed following the manufacturer’s instructions.Leptin, chemerin, IL6, IL10, and TNF-α were assessed using a multiplexing immunobead array platform according to the manufacturer’s instructions . Median fluorescence intensities were collected on a Bioplex-200 system, and data were processed using the Bio-Plex Manager Software version 6 , with five-parametric curve fitting.Standing height and bodyweight of the study participants were measured in a standardized manner, using a digital weight and height scale , with the participants standing erect without shoes and wearing light clothes. WHO growth charts were used to calculate BMI-for-age z-scores. Obesity was defined as BMI-for-age ≥+3 Standard Deviation (SD), while overweight was defined as BMI-for-age >+2 SD and <+3 SD.p < 0.05 were deemed to be significant.All measurements are reported as median (interquartile range: IQR). The associations between LRG1 and markers of interest, including HsCRP, leptin, chemerin, IL6, IL10, and TNF-α, were depicted graphically, and Spearman correlation coefficient was calculated. Multinomial logistic regression was used to determine the association between LRG1 and overweight or obesity. First, crude odds ratios (OR) were calculated, then we adjusted for sex and age group  to calculate adjusted odds ratios (AOR). Separate analyses were conducted while fitting LRG1, first as a continuous variable and then as a categorical variable. We used the Wald test to evaluate the statistical significance of these analyses. Associations with In conclusion, higher plasma LRG1 levels were observed in obese and overweight adolescents compared with their normal-weight counterparts, indicating that LRG1 was associated with increased obesity risk. Our findings also suggest that LRG1 might be an early obesity marker as the difference in LRG1 levels between overweight and obese groups was not significant. Moreover, a positive correlation was observed between LRG1 and other obesity markers, including HsCRP, leptin, and chemerin. These findings shed light on the importance of LRG1 in adolescent obesity. In addition, the results indicate that LRG1 could be used as a potential prognostic and diagnostic tool in adolescent obesity. Lastly, our findings suggest that LRG1, chemerin, leptin, and HsCRP might be part of a common pathway involved in the development of obesity, possibly through P38/MAPK signaling pathway.


Mansoa hirsuta was investigated, and in vitro assays and theoretical approaches were carried out to evaluate its antioxidant potential. The phytochemical screening detected saponins, organic acids, phenols, tannins, flavonoids, and alkaloids in extracts of leaves, branches, and roots. Through LC-MS analysis, the triterpenes oleanolic acid (m/z 455 [M-H]−) and ursolic acid (m/z 455 [M-H]−) were identified as the main bioactive components. The extracts of the leaves, branches, and roots revealed moderate antioxidant potential in the DPPH test and all extracts were more active in the ABTS test. The leaf extracts showed better antioxidant capacity, displaying IC50 values of 43.5 ± 0.14, 63.6 ± 0.54, and 56.1 ± 0.05 µg mL−1 for DPPH, ABTS, and kinetics assays, respectively. The leaf extract showed higher total flavonoid content (TFC) (5.12 ± 1.02 mg QR/g), followed by branches (3.16 ± 0.88 QR/g) and roots (2.04 ± 0.52 QR/g/g). The extract of the branches exhibited higher total phenolic content (TPC) (1.07 ± 0.77 GAE/g), followed by leaves (0.58 ± 0.30 GAE/g) and roots (0.19 ± 0.47 GAE/g). Pharmacophore and molecular docking analysis were performed in order to better understand the potential mechanism of the antioxidant activity of its major metabolites.In this work, a metabolic profile of  In recent years, a marked increase has been observed in the evaluation of naturally occurring bioactive compounds in the search for new drugs to counter oxidative stress ,2. AntioOxidative stress plays an essential role in the pathogenesis of chronic diseases such as cardiovascular disease, diabetes, neurodegenerative diseases, and cancer. Oxidative processes are fundamental metabolic processes for all living organisms , but freThe skin pigment melanin plays a critical role in skin protection against induced damage by UV and free radicals. Tyrosinase is the rate-limiting enzyme in the first two steps of melanogenesis. It catalyzes the hydroxylation of L-tyrosine into L-DOPA, and oxidation of L-DOPA to form the respective O-dopaquinone ,12.Different skin disorders  resulted from the abnormal accumulation of melanin. Therefore, tyrosinase inhibitors are essential to ensure a decrease in the content of melanin and to design and develop new depigmenting compounds useful in pharmaceutical areas ,12. AntiMansoa hirsuta DC. (Bignoniaceae), an edible plant known as garlic vine in Brazil and endemic to the semiarid region of Brazil, which stands out for its popularity and potential bioactive phytochemicals and/or functional foods. In traditional medicine, its leaves are used to control diabetes and to treat sore throats −) and ursolic acid (m/z 455 [M-H]−) were identified as the main bioactive components from M. hirsuta. We then used molecular docking to fit the oleanolic acid and ursolic acid into the active site of a target enzyme to identify possible correlations between the binding models and their antioxidant activities.However, there are few reports of experiments carried out with methanol extracts of Phytochemicals are produced by specific biochemical pathways that occur within plant cells and are used for plant defense and adaptation to environmental stress . PhytochM. hirsuta leaves presented saponins, steroids, triterpenoids, phenols, tannins, anthocyanins, anthocyanidins, flavonoids  and/or xanthones and their heteroside derivatives. In addition, metabolites including naphthoquinones −) and ursolic acid (m/z 455 [M-H]−) were identified as the main bioactive components of M. hirsuta, we undertook a molecular docking study. Three enzymes are used in this docking study: Tyrosinase, PRDX5 and Superoxide Dimutase (SOD1). Tyrosinase [To explore whether the established antioxidant activities of the triterpenes oleanolic acid (rosinase  is the mrosinase  as well rosinase  have antBacillus megaterium (PDB: 3NM8) [Mammalian Peroxiredoxin (PDB: 1HD2) [Docking poses were analyzed and compared to the standard Quercetin. First, docking was performed on crystal structure of tyrosinase from B: 3NM8) . TyrosinB: 1HD2) . The perB: 1HD2) . SuperoxThe two triterpenes were subjected to docking analysis, and the specificities of their interaction with these targets, as shown in Ligplots are shown in Ligplots in Ligplots show in Using the lowest energy conformers of Oleanolic acid and Ursolic acid, a pharmacophore model was generated . The genFisatom R-801 rotary evaporator, under reduced pressure with the aid of PRISMATEC BBV-132 vacuum pumps .The solvents used in the extraction and chromatographic analysis were of analytical and spectroscopic grade, including methanol, ethyl alcohol, acetic acid, and acetonitrile . The extracts were concentrated in a ®  and ultra-pure water (18.2 MΩ in an Elga Purelab Option-Q system) were used. The stationary phase used was Silica C18 .For the chromatographic and spectrometric experiments, high purity HPLC grade solvents purchased from Sigma AldrichAntioxidant analyses were carried out using DPPH, ABTS, Folin-Ciocalteu reagent, gallic acid, Trolox, quercetin and rutin purchased from Sigma Chemical Co. .In Silico Molecular Docking analyses were predicted and calculated using AutoDock Vina v.1.2.0 .M. hirsuta were collected in March and December 2020 at Sitio do Mocó, Coronel José Dias, near the municipality of São Raimundo Nonato-PI, , under the registration number in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) (A2CA781). After collection, leaves, branches, and roots were dried and ground at room temperature. Complete plant samples were sent to the Herbarium Graziela Barroso (TEPB) at UFPI, Teresina, PI, identified and deposited under voucher specimen (TEPB.32.277).Leaves, branches, and roots from Different parts of the plant were crushed in an industrial blender and milled, and yielded 378.77 g of dried leaves, 650.12 g of dried branches and 107.32 of dried roots. The phytochemical extractions were carried out by maceration with methanol until exhaustion, followed by solvent evaporation in a rotary evaporator at 40 °C and 180 bar pressure.Preliminary phytochemical evaluation of each methanol extract was performed using colorimetric tests to detect the presence/absence of specific classes of phytochemical constituents  .v/v); ethyl acetate/methanol 8:2 (v/v); ethyl acetate/methanol 7:3 (v/v); ethyl acetate/methanol 9:1 (v/v), hexane/ethyl acetate 8:2 (v/v); Chloroform/methanol 9:1 (v/v). The method was carried out using 60778-25EA silica gel thin layer chromatoplates, fluorescing at 254 nm  obtained from Sigma-Aldrich. The plates were developed by UV irradiation (254 nm and 366 nm) and with physical developer ceric sulfate. The results were expressed as the intensity of staining after developing the chromatoplate.Comparative Thin Layer Chromatography analysis using the following solvent systems: ethanol/ethyl acetate 8:2 , model LC20A, CBM-20 controller, UV-visible detector with “Diode Array” (DAD) model SPDM-20A, DGU-20A3 degasser and LC solutions software . A quantity of 1 mg of M. hirsuta extracts was injected, using a gradient method up to 50 min, and a mobile phase of a mixture of water + 0.1% acetic acid (A) + 100% acetonitrile (B), which were pumped at a rate 1.0 mL min−1, Stationary phase C18 column reversed phase , brand Macherey-Nagel  and membrane filtered 0.45 µm, the column oven was heated to 40 °C using a UV-visible detector chromatograph in 50 min.Concerning the HPLC-PDA analysis, the methanol extracts of leaves, branches, and roots of ® LC System equipment, LC-20AD pump, coupled to SPD-M20A Diode Array detector, and automatic injector (model SIL-20AHT), Communication module: CBM-20ª, Auto-MS mode. MS/MS, frag Ampl: 75%. Full: 50–1200 m/z. Mass Spectrometer: Amazon SL Bruker® . Ionization source: electrospray (ESI). Analyzer: ionTrap. HPLC method. The mobile phase was programmed in a mixture of methanol B and ultra-pure water A solvents. Exploratory gradient: 5–100% B (45 min), 100% B (45–55 min), 5% B (55–57 min) and from (57–68 min) remaining at 5% to re-equilibrate the column at a flow rate of 1 mL min−1. As stationary phase, a C-18 Phenomenex Gemini column  was used in the analytical mode, with extract concentration 1 mg mL−1.LC-MS analysis of the extracts were performed using a Shimadzu1H chemical shifts were referenced to the DMSO-d6 solvent peak , used to solubilize the samples.One-dimensional NMR spectra were obtained on a Bruker Avance 600 spectrometer  with a 5 mm TCI cryoprobe and a 14.1 T magnetic field, operating at 600 MHz for 1H. −1). After 20 min from the start of the reaction in the dark, the absorbance of each sample was measured at 517 nm using a UV-Vis spectrophotometer . The determinations were made using a negative control  and a positive control (quercetin) [50 parameter. IC50 values denote the concentration of the sample, which is required to scavenge 50% of DPPH free radicals Equation (1):Serial dilutions of each sample were prepared. 1.8 mL of DPPH solution (0.06 mM) were added to 200 μL of each sample at different concentrations  . The res• radical (IC50) was estimated using a simple linear regression model.From the percentage of inhibition, the concentration effective to inhibit 50% of the DPPH−1), EMGMh (10–70 μg mL−1) and EMRMh (30–80 μg mL−1) extracts, were mixed with 1.8 mL of ABTS•+ solution on an ELISA plate. After 6 min of dark reaction, the decrease in absorbance was measured at 734 nm. Trolox was used as a positive control. Determinations were carried out in triplicates. The ABTS•+ radical scavenging activity was expressed as a percentage using the same formula as the DPPH assay (Equation (1)). From the percentage inhibition obtained for the samples of methanol extracts from leaves, branches and roots, the mean inhibitory concentrations  in μg. mL−1 were determined [Twenty-microliter aliquots of different concentrations of samples of EMFMh  vs. time (X axis) results were plotted in graphs to generate kinetic curves [Similar to the DPPH method, in an Elisa plate, 1.8 mL of DPPH solution (0.06 mM) was added to 200 μL of each extract, referring to EMFMh, EMGMh, and EMRMh in a concentration of 100 μg mLc curves . The det−1), EMGMh (70 μg mL−1), and EMRMh (80 μg mL−1) were mixed with 1.8 mL of ABTS•+ (7 mM) solution in an Elisa board. At 6, 10, 20, 30 and 60 min the absorbance was measured at 734 nm in a spectrophotometer in the dark [For ABTS, 20 μL aliquots of different concentrations of samples from EMFMh (50 μg mLthe dark . At the −1). For the reaction, 2 mL of distilled water was mixed with 250 μL of Folin Ciocalteu reagent and 250 μL of the extracts. After the light protection period (8 min), 100 μL of sodium carbonate solution (10% w/v) was added. The solutions were mixed and allowed to stand in the dark for 1 h at room temperature. Absorbance was measured at 760 nm using a UV-Vis spectrophotometer. To determine the total phenolic content, gallic acid was used to make the standard calibration curve (10–160 μg μL−1). The absorbance of the samples was applied to the standard line equation , where y = absorbance and x = gallic acid concentration  was added to 1000 μL of 2% Aluminum Chloride (AlCl3) (diluted in methanol). The solutions were mixed and placed under light protection at room temperature (25 °C) for 1 h. The absorbance was measured at 420 nm using a UV-Vis spectrophotometer: SP-220, Biospectro . To determine the total flavonoids content, a calibration curve was constructed  with the standard rutin (10–160 μg mL−1). The total flavonoid content was expressed in mg of rutin equivalent (ER)/g per gram of extract dry weight [A volume of 1000 μL of each sample .Statistical analysis for antioxidant tests was performed in triplicate and data presented as means ± standard deviations (SD) as ICMolecular docking analysis was performed using Autodock Vina v.1.2.0  docking software .The receptor site was predicted using LigandScout (Inte: Ligand) Advanced software   [The resulting pocket grid consists of several clusters of grid points, rendered using an iso surface connecting the grid points to each other. The iso surface represents empty space that may be suitable for creating a pocket. The x-ray crystal structure of Tyrosinase from B: 3NM8) , Human PB: 3NM8) , and AtoB: 3NM8)  were retThe box center and size coordinates for (PDB: 3NM8) was −5.59208 Å × −3.33416 Å × 10.2111 Å and 31.5668 Å × 22.6662 Å × 23.0593 Å; for (PDB: 1HD2) was 18.9015 Å × 44.192 Å × 28.1802 Å and 12.81 Å × 17.1255 Å × 15.8749 Å; for (PDB: 2C9V) was 31.8729 Å × −0.108084 Å × 14.3963 Å and 13.8504 Å × 18.6613 Å × 35.181 Å around the active site.Default search parameters were used where number of binding modes were 10, exhaustiveness was 8, and maximum energy difference was 3 kcal/mol. Chimera 1.16, UCSF_USA  LigPlot+LigandScout by Inte Ligand, Advanced software (Wolber and Langer), Vienna, Austria, Europe  , followed by branches (EMGMh) and roots (EMRMh). These results also show the importance of selecting several methods to quantify the antioxidant activity of plant extracts. The presence of different phytochemicals observed in chemical profiles may justify pharmacological activities, such as their antioxidant potential. Also, an evaluation of the antioxidant activities of the triterpenes oleanolic acid and ursolic acid was identified as the main bioactive components of


The primary aim of this prospective study in clinical practice was to evaluate the effectiveness of HBVAXPRO®40 and FENDRIX® in this setting. Patients received HBVAXPRO®  or FENDRIX®  depending on availability. Clinical data and anti-HBs levels were collected at 2, 6 and 12 months. A total of 125 patients were included : 76 were vaccinated with HBVAXPRO® and 49 with FENDRIX®. There were no significant differences between the two vaccines. The overall response rates at 2, 6 and 12 months were 76.8, 72.8 and 59.2%, respectively. In the univariate analysis, active alcohol intake, alcohol etiology, liver cirrhosis and ultrasound signs of portal hypertension were associated with a lower response to vaccination, whereas in the multivariate analysis, liver cirrhosis was the only factor that significantly increased the likelihood of nonresponse (OR 10.5). HBVAXPRO® and FENDRIX® are good options for HBV vaccination in patients with chronic liver disease.Chronic liver disease results in a low response rate to the hepatitis B virus vaccine. Information on the efficacy of the double adjuvanted vaccine FENDRIX Cirrhotic patients present innate and adaptive immune dysfunction, which results in an increased risk of contracting infections . In thesHowever, the efficacy of both vaccines in this group of patients is lower than that in the healthy population, especially in advanced liver disease.The HBV vaccine has been shown to be effective in the immunocompetent adult population (patients under 40 years of age) with a 95% seroconversion rate ,7. The rSeroprotection was defined as having hepatitis B surface antibody (anti-HBs) titers equal to or greater than 10 U/mL . The proThere are several factors associated with a lower response to vaccination, such as smoking, male sex, obesity and age over 40 years ,10,11.In addition, the effectiveness of HBV vaccination has been shown to be lower in patients with chronic renal failure on dialysis , in patiChronic liver disease also results in a lower response to vaccination. Seroprotection at standard doses is low 16–20%) ,16,17. D–20% ,16,® vaccine  is a double adjuvant HBV vaccine containing the adjuvant 3-O-desacyl-4’-monophosphoryl lipid A and aluminium phosphate. Although the cost of this vaccine is higher than that of the nonadjuvanted vaccine , this adjuvanted vaccine could induce higher immunity in patients with chronic liver disease. In a randomized experimental study with 93 liver transplant candidates, the safety and immunogenicity of two doses of FENDRIX® vs. three double doses of ENGERIX B®  showed that the percentage of patients with seroprotective anti-HBs levels was significantly higher in the FENDRIX®  group (60%) versus the Engerix B group (32%) [® 40  is a single adjuvant HBV vaccine with aluminium hydroxyphosphate sulfate, approved for predialysis, peritoneal dialysis and hemodialysis patients aged ≥18 years.The FENDRIXup (32%) . On the ®  in patients with chronic liver disease in clinical practice.There are few studies evaluating the effectiveness of HBV vaccination in patients with chronic liver disease in clinical practice ,19,21,22® 40  and FENDRIX®  in patients with chronic liver disease in a real-life setting, (2) assess the effectiveness of both vaccines at 6 and 12 months and (3) assess the occurrence of associated HBV vaccination adverse effects (AEs).Thus, we aimed to (1) evaluate the effectiveness of the HBV vaccines HBVAXPRO® 40  and FENDRIX®  involving chronic liver disease patients with HBV-negative serologic tests. The study was conducted between September 2017 and August 2021.This is a prospective observational study in clinical practice using HBVAXPROhttp://clinicaltrials.gov .Noncirrhotic and cirrhotic liver disease patients with HBV-negative serologic tests were consecutively and prospectively included in the outpatient liver unit of the Hospital Universitari Mutua Terrassa. Liver disease was assessed by abnormal liver blood tests lasting for more than 6 months, elastography, noninvasive markers of fibrosis, ultrasound and biopsy, when available. The inclusion criteria were as follows: (1) chronic liver disease patients (noncirrhotic and cirrhotic) diagnosed by liver biopsy and/or noninvasive methods ; (2) HBV-negative serologic tests, including hepatitis B surface antigen (HBsAg), anti-HBs and hepatitis B core antibody (anti-HBc); and (3) 18 years old or older. The exclusion criteria were as follows: (1) allergy to vaccine components ; (2) active or past HBV infection; (3) patients previously vaccinated against HBV (regardless of response); (4) Child–Pugh class C; (5) conditions that cause immunosuppression ; (6) pregnancy or breastfeeding; or (7) previous HAV infection or anti-HAV vaccination. The study was approved by the institutional review board (TASDA_003) and complied with the provisions of the Good Clinical Practice guidelines and the Declaration of Helsinki. Written informed consent was obtained from all participants, in accordance with local institutional review board requirements. Vaccination and study assessments were performed at the Digestive Unit of Hospital Universitari Mutua Terrassa. The study was registered in the Clinical Trials Registry at Serological markers of HBV infection were measured in all outpatients with noncirrhotic and cirrhotic liver disease . After informed consent was obtained, all seronegative patients were prospectively included.® 40  or FENDRIX®  depending on the type of vaccine available to the Health Department of the Catalan Government.Patients were vaccinated with HBVAXPROClinical and blood tests and the presence of anti-HBs levels were collected at 2, 6 and 12 months after the last dose of vaccination.®  (40 µg of HBsAg) at 0, 1 and 6 months or FENDRIX®  (20 µg of HBsAg + 50 µg of -3-O-desacyl-4’-monophosphoryl lipid) at 0, 1, 2 and 6 months. Both were administered intramuscularly in the deltoid muscle  , liver fA classic response to HBV vaccination was defined as anti-HBs ≥ 10 U/mL, and a nonclassical response was defined as anti-HBs ≥ 100 U/mL determined two months after the last dose of HBV vaccination .® 40 and FENDRIX® 2 months after the last dose of HBV vaccination. Secondary endpoints were (1) HBV vaccination response at 6 and 12 months; (2) AEs related to HBV vaccination; and (3) assessment of factors associated with HBV effectiveness.The primary endpoint was the effectiveness of the HBV vaccines HBVAXPROQuantitative data are reported as the means ± standard deviations (SD), and categorical variables are presented as frequencies and percentages. Groups were compared using the t test for quantitative variables when appropriate, and Fisher’s exact test was used for categorical variables. A multivariate logistic regression analysis was performed to evaluate variables associated with the vaccine response. The level of significance was set at two-sided 5%. All calculations and analyses were performed with the SPSS 25.0 software package .® 40  and 57 received FENDRIX®  and 18 patients dropped out. Reasons for drop-out are shown in ® 40  group and 49 in the FENDRIX®  group.A flow chart for the study is provided in ® group  (p = 0.001). The median age was 61.8 years (39–83), 57.6% (n = 72) were male and 43.2% (n = 54) had liver cirrhosis, 75.9% of whom were Child–Pugh A and 24.1% Child–Pugh B.Baseline characteristics are depicted in The etiology of chronic liver disease was 35.2% alcohol, 28.8% metabolic-associated fatty liver disease, 20% hepatitis C virus and 16% autoimmune hepatitis/primary biliary cholangitis. Regarding other medical conditions, 21.6% had diabetes, 83.3% had a BMI ≥ 25, 68% were smokers and 32% had active alcohol abuse.n = 96)  response rate at two months was 72.4% (n = 55) (anti-HBs ≥ 10 U/mL) and 52.6% (n = 40) (anti-HBs ≥ 100 U/mL), whereas the FENDRIX®  response rate was 83.7% (n = 41) (anti-HBs at two months was 72.4% (n = 55)) (anti-HBs ≥ 10 U/mL). No significant differences between the two vaccine protocols were detected (anti-HBs n = 41). The geometric mean titers (GMTs) of anti-HBs with HBVAXPRO®  and FENDRIX®  were 59.5525 ± 11.1742 U/mL and 134.0293 ± 8.9071 U/mL, respectively. No differences were observed between the two different vaccines (p = 0.059).The overall response rate two months after the last dose of vaccination was 76.8% (n = 96) . The medp = 0.04), alcoholic etiology (p = 0.001), liver cirrhosis (p < 0.001), advanced fibrosis (F3–F4) by elastography (p < 0.001) and ultrasound signs of portal hypertension (p = 0.005) were associated with a lower response rate to vaccination in the univariate analysis (p < 0.001) was independently associated with a lower effectiveness of vaccination.Active alcohol intake  (anti-HBs ≥ 10 U/mL) and 43.2% (n = 54) (anti-HBs ≥ 100 U/mL). The median anti-HBs level in responders was 286 (2–1000) U/mL. There were no significant differences between the two vaccine protocols at six months (anti-HBs ≥ 10 U/mL p = 0.100) (anti-HBs ≥ 100 U/mL p = 0.356)  and FENDRIX®  were 40.9826 ± 10.5988 U/mL and 81.5455 ± 8.900 U/mL, respectively. No differences were observed between the two different vaccines (p = 0.104).At 6 months, the overall response rates were 72.8% (= 0.356) . The GMTn = 74) (anti-HBs ≥ 10 U/mL) and 41.9% (n = 52) (anti-HBs ≥ 100 U/mL). The median anti-HBs levels in responders were 250.7 (2–1000) U/mL. There were no significant differences between the two vaccine protocols at twelve months (anti-HBs ≥ 10 U/mL p = 0.576) (anti-HBs ≥ 100 U/mL p = 0.271)  and FENDRIX®  were 30.0746 ± 10.5905 U/mL and 56.8721 ± 10.1744 U/mL, respectively. No differences were observed between the two different vaccines (p = 0.143).At 12 months, the overall response rates were 59.2% (= 0.271) . The GMTp < 0.001) was independently associated with a lower effectiveness of vaccination.Diabetes, alcoholic etiology, advanced fibrosis (F3–F4), liver cirrhosis and ultrasound signs of portal hypertension were associated with a lower response rate to vaccination at 6 months. In the multivariate analysis, liver cirrhosis .  were found in the univariate and multivariate analyses . .There were no AEs reported during the study period.®, GlaxoSmithKline, Rixensart, Belguim and HBVAXPRO® 40  with an increased immunogenic response were used depending on the availability in the setting of the vaccination program of the Health Department of the Catalan autonomous community.The majority of studies assessing HBV vaccine response have only included liver transplant candidates ,21,22,25®  group and 72.4% in the HBVAXPRO® 40  group. There were also no differences in the GMT between the two vaccines, although a tendency to have higher values with the Fendrix vaccine was also observed, suggesting that it could have a better efficacy. In addition, vaccination efficacy, measured by anti-HBs levels, decreased over time .We found an overall good response rate (76.8%) two months after completing the vaccination protocol with both vaccination schedules: 83.7% in the FENDRIXThe HVB vaccination response rate in prior studies is heterogeneous due to the use of different vaccination regimens and the heterogeneity of the patients included. In cirrhotic patients, despite the use of double doses or accelerated regimens of standard HVB vaccines to increase efficacy, the response rate remains suboptimal 23–68%) 3–68% 2,,19,21,22,21,2219.®  40 µg at 0, 1 and 2 months. Finally, Aziz et al. [To date, few studies have included patients with chronic liver disease with different etiologies ,10,23. AThe good vaccination response obtained in our study could be in part explained by the fact that 56.8% of the patients included were noncirrhotic patients, emphasizing the need for early adherence to a vaccination program. In contrast to previous studies, we also included Child–Pugh B patients who demonstrated a worse vaccine response in patients with complicated cirrhosis. However, direct comparison to other studies is difficult due to the use of different vaccine doses, schedules and patient characteristics.Active alcohol intake, alcoholic etiology, liver cirrhosis and indirect signs of portal hypertension were associated with a lower response to HBV vaccination. At six months, diabetes was also associated with a lower immunization response. Several risk factors for nonresponse have been previously identified, including older age, male sex, smoking, obesity and diabetes ,10,29. OAlcohol consumption alters both innate and adaptive immunity and is associated with a decreased frequency of lymphocytes and an increased risk of both bacterial and viral infections . MoreoveLiver cirrhosis and advanced fibrosis are well-established factors influencing a low response to HBV vaccination. As previously mentioned, this aspect has been especially evaluated in patients with end-stage liver cirrhosis awaiting liver transplant, in which accelerated or double-dose vaccination must be considered . The lowAnti-HBs titers ≥ 10 U/mL are considered a reliable marker of protection against HBV infection. However, anti-HBs titers decline over time, as seen in the previous literature in other clinical settings . In our ® 40  or FENDRIX®  and did not have a control group. However, the study was designed in a clinical practice setting, and we did not find significant differences in either vaccine protocol. In addition, we did not include patients who were not immunized for HAV. In this group of patients, as recommended in clinical practice, we used a combined HAV/HBV vaccine [There are several limitations of this study. We did not randomize patients to receive either HBVAXPRO vaccine . Since t vaccine , these p® 40  and FENDRIX®  are effective and safe in patients with chronic liver disease. Indications for these vaccines should be expanded to enable vaccination of chronic liver disease patients in early stages.The results of our study show that HBVAXPRO


Paracentrotus lividus. The boss microstructure was identified as a Voronoi construction characterized by 82% concordance to the computed Voronoi models, a prevalence of hexagonal polygons, and a regularly organized seed distribution. This pattern is interpreted as an evolutionary solution for the construction of the echinoid skeleton using a lightweight microstructural design that optimizes the trabecular arrangement, maximizes the structural strength and minimizes the metabolic costs of secreting calcitic stereom. Hence, this identification is particularly valuable to improve the understanding of the mechanical function of the stereom as well as to effectively model and reconstruct similar structures in view of future applications in biomimetic technologies and designs.Repeated polygonal patterns are pervasive in natural forms and structures. These patterns provide inherent structural stability while optimizing strength-per-weight and minimizing construction costs. In echinoids (sea urchins), a visible regularity can be found in the endoskeleton, consisting of a lightweight and resistant micro-trabecular meshwork (stereom). This foam-like structure follows an intrinsic geometrical pattern that has never been investigated. This study aims to analyse and describe it by focusing on the boss of tubercles—spine attachment sites subject to strong mechanical stresses—in the common sea urchin  As stated by Galileo Galilei in the Assayer 1623): ‘the book of nature is written in the language of mathematics'. Repeated arrays of polygons or polyhedra recur in biological  and physico-chemical  systems 623: ‘the. In orgaPediastrum boryanum) [Cucumis sativus) epidermis [Drosophila imaginal discs [Many biological constructions can be specifically described as Voronoi models. A Voronoi model is a geometrical partitioning of two-dimensional or three-dimensional spaces into polygonal regions, known as Voronoi cells. These cells are generated around a pre-existing number of points, called seeds . Voronoioryanum) , chick eoryanum) , cucumbepidermis  and Drosal discs .et al. [Heterocentrotus mamillatus describing it as Voronoi-like pattern but without providing a specific assessment. Hence, to our knowledge, Voronoi structures have been neither specifically assessed in echinoids nor described in detail in their tests at a microstructural level.Echinoids possess a hierarchically organized lightweight endoskeleton with a unique mechanical design –37. Voroet al.  investigNumerous studies have been carried out on the echinoid endoskeleton architecture, with a particular focus on the variability in their highly porous microstructure and related mechanical properties ,37,42–56Paracentrotus lividus . In particular, the tubercle is the spine attachment site and is morphologically variable consisting of: (1) mamelon, a dome-like solid structure of imperforate stereom functioning as a ball-and-socket joint where the spine is articulated; (2) smooth platform, located between the summit of the boss and the mamelon base, which allows a circular movement of the spine and consists of imperforated stereom; (3) boss, a conical dome-like lightweight construction characterized by galleried stereom, where a hollow truncated cone of collagenous ligaments, called catch apparatus, enters the pores; (4) areola, the area surrounding the base of the boss, consisting of fine labyrinthic stereom where longitudinal muscle fibres are attached [The aim of this study was to identify and geometrically describe the stereom skeletal pattern located at the tubercle boss of primary spines in the sea urchin attached ,56,74–76Lamarck, 816. In pculated;  smooth pThe tubercle boss (TB) is a complex and mechanically stressed architecture that has attracted attention due to its rigorous microstructural regularity in contrast to the surrounding, less-organized trabecular mesh . A rigor. 2. 2.1Paracentrotus lividus  were collected by the scuba divers of the Stazione Zoologica Anton Dohrn in the Gulf of Naples, Italy (June 2019) from public and non-protected natural locations. Animal sampling was performed in full compliance with the European Union guidelines . The number of animals used for experimental purposes was reduced to minimum, and only samples required to obtain reliable data were used. Three tests  were digested in 0.1 N NaOH (for 1 week) to remove soft tissues. The samples were then washed three times in deionized water to remove caustic remains and air-dried for several days. Two types of interambulacral plates were extracted from the samples: (1) apical plates (N = 3), which represent the youngest plates in echinoid ontogeny and are small compared to older plates; and (2) ambital plates (N = 3), which represent the largest and most mature plates in echinoid ontogeny and are located at the ambitus . The plates were isolated, mounted on stubs and sputter-coated (Leica EM ACE 200) with a 10 nm platinum layer. Micrographs of the apical (Ap) and ambital (Am) TB stereom were recorded in four regions   (Specimens of ographs) , using a. 2.2Twenty-four SEM images of the TB stereom were recorded to a resolution of 410 × 1024 pixels. Micrographs were processed using Avizo software (v. 2019.1); Thermo Fisher Scientific, Waltham, MA, USA) and ImageJ Fiji software (v. 1.52e). The images were converted to binary images using an interactive manual thresholding to identify and separate the area of interest from the background. The binarized images were used for the skeleton analyses, pore analyses, assessment and geometrical characterization of the Voronoi pattern .. 2.3nodes (n), number and Euclidean coordinates ; (2) segments (s), number of segments; (3) connectivity (C), number of junctions per each node; (4) chord length (lc), length of the straight line between two nodes; (5) curved length (lt), length of the approximated curved course of a segment; (6) tortuosity (t), ratio between curved length and chord length t = lt/lc; (7) radius (r), mean value of all point radii describing a segment. Terminology follows Grun & Nebelsick and Avizo [SEM images were skeletonized using the Avizo ‘auto skeleton’ command . The skend Avizo .Figure . 2.4centroids (cn), centre points calculated as an average of the x and y coordinates of all the pixels in the selection; (2) total area (A0) of the selection in calibrated square units (μm2); (3) pore area (Apore) of the selection in calibrated square units (μm2); (4) perimeter (Perim), the length of the boundary of the selection calculated by its decomposition into individual selections; (5) circularity (Circ), shape descriptor calculated using equation (2.1)porosity (P), amount of pore area in the skeleton compared to the skeletal area investigated in toto (2).The pore spaces were analysed from binarized SEM images of the TB regions using the ‘Analyze particles’ command in Fiji . This pr. 2.5x1, x2, … } specified in Rd, a Voronoi cell C  of a point ix, can be generated as the set containing all the points closer to ix, than to any other seed [Iv) for each echinoid sample.A Voronoi model is a geometrical way of dividing space into a distinct number of regions or cells . As stather seed .2.3C and Voronoi (IV) images were compared using MATLAB R2020a . The images were translated into binary matrices (BWIS and BWIV), and, subsequently, a matrix of differences as absolute value (diff) was calculated using equation (2.4). This matrix quantifies the differences between the two images where all the concordant pixels assume the value 0, while the discordant pixels assume value 1. The discordance between the IS and IV images (equation (2.5)) was calculated by the ratio between the number of discordant pixels (4) and the total number of pixels (TP = 410 × 1024).The TB skeleton (. 2.7 (1)Number of seeds (nc), corresponding to the number of centroids . (2)Distance (d) between two seeds, calculated using equation (2.6). (3)Number of neighbour seeds (nns), the number of surrounding seeds computed for each seed as the number of cells sharing a boundary with the analysed seed cell. This was calculated via the nearest neighbour point analysis using R software  based on the centroid coordinates. The number of neighbour seeds and their distribution determines the number of cell sides (shape) in the Voronoi polygonal pattern ( pattern . It is w (4)Regularity (α), [ity (α), ,13 in coδ is the minimum distance calculated on all the pairwise distances between seeds and d0 is their distance given by equation (2.8):A0 is the total area and n represents the number of seeds.Where Once the overlap between the TB skeletal pattern and Voronoi model was estimated, the geometrical properties of the echinoid TB Voronoi pattern were investigated defining the following parameters:δ = d0 and α = 1; whereas δ and α are close to 0 for a completely random (Poisson Voronoi) model [ (5)Seed spatial distribution (ssd). Statistical analyses based on Ripley's K-function [complete spatial randomness (CSR) [function  were carss (CSR) . A map oss (CSR) . In thisWhere λ is the intensity of the point process of seeds, i.e. the mean number of points per unit area; E(h) denotes the expected number of further events within distance h of an arbitrary event. The K-function is usually estimated by equation (2.10):A| is the observation area, n the number of seeds, ijr the distance between the seeds ix and jx, I is the indicator function (i.e. I(ijr ≤ h) is equal to 1 if ijr ≤ h and is equal to 0 otherwise), and ijw are weights correcting the edge effect estimate (ijw is the proportion of the circumference of a circle centred at ix passing through jx contained in the observation region A).A regular hexagonal honeycomb is characterized by i) model ,13.(5) K(h) = πh2 and provides a benchmark against which it is possible to assess the pattern divergence from CSR. In aggregated patterns, K(h) tends to be larger than πh2 at distance h greater than the typical diameter of a cluster. Conversely, in regular patterns, K(h) is smaller than πh2 because each event tends to be surrounded by empty space [K(h) function for each of them in different h values. The band was obtained by computing the minimum and maximum values of K(h) on the simulated patterns for each value of h. The variable h is varying in 512 equally spaced points of the (discretized) interval , where H is equal to ¼ of the minimum dimension of the window of observation. This choice is recommended [h is large.The K-function of a homogeneous Poisson process is ty space ,79. The ommended ,80,81 to. 2.8Multivariate approaches were used to compare the plate types and plate regions in terms of their geometric characteristics among the 24 micrographs analysed. All the variables included in the analysis were numerical interval variables. They included (1) number of nodes, (2) number of segments, (3) connectivity, (4) curved length, (5) chord length, (6) radius, (7) tortuosity, (8) total stereom area, (9) pore area, (10) pore perimeter, (11) pore circularity, (12) stereom porosity, (13) divergence estimation of Voronoi patterns, (14) number of Voronoi seeds, (15) distance between Voronoi seeds, (16) regularity of Voronoi elements and (17) number of neighbour seeds. Since the variables were expressed in multiple types of units of measurement, any variances were not meaningfully comparable across variables. Consequently, data were z-standardized prior to analyses. A permutation multivariate analysis of variance (PERMANOVA)  based on. 3N = 24), and their interactions were also statistically insignificant (k = 2 dimensions), which showed an absence of groupings related to plate type or plate region  and 2417.33 ± 504.4 segments (N = 29 008). Node connectivity is 3.0 ± 0.0 (N = 18 275), curved length is 9.33 ± 0.7 µm (N = 29 008) and chord length is 9.05 ± 0.7 µm (N = 29 008). Segment tortuosity τ is 1.02 ± 0.0 (N = 29 008) with a radius of 2.55 ± 0.2 µm (N = 29 008)  and 2321.08 ± 465.3 segments (N = 27 853). Node connectivity is 3.0 ± 0.0 (N = 18 634), the curved length is 9.58 ± 0.8 µm (N = 27 853) and the chord length is 9.30 ± 0.8 µm (N = 27 853). Segment tortuosity τ is 1.02 ± 0.0 (N = 27 853) with a radius of 2.65 ± 0.7 µm (N = 27 853)  with a perimeter of 34.30 ± 5.9 µm (N = 10 241), porosity is 43.02 ± 7.2% (N = 10 241) ranging from 26 to 55% and circularity is 85.76 ± 3.1% (N = 10 241)  with a perimeter of 35.05 ± 6.7 µm (N = 10 180), porosity is 44.10 ± 8.5% (N = 10 180) ranging from 31 to 57% and circularity is 85.0 ± 3.0% (N = 10 180)  and Voronoi (IV) images indicated a divergence between these two tessellations of 18.0 ± 1.5% resulting in a concordance of 82.0 ± 1.5% (The matrix comparisons between the TB skeleton (0 ± 1.5% . The res0 ± 1.5%  (electro. 3.4N = 10 241), their distance is 90.54 ± 4.0 (N = 10 241) and regularity (α) is 0.47 ± 0.0 (N = 10 241) . In the TB ambital plates, the number of seeds is 848.33 ± 212.8 (N = 10 180), their distance is 91.06 ± 3.7 (N = 10 180) and regularity (α) is 0.45 ± 0.0 (N = 10 180) .In the TB apical plates, the number of seeds is 853.42 ± 183.8 (N = 20 421) ranging from 2 to 15 (N = 20 421). The neighbour data also correspond to the distribution of polygons constituting the TB Voronoi pattern in echinoids according to their shape. Thus, the median TB Voronoi cell sides conform with a constant equilibrium distribution of hexagons (44%), followed by pentagons (26%), heptagons (20%), quadrilaterals (5%), octagons (4%) and nonagons (1%) (The number of neighbor seeds is 6.0 ± 0.0 (ons (1%) .Figure K(h)-πh2 for TB stereom at apical and ambital plates was always located below the confidence band for CSR, obtained through a Monte Carlo simulation. The functions (blue lines) indicate that K(h), varying h, is smaller than πh2 and lies below the confidence band (dashed lines) predicted for CSR , retinal pigment and lung epithelial cells of chick embryo and blastular cell sheet of starfish (Asterina pectinifera) are typically described as Voronoi Dirichlet polygonal constructions, although they present deviations from the mathematical models [The image divergence estimation between the TB trabecular system and the generated Voronoi model revealed that the TB stereom geometrically diverges from a predicted Voronoi model by only 18%. Deviations from a computational generated Voronoi pattern resulting less than 20% can be interpreted as acceptable since naturally grown structures are subject to constraints such as genetic heritage and phenotypic plasticity. In fact, numerous patterns in nature, both in the plant and animal kingdom, such as coenobial green alga  . In natutruction . Moreovetruction ,86,87. Ih et al. . Nonetheh et al.  and cucuh et al. . Recentl4% . In n [et al. ,89,90–93 [et al. ,89. The  [et al. ,89. The  [et al. –96. Henc [et al. ,84.P. livius plates.A polygonal construction such as the Voronoi meshwork can vary from highly irregular to fully ordered, based on seed distance and disposition ,13. In tet al. [Numerous studies have dealt with two-dimensional and three-dimensional Voronoi constructions and their mechanical performance –15,26. Iet al.  reportedet al. . Thus, aEchinocyamus pusillus [The results achieved showed that TB trabeculae are uniformly long and possess little tortuosity, indicating that the trabeculae follow a direct course between two nodes. Similar results have been reported for the dwarf sea urchin pusillus . Linear pusillus  and strepusillus  and liteCirc) were close to 1, indicating that pore shapes approximate circles. Porosity is usually highly variable in the skeletal components of echinoids, e.g. ossicles of the buccal apparatus and spines [The pore analysis provided information on the TB pore area and its geometry. Porosity is around 44% on average, in accordance with the plate porosity of 30 to 50% reported in literature . The cird spines ,55,106. d spines . Recentld spines . In the d spines –76,97,98P. lividus. The microstructure of the echinoid stereom was demonstrated to be consistent with a Voronoi construction, providing a new perspective for investigating and modelling the different echinoid stereom structures, soft tissues disposition, mechanical skeletal adaptations, and structural growth mechanisms at two-dimensional and three-dimensional scale. Further quantitative documentation of microstructural patterns demonstrated by echinoids can also inspire and motivate the design implementation of various load carrying rod systems, in which lightweight and structural robustness are of prime importance. The echinoid stereom may be of broad biomimetic relevance to many applied fields, including engineering, architecture, biotechnology, and material sciences.In conclusion, this study provides critical analyses on the structure and design of the echinoid


Wulongzhiyangwan (WLZYW) is a Chinese prescription medicine for the treatment of pruritus, but its mechanism has not been clarified. The purpose of this study was to explore the mechanism of WLZYW in pruritus through network pharmacology analysis and experimental validation. The active components and corresponding targets of WLZYW were obtained from the Traditional Chinese Medicine Systematic Pharmacology (TCMSP) database. Pruritus-related targets were obtained from the GeneCards, TTD (Therapeutic Target Database), and DrugBank databases. The key compounds, core targets, main biological processes and signaling pathways related to WLZYW were identified by constructing and analyzing related networks. The binding affinity between WLZYW components and core targets was validated by AutoDock Vina software. In this study, RBL-2H3 cells were used to construct a degranulation model to simulate histamine-dependent pruritus. 10 chemical constituents, 235 targets and 3606 pruritus-related targets of WLZYW were obtained. Subsequently, 26 core targets were identified through analysis, VEGFA and AKT1 were the main candidates. A pathway enrichment analysis showed that overlapping targets were significantly enriched in the PI3K/AKT signaling pathway. A molecular docking analysis revealed tight binding of VEGF to three core compounds, kaempferol, luteolin and quercetin. Experiments showed that WZLYW inhibited mast cell degranulation, regulated VEGFa mRNA and protein expression levels by inhibiting PI3K/AKT and ERK1/2 signaling pathway activation. The mechanism of WZLYW in pruritus may be regulating VEGFa expression. Network pharmacology assays suggested that WLZYW downregulates VEGFa expression by regulating the PI3K/AKT and ERK1/2 signaling pathways in pruritis treatment. It often leads to great personal stress and financial burden. The pathogenesis of pruritus is generally classified into histaminergic and nonhistaminergic pathways2. The former is mediated by histamine secreted primarily by mast cells, basophils and keratinocytes3. Mast cell activation causes histamine pruritus involves cell degranulation following the cross linking of immunoglobulin E (IgE) with high-affinity IgE receptors (FcεRI). Then, other influencing factors are released, such as histamine, prostaglandin, NGF, VEGFa, TNF-α and leukotriene4. When the patient is diagnosed with chronic pruritus of unknown etiology, treatment should be in principle according to T helper 2 (Th2) type inflammation5.Itch is a common sensory experience that is prevalent in patients with inflammatory skin diseases, as well as in those with systemic and neuropathic conditions. Itching that lasts 6 weeks or longer is considered chronic and its estimated lifetime prevalence is 22.6%Zaocys dhumnades), Fang feng (Saposhnikovia divaricata), Shechuangzi (Cnidium monnieri (L.) Cuss.), Kushen (Sophora flavescens Aiton), Huangbai (Phellodendron amurense Rupr.), Cangshu (Atractylodes lancea (Thunb.) DC), Renshen (Panax ginseng C. A. Meyer), Mudanpi (tree peony bark), Shedanzhi (Snake bil), Bovis Calculus (Bos taurus domesticus Gmelin), and Danggui (Angelica sinensis). This formula is widely used in the treatment of pruritus in clinical practice.The Chinese Pharmacopoeia (2010 Edition) shows that Wulongzhiyangwan (WLZYW) is used to treat addictive rashes, rubella, and pruritus caused by the accumulation of heat rash on the skin, with symptoms that manifest as red spots, blistering rashes, and intolerable itching. WLZYW contains ten compounds, namely, Wushaoshe  have been used in China for thousands of years with good efficacy for certain diseases. Recently, researchers have gradually elucidated the effective components and targets of TCM. Network pharmacology is based on the similarity in structure and function between drugs, analyze disease-related genes in regulatory network of drug–component–target–disease, to explore the interactive relationships between target molecules and biological effector molecules in vivo. TCM are two or more mixed preparations, with complex regulatory networks. The ingredients in WLZYW formulation act on different targets and pathways, but some may act on the same target. In other words, the ingredients in the formula are synergistic. Network pharmacology integrates multidisciplinary technologies and contents, such as multidirectional pharmacology, computational biology, network analysis and related findings. The comprehensive network analysis of drug effects can be achieved by multitarget research strategiesIn this study, we simulate the action network of WLZYW on the basis of manufacturing drug component target pathway network, selected the core drug components and the most likely drug targets, and validated the findings through molecular docking. The workflow diagram is shown in Fig. Ten drugs in WLZYW formula correspond to 59 complex components, and 235 drug targets were selected by OB  ≥ 30% and DL (Drug Likeness) ≥ 0.18 following the drug-like principle . The corresponding secondary protein interaction network was obtained Fig. A, with 2nds Fig. . With an. The cor10. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed for the 26 target genes in the PPI core network, resulting in 2489 GO enriched terms , which used by researchers to understand all aspects of biomedicine, from protein synthesis to the roles in health and disease. Among the 26 potential targets, AKTI and VEGFa showed the highest degree of centrality (degree = 59) and were closely related to the pathways indicated in KEGG analysis. Therefore, we searched the three-dimensional structure of AKT1 and VEGFa in PDB as the protein receptor in docking experiments. The interaction targets of WLZYW with AKT1 were identified by PPI network, and the corresponding compound ligands were luteolin, quercetin, and kaempferol. PubChem software was used to score and select the ligand structure with the smallest free energy. Docking of AKT1 and VEGFa receptor and their corresponding small-molecule drug ligands were performed by AutoDock Vina (opensource molecular docking program). Based on semiflexible docking, the interaction energy results are shown in Table Kaempferol, luteolin, and quercetin bound effectively to the active pockets of VEGFA protein. Their binding energies were − 6.8, − 7, and − 6.9 kcal/mol, respectively Table . The resKaempferol, luteolin and quercetin effectively bound to the active pockets of AKT1 protein with binding energies of − 9.2, − 9.8 and − 10.1 kcal/mol, respectively, all were lower than − 7.0 Table D. Luteol0.5 µg/mL DNP-IgE was used to induce RBL-2H3 cells for 24 h, and then, DNP-BSA (100 ng/mL) was added as the secondary stimulation to cause degranulation and release inflammatory mediators. WLZYW (1 µg/mL to 100 µg/mL) was not toxic to RBL-2H3 cells , Shechuangzi-Cnidium monnieri (L.) Cuss., Kushen-Sophora flavescens Alt., Huangbai-Phellodendron amurense Rupr., Cangshu-Atractylodes Lancea (Thunb.) DC, Renshen-Panax ginseng C. A. Meyer, Mudanpi-Tree Peony Bark, Shedanzhi-Snake bil, Bovis Calculus-Bos taurus domesticus Gmelin, and Danggui-Angelica sinensis. They were searched in TCMSP database11 for drug screening, with OB ≥ 30% and OL ≥ 0.18, and obtained the targets name of all compounds. Symbol matching of target names were performed by human-derived gene information from Uniprot database for multi-database information docking.WLZYW contains ten herbs, namely, Wushaoshe-12: the keyword was pruritus, 16 pruritus-related targets were found. (2) GeneCards: the keyword was pruritus, 3596 pruritus-related targets were found. (3) DrugBank Online13: the keyword was pruritus, 9 pruritus-related targets were found.The retrieval of pruritus-related targets resulted in the collection of different genes associated with pruritus from the following resources. (1) Therapeutic Target Database (TTD)14. Finally, targets with score less than average were removed, after two times the core targets were selected.Pruritus targets and WLZYW targets were intersected by R 4.0.2 to generate coincident targets. Core target screening and analysis were performed by STRING databaseProtein–protein interaction (PPI) data were obtained from Mutual Interaction Gene/Protein Search Tool. The coincident targets were entered into a database with species restricted to Homo sapiens, and confidence values were higher than 0.4. The hypergeometric test was performed via the phyper function of R 4.0.2.Network construction was performed by two networks in this study. WLZYW compounds were connected with corresponding targets to establish compound-target network, network of pruritus targets was constructed by searching pruritus-related targets in different databases. The compound-targets network was crossed with the pruritus-related targets network to construct PPI network. Genes that did not cross were removed. A potential target–protein interaction network was constructed, and analyzed using modules to advance the core network. The networks were all made by Cytoscape 3.8.10, a software package used in the visualization and analysis for networks. For easily interpret large biological datasets, biological information is often visualized as graphs, such as nodes and edges. Nodes represent biomolecules, and edges between nodes represent certain relationship.15. The GO database includes BP, CC, and MF terms, were used to identify possible biological mechanisms based on high-throughput genomic or transcriptomic data16. KEGG is a reference knowledge base of biological interpretation for genome sequences and other high-throughput data. In addition, the bio-conductor cluster Profiler , enrich plot, and pathview were used to analyze potential target modules for GO and KEGG pathway analysis. These five R packages were also applied in the enrichment analysis of gene clusters17.DAVID , functional enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis for the targets networks of pruritus genes corresponding to WLZYW were performed. In addition, Pathview was used to obtain visualized KEGG imagesThe SDF formats of compounds were downloaded from Pubchem data and imported into Chemdraw 3D. The energy was minimized using MM2 module to obtain the lowest energy, and saved as mol2 file. VEGFA and AKT protein structures were downloaded from the Protein Data Bank (PDB) database with PDB IDs of 6zfi and 7nh5, respectively. Subsequently, Pymol was used for visualization respectively. Mgtools 1.5.6 was used to dehydrate, hydrogenate, calculate charge and combine non-polar hydrogen, the ligands and receptors were respectively stored as PDBQT. Autodock vina 1.1.2 was used to dock ligand with receptor, and the parameters were shown below. The high rated were visualized by PYMOL and Discovery Studio.−1 penicillin and 100 μg mL−1 streptomycin. The cells were cultured in a 37 °C, 5% CO2 incubator. RBL-2H3 (0.5 × 106 cells/mL) grew in multi-well plates. After the cells fused to 75%, added DNP-IgE  for 24 h, then washed with PBS. Cells and supernatants were collected after stimulation with 100 ng/mL DNP-BSA .RBL-2H3 cells were purchased from Procell (CHN). RBL-2H3 were cultured in DMEM medium containing 10% fetal bovine serum, 100 U mL5 cells/well and incubated for 24 h at 37 °C and 5% CO2. Cells were exposed to 1–1000 μg/mL WLZYW and further incubated for 24 h. The medium was replaced with 100 µL fresh medium containing 10% CCK8 , and cells were incubated for 1.5 h. The OD (450 nm) absorbance value in each well was determined by spectrophotometer . Cell viability rate (%) was calculated.CCK8 assay was performed to detect cell viability and proliferation. RBL-2H3 cells were seeded in the 96-well culture plates with 1 × 10Cells were seeded in 6-well plate culture dish. When cells fused to 75%, added DNP-IgE  for 24 h, washed with PBS, and replaced with WLZYW (10 μg/mL or 50 μg/mL). Cells were collected after stimulation with 100 ng/mL DNP-BSA for 10 min. Total RNA was extracted from cells using RNeasy Mini Kit , and 1000 μg template RNA was transcribed into cDNA by Superscript III RT-PCR Kit . Then, the cDNA was qRT-PCR via SYBR GREEN . The primer sequences are: TNF-α, Forward-CATCCGTTCTCTACCCAGCC; Reverse-CATCCGTTCTCTACCCAGCC. VEGFa, Forward-ACGAAAGCGCAAGAAATCCC; Reverse-GCAACGCGAGTCTGTGTTTT. GAPDH, Forward-TGAGATCAACGTGTTCCAGTG; Reverse-ACCAGATGAAATGTGCCCC.RBL-2H3 cells were sensitized with IgE (0.45 µg/mL) for 24 h and then treated with WLZYW (10 or 50 µg/mL) for 30 min. After cells were attacked by 100 ng/mL DNP-BSA for 4 h, supernatant was collected and the levels of TNF-α and VEGFa  were determined with ELISA kit according to the instructions.The proteins extracted from cells were measured with BCA Protein Assay Kit . 25 μg protein samples were boiled at 90 °C for 7 min, then loaded and subjected to electrophoresis in 12% FuturePAGE  gels at 160 V for 20 min. The membranes were probed with anti-PI3K/AKT antibody , anti-PI3K/AKT antibody , anti-p-ERK1/2 antibody , anti-ERK1/2 antibody , and mouse monoclonal antibody against tubulin  overnight at 4 °C. IRDye 680RD Donkey anti-Mouse (LI-COR USA) and IRDye 800CW Donkey anti-Rabbit (LI-COR USA) was used as secondary antibody. The visualization of protein bands was accomplished by Li-cor Odyssey infrared imaging system (LI-COR USA).t test, one-way ANOVA and Dunnett’s multiple comparisons test were used. In all analyses, p values less than 0.05 were considered significant.All experiments were repeated at least three times independently. Date was presented with mean ± SD, and statistical analysis was performed using Prism 8 . 18. Unfortunately, targeted treatments are limited, current treatments usually only relieve itching or reduce irritation caused by external factors, do not cure. Such as glucocorticoids were used to treat inflammatory skin pruritus19. In addition, patients need to prolong prescribing, which may lead to drug resistance and adverse side effects. Due to its complex mechanism, pruritus is rarely studied. In this study, in order to explore the therapeutic mechanism of WLZYW in pruritus, network pharmacology was used to find the key agents and target pathways by constructing ingredients-targets-pathway and other networks.Pruritus is the most common complaint encountered by dermatologists, and it causes psychological stress and insomnia. Moreover, in patients with atopic dermatitis, it results in skin barrier damage. Pruritis aggravates the progression of skin disease and increases sensitivity to antigens20. The activation of these molecules enhances genes expression, the most important is VEGF21. Binding of VEGFR to VEGF ligand will directly activate phosphotyrosine residues, such as Sck, PLC-γ, PI3K, Akt, and MAPK22. VEGFa is a versatile polypeptide that mediates endothelial cell specific responses, including induced proliferation and vascular leakage. Krause23 reported significant improvement in simple pruritus patients treated with bevacizumab . Moreover, serum levels of VEGF were significantly higher in patients with pruritus23, and correlated with severity assessed by physicians. VEGFa can effectively enhance vascular permeability, with molar efficacy approximately 1000-fold greater than that of histamine24. In addition, mast cells constitutionally express VEGF as preformed heparin-binding factor25 and can secrete this protein to respond to triggers, including IgE and antigen4.Now the mechanism of itching is thought that degranulation via Lyn in mast cells or basophils results in the phosphorylation and recruitment of Syk, which in turn activates the substrate LAT and NLAT signaling molecules, such as PI3K/AKT, ERK1/2, PLC, PKC, transcription factors, and NF-κB26. Flavonoid derivatives act as antivascular agents by interfering VEGF/VEGFR2 pathway27. Kaempferol inhibits VEGF secretion in a time-dependent manner by regulating the signal cascades of PI3K/AKT, MEK and ERK29. Quercetin can inhibit the secretion of angiogenic factor30. Kaempferol and quercetin effectively inhibit the development of IgE-mediated allergic inflammation31. Kaempferol prevents MC-mediated allergic diseases by attenuating Lyn activation32. ERK1/2 is regulated dose-dependently by luteolin, induced by inhibition of AKT and MAPK pathways33. Luteolin can inhibit the FcεRΙ- and MrGPrx2-mediated allergic reaction in vivo and in vitro, and reduce the serum levels of histamine, TNF-α, MCP-1, IL-8 and IL-13 in mice34.After screening drug targets network, 16 core targets were identified following multiple analyses of 160 common targets between WLZYW and pruritus. The results indicated that kaempferol, luteolin, and quercetin might be the key ingredients in WLZYW able to exert therapeutic effects against pruritus. All three are natural flavonoids found in many plants with therapeutic properties, such as antioxidant, anticancer, and anti-inflammatory35. LY294002 (Akt phosphorylation antagonist) can effectively treat atopic dermatitis in mice and significantly inhibit scratching behaviors36. These results suggested that Akt can be targeted in the treatment of pruritus. RBL-2H3 cell line is a useful in vitro model for studying the degranulation of mast cells induced by IgE–FcεRI37. ERK1/2 regulates the phosphorylation of cPLA2, as well as the production and release of arachidonic acid, which are the major factors of pruritus38. Through in vitro experiments, both concentrations of WLZYW significantly inhibited PI3K/AKT and ERK1/2 signaling pathways, and the degree of degranulation in the model reduced. To understand the relationship between two pathways and VEGFa, wortmannin (1 µM) and U0126 (2 µM) were added to inhibit the model. Western blot and PCR experiments proved WLZYW induced down-regulation of TNF-α and VEGFa mRNA and protein levels. In conclusion, WLZYW downregulates VEGFa expression by inhibiting the phosphorylation of PI3K/AKT and ERK1/2 pathways.GO and KEGG pathways enrichment results indicated that WLZYW might exert effects on pruritus by regulating PI3K-Akt (has04151) signaling pathway. Pathview revealed that WLZYW act not only on AKT, but also on the upstream and downstream of AKT pathway, as well as on VEGF and ERK1/2. Recently, in vivo experiments have confirmed the effects of p-AKT and p-ERK in chronic pruritus, enhance the expression of melatonin and improve the quality of sleepOur results suggest that the active components of WLZYW regulate mast cell activation in many ways, thereby inhibiting the release of sensitizing mediators caused by degranulation, which is main mechanism in the treatment of pruritus. The limitation of this study is that in vivo experimental verification was not carried out. In addition, the etiology of pruritus is complex, and it is difficult to simulate perfectly with only one model.Network pharmacology can demonstrate the pharmacological effects of WLZYW on pruritus via multiple components, multiple targets and multiple pathways. The molecular docking results suggest that the main pathway is PI3K/AKT, the main target is VEGFa. In vitro, WLZYW inhibits DNP-IgE/BSA-induced RBL-2H3 degranulation and regulates VEGFa expression by inhibiting PI3K/AKT and ERK1/2 pathways.Supplementary Legends.Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Figure 4.Supplementary Legends.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.Supplementary Table 7.