The Effect Of Jiedu Tongluo Tiaogan Decoction On The Apoptosis Of Islet Cell Based On The Study Of Autophagy Regulation

Objective:Explore the mechanism of Jiedu Tongluo Tiaogan Decoction(JTTD)based on autophagy regulation to protect the function of islet cell in type 2 diabetes mellitus(T2DM)SD rats through animal experiments.Methods:8-week-old SD male rats were fed with high-fat diet for 4 weeks and combined with a single intraperitoneal injection of streptozocin 40 mg.Kg^-1to induce animal model of T2DM.The traditional Chinese medicine groups were given the low-dose(1.5g.kg^-1.d^-1),middle-dose(4.5 g.kg^-1.d^-1)and high-dose(13.5 g.kg^-1.d^-1)of JTTD,and the western medicine group was given metformin hydrochloride(0.15 g.kg^-1.d^-1).Observe the general state,body weight,and water consumption of SD rats,and at the end of the fourth week of drug intervention,the SD rats were given oral glucose tolerance test(OGTT)and calculated the area under the curve(AUC).SD rats were put to death by anesthesia after 4weeks of intragastric administration and detected fasting blood glucose(FBG),glycosylated serum protein(GSP),triglyceride(TG),cholesterol(CHO),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),free fat acid(FFA).Fasting insulin(FINS)was detected by Elisa and calculated the insulin resistance index(HOMA-IR).HE staining and Masson staining were used to observe the pathological and morphological changes of the pancreas in SD rats.Immunohistochemical was used to detect the protein expression of Beclin-1 and LC3.Western Blot was used to detect the protein expression of Beclin-1,LC3,P62,Bax and Bcl-2.RT-PCR was used to detect the m RNA expression of Beclin-1,LC3,Bax,Bcl-2 and Caspase-3.TUNEL was used to detect the apoptosis rate of islet cell in the pancreas of SD rats.Results:1.Effects of JTTD on general state,body weight,amount of drinking water,glucose and lipid metabolism in SD ratsThe SD rats in the control group are generally in good condition,with steady weight gain and normal amount of drinking water.The model group are in poor general condition,with significant weight loss and increased water consumption.Compared with the model group,the JTTD and the metformin hydrochloride group have improved general status,reduced weight loss,and improved polydipsia(P<0.05,P<0.01).FBG and GSP of SD rats in the low,medium,and high dose groups of JTTD and the metformin hydrochloride group all improved significantly compared with the model group,and had a better hypoglycemic effect(P<0.05,P<0.01).Compared with the control group,the FINS level in the model group increased significantly(P<0.05).Compared with the model group,the FINS levels of JTTD and the metformin hydrochloride groups were significantly decreased(P<0.05,P<0.01).Compared with the control group,the HOMA-IR of SD rats in the model group was significantly increased(P<0.01).Compared with the model group,the JTTD and the metformin hydrochloride groups were significantly decreased(P<0.01).The OGTT-AUC results showed that the the model group was significantly increased(P<0.01),and the low,medium and metformin hydrochloride groups of the JTTD was significantly decreased(P<0.05,P<0.01).In terms of lipid metabolism,JTTD and metformin hydrochloride can reduce the levels of TG,CHO,LDL-C,and FFA in T2DM SD rats(P<0.05,P<0.01),and increase the level of HDL-C(P<0.01).2.Effect of JTTD on the pathological morphology of the pancreatic tissue in SD ratsHE staining of SD rats pancreatic tissue:the control group have complete structure,clear borders,tightly arranged,and rich cytoplasm;the model group are structurally disordered with unclear borders,irregular arrangements,less cytoplasm,and the cytoplasm of the pancreatic islets becomes lighter or vacuolated;the groups of JTTD and the metformin hydrochloride were improved compared with the model group.Masson staining of SD rat pancreas tissue:compared with the control group,the model group showed obvious fibrosis of the pancreas,with a large amount of collagen fiber deposition.The groups of JTTD and the metformin hydrochloride were significantly improved compared with the model group(P<0.05,P<0.01).3.Effect of JTTD on autophagy of pancreatic tissue in SD ratsBy immunohistochemistry,the protein expression levels of Beclin-1 and LC3 in the model group were decreased than the control group(P<0.01),and the protein expressions of Beclin-1 and LC3 in the JTTD and the metformin hydrochloride group were significantly higher than the model group(P<0.05,P<0.01).Using Western Blot,compared with the control group,the protein expression of Beclin-1 and LC3 II/I in the model group decreased(P<0.01),and the protein expression of P62 increased(P<0.01),Compared with the model group,JTTD and metformin hydrochloride can increase protein expression of Beclin-1 and LC3II/I(P<0.01),and decrease protein expression of P62(P<0.05,P<0.01).Using RT-PCR,compared with the control group,the m RNA expression of Beclin-1 and LC3 in the model group decreased(P<0.01).Compared with the model group,the m RNA expression of Beclin-1 and LC3 in the JTTD and the metformin hydrochloride group increased significantly(P<0.01).4.Effect of JTTD on apoptosis of pancreatic tissue in SD ratsUsing Western Blot,compared with the control group,the protein expression of Bax in the model group increased(P<0.01),the protein expression level of Bcl-2 decreased(P<0.01),and the ratio of Bax/Bcl-2 Increased(P<0.01),Compared with the model group,the JTTD and the metformin hydrochloride group can reduce the protein expression of Bax(P<0.01),increase the protein expression level of Bcl-2(P<0.01),and reduce Bax/Bcl-2ratio(P<0.01).Using RT-PCR,the expression of Bax and Caspase-3 m RNA in the model group was higher than that in the control group(P<0.01)and the m RNA expression of Bcl-2 was lower(P<0.05).Compared with the model group,the JTTD and the metformin hydrochloride group can reduce the m RNA expression of Bax and Caspase-3(P<0.01),increase the expression of Bcl-2 m RNA(P<0.01),and reduce Bax/Bcl-2 ratio(P<0.01).The TUNEL was used to detect the islet cell apoptosis rate in the pancreatic tissue.The apoptosis rate of the model group was significantly higher than that of the control group(P<0.01).Compared with the model group,the JTTD and the metformin hydrochloride group can reduce the pancreatic islet cell apoptosis rate in T2DM SD rats.Conclusion:1.JTTD can effectively alleviate the general state and weight loss trend of T2DM SD rats,reduce polydipsia,improve glucose and lipid metabolism,and it can alleviate the damage to the pathological structure of the pancreas in T2DM SD rats.2.JTTD can alleviate the apoptosis of islet?-cell in T2DM SD rats,and its protection of islet?-cell may be achieved by enhancing autophagy.