The Mechanism Of Gallic Acid Of Phyllanthus Emblica L. On Apoptosis Of Pancreatic Islet ? Cells Induced By High Glucose

Objective:Diabetes is an endocrine and metabolic disease,and its incidence has been increasing recently.The prevalence of type 1 diabetes increases by 2%-3%annually.High glucose induces islet?-cell apoptosis is one of the important pathological basis of diabetes.Phyllanthus emblica L.is a traditional Tibetan medicine for the treatment of diabetes.According to previous experimental results from research group,it shows that Gallic acid is an active ingredient in Phyllanthus emblica L.and also plays a role in inhibiting apoptosis of islet?cells.This study to be established an in vivo and in vitro model of high glucose-induced apoptosis of pancreatic islet?cells.Based on this model,we further studied the effect of gallic acid on pancreatic?-cell apoptosis and its mechanism,which could be Phyllanthus emblica L.and natural active product gallic acid.The follow-up research and development and research on the natural active products of traditional C hinese medicines for the treatment of type 1 diabetes provide experimental evidence.Methods:1.The contents of gallic acid in Phyllanthus emblica L.were determined by RP-HPLC.Chromatographic conditions:Agilent C18 ZORBAX SB-C18 column(4.6×250 mm,5?m);mobile phase:methanol-0.2%phosphoric acid(5:95);detection wavelength:273nm;column temperature:25°C;flow rate:1 mL/min.2.Effect of gallic acid in Phyllanthus emblica L.on diabetic model ratsEstablishment of Diabetic Rat Model:Seventy Wistar ma le rats were injected intraperitoneally with streptozotocin(STZ,50mg/kg),and blood glucose was measured after 3 days.Group dosing:Rats were randomized according to randomization:normal,model,positive(sitagliptin,10.5 mg/kg),gallic acid high,middle,and low dose groups(80,40,20 mg/kg),Rats were dosed for 6 weeks with gallic acid.After 6 weeks,blood was taken from the abdominal aorta and the pancreas was dissected.Determine serum and urine insulin levels by Elisa.Rat pancreatic tissue was stained with HE.Rat pancreatic tissue was stained with erythrochrome-orange G.Western Blot was performed to investigate the expression of TRX,TXNIP,Caspase-1,NLRP3 and NF-?B protein.Real-time PCR was performed to assess TRX,TXNIP,Caspase-1,NLRP3 and NF-?B mRNA expression.3.The effect of gallic acid in Phyllanthus emblica on INS-1 cells in high glucose environmentThe growth curve of INS-1 cells was determined by MTT assay.High-glucose model establishment:Rat insulinoma cell line INS-1 cells were used as in vitro targets to establish high-glucose-induced cell apoptosis models,and INS-1 cells were cultured with high-glucose medium to determine the optimal value for the next experiment.The effect of different concentrations of glucose and gallic acid on the activity of INS-1 cells was determined by MTT assay.Flow cytometry was used to detect the effect of GA on the apoptosis of INS-1 cells under high glucose condition.INS-1 cells were stained with AO/EB.Western Blot Blot was performed to investigate the expression of TRX,TXNIP,Caspase-1,NLRP3 and NF-?B protein.Real-time PCR was performed to assess TRX,TXNIP,Caspase-1,NLRP3 and NF-?B mRNA expression.Results:1.Gallic acid had a good linear relationship in the range of 2-22?g/?L(r~2=0.997).The average recovery was 97.2%.The content of gallic acid in Phyllanthus emblica L.was 1.21mg/g.2.The model was successful when 11.1 mmol/L?random blood glucose<30mmol/L.The results of Elisa assay showed that the serum insulin content in the model group was lower than that in the normal group,and the urinary insulin content was increased(p<0.05);gallic acid increased the serum insulin content and decreased the urinary content,The insulin content was statistically different(p<0.05 and p<0.01).The results of HE staining showed that:In the normal group,the pancreas had normal cell borders,cells were aggregated and distributed,and there was abundant cytoplasm;in the model group,the islet cells of the islet were uneven at the edges,the distribution was scattered,and the number of cells and cytoplasm was significantly reduced.After administration of gallic acid,the morphology of islet cells changed significantly.The results of aldehyde erythrochrome-orange G staining showed that the islet?-cells in the normal group had complete cell morphology,dark purple cytoplasm,and rich intracellular microparticles.After administration of gallic acid,the morphology of pancreatic islet?cells was significantly improved.Western Blot results showed that gallic acid can reduce the expression of TXNIP,N LRP3,NF-?B and Caspase-1 protein in pancreatic tissue of diabetic rats,and up-regulate the expression of TRX(p<0.01 and p<0.05).The results of RT-PCR showed that gallic acid could down-regulate the expression of TXNIP,NLRP3,NF-?B and Caspase-1mRNA and up-regulate the expression of TRX mRNA,which was significantly different(p<0.01 and p<0.05).3.According to the cell growth curve,the optimal seeding density of cells was 25000cells/well.Compared with the control group,the survival rate of INS-1 cells was significantly decreased after cultured for 48 h in a medium with glucose concentration of 25 mmol/L,indicating that the high glucose-induced cell apoptosis model was established successfully.Gallic acid(10,5,2.5?mol/L)had no significant difference in normal cells(p>0.05).Gallic acid increased the survival rate of apoptosis induced by high glucose,with statistical differences(p<0.01).Western Blot results showed that gallic acid can reduce the expression of TXNIP,NLRP3,NF-?B and Caspase-1 in INS-1 cells,and up-regulate the expression of TRX,which has statistical significance(p<0.01 and p<0.05).The results of RT-PCR showed that gallic acid could down-regulate the expression of TXNIP,NLRP3,NF-?B and Caspase-1 mRNA and up-regulate the expression of TRX mRNA,which was significantly different(p<0.01and p<0.05).Conclusion:The average content of gallic acid in Phyllanthus emblica was 1.21mg/g.Gallic acid in Phyllanthus emblica L.can affect the apoptosis of rat pancreatic islet cells induced by STZ and apoptosis of INS-1 cells induced by high concentration of glucose.It can decrease the expression of inflammatory factors and inhibit the apoptosis of pancreatic islet?cells.Reduce the inflammatory factor NF-?B,NLRP3,Caspase-1,inhibition of TXNIP and TRX binding.