| Hepatocyte injury was the most common pathological changes of clinical liver diseases. The inductive factors included virus infection, alcohol, chemical toxin, and so on. Its molecular mechanisms involved the production of free radical, lipoperoxide, calcium over-loaded and functional changes of monocytes/macrophages. All those factors are reciprocal, and act synergically, which lead to apoptosis, degeneration and necrosis of hepatocyte. Recently, it is believed that the liver is not only the biggest digestant gland in human body, but also one of the most important immune organs. The liver contains a large population of immune cells, such as dendritic cells and Kupffer cells, and takes part in immune response of human body. The uncontrolled functions of immune cells consequentially cause hepatocyte injury. The immune response can be man-madely divided into two parts, innate immunity and adaptive immunity. The innate immunity is the first line of defense, which can regulate the direction and intensity of adaptive immunity. The Kupffer cells, liver sinusoidal endothelial cells and dendrite cells in liver all can take part in the innate immunity, among which Kupffer cells are a member of the monocytes/ macrophages, the most numerous and have the strongest immune function. Therefore, our studies focused on the monocytes/macrophages to explore the mechanism of hepatocyte injury mediated their innate immunity.This dissertation included five sections. The innate immune functions of monocytes/macrophages were principally investigated through studying the TLR4 expression and its changes of signal transduction. The results indicated the innate immunity mediated by monocytes/macrophages play an important role in hepatocyte injury.Firstly, in order to study the relationship between the changes of TLR4 mRNA expression of peripheral blood monocytes and endotoxemia in patients of viral hepatitis, peripheral venous blood was obtained from hepatitis patients and health volunteers. The LPS concentration of plasma was detected by limulus lysate test. RT-PCR was used to detect the expressive change of TLR4 mRNA. The results showed that in the patients, LPS concentration of plasma was elevated in different degrees. In severe hepatitis group, the elevation of the LPS concentration was higher than that of other hepatitis group (P<0.05). The expression of the TLR4 mRNA in severe group was increased significantly compared with those of control group (P<0.05). However, in other hepatitis group, TLR4 mRNA expression was not significantly changed compared with those of control group ( P>0.05). The results indicated that endotoxemia was present in various types of the hepatitis patients in different degree, which was more prominent in severe hepatitis group. The expressions of TLR4 mRNA in peripheral blood monocytes in severe hepatitis patients were the highest, which might be correlated closely with hepatocyte injury.The purpose of second part is to study the relationship between the expressive change of TLR4 in the liver and hepatocyte injury in animal models of acute liver injury. The chemical toxins, D-Gal and LPS, were used to induce acute liver injury in rats. The degree of pathological changes in liver tissues was observed by HE staining. Serum transaminase (ALT and AST) and TLR4 in the liver and the apoptosis of liver cells were determined at different time points. We used specific inhibitor of NF-κB–PDTC to inhibit TLR4 downstream signal transduction, to study the influence of inhibition of TLR4 on liver injury. The results showed the serum transaminase level was elevated at 4, 8, 12, 24 hour time point after injection of D-Gal/LPS (P<0.05). The expression of TLR4 and the number of apoptotic cells in the liver increased gradually, which was higher than that of the normal control group (P<0.05). However, after treatment with PDTC, serum transaminase level, the expression of TLR4 and the number of apoptotic cells in the liver were lower than those of the model group (P<0.05). It indicated that TLR4 play an important role in acute liver injury through activating NF-κB pathway.The TLR4 expression of Kupffer cells is the main receptor which combined LPS in the liver. However, hepatocytes and liver sinusoidal endothelial cells were also expressed TLR4. To elucidate the expression of TLR4 in Kupffer cells play a role in acute liver injury, the rats were treated by Gadolinium Chloride (GdCl3) to specifically inhibit the function of Kupffer cells before the D-Gal/LPS administration, which did not affect the hepatocyte and other nonparenchymal cells. ED1 expression in liver tissues was observed by immunohistochemistry to identify the degree of inhibiting of Kupffer cells. Serum ALT, TNFαand IL-1βlevels were measured in different groups, and the expression of TLR4 in the liver was detected by Western blot. The number of ED1 positive cells in liver tissues of GdCl3 group were lower significantly than that of control group (P<0.05).Portal venous blood ALT, TNFαand IL-1βlevels was not elevated in rats receiving GdCl3 alone. Moreover, the degree of degeneration and necrosis of hepatocyte in D-Gal/LPS group was lower than that of LPS group (P<0.05). Serum ALT concentration was elevated in the D-Gal/LPS treated rats, and this increase was reduced with GdCl3 pretreatment. Similar results were observed with serum TNFαand IL-1βlevels (P<0.05). The expression of TLR4 in GdCl3 +LPS group was significantly lower than that of LPS group (P<0.05), but had no statistically significant difference compare with control group (P>0.05). Taken together, all results indicated that the TLR4 expression was decreased through inhibiting the activation of Kupffer cells stimulated by D-Gal/LPS in liver tissues, and Kupffer cells and its TLR4 play an important role in acute liver injury.The Kupffer cells activated by LPS can product not only the pro-inflammatory cytokines, such as IL-1 and TNFα,but also some kinds of chemotactic factors resulting in the rapid recruitment of immune cells to the liver. Expression of 5-lipoxygenase (5-LO) in the liver is basically restricted to Kupffer cells. 5-LO is the key enzyme that can catalyze oxygenation of arachidonic acid to 5(s)-hydroperoxy-6-trans-8, 11, 14-9-eicosatetraenoic (5-HPETE) and leukotrienes (LTs), which is one of the most strong chemokine of neutrophils. It is worthy of researching the synthesis and release of inflammatory mediators after TLR4 activation of Kupffer cells stimulating by LPS. The purified Kupffer cells were isolated from liver of Sprague-Dawley rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Percoll reagent. Then, those cells were treated by LPS, the production of TNFαwas assessed in culture supernatants by ELISA and that of nitric oxide (NO) by nitrate reducase method. 5-LO mRNA expression was assessed by semiquantitative RT-PCR. At the same time, Somatostatin (SST) was used to evaluate the immunity modulator's action on the functions of Kupffer cells. The results showed that vehicle-stimulated Kupffer cells produced a basal amount of No, TNFαand expressed 5-LO mRNA. Kupffer cells stimulated by LPS secreted significantly increased amounts of NO and TNFαand 5-LO mRNA (P<0.05). The secretions of NO and TNFαand 5-LO mRNA induced by LPS were inhibited by SST. The amount of NO and TNFαwas higher than control group at each time point, however 5-LO mRNA expression was still higher than that of control group at 12 and 24h (P<0.05), but had no statistically significant difference compare with control group at 48h (P>0.05). The data presented in this part study strongly supported the views that Kupffer cells can produce a great lot of inflammatory mediators induced by LPS and SST can modulates the Kupffer cells function through inhibiting No, TNFαproduction and 5-LO mRNA expression, which alleviate inflammatory filtration and in turn play roles in hepatocyte protection.Finally, in order to explore the direct effects of Kupffer cells activated by LPS on the metabolism and proliferation of rat hepatocytes in endotoxemia, the Kupffer cells were stimulated by LPS and Kupffer cell conditioned medium (KCCM) were collected and administrated to hepatocytes. After hepatocytes exposured to KCCM, transaminase (ALT and AST) and lactate dehydrogenate (LDH) were measured in the supernatants in different time points. Meanwhile, MTT colori metric assay was used to detect the action of KCCM on hepatocytes viability. The results showed that the concentrations of ALT, AST and LDH were gradually increased 2h after exposure (P<0.05). There was remarkable time-dependent relationship between KCCM and release of transaminase and LDH in the supernatants of rat primary cultured hepatocytes from 2h to 24h. The results of MTT suggested that hepatocytes was obviously injured at 12h and 24h(P<0.05). The above results indicated that the soluble factors released by Kupffer cells, which were activated by LPS, play a role in hepatocyte injury directly.Summary:Endotoxemia was present in various hepatitis groups, which was more prominent in severe hepatitis group. The expression of TLR4 mRNA in peripheral blood monocytes in severe hepatitis patients was the highest, which might be closely correlated with liver injury. Kupffer cells and the expression of TLR4 might be involved in acute liver injury. SST inhibits NO, TNFαproduction and 5-LO mRNA expression by LPS-stlimulated Kupffer cells which has protective effect on hepatocytes. The soluble factors released by Kupffer cells, which were activated by LPS, play a role in hepatocyte injury directly. |
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