Effect Of Chinese Traditional Medicine Ganfukang On Liver Fibrosis

Background and objective:Liver fibrosis is characterized by an accumulated deposition of extracellular matrix materials (ECMs) and the resulting reconstruction of liver parenchyma, companying the impairment of normal liver function. A clinical and experimental study has found that liver cells, hepatic stellate cells (HSC), kupffer cells and sinus endothelial cells all take part in the formation of hepatic fibrosis, in which HSC plays a very important role. Activation of HSC is commonly regarded as the major link of hepatic fibrosis and the main resource of synthesis of ECM. The main characteristic of activation of HSC is excessive proliferation of HSC. In addition,α-SMA is regarded as a marker of activation of HSC Activation of HSC can lead to excessive synthesis of collagen. HSC proliferation is stimulated by growth factors derived from hepatocytes, Kupffer cells, infiltrating macrophages, platelets, and activated HSCs themselves in paracrine and autocrine manners. In particular, PDGF(platelet-derived growth factor,PDGF), the most potent mitogen for HSCs, stimulates their DNA synthesis by activating extracellular signal-regulated kinas(ERK), phophatidylinositol 3-kinase (PI3K) and signal transducer and activator of transcription(STAT) pathways. Hence, PDGF play a very important role in proliferation, activation of HSC and synthesis of ECM. Traditional Chinese medicine has shown its own advantage in treating some difficult diseases. Developed by our department compound Ganfukang has been used as the traditional Chinese medicine for treating hepatic fibrosis. Clinical observations in our affiliated hospital have proved its effective. However, further study of its detailed anti-hepatic fibrosis mechanism is still needed. On the basis of theabove mentioned theory and research developments, our study with the cell culture as technical platform, was to observe the influence of serum collected from rats perfused with Ganfukang on activation and proliferation of HSC in vitro, and using transcription-polymerase chain reaction (RT-PCR) and image analysis technology to observe its influence on the expressions ofα-SMA, collagens I, III, using western blotting to observe its influence on the expressions of protein ERK, PI3K and STAT, particularly through disturbing platelet-derived growth factor dependent signaling pathways and cross-talk among these pathways.With the rapid development of proteomic methods, it is possible to find and identify the direct sites of Chinese medicine by using comparative proteomic methods. Two-dimensional gel electrophoresis (2-DE) is currently the most common analytical technique available for the study of protein expression patterns. Our study is to observe the influence of Chinese medicine on liver tissues and HSC by using 2-DE. In this way, we can supply the theory basement for drug development.Materials and methods:1 Cell culture and grouping for RT-PCRAccording to intervening factors, HSCs were divided into 5 groups, i.e. control group, model group, high dosage serum group, medium dosage serum group and low dosage serum group. Upper culture medium was removed after cultivated for 24 h. Control group serum:10% normal SD rat serum +DMEM, model group serum: 10% normal SD rat serum +DMEM+PDGF(final concentration 60ng/ml), high dosage group serum: 10% high dosage serum +DMEM+PDGF(final concentration 60ng/ml), medium dosage group serum: 10% medium dosage serum + DMEM + PDGF(final concentration 60ng/ml) and low dosage group serum: 10% low dosage serum +DMEM+PDGF(final concentration 60ng/ml) were accordingly added. Total RNA was extracted from HSCs. Expression of mRNA was determined by reverse transcription polymerase chain reaction2 Cell culture and grouping for western blottingHSC cells were randomly assorted for six groups, those were control group(10% normal SD rat serum +DMEM), model group(10% normal SD rat serum +DMEM), Chinese herb-treated (with 10%medium dosage serum )group, ERK blocker-treated (U0126, final concentration 10μmol/L)group, PI3K blocker-treated (LY-294002, final concentration 10μmol/L )group and STAT blocker-treated (Parthenolide, final concentration 50μmol/L )group, After the treatments of PDGF (10% normal SD rat serum +DMEM+PDGF:final concentration 60ng/ml),and Ganfukang (10% medium dosage serum + DMEM + PDGF:final concentration 60ng/ml) at defined concentration and time(12h, 24h,48h respectively). Cells of control, PDGF-treated or drug-treated, were harvested. Immunoblotting was used to determine the expressions of STAT, ERK and PI3K.3 Two-dimensional gel electrophoresisHSCs were assorted for three groups, those were control group, model group, Chinese herb-treated group; Live tissues were divided into 3 groups. Those were control group, model group and Chinese herb-treated group. All protein from those groups was extracted. The method of O'Farrell, with modification, was followed . For the first-dimension, isoelectric focusing was performed by using a vertical mini-IEF system (Jim-X Company) and using analytical SDS-PAGE carried out the second-dimension separation.Statistical analysisValues reported in the figures represent means±SD of three or more independent samples. The results were analyzed by the unpaired Student's t-test. Statistical significance was set at P<0.05.Results1 Effect of each group serum on collagen I, III mRNA expression of HSC in ratsBy using RT-PCR, we investigated effects of Ganfukang serum on collagen I, III mRNA expression. It shows that Ganfukang serum decreases collagen I, III mRNA expression of HSC in a concentration-dependent manner. PDGF can increase the expression of collagen I, III mRNA obviously compared with control group P<0.01. Ganfukang serum can decrease collagen I, III mRNA expression significantly compared with model group p<0.01,especially medium dosage serum has the most effective function on inhibition the expression of collagen I mRNA, which has the significant difference compared with low dosage group P<0.01,while there is no significant difference between high dosage group and medium dosage group.2 Effect of each group serum onα-SMA mRNA expression of HSC in ratsRT-PCR shows that Ganfukang serum decreasesα-SMA mRNA expression of HSC in a concentration-dependent manner. PDGF can increase the expression ofα-SMA mRNA obviously compared with control group P<0.01. Ganfukang serum can decreaseα-SMA mRNA expression significantly compared with model group p<0.01, especially medium dosage serum has the most effective function on inhibition the expression ofα-SMA mRNA. While there is no significant difference among these three different Ganfukang dosage groups.3 Protein expression of STAT, ERK and PI3K assay by western blottingWestern blots show the effects of Ganfukang, ERK blocker, PI3K blocker, STAT blocker on PDGF-induced STAT, ERK and PI3K protein levels. The protein levels increased by PDGF. However, The protein levels stimulated by PDGF were significantly inhibited not only by Ganfukang or it's own blocker, but also by the other two blockers.4 Comparison of the protein patterns of HSCsThe HSCs were harvested after 12-hour,24-hour and 48-hour treatments of PDGF and Ganfukang respectively. The proteins of control group, PDGF -treated group, and Ganfukang-treated group were extracted and prepared for comparative proteomic analysis. Within the applied pH range (PH 4.0~ 6.0), a protein load of 30μg per 8 cm strip was the optimal quantity for silver-stained gels. Gel scans were analyzed for spot numbers using PDQuest software. After treatments 12 hours, there were 188 spots that the gel of control group contained , 161 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 124 spots. After treatments 24 hours, there were 189 spots that the gel of control group contained,229 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 397 spots. After treatments 48 hours, there were 118 spots that the gel of control group contained,183 spots were found on the gel of PDGF-treated group, the gel of Ganfukang-treated group contained 81 spots. There are many kinds of alterations on protein spots in different time of treatment.5 Comparison of the protein patterns of liver tissuesFor the liver tissues,there were 217 spots that the gel of model groupcontained, the gel of Ganfukang-treated group contained 241 spots.Conclusions:1 The anti-fibrosis roles of Ganfukang compound maybe influence the function of PDGF by nonspecific action, thereby inhibit the transcription of I,III collagen,α-SMA mRNA and decrease the production of ECM.2 The activity of ERK can be inhibited by its inhibitor, while the activity of PI3K and STAT can also be inhibited by ERK inhibitor. So there maybe exist crosstalk among these three pathways. Such crosstalk may contribute to development of liver fibrosis.3 It was found that there are different protein spots in every different phase by comparing Gel scan among three model groups. The number of protein is changed as the time prolonged. This shows that the pathogenesis of liver fibrosis is involved with many kinds of protein.4 There is a significant difference at protein level between untreated and PDGF-, Ganfukang- treated cells. It suggests that the differential expression analysis of proteomes may be useful to further study on mechanism of Ganfukang, and search for new targets of anti-liver fibrosis medicine.