Effects Of Metabolic Endotoxemia On Hepatocytes Mitochondrial Functions In Hepatic Insulin Resistance

Objective: 1 Through the establishment of rat metabolic endotoxemia and liver insulin resistance model,to investigate the effects of metabolic endotoxemia on liver mitochondrial structure and energy metabolism;2 Through in vitro,to investigate the direct effects of LPS on mitochondria and energy metabolism in normal rat hepatocytes.Methods: In vivo experiment: Thirty male Sprague-Dawley rats were randomly divided into three groups(10 rats in each group): normal control group(NC group),high fructose group(HFD group,10% fructose water feeding),LPS group(300 ?g·kg-1·d-1 of LPS subcutaneous injection).The body of weight were measured and recorded every week.After 8 weeks of fasting,the rats were fasted for at least 12 hours,at the end of the intraperitoneal injection of glucose tolerance test,the abdominal aorta was collected by ether anesthesia,4 ? 3500 r/min centrifugal 10 min,after dispensing plasma placed at-40 ? preservation.The liver tissue was partially separated and fixed in 4% paraformaldehyde solution for 12 ~24 h,and then stained with hematoxylin eosin staining(H&E staining).The remaining samples were frozen in liquid nitrogen and then transferred to-80 ?.1 Intraperitoneal injection of glucose tolerance test(IPGTT)Rats were tested for body weight every week.At the end of 8 weeks,50% glucose solution(2 g/kg)was injected intraperitoneally.Blood samples were collected from the tail vein,and the blood glucose levels were measured by fast blood glucose analyzer(Roche activity type)before(0 min)and after injection(15 min 30 min,120 min)respectively.2 Plasma liver enzymes,LPS detection and evaluation of hepatic insulin resistance The determination of blood glucose and plasma levels of AST and ALT by enzyme method,the detection of plasma LPS by limulus test,the changes of insulin were detected by ELISA,and calculated insulin resistance index(Homeostasis model assessment-insulin resistance,HOMA-IR),the formula is: HOMA-IR= fasting blood glucose(FPG,mmol/L)* fasting insulin(FINS,EU/ml)/22.5.3 Detection of oxidative damage and energy metabolism The levels of GSH-PX in plasma were detected by enzyme method,and the expression of oxidative damage products(8-Ohd G,MDA,4-HNE)and energy metabolism indexes(ADP,ATP)in plasma were detected by the method of ELISA.4 Liver histopathology The liver tissue was removed from 4% paraformaldehyde fixative solution.After washing and dressing,the gradient alcohol was dehydrated,the xylene was transparent,and the conventional paraffin-embedded sections were cut for 5 ?m.H & E staining was performed and the histopathological changes were observed under light microscope.5 Western blot analysis Western blot was used to detect the expression of key proteins(p-IRS1Tyr632?IRS1?p-PI3KTyr458?PI3K)and mitochondrial membrane receptor protein(UCP2)in liver tissue.In vitro experiment: Using modified two-step collagenase perfusion,isolated rat hepatocytes cultured in vitro were randomly divided into four groups: normal control group(NC group,cultured by DMEM medium),high fructose group(HFD group,medium + 4.5 g/L fructose water),LPS group(medium + 10 mg/L LPS),fructose and LPS in the intervention group(H + L group,medium + 4.5 g/L fructose water + 10 mg/L LPS).After 20 h,the cell supernatant was drawn and dispensed,Hepatocytes by trypsin(containing EDTA)digestion and 2000 r/min centrifugal 3 min,the supernatant was discarded,Then transferred to-40 ?.1 Detection of oxidative damage and energy metabolism The expression of oxidative damage products(8-Ohd G,MDA,4-HNE)and energy metabolism indexes(ADP,ATP)in plasma were detected by the method of ELISA.2 Western blot analysis Western blot was used to detect the expression of key proteins(p-IRS1Tyr632?IRS1?p-PI3KTyr458?PI3K)and mitochondrial membrane receptor protein(UCP2)in hepatocytes.Results: In vivo experimental results: 1 Weight change and impaired glucose tolerance During the experiment,the rats in each group were in good condition.Compared with NC group,the body weight of HFD group and LPS group was significantly increased at 2~8 weeks(P<0.01,P<0.05).The blood glucose levels in HFD group and LPS group were significantly higher than that of NC group(P<0.01,P<0.05),indicating that glucose tolerance was abnormal in HFD group and LPS group.2 Plasma liver enzyme,LPS test and liver insulin resistance assessment results The levels of liver enzymes,FINS,FPG,LPS and HOMA-IR in HFD group and LPS group were significantly higher than those in NC group(P<0.01).3 Pathological changes of liver tissue NC group of liver was dark red,no greasy feeling,H & E staining can be seen under light microscope liver was radial arrangement.HFD group and LPS group of liver were significantly greasy feeling;microscopic liver capsule can be seen within the lipid droplets vacuoles and accompanied by balloon-like changes.4 Western blot results Compared with NC group,the expression of IRS1,PI3 K,p-IRS1Tyr632 / IRS1 and p-PI3KTyr458 / PI3 K in liver tissue of HFD group and LPS group were significantly decreased(P<0.01),UCP2 expression was significantly increased(P<0.01).In vitro experimental results: 1 Changes of Oxidative Damage Products and Energy Metabolic Indexes The levels of 8-Ohd G,MDA and 4-HNE in HFD group,LPS group and H + L group were significantly higher than those in NC group(P<0.01,P<0.05).Compared with NC group,ADP,ATP in group HFD,group LPS and group H+L were significantly decreased(P<0.05).2 Western blot results Compared with NC group,the expression of IRS1,PI3 K,p-IRS1Tyr632 / IRS1 and p-PI3KTyr458 / PI3 K in hepatocytes of HFD group,LPS group and H + L group were significantly decreased(P<0.01,P<0.05),UCP2 expression was significantly increased(P<0.01).Conclusion: 1 High fructose diet and subcutaneous injection of LPS can induce insulin resistance and metabolic endotoxemia in rats,and associated with oxidative stress and hepatocytes mitochondrial dysfunction.2 LPS can induce the formation of intermediate oxidation products,and decrease the removal rate,so that the liver is in a state of oxidative stress,and then destroy the mitochondrial structure of hepatocytes,affecting its energy metabolism,accelerate the development of insulin resistance and metabolic diseases.