| Banxia Xiexin Decoction comes from "Shang Han Lun",which has the effects of calming the heat and cold,eliminating spleen and dissolving knots,hardening pain,lowering diarrhea and applying phlegm and removing blood stasis.Previous researches have shown that Banxia Xiexin Decoction can inhibit the in vitro isletβ cell apoptosis and promote insulin secretion,but the specific mechanism needs to be further explored.This experiment combined with in vitro and in vivo experiments to further confirm the protective effect of Banxia Xiexin Decoction on islet cells and explore its mechanism.Objective:1.Combination of in vivo and in vitro experiments to observe the protection of Banxia Xiexin Decoction(BX)on islet cells for DM mice induced by high-fat diet combined with streptozocin(STZ)and t-butylhydroperoxide(TBHP)induced damage of MIN6 cells.2.To explore the protective mechanism of Banxia Xiexin Decoction for islet cells of DM mice and MIN6.Methods:1.Animal experiments:8-week-old SPF male C57BL/6 mice were randomly divided into 6 groups.After 8 weeks of high-fat diet feeding,55 mg/kg of STZ was injected intraperitoneally,and the injection was continued for 3 days for modeling.The experiment was divided into 6 groups:normal group,model group,metformin group,Banxia Xiexin Decoction low-dose group,Banxia Xiexin Decoction medium-dose group,Banxia Xiexin Decoction high-dose group.Gavage was continued for 7 weeks.And monitor body weight every 2 weeks.Blood was collected after 7 weeks,and the pancreas and liver were stored at-80℃ for future use.The weight of the mice was measured every 2 weeks,and the FBG was measured at 0,3,and 7 weeks.After 7 weeks,HbAlc,FINS and blood lipids(TC,TG,HDL-C,LDL-C)were detected.For the OGTT tset,after FBG was measured,mice were gavaged with 2g/kg glucose.Blood glucose was measured at 30min,60min,and 120min after the glucose load,and the OGTT curve was drawn according to the blood glucose value at each time point,and the AUC of the blood glucose was calculated.Pancreas HE staining and immunohistochemistry were performed.The TUNEL method was used to detect the islet cell apoptosis rate,and the the levels of MDA and SOD were detected too.The Western blot method was used to detect the PI3K/AKT/FOXO1 signal pathway and related protein expression.2.Cell experiment:The MIN6 cells were digested with 0.25%trypsin and then added with high-glucose DMEM culture solution,pipette with PBS and inoculate into a 96-well plate.The cell suspension was cultured in an incubator with 100μL per well.After the cells adhere to the wall,different concentrations of Banxia Xiexin Decoction(0.1,0.5,1,1.5,2.5,5,10 mg/ml)were added and incubated for 12 h.Then perform the MTT experiment,check the OD value with a microplate reader,and calculate the cell viability,to determine the concentration range of Banxia Xiexin Decoction.After the cells adhered to the wall,Banxia Xiexin Decoction(0.25,0.5,1.0 mg/ml)was added to the cells for 12 h.After the drug incubation,100μM TBHP was added to incubate for 2 h.After the TBHP treatment was completed,MTT experiments was to proceed,and the OD value was measured with a microplate reader to screen the final incubation concentration of Banxia Xiexin Decoction.The experiment was divided into the following 5 groups:the control group,the model group,and the Banxia Xiexin Decoction group,Banxia Xiexin Decoction+Inhibitor Group and the Inhibitor Group.Hoechst 33342 method and TUNEL method were used to detect the effect of Banxia Xiexin Decoction on TBHP-induced apoptosis.GSIS test was performed,and the insulin levels were measured by ELISA.The DCFH-DA probe was used to detect the intracellular ROS content,and the intracellular MDA,SOD,GSH-Px content were detected as well.Western blot was used to detect the expression of PI3K/AKT/FOXO1 signaling pathway and related proteins.Results:1.Animal experiment:(1)Effect of Banxia Xiexin Decoction on body weight of mice:After modeling,and the weight of the mice in the model group was always lower than those of other groups.The mice in the control group increased steadily.By the end of the 7th week,the weight of mice in the control group was significantly larger than that in the model group(P<0.05)and each drug intervention group(P>0.05).(2)Effect of Banxia Xiexin Decoction on glucose and lipid metabolism:After successful modeling,the blood glucose level of the control group was lower than that of the groups(P<0.05).After 3 and 7 weeks of gavage,the blood glucose level in the model group was significantly higher than that in the control group(P<0.05);compared with the model group,the blood glucose level in the Banxia Xiexin Decoction group was significantly lower than that in the model group(P<0.05).Compared with the control group,the model group content of HbA1C,TC,TG,and LDL in the model group increased significantly(P<0.05),and the HDL content decreased significantly(P<0.05).Compared with the model group,the content of HbA1C,TC and TG decreased significantly(P<0.05),and the HDL content increased significantly(P<0.05)in the metformin group and the Banxia Xiexin group.(4)Effect of Banxia Xiexin Decoction on insulin secretion and HOMA-β:Compared with the control group,the FINS and HOMA-β of the mice in the model group were significantly decreased(P<0.05);compared with the model group,the FINS and HOMA-B in the metformin group,the Banxia Xiexin Decoction medium and and the Banxia Xiexin group increased.(5)Effect of Banxia Xiexin Decoction on OGTT and AUCOTTT:Compared with the control group,the blood glucose level in the model group significantly increased at each time point before and after glucose administration(P<0.05).At 0 minin the glucose tolerance test,compared with the model group,the blood glucose levels of the metformin group,the Banxia Xiexin Decoction group significantly decreased(P<0.05).At 30 minutes,the blood glucose levels of each group significantly increased.When the glucose tolerance test was performed for 60 minutes,compared with the model group,the blood glucose levels of the metformin group,Banxia Xiexin Decoction groups began to decrease significantly(P<0.05).At 120 minutes,compared with the model group,the blood glucose levels of the metformin group,the Banxia Xiexin Decoction groups significantly decreased(P<0.05).Compared with the control group,the AUCOTTT of the model group mice increased significantly(P<0.05);compared with the model group,the AUCOTTT of the metformin group,Banxia Xiexin Decoction groups all significantly decreased(P<0.05).(6)Effect of Banxia Xiexin Decoction on the morphology of pancreas(HE staining method):The pancreas of the control group were round or oval cell clusters with different cell cluster sizes,complete structure,regularity,and clear edges.They are scattered between pancreatic acinars,and the number of cell clusters is large with uniform size.While in the model group,the number of islet cell clusters decreased,the area shrinked with irregular shape,unclear edge and disordered structur.Compared with the model group,the number of islet cells of each drug intervention group increased,and the morphological structure improved.(7)Effect of Banxia Xiexin Decoction on Apoptosis of Islet β Cells:Compared with the control group,the apoptosis rate of the mice in the model group was significantly increased(P<0.05);compared with the model group,the apoptosis rates of the metformin group,Banxia Xiexin Decoction groups were decreased(P<0.05).(8)Effect of Banxia Xiexin Decoction on oxidative stress indicators and inflammatory factors:Compared with the control group,the MDA contents in the model group were significantly increased(P<0.05),and the SOD content was significantly decreased.(P<0.05);Compared with the model group,the MDA contents in the metformin group,the Banxia Xiexin Decoction groups all decreased significantly(P<0.05);the SOD contents were all significant increase(P<0.05)(9)Effect of Banxia Xiexin Decoction on PI3K/AKT/FOXO1 signal pathway and related protein expression:compared with the control group,p-AKT/AKT;p-FOXO1/FOXO1,Pdx-1/β-actin and MafA/β-actin in the model group decreased significantly(P<0.05);the ratios of Caspase-3/β-actin,Bax/β-actin,and P27/β-actin increased significantly(P<0.05).Compared with the model group,the p-AKT/AKT,p-FOXO1/FOXO1,Pdx-1/13-actin,and MafA/β-actin ratios in the metformin group and the Banxia Xiexin Decoction groups were significantly increased(P<0.05);Caspase-3/β-actin,Bax/β-actin,and P27/13-actin ratios decreased significantly(P<0.05).2.Cell experiment:(1)The toxicity Effect of Banxia Xiexin Decoction on MIN6 cells:When the concentration of Banxia Xiexin Decoction was≥2.5 mg/ml,the viability of MIN6 cells decreased significantly.Therefore,the following experiments were performed at three concentrations of 0.25,0.5,and 1.0 mg/ml.(2)Effect of Banxia Xiexin Decoction on the cell viability of MIN6 cells:Compared with the control group,the cell viability of the model group significantly decreased(P<0.05).Compared with the model group,the cell viability of the BX0.5 dose group and BX1.0 dose group was significantly increased(P<0.05),and there was no significant difference between the BX0.5 dose group and the BX1.0 dose group(P>0.05),The cell viability of cells in the BX0.5 dose group was slightly higher.Therefore,the Banxia Xiexin Decoction 0.5 mg/ml group was selected as the Banxia Xiexin Decoction drug intervention group.(3)Effect of Banxia Xiexin Decoction on TBHP-induced MIN6 cell apoptosis:Hoechst 33342 staining showed that the cells in the control group were intact and the nuclei were uniformly stained.After TBHP stimulation,the cells irregularly condense and aggregate,showing dense dense staining,with the nucleus dividing into fragments.Compared with the model group,the nuclear shrinkage,dense dense staining,and reduced cell debris of the Banxia Xiexin Decoction group improved cell morphology.Compared with the control group,the apoptosis rate of the model group increased significantly(P<0.05).Compared with the model group,pretreatment with Banxia Xiexin Decoction for 12 h can significantly reduce the apoptosis rate(P<0.05).(4)Effect of Banxia Xiexin Decoction on glucose-stimulated insulin secretion:The function of MIN6 cells in the model group was impaired,compared with the 2.8mM model group,glucose-stimulated insulin secretion in the 16.7mM model group did not increase significantly(P>0.05).However,the glucose-stimulated insulin secretion of the 16.7 mM Banxia Xiexin Decoction group was significantly increased after pretreatment of Banxia Xiexin Decoction for 12 hours,which improved the function of islet cells(P<0.05).(5)Effect of Banxia Xiexin Decoction on oxidative stress indicators of MIN6 cells:Compared with the control group,the contents of ROS and MDA in the model group increased significantly(P<0.05);the contents of SOD and GSH-Px decreased significantly(P<0.05).Compared with the model group,the intracellular ROS and MDA contents in the Banxia Xiexin Decoction group were significantly reduced(P<0.05);the SOD and GSH-Px contents were significantly increased(P<0.05).(6)Effect of Banxia Xiexin Decoction on PI3K/AKT/FOXO1 signaling pathway and expression of related proteins:Compared with the control group,p-AKT/AKT,p-FOXO1/FOXO1,Pdx-1/β-actin in the model group decreased significantly(P<0.05);the Caspase-3/β-actin,Bax/β-actin,and P27/β-actin ratios increased significantly(P<0.05).Compared with the model group,the p-AKT/AKT,p-FOXO1/FOXO1,Pdx-1/β-actin ratios in the Banxia Xiexin Decoction group were significantly increased(P<0.05);Caspase-3/β-actin,Bax/β-actin and P27/β-actin ratios decreased significantly(P<0.05).After the PI3K inhibitor was added,the effect of Banxia Xiexin Decoction increasing the expression of p-AKT/AKT ratio was offset.Conclusion:1.Banxia Xiexin Decoction can significantly improve glucose and lipid metabolism,pancreatic islet function,and pancreatic tissue damage in DM mice.It can significantly inhibit islet β cell apoptosis,and promote insulin secretion.2.Banxia Xiexin Decoction has the effects of inhibiting MIN6 cell apoptosis,promoting insulin secretion,and protecting MIN6 cells.3.The protective effect of Banxia Xiexin Decoction on DM mice and MIN6 islet cells may be through inhibiting oxidative stress and enhancing the body’ s antioxidant capacity;activating the PI3K/AKT/FOXO1 signal pathway and influencing the related downstream expression,such as Caspase-3,Bax,P27,MafA and Pdx-1 proteins.Innovation1.This study proved that Banxia Xiexin Decoction can improve DM mice and MIN6 islet β cell apoptosis,promote insulin secretion,protect islet cells,and provided an experimental basis for the clinical application of Banxia Xiexin Decoction.2.By combining in vivo and in vitro experiments,it was confirmed that Banxia Xiexin Decoction play a role in protecting islet cells by reducing the oxidative stress injury;activating the PI3K/AKT/FOXO1 signaling pathway and producing a cascade reaction,affecting downstream Caspase-3,Bax,P27,MafA and Pdx-1 protein expression. |
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