Mechanism By Which Of Multiple Small Dosage LPS Treatment Relieves Chronic Hepatic Injury

Endotoxin is named as lipopolysaccharide (LPS), which is the main component in the outer membrane of Gram-negative bacteria, and is the most important pathogenic factor of this kind of bacteria. LPS can activate mononuclear phagocytic system (MPS), leading to the release of multiple pro-inflammatory mediators including tumor necrosis factor-a (TNF-α), leukotriene, et al. AS a result, hepatic injury and even liver function failure occurrence. However, LPS stimulation can induce a protective mechanism, the endotoxin tolerance. Endotoxin tolerance is defined as a reduced capacity of the host or of cultured monocyte/macrophage to respond to LPS activation following a first exposure to the stimulus of smaller LPS. Now, endotoxin tolerance is known as a negative feedback regulation and an adaptive response, which prevents host from continuous over-responding to LPS stimulation. So endotoxin tolerance is also an important part of host defence. That is important for preventing and curing hepatic injury if endogenous anti-damage mechanism can removed to bring into full play anti-infection, and can not initiate excessive inflammatory reaction. Many animal experiments in our laboratory and clinic researches show that there is intestinal endotoxemia (IETM) in the animals and patients with various acute and chronic hepatitis, cirrhosis and fulminant hepatitis. The investigations show that low dose LPS treatment can relief acute hepatic injury. However, the relationship and mechanism between chronic hepatitis and low dose LPS treatment many times remains to be further explored.The scientific research was carried out in three parts.The first part:effect of low dose LPS treatment on CCl4-induced chronic hepatic injury and the change of LPS signal transduction.The second part:mechanism and effect of low dose LPS treatment on nonalcoholic fatty liver.Part One Effect of low dose LPS treatment on CCl4-induced chronic hepatic injury and the change of LPS signal transductionThere are five experiments in this part.Experiment 1 Effect of low dose LPS treatment on CCl4-induced chronic hepatic injuryThe male Wistar rats were divided randomly into liver-injury group which injected 0.5ml CCl4/100g first, three days later injected 0.3ml 40% CCl4 and 60% olive oil, LPS treatment group which injected LPS 0.05mg/100g before the day injected CCl4 and received high fat diet as liver injury group, and normal control group which received normal diet. The blood from various animals was sterility collected for determination of plasma endo toxin and ALT. The part of liver tissue was divided for determination of TNF-α. The liver tissue sections were prepared for surveying pathologic changes in the liver. The results have shown that the trend of endotoxin and ALT changes in plasma and TNF-a in the liver tissue was similar. They rose obviously in rats with chronic hepatic injury than in normal control. But ALT in plasma and TNF-αin liver homogenate after treated by small dosage LPS were lower obviously than in the liver-injury group. Histology with hematoxylin and eosin staining has shown that hepatocyte presented severe steatosis with cellular nucleuses were crushed to the margin of cell which were similar to adipocyte, and there were spotty necrosis and focal necrosis and lymphocytes which infiltrated in the zone of necrosis. In contrast, steatosis of hepatocyte was relieved obviously with smaller lipid droplet, and there were fewer lymphocytes which infiltrated in the liver tissue of LPS treatment group. These results suggest that there is IETM in the animals with hepatic injury induced by CCl4, and small dosage treatment can relive the hepatic injury.Experiment 2 Role of TLR4 in the development of low dose LPS treatment relieves CCl4-induced chronic hepatic injuryThe expression of TLR4 was determined with Western blotting. The experimental animals were the same as experiment one. The results have shown that the animals with chronic hepatic injury have a higher expression of TLR4 than the normal control. The expression of TLR4 decreased significantly in the liver of LPS treatment group.Experiment 3 The expression of p38MAPK in the development of low dose LPS treatment relieves CCl4-induced chronic hepatic injuryThe aim of this experiment is to investigate the change of expression of p38MAPK in liver tissues. The results have shown that the expression of p38MAPK were the same, and the expression of pp38MAPK were higher significantly in injury group than LPS treatment group.Experiment 4 The expression of NF-κB and IκB in the development of low dose LPS treatment relieves CCl4-induced chronic hepatic injuryThe experimental animals were the same as experiment one. The goal of this study was to survey the change of expression of NF-κB and IκB in livers. The injury group had a higher NF-κB expression and a lower IκB expression, and the results were opposite in LPS treatment group.Experiment 4 The expression of IRAK-M in the development of low dose LPS treatment relieves CCl4-induced chronic hepatic injuryIn this experiment we investigate the expression of IRAK-M in every group liver. The results indicate that small dosage LPS treatment can enhance significantly the expression of IRAK-M in LPS group than it in injury group.The data of the first part could be summarized as follows:①An cooperative action of intestinal endotoxin and CCl4 may be a major pathogenesis of chronic hepatic injury because of IETM formed for the duration of hepatic injury.②Multiple small dosage LPS can relieve CCl4_induced chronic hepatic injury.③Decreased expression and down-regulated function of TLR4 in the liver may be an important part of mechanism by which small LPS treatment relieves CCl4_induced chronic hepatic injury.④Because LPS treatment interfere MAPK signalling transduction pathway by up-regulation IRAK-M level, production of TNF-αdecreased and result in CCl4_induced chronic hepatic injury relived.Part Two The study about the effect and the mechanism of low dose LPS treatment on nonalcoholic fatty liverThere are two experiments in this part.Experiment 6 The effect of low dose LPS treatment on high-sucrose and high-fat induced nonalcoholic fatty liverThe male Wistar rats were divided randomly into liver-injury group which received high-sucrose and high-fat diet, LPS treatment group which injected LPS 0.05mg/100g every other day and received the same diet as liver injury group, and normal control group which received normal diet. The blood from various animals was sterility collected for determination of plasma ET,ALT,TNF-α,IL-10,APN,FPG,FINS at the fourth week and the ninth week. The left hepatic lobe was mounted in 10% formalin solution and paraffin imbedding, which was prepared to investigate hepatic pathologic change by HE-stained and apoptosis of liver cells by TUNEL-stained. The results have shown that ET and ALT were higher slightly than normal control in the fourth group, and in the ninth group they were higher significantly than normal control, it indicate that there was intestinal endotoximia in injury group. Histology with hematoxylin and eosin staining has shown that hepatocyte presented severe steatosis with cellular nucleuses were crushed to the margin of cell which were similar to adipocyte, and there were spotty necrosis and focal necrosis and lymphocytes which infiltrated in the zone of necrosis. In contrast, steatosis of hepatocyte was relieved obviously with smaller lipid droplet, and there were fewer lymphocytes which infiltrated in the liver tissue of LPS treatment group. TH1/TH2 decreased because the proinflammatory factor TNF-a level increased and antiinflmmatory factor IL-10 decreased. The hepatocyte, as the target cell of LPS, its apoptosis rate in LPS treatment was lower than injury group.Experiment 7 The effect of low dose LPS treatment on IR and apoptosis of beta cell of isletEmploying TUNEL-stained, we attempt to demonstrate whether or not apoptosis of beta cell of islet in the animal with NASH. The results have shown that apoptosis of beta cell of islet and FIRI decreased than injury group. But there was not significance of difference of FIRI, which may be related to test duration.The data of the second part could be summarized as follows:①Intestinal endotoxin may be a major pathogenesis of chronic hepatic injury because of IETM formed for the duration of hepatic injury.②Multiple small dosage LPS can alleviat high-sucrose and high-fat induced NASH.③Decreased TH1/Th2 may be an important part of mechanism by which small LPS treatment relieves chronic hepatic injury.④Decreased apoptosis of hepatocyte was related to hepatic injury alleviate.⑤Multiple small dosage LPS treatment could result in apoptosis of beta cell of islet and FIRI decrease.The series of experiments have indicated that1. This study indicate that multiple small dosage LPS can alleviate chronic hepatic injury resulted from CCl4 or high-sucrose and high-fat. 2. The change of acceptor and signaling transduction pathway which result in pre-inflammatory cytokines, especially TNF-α, decreased may be correlated with multiple small dosage LPS alleviates CCl4_induced chronic hepatic injury.3. Role of macrophages in NASH should be valued. M1/M2 decreased may be related to that multiple dosage LPS treatment relieves NASH resulted from high-sucrose and high-fat and related FIRI and beta cell of islet apoptosis decreased.4. The study show that hepatic injury induced by CCl4 or high-sucrose or high-fat was close correlated with activating macrophages. And it may be an important pathogenesis that multiple small dosage LPS treatment inhibits the secretion of macrophages.