| Liver fibrosis is formed in almost all the chronic liver injury patients with the characterized of nodules and liver contraction and finally ended withcirrhosis or liver cancer.Liver fibrosis is the basis of all complications in the telophase of liver disease, including portal hypertension, ascites, hepatic encephalopathy, synthesis and metabolic disorders. Liver fibrosis can damagethe function of liver cells and increase the portal vein pressure. At present, the general principle of the formation of liver fibrosis was considered that: Liver fibrosis is the repair response after a chronic, self-limited damage of liver. Activated hepatic stellate cells were the main cell in the formation of liver fibrosis. The activation of hepatic stellate cells is the key point of the fomation of liver fibrosis.Hepatic stellate cells, lie between liver cells and sinus endothelial cells, the main cells which produce extracellular matrix in the normal and fibrosis liver, and the main cells of storing retinaldehyde derivatives. In the process of the activation of stellate cells, the quiescent hepatic stellate cells with copious vitamin A were transtormed to the cells which could induce fibrosis and its morphological characteristics were the augumentation of rough endoplasmic reticulum, the decrease vitamin A particle, corrugation aryotheca, the appearance of contractility cellosilk and cell proliferation. The most direct way of the formation of Activated hepatic stellate cells to liver fibrosis was to increase the content of extracellular matrix. Extracellular matrix was a series of macromolecules which pariticitated the constraction of normal liver and fibrosis liver interstitial framework, including the family of several structures and support molecular: the collagen, the collagen glycoprotein and matrix combining growth factor, mucopolysaccharide, proteoglycan and matrix cells protein. The nature and content of matrix components were different from normal liver in liver fibrosis, its collagen total content increased 310 times. In liver damage area, these changes accelerated the activation of hepatic stellate cells and the formation speed of fibrosis. Further more, the activated hepatic stellate cells expressed integrin receptor also help matrix components of deposits, which eventually lead to extracellular matrix synthesis increase, degradation decline, and resulted in the extracellular matrix of excessive deposition.Researches showed that fibrosis can be reversed in many diseases such as alcohol withdrawal; the liver fibrosis can be significant reversed in alcoholic liver disease. Patients with chronic hepatitis b patients in application disease-resistant poison lamivudine after liver fibrosis degree is improved markedly. After application of ribavirin, the fibrosis progression was slowdown in patients with hepatitics.At present, hepatic fibrosis treatment mainly focus on: (1) To cure primary diseases such as chronic viral infection; alcohol withdrawal and poisonous drug discontinuation, to remove excess iron and copper, to remove biliopancreatic obstruction, etc. (2) To ease inflammation and immune response. (3)To restrain stellate cell activation including the antiproliferative of stellate cells; To promote stellate cell apoptosis, resist fibrogenesis inhibit collagen synthesis of extracellular matrix, and increase the degradation of extracellular matrix.Hepatic stellate cells were the target of treatment of hepatic fibrosis. Firstly, antioxidants such as tocopherol, silymarin etc, could reduce liver damage through protective liver cells and inhibit kupffer cells. Secondly, the material was to restrain the production of matrix, which mainly through blocking matrix synthesis and processing or indirectly inhibiting activity of TGF-β. Neutralize transformation growth factor can accelerat matrix biodegradation and inhibit matrix production. The focus was transformation growth factor (beta) antagonists, such as flabby peptide, etc. Ppar-gamma was expressed in stellate cells, whose ligand can degrade stellate cell activity. In the activated hepatic stellate cells, and the expressed ppar-gamma was declined significantly. Up-regulated Ppar-gamma expression could make hepatic stellate cells from activted state to rested state, so that inhibit hepatic stellate cell activity. In the study, the mechanism of action of HuaZhuojiedu Formula was observed by its action on TGF - beta and Ppar-gamma.TCM regarded hepatic fibrosis and cirrhosis as "ZhengJia" "GuZhang" "JiJu". It is used to be considered as the results of blood stasis sluggish, treated by promoting blood circulation to remove blood stasis. In the clinical practice, Mr Li (Diangui) considered turbidity poison playing a key role in the formation of liver fibrosis. Turbidity Poison could affect the liver function and lead to qiji yuzhi. The cause of liver fibrosis was Turbidity Poison and asthenia. Guben and QuXie were important in treatment of liver fibrosis. The effectiveness of HuaZhuojiedu formula was satisfactory after clinical verification, but the mechanism of its action pathway is unclear. In this study, the mechanism of action of HuaZhuojiedu formula on hepatic stellate cells would be observated in the cell level.In the treatment of liver fibrosis, according to TCM treatment principle, those tonify hepatorenal drugs would be applicated besides Huazhuojiedu, soft liver improving quality, promoting blood circulation to remove blood stasis, and psoralea corylifolia main applicated in the treatment of liver fibrosis. In the treatment of liver fibrosis, medicine such as natural flavonoids silymarin and curcumin could inhibit hepatic stellate cell proliferation, differentiation and extracellular matrix synthesis and prevent the formation of hepatic fibrosis and cirrhosis. In the former research psoralea was found also had much similar effect with curcumin, we assume that psoralen may also have similar function in preventing liver fibrosis, so in this study the function of psoralen on hepatic stellate cells will be observed. This study would be divided into five parts to explore the mechanism of action of HuaZhuojiedu formula and the effect of its anti-oxidant psoralen on the prevention of liver fibrosis, and to find the correlation of ZhuoDu and oxidative stress. Objective: HuaZhuojiedu Formula is a popular folk medicine which has been used for treatment of liver fibrosis in clinic. The mechanism was unclear.In the study, the effect of HuaZhuojiedu Formula medicated serum on the proliferation and apoptosis of hepatic stellate cells in vitro was analyzed, in order to explore its mechanism of action.Methods: SD rats, choosed to produce the medicated serum, were divided into five groups: interferon group (positive group), HuaZhuojiedu Formula large, medium and small dose groups and control group. Hepatic stellate cells were cultivated in vitro, hepatic stellate cell proliferation was observed after addition of each medicated serum 24 hours, 48 hours, 72 hours, using the MTT method, and hepatic stellate cell apoptosis was analysed after cultivating 48 hours using flow cytometric analysis.Results: After cultivated 24h, compared with control group, HSC-T6 proliferation have been inhibited in the HuaZhuojiedu high-dose group (0.216±0.051) and positive group (0.232±0.029) (P<0.05). After cultivated 48h, HSC-T6 proliferation have been inhibited in HuaZhuojiedu groups, especially in high-dose group (0.160±0.044) (P<0.05), but the effect of inhibition on HSC was indiscriminately between HuaZhuojiedu high-dose group and positive group. After cultivated 72h, the inhibition action of HuaZhuojiedu high-dose group was better than positive group (P<0.05).Flow cytometric analysis showed that: The apoptosis rate of HSC was about 2.68% in control group after 48h,the apoptosis rate was higher in positive control 15.78% and HuaZhuojiedu high-dose group than that in control group (P<0.05). Comared with positive group, the apoptosis rate of HuaZhuojiedu high-dose group was 20.02% higher (P<0.05) than positive group. The apoptosis rate of HuaZhuojiedu middle-dose group 13.38% was indiscriminately with positive control group (P>0.05), and in the small-dose group, the apoptpsis rate was 12.88% lower than positive control group (P <0.05).Conclusions: HuaZhuojiedu Formula medicated serum can inhibit hepatic stellate cells proliferation and induce apoptosis in a dose-response Part one Effect of HuaZhuojiedu Formula medicated serum on the proliferation and apoptosis of Hepatic Stellate Cells in vitro relation, namely the inhibiting proliferation and the concentration of apoptosis role to promote the stronger. Compared with positive group, HuaZhuojiedu Formula medicated serum restrains hepatic stellate cell proliferation and induced the apoptosis more effectively.Part two Effect of HuaZhuojiedu Formula medicated serum on Extr-acellular Matrix Secretion of Hepatic Stellate cells in VitroObjective: To observe the effect of HuaZhuojiedu Formula medicated serum on extracellular matrix secretion of hepatic stellate cells in vitro.Methods: HSC-T6 was incubated with different medicated serum for 48 hours. Collagen type I, collagen typeⅢ, layer adhesion protein, hyaluronic acid was determined by enzyme-linked immunosorbent adsorption method (ELISA).Results: Addition of HuaZhuojiedu Formula medicated serum into culture medium inhibited Type I collage, hyaluronic acid and laminin secretion. Type I collagen, the content ofⅢcollagen type in high-dose group (117.9±17.0 ng·mL-1, 56.3±6.2 ng·mL-1)were lowest, and its function is obviously superior (plus or minus 158.0±15.2 ng·mL-1 170.9±6.3 ng·mL-1) than small dose group (162.6±1.98 ng·mL-1, 79.6±5.9 ng·mL-1) and control group (148.3±17.8 ng·mL-1, 164.3±8.8 ng·mL-1) (P < 0.05). Layer adhesion protein, hyaluronic acid content in high-dose group (60.1±4.5 ng·mL-1, 31.2±4.2 ng·mL-1) and positive control my monitor section (60.0±5.1 ng·mL-1, 32.8±4.1ng·mL-1) were lower, there was no obvious difference between the two groups (P > 0.05).Conclusion: HuaZhuojiedu Formula medicated serum can reduce the secretion of the extracellular matrix of hepatic stellate cells in vitro.Part three Effect of HuaZhuojiedu Formula medicated serum on expression of Transforming Growth Factor- beta1 and Protein Smads in Hepatic Stellate CellsObjective: To investigate the effect HuaZhuojiedu Formula medicated serum on the expresion of TGF-β1 mRNA and Smad4 mRNA. Methods: Effect of different volume HuaZhuojiedu Formula medicated serum on TGF-β1 mRNA and Smad4 mRNA were detected through RT-PCR method.Results: After treated with HuaZhuojiedu Formula medicated serum for 48h, Contrasted to the black control group, the expression of TGF-βmRNA of HSC were significantly decreased in high-dose HuaZhuojiedu Formula (P<0.05), and the expressions of Smad4 mRNA was down-regulated (P<0.05). Conclusion: HuaZhuojiedu Formula medicated serums inhibited hepatic stellate cell proliferation and promoted its apoptosis and reduced synthesis of extracellular matrix, which action mechanism possibly was via down-regulated TGF -β1 mRNA and Smad4 mRNAPart four Effect of HuaZhuojiedu Formula medicated serum on expression of Ppar-gamma mRNA influenceObjective: Aim to study the effect of HuaZhuojiedu Formula medicated serum on peroxisome proliferator-activated receptorγ(PPARγ) of hepatic stellate cells (HSC) and the correlation between PPARγand TGF-β.Methods: The effect of HuaZhuojiedu Formula medicated serum on the express of PPARγmRNA in HSC was observed by applying RT-PCR.Results: At the transcription and translation level, HuaZhuojiedu Formula medicated serum groups upregulated the express of PPARγmRNA (P<0.05), the express of PPARγmRNA was significiently higher in high-dose group than that in other groups (P<0.05).Conclusion: HuaZhuojiedu Formula medicated serum can upregulate the expression of PPAR gamma mRNA in hepatic stellate cells especialy in HuaZhuojiedu Formula medicated serum high-dose group, which showed that the interferen of HuaZhuojiedu Formula medicated serum in the signal pathway of TGF-β/Smad is funcional through upregulating the express of PPARγmRNA. Part five Effect of Psoralen on the proliferation and activation of Hepatic Stellate CellObjective: To investigate the effect of antioxidant psoralen on the proliferation and lipid peroxidation of oxidative stress in rat hepatic stellate cells in vitro.Methods: HSC-T6 was incubated with H2O2, which were treated with psoralen.MTT colorimetry was used for assaying proliferation of HSC. Intracellular MDA, SOD, GSH and GSH-PX levels in the culture media were measured by ELISA reagent boxes, collagen I was determined by ELISA.Results: Comapred with model group, Psoralen significantly decreased the number of HSC in dose of 10μmol·L-1, 1μmol·L-1, 0.1μmol·L-1(P<0.05) especially at 48h and psoralen in the concentration of 10mM, 1mM, 0.1 mM groups could decrease the content of collagen type I and intracellular MDA and GSH (P<0.05), and increased the activity of SOD and GSH-PX (P<0.05). psoralen in the concentration of 10 mM, 1mM, 0.1 mM groups can improve SOD and GSH - PX activity (P < 0.05), reduce the content of MDA and GSH (P < 0.05), and decrease the content of collagen type I(P < 0.05).Conclusion: Psoralen could inhibit the proliferation and oxidative stress of HSC. Psoralen was one of material basis of HuaZhuojiedu Formula which prevents liver fibrosis. The mechanism of ZhuoDu induce the liver fibrosis may be correlate with oxidative stress.HuaZhuodu was related to anti-oxidant stress.General Introduction1 HuaZhuojiedu Formula medicated serum can inhibit hepatic stellate cell proliferation.2 HuaZhuojiedu Formula medicated serums can promote hepatic stellate cell apoptosis.3 HuaZhuojiedu Formula medicated serums can restrain the secretion of collagen type I,Ⅲcollagen type, hyaluronic acid and layer adhesion protein.4 In HuaZhuojiedu Formula high-dose medicated serum group, expression of TGF -β1mRNA was decreased obviously in HSC. 5 In HuaZhuojiedu Formula high-dose medicated serum group, expression of Smad4 mRNA was lower obviously than that in medium and small dose group (P < 0.05).6 In HuaZhuojiedu Formula high-dose medicated serum group, expression of PPAR gamma mRNA upgrated obviously more than that in the control group and positive.7 The preventive effect of HuaZhuojiedu Formula on hepatic fibrosis could be implement through upregulating PPARγmRNA and interventing TGF - beta/Smad4 signaling pathways in stellate cells, promote the stellate cell apoptosis and inhibit stellate cell proliferation and reduce secrete extracellular matrix content.8 In different concentrations, psoralen significantly inhibits the proliferation of HSC-T6, especially in 10mM at 48h, 72 h, in the concentration-response relation.Psoralen could inhibit the proliferation and oxidative stress of HSC. Psoralen was one of material basis of HuaZhuojiedu Formula which prevents liver fibrosis.
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