| BackgroundDiabetes is one of the most common clinical endocrine metabolic disease of which more than 90%is type 2 diabetes mellitus(T2DM).According to the latest survey data released by International Diabetes Federation in 2019,about 463 million adults aged 20 to 79 was suffering from diabetes in the world,with the prevalence rate of 9.3%.The data is expected to reach 578 million(10.2%)by 2030.Insulin resistance(IR)is the initiating factor of T2DM and runs through the whole process of the development of T2DM.Active improvement of IR has become a key strategy in the treatment of T2DM.Biguanides and thiazolidinediones are commonly used to improve IR in clinical therapy and often cause side effects.Besides,treatment failure might occur after long-term use.Traditional Chinese Medicine(TCM)has distinct advantages in treating T2DM.According to the theory of TCM,Yin deficiency and heat accumulation are the basic pathogenesis of Xiaoke disease.Professor Lin Lan has established nourishing Yin and clearing heat as the basic treatment method for T2DM-IR with Yin-deficient and heat-rich and taken Qingrun Formula(rhizoma anemarrhenae,cortex phellodendri,cortex lycii radicis etc,QRF)as the core prescription for the treatment which achieved good efficacy.The previous study of our research group found that QRF could reduce the inflammatory reaction and oxidative stress of T2DM rats to improve IR,but its in-depth molecular mechanism still needs to be further explored.A large number of studies have confirmed that T2DM is a low-grade inflammatory state of the body to some extent.The release of inflammatory factors could interfere with the insulin signaling pathway,leading to the occurrence of IR.SIRT1/NF-?B signaling pathway plays a key role in mediating liver inflammatory reaction.Micro Ribonucleic Acid(microRNA)is an important family of gene regulators in non-coding RNA which could affect the expression of target genes at the transcriptional or post-transcriptional levels by completely or incompletely binding to the 3' untranslated region of target genes.Recent studies have shown that a variety of microRNAs express abnormally in T2DM-IR among which miR-34a is closely related to the imbalance of glucose and lipid metabolism in the liver.And miR-34a could directly target and negatively regulate the expression of SIRT1 as its upstream.Therefore,this study focused on miR-34 regulating SIRT1/NF-?B signaling pathway in the incidence of heptic IR of T2DM and intervention mechanism of QRF funded by the national natural science foundation project of "Investigating molecular mechanism of nourishing yin and clearing heat method treating diabetic insulin resistance based on miRNA regulating signal pathway of SIRT1/NF-?B"(No.81573792)on the basis of previous work.So as to explore further effective therapy targets and provide strong theoretical and experimental basis for the prevention and treatment of T2DM by TCM.Objective(1)To observe the effect of Qingrun Formula on improving IR in T2DM rats and IR-HepG2 cells through in vitro and in vivo experiments;(2)To clarify the role of miR-34a regulating SIRT1/NF-?B signaling pathway in the pathogenesis of T2DM-IR and to explore the molecular mechanism of Qingrun Formula to improve T2DM-IR from this perspective.Methods(1)In vivo experimentsSD rats were used as the research objects in this study.T2DM rats model were established by feeding high fat diet in combination with small dose STZ intraperitoneal injection.The rats were divided into control group,model group,QRF high-dose group,QRF middle-dose group,QRF low-dose group and metformin group,Each group contained ten rats and was given corresponding drug intervention for eight weeks.The general status of the rats was observed in the process.The the body weight of the rats was recorded and the fasting blood glucose(FBG)was detected before drug intervention and at the end of second,fourth,sixth,eighth week after drug intervention.The oral glucose tolerance test(OGTT)was conducted at the end of seventh week after drug intervention,and the gulcose area under curve(GAUC)was calculated.Blood was collected from the abdominal aorta and liver tissues were clipped after drug intervention.The level of fasting serum insulin(FINS)was detected by radioimmunoassay and the homeostasis model assessment-insulin resistance(HOMA-IR)was calculate.Blood lipids(TC,TG,HDL-C,LDL-C)level was detected by oxidase method and direct method.The content of hepatic glycogen was detected by anthrone method.Pathologic changes of liver tissues were observed by HE staining.The ultrastructure of liver was observed by transmission electron microscope.The level of TNF-?,IL-6 was detected by ELISA.The expression level of SIRT1,NF-?B,IRS1,GLUT4 was detected by Western blot.The expression level of miR-34a,SIRT1mRNA,NF-?BmRNA,IRS1mRNA,GLUT4mRNA was detected by RT-PCR.(2)In vitro experimentsHepG2 cells were used as the research objects in this study.Proliferation activity of HepG2 cells was detected by CCK8 method after intervention with different concentrations of QRF for 24h and the intervention concentration of QRF was screened.IR-HepG2 cell model was established with 1×10-6mol/L insulin induction for 36h.It was divided into control group,model group,QRF group and metformin group.Each group was given corresponding drug intervention.Glucose consumption was detected by glucose oxidase assay.Glycogen content was detected by anthrone assay.The level of TNF-?,IL-6 was detected by ELISA.The expression level of SIRT1,NF-?B,IRS1,GLUT4 was detected by Western blot.The expression level of miR-34a,SIRT1mRNA,NF-?BumRNA,IRS1mRNA,GLUT4mRNA was detected by RT-PCR.MiR-34a inhibitor was transfected into by HepG2 cells by liposome transfection.The expression levels of corresponding proteins and RNA were detected by Western blot and RT-PCR.Results1.In vivo experiments(1)Compared with the control group,the levels of FBG,TC,TG LDL-C,FINS,HOMA-IR,GAUC in the model group were significantly increased(p<0.05)and liver glycogen contents was significantly decreased(p<0.05).Compared with the model group,the weight of rats in QRF middle-dose group increased significantly at the eighth week(p<0.05).The level of FBG in QRF high-dose group decreased significantly at the eighth week(p<0.05).The levels of FINS and HOMA-IR in QRF low-dose group and QRF high-dose group decreased significantly(p<0.05).The level of GAUC in each dose group of QRF decreased.But there was no significant difference(p>0.05).The levels of TC,TG and LDL-C in each dose group of QRF were significantly reduced(p<0.05).The level of liver glycogen in QRF high-dose group of was significantly increased(p<0.01).Observation by HE staining showed that the liver tissues in model group appeared obviously fatty degeneration and vacuoles degeneration.Observation of the ultrastructure by transmission electron microscope showed that the liver cells in model group appeared injury of organelles such as mitochondrial endoplasmic reticulum and scattered lipid droplets.The pathological changes of each dose group of QRF appeared different degrees of improvement compared with model group.(2)Results of ELISA showed that the levels of TNF-? and IL-6 were significantly increased in the model group compared with the control group(p<0.01).Compared with the model group,the level of TNF-? in QRF high-dose group was significantly decreased(p<0.05)and the level of IL-6 in QRF middle-dose group and QRF high-dose group significantly decreased(p<0.05).Results of Western blot showed that,compared with the control group,the expression levels of SIRT1 and GLUT4 in the model group were significantly down-regulated(p<0.05),the expression level of IRS1 was down-regulated,but the differences were not significant(p>0.05),and the expression level of NF-?B was significantly up-regulated(p<0.01).Compared with the model group,the expression level of NF-?B in each dose of QRF was significantly down-regulated(p<0.05),the GLUT4 protein expression level in QRF high-dose group was significantly up-regulated(p<0.05),and the expression levels of SIRT1 and IRS 1 in each dose of QRF were up-regulated,but the differences were not significant(p>0.05).Results of RT-PCR showed that,compared with the control group,the expression levels of miR-34a and NF-?BmRNA in the model group were significantly up-regulated(p<0.01),and the expression levels of SIRT1mRNA,IRS1mRNA and GLUT4mRNA were significantly down-regulated(p<0.05).Compared with the model group,the expression levels of SIRT1mRNA and IRS1mRNA in QRF high-dose group were significantly up-regulated(p<0.05),the expression levels of NF-?BmRNA were significantly down-regulated(p<0.05),the expression levels of miR-34a were down-regulated in QRF low-dose group and QRF high-dose group,and the expression levels of GLUT4mRNA were up-regulated,but the differences were not significant(p>0.05).2.In vitro experiments(1)QRF has a certain inhibitory effect on the proliferation of HepG2 cells after the intervention.The cell proliferation activity is still(94.15±6.23)%when the intervention concentration of QRF is 5mg/mL and chose it as the working concentration.The glucose consumption of HepG2 cells was significantly reduced(p<0.01)after the induced by 1×10-6mol/L insulin for 36h and the glycogen content was significantly reduced(p<0.01),suggesting that the IR-HepG2 cell model was induced successfully.Intervention of QRF could improve the glucose consumption of IR-HepG2 cells,but the difference was not significant compared with the model group(p>0.05),and the glycogen content of IR-HepG2 cells could be significantly increased(p<0.05).(2)Results of ELISA showed that the levels of TNF-? and IL-6 were significantly increased in the model group compared with the control group(p<0.01).Compared with the model group,the levels of TNF-? and IL-6 in QRF group were significantly reduced(p<0.01).Results of Western blot showed that compared with the normal group,the expression levels of SIRT1,IRS1 and GLUT4 in the model group were significantly down-regulated(p<0.01),and the expression level of NF-?B was significantly up-regulated(p<0.01).Compared with the model group,the expression levels of SIRT1 and GLUT4 in QRF group were significantly up-regulated(p<0.05),the expression level of IRS 1 was up-regulated,and the expression level of NF-?B was down-regulated,but the differences were not significant(p>0.05).Results of RT-PCR showed that compared with the normal group,the expression levels of miR-34a and NF-?BmRNA in the model group were significantly up-regulated(p<0.01),and the expression levels of SIRT1mRNA,IRS1mRNA and GLUT4mRNA were significantly down-regulated(p<0.01).Compared with the model group,the expression level of NF-?BmRNA in QRF group was significantly down-regulated(p<0.05),the expression level of miR-34a was down-regulated,but the difference was not significant(p>0.05),the expression level of SIRT1 mRNA was significantly up-regulated(p<0.01),and the expression levels of IRS1mRNA and GLUT4mRNA were up-regulated,but the difference was not significant(p>0.05).After miR-34a inhibitor transfeced into HepG2 cells,results of Western blot showed that the expression levels of SIRT1,IRS1 and GLUT4 in inhibitor group were up-regulated and the expression level of NF-?B was down-regulated,but the difference was not significant(p>0.05).Results of RT-PCR showed that the expression level of miR-34a in inhibitor group was significantly down-regulated(p<0.01),the expression level of SIRT1mRNA was significantly up-regulated(p<0.05),the expression levels of IRS1mRNA,GLUT4mRNA and NF-?B mRNA were up-regulated,but the differences were not significant(p>0.05).Conclusion(1)The Qingrun Formula could reduce glucose and lipid metabolism disorders in T2DM rats,improve IR status,and alleviate liver pathological injury while it could also increase the ability of glucose uptake and glycogen synthesis for IR-HepG2 cells and improve IR status.(2)The mechanism for Qingrun Formula to improve hepatic insulin resistance in T2DM might be realized by inhibiting the expression of miR-34a,activating the SIRT1/NF-?B signaling pathway,reducing the damage of liver inflammatory reaction to insulin signal transduction pathway,and improving insulin sensitivity. |
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