| Liver, the largest solid organ in the body, plays an important role in metabolism, biotransformation (drug and toxin), secretion and excretion (bile), coagulation and anticoagulation, immune defense and so forth. Chronic liver diseases represent a significant public health problem with a worldwide mortality of around800,000deaths per year. Liver injury as the common disease in almost all kinds of liver diseases, such as viral hepatitis, drug induced liver injury (DILI) and etc. Severe liver injury will lead to liver fibrosis, cirrhosis and further end up with liver failure. Currently the only effective therapeutic treatment for liver fibrosis is liver transplantation which impact is still limited due to unsatisfied availability, suitability and long-term effectiveness.Bone morphogenetic protein (BMP) was originally identified by Urist and extracted from human bone tissue in1965. It belongs to the transforming growth factor beta (TGF-β3) superfamily involved in wide variety of biological functions such as bone formation, chondrogenesis, cell proliferation, differentiation and apoptosis. Also, it is related to the development of heart, liver, kidney, lung, nerve, and muscle as well as iron metabolism. Regarding to the liver, BMP4is plays a role in hepatic embryonic development, liver formation, hepatocyte proliferation, differentiation, liver injury and liver fibrosis. Although BMP4exhibits a wide variety of biological activities, its role in the liver still remains to be investigated.Si Ni Powder was documented by ZhongJing Zhang in "Treatise on Febrile Diseases" which consists of Chaihu, Zhishi, Baishao and Gancao. It has the function of smoothing flow of liver qi and regulating spleen. According to modern research, it has been demonstrated that Si Ni Powder could significantly improve hepatic microcirculation, protect hepatocytes and anti-hepatic fibrosis. The book "Ben Jing" documents Chaihu as the sovereign drug involved in the function of smoothing flow of liver qi and dispersing blocked heat. Saikosaponin a (SSa) is the major medicinal component of Chaihu. It has been demonstrated that SSa exhibits anti-inflammatory, anti-proliferation of hepatocarcinoma cells and improves hepatic antioxidant capacity.Based on the theory and research above, we believe that the effect of BMP4in liver injury has to be investigated since it plays a very important role in liver diseases. In addition, the mechanism of SSa in treating liver fibrosis should be further elucidated. Also, it has to be revealed the relationship between liver fibrosis treated by SSa and the regulating effect of BMP4in the liver.Therefore, this project consist of three parts:1. the regulation of BMP4in D-gal induced liver injury and its signaling transduction mechanism. The aim of this part is to investigate the effect of hepatotoxic D-gal on Huh7cell morphology, the effect of D-gal and BMP4on Huh7cell proliferation, the expression of BMP4mRNA and protein in liver injury induced by D-gal, the signal transduction pathway of BMP4and the role of BMP4on Huh7cell differentiation.2. Screening the effect of SSa, PF, HES, NRG, GA on BMP4expression in LX-2cells.3. Regulation of bone morphogenetic protein4during hepatic stellate cell activation by Saikosaponin A. Investigate the expression of BMP4in hepatic stellate cells and its role in hepatic stellate cell activation, proliferation and apoptosis. The main results are showing below:1. Regulation of BMP4in D-gal induced liver injury and its signaling transduction mechanism.At the concentrations used, D-Gal did not induce significant change of Huh-7cell morphology. D-Gal inhibited HuH-7cell proliferation and BMP4as well as its antibody did not affect cell proliferation. In addition, BMP4did not reverse D-gal induced inhibition in the cells. Moreover, D-Gal induced BMP4mRNA expression in a time and dose dependent manner. BMP4mRNA level reached peak at6hours treatment of D-gal and gradually decreased to normal level at48hours. Furthermore, BMP4induced phosphorylation of both SMadl and ERK1/2but did not affect mRNA abundances of albumin and CK-19in HuH-7cells. Conclusion: D-Gal significantly inhibits HuH-7cell proliferation and induces bone morphogenetic protein4expression at the same time.2. The screening results of the effect of SSa, PF, HES, NRG, GA on BMP4expression in LX-2cells. PF, HES、NRG、GA did not induced BMP4mRNA expression in24hours. In addition, SSa significantly inhibited BMP4mRNA expression. Meanwhile, SSa significantly inhibited BMP4protein expression while HES increased BMP4protein expression.3. Regulation of bone morphogenetic protein4during hepatic stellate cell activation by Saikosaponin A.Both mRNA and protein levels of BMP-4were significantly inhibited by5μM SSa treatment in human hepatic stellate cell (LX-2). SSa significantly inhibited LX-2proliferation at the concentration of5μM while BMP-4had no effect on LX-2proliferation. BMP-4increased a-SMA expression in LX-2while SSa reduced a-SMA expression. In addition SSa could neutralize the effect of BMP-4on a-SMA expression. Bax protein expression was induced by5μM SSa in LX-2. Conclusion: SSa could down-regulate BMP-4expression and inhibit hepatic stellate cell activation.In conclusion, BMP4plays a very important role in liver injury. And SSa is the major component in Si Ni Powder which could inhibit BMP4expression. Moreover, the inhibitory effect of SSa on hepatic stellate cell proliferation, activation and its effect of inducing hepatic stellate cell apoptosis could contribute to its therapeutic effect on liver fibrosis. |
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