Study On The Mechanism Of Yitangkang Improving IR In T2DM Rats Based On FXR Regulating PI3K/AKT/GSK-3β Signaling Pathwa

Purpose:In this study,the IR model of T2DM rats was established to observe the effects of Chinese herbal compound Yitangkang on body weight,blood glucose,insulin sensitivity,and the expression of key proteins in the bile acid nuclear receptor FXR and liver PI3K/AKT/GSK-3βsignaling pathway.To explore the mechanism of Chinese medicine compound Yitangkang in the treatment of T2DM and improve IR.Material and method:From 96 Wistar male rats,16 were randomly selected as normal control group and fed with normal diet,the rest 80 Wistar rats were fed with high-sugar and high-fat diet,and were injected intraperitoneally with streptozotocin（STZ）after 8 consecutive weeks to construct T2DM rats.IR model.Tail tip blood was collected to detect the blood sugar of the model rats,and the hyperinsulinemic-euglycemic clamp experiment was conducted to screen out the successful model rats and divided them into the model control group,Yitangkang high,middle,and middle groups according to the random number table method.There were 5 groups in the low-dose group and the chenodeoxycholic acid（CDCA）group,with different interventions.Yitangkang high,medium and low dose groups were respectively given different doses of Yitangkang traditional Chinese medicine decoction(10ml/kg·d^-1)by gavage,while CDCA group was given CDCA preparation solution(2ml/kg·d^-1)by gavage plus Normal saline(8ml/kg·d^-1)was administered by gavage,while normal control group and model control group were given normal saline(10ml/kg·d^-1)by gavage,once a day for 8 consecutive weeks.During the whole process of the experiment,the general conditions of the 6 groups of rats were observed and recorded,and the body weight and fasting blood glucose（FPG）values of the rats were detected before the intervention and at the 2nd,4th,6th,and 8th weeks after the intervention.At the end of the 8th week,hyperinsulinemia-euglycemic clamp test was used to detect insulin sensitivity;HE staining and PAS staining were performed on liver tissue to observe the pathological changes and hepatic glycogen synthesis;immunofluorescence method was used to detect FXR,GSK-The expression of 3β;Western-Blot and RT-q PCR were used to detect the expression of IRS1,PI3K,AKT,GSK-3β,GS and other related proteins and genes.Results:1.Model evaluation results:4 rats died within 72 hours after injection of STZ in 80 Wistar rats,and the remaining 76 rats were tested.In the end,a total of 73 rats met the requirements,and the modeling success rate was 76.04%.2.The general condition of the rats:The natural control group rats is good,and the skin is bright,and the amount of drinking water and normal is normal.After successful modeling,the rats in the model control group were in a sluggish mental state,their skin was yellow,rough and less shiny,their diet and water intake increased significantly,and the amount of urination increased.Compared with the model control group,Yitangkang high,medium and low dose groups and CDCA group,after drug intervention,the mental state of the rats improved,the fur color and gloss were improved,the amount of food and water and the amount of urine were different to different degrees reduce.3.Body weight of rats:Before the intervention,compared with the normal control group,the body weights of the other 5 groups were significantly increased（P<0.01）.After the 6th week of intervention,compared with the model control group,the body weight of the rats in the Yitangkang high-dose group and CDCA group increased significantly（P<0.05）.After the 8th week of intervention,compared with the model control group,the body weight of the rats in the Yitangkang high-dose,middle-dose and CDCA groups increased significantly（P<0.05 or P<0.01）;The body weight of rats in high-dose Yitangkang group and CDCA group increased significantly（P<0.01）.4.FBG situation of rats:Before the intervention,compared with the normal control group,the FBG of the other five groups was significantly increased（P<0.01）.After the 2nd,4th,6th and8th weeks of intervention,compared with the model control group,the FBG of Yitangkang high,medium and low dose groups and CDCA group were significantly decreased（P<0.05 or P<0.01）.After the 4th week of intervention,compared with the low-dose Yitangkang group,the high-dose Yitangkang group and the CDCA group decreased significantly（P<0.01）.After the 6th and 8th week of intervention,compared with the low-dose Yitangkang group,the Yitangkang high-dose,middle-dose and CDCA groups were significantly decreased（P<0.05or P<0.01）.5.Insulin sensitivity of rats:After 8 weeks of intervention,compared with the normal control group,the steady-state blood glucose level（BG）of the remaining 5 groups of rats was significantly increased（P<0.01）,and the glucose infusion rate（GIR）was significantly decreased（P<0.01）.Compared with the model control group,the BG value of Yitangkang high-dose,middle-dose and CDCA groups decreased significantly（P<0.01）,while the GIR value increased significantly（P<0.05 or P<0.01）.Compared with the low-dose Yitangkang group,the BG value of the high-dose Yitangkang group and the CDCA group decreased significantly（P<0.01）,while the GIR value increased significantly（P<0.05）.6.The results of HE staining showed that compared with the model control group,the volume of hepatocytes in the middle and low-dose Yitangkang groups was slightly reduced and slightly swollen,and the cytoplasm still contained a small amount of fat vacuoles,and the steatosis was alleviated;the high-dose Yitangkang group and CDCA The volume of hepatocytes in the rats in the group was significantly reduced,the swelling was reduced,the arrangement was more regular,the fat vacuoles were significantly reduced,and the steatosis was significantly reduced.7.The results of PAS staining showed that,compared with the model group,the color of PAS staining in the CDCA group,Yitangkang high-dose,medium-dose and low-dose groups became lighter to varying degrees,and the CDCA group and the Yitangkang high-dose group had more significant changes.8.The results of immunofluorescence showed that compared with the normal control group,the expression of FXR in the model group decreased,while the expression of GSK-3βincreased;compared with the model control group,the expression of FXR in the Yitangkang high,medium and low dose groups and the CDCA group increased,GSK-3βexpression decreased,and the most obvious changes were in the CDCA group and the Yitangkang high-dose group.9.Western-Blot results showed that compared with the normal control group,the expression levels of FXR,IRS1,PI3K,AKT and GS proteins in the liver tissue of the model control group were significantly decreased（P<0.05 or P<0.01）,and the protein expression level of GSK-3βwas significantly increased（P<0.05 or P<0.01）;compared with the model control group,the levels of FXR,IRS1,PI3K,AKT,and GS in Yitangkang high-and medium-dose groups and CDCA groups were significantly increased（P<0.05 or P<0.01）,and the levels of GSK-3βwere significantly increased（P<0.05 or P<0.01）.Compared with the low-dose Yitangkang group,the levels of FXR,IRS1,PI3K,AKT and GS in the Yitangkang high-dose group and CDCA group were significantly increased（P<0.05 or P<0.01）,GSK-3βlevels were significantly decreased（P<0.01）.10.The results of RT-q PCR showed that compared with the normal control group,the m RNA levels of FXR,IRS1,PI3K,AKT and GS in the liver tissue of the model control group were significantly decreased（P<0.01）,and the GSK-3βm RNA level was significantly increased（P<0.01）.Compared with Yitangkang high and medium dose groups and CDCA groups,the m RNA levels of FXR,IRS1,PI3K,AKT and GS were significantly increased（P<0.01）,and the m RNA levels of GSK-3βprotein were significantly decreased（P<0.01）;Compared with the low-dose Yitangkang group,the levels of FXR,IRS1,PI3K,AKT,and GS m RNA in the high-dose Yitangkang group and CDCA group were significantly increased（P<0.05 or P<0.01）,and the levels of GSK-3βm RNA were decreased to varying degrees.（P<0.01）.Conclusion:1.The traditional Chinese medicine compound Yitangkang can improve the body weight,mental state,fur color and gloss of T2DM rats,and reduce the amount of water and urine in their diet.2.The traditional Chinese medicine compound Yitangkang can reduce blood sugar and reduce insulin resistance in T2DM rats.3.The traditional Chinese medicine compound Yitangkang can alleviate the pathological changes of liver in T2DM rats,improve steatosis,and promote glycogen synthesis.4.The traditional Chinese medicine compound Yitangkang can activate the bile acid nuclear receptor FXR.5.The traditional Chinese medicine compound Yitangkang can promote hepatic glycogen synthesis and improve insulin resistance in T2DM rats by up-regulating the expression of FXR and activating the PI3K/AKT/GSK-3βpathway.