| Background and objective:Hepatic fibrosis is a kind of progressive pathologic process in chronic liverdiseases, which results in hepatic cirrhosis or hepatic cancer. It is a compensatorywound-healing response to multiple injuries. The mechanisms of hepatic fibrosis arevery complicated, including the interactions between cells and cells, or cells and matrixs,with lots of cytokines involved. Thus, the molecular mechanisms of hepatic fibrosis arethe hot contents.Connective tissue growth factor (CTGF) is a protein with multiple effects, such asregulation of cellular proliferation, differentiation, and adhesion. So, CTGF playsimportant roles in tissue wound-healing responses and extra cellular matrix (ECM)producing processes. It was confirmed that CTGF was a positive-effect cytokine inhepatic fibrosis. Integrins are receptors located in cellular surface of hepatic stellatecells (HSCs), it was found that CTGF could activate its following cellular signals bycombining with integrins. Focal adhesion kinase (FAK) is a key factor in the integrinsignaling systems, its activation can start the downstream cascade signals. However, themechanisms of the pro-fibrosis effects of CTGF in hepatic fibrosis remain unclear, andthere are no reports about the relationship between CTGF/integrin/FAK signal pathwayand hepatic fibrosis yet.Traditional Chinese medicines have some therapeutic achievements in thetreatment of chronic liver diseases. The compound prescription Gan-Fu-Kang (GFK)has some superiority in hepatic fibrosis treatment, including lots of effectiveconstituents and multiple biological targets. Our previous studies found that GFK couldimprove liver functions, attenuate inflammatory responses, and slow down theprogression of hepatic fibrosis. However, the molecular mechanisms of the protectiveeffects need further research. The aim of this study: First, to observe the expression of CTGF, integrinα5β1,fibronectin, FAK, Akt, mammalian target of rapamycin (mTOR), and ribosomal proteinS6kinase (P70S6K), which are involved in CTGF/integrin/FAK signal pathway inhepatic fibrosis; Second, to study the effects of GFK on the activation ofCTGF/integrin/FAK signal pathway and make further researches on the moleculartherapeutic mechanisms of GFK.Methods:The rats (n=50) were randomly divided into five groups as follows: normal group,model group, GFK high-dose treatment group, GFK medium-dose group and GFKlow-dose group. Rats with liver fibrosis were induced by repeated injection of CCl4(0.5mg/kg in a vehicle of olive oil, s.c., twice per week). Rats in the control group weretreated with olive oil of the same volume.8weeks later, the rats in the three GFKtreatment groups were given GFK31.25,312.5,3125mg/kg, p.o., respectively per day.All the rats were sacrificed at the end of20th week.Rat HSCs were stimulated by acetaldehyde: Continous cultured HSCs weredivided into three groups randomly: control group: HSCs were incubated with bloodserum (10%) from the rats in the control group; model group: HSCs stimulated byacetaldehyde (200μmol/L) were incubated with blood serum (10%) from the rats in thecontrol group; GFK treatment group (stimulated by acetaldehyde+blood serum ofGFK): HSCs stimulated by acetaldehyde (200μmol/L) were incubated with blood serumof GFK (10%).Expression of the key factors in CTGF/integrinα5β1/FAK in hepatic fibrosis ratsand HSCs were analyzed by RT-PCR, IHC, and western blot.Results:1. The expression of CTGF, fibronectin, integrinα5β1in hepatic fibrosis and theinhibition effect of GFKThe results in RT-PCR, western-blot, and IHC showed that the mRNA and proteinlevels of CTGF, fibronectin, integrinα5, and integrinβ1in the model group wereincreased significantly compared with the control group (P﹤0.01); While they weredecreased obviously after GFK treatment (P﹤0.01).In the cultured HSCs, the mRNA level of CTGF, fibronectin, integrinα5, andintegrinβ1in the model group (stimulated by acetaldehyde) was up-regulatedsignificantly when it was compared with the normal group (P﹤0.01); However, it wasdown-regulated in the GFK treatment group compared to the model group (P﹤0.01). 2. The role of FAK/Akt signal pathway in hepatic fibrosis and the regulative effectof GFKComparing with the normal group, the expression of phospho-FAK andphospho-Akt in the livers of CCl4rats was obviously increased (P﹤0.01); And thephosphorylation levels of both FAK and Akt were decreased in the GFK treatmentgroup (P﹤0.01). At the same time, the gene expression of α-smooth muscle actin(α-SMA) in RT-PCR showed the similar results with other factors in this study.The mRNA level of FAK, Akt, and α-SMA in the HSCs of model group wassignificantly up-regulated (P﹤0.01); And it was down-regulated after GFK treatmentcompared with the model group (P﹤0.01).3. The expression of mTOR/P70S6K signal pathway in hepatic fibrosis and theattenuating effect of GFK on mTOR/P70S6K expressionThe mRNA level of mTOR, P70S6K, collagen I, and collagen III in the CCl4ratlivers was significantly higher than the level of above factors in control group (P﹤0.01);While GFK attenuated the expression of these factors obviously in the GFK treated ratlivers (P﹤0.01).The mRNA and protein levels of cyclinD1were increased in the model groupsignificantly (P﹤0.01); While they were decreased after GFK treatment compared withthe model group (P﹤0.01).It was rather lower of the mRNA level of mTOR, P70S6K, cyclinD1, collagen I,and collagen III in the HSCs of control group; While it was up-regulated in the modelgroup (P﹤0.01); After incubated with blood serum of GFK, the mRNA level of abovefactors was down-regulated obviously (P﹤0.01).Conclusion:1. The activation of CTGF/integrinα5β1/FAK signal cascade plays an importantrole in CCl4-induced hepatic fibrosis.2. The proliferation and activation of cultured HSCs are associated with the effectof CTGF/integrinα5β1/FAK signal pathway.3. The molecular mechanisms of the anti-fibrosis activity of GFK may be due to itseffects against CTGF/integrinα5β1/FAk signal pathway. |
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