Oxidative stress causes the progression of diabetes and its complications; thus, maintaining the balance between reactive oxygen species produced by hyperglycemia and the antioxidant defense system is important. We herein examined the antioxidant potential of non-extractable fractions of dried persimmon (NEP) against oxidative stress in diabetic rats. Rats with streptozotocin-induced type 1 diabetes (50 mg/kg body weight) were administered NEP for 9 weeks. Antioxidant enzyme activities and concentration of antioxidants in liver tissues were analyzed with a microplate reader. Extensor digitorum longus (EDL) and soleus muscle fibers were stained with succinate dehydrogenase and muscle fiber sizes were measured. The administration of NEP increased the body weight of diabetes rats. Regarding antioxidant activities, the oxygen radical absorbance capacity and superoxide dismutase activity in liver tissues significantly increased. In addition, increases in glutathione peroxidase activity in liver tissues and reductions in the cross-sectional area of EDL muscle fibers were significantly suppressed. In these results, NEP improved the antioxidant defense system in the liver tissues of diabetic rats, in addition to attenuating of muscle fibers atrophy against oxidative damage induced by hyperglycemia. Diospyros kaki Thunb.) belongs to the Ebenaceae family and is rich in vitamins, minerals, fiber, and bioactive compounds. Persimmon is generally classified into astringent or non-astringent varieties. The fruit of astringent persimmon is rich in soluble condensed tannin [Fruits and vegetables are rich in vitamins, minerals, fiber, and natural antioxidants. The sustained intake of fruits and vegetables has been shown to reduce the risk of diseases, such as cancer, hypertension, and cardiovascular diseases ,2,3. Perd tannin  and, thud tannin ,6. Previd tannin , exhibitd tannin , and exed tannin ,10. Furtd tannin .2O2), hydroxyl radical, singlet oxygen, NO, etc. They react to DNA, proteins, lipids, etc., and cause dysfunction. ROS are also produced by environmental factors, such as smoking, UV light, radiation, and air pollution [Diabetes mellitus is a major health issue worldwide. Over 10.5% of the world’s population suffers from diabetes, which is projected to increase to 12.2% by 2045 ,14,15, wollution . Increasollution ,20,21. Oollution . The maiThe antioxidant activities of fruits are generally evaluated using their soluble extracts, but few assessments have been conducted on non-extractable fractions. However, it is possible that the non-extractable fraction of dried persimmon (NEP) is rich in condensed tannin. We previously demonstrated that NEPs exhibited high antioxidant activity in vitro and in vivo . TherefoTrolox  and fluorescein sodium salt were purchased from Sigma-Aldrich Japan Co., Ltd. . 2,2′-Azobis (2-amidinopropane) dihydrochloride (AAPH) was obtained from FUJIFILM Wako Pure Chemical Corp. . All reagents were of analytical grade.TM X4 . Absorbance measurements of the concentration of H2O2 remaining after the effects of catalase were performed using the Shimadzu UV mini-1240 spectrophotometer .HPLC analysis was performed using a Shimadzu LC-10AD VP multisolvent delivery system equipped with a SIL-10AD VP auto injector and SPD-M10A VP photodiode array detector . A Diaion HP-20  was used for the column chromatography. Oxygen radical absorbance capacity (ORAC), SOD, and GPx activities, and concentrations of total glutathione and oxidized glutathione (GSSG) were measured using the PerkinElmer 2030 Multilabel Microplate Reader ARVODiospyros kaki Thunb.) was harvested in Yoshino-gun, Nara, Japan. Fruit was peeled and sulfureted to prevent discoloration and oxidation and then processed into dried persimmon after air-drying for one month.The “Hohlenbo” variety of astringent persimmon  was cut into small pieces and homogenized with 14.8 L of 90% aqueous ethanol (EtOH). The extract was filtered from the homogenate and 90% aqueous EtOH was added to the residue. These extracting and filtering procedures were repeated three times. The extracted residue was dried at room temperature to obtain NEP (769.3 g).Diospyros kaki Thunb., cv. “Hiratane-nashi” and “Tone-wase”) harvested in Nara, Japan, were used for tannin extraction. “Hiratane-nashi” was designated as a protected species  in 1962. “Tone-wase”, a mutated variety of ”Hiratane-nashi”, was registered  in 1980. The species of persimmon were identified by Dr. Sadahiro Hamasaki , and the extraction of tannin was conducted using a modified method from his study [w/v) for 5 days for the insolubilization of tannin. The ethanol-treated persimmons were crushed into small pieces and soaked in water for 2 days at room temperature. The supernatant contained soluble components, such as sugars, and the residue contained insoluble tannin. After discarding the supernatant, water was added to the residue. The suspension was heated at 120 °C for 30 min to exchange insoluble tannin for soluble tannin. The suspension was filtered, evaporated in vacuo, and dried at 160 °C to obtain a powder of persimmon-derived tannin. The batch of this powder contained 75.5% condensed tannin, measured as epigallocatechin gallate equivalent based on the Folin–Ciocalteau method. Persimmon-derived tannin was stored at −20 °C until used.The procedure for persimmon-derived tannin extraction has been described previously ,24,25. Iis study . Immatur2O as an eluting solution, followed by elution with 100% MeOH. The H2O eluate was neutralized with NaOH, and ethyl acetate (EtOAc) was added to the partition between EtOAc and H2O. The EtOAc layer was evaporated in vacuo to obtain the fractions of hydrolyzed NEP or persimmon-derived tannin for HPLC analysis.Portions (1 g) of NEP or persimmon-derived tannin were combined with 50 mL of a 1.2 M HCl-50% methanol (MeOH) solution and heated at 90 °C for 3 h. The treated suspensions were centrifuged at 3000 rpm at room temperature for 15 min to obtain the supernatant. The same solution was added to the residue, and they were heated again at 90 °C for 30 min. The suspensions were centrifuged under the same conditions to obtain another supernatant. The combined supernatant was subjected to Diaion HP-20 column chromatography using HEach fraction of NEP or persimmon-derived tannin was dissolved in 2 mL of 100% MeOH and filtered through 0.45 µm disposable membrane filters . The conditions of HPLC were as follows: column, RPAQUEOUS-AR-5, 4.6 × 250 mm, ; column temp., 40 °C; mobile phase, A = 0.1% formic acid, B = 0.1% formic acid in 30% acetonitrile; flow rate, 1.0 mL/min; gradient, 0.00 min, %B = 0; 5.00 min, %B = 20.0; 10.00 min, %B = 21.0; 20.00 min, %B = 23.0; 25.00 min, %B = 25.0; 30.00 min, %B = 5.0; 35.00 min, %B = 0; 40.00 min, %B = 0; injection volume, 5µL; detection, photodiode array (190–600 nm).Type 1 diabetes was induced in eight-week-old male Wistar rats with streptozotocin (50 mg/kg body weight) and normal rats were obtained from Japan SLC . Normal rats were fed an AIN-93G-modified basal diet  (NOC group). Diabetic rats were randomly divided into two groups of six animals each, as follows: (i) diabetic rats fed an AIN-93G-modified basal diet (DMC group); (ii) diabetic rats fed a basal diet supplemented with 5% NEP instead of cellulose (DMP group). Each animal was individually housed. Study diet compositions are shown in ad libitum. Food intake and the body weights of all groups were monitored daily for 9 weeks. Blood was collected from the tail vein weekly, and plasma was isolated and stored at −80 °C until use. Rats were killed after 9 weeks, and the liver and skeletal muscle tissues (extensor digitorum longus (EDL) and soleus muscles) were isolated. Isolated liver tissues were perfused with ice-cold physiological saline, immediately frozen in a dry-ice acetone bath, and stored at −80 °C until use. Isolated muscles were frozen in liquid N2 and stored at −80 °C until use.Animals were fed g at 10 °C for 5 min. The supernatants were diluted 1:6.25 (v/v) in 75 mM phosphate buffer (pH 7.4) to obtain the plasma solution. Plasma solutions were further diluted two- to eightfold in 75 mM phosphate buffer (pH 7.4).Plasma samples were removed from storage at −80 °C and thawed under running water. Aliquots (50 µL) were inserted into tubes and 100 µL of EtOH and 50 µL of H2O were added. After shaking for 30 s using a vortex, 200 µL of 0.75 M metaphoric acid was added. The mixtures were centrifuged at 210× The ORAC of each sample was assessed according to a previously reported method ,28,29,30ORAC was measured at 37 °C every 2 min for 90 min. The area under the fluorescence curve was calculated, and the ORAC of each sample was expressed as units for 1 µmol equivalent of Trolox.w/v) of PBS (pH 7.5)  with 0.5% Triton X-100 . Suspensions were then homogenized, and the resulting homogenates were centrifuged at 22,360× g at 4 °C for 10 min. Supernatants were stored at −80 °C until use. Prior to the ORAC assay, supernatants were thawed under running water and further diluted 32-fold in 75 mM phosphate buffer (pH 7.4). The ORAC of diluted supernatant solutions was measured as described above (Liver tissues were weighed to approximately 300 mg and added to 10 volumes (ed above .w/v) of 20 mM HEPES buffer (pH 7.2)  with 1 mM EGTA , 210 mM mannitol , and 70 mM sucrose . Suspensions were then homogenized, and the resulting homogenates were centrifuged at 1500× g at 4 °C for 5 min. Supernatants were further centrifuged at 14,000× g at room temperature for 15 min to concentrate the samples using an Amicon concentrator  with a molecular weight cut-off of 10,000. The samples obtained were stored at −80 °C until used. Prior to the SOD activity assay, filtrates were thawed under running water.Liver tissues were weighed to approximately 100 mg and added to 10 volumes .Liver tissues were weighed to approximately 100 mg and added to 350 µL of PBS with 0.5% Triton X-100 and 1% protease inhibitor . Suspensions were then pulverized with the Micro Smash Ms-100R bead beater (TOMY Japan) at 4500 rpm at 4 °C for 30 s. The resulting solutions were centrifuged at 11,000× 2O2 remaining after the effects of catalase. One unit of catalase was expressed as units required for 1 µmol of H2O2 to decompose to oxygen and water per minute with a substrate concentration of 10 mM H2O2.Catalase activity was assessed using a previously reported method ,33 with w/v) of 50 mM Tris-HCl buffer (pH 7.5)  with 5 mM EDTA  and 1 mM 2-mercaptoethanol . Suspensions were then homogenized, and the resulting homogenates were centrifuged at 8000× g at 4 °C for 10 min. Supernatants were stored at −80 °C until used. Prior to the GPx activity assay, supernatants were thawed under running water. Supernatants were further diluted 10-fold in phosphate buffer with EDTA and 4 mM NaN3 (pH 7.0).Liver tissues were weighed to approximately 400 mg and added to 5 volumes . This assay was performed according to the manufacturer’s instructions. One unit of GPx-1 was expressed as the amount of enzyme needed to catalyze the oxidation of 1 µmol reduced glutathione (GSH) to GSSG per minute.The GPx activity assay is an adaptation of the method developed by Paglia and Valentine . Its actw/v) of 5% 5-sulfosalicylic acid solution . Suspensions were then homogenized, and the resulting homogenates were centrifuged at 8000× g at 4 °C for 10 min to remove proteins. Supernatants were stored at −80 °C until used. Prior to the total glutathione and GSSG assay, supernatants were thawed under running water. Supernatants were then further diluted 50-fold in ultrapure water for total glutathione and 5-fold in ultrapure water for GSSG.Liver tissues were weighed to approximately 100 mg and added to 10 volumes  method with bovine serum albumin as a standard . The ORAC and SOD, catalase, and GPx activities of liver tissues were normalized to the protein concentrations of the homogenates.To measure the CSA of EDL and soleus muscle fibers, 10 µm thick transverse sections of muscle were stained with succinate dehydrogenase (SDH) and examined under a light microscope . SDH staining is a method that uses the enzyme activity involved in energy production in mitochondria and classifies muscle fibers into slow-twitch oxidative (SO) plus fast-twitch oxidative glycolytic (FOG) and fast-twitch glycolytic (FG). Muscle fiber sizes were measured using ImageJ .p < 0.05.Each sample in all assays was measured in duplicate, and data were expressed as means ± standard deviations. Statistical analyses were performed using the Mann–Whitney U test and differences were considered to be significant at It was speculated that NEPs are rich in condensed tannin. Therefore, HPLC analysis was performed to confirm the existence of tannin in NEPs. HPLC chromatograms of hydrolyzed NEP and persimmon-derived tannin are shown in p < 0.001) than in the two other groups of diabetic rats (DMC and DMP groups). The body weight after 3 weeks was significantly higher in the DMP group than in the DMC group (p < 0.05); however, the body weight after 9 weeks did not significantly differ between DMC and DMP groups  in the two groups of diabetic rats (DMC and DMP groups) than in the NOC group (p < 0.05) in the DMP group than in the two other groups (NOC and DMC groups)  B.p < 0.05) in the DMP group than in the NOC and DMC group (p < 0.05) in the DMC group than in the NOC group; however, the increase observed in the DMP group was slightly smaller than that in the DMC group  in the DMP group than in the NOC and DMC groups (p < 0.05) higher in the DMP group than in the NOC and DMC groups  scavenging activity of biological samples ,28,29,302O2 into water and oxygen. GPx detoxifies H2O2 and lipid peroxides [2O2. GSSG is regenerated into GSH by glutathione reductase. Since total glutathione concentrations and the GSH/GSSG ratio are altered by oxidative stress, they are used as an indicator of oxidative stress.Oxidative stress markers are broadly classified into three types: antioxidant enzymes, antioxidants, and products produced in vivo by ROS. Antioxidant enzymes, including SOD, catalase, and GPx, remove ROS. SOD catalyzes the degradation of the superoxide radical anion. Catalase disproportionates Heroxides . When theroxides . There a2O2 concentrations have been shown to inactivate SOD [2O2. However, SOD and catalase activities were higher in the DMP group than in the DMC group (2O2 concentrations by hyperglycemia and decreases in catalase activity. GPx activity was slightly lower in the DMP group than in the DMC group (2O2 but also organic peroxides, using GSH as a cofactor. Since the total glutathione concentration was lower in the DMP group (In the present study, SOD and catalase activities were slightly lower in the DMC group than in the NOC group A,B. Incrvate SOD . The decMC group A,B, indiMC group C and appMC group C. GPx deMP group B and GSSMP group D, GPx maThe GSH/GSSG ratio and total glutathione, GSH, and GSSG concentrations did not significantly differ between the DMC and NOC groups , which iSkeletal muscle fibers are classified into three basic types: SO, FG, and FOG types . The natIn the present study, the CSAs of all muscle fiber types in the EDL and soleus muscles were significantly lower in the DMC group than in the NOC group . AnderseThe present study examined the antioxidant potential of NEP against oxidative stress in the liver and muscle fibers in streptozotocin-induced diabetic rats. The administration of NEP increased the ORAC of liver tissues and SOD activity in diabetic rats. In addition, reductions in the CSA of EDL muscle fiber types were suppressed.


Purpose: Methimazole is an anti-thyroid agent, especially as main therapy option for Graves’ disease in children and adults. Drug induced pancreatitis is one of the known adverse effect of methimazole mentioned in case reports. However, the detailed molecular mechanisms of methimazole-induced pancreatitis are still unclear. In this study, the aim is to investigate the adverse effect of methimazole on pancreas cell stress mechanism and apoptosis.Methods: Cytotoxicity was evaluated in human pancreas/duct (PANC-1) cell line. Total oxidant (TOS) and antioxidant status (TAS) for oxidative stress index, glutathione (GSH) level and endoplasmic reticulum (ER) stress biomarkers were evaluated by ELISA. Reactive oxygen species (ROS) levels and apoptosis were evaluated by flow-cytometer. Results: The 30% inhibition rate concentration (IC30) value was determined as 53 mM in PANC1 cells. The exposure concentrations were in the range of 0-40 mM for 48 hours. Methimazole might induce cellular stress conditions. ROS production increases depending on concentration, and this increase shows parallelism with the increase in ER stress biomarkers such as TOS, ERN1 and CASPASE12. Conversely, there was no significant difference between control and exposure groups in terms of apoptosis. Conclusion: In conclusion, methimazole might have triggered the mechanisms of inflammation or autophagy in the pancreatic cells. However, there is still a need for in vitro and in vivo studies including other cellular parameters related to apoptosis. Thus, methimazole is one of the options that could be safely use in both adults and children.2 Adverse effects such as hypersensitivity, decrease in the number of leukocyte, agranulocytosis, gastrointestinal symptoms and hepatic dysfunction can occur in patients with methimazole treatment. Furthermore, the patients with acute pancreatitis induced methimazole reported abdominal pain, increased pancreatic enzyme levels and imaging findings like pancreas swelling in case-reports.6 Acute pancreatitis was added to the list of adverse effects of methimazole based on six case reports in 2019; also, methimazole product information was changed as including acute pancreatitis with European Medicine Agency (EMA) warning.1 Methimazole, carbimazole (prodrug of methimazole) and propylthiouracil are three thionamide compounds used in hyperthyroidism therapy.7 One of the main molecular mechanism underlying acute pancreatitis is oxidative stress. The increased cellular reactive oxygen species (ROS) causes the death of pancreatic cells.8 It has been reported that cellular calcium level changes, mitochondrial membrane permeability changes, endoplasmic reticulum (ER) stress, unfolded protein response (UPR), apoptosis and autophagy are main underlying molecular mechanisms of acute pancreatitis pathogenesis. Pancreatic cells are under high risk of ER stress due to its higher protein synthesis capacity. During the ER stress in acinar cells, the UPR system is activated to restore cellular homeostasis. Drug induced acute pancreatitis occurs rarely; however, it could be life threatening.9 ERN1 induction in the pancreas cells for different reasons may cause cell death.10 One of the key factor for severity of pancreatitis is the increased pancreatic cell death. caspase 12, one of the important cytoplasmic caspase, forms a link between ER stress and apoptosis. When caspase 12 activates UPR signaling via ER stress, it in turn activates important caspase 9 and caspase 3 resulting in apoptosis.12 The three functional pathways of get involved with three main pathways such as ERN1, activating transcription factor 6 (ATF6) and protein kinase RNA-like ER kinase (PERK) pathways. Glucose-regulated protein 78 (Grp78), Grp90 and heat shock protein 90 (Hsp90) are the main regulator of UPR signaling. Growth arrest- and DNA damage-inducible gene 153 (DDIT3) is the downstream effector protein regulating gene expression in the cell during UPR signaling.16 The underlying molecular mechanism of methimazole induced acute pancreatitis has been not clarified yet. Thus, in this study, we aimed to observe toxic effect mechanism of methimazole in human pancreas duct epithelioid carcinoma cells, by assessing the cellular cytotoxicity, oxidative stress, ER stress and apoptosis. PANC-1 cells have optimal cell proliferation, gene expression and adhesion profiles to study on pancreas functions and these cell types were used in many studies. Methimazole was purchased from Chemie Uetikon GmbH . Human pancreas/duct (PANC-1) cell line (CRL-1469) was obtained from the American Type Culture Collection . ELISA kits for total antioxidant status (TAS), total oxidant status (TOS), ERN1, DDIT3, Grp78, Hsp90, and caspase12 from Elabscience Biotechnology Co. Ltd . Glutathione (GSH) Elisa Kit was obtained from YH Biosearch laboratory . FITC Annexin V Apoptosis Detection Kit with propidium iodide (PI) was from BioLegend . The cell culture chemicals and all other cell culture supplements were from Multicell Wisent .2. For cytotoxicity assessment, the methimazole concentrations were in the range of 0-200 mM for 48h exposure. The medium was used for control. After 48 hours exposure, the optical densities (ODs) were measured at 590 nm by a microplate spectrophotometer reader , and IC30 was calculated. The PANC-1 cells were nourished in Dulbecco’s modified eagle medium: nutrient mixture F-12 (DMEM F-12) media include 10% fetal bovine serum (FBS) and 100 U/100 μg/mL penicillin/streptomycin at 37°C in a humidified incubator with 5% CO30 value, the cells were exposed to 20, 30 and 40 mM methimazole for 48 hours with control group for oxidative stress, ER stress and apoptosis evaluation. All assay was done in triplicates and performed in three independent days. Considering IC2DCF-DA) assay with flow-cytometer.18 The cells (1 × 105) were seeded in 6-well plates, and incubated for 24 hours. The fluorescence intensities were measured with ACEA NovoCyte flow-cytometer  at 488 nm. The results were expressed as the percentage of median fluorescence intensity. The GSH, TAS and TOS levels were measured by Elisa kits according to manufacturer’s instructions. The GSH levels were expressed as μmol/104 cell. The TAS and TOS levels were expressed as U/mL. The ERN1, DDIT3, Grp78, Hsp90 and caspase12 levels were assessed with Elisa kits according to the manufacturer’s instructions. The results were expressed as ng/mL. Annexin V Apoptosis Detection Kit with PI was used to investigate the apoptosis/necrosis pattern of the cells by flow-cytometer ACEA NovoCyte flow-cytometer at 488 nm according to manufacturer’s instructions. The results were expressed as the percent of the total cell amount. The cellular ROS generation was assessed by 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate . TAS, TOS, ERN1, DDIT3, Grp78, Hsp90 and caspase 12 results statistical analysis were performed with one-way ANOVA post hoc Dunnett’s t test by using SPSS version 20.0 , and the results were given as mean ± standard deviation (SD). P ≤ 0.05 was the level of significance. For the statistically analysis, H20 It is important to determine the etiological causes of acute pancreatitis for appropriate treatment and follow-up. The most common causes of acute pancreatitis are biliary, alcohol, hypertriglyceridemia, hypercalcemia, drug-related, autoimmune, hereditary/genetic and anatomical anomalies.21 The annual incidence of drug-induced acute pancreatitis is 0.1-5%. More than 500 drugs triggering acute pancreatitis were listed by World Health Organization (WHO) and the number is rising in each passing year. The mechanisms of drug-induced acute pancreatitis are generally based on case reports, case-control studies, animal studies, and other experimental data. The potential mechanisms for drug-induced acute pancreatitis include pancreatic duct narrowing, cytotoxic and metabolic effects, accumulation of toxic metabolites or mediators, and hypersensitivity reactions. However, the mechanism of drug induced acute pancreatitis has not been clarified yet. Various methods have been proposed to assess the causality in drug-induced acute pancreatitis. Currently, the drug-induced pancreatitis classification system is widely used for uniform assessment of causality in pancreatoxicity.27 There are limited methimazole induced acute pancreatitis case reports in the literature.30 However, this is the first study to evaluate oxidative and ER stress mechanisms role in methimazole induced acute pancreatitis The cytotoxic effect of methimazole on the hepatocytes was evaluated by Heidari et al,31 isolated from male Sprague-Dawley rats, for 2 hours exposure in the range of 1-15 mM. A concentration-dependent cytotoxic effect was detected in hepatocytes isolated from rats with trypan blue assessment and the IC50 value was found as 10 mM. In the present study, it was observed that methimazole decreased the cell viability depending on concentration with 48h exposure in PANC-1 cells. IC30 value of methimazole was 53 mM . However, the TOS levels increased significantly at only 40 mM . On the other hand, there was no significant difference as compared with control group in terms of the GSH and TAS levels . In addition, methimazole increased ROS levels 1.8-fold compared to the control group at 10 mM (P < 0.05). In another study,33 methimazole induced serious liver damage and GSH depletion induced significantly (2.5-fold) at 200 mg/kg in Swiss albino mouse (P < 0.05). Vickers et al34 compared 8 drugs, including methimazole, in an ex vivo 3D multicellular human liver section model. They observed that methimazole increased GSH levels by 15%-37% at 24 hours, 11%-49% at 48 hours and 30%-49% at 72 hours at 500 μM. Kobayashi et al35 found there was a 1.5-fold significant decrease in the amount of GSH in the hepatic tissue in BALB/c female mice 6 hours after exposure to 450 mg/kg oral methimazole. Niknahad et al36 observed a significant increase in ROS levels in the liver mitochondria isolated after exposure to ≤ 400 mg/kg methimazole in BALB/c male mice (P < 0.001). In vivo evaluation revealed that the amount of mitochondrial ROS was higher compared to control animals (P < 0.001). In our study, GSH amounts were 3.31 and 3.24 μmol per 104 cells at 20 and 40 mM concentrations in PANC-1 cell lines (P > 0.05). A significant increase (5.3-fold) was found in the ROS level at 40 mM (P < 0.05) compared to control group. It has been reported that the producing reactive metabolites play important role in methimazole induced liver injury. Heidari et al37 This is the first study to evaluate methimazole treatment effects on ROS, ER stress and apoptosis in pancreas cells. According to the results of our study, methimazole induces ER stress mechanism via ERN1 pathway that could be associated with pancreatitis. However, cell death was not induced. Given that the changes in autophagy and inflammation markers were not examined in this study, the last step leading to pancreatitis in pancreatic cells could not be clarified. In the present study, no significant difference was found between groups in parameters of DDIT3, Hsp90 and Grp78. In 40 mM group, ERN1 and caspase 12 levels significantly increased in the order of 4.7- and 1.4-fold, respectively. We suggest that methimazole may induce ER stress pathway in pancreas cells via ERN1 pathway . Althougin vitro and in vivo studies that determine the relationship between methimazole and its metabolites with the risk of pancreatitis. In this study, we evaluated the toxicity of methimazole in PANC-1 cell line through oxidative stress and endoplasmic reticulum stress parameters. According to cell viability study result, the IC30 value of methimazole was calculated as 53 mM for 48 hours. ROS, TOS, ERN1 and CASPASE12, cellular stress pathways biomarkers, levels increased with methimazole. However, it was observed that although cellular stress markers increased, apoptosis was not induced in cells. These results suggest that the effect of methimazole on the pancreas is not mediated by induction of apoptosis. In summary, the data obtained from our study, in which basic evaluations were made for elucidating the mechanisms underlying the risk of methimazole induced acute pancreatitis, need to be supported by further  The present work was supported by the Research Fund of Istanbul University (Project No. 36298).Conceptualization: Özge Yazıcı, Mehtap Kara, Tuğçe Boran, Gül Özhan.Data curation: Özge Yazıcı, Mehtap Kara, Tuğçe Boran.Formal Analysis: Özge Yazıcı.Funding acquisition: Gül Özhan.Investigation: Özge Yazıcı, Gül Özhan.Methodology: Özge Yazıcı, Tuğçe Boran.Project administration: Özge Yazıcı, Gül Özhan.Resources: Özge Yazıcı.Software: Özge Yazıcı, Tuğçe Boran.Supervision: Tuğçe Boran, Gül Özhan.Validation: Özge Yazıcı, Tuğçe Boran.Visualization: Özge Yazıcı, Mehtap Kara, Tuğçe Boran.Writing – original draft: Özge Yazıcı, MMehtap Kara, Tuğçe Boran.Writing – review & editing: Özge Yazıcı, Mehtap Kara, Tuğçe Boran, Gül Özhan. Not applicable. The authors declare that there are no financial competing interests.


Moreover, 31 compounds and 100 potential target genes were employed for the Kyoto Encyclopedia of Genes and Genomes (KEGG) Markup Language (KGML) network analysis to explore the core signaling pathways for the progression of SAE. The integrative pathway analysis showed that various dysregulated metabolism pathways, including lipids metabolism, amino acids, glucose and nucleotides, inflammation-related pathways, and deregulated synapses, were tightly associated with hippocampus dysfunction at early SAE. These findings provide a landscape for understanding the pathophysiological mechanisms of the hippocampus in the progression of SAE and pave the way to identify therapeutic targets in future studies.Sepsis-associated encephalopathy (SAE) is an intricated complication of sepsis that brings abnormal emotional and memory dysfunction and increases patients’ mortality. Patients’ alterations and abnormal function seen in SAE occur in the hippocampus, the primary brain region responsible for memory and emotional control, but the underlying pathophysiological mechanisms remain unclear. In the current study, we employed an integrative analysis combining the RNA-seq-based transcriptomics and liquid chromatography/mass spectrometry (LC-MS)-based metabolomics to comprehensively obtain the enriched genes and metabolites and their core network pathways in the endotoxin (LPS)-injected SAE mice model. As a result, SAE mice exhibited behavioral changes, and their hippocampus showed upregulated inflammatory cytokines and morphological alterations. The omics analysis identified 81 differentially expressed metabolites (variable importance in projection [VIP] > 1 and  Sepsis and septic shock are prevalent and severe symptoms in an intensive care unit (ICU). They account for 25%–80% of mortality worldwide  and usuaSepsis initiates a series of systemic inflammatory responses leading to deregulated peripheral inflammation, neuroinflammation, ischemic damage, oxidative stress, and metabolic disorders, disrupting the integrity of the brain–blood barrier (BBB) and, thus, causing the progression of SAE . FurtherMultiple omics approaches, such as expression array, transcriptomic, and metabolomic, have been employed to elucidate the biological events from different brain regions or spheres to uncover the etiology and molecular mechanisms of cognitive or neurofunction impairment. However, single approaches have gained fewer insights into the complicated mechanisms . In contIn the SAE-related study, single omics, including metabolomics and transcriptomics, have been widely used to decipher the molecular mechanisms in the hippocampus . NeverthEscherichia coli (O111:B4), #L2630, purity >99%, with high-purity quality Standards M300) was commercially available from Sigma-Aldrich .All chemicals and solvents were analytical or HPLC-grade. Acetonitrile, methanol, and formic acid were purchased from CNW Technologies GmbH . L-2-chlorophenylalanine was obtained from Shanghai Hengchuang Biotechnology Co., Ltd. , and LPS  or saline was randomly administered to mice. All procedures were performed according to regulations for animal experimentation and approved by the ethics committee of Tongren Hospital, Shanghai Jiao Tong University School of Medicine. After 8 h or 24 h post-injection, mice were killed, with the numbers of animals for histology analysis, omics, or biochemical analysis of the hippocampus being described below.C57BL/6J  from SPF facility were purchased from Model Animal Research Center of Nanjing University  and maintained in an animal facility with free access to food and n = 9 per group) with Student’s t-test.The open-field test (OFT) widely examined rodent locomotor activity and exploratory behaviors. All open-field testing took place inside an arena (50 cm [l] × 50 cm [w] × 40 cm [h]). The arena was divided into the center and peripheral regions by the VisuTrack system . Mice that were injected with LPS (10 mg/kg body weight) and controls  for 24 h were removed from their home-cage by the tail and placed directly into the open field center. Tracking/recording was initiated upon the first locomotion grid beam break and lasted 5 min. The trajectory of each mouse was analyzed with the VisuTrack system. Total distance, speed, and freezing time were recorded to evaluate the movement ability of mice, and center entries in the central region were measured to detect mice’s anxiety. The data were interpreted as mean ± SEM (standard error of the mean) (n = 3 per group) and metabolites analysis (n = 6 per group). Finally, entire brain coronal sections (5 µm) were used for the Nissl staining or hematoxylin-eosin staining (n = 3 mice per group). These stainings were performed as described previously  with RNeasy Mini Kit (Qiagen#74106). RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer . RNA integrity was analyzed by Agilent 2100 bioanalyzer (RIN >6.0 and 28S/18S ≥ 0.7); thus, it could qualify for subsequent library construction using NEBNext Ultra RNA Library Prep kit  according to the manufacturer’s instructions.The libraries were sequenced on an Illumina NovaSeq 5,000 platform, and 150-bp paired-end reads were generated. In total, 45.18–49.38 Mb raw reads for each sample were generated. Raw data (raw reads) of fastq format were firstly processed using Trimmomatic , and thehttps://cloud.oebiotech.cn).The clean reads were mapped to the mouse genome (GRCm38.p6) using HISAT2 . We calcs (KEGG)  pathway http://software.broadinstitute.org/gsea/index.jsp) was used to further screen the signaling pathways associated with SAE  and 400 μL of methanol: water (4:1 = vol: vol) were added to each hippocampal sample (30 mg/group sample), and ground with a grinder machine . Mixtures of the sample were extracted by ultrasonication for 10 min in the ice water bath and kept at −20°C for 30 min. The extraction was centrifuged at 13,000 rpm and 4°C for 10 min. Next, 300 μL of supernatant was transformed into a liquid chromatography/mass spectrometry (LC-MS) injection vial to evaporate and dissolved again in 200 μL methanol: water (4:1 = vol: vol) by sonification (vortex 30 s and sonification 3 min). The samples were kept at −20°C for 2 h and then centrifuged at 13,000 rpm and 4°C for 10 min. The supernatants (150 μL) from each tube were collected using crystal syringes, filtered through 0.22-μm filters, and transferred to LC vials. The vials were stored at −80°C until LC-MS analysis. Of note, all reagents for extraction were precooled at −20°C.The liquid chromatography was performed using a Dionex U3000 UHPLC equipped with ACQUITY UPLC HSS T3 column . The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The analysis was performed with elution gradient as follows: 0.0–2.0 min, 5% B; 2.0–4.0 min, 5%–25% B; 4.0–8.0 min, 25%–50% B; 8.0–10.0 min, 50%–80% B; 10.0–14.0 min, 80%–100% B; 14.0–15.0 min, 100% B; 15.0–15.1 min, 100%–5% B, 15.1–16.0 min, 5% B. The column temperature was 45°C. The auto-sampler temperature was 4°C, and the injection volume was 2 ml.The QE mass spectrometer (ThermoFisher) was used to acquire MS/MS spectra in information-dependent acquisition (IDA) mode in the control of the acquisition software . The acquisition software continuously evaluates the full scan MS spectrum in this mode. The ESI source conditions were set as follows: sheath gas flow rate of 35 Arb, Aux gas flow rate of 8 Arb, capillary temperature of 320°C, full MS resolution of 70,000, MS/MS resolution of 17,500, collision energy of 10/20/40 eV in NCE mode, and spray Voltage of 3.8 kV (positive) or −3.0 kV (negative).The acquired LC-MS raw data were analyzed by Progenesis QI v2.3 software  with the following parameters: the precursor tolerance of 5 ppm/10 ppm, fragment tolerance of 10 ppm/20 ppm, and retention time (RT) tolerance of 0.02 min. The product ion threshold was 5%. Internal standard detection parameters were deselected for peak RT alignment, isotopic peaks were excluded for analysis, and the noise elimination level was set at 10.00. The minimum intensity was set to 15% of the base peak intensity. The Excel file was obtained with three-dimension datasets including m/z, peak RT, and peak intensities, and RT-m/z pairs were used as the identifier for each ion. The resulting matrix was further reduced by removing any peaks with a missing value (ion intensity = 0) in >50% of samples. The internal standard was used for data quality control (reproducibility).http://www.hmdb.ca/), Lipidmaps (v2.3) (http://www.lipidmaps.org/), and self-built database (Luming Biotech). The resulting positive and negative ion scans were combined into a dataset for further analysis with R ropls package. Principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis OPLS-DA were performed to visualize the metabolic alterations among experimental groups. The Hotelling’s T2 region, shown as an ellipse in score plots of the models, defined the 95% confidence interval of the modeled variation. Variable importance in the projection (VIP) ranked the overall contribution of each variable to the OPLS-DA model, and those variables with VIP >1 were considered relevant for group discrimination. In this study, the default 7-round cross-validation was applied, with 1/seventh of the samples being excluded from the mathematical model in each round to guard against overfitting. The criteria for differentially expressed metabolites (DEMs) were VIP of >1.0 obtained from the OPLS-DA model and p of <0.05 from a two-tailed Student’s t-test on the normalized peak areas.Metabolites were identified by progenesis QI software based on public databases, such as HMDB  network analysis. Pearson correlation coefficients were calculated for transcriptome and metabolome data integration . BrieflyStatistical analysis was performed on raw data for each group by unpaired Student’s t-test, using GraphPad Prism 8. Data were shown as mean ± SEM. P-values referred to the probability of the null hypothesis meaning that the means do not differ. A P of <0.05 was considered significant, and a p of ≥0.05 was not significant.Tnf, IL1β, and Il6 mRNA decreased at 24 h LPS post-injection compared to 8 h  or saline by open-field test. The results showed behavioral changes, i.e., a decrease in total distance travel, speed, and center entries, but an increase in freezing time −H in LPSn = 6 mice per group) to determine the alterations of metabolites. A total of 5,109 metabolites  were detected by LC-MS and subsequently used for multivariate analysis. The PCA and OPLS-DA score plot showed that the SAE group was discriminated from the saline group , nucleosides, nucleotides, and analogs (n = 17), organic acids and derivatives (n = 25), and others (n = 23), glucose metabolism (n = 11), lipid metabolism (n = 14), purine metabolism (n = 11) and pyrimidine metabolism (n = 8), and other uncategorized metabolisms (We then performed the OPLS-DA test and identified 149 upregulated and 199 downregulated DEMs between the SAE group (S1−S6) and the control group (Ctrl1−6) under the criteria of VIP >1 and p < 0.05 . Of thesd others . They ard others , which sabolisms ; Table 1The DEMs matched in the KEGG database were significantly enriched in glycolysis/gluconeogenesis, regulation of lipolysis, amino acids metabolism, nucleotide metabolism , synapse function , and signal transduction  . AdditioWe next performed RNA-seq-based profiles analysis using the same batch of hippocampus tissues as in LC-MS metabolomics analysis to uncover the molecular basis of the hippocampus from SAE mice. A total of 41703102–4411552 new reads were sequenced, mapped into the mouse genome, and deposited in the NCBI sequence read archive .n = 3) and control   . Next, w, n = 3)  and iden, n = 3) . Moreove, n = 3) . These dKEGG analysis was performed to obtain the top 20 significant pathway terms with the upregulated or downregulated DEGs. For the upregulated DEGs, the KEGG term included most of the inflammatory response, cytokine signaling pathways, necroptosis, and apoptosis in the hippocampus . In addiA core genes-metabolites regulation network must decipher the significant clustered genes and metabolites responsible for the SAE progression. According to the KEGG enrichment analysis with the RNA-seq and metabolomics data, we observed that 392 DEGs and 31 DEMs in a cluster of signaling pathways were highly represented in the SAE group. The top 100 expression DEGs and top 100 DEMs (only 31 DEMs) were used for Pearson correlation to distinguish the regulations among the DEGs and DEMs. The correlation heatmap shows the calculated correlation coefficient results . AdditioThe development of acute sepsis in patients can cause septic shock and further develop as SAE . In thisMetabolomics has been vigorously used to explore the enriched metabolites and related metabolic pathways in the development of SAE , which lThe lipids altered in sepsis have been documented in many studies . In the ALOX5, CYP1B1, CYP27B1, CYP3A13, PLA2G4E, SOAT2, UGT1A6A, and UGT1A7C) or downregulated  in the hippocampus tissues of SAE  were accumulated in the hippocampus of SAE. Moreover, lipids and steroids metabolism-related genes were detected to be upregulated  are tightly related to arachidonic acid metabolism and isovaleric acid, which is associated with cognitive impairment and systemic inflammation , and wernd PLAUR  were upr -MS-based metabolomics analyses . In ligh2+ homeostasis, and neurogenesis  decreased and subsequently increased L-lactic acid production, indicating that glycolysis was reinforced for a metabolic adaptation to the inflammatory response in the SAE-grouped hippocampus. However, several metabolites (fumaric acid and ascorbic acid) holding anti-inflammatory and/or neuroprotective effects were decreased, and the oxidative stress metabolite gluconic acid was increased in the SAE group .Regarding the nucleotides, the level of purine nucleobases (guanosine and adenine) and pyridine nucleobases  decreased in the SAE hippocampus, indicating that their pyrimidine pathway was impaired and in shortage of energy. Uracil has been shown as the intermediate metabolite to neuroprotection and neuroinflammation . AdenineBehavior abnormality and cognitive dysfunction are common characteristics of SAE progression . SynaptiAlthough our study depicted that dysregulated neuroinflammation, metabolism, and synapse pathways were tightly associated with the hippocampus at the early SAE, several key predictions, such as the lipid mediator, and amino acids, need further functional validation. Furthermore, it is still a challenge to directly monitor the cognitive behaviors of SAE rodents at the early stages  because In summary, RNA-seq-based transcriptomics and LC-MS-based metabolomics were gathered for the first time to obtain a comprehensive landscape of the hippocampus of SAE. Our integrated studies showed that various dysregulated metabolism pathways, including lipids metabolism, amino acid, glucose, and nucleotide metabolism, inflammation-related pathways, and deregulated synapses were possible mechanisms of SAE progression in the hippocampus. This study provided comprehensive omics data analysis to understand the possible pathophysiological mechanism of the hippocampus in the early phase of SAE. Furthermore, it might pioneer the SAE field to identify future therapeutic targets for brain protection.


Amaryllis vittata (L.) leaf and bulb extracts in order to determine their biological significance and use the toxic plants for human health benefits. The formation of silver nanoparticles was detected by a change in color from whitish to brown for bulb-AgNPs and from light green to dark brown for leaf-AgNPs. For the optimization of silver nanoparticles, various experimental physicochemical parameters such as pH, temperature, and salt were determined. UV-vis spectroscopy, Fourier transform infrared spectroscopy, X-ray dispersion spectroscopy, scanning electron microscopy, and energy dispersion spectroscopy analysis were used to characterize nanoparticles. Despite the fact that flavonoids in plant extracts were implicated in the reduction and capping procedure, the prepared nanoparticles demonstrated maximum absorbency between 400 and 500 nm. SEM analysis confirmed the preparation of monodispersed spherical crystalline particles with fcc structure. The bioinspired nanoparticles were found to show effective insecticidal activity against Tribolium castaneum and phytotoxic activity against Lemna aequincotialis. In comparison to plant extracts alone, the tested fabricated nanoparticles showed significant potential to scavenge free radicals and relieve pain. Antibacterial testing against human pathogenic strains, i.e., Escherichia coli and Pseudomonas aureginosa, and antifungal testing against Aspergillus niger revealed the significant potential for microbe resistance using AgNPs. As a result of the findings, the tested silver nanoparticles demonstrated promising potential for developing new and effective pharmacological and agricultural medications. Furthermore, the effects of biogenic AgNPs on an in vitro culture of Solanum tuberosum L. plants were investigated, and the findings indicated that bulb-AgNPs and leaf-AgNPs produced biomass and induced antioxidants via their active constituents. As a result, bulb-AgNPs and leaf-AgNPs may be recommended for use in Solanum tuberosum L. tissue culture for biomass fabrication and metabolic induction.The current study sought to synthesize silver nanoparticles (AgNPs) from  Aloe vera, Jatropha curcas, Citrus aurantium (Orange blossom), Solanum melongena (Eggplant), and Trifolium resupinatum [Amaryllis vittata (L.) Herit., a poisonous plant, was selected to synthesize silver nanoparticles, which contain flavonoids, alkaloids, and coumarins and have power over carcinogenic, oxidation, and inflammatory effects. Nanotechnology is known as the art of synthesizing particles with at least one dimension in the range of 1–100 nm, resulting in large surface-to-volume ratios. Nanotechnology is rapidly emerging in food, pharmaceutics ,3. The uupinatum , are knoAmaryllis vittata belongs to the family Amaryllidaceae and is represented by a single genus Amaryllis  for future studies.The aerial parts of 3 solution  was prepared in a 250 mL volumetric flask using distilled water.One millimolar AgNOA. vittata in 500 mL ethanol in a beaker. The beaker was kept at room temperature for 7 days with occasional shaking. Then, the sample was filtered through filter paper. The filtrate was subjected to evaporation with the help of a rotary evaporator at 60 °C; the concentrated solution was transferred to Petri plates and placed in a water bath at 60 °C for complete drying. For the preparation of aqueous bulb extract, 25 g of chopped bulb were added to 100 mL distilled water in a flask and boiled in Kjeldhal apparatus at 50 °C for 10 min. Then, the sample was filtered through filter paper, and the filtrate was used as a reducing agent for silver nanoparticle synthesis.Leaf extract was prepared by adding 100 g of leaf powdered All samples were placed on a magnetic stirrer at room temperature for 24 h. The appearance of brown color indicated the reduction of silver ions and the formation of nanoparticles. Color change for leaf-capped AgNPs was observed after 10 min of reaction, while for bulb-capped AgNPs, color change was noted soon after mixing the reactants. The rapid change in color for leaves and bulb extracts was observed in the ratio 1:5 as compared to all other samples of various salt to plant ratios. Maximum peak absorbency was observed with the help of Uv-vis spectroscopy.In the present work, the determination of various optimization parameters, i.e., temperature effect, pH effect, and NaCl concentration effect, were carried out upon the biofabrication of AgNPs.A. vittata as reducing agents, and AgNO3 solution was confirmed with the help of UV-vis spectrometry analysis. The synthesized silver nanoparticles were tested for absorbency against distilled water as a blank. UV-vis spectroscopy was carried out using a UV-1602 double beam UV-vis spectrometer with a resolution of 1nm to monitor the synthesis of silver nanoparticles. The spectral absorbency was evaluated with wavelengths ranging from 300 nm to 800 nm for AgNPs fabricated from the reaction mixture in different ratios such as 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6, respectively.AgNPs were synthesized using leaf and bulb extracts of −1 in the range of 4000 cm−1–0 cm−1. The bulb and leaf extracts of A. vittata were then subjected to FT-IR spectroscopy, respectively, using the same procedure as for nanoparticles of A. vittata bulb and leaf extract, i.e., using the “Perkin Elmer spectrometer FT-IR SPECTRUM ONE” at a resolution of 4 cm−1 in the range of 4000 cm−1–0 cm−1.The silver nanoparticles were dried and mixed with KBr and pressed by a Hydraulic pellet press to prepare the sample pellet. The sample was subjected to FT-IR spectroscopy using the “PerkinElmer spectrometer FT-IR SPECTRUM ONE”  at Department of Chemistry, Bacha Khan University, Charsadda, Pakistan, via a resolution of 4 cmA. vittata were analyzed using a JEOL JDX 3532 X-ray diffractometer  at the University of Peshawer. The diffraction pattern of biogenic nanoparticles was recorded by a nickel monochromator filtering the wave at a tube voltage of 40 KV and tube current of 30 mA with cu-kα radiation (λ = 1.5406 A0). The average diameter of nanoparticles was calculated from the line width of the maximum intensity reflection peak. The size of Ag nanoparticles was calculated through Scherer’s Equation (1).0), and K (0.89) is Scherer’s coefficient. Θ is Bragg’s angle, and β is the full width at half maximum (FWHM) in the radius.The X-ray diffraction pattern of Ag nanoparticles synthesized from bulk and leaf extract of A. vittata were completely air-dried at 35 °C with the help of a vacuum drier, then a small droplet of leaf-AgNPs and bulb-AgNPs were dropped on a carbon-coated SEM grid, respectively, and allowed to dry. The samples were then subjected to silver coating with the help of a spi-module sputter coater (Auto fine coater), and morphological features were analyzed by scanning electron microscopy using FE-SEM (JSM-5910-JEOL)  at the University of Peshawer.Silver nanoparticles prepared from the leaf and bulb extract of EDS assessment was applied to observe the confirmation of elemental Ag present in AgNPs. The sample was prepared by centrifugation of nanoparticles solution at 14,800 rpm for 15 min and allowed to dry at 35 °C by vacuum drier. The dry powder mass (nanoparticles) was then subjected to energy-dispersion spectroscopy analysis with the help of a thermal EDS instrument attached to an Oxford Inca 200 SEM,  at the University of Peshawer.Tribolium castaneum. All samples were tested in Petri plates under aseptic conditions with two replicates. The filter papers were cut according to the size of Petri dishes and were placed on plates. Approximately 1.5 mg of sample were dissolved in 1.5 mL solvent (distilled water) serving as stock solution. Three replicates of sterilized Petri dishes were incubated with 100 µL, 500 µL, and 1000 µL solution over filter paper with the help of a micropipette. The plates were kept for 24 h at 25 °C to evaporate the solvent completely. After 24 h, stored insects obtained from a pharmacognosy lab were deposited on each Petri plate. After the evaporation of the solvent, Tribolium castaneum  were placed on each plate (test and control) with the help of a clean brush. Healthy insects of the same age and size were selected. The plates were incubated at 27 °C for 24 h with 60% relative humidity in the growth chamber. The number of living insects was counted and examined daily during incubation. The Petri plates remained there for 3 days. The number of insects/plates was counted and recorded on the third day. The result was analyzed using (SPSS) in terms of percentage. The mortality percentage was calculated with the help of the following Equation (2).Leaf and bulb extracts and silver nanoparticles were tested for insecticidal activity against The insecticidal activity was determined using the following method .A. vittata. The experiment was carried out in Petri dishes in three different concentrations  with three replicates. The Petri dishes were autoclaved at 121 °C for 24 hrs. Lemna aequinoctialis was used as a test plant, with 10 plants per Petri dish. The extracts (1 mg) were dissolved in 1 mL distilled water and served as a stock solution (1000 ppm). Three concentrations, i.e., 50, 100, and 500 µL, were prepared from 1000 ppm solution. Ten plants containing rosettes of three fronds were kept on each plate, and samples were added to all plates containing Lemna aequinoctialis. The same protocol was followed for bulb extract, leaf-AgNPs, and bulb-AgNPs. Other plates were supplemented with L. medium, and reference growth inhibitor (Atrazine) served as a negative and positive control, respectively. All plates were kept in a growth chamber at 28 °C for seven days. The number of fronds per plate was counted and recorded on the seventh day.Phytotoxic activity analysis was carried out to evaluate the phytotoxic potential of silver nanoparticles synthesized using leaf and bulb extracts of Percentage growth inhibition was recorded with reference to the negative control using Equation (3).Phytotoxic activity analysis was carried out following the standard protocols of Ref. .A. vittata was evaluated using DPPH (1-diphenyl-2-picrylhydrazyl) solution [w/v DPPH solution was prepared in 99.9% methanol, and various concentrations of all samples were added to 2 mL fresh solution of DPPH in test tubes, respectively, so that the final volume reached 3 mL. Samples were shaken vigorously and incubated at room temperature for 60 min in the dark. For standard antioxidant drugs, ascorbic acid was used, and the same protocol was followed for the preparation of different concentrations of the standard drug. After incubation for 60 min, the absorbency of all samples was calculated at 517 nm against the blank (methanol) solution using a UV-spectrophotometer. The low absorbency value of the spectrophotometer revealed high free radical scavenging potential, while higher absorbency showed low scavenging activity. The percentage of antioxidant potential for all samples was calculated via Equation (4).A comparative antioxidant assay of silver nanoparticles and plant extracts of solution ,19,20. NA comparative antibacterial assay was performed against pathogenic bacterial strains using the disc-diffusion (Kirby–Baurer) method. All samples with three concentrations  were prepared in distilled water. The experiment was carried out in triplicate. For sterilization, all the experimental instruments and nutrient agar were autoclaved at 121 °C and 17 lb pressure for one hour. The media was transferred into sterilized Petri dishes after sterilization and kept overnight at room temperature for solidification of media and to check for any contamination. A 10 µL bacterial suspension was poured on solidified media with the help of a micropipette and was spread with a glass spreader. Four paper discs were placed on the medium in each plate and subjected to different concentrations of leaf-AgNPs, bulb-AgNPs, and plant (leaf and bulb) extracts. The standard drug (streptomycin) and distilled water were used as +ve and −ve controls, respectively. All plates were incubated at 25 °C for 24 h. Antibacterial activity was observed by measuring inhibition zones with an ordinary scale ,21. Aspergillus niger. Analysis was conducted following the well diffusion method [Solanum tuberosum L. dextrose agar) was prepared by adding nutrient agar and dextrose in Solanum tuberosum L. juice. All experimental equipment and PDA media were sterilized in an autoclave at 121 °C at 17 lb for one hour. To each sterilized plate, 20 mL molten media were transferred and left overnight to check for any contamination. In total, 10 fungal strains (A. niger) suspension were poured onto solidifying media using a micropipette and spread with a glass spreader. Four wells (5 mm each) were formed using a sterilized borer in each Petri dish and were labeled as a positive control, negative control, nanoparticles, and plant extracts, respectively. Various concentrations of all samples were poured into the respective wells. For positive and negative controls, clotrimazole  and distilled water were used, respectively. All Petri dishes were incubated at 25 °C for three days. Finally, the inhibition zones of the samples were measured using a scale and all zones were recorded in millimeters (mm). Biofabricated silver nanoparticles and plant extract were comparatively assessed for antifungal properties against n method . All samv/v). The second group of mice was taken as the disease group and only injected with acetic acid. Then, 1 mL of acetic acid was injected into all mice of the third, fourth, fifth, and sixth groups. Ten minutes after then injection acetic acid solution, writhing was counted for 5 min. After writhing, the third group of mice were injected with 1 mL of paracetamol solution. The mice of the fourth, fifth, and sixth groups were subjected to 1 mL leaf ethanolic extract solution at doses of 50 mg/kg, 100 mg/kg, and 500 mg/kg, respectively. The same protocol was followed for bulb extract and leaf and bulb synthesized silver nanoparticles. After treatment with the samples, the inhibition of writhing was determined [To evaluate the significance of the tested samples and determine their capabilities to relieve pain, an acetic acid-induced writhing test following ARRIVE was conducted. The design of the study was approved by the Ethical Committee of Abdul Wali Khan University Mardan, KPK, Pakistan (AWKUM⁄Bot⁄2019⁄1672 (16 December 2019)). Swiss albino mice were evenly arranged into 6 groups. The experimental animals of the first group were considered the control group and injected with 1 mL of normal saline (1% Solanum tuberosum L. (potato) were collected from the “National Institute for Genomics and Advanced Biotechnology” (NIGAB), Islamabad (33°43′17.33″ N and longitude of 73°02′35.84″ E), Pakistan. Solanum tuberosum L. shoots with axillary buds were surface sterilized using 50% Clorox and 5.5% NaOCl, followed by washing with 70% ethanol and 1–2 drops of tween 20. The internodal explants were finally washed (3×) with distilled water for 10 min each. Uniformly sized axillary bud segments (2 cm each) enclosing the meristem were inoculated on Murashige and Skoog [−2 s−1 at 25 °C, photoperiod 16/8 h, relative humidity of 55–66%) until micro-propagation plantlets were grown. Uniform cuttings of each explant with two axillary buds were sub-cultured in an ethanol-sterilized chamber with laminar air flow and propagated in an MS medium supplemented with final concentrations of leaf-AuNPs and bulb-AgNPs (4 ppm each). Five cuttings of explants were sub-cultured in each glass jar, which were tightly sealed with lids and kept in the controlled conditions mentioned above. All sub-cultured plantlets were placed in a growth chamber until the day when phenotypic differences in biomass among the treatments were visibly noticed. Then, the plantlets were harvested after 30 days since first subculturing and used for subsequent experiments.Healthy internodal explants of the Astrix variety of nd Skoog  media, 3For the determination of fresh weight (FW), the aggregate of plantlets was harvested from all the respective treatments, washed with sterilized distilled water, and placed between filter papers to remove extra water. For dry weight (DW), each aggregate of plantlets was oven-dried (60 °C) for 24 h. 3) method as described by Ref. [For total phenolic contents (TPC) determination, the Folin–Ciocalteu method was followed  using ga by Ref. . QuercetIn this equation, AE is the absorbance of the solution when a particular concentration of extract is added, and AD is the absorbance of the DPPH solution when nothing was added to this solution.For antioxidative enzyme activities, the extract was prepared from fresh plant tissue by homogenizing with ice-cold 0.5 M Tris–HCl (pH 6.8) buffer, according to the method of Abbasi et al. . Centrifp ≤ 0.05).Experiments were performed in a completely randomized design. One-way analysis of variance (ANOVA) using SPSS ver. 16.0 software  was performed for data analysis (Origanum vulgare) and Ref. [Allium sativum) and Ref. [Allium cepa) using bulb extract as a reducing and capping agent for silver ions. Changes in color of the sample solutions from light green and whitish to dark brown revealed AgNPs formation, as shown in After adding leaf ethanolic extract to 1 mM silver nitrate solution, the color of the solution changed from light green to dark brown, indicating the synthesis of silver nanoparticles and Ref.   and Ref. [Hippophea rhamnoides Linn.). Their results observed that the favorable temperature for the biofabrication of silver nanoparticles was recorded as 40 °C and 75 °C, respectively. It was observed from leaf-capped AgNPs that with an increase in temperature in the range of 30–90 °C, the nanoparticle formation rate was increased, i.e., a maximum absorbency peak was observed at high temperatures with reduced size, as shown in Asalmalia malabaric was conducted by Murali et al. [A. vittata, respectively. For both leaf and bulb-capped AgNPs, it was found from UV absorbance that with a higher concentration of NaCl, AgNPs showed low stability, and their stability was affected , (200), (220), and (311), respectively. For leaf extract mediated nanoparticles, the XRD diffraction pattern showed diffraction peaks at 37.9, 46.1, 64.61, and 76.9 in 2Ø with corresponding planes at 111, 200, 220, and 311, respectively [The biogenic AgNPs were synthesized from biological moieties for the FTIR measurements to identify the ethanolic leaf extract and aqueous bulb extract biomolecules and their possible involvement. The FTIR spectra of dried leaf-capped AgNPs are shown in −1 A. The reectively   observed in bulb-capped AgNPs at 1000 µg/mL with a 56.6% mortality rate, followed by leaf-capped nanoparticles which show the highest significant (p < 0.01) mortality rate of 50% at 1000 µg/mL. Leaf extract showed a significant (p < 0.05) mortality rate of 46.67% at 1000 µg/mL, while bulb aqueous extract showed a significant (p < 0.05) mortality rate of 43.34% at 1000 µg/mL (Tribolium castaneum (wheat insect), analysis for the evaluation of the insecticidal activity of silver nanoparticles was carried out using malathion [Myriostachya wightiana [Insecticidal activity evaluation was carried out using silver nanoparticles prepared from leaf and bulb extract using 00 µg/mL A. Using alathion  and Myriightiana . The resightiana .A. vittata at 50 µg/mL, 100 µg/mL, and 500 µg/mL concentrations. All samples showed a significant inhibition rate to Lemna aequincotialis. It was observed that the highest inhibition rate with significance (p < 0.0001) was observed by bulb-capped silver nanoparticles (96.6%) at 500 µg/mL, followed by Atrazine (standard herbicide) which non-significantly showed 93.3% inhibition at 500 µg/mL. Bulb aqueous extract as compared to bulb-AgNPs significantly (p < 0.001) showed an 86.6% inhibition rate at 500 µg/mL. It was also concluded from the result that leaf-capped AgNPs showed the highest significant (p < 0.0001) inhibition rate of 86.6% at 500 µL/mL while leaf extract showed a significant (p < 0.001) 70% inhibition rate at 500 µg/mL  in leaf-synthesized silver nanoparticles with 92% inhibition at 100 µg/1 mL, followed by ascorbic acid, which showed the highest significant (p < 0.0001) inhibition of 87%. Nanoparticles synthesized using aqueous bulb extract showed the highest significant (p < 0.0001) inhibition rate of 69.6 at 100 µg/1 mL compared to aqueous bulb extract, which showed a significant (p < 0.0001) maximum inhibition rate of 48.7%  on the surface of these nanoparticles causing the reduction and capping of silver ions. These functional groups play an active role in neutralizing free radicals documented by Keshari et al. [The highest ability of DPPH radical inhibition was found to be significant  extracts at 100 µL/mL (p < 0.0001), showing the highest zone of inhibition (20 mm) against Staphylococcus aureus and lowest inhibition (16 mm) against Pseudomonas aureginosa. This is significant (p < 0.0001) compared to leaf extract, which showed a high inhibition zone (18 mm) against Staphylococcus aureus while showing no inhibition against Pseudomonas aureginosa. Silver nanoparticles prepared from bulb extract showed a significant (p < 0.0001) high inhibition zone (19 mm) against Pseudomonas aureginosa (Staphylococcus aureus. Only aqueous bulb extract showed a significant (p < 0.0001) 14 mm inhibition zone against Staphylococcus aureus while showing no inhibitory zone against Pseudomonas aureginosa (p < 0.0001) maximum zone of inhibition (24 mm) against Staphylococcus aureus and 22 mm against Pseudomonas aureginosa compared to plant extracts and silver nanoparticles , and it was concluded that these nanoparticles have significant potential against these pathogenic strains as reflected in the literature of Rao et al. [Klebsiella pneumonia, Pseudomonas aeruginosa, and Escherichia coli. The study observed that the highest inhibition was showed by Klebsiella pneumonia (15 mm zone of inhibition) E. coli and P. aeruginosa showed 10 mm and 8 mm inhibition, respectively. Leaf-AgNPs and bulb-AgNPs showed good antibacterial potential against pathogenic bacterial strains, as shown in The antibacterial activity of silver nanoparticles synthesized by o et al.  who workA. vittata plant was evaluated by a well diffusion method against Aspergillus niger. The potential was calculated by the presence or absence of inhibition zones measured in millimeters. It was observed that biofabricated silver nanoparticles showed a significant zone of inhibition compared to plant (leaf and bulb) extracts. This activity showed that bulb-synthesized silver nanoparticles significantly (p < 0.0001) showed the highest zone of inhibition (15 mm) at 100 µL/mL and 12 mm at 50 µL/mL against Aspergillus niger, followed by clotrimazole  which showed a 12 mm maximum zone of significant (p < 0.0001) inhibition. Bulb extract showed a 3 mm significant (p < 0.0001) maximum zone of inhibition at 100 µL/mL. Silver nanoparticles prepared using leaf extract showed a significantly (p < 0.0001) high inhibition zone (11 mm) at 100 µL/mL and 9 mm at 50 µL/mL as in p < 0.0001) against Aspergillus niger compared to leaf extract which showed a significant (p < 0.0001) maximum zone of inhibition of 5 mm at 100 µL/mL  percentage of inhibition was shown by leaf-synthesized silver nanoparticles (93%) at 500 µL/mL in which writhing was reduced to 2.75 ± 0.75 * followed by standard drug paracetamol, which showed significant (p < 0.05) inhibition of 76.77%, in which writhing was reduced to 9 ± 3.0 **. Leaf ethanolic extract showed the highest significant (p < 0.05) inhibition rate of 80.64% at 500 µg/mL, which reduced the writhing rate to 7.5 ± 1.19 **. In a comparative study of bulb extract and bulb-capped nanoparticles, the highest %inhibition with significance (p < 0.05) was observed by bulb nanoparticles (91%), while bulb extract showed the highest significant (p < 0.05) inhibition of 77.7%, reducing writhing to 3.5 ± 2.5 * and 8.75 ± 1.88 **, respectively (Dryopteris blanfordii. Their result showed 45% inhibition compared to the standard drug (Aspirin), which showed 40.43% inhibition. As compared to the plant extract of A. vittata, silver nanoparticles biofabricated using these extracts determined effective analgesic potential. Additionally, this study revealed the active role of synergistic potential of plant secondary metabolites and AgNPs.The highest significant  increased upon supplementing the leaf-AgNPs (8.1 ± 0.4 cm) and bulb-AgNPs (9.4 ± 0.7 cm) compared to the control (5.9 ± 0.5 cm). Fresh weight was significantly (p < 0.05) increased upon supplementing the leaf-AgNPs (2.85 ± 0.09 g) and bulb-AgNPs (3.54 ± 0.07 g) compared to the control (1.6 ± 1.25 g). Dry weight was significantly (p < 0.05) increased upon supplementing the leaf-AgNPs (0.65 ± 0.004 g) and bulb-AgNPs (0.082 ± 0.005 g) compared to the control (04 ± 0.003 g). Similarly, the MS media supplemented with leaf-AgNPs and bulb-AgNPs showed significantly (p < 0.05) higher total flavonoid content (TFC), total phenolic content (TPC), and DPPH free radical scavenging activity compared to the control (p < 0.05) increased upon supplementing the leaf-AgNPs (3.05 ± 0.155 µg/mL) and bulb-AgNPs (3.5 ± 0.2 µg/mL) compared to the control (2.08 ± 0.27 µg/mL). TPC was significantly (p < 0.05) increased upon supplementing the leaf-AgNPs (1.4 ± 0.044 µg/mL) and bulb-AgNPs (2 ± 0.09 µg/mL) compared to the control (0.703 ± 0.04 µg/mL). DPPH free radical scavenging activity (%) was significantly (p < 0.05) increased upon supplementing the leaf-AgNPs (80 ± 3.8 µg/mL) and bulb-AgNPs (88 ± 3.9 µg/mL) compared to the control (52 ± 3.3 µg/mL).In vitro micropropagation, growth response, and biomass production of Solanum tuberosum L. supplemented with leaf-AgNPs (4 ppm) and bulb-AgNPs (4 ppm) were evaluated in MS media. Both types of biogenic AgNPs positively induced micropropagation and biomass production in terms of an overall increase in shoot length and fresh and dry weight compared to the control (without AgNPs) A–C. Shoo control D–F. TFC p < 0.05) increased in in vitro growth plantlets of Solanum tuberosum L. supplemented with leaf-AgNPs and bulb-AgNPs compared to control cultures (p < 0.05) increased upon supplementing the leaf-AgNPs (6.9 ± 3.8 U/mg protein) and bulb-AgNPs (7.2 ± 0.34 U/mg protein) compared to the control (3 ± 0.49 U/mg protein). Peroxidase (POD) was significantly (p < 0.05) increased upon supplementing the leaf-AgNPs (4.3 ± 0.22 µg/mL) and bulb-AgNPs (6.5 ± 0.24 U/mg protein) compared to the control (1.8 ± 0.35 U/mg protein). Ascorbate peroxidase (APX) was significantly (p < 0.05) increased upon supplementing the leaf-AgNPs (4.8 ± 0.23 U/mg protein) and bulb-AgNPs (4.6 ± 1.3 U/mg protein) compared to the control (2.7 ± 0.48 U/mg protein) in Solanum tuberosum L.es produce bioactive and defense-related secondary metabolites, including glycoalkaloids, calystegines alkaloids, Steroidal Alkaloids, protease inhibitors, lectins, phenolic compounds, flavonoids, and antioxidant enzymes. These bioactive secondary metabolites have either beneficial effects on diet or show phytopathogenic effects on plant survival upon infections as stated by Kaunda et al. [Antioxidative enzymes such as phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD) were significantly  Herit is an effective source for the fabrication of AgNPs. This was confirmed by the conversion of the green color of leaf ethanolic extract and the whitish color of aqueous bulb extract to brown color. The leaf and bulb extract of A. vittata plants has great capability for reducing silver nitrate solution, resulting in the formation of silver nanoparticles. The UV-vis spectroscopy of the solution with leaf extract and silver nitrate solution, as well as bulb aqueous extract and silver nitrate solution, observed maximum absorbency in the range of 400–500 nm, which confirms the formation of AgNPs. X-ray diffraction analysis indicated that nanoparticles are face-centered cubic (FCC) in structure. The generated nanoparticles were found to be crystalline spherical and biofunctionalized with organic molecules. Energy dispersion spectroscopy indicated the confirmation of the presence of the Ag element. The highest insecticidal and phytotoxic activities were recorded in leaf-AgNPs and bulb-AgNPs nanoparticles using A. vittata along with antimicrobial potential against pathogenic microbes. Both the tested samples offered significant potential to scavenge the free radicals. The acetic acid-induced writhing activity confirmed that leaf-AgNPs and bulb-AgNPs have the potential for analgesic properties. It was concluded from the above research that these biofabricated nanoparticles have significant pharmacological importance and can be used in designing and processing new drugs used in the pharmacological and agricultural fields. Additionally, this study proved an emerging application of nanotechnology in agriculture for the establishment of viable biomass production and beneficial secondary metabolite induction.We used the green and simple procedure for the synthesis of AgNPs at atmospheric pressure and room temperature. The whole method is said to be “green” because of the reduced energy consumption required (reaction at atmospheric pressure and room temperature).


Raltitrexed is a specific inhibitor of thymidylate synthase and a potential chemotherapeutic agent for the treatment of advanced gastric cancer. In this study, we investigated the effect of raltitrexed on the proliferation of HGC-27 human gastric cancer cells and its potential underlying molecular mechanism(s).RT-qPCR and western blotting were used to quantify RSK4 levels. Colony formation and flow cytometry assays were used to assess HGC-27 cell proliferation, cell cycle progression, mitochondrial membrane potential, and apoptosis. The expression of cell cycle and apoptosis markers were determined by western blotting.Our results demonstrate that raltitrexed upregulated RSK4 mRNA and protein levels in HGC-27 cells. Moreover, raltitrexed significantly inhibited tumor cell colony formation, arrested the cell cycle, decreased the mitochondrial membrane potential, and induced apoptosis. We observed that raltitrexed was capable of upregulating the expression of Bax, cyclin A1, and CDK3, and downregulating the expression of Bcl-2 and cleaved caspase-3. Importantly, siRNA-mediated RSK4 knockdown significantly reduced the inhibitory effect of raltitrexed on cell proliferation and its promotion of cell apoptosis. Moreover, silencing of RSK4 inhibited the raltitrexed-induced upregulation of cytochrome C. In addition, the changes in molecular markers related to the cell cycle and apoptosis induced by raltitrexed were reduced upon RSK4 depletion.Our study shows that RSK4 is a key target of raltitrexed in the regulation of gastric cancer cell proliferation, cell cycle progression, and apoptosis.The online version contains supplementary material available at 10.1186/s40360-022-00605-2. Gastric cancer is the fifth most common malignant tumor worldwide . AlthougRaltitrexed, also known as ZD1694 or Tomudex®, is a specific thymidylate synthase (TS) inhibitor that can reduce the proliferation of tumor cells by arresting the cell cycle in the G0/G1 phase and by inducing tumor cell apoptosis through the mitochondrial pathway , 6. ClinRSK4, also known as RPS6KA6, belongs to the p90 ribosomal protein S6 kinase (RSK) family, and it plays an essential role in cell proliferation, migration, and invasion , 10. RSKThe gastric cancer cell line HGC-27 was purchased from Xiamen Immocell Biotechnology Co., Ltd.  and maintained in high-glucose Dulbecco’s Modified Eagle’s Medium  supplemented with 100 U/mL penicillin/streptomycin , and 10% fetal bovine serum .Small interfering RNA targeting RSK4 (siRSK4) was designed based on the human RSK4 sequence (GenBank accession number: NM_014496.5). siRSK4 sense: 5′-UUUACCUUGUUACGGAUUUAA-3′, and siRSK4 antisense: 5′-UUAAAUCCGUAACAAGGUAAA-3′. HGC-27 cells underwent four types of treatment: dimethyl sulfoxide (DMSO) + siNC, DMSO + siRSK4, raltitrexed + siNC, and raltitrexed + siRSK4. In the first two groups, the cells were transfected with negative control siNC or siRSK4, respectively, and then treated with 0.1% DMSO for 48 h. In contrast, cells in the last two groups were transfected with negative control siNC or siRSK4, respectively, and subsequently treated with 0.5 µg/mL Raltitrexed  for 48 h. The transfections were performed using Lipofectamine™ 3000 reagent  following the standard manufacturer’s recommendations.HGC-27 cells were seeded at a density of 500 cells/well in 6-well plates at 48 h post-transfection. After subculturing for two weeks at 37 °C, the cells were fixed with 4% paraformaldehyde (PFA) for 10 min and subsequently stained with 0.5% crystal violet for 30 min at room temperature. The stained cells were imaged and counted after several washes.Cells were harvested and stained with 20 μg/mL propidium iodide (PI) in the dark at 28 °C for 30 min. The cells were then washed with phosphate-buffered saline (PBS) before being analyzed using a NovoCyte® flow cytometer .Trypsin (0.25%) without EDTA was used to harvest both floating and adherent cells. The cells were then washed once with PBS and stained with annexin V-FITC according to the manufacturer’s instructions . Finally, the fluorescent signals were detected and analyzed using a NovoCyte® flow cytometer (Agilent).Total protein was extracted using cold RIPA buffer , and bicinchoninic acid (BCA) solution  was used to determine the protein concentration following the manufacturer’s protocol. The proteins were then subjected to 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes . The membranes containing the electrophoresed proteins were blocked with 5% non-fat milk at 25 °C for 2 h and then incubated with the appropriate primary antibodies against RSK4 , cleaved caspase-3 , Bax , Bcl-2 , cyclin A1 , CDK2  and GAPDH  at 25 °C for 2 h. After three washes with Tris–HCl buffer, the membranes were probed with horseradish peroxidase (HRP)-conjugated secondary antibodies against mouse IgG or rabbit IgG at 25 °C for 1 h. The membranes were washed several times before signal detection.At 48 h post-transfection, measurement of the mitochondrial membrane potential was performed using a JC-1 Mitochondrial Membrane Potential Kit  according to the manufacturer’s suggestions and a previous description . The cel−ΔΔCq formula. The primers used in this study were designed based on the human RSK4 sequence (GenBank Accession Number: NM_014496.5) and 18S rRNA (GenBank Accession Number: NR_145820.1) and were as follows: 18S rRNA Forward: 5′-CGACGACCCATTCGAACGTCT-3′; 18S rRNA Reverse: 5′-CTCTCCGGAATCGAACCCTGA-3′; RSK4 Forward: 5′-CCTCCTTTCAAACCTGCTTCTGG-3′; RSK4 Reverse: 5′-GCTGATGAGCATTTGCACTGGC-3′.Total RNA was isolated using an RNA isolation kit , and the resulting RNA was reverse transcribed using a reverse transcription kit . Target genes were amplified using a ChamQ SYBR® qPCR Master Mix Kit , and the signals were detected using an iQ™5 real-time PCR system . The expression levels of the various target genes were normalized to the level of 18S rRNA using the standard 2t-test for comparison between two groups. One-way ANOVA was performed to compare differences among multiple groups. GraphPad Prism software (version 8.2.1) was used to generate figures, and the data are presented as means ± s.d. P-values less than 0.05 were considered statistically significant. All experiments were performed independently at least three times.Statistical analyses were performed using SPSS software (Version 22.0). Statistical significance was calculated using the unpaired Student’s RSK4 was significantly upregulated in HGC-27 cells upon treatment with raltitrexed for 48 h in a dose-dependent manner compared with the DMSO control group , which is an indicator of the mitochondrial membrane potential, decreased when cells were stimulated with raltitrexed, whereas it increased when they were transfected with siRNA against RSK4 Fig. A, B. In Finally, we examined the function of RSK4 in regulating cell cycle arrest and apoptosis induced by raltitrexed at the molecular level. The protein levels of cell cycle and apoptosis molecular markers were determined by western blotting. This showed that RSK4 knockdown significantly increased the basal protein levels of the apoptosis resistance marker Bcl-2 and cell cycle markers cyclin A1 and CDK2, and decreased the protein levels of cytochrome C, cleaved caspase-3, and Bax Fig. A, B. RalGastric cancer is one of the most common cancers worldwide and its etiology is associated with numerous factors , 17. EarIndeed, it has been shown that raltitrexed can lead to apoptosis, reduction of mitochondrial membrane potential, and G0/G1 cell cycle arrest in SGC7901 cells . In lineRSK4 gene transcription and, thereby, lead to increased RSK4 protein levels. However, the underlying mechanism requires further investigation. Studies have shown that RSK4 levels are suppressed in a variety of cancers, including breast, colorectal, and gastric cancer, and overexpression of RSK4 potentiates anti-tumor effects [Our study also reported that raltitrexed upregulated the mRNA and protein levels of RSK4 in HGC-27 cells, indicating that it might affect  effects , 13, 24. effects . Moreove effects . Taken tAdditionally, another study showed that the levels of Bcl-2, cyclin A1, and CDK2 were downregulated, and Bax expression was upregulated by overexpression of RSK4 in gastric cancer cells , which iApoptosis is a spontaneous process of programmed cell death. There are three main signal transduction pathways involved in this process: mitochondrial, death receptor, and endoplasmic reticulum signal transduction pathways . MoreoveRaltitrexed is an inhibitor of TS, which is the rate-limiting enzyme in pyrimidine deoxynucleotide biosynthesis, and inhibition of TS inhibits the cell cycle . While sIn conclusion, our data illustrate that raltitrexed inhibits the growth of HGC-27 cells and regulates the expression of Bax, cytochrome C, Bcl-2, cyclin A1, CDK2, and cleaved caspase-3 by upregulating RSK4 levels, leading to G0/G1 cell cycle arrest, reduction of the mitochondrial membrane potential, and cell apoptosis. However, further in vivo research and clinical investigations are needed to elucidate the fundamental basis for raltitrexed treatment.Additional file 1. Original images. Figure 1B. Raltitrexed upregulated the protein level of RSK4. Figure 6A. Raltitrexed regulated expressions ofcell cycle- and apoptosis-related by increasing RSK4.


Helianthus annuus) is crucial and one way to accomplish this feat is to have an understanding of the beneficial bacteria of sunflower rhizosphere. Similarly, the respiratory response of these bacteria needs to be studied to understand their roles in the ecosystem. This study was therefore conceptualized to gain insights into the effects of soil properties and carbon substrate utilization on bacterial community diversity of sunflower rhizosphere grown in Ditsobottla and Kraaipan, North West Province, South Africa. Extracted DNA from sunflower rhizosphere and bulk soils was subjected to 16S amplicon sequencing. Significant differences were observed in the alpha and beta diversities of the soil bacterial communities (p < 0.05). At the order level, among all the bacterial taxa captured in the farms, Bacillales were the most dominant. The abundance of Lactobacillales, Bacillales, Rhizobiales, Enterobacteriales, Burkholderiales, Flavobacteriales, Sphingomonadales, Myxococcales, and Nitrosomonadales obtained from Ditsobottla rhizosphere soil (R1) was positively influenced by organic matter (OM), while the abundance of Planctomycetales, Cytophagales, Gemmatimonadales, Nitrospirales and Caulobacteriales from Kraaipan rhizosphere soil (R2) was positively influenced by total N and pH. Bacterial communities of all the soil samples utilized the different carbon substrates  as an energy source. Significant differences (p < 0.05) were only observed in tryptophan and methionine amended soils. Unclassified bacteria were also captured in this study, such bacteria can further be harnessed for sustainable production of sunflower and other agricultural crops.The sustainable production of sunflower (The online version contains supplementary material available at 10.1186/s13568-022-01388-9. Over a decade, studies have revealed that plant-microbial interactions are crucial for plant growth and health, and these interactions are of immense importance in comprehending nature conservation and agricultural sustainability  is between 2 and 20 °C, while autumn (March–May) and spring (September–November) annual temperatures are mainly between 13 and 25 °C and 19–30 °C respectively.o 2′41.202″ S 25o 57′ 47. 49″ E) and Kraaipan (26o 17′24.186″ S 25o 13′33.258″ E) were cautiously selected based on the agricultural practices of the farms  soils were collected from both farms. Two replicate soil samples each were obtained for Ditsobottla and Kraaipan rhizosphere soils (R1 and R2 respectively), and each replicate was composed of soils collected from 20 plants after germination at a depth of 0–15 cm on different plots having radius of 3 cm away from the plants, and afterward pooled together to make a composite sample as described by Oberholster et al. . SimilarThe soil samples were collected into sterile plastic bags, then transferred into an ice packed container and conveyed to the laboratory immediately. A mesh sieve (2-mm pore size) was used to sieve the soil samples, while the sieved soil samples were divided into three portions: one portion was used for 16S amplicon sequencing, which was stored at – 20 °C, the second portion was stored at 4 °C for physiochemical analyses, while the third portion was used for CLPP.A Jenway 3520 pH-meter  was used to determine soil pH after mixing 2 g of the soil in 10 ml deionized water. Soil moisture content was quantified by oven-drying the soil samples at 105 °C for 24 h  was used for the extraction of the DNA following manufacturer’s instruction. The extracted DNA samples were sent to MR DNA Laboratory  for 16S amplicon sequencing. The 16S rRNA gene variable region V4 for bacterial community was sequenced using an Illumina MiSeq sequencer (Illumina NovaSeq 6000 system). The PCR primers 515 F and 806R were used, and then paired-ends reads of 312 bp were obtained.The forward and reverse sequences were obtained after sequencing was performed on the Illumina MiSeq sequencer using a paired-end technique. The raw sequence reads were uploaded into MG-RAST, an online server . OTUs were clustered at 3% divergence (97% similarity). Equitability_J, Fisher_alpha, Berger-Parker and Chao-1 were used to estimate species richness. Taxonomic richness was expressed as OTU number. Furthermore, statistical analysis was carried out using the mean of the relative abundance for all the replicates of each sampling site. The sequences have been deposited on the National Center for Biotechnology Information SRA dataset under the accession number PRJNA672856.The bacterial community level physiological profiles of the soil samples were determined using the MicroResp™ method  and 3 carboxylic acids  were used to determine the physiological profiles, while distilled water served as the control. Sterile deionized water was used to measure individual basal respiration. Each substrate dissolved in sterile deionized water was added to 4 replicated wells. Detection plates containing cresol red (12.5 µg/ml), potassium chloride (150 mM) and sodium bicarbonate (2.5 mM) were read at absorbance wavelength 570 nm, and immediately placed on the MicroResp™ seal, and then incubated at 25 °C in the dark for 6 h as recommended in the instruction manual. After incubation, the modification in optical density (OD) was determined on a spectrophotometer microplate reader  at a wavelength of 570 nm. The rate of individual CO2 respiration per gram of soil was calculated using the formula provided in the MicroResp™ manual . Total respiration rate was obtained by following the method of Colombo et al. (Eleven (11) different carbon substrates, consisting of 3 amino acids , 6 carbohydrates  graphs were used to evaluate the relationship between bacterial communities and the measured physiochemical parameters, which were plotted using CANOCO version 5.0 . The effects of pH, total N and OM on the bacterial community distribution were determined using Canonical Correspondence analysis (CCA). Graphs and one-way ANOVA for functional measurements were achieved using GraphPad Prism 7 .Microsoft Excel Software was used to analyze the abundance and distribution of the key rhizosphere bacterial communities at the class and order level. Also, Shinyheatmap online tool (www1.heatmapper.ca/expression/) was used for the plotting of heatmaps using the relative abundance values. Equitability_J, Fisher_alpha, Berger-Parker and Chao-1 indices for diversity assessment were used for samples across rhizosphere and bulk soil samples, and the comparison of these indices was performed using Kruskal–Wallis test. The analyses were carried out using PAST version 3.20  and 80,404,565 (R2) sequence reads for sunflower rhizosphere soils and 50,642,798 (B1) and 74,956,208 (B2) sequence reads for bulk soil samples. After performing quality control (QC) using MG-RAST software, the estimated sequence were 8,362,450 (R1) with a mean G + C content of 56%, and R2 had a mean sequence of 8,310,524 with a mean G + C content of 55.5%, while the mean sequence of B1 sample was 5,824,673 with a mean G + C content of 54.5%. Whereas, B2 had a mean sequence of 9,525,300 with a mean G + C content of 57%. Additionally, 29,112 (R1) and 272,663 (R2) sequence reads contained predicted proteins with unknown functions from the sunflower rhizosphere soils, while 173,859 (B1) and 254,030 (B2) sequence reads contained predicted proteins with unidentified functional categories from sunflower bulk soil samples.http://bioinformatics.psb.ugent.be/cgi-bin/liste/Venn/calculate_venn.htpl p < 0.05) (Table p = 0.01). The PCoA graph revealed a clear difference in the relative abundance of bacterial groups between R1 and B1 sites as compared to R2 and B2 sites .The diversity of the structural categories was examined using the equitability_J, Fisher_alpha, Berger-Parker and Chao-1, and the results revealed that they differ significantly . Bacilli were higher in all soil samples  with relative abundance values of 38.18, 30.66, 28.85 and 37.43 respectively. Gammaproteobacteria were dominant in R1, B1, and R2 soil samples with relative abundance values of 23.74, 28.36, and 21.96 respectively. While Actinobacteria (18.05) were recorded to be more abundant in bulk soil sample (B2). Cytophagia was exclusively detected in the rhizosphere soil (R2). Whereas, Actinobacteria, Clostridia, Spingobacteria, Gemmatimonadetes, Deltaproteobacteria, Negativicutes, Thermamicrobia, Solibacteres, Nitrospira and Spartobacteria were abundant in the bulk soil sample (B2).Sequence tags were assigned to rhizosphere and bulk soil samples into diverse taxa via rapid metagenomic annotations using MG-RAST software. At the class level, the relative abundance of bacterial class for each soil sample is presented in Fig. Bacillales were higher in R1 (36.24) than the bulk soil (B1 = 29.28). On the other hand, B2 (37.00) had higher mean relative abundance than the rhizosphere soil (R2 = 26.99). Pseudomonadales were higher in bulk soil (B1 = 26.94) than in the R1 (22.64). Also, Pseudomonadales in R2 (21.10) were more than those of B2 (7.35). Actinomycetales were higher in B2 (7.41) than R1, B1, and R2. From the heatmap Z score color intensity, Lactobacillales, Burkholdeiales, Enterobacteriales, Rhizobiales, Sphingomonadales, Myxococcales, Nitrosomonadales, and Flavobacteriales were higher in the R1 than in other soil samples. Spingobacteriale and Herpetosiphonales were relatively more abundant in B2. Lactobacillales and Nitrospirales were more dominant in R2. Rubrobacterales, Caulobacterales, Gemmatimononadales, Pleurocapsales, Cytophagales and Rhodocyclales were highly abundant in the corresponding bulk soil (B2)  positively correlated with les Fig. . The soi positive2 produced   showed different carbon substrate utilization and respiration rates as depicted in Figs. ced Fig. . However5) Figs. , 7 and 8−1 h−1 CO2–C), while for the two bulk soils, B2 showed a higher total respiration rate (23.80 µg g−1 h−1 CO2–C). The respiration responses from distilled water utilization  were 4.50, 7.23, 6.38 and 8.19 µg g−1 h−1 CO2–C for R1, B1, R2 and B2 respectively.The influence of the carbon substrate utilization on the relative abundance of bacterial species at the order level was determined using CCA  composition and abundance  in the structural diversity ‘between’ samples. The significant variation in the bacterial diversity observed between samples in this study can partly be attributed to the differences in the agricultural practices  in their cellular structures  of these endospore-forming bacteria, with spo0A present in the microbiome of mammals  and pH on five bacteria groups at the order level indicates that an increase in the soil total N and pH would lead to a direct increase in the bacterial species abundance of the five taxa. These bacteria were captured from R2 Fig. . From Fi2 released or produced (respiration rate)  and a disaccharide (sucrose) had a positive influence on les Fig. . Citric les Fig. .Actinomycelales, Acidobacteriales, Rhodocyclales, Rubrobacterales, and Pleurocapsales  , while at the order level, among all the bacterial taxa captured in the two farms, Bacillales were the most dominant. Bacterial communities of R1, B1, R2 and B2 soils were found to use the different carbon substrates as an energy source, but significant differences were only observed in tryptophan and methionine amended soils. The utilization of most of the carbon substrates positively influenced the soil bacterial communities. The presence of unclassified bacteria in this study, calls for a further effort to capture and identify these unknown bacteria, since it may be possible to discover novel bacterial species with plant growth promoting functionality.Finally, at a probability level of 0.05, significant differences were observed for the alpha and beta diversity of the soil bacterial communities. At the class level, Additional file 1: Figure S1. The influence of pH, total N (%) and OM (%) on sunflower rhizosphere and bulk soils obtained from Ditsobottla and Kraaipan farms using Canonical correspondence analysis. N nitrate; % percentage; OM organic matter; R1 Ditsobottla rhizosphere soil; B1 Ditsobottla bulk soil; R2 Kraaipan rhizosphere soil; B2 Kraaipan bulk soil. Figure S2. Effects of soil type on soil functional properties as measured for distilled water  and sum of the individual respiration rate of the 11 carbon substrates  used in the CLPP assay. Number of replicates (n) = 2. R1 Ditsobottla rhizosphere soil; B1 Ditsobottla bulk soil; R2 Kraaipan rhizosphere soil; B2 Kraaipan bulk soil. Figure S3. The influence of pH, total N (%) and OM (%) on carbon substrate utilization by bacterial components of sunflower rhizosphere and bulk soils obtained from Ditsobottla and Kraaipan farms using canonical correspondence analysis. N nitrate; % percentage; OM organic matter.Additional file 2: Table S1. Agricultural history of Ditsobottla and Kraaipan farms. Table S2. Forward selection of soil physio-chemical components that best described difference in bacterial components between sunflower rhizosphere and bulk soils of Ditsobottla and Kraaipan farms. Table S3. Forward selection of soil carbon substrates that best described difference in bacterial components between sunflower rhizosphere and bulk soils of Ditsobottla and Kraaipan farms. Table S4. Forward selection of soil physio-chemical components that best described difference in carbon substrate utilization between sunflower rhizosphere and bulk soils of Ditsobottla and Kraaipan farms.