化瘀通络中药对肝纤维化大鼠的治疗作用及其对血小板衍生生长因子（PDGF）及Ras信号转导通路的影响

  本文关键词：化瘀通络中药对肝纤维化大鼠的治疗作用及其对血小板衍生生长因子（PDGF）及Ras信号转导通路的影响，由笔耕文化传播整理发布。

        目的：肝纤维化（Hepatic fibrosis，HF）是各种慢性肝病发展为肝硬化的一个动态过程及共同病理途径，是肝脏纤维结缔组织过度沉积的结果。肝纤维化是机体对慢性损伤的主动性修复反应,以肝脏细胞外基质（Extra-cellular matrix，ECM）的增生与沉积为特征,形态上表现为肝窦毛细血管化与肝小叶内纤维化，功能上可引起肝功能减退、门静脉高压等，甚至发展成为肝癌，严重影响患者的生命健康。因此，及时治疗阻断肝纤维化的进一步发展意义重大。很多细胞因子参与了肝纤维化的发生发展，血小板衍生生长因子（Platelet derivative growth factor，PDGF）是已知的最强的促肝星状细胞（Hepatic stellate cell，HSC）增殖分化的细胞因子，它能够强烈刺激HSC的增殖、迁移,转化为肌成纤维细胞,促使胶原产生、沉积,在肝纤维化的发生发展中意义重大。国内外学者对肝纤维化的发病机制进行了大量研究，但临床上阻断肝纤维化仍是一个难题，近年来开展的中医药抗肝纤维化的临床和基础研究显示了一定的优势和较大的潜力。化瘀通络中药鳖甲煎丸出自张仲景《金匾要略》，具有化瘀通络、软坚散结之功效，攻补兼施，作用全面。大量实验证实，鳖甲煎丸应对肝纤维化的复杂病机可通过多途径、多靶点的发挥中医药抗肝纤维化的作用。本研究通过对SD大鼠采用皮下注射四氯化碳（CCL4）的方法建立肝纤维化大鼠模型，应用化瘀通络中成药鳖甲煎丸对该模型进行干预，通过观察实验动物的一般情况，检测血清层粘连蛋白（LN）、Ⅳ型胶原（Col-Ⅳ），血清谷丙转氨酶（ALT）、谷草转氨酶（AST）、碱性磷酸酶（ALP）、血清白蛋白（ALB）的含量，观察肝脏病理学形态，检测肝组织PDGF水平，肝组织Ras、细胞外信号调节激酶（Extracellular signal regulated kinase，ERK1）水平，探讨化瘀通络中药对肝纤维化的防治作用及其可能作用机制，为临床上治疗以“气阴两虚、瘀血阻络”为基本病因病机的“癥积”提供实验依据。方法：清洁级健康雄性SD大鼠48只，体重280-320g,适应性饲养一周后按体重随机分为正常对照组（Control）、模型组（Model）、西药组（Thewestern medicine group，简称XY）、化瘀通络中药低剂量组（Low dosetreated）简称ZY（L）、中剂量组（Middle dose treated）简称ZY（M）、高剂量组（High dose treated）简称ZY（G）。除正常组外，模型组、中药各组、西药组均于实验第1天每只大鼠皮下注射40%CCl4花生油5ml/kg，此后每3天注射3ml/kg，同时从实验第一天起，中药各组分别给予化瘀通络中药低、中、高剂量组大鼠鳖甲煎丸0.55g/（kg.d）、1.1g/（kg.d）、2.2g/（kg.d）灌胃，西药组给予秋水仙碱０.3mg/kｇ灌胃，每日一次。从实验第四周末开始，每周末随机处死模型组大鼠，光镜下观察肝脏病理学改变，实验第6周造模成功。造模成功后给予中药各组大鼠及西药组大鼠继续灌胃5周，每日一次。从实验第一天开始，模型组大鼠给予蒸馏水灌胃，共11周。整个实验中，正常组大鼠皮下注射等量花生油，并给予蒸馏水灌胃,每日1次，共6周,6周停止皮下注射花生油，继续给予蒸馏水灌胃5周。于实验第11周末，禁食12小时，水合氯醛麻醉，股动脉放血，分离血清，测定血清ALT、AST、ALP、ALB水平；放射免疫法检测血清LN、Col-Ⅳ水平；摘取大鼠肝脏，留取肝组织，置于4%多聚甲醛固定液中，应用HE染色及Masson染色观察各组大鼠的肝脏病理形态学变化；留取肝组织于液氮速冻后置于-800C低温冰箱内保存，反转录聚合酶联反应（reverse transcription-polymerase chainreaction RT-PCR）法检测肝组织中PDGF含量、Ras蛋白水平；免疫组化方法检测肝组织中ERK1的表达。结果：1各组大鼠的一般情况在实验过程中，正常组除外，其余各组均有死亡情况:模型组死亡3只，中药高剂量组死亡1只，中药低、中剂量组各死亡2只，西药组死亡3只。正常组大鼠体重呈进行性增长，饮食正常，反应敏捷，眼光有神，毛发有光泽。其余各组大鼠一般状况较差，反应迟钝，皮毛松弛，毛色暗淡，潮湿成缕，其中模型组个别大鼠出现双目失明，且脾气急躁易怒，易激惹，形体消瘦。2各组大鼠肝脏病理形态学改变正常组大鼠肝脏形态正常，包膜完整，色淡红，切面光滑整齐。肝小叶结构清晰，肝细胞多为单核，肝板呈条索状，肝细胞围绕中央静脉呈放射状排列。模型组大鼠，肝脏形态异常，表面毛糙、质脆，切面有沙粒感。大鼠肝细胞肿胀变大，肝细胞广泛坏死，肝脏汇管区扩大及炎细胞浸润，肝小叶结构破坏，汇管区纤维结缔组织增生明显，将肝小叶包绕成大小不等的肝细胞团，形成假小叶。化瘀通络中药各剂量组肝小叶结构较清晰，肝窦轻度扩张，肝细胞中度坏死，汇管区纤维结缔组织中度增生，假小叶形成明显较少。西药组肝脏病理变化介于模型组与中药组之间。提示，化瘀通络中药可改善肝纤维化大鼠的肝脏纤维化程度，且相对于西药秋水仙碱效果更明显。3各组大鼠血清ALT、AST、ALP、ALB水平各组大鼠血清ALT、AST、ALP含量结果显示：与正常组相比，模型组、西药组、中药低、中剂量组大鼠血清ALT、AST、ALP含量明显升高（P<0.05）,中药高剂量组与正常组ALT、AST、ALP含量差异无统计学意义；与模型组相比，中药各剂量组、西药组血清ALT、AST、ALP明显降低（P<0.05）；与西药组相比，中药高剂量组血清ALT、AST、ALP含量明显减低（P<0.05），中药低剂量组含量较高（P<0.05），中药中剂量组与之差异无统计学意义；中药各剂量组之间相比，中药低剂量组血清ALT、AST、ALP含量高，中药高剂量组含量最低，中药中剂量组居中，中药各剂量组间存在显著性差异（P<0.05）。提示，化瘀通络中药可明显降低肝纤维化大鼠血清ALT、AST、ALP水平，有助于肝纤维化的治疗。各组大鼠血清ALB含量的结果显示：与正常组相比，模型组、西药组、中药低、中剂量组大鼠血清ALB含量明显降低（P<0.05）,中药高剂量组与正常组差异无统计学意义；与模型组相比，西药组、中药高、中剂量组血清ALB含量明显升高（P<0.05），中药低剂量组与之差别无统计学意义；与西药组相比，中药高剂量组血清ALB含量明显升高，中药低剂量组血清ALB水平降低（P<0.05），中药中剂量组与之相比差别无统计学意义；中药各剂量组间血清ALB水平两两比较差异，其中，中药高剂量组血清ALB含量最高，中药中剂量组次之，中药低剂量组含量最低（P<0.05）。提示，化瘀通络中药可使血清ALB水平下降趋势得到扭转，有助于肝纤维化的治疗。4各组大鼠血清LN、Col-Ⅳ水平各组大鼠血清LN、Col-Ⅳ含量结果示：与正常组相比，模型组、西药组、中药低、中剂量组大鼠血清LN、Col-Ⅳ含量明显升高（P<0.05）,中药高剂量组与正常组差异无统计学意义；与模型组相比，中药各剂量组、西药组血清LN、Col-Ⅳ明显降低（P<0.05）；与西药组相比，中药高剂量组血清LN、Col-Ⅳ含量明显减低（P<0.05），中药低剂量组含量较高（P<0.05），中药中剂量组与之差异无统计学意义；中药各剂量组之间相比，中药低剂量组血清LN、Col-Ⅳ含量高，中药高剂量组含量最低，中药中剂量组居中，中药各剂量组间存在显著性差异（P<0.05）。此结果提示，化瘀通络中药可明显降低肝纤维化大鼠血清LN、Col-Ⅳ含量，此可能为其治疗肝纤维化的机制之一。5化瘀通络中药对肝纤维化大鼠Ras/ERK信号转导途径的影响5.1各组大鼠肝脏组织PDGF mRNA含量、Ras蛋白mRNA水平各组大鼠肝组织PDGF mRNA、Ras mRNA含量的表达结果显示：与正常组相比，其余各组PDGF mRNA、Ras mRNA含量均升高（P<0.05），其中模型组含量最高，中药低剂量组次之，西药组、中药中剂量组再次之，中药高剂量组含量表达最低（P<0.05）；与模型组相比，余各组肝组织PDGFmRNA、Ras mRNA含量的表达均较低（P<0.05）；与西药组相比，中药中剂量组与之差异无统计学意义；中药各剂量组之间比较有统计学意义（P<0.05），其中，中药低剂量组表达较高，中药中剂量组次之，中药高剂量组肝组织PDGF mRNA、Ras mRNA含量的表达最低。5.2各组大鼠肝组织ERK1含量表达正常组大鼠肝组织中ERK1呈棕黄色弱阳性表达，主要可见于小叶中央静脉四周、汇管区及肝索Disse腔间隙中，高倍镜下观察可见ERK1位于Disse间隙的肝星状细胞胞质中，肝细胞中未见表达；模型组大鼠肝组织中ERK1表达明显增多、增强，汇管区、小叶中央静脉周围及肝小叶内均可见大量星芒状、条索状ERK1阳性表达，高倍镜下观察可见肝星状细胞胞质、胞核为ERK1阳性表达的分布区域；其余西药组、中药各剂量组阳性染色程度较模型组明显减轻，纤维隔内间质细胞及炎细胞阳性染色程度减轻，ERK1阳性细胞数目减少，其中中药高剂量组ERK1阳性程度最低，，西药组、中药中剂量组之间差异无统计学意义（P>0.05），中药低剂量组ERK1阳性程度较中药高、中剂量组稍高，但较模型组较低（P<0.05）。提示，化瘀通络中药可明显降低肝组织中PDGF mRNA的含量并减少Ras蛋白mRNA的表达，明显减少大鼠肝组织ERK1的表达，显著改善大鼠抗肝纤维化能力，此可能为其治疗肝纤维化的机制之一。结论：1化瘀通络中药可改善肝纤维化大鼠的一般状况，改善肝功能。2化瘀通络中药可明显改善肝纤维化大鼠肝脏纤维化程度，对于肝纤维化有良好的治疗作用。3化瘀通络中药可明显降低肝纤维化大鼠血清LN、Col-Ⅳ水平，减少二者在肝Disse间隙沉积,从而减少ECM蓄积,此可能为其治疗肝纤维化的机制之一。4化瘀通络中药可明显下调肝纤维化大鼠肝脏组织中PDGF mRNA、Ras蛋白mRNA以及ERK1的表达，提示其抗肝纤维化作用可能是通过激活Ras ERK1/2信号转导通路实现的

    Objective: hepatic fibrosis is a dynamic process and pathology whichcan developing to cirrhosis by various chronic liver disease, it’s the result ofliver fibre connective tissue over sedimentation. Liver fibrosis is self recoveryreaction of organism to chronic damage, based on the feature of liverextra-cellular matrix hyperplasia and sedimentation, blood capillarization andhepatic lobule fibrosis is shown in the hepatic sinus, liver function slack up,hyperpiesia of portal vein, such as splenomegaly, ascites… in the functionality,even worse, it develops to liver cancer and influences patients quality of lifeseriously, therefore, treatment in time and blocking the further development ofliver fibrosis is significant. Lots of cell factor participated in the generationand development of liver fibrosis, PDGF is a strongest cell factor currentlywhich promotes the HSC increase and differentiation. It irritates increase,moving and inversion to the myofibroblast of HSC strongly, inducing thegeneration of collagen, sedimentation is significant in the liver fibrosisdevelopment. The researchers at home and abroad conducted a mass of studiesfor the liver fibrosis, but blocking of liver fibrosis is still difficult in clinical,recently, Using the Chinese traditional medicine to prevent the liver fibrosisindicates the certain advantage and great potential in clinical as well as baseresearch. Chinese traditional medicine Biejiajianwan comes from Jinguiyaoluewhich wrote by Zhang zhongjing, it promotes the blood circulate, removingthe blood stasis, using the gentle method and force, attack the virus andreplenish the body both together, and covered all. Demonstrated by a greatnumber of experiment, Biejiajianwan as a kind of Chinese traditional medicinehas the function for liver fibrosis complex pathogenesis via muti-pathway and muti-target spots to anti the liver fibrosis. This researchh will take thesubcutaneous injection of CCL4method for SD mouse to establish the liverfibrosis mouse module, Biejiajianwan, which promote the blood circulate andremove the blood stasis is put in use to interpose for the model. Observing thegeneral situation of experiment animal, inspect the content of LN, Col-IV, ALT,AST, ALP, ALB. Observing the liver nosology shape, inspecting liver tissuelevel of PDGF, liver tissue Ras/extracellular signal regulated kinase，ERK1,discuss the prevent function and possible affective principle of Chinesetraditional medicine which promote the blood circulate and remove the bloodstasis. It provide the experiment basis to cure the Ganji cause byextravasated blood blocking, upright deficient which defined as basicpathogeny.Methods:48healthy male SD rats, weighted280-320g, after raising oneweek, split to6groups: control, model,the western medicine group,Biejiajianwan low dose treated, middle dose treated, high dose treated. Exceptthe control group, every rats of other groups will be injected the40%CCL4peanut oil5mL/Kg in the subcutaneous from the first day, then, inject3ml/kgevery3days, meanwhile, Biejiajianwan0.55g/（kg.d）,1.1g/（kg.d）,2.2g/（kg.d）will be given to stomach for every Chinese traditional medicine lowdose,middle dose and high dose group except the western medicine group, thecolchicin0.3mg/kg will be given to the stomach for western medicine group,once per day. At the beginning of the forth weekends, execute the rats in themodel randomly, and observe the liver nosology change, the modelsuccessfully created after sixth week from experiment. Then, medicine will begiven to the stomach of rats in each Chinese medicine group for five weeks,once per day. The distilled water is put into the stomach of rats in model groupfor11weeks. The same quantity of Peanut oil is shot to the subcutaneous ofrats in control group and distilled water is put into the stomach, once per day,at the total of6weeks, after6weeks, stop the peanut oil injection and putdistilled water in the stomach for5weeks. At the end of11thweek, abrosia12hours, arteria cruralis Blood Sampling are separated, measuring the serum ALT, AST, ALP, ALB level, detect the serum LN, Col-IV level, according toradioimmunoassay, take the liver, put the liver tissue in the4%paraformaldehyde, dyed by HE and Masson, observe liver nosology change ofmouse in each group, then,preserve the liver tissue in the refrigerator with-80degree after quick-freezing by the liquid nitrogen, inspect the content of PDGF,Ras protein level in the liver tissue in accordance with reversetranscription-polymerase chain reaction RT-PCR.Results:1General condition of experimental ratsDuring the experiment, the death happened in each group except thecontrol group,3rats dead in model group,1dead in high dose medicinetreated,2in low dose treated group and middle dose treated group,3inwestern medicine group. The weights of rats in the control group areincreasing, regulate diet, quickly react, bright eye, smoothly hair. Thecondition of the rats in other groups are poor, slow in reacting, loose hair anddamp, dull color, some of the rats in the model group blind and bad temper,skinny figure.2The pathology changes of the rat,hepatic tissue in different groupIn the normal group, integrate diolame, light red color, smooth and tidysection. hepatic lobule structure clear, most of the liver cell are mononuclear,strip liver surface, liver cell shows radial pattern and surround the center vein.Regarding on the model group, the shape of liver is abnormally, coarse surface,feels like the sand particle. Liver cell become swell and getting bigger, most ofcells thanatosis, the vessels expanded and inflammation cells found, thestructure of liver lobule damaged, fibers connective tissue in the veins area hasobvious hyperplasia, the liver lobule is surrounded to different size of livercell bunches and take shape to pseudolobule. The liver lobule in the each doseof Chinese medicine group which can promote the blood circulate and removethe stasis has clear structure. Liver sinus expand slightly, liver cells necrotic.fibers connective tissue in the veins area has moderate hyperplasia, theformation of pseudolobule decreasing distinctly, the liver nosology change of western medicine group is in the middle of western group and Chinesemedicine group. Note: the medicine which promote the blood circulate andremove the stasis improves the liver fibrosis level in the liver fibrosis rats, andfunction better than western medicine colchicines.3The levels of ALT, AST, ALP, and ALB in the serum of different groupsThe level of ALT, AST and ALP in the serum of different groups display:compared with normal group, the content of ALT, AST, ALP in the rats serumincrease obviously （P<0.05） in model group, western medicine group andChinese medicine low dose group and Chinese medicine middle dose group,and there were no significant differences between the normal group andChinese medicine high dose treated group （p>0.05）. Compare with modelgroup, the serum ALT, AST, ALP content decrease obviously （P<0.05） in eachChinese medicine group and western medicine group. Compare with westernmedicine group, serum ALT, AST, ALP content decrease obviously （P<0.05）in Chinese medicine high dose treated group, more content in the Chinesemedicine low dose treated group（P<0.05）, there is no meaning to comparewith Chinese medicine middle dose treated group（p>0.05）. compare in eachgroups of Chinese medicine group, the serum ALT、AST、ALP content mostthan others in low dose group, and the serum ALT、AST、ALP content lest thanothers in high dose group, the middle dose group in the middle place, distinctdiscrepancy exist in each Chinese medicine dose group （P<0.05）, Note: theChinese traditional medicine which promote the blood circulate and removethe stasis can decrease serum ALT, AST, ALP level of live fibrosis rats,improve the treatment of liver fibrosis.The level of ALB in the serum of different groups display: comparedwith normal group, the serum ALB declined obviously （P<0.05） in modelgroup, western medicine group, Chinese medicine low dose treated group andmiddle dose treated group, and there were no significant differences betweenthe normal group and Chinese medicine high dose treated group （p>0.05）.compared with model group, serum ALB content increase obviously （P<0.05）in western medicine group, Chinese medicine high dose treated group and middle dose group, There isn’t any meaning to compare discrepancy withChinese medicine low dose treated group. compared with western medicinegroup, serum ALB content increase obviously in Chinese medicine high dosetreated group, serum ALB content level decrease （P<0.05） in Chinesemedicine middle dose treated group, there isn’t any meaning to compare withmiddle dose group. there is discrepancy existing among each Chinesemedicine group serum ALB level. There into, high dose treated has the mostserum ALB content, middle dose group follows in second place, low dosetreated group has the lowest content （P<0.05）, Note: the medicine whichpromote the blood circulate and remove the stasis can turnaround the serumALB level decreasing trend, and improve the treatment for liver fibrosis.4The levels of LN and Col-IV in the serum of different groupsThe rats serum LN, Col-IV content indicates: compare with normal group,rats serum LN, Col-IV content in model, western, Chinese medicine low doseand middle dose increase obviously （P<0.05）,, and there were no significantdifferences between the normal group and Chinese medicine high dose treatedgroup （p>0.05）. compare with western medicine, the serum LN, Col-IVcontent decrease obviously （P<0.05） in the Chinese medicine high dose treatedgroup, higher content level in low dose medicine group （P<0.05）, no meaningto compare the discrepancy with middle dose treated group（p>0.05）. compareamong each dose medicine treated group, serum in Chinese medicine low dosetreated group has highest LN, Col-IV content, high dose group has lowestcontent and middle dose group in the middle place, the obvious discrepancyexisting in each dose treated medicine group（P<0.05）.The conclusion indicatesthat Chinese traditional medicine which promote the blood circulate andremove stasis can decrease the liver fibrosis serum LN, Col-IV content, maybethis is one of the method which treat liver fibrosis.5The levels of PDGF mRNA and Ras protein mRNA in the hepatic tissue ofdifferent groupsLiver tissue PDGF mRNA, Ras mRNA content indicates: compare withnormal group, the content of PDGF mRNA, Ras mRNA increased （P<0.05） in each group, thereinto, model group has the highest content, Chinese medicinelow dose group follows, western medicine, middle dose group at the middleplace, high dose group has the lowest content （P<0.05）. compare with themodel group, PDGF mRNA, Ras mRNA is shown as lower content in the livertissue, compare with western medicine, no meaning to compare thediscrepancy with Chinese medicine middle dose group（P>0.05）. compareamong each dose Chinese medicine group has the statistics meaning（P<0.05）,thereinto, low dose has the highest content, middle dose follows, liver tissuehas lowest PDGF mRNA, Ras mRNA content in the Chinese medicine highdose treated group.6The ERK1content of liver tissue in each group indicationIn the normal group, ERK1in liver tissue shows slight positive andyellow color, especially surround center vein of liver lobule, manifold, andDisse chamber, ERK1in the cytoplasm of liver star shape cells observed byhigh-power microscope, liver cells aren’t shown; regarding on the modelgroup, ERK1in the liver tissue increasing, enhance, ERK1as star shape andstrip shape is shows as positive surrounding the center vein and in the liverlobule，positive ERK1is shown in the area of the liver star cytoplasm andnucleus observed by high-power microscope, in western medicine and eachChinese medicine group, positive dyeing degree is decreased obviously,dyeing degree of interstitial cell between fibro and inflammatory cells positivedecrease, quantity of ERK1positive cells decrease, thereinto, ERK1has thelowest positive in Chinese medicine high dose group, and the highest positivein Chinese medicine low dose group, and no meaning to compare amongwestern and Chinese medicine middle dose. Note: the Chinese medicine whichpromote the blood circulate and remove stasis can decrease the PDGF mRNAcontent in the liver tissue and decrease the mRNA in the Ras protein obviously,decrease the ERK1in the rats liver tissue, improve the capability of anti liverfibrosis, maybe this is one of methods to cure liver fibrosis.Conclusions:1The Chinese traditional medicine which promote blood circulate and remove the stasis can improve the general situation of rats liver fibrosis, andimprove the liver function.2The Chinese traditional medicine which promote the blood circulateand remove the stasis can improve the rats liver fibrosis level, it’s a welltreatment for liver fibrosis.3The Chinese traditional medicine which promote blood circulate andremove the stasis can decrease the serum LN, Col-IV levels, decrease thesedimentation in the liver Disse, therefore, decrease the accumulation of ECM,maybe this is one of methods to cure liver fibrosis.4The Chinese traditional medicine which promote blood circulate andremove the stasis can decrease the PDGF mRNA and Ras protein mRNA inthe liver tissue of liver fibrosis, decrease the ERK1in the rats liver tissueobviously, improve the capacity anti liver fibrosis via activateRas/ERK1/2signal transmit route to achieve this, maybe this is one of themethods to prevent liver fibrosis.

        化瘀通络中药对肝纤维化大鼠的治疗作用及其对血小板衍生生长因子（PDGF）及Ras信号转导通路的影响

中文摘要4-9英文摘要9-15前言16-17材料与方法17-24结果24-28附图28-37附表37-40讨论40-49结论49-50参考文献50-54综述 血小板衍生生长因子在肝纤维化发生中的作用54-64    参考文献59-64致谢64-65个人简历65

  本文关键词：化瘀通络中药对肝纤维化大鼠的治疗作用及其对血小板衍生生长因子（PDGF）及Ras信号转导通路的影响，由笔耕文化传播整理发布。