Research Of Ethyl Pyruvate Protect Rats From CCl₄-induced Liver Fibrosis

Liver fibrosis is the dynamic process that the body repairs damage made by various stimuli, while it is also a state that the production and degradation imbalance of extracellular matrix(ECM) leads to the excessive deposition of ECM.The balance of matrix metalloproteinases(MMPs) and tissue inhibitor of matrix metalloproteinases(TIMPs) could affect ECM degradation. As the main effective cells of liver fibrogenesis,hepatic stellate cells(HSCs) interact with other intrahepatic host cells, such as liver cells,macrophages and endothelial cells to promote the production of ECM and development of fibrosis. Several signaling pathways are essential to HSCs survival and activation.These pathways are regulated by many factors from HSCs secretion and paracrine. In HSCs, Toll-like receptor 4(TLR4) signaling induces the expression of inflammatory chemokines and adhesion molecules, while nuclear transcription factors(NF-κB) are activated through cell response to various stimulus. Therefore, they play a major role in chronic liver disease. Ethyl pyruvate(EP) as anti-inflammatory factor plays a role of protection in the various damage model.ObjectiveLiver fibrosis model is established by subcutaneous injection of carbon tetrachloride(CCl4). We observe the role of ethyl pyruvate at different time points of liver fibrosis progression. At the same time, the expression of hepatic stellate cell activation markers(α-SMA), TLR4, NF-κB, MMPs, TIMPs and collagen I, III, IV were detected. We preliminarily discussed the mechanism that EP inhibits HSCs activation and accelerate collagen degradation which are good for reversing liver fibrosis.Materials and MethodsHealthy male Sprague-Dawley rats were divided into three groups: Control group,CCl4 group and CCl4+EP group. Mixture of CCl4 and oil was intraperitoneally injected twice a week to obtain the liver fibrosis model, 8 weeks total. EP was injected once every day. There are 4 observation time points: 2, 4, 6 and 8 weeks. Liver samples were acquired 48 h after CCl4 injection at each time points.The ascites were observed. HE, MASSON staining and ISHAK score were used to assess the liver pathological changes and the fibrosis degree. The mRNA expression ofTLR4, NF-κB, MMPs, TIMPs and collagen I, III, IV were assessed by Q-PCR.Contents of collagenn IV, TIMP2, MMP2, TLR4 and α-SMA were determined using enzyme-linked immunosorbent assay(ELISA) kits. Positive cells and location ofα-SMA, TLR4, NF-κB, MMPs, TIMPs and collagen I, III, IV were determined by immunohistochemistry.Results1. Rats wrer injected CCl4 by subcutaneous and were injected EP intraperitoneal(40 mg/kg, daily),.We obain samples at 2, 4, 6, 8 weeks 4 time points, EP obviously improved diet and psychosis of rats and their weights increased compared with CCl4 group.Skin lesions can heal in the short term.Rats were treated by EP which significantly decreased serum levels of ALT, AST.2. After CCl4 injection, pathological of liver tissues were changed with liver cell edema, steatusis,pseudolobule and disorder of liver tissue structure. After EP,the damage of of liver tissue were significantly reduced. EP could reduce the collagenn fiber area3. In the model of CCl4-induced liver fibrosis,HSCs activation,.After EP, markers of HSCs activation,a- SMA significantly reduced4. Immunohistochemical results showed that the activation of HSCs and MyD88 were located in perisinusoidal space, the positive expression of TLR4, NF-κB, MMPs and TIMPs were located in hepatic cells.5. The results of QPCR illustrated that the mRNA expression of α-SMA, TLR4,NF-κB, MMPs, TIMPs and collagen 1, 3,4 were significantly increased compared with control group. There have no meaning at 2w. EP significantly decreased their expression levels compared to CCl4 group.6. The ELISA results were consistent with the corresponding Q-PCR results.Conclusion1、Intraperitoneal injection of EP every day plays a protective role on liver fibrosis damage.2、EP could inhibited CCl4-induced HSCs activation.3、The protective role of EP may be related to the TLR4/NF-κB signalinginhibition, HSCs and reduce inflammation to play a role of anti-fibrosis.4、EP up-regulated MMPs/TIMPs ratio to accelerate collagenn degradation.