| ObjectiveTo observe the effect of Gegenqinlian Decoction on obese T2DM model rats with hypoxia and insulin resistance of IR-L02 cells induced by hypoxia through in vivo and in vitro experiments.Starting from the insulin signaling pathway IRS-1/PI3K/AKT,the mechanism of improving hypoxia to insulin resistance by Gegenqinlian Decoction was discussed.Methods1.In vivo experiment:A total of 84 SPF male SD rats,including 12 in the normal group and 72 in the high fat group,were fed the normal group with normal diet and the high fat group with high fat diet.The body weight,body length and Lee’s index were measured weekly,and the levels of fasting blood glucose,insulin and hypoxia-inducing factor were measured every 2 weeks.According to the insulin level,insulin resistance index and the expression of hypoxia-inducible factor-2α,the model of insulin resistance with hypoxia in type 2 diabetes mellitus was established（film formation rate was 50%）.（1）Before administration:12 rats from normal group and 72 rats from high fat group were selected,and the body weight and body length of the two groups were observed at each time point;Serum fasting blood glucose（FBG）,insulin levels（FINS）,serum hypoxia-induced-factor-1a（HIF-1α）,hypoxia-induced-factor-2α（HIF-2α）and erythropoietin（EPO）were detected at different time points.The area under the curve of oral glucose tolerance was calculated by OGTT test.After membrane formation,Six rats from normal group and six rats from high fat group were sampled.Then,samples were collected from the two groups of rats.Abdominal aorta blood was collected,and The levels of TC,TG,LDL-C and HDL-C in blood lipid were detected by automatic biochemical instrument.Lipid deposition in liver tissue was observed by oil red staining.The pathological changes of liver and kidney were observed by HE staining.（2）After administration:30 SD rats successfully modeled were randomly divided into 5 groups:model group,Gegenqinlian decoction low-dose,medium-dose and high-dose groups and rosiglitazone group,with 6 rats in each group,and continued to be fed high-fat diet.The other 6 rats continued as the normal control group and were fed ordinary diet.Gegenqinlian Decoction low-dose,medium-dose and high-dose groups were given intragastric administration of 1.65g/kg,4.65g/kg and 14.85g/kg;rosiglitazone group was given intragastric administration of 5mg/kg;normal group and model group were given intragastric administration of normal saline at the same volume,once a day.The changes of body weight and fasting blood glucose（FBG）in each group were monitored after administration.The OGTT test was performed after the drug efficacy showed,and the area AUC under the glucose tolerance curve was calculated.Blood samples were collected from abdominal aorta to detect serum fasting blood glucose（FBG）and insulin levels（FINS）to evaluate insulin resistance index（HOMA-IR）.Using ELISA to detect serum hypoxia inducing factor-2α（HIF-2α）and erythropoietin（EPO）,oxidative stress index（SOD,MDA）and inflammatory factor（IL-1b,IL-10）and colorimetry to detect hematic fat four（TC,TG,LDL,HDL-C）level;The Scr,BUN and UA of renal function were detected by automatic biochemical analyzer.Lipid deposition in liver tissue was observed by oil red staining.The pathological changes of liver and kidney were observed by HE staining.Immunohistochemistry was used to detect the expression of liver inflammatory factor proteins,and it was clear that Gegenqinlian decoction could improve the hypoxia,glucose and lipid metabolism of T2DM insulin resistance rats with hypoxia,and improve oxidative stress,inflammatory indicators,pathological changes of liver tissue and lipid deposition.To further verify the mechanism of Gegenqinlian Decoction in improving insulin resistance accompanied by hypoxia.By real-time fluorescent quantitative PCR method to detect liver,tissue HIF-2α,PI3K,AKT,GSK-3β and IRS-1 mRNA expression quantity relatively.Using Westernblot method to detect liver,kidney tissue HIF-2α,the IRS-1,p-IRS-1（Ser307）,PI3K,p-PI3K,AKT,p-AKT（Ser473）,GSK-3β and p-GSK-3β protein expression level.2.In vitro experiment:Preparation of medicated serum from Gegenqinlian Decoction:Sixty male SD rats were randomly divided into normal group and Gegenqinlian decoction（GQD）group,with 30 rats in each group.The Gegenqinlian decoction group was given gavage of 25g·kg-1,and the normal group was given gavage of equal volume of normal saline,once a day for 7 consecutive days,to prepare blank serum and GQD-containing serum.Hypoxia-induced IR-L02 cell model:The glucose consumption and cell viability of the supernatant of L02 hepatocytes were measured at 6,12,18,24 and 48 h after anoxia,respectively.The reduced glucose consumption and unchanged cell viability were used as the basis for judging the success of the anoxia-induced IR-L02 cell model,and the optimal time point was selected to establish the anoxia-induced IR-L02 cell model.In order to further verify the IR-L02 cell model caused by hypoxia,different concentrations of insulin were added to stimulate each group under hypoxia condition,and the changes of cell sugar consumption and cell vitality were further verified.Effect and mechanism of GQD on hypoxia-induced IR-L02 cells:Cells were divided into normal oxygen group,hypoxia group,GQD low,medium,high（5,10,15%drug-containing serum）dose groups,rosiglitazone group.The cells in the normal-oxygen group were cultured in the normal-oxygen constant temperature incubator（37℃,5%CO2）,and the cells in the other groups were cultured in the anoxic incubator（37℃,5%CO2,94%N2,1%O2）.Cell vitality was detected by CCK-8 method,and glucose oxidase method was used to detect the glucose consumption of cells in each group.Test each cell oxidative stress indicators（SOD,MDA）and inflammatory factor（IL-1b,IL-10）level,Using Western blot method to detect each cell HIF-2α,IRS-1,p-IRS-1（Ser307）,PI3K,p-PI3K,AKT,p-AKT（Ser473）,GSK-3β and p-GSK-3β protein expression level;By real-time fluorescent quantitative PCR method to detect each cell HIF-2α,IRS-1,PI3K,AKT,and GSK-3β mRNA expression quantity relatively.The mechanism of hypoxia-induced IR-L02 cells was further clarified:In order to further verify that hypoxia can inhibit insulin signaling pathway and cause insulin resistance of L02 cells,a response experiment was conducted.Cells in hypoxia group were subjected to HIF-2αgene interference and divided into simple hypoxia group,hypoxia meaningless interference group（NC-si RNA）,hypoxia+HIF-2α-sirna,hypoxia+HIF-2α-sirna+15%GQD drug-containing serum group.By real-time fluorescent quantitative PCR method to detect the IRS-1,PI3K,and AKT,GSK-3β mRNA expression,the relationship between hypoxia and insulin signaling pathway,explore the mechanism of hypoxia-L02 cells to IR.Results:1.In vivo experiments（1）Before administration,at week 13,compared with normal control group,body weight,body length,Lee’s index,FBG,FINS,HOMA-IR and HIF-2αof rats in high fat group were rised significantly（P<0.01）.HIF-1αshowed an upward trend at different time points but had no statistical significance.The results of OGTT experiment showed that compared with the normal group,the blood glucose of the high fat group increased at different time points,reached the peak at 15-60 min,and fell back at 60-120 min but was still higher than the blood glucose level of 0 min.The area AUC under the curve increased significantly（P<0.01）indicating that the glucose tolerance of the high fat group was decreased.Compared with normal group,TC and TG of rats in high fat group were increased,but LDL-C and HDL-C levels were not significantly changed.Oil red staining showed lipid deposition in liver and mild pathological changes in liver and kidney tissues.（2）After administration,compared with normal group,body weight,FBG,FINS,HOMA-IR and HIF-2αin model group were significantly increased（P<0.05,P<0.01）,serum levels of TC,TG and LDL-C were increased and HDL-C levels were decreased（P<0.05）.Serum renal function indexes Scr,BUN and UA were not significantly different.Oil red staining showed lipid deposition in liver and pathological changes in liver and kidney.Compared with model group,the body weight,FBG,FINS,HOMA-IR and HIF-2αof rats in administration group were significantly decreased（P<0.05,P<0.01）,the levels of TC,TG and LDL-C in serum were decreased and the levels of HDL-C were increased（P<0.05,P<0.01）.These results indicated that GQD could reduce blood glucose and insulin levels in model rats,improve IR resistance index,improve hypoxia in model rats,improve histopathologic morphology of liver and kidney,and reduce lipid deposition in liver.（3）Compared with normal group,IL-1b and MDA were increased in liver and kidney tissues of rats in model group,while SOD and IL-10 were decreased（P<0.01）.Immunohistochemical results showed that TNF-a protein expression was increased and IL-10 protein expression was decreased in liver tissues,indicating that oxidative stress and inflammation occurred in rats in model group;Compared with model group,IL-1b and MDA were decreased,SOD and IL-10 were increased in liver and kidney tissue of rats in GQD administration group（P<0.05,P<0.01）,and the expression of TNF-a protein was decreased and IL-10 protein was increased in liver tissue by immunohistochemical results,indicating that GQD can improve the oxidative stress and inflammatory response in early stage of T2DM obesity,and the effect of high dose is the best.（4）Real-time fluorescent quantitative PCR results showed:Compared with normal group,model group liver tissue HIF-2α,GSK-3β mRNA expression of relative quantity increases,IRS-1,PI3K,AKTmRNA relative expression decreased（P<0.05,P<0.01）;Compared with model group,medication group of liver tissue HIF-2α,GSK-3βmRNA expression decreased relatively,IRS-1,PI3K,and AKT mRNArelative expression increased（P<0.05,P<0.01）.（5）Westernblot results showed:Compared with normal group,HIF-2α,p-GSK-3β/GSK-3β protein expression levels in liver and kidney tissues of model group were increased.The protein expression levels of p-IRS-1/IRS-1、p-PI3K/PI3K、p-AKT/AKT were decreased（P<0.05,P<0.01）;Compared with model group,HIF-2α,p-GSK-3β/GSK-3β protein expression levels in liver and kidney tissues of GQD administration group decreased,while p-IRS-1/IRS-1、p-PI3K/PI3K、p-AKT/AKT protein expression levels increased（P<0.05,P<0.01）.2.In vitro experimentL02 cells were hypoxic（1%oxygen concentration）for 18h,the consumption of glucose was significantly increased（P<0.01）,and the cell viability was unchanged,which proved that anoxia caused the establishment of IR-L02 cell model.After low,medium and high doses of GQD and rosiglitazone intervention,the experimental results showed that:（1）Compared with normal oxygen group,cell sugar consumption in hypoxia group was decreased（P<0.01）.Compared with the hypoxia group,the sugar consumption in the administration group was increased（P<0.05,P<0.01）.（2）Compared with normal oxygen group,IL-1b and MDA were increased,while SOD and IL-10 were decreased in hypoxia group;Compared with model group,IL-1b and MDA were decreased,while SOD and IL-10 were increased in medication group.（3）q RT-PCR results showed:Compared with normal oxygen group,the mRNA expression levels of HIF-2αand GSK-3β in hypoxia group were increased,while the mRNA expression levels of IRS-1,PI3K and AKT were decreased（P<0.05）;Compared with the model group,the mRNA expression levels of HIF-2αand GSK-3β in the medication group were decreased,while the mRNA expression levels of IRS-1,PI3K and AKT were increased（P<0.05,P<0.01）.（4）q RT-PCR results showed that there was no difference in HIF-2αmRNA expression level in the hypoxia meaningless interference group compared with the simple hypoxia group（P>0.05）,HIF-2αand GSK-3β mRNA expression levels were decreased in hypoxia+HIF-2α-si RNA group,and the mRNA expression levels of IRS-1,PI3K and AKT were increased（P<0.05）.Compared with hypoxia+HIF-2α-si RNA group,mRNA expression levels of HIF-2α-si RNA and GSK-3β in hypoxia+HIF-2α-si RNA+15%GQD group were further decreased,while mRNA expression levels of IRS-1,PI3K and AKT were further increased（P<0.05）,These results indicated that the insulin pathway IRS-1/PI3K/AKT/GSK-3β was also changed when the cells in hypoxia group interfered with HIF-2α,suggesting that the insulin pathway IRS-1/PI3K/AKT/GSK-3β might be related to the IR of L02 cells caused by hypoxia.After GQD administration,insulin signaling pathway can be activated to improve IR in L02 cells.（5）Westernblot results showed:Compared with the normal oxygen group,the protein expression levels of HIF-2αand p-GSK-3β/GSK-3β in hypoxia group were increased,while the protein expression levels of I p-IRS-1/IRS-1、p-PI3K/PI3K、p-AKT/AKT in hypoxia group were decreas（P<0.05,P<0.01）;Compared with model group,the protein expression levels of HIF-2αand p-GSK-3β/GSK-3β in GQD administration group were decreased,while the protein expression levels of p-IRS-1/IRS-1、p-PI3K/PI3K、p-AKT/AKT were increased（P<0.05,P<0.01）.Conclusion:1.Gegenqinlian decoction can improve glucose and lipid metabolism,improve hypoxia,improve insulin sensitivity and improve IR in obese rats with hypoxia in early T2DM stage.2.Gegenqinlian decoction can improve the indexes of oxidative stress and inflammatory factors in obese T2DM rats with hypoxia,reduce oxidative stress damage and inflammatory response caused by hypoxia,and improve IR in hypoxia rats.3.Gegenqinlian decoction can reduce HIF-2αexpression of IR in obese pre-T2DM rats with hypoxia,and activate IRS-1/PI3K/AKT signaling pathway to improve IR in hypoxia rats.4.Gegenqinlian Decoction can improve the indexes of IR-L02 oxidative stress and inflammatory factors caused by hypoxia,reduce the oxidative stress damage and inflammatory response caused by hypoxia,and improve the IR of L02 cells in the hypoxia group.5.Hypoxia can cause insulin resistance L02 cells and its mechanism may be related to IRS-1/PI3K/AKT/GSK-3β.6.Gegenqinlian decoction can improve HIF-2αexpression in IR-L02 cells induced by hypoxia,activate IRS-1/PI3K/AKT/GSK-3β signaling pathway,and improve IR in L02 cells induced by hypoxia. |
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