Effect Of Lipopolysaccharide On Pathogenesis Of High Fat And High Sugar-induced Non-alcoholic Steatohepatitis In Rats

Objective:To investigate the role that LPS plays in non-alcoholic steatohepatitis(non- alcoholic steatohepatitis, NASH) in the “two-hit” theory, the NASH model in rats were induced by a high-fat and high-sugar diet, then the plasma lipopolysaccharide(LPS), alanine aminotransferase(ALT), tumor necrosis factor-α(TNF-α), macrophage marker(CD68), the expression of glucose-regulated protein 78(GRP78), and hepatocyte apoptosis were detected.Methods:1. 30 male Sprague-Dawley rats were fed normally for 3 days. 20 of them were divided randomly into 4 groups: Synthetic diet + saline group(n=5), Synthetic diet + LPS group(n=5), Regular diet + LPS group(n=5), Regular diet + saline group(n=5). In addition, we set up a 5 week synthetic diet group(n=5) and regular diet group(n=5) to observe the short term impact on the rats’ liver as a results of high-fat and high-sugar diet.The synthetic diet consists of 68.75% standard diet, 20% lard, 10% sucrose, 1% cholesterol,0.25% bile acid. On the 6th week, the rats underwent subcutaneous injection LPS(0.5mg /Kg) or the same volume of normal saline every second day. All rats had free access to food and water. At the end of 9th week the animals were anesthetized via intraperitoneal injection with 1% sodium pentobarbital(3ml/Kg). Abdominal aortic blood samples were collected for analysis under a special pathogen and heat-free condition, plasma were obtained after centrifugation at the speed of 3500 rpm., Fixed the part of the left hepatic lobe with 10% formalin, adding to undergo the procedures of paraffin embedding and5μmsections, spare liver tissues were saved at- 70℃ freezer.2. The liver tissue sections of 5 week group were stained with HE in order to observe the pathological changes of the liver tissue under light microscope. We measured plasma TNF-α levels using radioimmunoassay; detected plasma ALT activity by Lai2,4-dinitrobenzene reitman hydrazine colorimetric method; tested plasma LPS levels with the limulus reagentassay. And immunohistochemical method was used for the expression of CD68(macrophage marker), the TUNEL assay for hepatocyte apoptosis level, Western blot assay for measuring the expression of GRP78 protein.Results:1. Under the light microscope, hepatic lobule structure is complete, liver cell nucleus are located in the center, cells radially centered around the central vein, neat and funicular arranged without lipid droplets in the liver cells of regular diet group. While in 5 week synthetic diet group, we observed steatosis in the hepatic cells with small vesicant liposome accounting for the majority of the liver cells, while infiltration by inflammatory cells was not obvious.2. LPS, ALT, TNF-α levels were significantly higher in rats fed a synthetic diet + LPS relative to the levels in the rats fed a synthetic diet + saline and regular diet + LPS(p<0.05).3. In the treatment groups with LPS added to the synthetic diet and fed for 9 weeks we found that the levels of CD68 positive mononuclear macrophage infiltration were significantly higher relative to the group of regular diet + LPS and Synthetic diet + saline(0.140±0.018, 0.053±0.018, 0.100±0.017).4. In the synthetic diet group added LPS treatment, the expression of GRP78 protein was significantly higher than regular diet + LPS group and synthetic diet + saline group(p<0.05); the result of synthetic diet + saline group was higher than regular diet + saline group(p<0.05); There is no significant difference between regular diet + LPS group and regular diet + saline group(p>0.05).5. In a separate comparison of the hepatocyte apoptosis in the synthetic diet + LPS group relative to those in the synthetic diet + saline group and those in the regular diet +LPS group were significantly higher(p<0.05). There is no significant difference of apoptosis rate between the regular diet + LPS group and regular diet + saline group(p>0.05).6. Applied Pearson’s correlation analysis, LPS and ALT were significantly positive correlation, r=0.78, p<0.05; CD68 and TNF-α expression significantly correlated, r=0.64,p<0.05, GRP78 expression and apoptosis of liver cells was significantly positive correlation, r = 0.77, p<0.05.Conclusion:1. Non-alcoholic steatohepatitis(NASH) rat model can be induced by high-fat and high-sugar diet;2. In the “two-hit” theory, can be the second hit playing an important role in the occurrence and development of NASH;3. LPS activated macrophages to induce the large release of TNF-α;4. The endoplasmic reticulum stress(ERS) exists in NASH rats, maybe TNF-α is a crucial factor to stimulate it. Even TNF-α involves in the pathological process of liver cell apoptosis.