Effect Of Intestinal Endotoxemia On Insulin Resistance In The Development Of Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is the clinic-pathology syndrome that hepatic tissue pathological change is similar to alcoholic fatty liver disease without chronic alcohol abuse. It includes pure fatty liver, fatty hepatitis, fatty fibrosis and hepatic cirrhosis. NAFLD has become a hot topic in medical research due to its higher morbility and lower age.However, further studies is necessary on how and why it occurs. The publicly-accepted'two hits'theory holds that insulin resistance (IR) involved in the'first hit'during the formation of pure fatty liver; and the'second hit'through oxidative stress and lipid peroxidation, which further worsen fatty hepatitis.Now sufficient evidences indicate intestinal endotoxemia (IETM) plays a key role during the occurrence and development of NAFLD. Thus, the following experiments were carried out so as to investigate the mechanism and relationship between IETM and IR during the course of NAFLD.The experiments were divided into three parts:PartⅠInducing NAFLD rat model complicated with IETM and IRRats were fed with special stuff containing 20% corn oil in Experiment 1, 2. The body weight, liver index, liver tissue Tch and TG, blood plasma ALT and ET increased obviously at the end of the 8th week. HA started to elevate at the end of 12th week. FIRI rised obviously at the end of the 14th week. The conclusion combined with observation of histomorphology show that pure fatty liver was induced from the end of the 8th week to the end of the 10th week, and developed into fatty hepatitis from the end of the 12th week to the end of the 14th week and formed fatty fibrosis at the end of the 16th week. NAFLD rat model combining with IETM and IR was induced at the end of the 14th week. But each indicator in the group intervened with GLY were lower than those in the 16 week group. It indicated that NAFLD was alleviated obviously along with relief of IETM and IR. IETM and IR are closely related with each other during the course of NAFLD.PartⅡEffect of LPS on IR of adipose cell and liver cellThe 3T3-L1 adipose precursor cell differentiated into ripe adipose cell induced by specificity inductor after 8 days, which had been proved by oil red stained in experiment 3. Sugar, insulin and IRI in medium and expression of IRS-1mRNA and PPARγmRNA were observed. It indicated that cell IR model may be induced successfully by high concentration of TNFα(20ng/ml)for 24 hours. IR of adipose cell was controlled obviously by Gly compared with euglycemic agent rosiglitazone. This further provided evidence to the result of experiment 2. In other words, there was close relationship between IETM and IR, and they may play significant role in the occurrence and development of NAFLD. Then preventive and therapeutic effect of Gly on NAFLD may be realized by IETM antagonisis to control IR of fatty tissue through rivaliry.Adipose cell sugar, insulin and IRI incresed after being treated by LPS. Glucose uptake in two conditions and expression of IRS-1mRNA and PPARγmRNA lowered in experiment 4. The result of small dosage and frequent LPS fed group was similar to the TNFαgroup. It indicate small dosage and frequent LPS fed may imitate the pattern of IETM in vivo and excite adipose cell to create the similar enviroment of high concentration TNFαwhich can induce adipose cell to form IR.The content of sugar, insulin, IRI, glycogen, glycogen particle dyed with PAS and glucose uptake of medium in every group in experiment 5,6 were observed. The result indicated that IR model was copied successfully with high concentration insulin(5×10-7mol/L)on HepG2 cell for 24 hours, it continued to 48 hours. IR of HepG2 cell intervend with Gly was controlled, which confirmed the results in PartⅠand PartⅡ, that is there was close relationship between IETM and IR and they played important role during development of NAFLD. IR of HepG2 cell was not obvious in small dosage and frequent LPS administration group and the group administrated with equal amount TNFαin experiment 3. IR was obvious when administrated with high concentration insulin simultaneously. It indicated that IETM and IR affectd each other in a vicious circle and promoted IR of liver cell during the development of NAFLD.PartⅢStudy relation of IETM and IR during the development of NAFLDRats were fed with a great quantity of unsaturated fat acid, saturated fat acid and high glucose and fat diet for 9 weeks, and were observed BW plot, liver index, TG in blood serum and hepatic tissue homogenate, plasma FFA, ALT, FBS,FINS and FIRI, histology observation, liver steatosis comparison, inflammation activity counts comparison, dyed with sudanⅣultramicrostructurely. The result indicated that rat liver has become ballooning degenerationat the end of the 4th week, then became pure fatty liver and fatty hepatitis complicated with IETM and IR at the end of the 9th week. Abundant abdomen fat, large fat area, surrounding macrophage infiltrating, strong positive expression of TNFαin adipose cell were proceeded with the development of NAFLD. It indicated that original resource of TNFαwas abdomen fat and inflammation cell during the development of NAFLD.The study indicated that: IR is a beginning factor, and IETM is a indispensible trigger factor, the two accompany during the course of NAFLD with TNFαas a connection.The main conclusions are as follows:1. NAFLD animal model complicated with IETM and IR can be induced by unsaturated fat acid diet, saturated fat acid or high glucose and fat diet.2. IR can be induced by LPS in 3T3-L1 lipocyte and HepG2 hepatocyte in vitro culture.3. The cytokine such as TNFαcan be released by macrophagus (including lipocyte) induced by LPS , which can cause IR and aggravate oxidative stress simultaneously. So, we consider that LPS is the trigger factor to NAFLD.4. Gly is proved to be the effective antagonists for endotoxin in this study which provided something new for the prevention and therapy of NAFLD.