| Objective:Apoptosis caused by islet cell dysfunction is an important pathogenic factor of T2 DM.As one of the main signaling pathways of ER stress,PERK-eIF2α-CHOP signaling pathway can participate in apoptosis induced by glycolipid toxicity,thus aggravating islet injury.It is not clear whether this pathway is involved in the protective effect of Jianpi Xiaoke Decoction on islet cells.Therefore,this study established in vivo and in vitro models to explore the possible mechanism of endoplasmic reticulum stress PERK-eIF2α-CHOP pathway in Jianpi Xiaoke formula to protect islet cell function.Method:In vivo experiment: Wistar rats were randomly divided into normal control group(Con group),T2 DM Model group(Model group),Jianpi Xiaoke Decoction group(Jpxk group)and Lilaglutide positive control group(Li group).In addition to conventional feeding in Con group,other rats were jointly established with streptozotocin(STZ)combined with high-sugar and high-fat diet.After successful modeling,the rats were given corresponding drug intervention 8 weeks later.1.Fasting blood glucose(FBG),serum insulin(Fins)and glycosylated hemoglobin(Hb A1c)were measured.2.The expressions of PERK,eIF2α and CHOP in pancreas were determined by immunohistochemistry.3.Serum levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in rats;4.Determination of the contents of triglyceride(TG),total cholesterol(TC),low density lipoprotein(LDL-c)and high density lipoprotein(HDL-c).In vitro experiment: min6 cells were randomly divided into normal control group(Con group,5.5mmol/L glucose),Model group(Model group,25mmol/L glucose),Jianpi Xiaoke Decoction group(Jpxk group,25mmol/L+ Jianpi Xiaoke Decoction with optimal intervention concentration0.001mg/ml)and Lilaplutide positive control group(Li group,25mmol/L+ Liraglutide at optimal intervention concentration10nmol/L).1.The cell viability of each group was detected by CCK8 method.2.Insulin secretion was detected by enzyme-linked immunosorbent assay(ELISA).3.Apoptosis was detected by Annexin V-FITC/PI double staining flow cytometry and Hoechst33258 staining.4.The expression levels of GRP78,PERK,eIF2α,ATF4,CHOP and Caspase12 were detected by Western Blot.5.m RNA expressions of PERK,eIF2α and CHOP were detected by quantitative fluorescence polymerase chain reaction(q PCR).Results:In vivo experiment:1.Compared with Con group,the daily food intake and water intake of rats in Model group were increased,the excreta was significantly increased,the mental state was poor,the body size was emaciated,and the blood glucose value was stable more than16.8mmol/L,indicating the success of modeling.Compared with normal group,body weight,FBG and Hb A1 c in Model group were increased(P<0.01),Fins content was decreased(P<0.01),TC,TG and LDL-C were increased(P<0.01),and HDL-C was decreased(P>0.05).The contents of inflammatory cytokines IL-6 and TNF-α were increased(P<0.01);Immunohistochemistry showed that the positive expression rates of PERK,eIF2α and CHOP increased in the pancreas,most of which were located in the central region of the islet,with a small amount of distribution in the periphery.2.Compared with Model group,after treatment with Jianpi Xiaoke Decoction,the mental state of rats was significantly improved,diet and urine volume decreased,and body weight increased(P<0.01);FBG and Hb A1 c were decreased(P<0.01),Fins content was increased(P>0.05),TC and LDL-C were decreased(P<0.01),TG was decreased(P<0.05)and HDL-C was increased(P>0.05).The contents of IL-6 and TNF-α were decreased(P<0.01).Immunohistochemistry showed that the positive expression rates of PERK,eIF2α and CHOP decreased in pancreas.3.Compared with Model group,after liraglutide treatment,DM rats showed improved symptoms,good spirits,and increased body weight(P<0.01);FBG and Hb A1 c were decreased(P<0.01),Fins content was increased(P>0.05),TC and LDL-C were decreased(P<0.01),TG was decreased(P<0.05)and HDL-C was increased(P>0.05).The contents of IL-6 and TNF-α decreased(P<0.05);Immunohistochemistry showed that the positive expression rates of PERK,eIF2α and CHOP decreased in pancreas.4.Compared with Li group,the levels of body weight,FBG,Hb A1 c,Fins,blood lipid and inflammatory factors in Jianpi Xiaoke Decoction rats were not significantly increased or decreased(P>0.05),and the positive expression rates of PERK,eIF2α and CHOP in pancreas were similar.In vitro experiment:1.Compared with Con group,the activity of min6 cells in Model group was significantly decreased(P<0.01);Insulin level decreased(P<0.01);The proportion of apoptotic cells increased(P<0.01),and the number of apoptotic bodies increased,chromatin and nucleus condensation;The expression of GRP78,PERK,eIF2α,CHOP and Caspase12 proteins in endoplasmic reticulum stress pathway was up-regulated(P<0.01),while the expression of ATF4 protein was increased(P< 0.05).PERK,eIF2α and CHOP m RNA expressions were increased(P<0.01).2.Compared with Model group,Jianpi Xiaoke Fang dry prognosis,min6 cell activity increased(P<0.01);Insulin level increased(P<0.05);The proportion of apoptotic cells was decreased(P<0.05),and the apoptotic corpuscles in min6 cells were decreased.The protein expressions of endoplasmic reticulum stress pathway PERK,eIF2α and CHOP were down-regulated(P<0.01),and the protein expressions of GRP78 and Caspase12 were decreased(P< 0.05).The m RNA expressions of PERK and eIF2α were decreased(P<0.01),and those of CHOP were decreased(P<0.05).3.Compared with Model group,the activity of min6 cells was increased after liraglutide intervention(P<0.05);Cell apoptosis rate was decreased(P<0.01)and cell shrinkage and agglutination were decreased.PERK expression increased(P >0.05),eIF2αprotein expression decreased(P<0.01),and GRP78 and Caspase12 protein expression decreased(P< 0.05).PERK m RNA expression was decreased(P<0.01)and CHOP m RNA expression was decreased(P<0.05).4.Compared with Li group,Jianpi Xiaoke decoction significantly decreased PERK and CHOP protein expression(P<0.05),and the effect of decreasing eIF2α and CHOP m RNA was better than liraglutide.Conclusion:1.Invigorating the spleen and relieving thirst can reduce FBG,body weight,Hb A1 c,improve glucose and lipid metabolism and islet function in rats.2.Jianpi Xiaoke can reduce inflammatory response by reducing the expression of inflammatory factors IL-6 and TNF-α,and has a certain anti-inflammatory effect.3.Jianpi Xiaoke Decoction may improve endoplasmic reticulum stress to alleviate pancreatic β cell apoptosis by inhibiting PERK,eIF2α and CHOP activities.PERKEif2α-CHOP signaling pathway may be one of the main mechanisms for Jianpi Xiaoke Decoction to play its role. |
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