The Study Of Anti-fibrosis Ⅰ Herbal Compound On Hepatic Fibrosis And Portal Hypertension Induced By CCl₄ And Its Molecular Mechanism In Vitro

Hepatic fibrosis, which may ultimately lead to cirrhosis, is the pathological base of all the chronic hepatic diseases, and is characterized by the net accumulation of extracellular matrix (ECM). Lots of data have indicated that the activation of hepatic stellate cell (HSC) is the cytological base and central tache of the hepatic fibrosis. Activated HSCs are the main cells and the basis to synthesize extracellular matrix (ECM). So activated HSCs are the key to the formation of hepatic fibrosis. Thus HSCs are chosed to be remedial target for much more anti-fibrosis medicaments. In the course of HSCs activation, cytokine plays an important role, however, transforming growth factor-β₁ (TGF-β₁) is the strongest factor to facilitate the activation of HSCs. The contractibility and relaxation of HSC is adjusted by various vasoactive mediators, including endothelin-1(ET-1) and nitric oxide(NO). After HSC is activated, the expression of α-smooth muscle actin (α-SMA) increases, the capacity to contract increases, and which causes blood vessel resistance and portal hypertension. It is possible for hepatic fibrosis to reverse, the treatment of anti-fibrosis can stop as well as reverse the progression of the fibrosis. The therapeutic effects of Western medicine are no ideal, while Chinese traditional medicine has shown the characteristic of more taches and targets to treat hepatic fibrosis in clinic and experiment recently, but there are still plenty of deficiency in the mechanism of Chinese medicine at present. A large number of studies have been done to explore mechanism only in clinic or histology, so it is important to investigate the mechanism of hepatic fibrosis and seek effective anti-fibrosis drugs from the molecular level. TGF-β₁ antibody was used to block the effect of TGF-β₁ to explore the correlation among TGF-β₁, ET-1 and NO.This will give some new clues to explore the mechanism and therapy of fibrosis by blocking the effect of TGF-β₁ . On the basis of animal experiment ,This study was performed using the serum pharmacological method, then the change in intracellular calcium was observed with laser scanning confocal microscope, in order to further study the possible mechanism of anti-fibrosisâ… Chinese medicine to prevent and treat hepatic fibrosis and portal vein hypertention from cellular and molecular level. At the same time, to identify differential genes between the normal liver tissue and the hepatic fibrosis tissue in rat and to demonstrate the molecular mechanism from gene with mRNA differential display PCR(DD-PCR). Part 1 The effects of anti-fibrosisâ… herbal compound drug serum on hepatic stellate cells stimulated by carbon tetrachloride in rats Objective: To investigate the effect of anti-fibrosisâ… herbal compound on the proliferation, collagen synthesization and TGF-β₁ in hepatic stellate cells stimulated by carbon tetrachloride (CCl₄), to further explore its mechanism from cellular and molecular level. Methods: HSC strains were separated from rats with hepatocirrhosis induced by CCl4, and well cultured to gain eternal life, phenotypes were activated HSCs. The herbs were boiled with water and extracted by alcohol, whose solution was infused in rats to prepare drug serum, rat serum with physiological saline was prepared for the negative control in the same way. After HSCs were treated with 10mmol /L CCl₄ for 30min, rat serum and drug serum was added respectively, the final concentration (volume fraction) was 0, 5 mL/L ,10 mL/L ,20 mL/L , 0 was the control group, 48 h later, the cell proliferation was determined with MTT assay, cell cycle was measured with Flow Cytometry. HSCs were treated with 15mL/L drug serum , MTT assays were employed to estimate the proliferation of HSCs at different timepoint(12～48h). The mRNA expression of typeâ… procollagen and TGF-β₁ was analyzed by RT-PCR. Typeâ… collagen and TGF-β₁ protein productions were semiquantified by immunocytochemistry stain and image analysis system. Results: Within the range of 10mmol/L CCl₄ and 5²0mL/L drug serum, there are no evident influence on shapes of HSCs. MTT experiment showed that various concentrations of drug serum could inhibit the proliferation of HSC stimulated by CCl₄, and in a dose depend manner, but the cell proliferation in 20mL/L drug serum(0.210±0.015)was higher than in the untreated control group( 0.166±0.003) (P<0.05). The MTT transform of HSC was not significant different between various dosages rat serum group and CCl₄ group (p>0.05), while which in same dosage(15mL/L) drug serum was remarkable different at different timepoint (12h,24h,48h) (0.226,0.365,0.251) and lower than that in CCl₄ group(0.278,0.418, 0.470) respectively(P<0.05). Flow Cytometry results showed cell percent in G0/G1 phase in drug serum group(57.80±4.07) increased markedly , compared with that in CCl₄ group( 34.13±3.28)(P<0.05), which in S phase decreased markedly(31.68±3.28 vs 52.25±4.12)(P<0.05), however, which in M phase had not significant change.Compared with untreated control group(0.66±0.04 and 0.86±0.05),TGF-β₁ and typeâ… Collagen mRNA increased significantly in CCl₄ group(1.83±0.12 and 2.43±0.11) (P<0.05) .Whereas compared with CCl₄ group , drug serum reduced TGF-β₁ and typeâ… Collagen mRNA expressions, and there was significantly different (P<0.05), which was 1.63±0.07and 2.32±0.13 in 5mL/L drug serum, 0.84±0.04 and 1.19±0.06 in 20mL/L drug serum. Drug serum remarkably decreased the protein expression of TGF-β1 in HSC stimulated by CCl₄(P<0.05), as its dose increased, TGF-β₁ significantly fell, meanwhile, drug serum inhibited the typeâ… collagen secretion. TGF-β₁, typeâ… collagen had no significant difference between rat serum group and CCl₄ group. Conclusion: Anti-fibrosisâ… herbal compound not only directly inhibited the proliferation and the typeâ… collagen secretion of HSC induced by CCl₄, but also down-regulated TGF-β₁ mRNA and protein expression, restrained side secretion and self-secretion of actived HSC, thereby reduced the synthesization of extrac cellular matrix, possessed integration predominance of antifibrosis.Part 2 The effects of anti-fibrosisâ… herbal compound drug serum on NO and ET-1 of hepatic stellate cells stimulated by carbon tetrachloride in rats Objective:To examine the influences of TGF-β₁ antibidy on ET-1 and NO in HSC, to explore the relationship among cytokines and study its mechanism in hepatic fibrosis and portal hypertension. As viewed from cytokines, illuminate the mechanism of anti-fibrosisâ… herbal compound against hepatic fibrosis and portal hypertension , to search a new feasible therapeutic approach. Methods: Suspending liquid of cells at a density of 1×10⁵ cells/mL was plated in culture dish, cultured under 37℃,5 % CO₂ condition for 24h, divided into groups as following, 4 dishes in each group (1) HSC control group; (2) HSC+TGF-β₁ antibody(W/V)5,10,15,20μg /mL, and continuously cultured 24h, collected cell culture liquid, nitric oxide synthase (NOS) activity was measured using colorimetry, NO level was determined by nitrate reductase technique, ET-1 content in the medium was surveyed by enzyme linked immunosorbant assay(ELISA) method. HSCs stimulated by CCl₄ were treated with drug serum or/and TGF-β₁ antibody, ET-1and iNOS mRNA were determined with RT-PCR.The cells were fixed by 4% paraformaldehyde for test HSC ET-1 and iNOS expression by immunocytochemical method. Results: TGF-β₁ antibody markedly inhibited HSC ET-1 secretion in a dose depend manner. 5²0 /mL TGF-β₁ antibody reduced HSC ET-1 contents from 6.90±0.58 to 5.33±0.27, 3.69±0.33 and 2.87±0.54 respectively, which were lower than that in the control group(8.50±0.46) (P<0.05), and there was significant different for multiple comparison between differnet dosage groups(P<0.05). Simultaneously, TGF-β₁ antibody increased HSC NOS activity , NO level in supernatant were increased in parallel from 71.10±6.54 to 80.68±5.44, 88.58±2.84 and 93.62±3.53, which had significant difference between different dose TGF-β₁ antibody group andcontrol group(46.75±4.72) (P<0.05), as TGF-β₁ antibody dose increased, NOS activity and NO synthesis increased, but which between in 20μg /mL and 15μg /mL TGF-β₁ antibody had no significant difference(P>0.05). The effects of drug serum and TGF-β₁ antibody on ET-1and iNOS gene and protein in HSCs stimulated by CCl₄ were examined by RT-PCR and immunocytochemical method. ET-1 mRNA expression and content in HSCs stimulated by CCl4 (7.09±1.71 and 42.44±2.58)markedly increased, and were notable higher than that in control group (0.56±0.28 and 12.25±1.56) (P<0.05), which were inhibited by drug serum and TGF-β₁ antibody ,and inhibitory effect of combined application was the strongest in HSC stimulated CCl4. Meanwhile, iNOS mRNA and protein expression (0.96±0.16 and 5.31±0.86) slightly raised when compared with control group(0.63±0.19 and 4.66±1.12), but difference had no significance(P>0.05), which in drug serum and/or TGF-β₁ antibody were higher than in CCl4 group and control group, and the expression of NOS in combined group was higher than in single group(P<0.05). Conclusion: After TGF-β₁ antibody blocked TGF-β₁ function of HSC in vitro, ET-1 content significantly decreased , NOS activity increased, NO level in supernatant increased in parallel. Anti-fibrosisâ… herbs decreased ET-1 level and increased NO secretion in HSC stimulated by CCl₄ to restrain HSC contraction , further to reduce sinusoidal resistance and portal pressure. Part 3 Effects of drug serum of anti-fibrosisâ… herbal compound on calcium in hepatic stellate cell and its molecular mechanism Objective: The contractility of hepatic stellate cell may play an important role in the regulation of hepatic microcirculation and the development of portal hypertension. The aim of this study is to investigate the effects of anti-fibrosis I herbal compound on intracellular Ca²⁺ in activated hepatic stellate cell, and to try to survey its molecular mechanism in treatmentand prevention of hepatic fibrosis and portal hypertension. Methods: The activated hepatic stellate cell lines were plated on small glass cover slips in 24 wells culture dishes at a density of 5×10⁶ /mL, which treated with carbon tetrachloride(CCl4), TGF-β₁ antibody , rat serum and drug serum of anti-fibrosis I herbal compound respectively and incubated in RPMI-1640 media for 24 h. After the cells were loaded with fluorescence indicator Fluo-3/AM, intracellular Ca²⁺ was measured with laser scanning confocal microscopy(LSCM).The dynamic changes of intracellular Ca²⁺, stimulated by the different dosage carbon tetrachloride, TGF-β₁ antibody and the drug serum and their turn, interval time under orthogonal design, were determined by laser scanning confocal microscopy. According to results of orthogonal trial, the method of range analysis was used to find the best level of each factor, with which, the mechanism of anti-fibrosisâ… was studied. Firstly HSCs was treated with CCl₄ for certain time, before and after addition of TGF-β₁ antibody for certain time, drug serum was added into HSCs, 24 h later, the HSCs were loaded and the change of intracellular Ca²⁺ was observed. Using TCS SP2 data-image manage software with LSCM calculated the average fluorescence intensity of the whole cell. The concentration of HSC Ca²⁺ was measured in terms of intracellular fluorescent intensity. The larger the fluorescence intensity, the higher the Ca²⁺ concentration. Results: After carbon tetrachloride stimulation, intracellular Ca²⁺ of activated hepatic stellate cell increased significantly when the dosage of CCl4 from 5mmol/L to 15mmol/L, CCl₄ could make Ca²⁺ of HSCs overload, fluorescence intensity increased, what's more, the higher the CCl4 dosage, the stronger the fluorescence intensity, the higher the Ca²⁺ concentration (F=760.602 , P<0.05). However, treating HSCs with 5 mL/L ²0 mL/L drug serum, the Ca²⁺ dropped (F=554.962 , P<0.05); with 5μg /mL ²0μg /mL TGF-β₁ antibody, the Ca²⁺ dropped too( F=39.393 , P<0.05). The higher drug serum or TGF-β1 antibody dosage, the less Ca²⁺ concentration, but intracellular Ca²⁺ wasn't significantly different between rat serum without anti-fibrosisâ… and untreated group(control group).The result of orthogonaltrial showed the intracellular Ca²⁺ were significantly different in different dosage of carbon tetrachloride, Anti-fibrosis I drug serum,TGF-β₁ antibody and different turn of these substance(P<0.05), but there is no significance in their interval time between CCl₄ and TGF-β₁ antibody, CCl₄ and anti-fibrosis I drug serum(P>0.05). The results of range analysis showed that the most important matter that affected intracellular Ca²⁺ was CCl₄, the second important were anti-fibrosis I drug serum , TGF-β₁ antibody. According to results of orthogonal trial, the best level of each factor was 15 mmol/L CCl₄, 20 mL/L drug serum, 20μg /mL TGF-β₁ antibody. After blocking the effects of TGF-β₁ with TGF-β₁ antibody , anti-fibrosisâ… drug serum decreased Ca²⁺ most significantly. If drug serum prior to TGF-β₁ antibody was added into HSCs, the Ca²⁺ was higher than the former, but both were lower than Ca²⁺ in HSC treated with drug serum and TGF-β₁ antibody alone. These results suggested Anti-fibrosisâ… drug serum might induce HSCs relaxation by reducing Ca²⁺ in activated HSCs, and the mechanism was independent of decreasing TGF-β₁ effects. Conclusion: Anti-fibrosis I herbal compound may treat hepatic fibrosis and decrease portal hypertension by inhibiting activated hepatic stellate cell contractility through decreases intracellular Ca²⁺,but the molecular mechanism was independent of blocking TGF-β₁ effects. Part 4 Differential expression analysis of the liver fibrosis related genes in rats Objective: To identify differential genes between the normal liver tissue and the hepatic fibrosis tissue in rat and to demonstrate the molecular mechanism from gene. Methods: The hepatic fibrosis model of rats was established by injecting CCl₄, and was approved to be chronic hepatic fibrosis by pathology, the rats in control group were injected with physiological saline by the same means. mRNA differential display PCR(DD-PCR) was used. Both total RNA were...