“浊毒”立论组方经相关信号通路抑制DMN致肝纤维化及HSC活化的作用研究
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摘要
肝纤维化（liver fibrosis/hepatic fibrosis）是各种慢性肝病向肝硬化发展所共有的病理改变和必经途径，虽是可逆的病理过程，但治疗上仍缺乏有效、副作用小的药物。导师李佃贵教授首创了中医“浊毒”理论，他经多年临证认为，浊毒内蕴是肝纤维化的主要病机之一。以化浊解毒法为指导，选用中药组成化浊解毒方治疗肝纤维化取得了满意的疗效，但其治疗机制尚未明确。肝星状细胞（hepatic stellate cell,HSC）作为成纤维细胞/肌成纤维细胞的主要来源，是肝纤维化发生发展的中心环节，细胞外基质(extracellular matrix, ECM）的合成与降解失衡是肝纤维化的主要病理改变。多种细胞因子、生长因子、趋化因子是肝纤维化的关键因子，多条信号通路参与了肝纤维化的调控过程，它们被确认为肝纤维化形成的重要靶点。本论文分三个部分，从体内、体外不同水平上选取部分关键靶点，观察了化浊解毒方抗肝纤维化的作用和机制，为化浊解毒法和“浊毒”理论治疗肝纤维化提供了依据。
第一部分化浊解毒方经JAK/STAT通路抑制DMN致大鼠肝纤维化的作用研究
目的：通过观察化浊解毒法组方对二甲基亚硝胺（DMN）致大鼠肝纤维化模型中肝组织Ⅰ型胶原蛋白（ColⅠ)、Ⅲ型胶原蛋白（ColⅢ) mRNA以及JAK2/STAT3蛋白的表达情况，评价化浊解毒方对大鼠肝纤维化的调节作用，探讨其可能的作用机制。
方法：清洁级SD雄性大鼠70只，分为4组，即：正常对照组（A组）、模型对照组（B组）、复方鳖甲软肝片组（C组）、化浊解毒方组（分为等效、二倍、四倍剂量组，即D1、D2、D3组）、，除A组外,各组均予1%DMN10mg/kg腹腔内注射，实验第1周连续造模3天，第2到6周，每周连续造模2天，第5到8周，每天等体积灌胃给与不同药物。第8周末处死大鼠并取新鲜肝组织，用Masson染色观察肝组织病理情况；运用RT-PCR法检测肝组织中ColⅠ、ColⅢmRNA的表达；用Western-blot法检测JAK2/STAT3蛋白表达情况。
结果：
1肝组织及病理学情况观察：肉眼观察A组大鼠肝脏表面光滑、质韧，色鲜红；B组大鼠肝脏表面有颗粒状结节，质硬；D2组受试大鼠肝脏表面散在细砂粒样结节，质稍硬，色暗红。Masson染色情况显示：A组大鼠肝小叶结构完整，肝索排列整齐；B组纤维纵膈较多，纤维组织增粗、包绕肝小叶；D1、D2和C组纤维纵膈均较B组减轻，纤维条索变细；D3组纤维条索较粗。
2化浊解毒方对大鼠肝组织中ColⅠ、Col ⅢmRNA表达的影响：与A组相比，B组ColⅠ、Col Ⅲ表达均明显增多（P <0.01)。与B组相比， C及D1组ColⅠ、Col Ⅲ表达均降低（P <0.05)；D2组ColⅠ、Col Ⅲ表达显著降低（P <0.01)；D3组ColⅠ、Col Ⅲ表达降低不明显（P>0.05)。
3化浊解毒方对大鼠肝组织中JAK2、STAT3蛋白表达的影响：与A组相比，B组JAK2、STAT3蛋白表达均明显增多（P <0.01)。与B组相比，D1组JAK2蛋白表达降低（P <0.05)；C、D2组JAK2蛋白表达显著降低（P <0.01)；除C组外，各给药组STAT3蛋白表达较B组差异均无统计学意义（P>0.05)。
结论：化浊解毒方能够改善DMN致大鼠肝纤维化的程度，可以不同程度的改善肝纤维化大鼠肝组织ColⅠ、ColⅢ的情况，以二倍剂量效果最为明显。通过降低JAK2蛋白表达，从而调节JAK2/STAT3通路，可能是其发挥抗肝纤维化的作用机制之一。
第二部分化浊解毒方调节PDGF、CTGF、MCP-1和IL-13抑制DMN致大鼠肝纤维化的作用研究
目的：通过观察以化浊解毒法组方对DMN致大鼠肝纤维化模型中肝组织血管内皮生长因子-BB（PDGF-BB）、血清中转化生长因子-β1（TGF-β1）、结缔组织生长因子（CTGF）、单核细胞趋化蛋白-1（MCP-1）和白细胞介素-13（IL-13）含量的影响，探讨化浊解毒方经细胞因子抗肝纤维化的作用和机制。
方法：运用RT-PCR法检测肝组织中PDGF-BB mRNA的表达，运用ELISA法检测大鼠血清中CTGF、MCP-1、IL-13的含量。
结果：
1化浊解毒方对大鼠肝组织PDGF-BB mRNA表达的影响：与A组比较，B组PDGF-BB mRNA的表达显著增加（P <0.01）；与B组比较，C、D1组PDGF-BB mRNA表达均降低（P <0.05）；D2组的PDGF-BB mRNA降低更为显著（P <0.01）；D3组与B组PDGF-BB mRNA的表达差异无统计学意义（P>0.05）；D3组PDGF-BB mRNA的表达较D2组显著升高（P <0.01）。
2化浊解毒方对大鼠血清中CTGF含量的影响：与A组相比，B组CTGF含量增多（P <0.05)；与B组相比，各给药组大鼠血清CTGF含量均降低（P <0.05)；与C组相比，D3组含量有增多趋势，但无统计学意义（P=0.063>0.05)；D2组CTGF含量较C组、D1、D3组均降低（P <0.05)。
3化浊解毒方对大鼠血清中MCP-1含量的影响：与A组相比，B组MCP-1表达明显增多（P <0.01)；与B相比，除D3组外（P>0.05)，各治疗组MCP-1含量均明显降低（P <0.01)；C组MCP-1含量较D1、D3组降低（P <0.01，P <0.05)。
4化浊解毒方对大鼠血清中IL-13含量的影响：与A组相比，B组IL-13含量明显增多（P <0.01)；与B组相比，各给药组大鼠血清IL-13含量均明显降低（P <0.01)；C组IL-13含量较D3组显著降低（P <0.01)；D2组IL-13含量与D1、D3组相比显著降低（P <0.01，P <0.05)。
结论：化浊解毒方可减少DMN致肝纤维化大鼠肝组织中PDGF-BB的表达及血清中CTGF、MCP-1、IL-13的含量，推测上述细胞因子是化浊解毒方抗肝纤维化的作用靶点之一。
第三部分化浊解毒方含药血清经MAPK及PI3K/AKT通路抑制大鼠HSC活化的作用研究
目的：运用血清药理学方法观察大鼠HSC中p-p38、p-JNK、PI3K及p-AKT蛋白的表达，评价化浊解毒方大鼠含药血清对MAPK、PI3K/AKT信号转导通路的作用，探讨化浊解毒方抗肝纤维化可能的分子水平作用机制。
方法：选取健康成年雄性SD大鼠，按分组要求灌胃给予相应药物10天后制备含药血清，分为：正常对照组（A组）、模型对照组（B组）、阳性对照组（C组）、化浊解毒方含药血清等效剂量(D1)和二倍剂量（D2）组，B、C、D1、D2组均先行加入TGF-β1干预。各组加入相应血清培养48h后，利用Western-blot法检测p-p38、p-JNK、PI3K及p-AKT蛋白的表达。
结果：
1p-p38蛋白的表达：与A组比较，B组p-p38蛋白的表达明显增强（P <0.05）；与B组比较，各给药组p-p38蛋白表达显著降低（P <0.05）；与C组比较，D1组p-p38蛋白表达有增高趋势，但差异无统计学意义（P=0.051>0.05），D2组较C组及D1组p-p38蛋白表达均降低（P <0.05）。
2p-JNK蛋白的表达：与A组比较，B组p-JNK蛋白的表达增强（P <0.05）；与B组比较，各给药组p-JNK蛋白的表达降低（P <0.05）。与C组比较，D1、D2组p-JNK蛋白的表达均降低（P <0.05）；D2组p-JNK蛋白的表达低于D1组（P <0.05）。
3PI3K蛋白的表达：与A组比较，各受试组PI3K蛋白表达均升高（P<0.05）；与B组比较，D2组PI3K蛋白表达显著降低（P <0.01）；D2组PI3K蛋白表达低于C组和D1组（P <0.05）。
4p-AKT蛋白的表达：与A组比较，各受试组p-AKT蛋白的表达升高（P <0.05）；各给药组p-AKT蛋白表达均较B组降低，差异有统计学意义（P <0.05），其中D2组差异有显著性（P <0.01）；D2与D1组比较，p-AKT蛋白表达降低，（P <0.05）。
结论：化浊解毒方含药血清能够降低p-p38、p-JNK、PI3K、p-AKT蛋白在TGF-β1诱导的HSC中的表达，从而调控p38、JNK及PI3K/AKT信号通路的传导，推测化浊解毒方发挥抗肝纤维化的分子水平作用机制与调节MAPK及PI3K/AKT信号通路有关。
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 Rats
Objective: 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-13
Objective: 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 Pathways
Objective: 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.
第一部分化浊解毒方经JAK/STAT通路抑制DMN致大鼠肝纤维化的作用研究
目的：通过观察化浊解毒法组方对二甲基亚硝胺（DMN）致大鼠肝纤维化模型中肝组织Ⅰ型胶原蛋白（ColⅠ)、Ⅲ型胶原蛋白（ColⅢ) mRNA以及JAK2/STAT3蛋白的表达情况，评价化浊解毒方对大鼠肝纤维化的调节作用，探讨其可能的作用机制。
方法：清洁级SD雄性大鼠70只，分为4组，即：正常对照组（A组）、模型对照组（B组）、复方鳖甲软肝片组（C组）、化浊解毒方组（分为等效、二倍、四倍剂量组，即D1、D2、D3组）、，除A组外,各组均予1%DMN10mg/kg腹腔内注射，实验第1周连续造模3天，第2到6周，每周连续造模2天，第5到8周，每天等体积灌胃给与不同药物。第8周末处死大鼠并取新鲜肝组织，用Masson染色观察肝组织病理情况；运用RT-PCR法检测肝组织中ColⅠ、ColⅢmRNA的表达；用Western-blot法检测JAK2/STAT3蛋白表达情况。
结果：
1肝组织及病理学情况观察：肉眼观察A组大鼠肝脏表面光滑、质韧，色鲜红；B组大鼠肝脏表面有颗粒状结节，质硬；D2组受试大鼠肝脏表面散在细砂粒样结节，质稍硬，色暗红。Masson染色情况显示：A组大鼠肝小叶结构完整，肝索排列整齐；B组纤维纵膈较多，纤维组织增粗、包绕肝小叶；D1、D2和C组纤维纵膈均较B组减轻，纤维条索变细；D3组纤维条索较粗。
2化浊解毒方对大鼠肝组织中ColⅠ、Col ⅢmRNA表达的影响：与A组相比，B组ColⅠ、Col Ⅲ表达均明显增多（P <0.01)。与B组相比， C及D1组ColⅠ、Col Ⅲ表达均降低（P <0.05)；D2组ColⅠ、Col Ⅲ表达显著降低（P <0.01)；D3组ColⅠ、Col Ⅲ表达降低不明显（P>0.05)。
3化浊解毒方对大鼠肝组织中JAK2、STAT3蛋白表达的影响：与A组相比，B组JAK2、STAT3蛋白表达均明显增多（P <0.01)。与B组相比，D1组JAK2蛋白表达降低（P <0.05)；C、D2组JAK2蛋白表达显著降低（P <0.01)；除C组外，各给药组STAT3蛋白表达较B组差异均无统计学意义（P>0.05)。
结论：化浊解毒方能够改善DMN致大鼠肝纤维化的程度，可以不同程度的改善肝纤维化大鼠肝组织ColⅠ、ColⅢ的情况，以二倍剂量效果最为明显。通过降低JAK2蛋白表达，从而调节JAK2/STAT3通路，可能是其发挥抗肝纤维化的作用机制之一。
第二部分化浊解毒方调节PDGF、CTGF、MCP-1和IL-13抑制DMN致大鼠肝纤维化的作用研究
目的：通过观察以化浊解毒法组方对DMN致大鼠肝纤维化模型中肝组织血管内皮生长因子-BB（PDGF-BB）、血清中转化生长因子-β1（TGF-β1）、结缔组织生长因子（CTGF）、单核细胞趋化蛋白-1（MCP-1）和白细胞介素-13（IL-13）含量的影响，探讨化浊解毒方经细胞因子抗肝纤维化的作用和机制。
方法：运用RT-PCR法检测肝组织中PDGF-BB mRNA的表达，运用ELISA法检测大鼠血清中CTGF、MCP-1、IL-13的含量。
结果：
1化浊解毒方对大鼠肝组织PDGF-BB mRNA表达的影响：与A组比较，B组PDGF-BB mRNA的表达显著增加（P <0.01）；与B组比较，C、D1组PDGF-BB mRNA表达均降低（P <0.05）；D2组的PDGF-BB mRNA降低更为显著（P <0.01）；D3组与B组PDGF-BB mRNA的表达差异无统计学意义（P>0.05）；D3组PDGF-BB mRNA的表达较D2组显著升高（P <0.01）。
2化浊解毒方对大鼠血清中CTGF含量的影响：与A组相比，B组CTGF含量增多（P <0.05)；与B组相比，各给药组大鼠血清CTGF含量均降低（P <0.05)；与C组相比，D3组含量有增多趋势，但无统计学意义（P=0.063>0.05)；D2组CTGF含量较C组、D1、D3组均降低（P <0.05)。
3化浊解毒方对大鼠血清中MCP-1含量的影响：与A组相比，B组MCP-1表达明显增多（P <0.01)；与B相比，除D3组外（P>0.05)，各治疗组MCP-1含量均明显降低（P <0.01)；C组MCP-1含量较D1、D3组降低（P <0.01，P <0.05)。
4化浊解毒方对大鼠血清中IL-13含量的影响：与A组相比，B组IL-13含量明显增多（P <0.01)；与B组相比，各给药组大鼠血清IL-13含量均明显降低（P <0.01)；C组IL-13含量较D3组显著降低（P <0.01)；D2组IL-13含量与D1、D3组相比显著降低（P <0.01，P <0.05)。
结论：化浊解毒方可减少DMN致肝纤维化大鼠肝组织中PDGF-BB的表达及血清中CTGF、MCP-1、IL-13的含量，推测上述细胞因子是化浊解毒方抗肝纤维化的作用靶点之一。
第三部分化浊解毒方含药血清经MAPK及PI3K/AKT通路抑制大鼠HSC活化的作用研究
目的：运用血清药理学方法观察大鼠HSC中p-p38、p-JNK、PI3K及p-AKT蛋白的表达，评价化浊解毒方大鼠含药血清对MAPK、PI3K/AKT信号转导通路的作用，探讨化浊解毒方抗肝纤维化可能的分子水平作用机制。
方法：选取健康成年雄性SD大鼠，按分组要求灌胃给予相应药物10天后制备含药血清，分为：正常对照组（A组）、模型对照组（B组）、阳性对照组（C组）、化浊解毒方含药血清等效剂量(D1)和二倍剂量（D2）组，B、C、D1、D2组均先行加入TGF-β1干预。各组加入相应血清培养48h后，利用Western-blot法检测p-p38、p-JNK、PI3K及p-AKT蛋白的表达。
结果：
1p-p38蛋白的表达：与A组比较，B组p-p38蛋白的表达明显增强（P <0.05）；与B组比较，各给药组p-p38蛋白表达显著降低（P <0.05）；与C组比较，D1组p-p38蛋白表达有增高趋势，但差异无统计学意义（P=0.051>0.05），D2组较C组及D1组p-p38蛋白表达均降低（P <0.05）。
2p-JNK蛋白的表达：与A组比较，B组p-JNK蛋白的表达增强（P <0.05）；与B组比较，各给药组p-JNK蛋白的表达降低（P <0.05）。与C组比较，D1、D2组p-JNK蛋白的表达均降低（P <0.05）；D2组p-JNK蛋白的表达低于D1组（P <0.05）。
3PI3K蛋白的表达：与A组比较，各受试组PI3K蛋白表达均升高（P<0.05）；与B组比较，D2组PI3K蛋白表达显著降低（P <0.01）；D2组PI3K蛋白表达低于C组和D1组（P <0.05）。
4p-AKT蛋白的表达：与A组比较，各受试组p-AKT蛋白的表达升高（P <0.05）；各给药组p-AKT蛋白表达均较B组降低，差异有统计学意义（P <0.05），其中D2组差异有显著性（P <0.01）；D2与D1组比较，p-AKT蛋白表达降低，（P <0.05）。
结论：化浊解毒方含药血清能够降低p-p38、p-JNK、PI3K、p-AKT蛋白在TGF-β1诱导的HSC中的表达，从而调控p38、JNK及PI3K/AKT信号通路的传导，推测化浊解毒方发挥抗肝纤维化的分子水平作用机制与调节MAPK及PI3K/AKT信号通路有关。
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 Rats
Objective: 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-13
Objective: 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 Pathways
Objective: 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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