Effect Of Fuzhenghuayu Decoction On Hepatic Stellate Cell And Liver Sinusoidal Endothelial Cell

Background:There are many patients with liver diseases in China, almost accounting for one-third of the world patients, patients with chronic hepatitis B will develop to liver cirrhosis even liver cancer,which is a serious threat to the patients. fibrosis is a common pathological process of chronic liver diseases, it is reversible, so we can seek effective treatments to slow liver fibrosis developing to cirrhosis,through exploring the mechanisms of liver fibrosis.The activation of hepatic stellate cells (HSCs) is generally considered to be a vital event in liver fibrosis, which results in deposition of a large deal extracellular matrix (ECM) in the liver,and HSCs do not secret enough matrix metalloproteinases and excessive to the inhibitor,so fibrosis forms.The early activation of HSC, mainly caused by paracrine cytokines by liver Kupffer cells, sinusoidal endothelial cells and platelet, and the late activation is mainly caused by autocrine of activated HSC, which result in a steady production of ECM, in which transforming growth factor beta (TGF-β1),Platelet-derived growth factor (PDGF) and connective tissue growth factor (CTGF) play an important roles in liver fibrosis.In liver fibrosis, sinusoidal endothelial cells (SECs) are another important cells, SEC is a special vascular endothelial cell, they have great differences with the normal vascular endothelial cells in morphology and functions, firstly there are many fenestraes arranging in sieve-like structure on SEC cytoplasm, In favor of exchanging ingredients between the liver cells and the sinusoids. secondly, sinusoids is a discontinuous capillary, which Lacks subendothelial basement membrane, only a little fibrous connective tissue.in liver fibrosis, liver sinusoidal endothelial cells become capillarization, with lacking fenestraes and forming subendothelial basement membrane, meantime, the phenotypes also change greatly, by lossing expressions of CD4、CD14and so on, and appearance of vascular endothelial markers, such as Von Willebrand factor (vWF), CD31, CD34and so on.At present, liver fibrosis has no effective treatments, Traditional Chinese medicine, due to its characteristics of multi-channel, multi-target and few side effects, has achieved good effects in liver fibrosis. Fuzhenghuayu(FZHY) decoction contains Salvia, Cordyceps sinensis, Walnuts, Gynostemma, Schisandra, Pine pollen,which is formulated on the basis of Chinese medicine theory"yuxuezuluo,zhengqixuruo", it can resist to lipid peroxidation, inhibite production of cytokines and activation of HSC, and reduce synthesis of collagen.our experiment studies the effects of FZHY on HSCs and SECs in liver fibrosis, and provide more evidences for the application of FZHY recipe, gamma-interferon (IFN-y) and beta-estrogen are extensivly researched, and have good effects on liver fibrosis, we use IFN-y and (3-estradiol as positive control drugs.Objective:To study the effects of FZHY on hepatic stellate cells and sinusoidal endothelial cell in hepatic fibrosis, and confirm the anti-fibrosis effect from the cellular level.Method:we preparated FZHY powder suspension at0.46g/10ml dose, and gavaged SD rats at0.46g/kg body weight, gave the first gavage two hours later, and took the second gavage, then collected drug serum after one hour later, meanwhile gave distilled water to control rat in the same condition and collected control serum, we separately added10%FZHY drug serum and control serum to DMEM medium as FZHY group(319group) and control group, IFN-y and β-estradiol were respectively added to control group as positive control groups, then coculture respectively with hepatic stellate cells and liver sinusoidal endothelial cells for48hours, lastly we studied the effects of FZHY in the following ways:1. detected HSC expression of a-SMA, Desmin by Immunocytochemistry;2detected HSC expression of a-SMA, Desmin, TGF-β1and CTGF by western blot;3detected the proliferation of HSC through flow cytometry;4. detected expression of vWF and CD31in SEC by Immunocytochemistry and Western blot;5. scanning electron microscopy observe the changes of SEC fenestraes structure.Results:1HSC expressing a-SMA and Desmin decreased in319group compared with control group by Immunocytochemistry, and the difference was significant (P<0.05), but no significant differences among drug groups (P>0.05);2. Western blot showed that HSC expressed a-SMA, Desmin, TGF-β1and CTGF in319group less than in control group, and the difference was significant (P<0.05), but no significant difference among drug groups (P>0.05);3.Flow cytometry showed that319group decreased the proliferation of HSC, and there were significant differences between the experiment group and control group (P<0.05) but no significant difference among drug groups (P>0.05);4Immunocytochemistry and Western blot showed that SEC expressed vWF and CD31in319group less than in the control group,and had significant difference(P<0.05), but no significant difference between drug groups (P>0.05);5.Scanning electron microscopy showed that SEC lossed most fenestraes in control group,but a lot of fenestraes re-emergence in319group.Conclusion:1.FZHY decoction could reduce activated HSC expression of a-SMA, Desmin, CTGF,TGF-(31and proliferation activity.2. FZHY recipe could reduce SEC expression of vWF,CD31and increased cell surface fenestraes, then reversed hepatic sinusoids capillarization.3.FZHY recipe may resist liver fibrosis by down-regulation secretion of ECM and inhibition proliferation of HSC and reversal of sinusoids capillarization.