| Hepatic fibrosis is the final common pathway and pathological changefor a multitude of chronic liver injuries to cirrhosis. Though the pathologicalprocess is reversible, there is a lack of effective drugs with low side effectclinically. Professor Li Diangui who initiated the Traditional ChineseMedicin（eTCM）“Turbidity toxin” theory deems that internal turbidity toxin isone of the main pathogenesis of hepatic fibrosis, according to his clinicalexperience for many years. Guided by the theory of resolving turbidity andtoxin, the prescription composed of Chinese herbs has showed satisfactoryeffects on treating hepatic fibrosis, while the mechanism of action is stillunclear. Hepatic stellate cells(HSC) are the main source of fibroblasts/myofibroblast, and the transformation between the two kinds of cells are thekey link in the process of hepatic fibrosis. The imbalance between synthesisand degradation of extracellular matrix (ECM) is the main pathologicalchange of hepatic fibrosis. The process of hepatic fibrosis is mediated bymultiple signaling pathways and many key factors including multiplecytokines, growth factors and chemotactic factors, all of which are deemed tobe the important targets of hepatic fibrosis. In this study, selecting several keytargets and dividing into three parts, we observed the effect of Huazhuojieduprescription on inhibiting fibrogenesis and its mechanism from different levels,which provides theoretical foundation for the “resolving turbidity and toxin”and “Turbidity toxin” theory in treating hepatic fibrosis.Part1Study on the Effect of Huazhuojiedu Prescription on InhibitingDMN-induced Hepatic Fibrosis via JAK/STAT Signaling Pathway in RatsObjective: To assess the effect of Huazhuojiedu prescription on hepatic fibrosis and discuss the possible action mechanism, we observed theexpression of ColⅠ and ColⅢ mRNA and JAK2/STAT3protein in hepatictissue of rats with DMN-induced hepatic fibrosis.Methods:70male SD rats were divided into4groups, namely normalgroup (Group A), model group (Group B), positive group treated with FufangBiejia Ruangan Pian (Group C), group treated with Huazhuojiedu prescription(subdivided into Sub-group D1, Sub-group D2and Sub-group D3respectivelytreated with equivalent, double and quadrupl dosages). Except Group A, allother groups were intraperitoneally injected with10mg/kg1%DMN. DuringW1of the study, models were made for consecutive3days; during each weekfrom W2to W6, models were made for consecutive2days; during each dayfrom W5to W8, models were administered with different drugsintragastrically in equal volumes. At the end of W8, rats were killed forextracting fresh hepatic tissues, we observed the pathological changes afterMasson staining, and tested the expression of ColⅠ and ColⅢmRNA byRT-PCR and expression of JAK2/STAT3protein in hepatic tissues byWestern-blot.Results:1Hepatic tissue and pathological observations: it was visually observedthat the surface of liver in the rats of Group A was smooth, tenacious andbright red, the surface of liver in rats of Group B contained granular nodes andis hard in texture, the surface of liver in tested rats of Sub-group D2wasinterspersed with fine grain shaped nodes, slightly hard in texture and dark red.Masson staining showed that the hepatic lobule in rats of Group A wasstructurally complete, hepatic cord was arranged in an orderly manner, hepaticlobule in rats of Group B was obviously structurally damaged with fibroustexture thickening and encompassing the hepatic lobule and the hepatic lobulein rats of Sub-group D2was significantly less structurally damaged withfibrous strips thinning.2Effect of Huazhuojiedu prescription on expression levels of ColⅠ andCol Ⅲ mRNA in rat hepatic tissues: Compared to Group A, both Col Ⅰand Col ⅢmRNA in Group B were significantly more expressed (P <0.01).Compared to Group B, both Col Ⅰ and ColⅢmRNA in Sub-group D2weresignificantly less expressed (P <0.01), both Col Ⅰ and ColⅢmRNA in GroupC and Sub-group D1were less expressed(P<0.05), both ColⅠ and ColⅢmRNA in Sub-group D3were insignificantly less expressed and thedifference was statistically not significant (P>0.05).3Effect of Huazhuojiedu prescription on expression of JAK2and STAT3protein: Compared to Group A, both JAK2and STAT3protein in Group Bwere significantly more expressed (P <0.01). Compared to Group B, JAK2protein in Sub-group D1were significantly less expressed (P <0.05), JAK2protein in both Group C and Sub-group D2were less expressed and thedifference was statistically significant (P <0.01), STAT3protein in grouptreated with Huazhuojiedu prescription were insignificantly less expressed andthe difference was statistically not significant (P>0.05).Conclusions: The Huazhuojiedu prescription can improve theDMN-caused hepatic fibrosis in rats and the expression of ColⅠand Col ⅢmRNA from hepatic tissues in rats with hepatic fibrosis to different extents,and the most obvious effect was observed in double dosage. Decreasing theexpression of JAK2protein to medication the JAK2/STAT3signaling pathwaymay be one of the mechanisms of anti-hepatic fibrosis action of thisprescription.Part2Study on the Effect of Huazhuojiedu Prescription on InhibitingDMN-induced Hepatic Fibrosis in Rats by PDGF,CTGF,MCP-1andIL-13Objective: To discuss the anti-hepatic fibrosis action and mechanism ofthe Huazhuojiedu prescription by observing its effects on PDGF-BB in hepatictissues and CTGF, MCP-1, IL-13in serum from models of DMN-inducedhepatic fibrosis in rats.Methods: the expression of PDGF-BB in the hepatic tissues was testedby RT-PCR method and the concentrations of CTGF, MCP-1and IL-13in rat serum were measured by ELISA method.Results:1Effect of Huazhuojiedu prescription on expression of PDGF-BB mRNAin hepatic tissues from rats with hepatic fibrosis: Compared to Group A,PDGF-BB mRNA in Group B was significantly more expressed (P <0.01).Compared to Group B, levels of PDGF-BB mRNA in both Group C andSub-group D1decreased (P <0.05). The level of PDGF-BB mRNA inSub-group D1significantly decreased (P <0.01). The difference betweenSub-group D3and Group B in expression of PDGF-BB mRNA wasstatistically not significant (P>0.05). PDGF-BB mRNA in Sub-group D3wasmore expressed than that in Sub-group D2(P <0.01).2Effect of Huazhuojiedu prescription on level of CTGF in rat serum:Compared to Group A, CTGF in Group B was significantly more expressed (P<0.05). Compared to Group B, CTGF in serum of rats of all of Group C andSub-groups D1, D2and D3was significantly less expressed (P <0.05). Thedifference between Group C and Sub-group D3in expression of CTGF wasstatistically not significant（P=0.063>0.05). CTGF in Sub-group D2wassignificantly less expressed than those in Sub-groups D1and D3(P <0.05).3Effect of Huazhuojiedu prescription on the level of MCP-1in rat serum:Compared to Group A, MCP-1in Group B was significantly more expressed(P <0.01). Compared to Group B, MCP-1in all other sub-groups undertreatment except Sub-group D3was significantly less expressed (P <0.01).MCP-1in Group C was less expressed than that in Sub-groups D1and D3(P<0.01, P <0.05).4Effect of Huazhuojiedu prescription on level of IL-13in rat serum:Compared to Group A, IL-13in Group B was significantly more expressed (P<0.01). Compared to Group B, IL-13in serum of rats of Group C andSub-groups D1, D2and D3was significantly less expressed (P <0.01), IL-13in Group C was significantly less expressed than that in Sub-group D3(P <0.01), IL-13in Sub-group D2is significantly less expressed than those inSub-groups D1and D3(P <0.01, P <0.05). Conclusions: The Huazhuojiedu prescription can decrease the expressionof PDGF-BB in hepatic tissues and the level of CTGF,MCP-1and IL-13inserum with DMN-induced hepatic fibrosis in rats, so we deemed that theabove cell factors were the targets of anti-hepatic fibrosis action of thisprescription.Part3Study on the Effects of Huazhuojiedu Prescription on Suppressionof HSC Activation in Rats via MAPK and PI3K/AKT PathwaysObjective: To assess the effect of Huazhuojiedu prescription containedrat serum on the MAPK and PI3K/AKT signaling pathways and discuss thepossible anti-hepatic fibrosis action mechanism of the Huazhuojieduprescription at molecular level by observing the expressions of p-p38, p-JNK,PI3K and p-AKT in rat HSCs using the serum pharmacological method.Methods: Healthy adult male SD rats were selected and administeredwith different drugs intragastrically according to the following groups, and thecontaining-drug serum was prepared after10Days: normal group (Group A),model group (Group B), positive group (Group C), sub-group (D1) withequivalent dosage of Huazhuojiedu prescription in serum and sub-group (D2)with twice dosage in serum. TGF-β1was firstly added into the serums inGroup B and C and Sub-groups D1and D2. Afterwards, the unactivated ratHSCs were cultivated in vitro with the serums of all the groups.Throughcultivation in vitro for48h after adding with the corresponding serum in eachgroup, the expressions of p-p38, p-JNK, PI3K and p-AKT protein were testedby Western-blot method.Results:1Expression of p-p38: Compared to Group A, p-p38in Group B wassignificantly more expressed (P <0.05). Compared to Group B, p-p38in all ofGroup C and Sub-groups D1and D2was significantly less expressed (P <0.05). Compared to Group C, p-p38in Sub-group D1tended to be moreexpressed but the difference was statistically not significant (P=0.051>0.05).Compared to Sub-group D1and Group C, p-p38in Sub-group D2was less expressed (P <0.05).2Expression of p-JNK: Compared to Group A, p-JNK in Group B wassignificantly more expressed (P <0.05). Compared to Group B, p-JNK in allof Group C and Sub-groups D1and D2was significantly less expressed (P <0.05). Compared to Group C, p-JNK in Sub-group D1and D2was lessexpressed (P <0.05). Compared to Sub-group D1, p-JNK in Sub-group D2was less expressed (P <0.05).3Expression of PI3K: Compared to Group A, PI3K in each group(sub-group) was significantly more expressed (P <0.05). Compared to GroupB, PI3K in Sub-group D2was significantly less expressed (P <0.01).Compared to Group C and Sub-group D1, PI3K in Sub-group D2wassignificantly less expressed (P <0.05).4Expression of p-AKT: Compared with Group A, p-AKT proteinexpression in each group was obviously increased (P <0.05). Compared withGroup B, p-AKT protein expression in Group C and Sub-group D1and D2were obviously decreased (P <0.05), especially the decrease in Sub-group D2was significant (P<0.01). Compared with Sub-group D1, p-AKT proteinexpression in Sub-group D2was obviously decreased (P <0.05).Conclusions: The Huazhuojiedu prescription can decrease theexpressions of p-p38, p-JNK, PI3K and p-AKT in TGF-β1-induced HSC, andmedication the p38, JNK and PI3K/AKT signal transmit pathways, so weconcluded that the mechanism of anti-hepatic fibrosis action of thisprescription at molecular level may lie in the medicating of the MAPK andPI3K/AKT signal transmit pathways. |
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