肝癌前病变中医证候特点及抗纤抑癌方对其HGF和c-Met mRNA调控作用研究
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摘要
目的
原发性肝癌(primary hepatic carcinoma, PHC)是一类发生率和死亡率均很高的疾病,严重威胁着人类健康。大多数肝癌患者都与乙型肝炎病毒(hepatitis B virus, HBV)感染有关。合并有HBV感染的肝硬化,尤其是伴随异型增生结节出现者,其发展为肝癌的几率高,被视为肝癌前病变。虽然近几十年PHC的治疗方法得到了一定发展和完善,但其临床疗效仍未得到明显的提高。阻止或延缓肝癌前病变的发生发展,预防肝癌疾病的发生,就显得具有非常重要的现实意义。本课题在导师前期的临床及实验的工作基础之上,针对肝癌前病变(乙型肝炎肝硬化腹水(肝内结节>1cm))患者的中医证型做初步研究,并从肝细胞生长因子(hepatic growth factor, HGF)及其受体(c-Met)与α平滑肌肌动蛋白(a-smooth muscle actin,α-SMA)之间的相互关系去探讨抗纤抑癌方防治肝癌前病变的作用机制。
方法
1临床研究广泛查阅相关文献,收集乙型肝炎肝硬化腹水的中医症状,建立临床流调问卷条目池,咨询相关专家并根据相关标准,确定所需调查的中医症状条目池以及相应的症状分级量化标准,最终形成中医证候临床调查问卷表。按统一问卷表收集乙型肝炎肝硬化腹水(肝内结节>1cm)患者的临床证候资料,应用无监督的因子分析方法研究其证候特点,并对所得出的证型与临床指标之间的相关性进行初步探讨。
2实验研究建立二乙基亚硝胺(diethylnirtosamine, DEN)诱导的大鼠肝癌前病变动物模型,采用抗纤抑癌方进行干预,并分别以复方鳖甲软肝片、秋水仙碱片作为对照,比较各组大鼠在一般情况及肝脏组织病理形态的情况,并应用免疫组化、酶联免疫及实时荧光定量PCR(real-time fluorescence quantitative PCR, RQ-PCR)等方法检测各组大鼠肝脏的胎盘型谷胱甘肽转移酶(glutathione-S-transferases-π, GST-Pi)、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA).甲胎蛋白(alpha-fetoprotein, AFP)、AFP-L3、α-SMA、HGF及c-Met等方面指标的情况。
结果
1临床研究对所收集到的134例乙型肝炎肝硬化腹水(肝内结节>1cm)患者的临床资料进行频数分析得出,乙型肝炎肝硬化腹水(肝内结节>lcm)发病年龄高峰在51～60岁,肝硬化病史在5年以内所占比例为67.16%,出现腹水的病史在5年以内的比例达85.07%,62.69%的患者ALB低于正常值,右侧卧位腹水深度以<3cm出现的频率最高,达到35.82%。对所收集到的临床症状通过无监督的因子分析得出,乙型肝炎肝硬化腹水(肝内结节>1cm)患者的证候可归纳为肝胆湿热兼脾虚、肝郁脾虚兼水湿内蕴、气郁化火兼血瘀、气虚血瘀、痰瘀互结、气滞湿阻、肝郁气滞、湿邪困脾、脾虚失运和肝肾亏虚兼瘀血内阻这十个证型。经与临床资料做相关分析得出,各证型与患者是否抗病毒治疗、PT及WBC有相关性。
2实验研究在大鼠的一般情况上,模型组大鼠在予DEN灌胃第3周以后开始出现一些相关的病态表现,各治疗组大鼠也有类似表现,但程度较模型组轻。第12周后除正常组之外的各组开始陆续出现死亡。至实验结束,模型组共死亡6只,抗纤抑癌组死亡2只(其中1只是因第2周时给药不慎致死),鳖甲软肝组死亡4只,秋水仙碱组死亡4只,抗纤抑癌组死亡率最低。在大鼠肝脏的大体形态上,模型组大鼠肝脏颜色暗红,无光泽,表面凹凸不平,可见弥漫性再生结节形成；抗纤抑癌组大鼠肝脏表面相对光滑、平整,结节较少、较小；鳖甲软肝脏及秋水仙碱组大鼠肝脏的表现介于模型组和抗纤抑癌组之间。经HE染色显示,模型组大鼠肝索排列紊乱,在门脉区有大量的结缔组织增生,胶原纤维增生明显,广泛假小叶形成,可见大量肝细胞异型增生灶及异型增生结节,肝细胞大小不等,核仁明显,细胞核具有多形性。与模型组相比,抗纤抑癌组在肝细胞变性坏死、结缔组织增生程度及肝细胞异型增生灶和异型增生结节数目上均较轻；而鳖甲软肝组、秋水仙碱组大鼠的情况介于两者之间。Masson染色显示各组大鼠肝纤维化的情况与HE类似,而胶原纤维着色更为明显。免疫组化结果显示,与模型组相比,抗纤抑癌组能明显减少GST-Pi阳性灶数及其面积,同时也降低了PCNA阳性细胞标记指数及α-SMA表达量,有统计学差异(P<0.01)；抗纤抑癌组与秋水仙碱组相比较,在GST-Pi阳性灶数、PCNA阳性细胞标记指数及α-SMA表达量具有显著性差异(P<0.05),抗纤抑癌组优于秋水仙碱组；抗纤抑癌组与鳖甲软肝组比较均无显著性差异(P>0.05)。酶联免疫结果显示,与模型组相比,抗纤抑癌组在AFP和AFP-L3的表达量上均有明显降低,有统计学差异(P<0.05及P<0.01),在AFP-L3与AFP的比值上亦较模型组表达低,但没有统计学差异(P>0.05)；抗纤抑癌组与秋水仙碱组相比较,在AFP-L3的表达量上有显著性差异(P<0.05)；抗纤抑癌组与鳖甲软肝组比较均无显著性差异(P>0.05)。RQ-PCR结果显示,HGF mRNA的相对表达量抗纤抑癌组与模型组比较有统计学差异(P<0.05),与秋水仙碱组比较亦有统计学差异(P<0.05),抗纤抑癌方能显著提高肝癌前病变大鼠HGF mRNA的表达水平。c-Met mRNA在DEN造模的各组中均低表达,各组间无统计学差异(P>0.05)。
结论
肝癌前病变(乙型肝炎肝硬化腹水(肝内结节>1cm))患者的中医证型可初步归纳为肝胆湿热兼脾虚、肝郁脾虚兼水湿内蕴、气郁化火兼血瘀、气虚血瘀、痰瘀互结、气滞湿阻、肝郁气滞、湿邪困脾、脾虚失运和肝肾亏虚兼瘀血内阻这十个证型。各证型与患者是否抗病毒治疗、PT及WBC有相关性。抗纤抑癌方能够改善肝癌前病变大鼠的相关指标,有一定抗纤维化、延缓肝癌前病变的作用,其作用机制是通过上调HGF mRNA的表达,抑制肝星形细胞(hepatic stellate cell, HSC)活化,促进细胞外基质(extracelluar matrix, ECM)降解,而起到相应作用。
Objective
Primary liver cancer (PHC) is a kind of disease with high incidence and mortality. It is a serious threat to human health. The majority of patients with liver cancer are associated with the infection of hepatitis B virus (HBV). The liver cirrhosis with HBV infection, especially intercurrent with dysplastic nodule, have high probability of developing into liver cancer. It has been regarded as the hepatic precancerous lesion. In recent decades, although PHC treatments have been developed and perfected, but its clinical efficacy has not been significantly improved. Therefore, it has a very important practical significance to prevent or delay the formation and development of liver cancer by interfering in the stagy of precancerous lession. Based on tutor's clinical and experimental work, we tried to do a preliminary study about the syndrome law of TCM in the ascites with hepatitis B cirrhosis (intrahepatic nodular>1cm), and to investigate the mechanism of prevention and treatment of hepatic precancerous lesion with Kangxianyiai Decoction by Means of observing the relationship between hepatocyte growth factor(HGF) and its receptor(c-Met) and alpha smooth muscle actin(α-SMA).
Methods
1 Clinical research Based on extensively reviewing the relevant literature, collected the clinical symptoms of TCM of the ascites with hepatitis B cirrhosis, then established an initially clinical epidemiological survey questionnaire. At last, determined the required clinical symptoms of the survey questionnaire item pool in TCM, and made the corresponding classification of quantitative criteria symptoms by relevant expert advice and feasibility analysis, repeated discussions and seminars and clinical research guidelines. Used uniform questionnaire to collect 134 cases clinical syndromes data of the ascites with hepatitis B cirrhosis (intrahepatic nodular>1cm). We use the method of factor analysis to investigate its characteristics of TCM syndrome, and to discuss the relationship between the objective indexes with the characteristics of TCM.
2 Experimental research Established the animal models of diethylnitrosamine(DEN)-induced hepatic precancerous lesion, and used Kangxianyiai (KXYA) Decoction to treat. Fufangbiejiaruangan(BJRG) tablets and colchicine tablets were taken as control drug. Compared the difference of rats in each group on the general situation and the liver pathological change, and determined the expression levels of glutathione-S-transferases-π(GST-Pi), of proliferating cell nuclear antigen(PCNA), alpha-fetoprotein(AFP), AFP-L3,α-SMA, HGF and c-Met in the liver of the rats in each group by using immunohistochemistry, Enzyme-linked immunoassay(ELISA) and real-time fluorescence quantitative PCR (RQ-PCR), so as to reveal the mechanism of intervention on hepatic precancerous lesion by Kangxianyiai decoction.
Results
1The results of clinical research Used collected clinical data of 134 cases of the ascites with hepatitis B cirrhosis (intrahepatic nodular>lcm) to do the frequency analysis derived:The peak age incidence of the ascites with hepatitis B cirrhosis (intrahepatic nodular>lcm) was between 51 and 60 years old; the time suffering from liver cirrhosis less than five years occupied 67.16%; the proportion that the history of ascites appears less than five years was 85.07%; 62.69% patients with ALB value lower than normal, and the ascitic depth of right lateral position less than 3cm was the highest frequency, accounted for 35.82%. By means of the unsupervised factor analysis to analyze the collected clinical symptoms, the characteristics of TCM syndrome of the ascites with hepatitis B cirrhosis (intrahepatic nodular>1cm) can be summarized as:the syndrome of dampness and heat in the liver and the gallbladder with spleen deficiency, the syndrome of stagnation of liver qi and deficiency of the spleen with water retention, the syndrome of fire due to qi stagnation with blood stasis, the syndrome of qi deficiency and blood stasis, the syndrome of qi and damp stagnation, the syndrome of stagnation of liver-qi, the syndrome of damp pathogen obstructing the spleen, the syndromes of dysfunction in transport due to spleen deficiency, the syndromes of insufficiency of the liver and the kidney with internal block due to blood stasis. The correlation analysis between syndrome characteristics and clinical data obtained has being done. The factors such as antiviral therapy affect the distribution of syndrome characteristics.
2The results of experimental research On the general condition, the model group begun to present some pathological behaviors after pouring the DEN into stomach for three weeks. Every treatment group had such similar condition but was lighter than the model group. In the twelfth week, the rats in every group died in succession except the negative control group. At the end of the experiment,6 rats in the model group were dead while 2 rats in KXYA group died (one of the rats was due to the careless drug delivery in the second week).4 rats in the BJRG group were dead and 4 rats in the group of colchicin died too. In conclusion, the fatality rate was lowest in the KXYA group. On the Gross morphology, liver surface was rough, and diffuse nodules formed in model group. It was found by pathological examination that liver cell necrosis, collagen fibers hyperplasia obviously, false lobular formed widely, liver cell volume increased, and the nucleus was pleomorphic. The pathological performances of liver of the BJRG group and the colchicine group were similar to the model group's. There were no obviously rugosity in the liver surface of the KXYA group, and the nodules were smalerl than these of model group. Masson's trichrome stain showed that the condition of liver fibrosis in each rat group was similar with HE's results, but with more obvious the coloring of collagen fibers.
The results of immunohistochemistry showed that:Compared with the model group, the KXYA group significantly reduced the GST-Pi positive foci area and number, while also reducing the PCNA-positive cell labeling index, and the expression levels of a-SMA, which has a remarkable statistical significance(P<0.01). The results of ELISA showed that: Compared with the model group, the KXYA group can significantly reduce the expression levels of AFP and AFP-L3, which has a remarkable statistical significance(P<0.05 and P<0.01); the ratio of AFP-L3 and AFP is also lower than the model control group, but the difference has no statistical significance(P>0.05). The results of RQ-PCR showed that:There were significant difference in the relative expression levels of HGF mRNA between KXYA group and model control group(P<0.05). Kangxianyiai Decoction can significantly increase the expression level of HGF mRNA in rat hepatic precancerous lesion. c-Met mRNA were all low expression in the each group of DEN-induce hepatic precancerous lesion, and the difference in each group has no statistical significance(P>0.05).
Conclusions
the characteristics of TCM syndrome of the ascites with hepatitis B cirrhosis (intrahepatic nodular>1cm) were:the syndrome of dampness and heat in the liver and the gallbladder with spleen deficiency, the syndrome of stagnation of liver qi and deficiency of the spleen with water retention, the syndrome of fire due to qi stagnation with blood stasis, the syndrome of qi deficiency and blood stasis, the syndrome of qi and damp stagnation, the syndrome of stagnation of liver-qi, the syndrome of damp pathogen obstructing the spleen, the syndromes of dysfunction in transport due to spleen deficiency, the syndromes of insufficiency of the liver and the kidney with internal block due to blood stasis. The characteristics of TCM syndrome have relativity with antiviral therapy, PT, WBC.
It's effective to reduce the extent of liver cell necrosis, collagen fibers hyperplasia and liver cell dyplasia, delay the occurrence and development of hepatic precancerous lesion by Kangxianyiai Decoction. The mechanism of action related to increased expression of HGF mRNA, and inhibited activation of hepatic stellate cells (HSC), promoted degradation of extracellular matrix(ECM).
原发性肝癌(primary hepatic carcinoma, PHC)是一类发生率和死亡率均很高的疾病,严重威胁着人类健康。大多数肝癌患者都与乙型肝炎病毒(hepatitis B virus, HBV)感染有关。合并有HBV感染的肝硬化,尤其是伴随异型增生结节出现者,其发展为肝癌的几率高,被视为肝癌前病变。虽然近几十年PHC的治疗方法得到了一定发展和完善,但其临床疗效仍未得到明显的提高。阻止或延缓肝癌前病变的发生发展,预防肝癌疾病的发生,就显得具有非常重要的现实意义。本课题在导师前期的临床及实验的工作基础之上,针对肝癌前病变(乙型肝炎肝硬化腹水(肝内结节>1cm))患者的中医证型做初步研究,并从肝细胞生长因子(hepatic growth factor, HGF)及其受体(c-Met)与α平滑肌肌动蛋白(a-smooth muscle actin,α-SMA)之间的相互关系去探讨抗纤抑癌方防治肝癌前病变的作用机制。
方法
1临床研究广泛查阅相关文献,收集乙型肝炎肝硬化腹水的中医症状,建立临床流调问卷条目池,咨询相关专家并根据相关标准,确定所需调查的中医症状条目池以及相应的症状分级量化标准,最终形成中医证候临床调查问卷表。按统一问卷表收集乙型肝炎肝硬化腹水(肝内结节>1cm)患者的临床证候资料,应用无监督的因子分析方法研究其证候特点,并对所得出的证型与临床指标之间的相关性进行初步探讨。
2实验研究建立二乙基亚硝胺(diethylnirtosamine, DEN)诱导的大鼠肝癌前病变动物模型,采用抗纤抑癌方进行干预,并分别以复方鳖甲软肝片、秋水仙碱片作为对照,比较各组大鼠在一般情况及肝脏组织病理形态的情况,并应用免疫组化、酶联免疫及实时荧光定量PCR(real-time fluorescence quantitative PCR, RQ-PCR)等方法检测各组大鼠肝脏的胎盘型谷胱甘肽转移酶(glutathione-S-transferases-π, GST-Pi)、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA).甲胎蛋白(alpha-fetoprotein, AFP)、AFP-L3、α-SMA、HGF及c-Met等方面指标的情况。
结果
1临床研究对所收集到的134例乙型肝炎肝硬化腹水(肝内结节>1cm)患者的临床资料进行频数分析得出,乙型肝炎肝硬化腹水(肝内结节>lcm)发病年龄高峰在51～60岁,肝硬化病史在5年以内所占比例为67.16%,出现腹水的病史在5年以内的比例达85.07%,62.69%的患者ALB低于正常值,右侧卧位腹水深度以<3cm出现的频率最高,达到35.82%。对所收集到的临床症状通过无监督的因子分析得出,乙型肝炎肝硬化腹水(肝内结节>1cm)患者的证候可归纳为肝胆湿热兼脾虚、肝郁脾虚兼水湿内蕴、气郁化火兼血瘀、气虚血瘀、痰瘀互结、气滞湿阻、肝郁气滞、湿邪困脾、脾虚失运和肝肾亏虚兼瘀血内阻这十个证型。经与临床资料做相关分析得出,各证型与患者是否抗病毒治疗、PT及WBC有相关性。
2实验研究在大鼠的一般情况上,模型组大鼠在予DEN灌胃第3周以后开始出现一些相关的病态表现,各治疗组大鼠也有类似表现,但程度较模型组轻。第12周后除正常组之外的各组开始陆续出现死亡。至实验结束,模型组共死亡6只,抗纤抑癌组死亡2只(其中1只是因第2周时给药不慎致死),鳖甲软肝组死亡4只,秋水仙碱组死亡4只,抗纤抑癌组死亡率最低。在大鼠肝脏的大体形态上,模型组大鼠肝脏颜色暗红,无光泽,表面凹凸不平,可见弥漫性再生结节形成；抗纤抑癌组大鼠肝脏表面相对光滑、平整,结节较少、较小；鳖甲软肝脏及秋水仙碱组大鼠肝脏的表现介于模型组和抗纤抑癌组之间。经HE染色显示,模型组大鼠肝索排列紊乱,在门脉区有大量的结缔组织增生,胶原纤维增生明显,广泛假小叶形成,可见大量肝细胞异型增生灶及异型增生结节,肝细胞大小不等,核仁明显,细胞核具有多形性。与模型组相比,抗纤抑癌组在肝细胞变性坏死、结缔组织增生程度及肝细胞异型增生灶和异型增生结节数目上均较轻；而鳖甲软肝组、秋水仙碱组大鼠的情况介于两者之间。Masson染色显示各组大鼠肝纤维化的情况与HE类似,而胶原纤维着色更为明显。免疫组化结果显示,与模型组相比,抗纤抑癌组能明显减少GST-Pi阳性灶数及其面积,同时也降低了PCNA阳性细胞标记指数及α-SMA表达量,有统计学差异(P<0.01)；抗纤抑癌组与秋水仙碱组相比较,在GST-Pi阳性灶数、PCNA阳性细胞标记指数及α-SMA表达量具有显著性差异(P<0.05),抗纤抑癌组优于秋水仙碱组；抗纤抑癌组与鳖甲软肝组比较均无显著性差异(P>0.05)。酶联免疫结果显示,与模型组相比,抗纤抑癌组在AFP和AFP-L3的表达量上均有明显降低,有统计学差异(P<0.05及P<0.01),在AFP-L3与AFP的比值上亦较模型组表达低,但没有统计学差异(P>0.05)；抗纤抑癌组与秋水仙碱组相比较,在AFP-L3的表达量上有显著性差异(P<0.05)；抗纤抑癌组与鳖甲软肝组比较均无显著性差异(P>0.05)。RQ-PCR结果显示,HGF mRNA的相对表达量抗纤抑癌组与模型组比较有统计学差异(P<0.05),与秋水仙碱组比较亦有统计学差异(P<0.05),抗纤抑癌方能显著提高肝癌前病变大鼠HGF mRNA的表达水平。c-Met mRNA在DEN造模的各组中均低表达,各组间无统计学差异(P>0.05)。
结论
肝癌前病变(乙型肝炎肝硬化腹水(肝内结节>1cm))患者的中医证型可初步归纳为肝胆湿热兼脾虚、肝郁脾虚兼水湿内蕴、气郁化火兼血瘀、气虚血瘀、痰瘀互结、气滞湿阻、肝郁气滞、湿邪困脾、脾虚失运和肝肾亏虚兼瘀血内阻这十个证型。各证型与患者是否抗病毒治疗、PT及WBC有相关性。抗纤抑癌方能够改善肝癌前病变大鼠的相关指标,有一定抗纤维化、延缓肝癌前病变的作用,其作用机制是通过上调HGF mRNA的表达,抑制肝星形细胞(hepatic stellate cell, HSC)活化,促进细胞外基质(extracelluar matrix, ECM)降解,而起到相应作用。
Objective
Primary liver cancer (PHC) is a kind of disease with high incidence and mortality. It is a serious threat to human health. The majority of patients with liver cancer are associated with the infection of hepatitis B virus (HBV). The liver cirrhosis with HBV infection, especially intercurrent with dysplastic nodule, have high probability of developing into liver cancer. It has been regarded as the hepatic precancerous lesion. In recent decades, although PHC treatments have been developed and perfected, but its clinical efficacy has not been significantly improved. Therefore, it has a very important practical significance to prevent or delay the formation and development of liver cancer by interfering in the stagy of precancerous lession. Based on tutor's clinical and experimental work, we tried to do a preliminary study about the syndrome law of TCM in the ascites with hepatitis B cirrhosis (intrahepatic nodular>1cm), and to investigate the mechanism of prevention and treatment of hepatic precancerous lesion with Kangxianyiai Decoction by Means of observing the relationship between hepatocyte growth factor(HGF) and its receptor(c-Met) and alpha smooth muscle actin(α-SMA).
Methods
1 Clinical research Based on extensively reviewing the relevant literature, collected the clinical symptoms of TCM of the ascites with hepatitis B cirrhosis, then established an initially clinical epidemiological survey questionnaire. At last, determined the required clinical symptoms of the survey questionnaire item pool in TCM, and made the corresponding classification of quantitative criteria symptoms by relevant expert advice and feasibility analysis, repeated discussions and seminars and clinical research guidelines. Used uniform questionnaire to collect 134 cases clinical syndromes data of the ascites with hepatitis B cirrhosis (intrahepatic nodular>1cm). We use the method of factor analysis to investigate its characteristics of TCM syndrome, and to discuss the relationship between the objective indexes with the characteristics of TCM.
2 Experimental research Established the animal models of diethylnitrosamine(DEN)-induced hepatic precancerous lesion, and used Kangxianyiai (KXYA) Decoction to treat. Fufangbiejiaruangan(BJRG) tablets and colchicine tablets were taken as control drug. Compared the difference of rats in each group on the general situation and the liver pathological change, and determined the expression levels of glutathione-S-transferases-π(GST-Pi), of proliferating cell nuclear antigen(PCNA), alpha-fetoprotein(AFP), AFP-L3,α-SMA, HGF and c-Met in the liver of the rats in each group by using immunohistochemistry, Enzyme-linked immunoassay(ELISA) and real-time fluorescence quantitative PCR (RQ-PCR), so as to reveal the mechanism of intervention on hepatic precancerous lesion by Kangxianyiai decoction.
Results
1The results of clinical research Used collected clinical data of 134 cases of the ascites with hepatitis B cirrhosis (intrahepatic nodular>lcm) to do the frequency analysis derived:The peak age incidence of the ascites with hepatitis B cirrhosis (intrahepatic nodular>lcm) was between 51 and 60 years old; the time suffering from liver cirrhosis less than five years occupied 67.16%; the proportion that the history of ascites appears less than five years was 85.07%; 62.69% patients with ALB value lower than normal, and the ascitic depth of right lateral position less than 3cm was the highest frequency, accounted for 35.82%. By means of the unsupervised factor analysis to analyze the collected clinical symptoms, the characteristics of TCM syndrome of the ascites with hepatitis B cirrhosis (intrahepatic nodular>1cm) can be summarized as:the syndrome of dampness and heat in the liver and the gallbladder with spleen deficiency, the syndrome of stagnation of liver qi and deficiency of the spleen with water retention, the syndrome of fire due to qi stagnation with blood stasis, the syndrome of qi deficiency and blood stasis, the syndrome of qi and damp stagnation, the syndrome of stagnation of liver-qi, the syndrome of damp pathogen obstructing the spleen, the syndromes of dysfunction in transport due to spleen deficiency, the syndromes of insufficiency of the liver and the kidney with internal block due to blood stasis. The correlation analysis between syndrome characteristics and clinical data obtained has being done. The factors such as antiviral therapy affect the distribution of syndrome characteristics.
2The results of experimental research On the general condition, the model group begun to present some pathological behaviors after pouring the DEN into stomach for three weeks. Every treatment group had such similar condition but was lighter than the model group. In the twelfth week, the rats in every group died in succession except the negative control group. At the end of the experiment,6 rats in the model group were dead while 2 rats in KXYA group died (one of the rats was due to the careless drug delivery in the second week).4 rats in the BJRG group were dead and 4 rats in the group of colchicin died too. In conclusion, the fatality rate was lowest in the KXYA group. On the Gross morphology, liver surface was rough, and diffuse nodules formed in model group. It was found by pathological examination that liver cell necrosis, collagen fibers hyperplasia obviously, false lobular formed widely, liver cell volume increased, and the nucleus was pleomorphic. The pathological performances of liver of the BJRG group and the colchicine group were similar to the model group's. There were no obviously rugosity in the liver surface of the KXYA group, and the nodules were smalerl than these of model group. Masson's trichrome stain showed that the condition of liver fibrosis in each rat group was similar with HE's results, but with more obvious the coloring of collagen fibers.
The results of immunohistochemistry showed that:Compared with the model group, the KXYA group significantly reduced the GST-Pi positive foci area and number, while also reducing the PCNA-positive cell labeling index, and the expression levels of a-SMA, which has a remarkable statistical significance(P<0.01). The results of ELISA showed that: Compared with the model group, the KXYA group can significantly reduce the expression levels of AFP and AFP-L3, which has a remarkable statistical significance(P<0.05 and P<0.01); the ratio of AFP-L3 and AFP is also lower than the model control group, but the difference has no statistical significance(P>0.05). The results of RQ-PCR showed that:There were significant difference in the relative expression levels of HGF mRNA between KXYA group and model control group(P<0.05). Kangxianyiai Decoction can significantly increase the expression level of HGF mRNA in rat hepatic precancerous lesion. c-Met mRNA were all low expression in the each group of DEN-induce hepatic precancerous lesion, and the difference in each group has no statistical significance(P>0.05).
Conclusions
the characteristics of TCM syndrome of the ascites with hepatitis B cirrhosis (intrahepatic nodular>1cm) were:the syndrome of dampness and heat in the liver and the gallbladder with spleen deficiency, the syndrome of stagnation of liver qi and deficiency of the spleen with water retention, the syndrome of fire due to qi stagnation with blood stasis, the syndrome of qi deficiency and blood stasis, the syndrome of qi and damp stagnation, the syndrome of stagnation of liver-qi, the syndrome of damp pathogen obstructing the spleen, the syndromes of dysfunction in transport due to spleen deficiency, the syndromes of insufficiency of the liver and the kidney with internal block due to blood stasis. The characteristics of TCM syndrome have relativity with antiviral therapy, PT, WBC.
It's effective to reduce the extent of liver cell necrosis, collagen fibers hyperplasia and liver cell dyplasia, delay the occurrence and development of hepatic precancerous lesion by Kangxianyiai Decoction. The mechanism of action related to increased expression of HGF mRNA, and inhibited activation of hepatic stellate cells (HSC), promoted degradation of extracellular matrix(ECM).
引文
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