以“浊毒”立论论治肝纤维化的理论初探及实验研究
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摘要
肝纤维化(Hepatic fibrosis,HF)是指各种致病因素所引起的肝脏慢性损害和炎症,是多种慢性肝病共同的病理基础,是肝脏细胞外基质(extracellular matrix,ECM)合成和降解失衡、导致其过度沉积的结果,也是慢性肝炎进一步向肝硬化、肝癌发展的重要环节。
以往的研究局限于将肝纤维化归为一种被动的静止状态,随着分子生物学研究方法和技术的日新月异,人们在肝星状细胞(hepatic stellate cells, HSC)活化、多种动物模型的建立、细胞因子信号传导通路、基因靶向治疗等方面取得了较大的进展。转化生长因子-β1(transforming growth factor- beta1-1,TGF-β1)、基质金属蛋白酶抑制因子-1(tissue inhibit of metallo -Proteinase,TIMP-1)、ECM成分等已经成为研究的热点。
目前研究的抗纤维化药物主要包括细胞保护类、HSC活化抑制类、细胞因子活性类、抑制ECM合成及分泌类、促进ECM降解类、中药单体和复方等。但肝纤维化是一个复杂的病理过程,临床上至今缺乏确切特效的抗纤维化药物与方法。
中医药治疗疾病具有多方位、多环节、多靶点的特点,在肝纤维化的预防和治疗方面有其独特的优势。在当前形势下,寻找能够改善HF状态的中药或者中药提取物,并对其相关机制进行探索,不仅有利于中药现代化的研究,也将对HF治疗产生深远的影响。
我们通过观察化浊解毒益气方对猪血清诱导的HF大鼠的干预研究,探讨肝纤维化的病理机制,为肝纤维化的治疗提供理论及实验依据。
目的:以“浊毒”理论为依据探讨其在肝纤维化治疗中的地位,探讨化浊解毒益气方对肝纤维化大鼠的影响及其可能的作用机制。主要内容包括以“浊毒”立论论治肝纤维化的理论初探;化浊解毒益气方对肝纤维化大鼠血清AST、ALT、ALB、TP和病理形态学的影响;化浊解毒益气方对肝纤维化大鼠血清HA、LN、PCⅢ、Ⅳ型胶原和病理形态学的影响;化浊解毒益气方对肝纤维化大鼠肝组织TGF-β1表达的影响;化浊解毒益气方对肝纤维化大鼠肝组织TIMP-1表达的影响。
方法:清洁级Wistar大鼠60只,雄性,体重200~220g。适应性喂养一周后随机分为正常对照组、模型对照组、秋水仙碱预防组(简称西药预防组)、化浊解毒益气方预防组(简称中药预防组)、秋水仙碱治疗组(简称西药治疗组)、化浊解毒益气方治疗组(简称中药治疗组)六组,每组10只。除正常对照组外,其余各组均采用未灭活的猪血清腹腔注射,每次0.5ml/只,每周两次,连续8周。秋水仙碱预防组和中药预防组于造模的同时给药,每天上午灌胃一次,每次2ml,连续12周。秋水仙碱治疗组和中药治疗组于造模的第8周末给药,每天上午灌胃一次,每次2ml,连续4周。正常对照组给予等量生理盐水。
各组分别于造模的第8周末、第12周末处死5只大鼠,腹主动脉取血,分离血清,测定各组大鼠干预后血清中丙氨酸氨基转移酶(ALT)、天门冬氨基转移酶(AST)、白蛋白(ALB)、总蛋白(TP)、透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(CⅣ)水平。其中ALT、AST用赖氏法测定;ALB用溴甲酚绿比色法测定;TP用双缩脲法测定;HA、LN、PCⅢ、CⅣ用放射免疫分析法测定。留取大鼠肝组织,采用苏木精-伊红染色和Masson染色(胶原纤维特殊染色法)观察肝组织病理形态学的变化。采用免疫组化和RT-PCR技术测定肝组织TGF-β1、TIMP-1蛋白和mRNA表达情况。所得数据经过整理,采用SPSS统计软件进行t检验或方差分析。
结果:
1化浊解毒益气方对肝纤维化大鼠血清AST、ALT、ALB、TP和病理形态学的影响
1.1化浊解毒益气方对肝纤维化大鼠血清AST、ALT、ALB、TP的影响
与正常对照组相比,模型组大鼠血清ALT、AST升高,ALB、TP降低,差异有统计学意义(P<0.05)。与模型对照组相比,第8周末,西药预防组和中药预防组各组大鼠血清ALT、AST下降,TP、ALB升高,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物处理组大鼠血清ALT、AST下降,TP、ALB升高,差异有统计学意义(P<0.05),且中药预防组优于西药预防组,中药治疗组优于西药治疗组(P<0.05)。但是西药处理组之间、中药处理组之间各组大鼠血清指标差异均无统计学意义(P>0.05)。
1.2化浊解毒益气方对肝纤维化大鼠肝脏病理形态学的影响
正常组肝小叶结构完整,肝细胞围绕中央静脉呈放射状排列,无变性坏死,无纤维组织增生。模型组肝小叶结构严重破坏,肝细胞广泛脂肪变性、坏死伴炎性细胞浸润,纤维组织大量增生,个别动物可见假小叶形成。第8周末,各药物预防组肝小叶结构受损轻,可见肝细胞脂肪变性及炎性细胞浸润,但较模型组显著减轻。胶原纤维增生较模型组减轻,主要散在于肝血窦和disse间隙内,无假小叶形成,中药预防组优于西药预防组。第12周末,各药物干预组肝脏损伤程度均较模型组轻,西药治疗组病理改变与西药预防组相似,中药治疗组病理改变与中药预防组相似。且中药预防组优于西药预防组,中药治疗组优于西药治疗组。
2化浊解毒益气方对肝纤维化大鼠血清HA、LN、PCⅢ、Ⅳ型胶原和病理形态学的影响
2.1化浊解毒益气方对肝纤维化大鼠血清HA、LN、PCⅢ、Ⅳ型胶原的影响
与正常对照组相比,模型组大鼠血清HA、LN、PCⅢ、Ⅳ型胶原升高,差异有统计学意义(P<0.05)。与模型对照组相比,第8周末,西药预防组和中药预防组各组大鼠血清HA、LN、PCⅢ、Ⅳ型胶原下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物处理组大鼠血清HA、LN、PCⅢ、Ⅳ型胶原下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组,中药治疗组优于西药治疗组(P<0.05)。但是西药处理组之间、中药处理组之间各组大鼠血清指标差异均无统计学意义(P>0.05)。
2.2化浊解毒益气方对肝纤维化大鼠病理形态学的影响
正常组肝小叶结构完整,肝细胞围绕中央静脉呈放射状排列,无变性坏死,无纤维组织增生。模型组肝小叶结构严重破坏,肝细胞广泛脂肪变性、坏死伴炎性细胞浸润,绿色纤维组织大量增生,个别动物可见假小叶形成。8周末,各预防组肝小叶结构受损轻,可见肝细胞脂肪变性及炎性细胞浸润,但较模型组显著减轻。胶原纤维增生较模型组减轻,主要散在于肝血窦和disse间隙,无假小叶形成。且中药预防组优于西药预防组。12周末,各药物干预组肝脏损伤程度均较模型组显著减轻,且中药预防组优于西药预防组。中药治疗组优于西药治疗组。
3化浊解毒益气方对肝纤维化大鼠肝组织TGF-β1表达的影响
3.1化浊解毒益气方对肝纤维化大鼠肝组织TGF-β1蛋白表达的影响。
正常组大鼠肝组织见少许TGF-β1阳性表达细胞,模型组大鼠肝组织中表达的阳性细胞比例显著高于正常组,表现为不均匀的黄褐色颗粒状物。与正常对照组相比,模型组大鼠肝组织TGF-β1蛋白表达增高,差异有统计学意义(P<0.05)。8周末,与模型对照组相比,西药预防组和中药预防组两组大鼠肝组织TGF-β1蛋白表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物干预组大鼠肝组织TGF-β1蛋白表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05),中药治疗组优于西药治疗组。但是西药预防组与治疗组之间、中药预防组和治疗组之间各组大鼠肝组织TGF-β1蛋白表达差异均无统计学意义(P>0.05)。
3.2化浊解毒益气方对肝纤维化大鼠肝组织TGF-β1mRNA表达的影响
与正常对照组相比,模型组大鼠肝组织TGF-β1mRNA表达增高,差异有统计学意义(P<0.05)。8周末,与模型对照组相比,西药预防组和中药预防组两组大鼠肝组织TGF-β1mRNA表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物干预组大鼠肝组织TGF-β1mRNA表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05),中药治疗组优于西药治疗组。但是西药预防组与治疗组之间、中药预防组和治疗组之间各组大鼠肝组织TGF-β1mRNA表达差异均无统计学意义(P>0.05)。
4化浊解毒益气方对肝纤维化大鼠肝组织TIMP-1表达的影响
4.1化浊解毒益气方对肝纤维化大鼠肝组织TIMP-1蛋白表达的影响
正常组大鼠肝组织见少许TIMP-1阳性表达细胞,模型组大鼠肝组织中表达的阳性细胞比例显著高于正常组,表现为不均匀的黄褐色颗粒状物。与正常对照组相比,模型组大鼠肝组织TIMP-1蛋白表达增高,差异有统计学意义(P<0.05)。8周末,与模型对照组相比,西药预防组和中药预防组两组大鼠肝组织TIMP-1蛋白表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物干预组大鼠肝组织TIMP-1蛋白表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05),中药治疗组优于西药治疗组。但是西药预防组与治疗组之间、中药预防组和治疗组之间各组大鼠肝组织TIMP-1蛋白表达差异均无统计学意义(P>0.05)。
4.2化浊解毒益气方对肝纤维化大鼠肝组织TIMP-1mRNA表达的影响
与正常对照组相比,模型组大鼠肝组织TIMP-1mRNA表达增高,差异有统计学意义(P<0.05)。8周末,与模型对照组相比,西药预防组和中药预防组大鼠肝组织TIMP-1mRNA表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物干预组大鼠肝组织TIMP-1mRNA表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05),中药治疗组优于西药治疗组。但是西药预防组与治疗组之间、中药预防组和治疗组之间各组大鼠肝组织TIMP-1mRNA表达差异均无统计学意义(P>0.05)。
结论:
1“浊毒”理论的提出对肝纤维化的中医病因病机分析具有重要意义,在肝纤维化的诊断和治疗中,占有重要的地位。
2化浊解毒益气方能有效改善肝纤维化大鼠肝组织病理改变、减轻肝细胞的损伤,减少胶原纤维的生成,从而发挥其抗纤维化的作用。
3化浊解毒益气方能显著降低大鼠血清AST、ALT含量,升高TP、ALB含量,减轻肝细胞的损伤,保护肝功能,通过保肝、抗炎而发挥其抗纤维化的作用。
4化浊解毒益气方能显著降低大鼠血清HA、LN、PCⅢ型胶原、Ⅳ型胶原含量,减少细胞外基质的生成,从而发挥其抗纤维化的作用。
5化浊解毒益气方能显著降低大鼠肝组织TGF-β1蛋白和mRNA的表达,抑制肝星状细胞的活化,通过调控相关细胞因子生物作用,促进细胞外基质的降解。
6化浊解毒益气方能显著降低大鼠肝组织TIMP-1蛋白和mRNA的表达,通过调节MMPS和TIMPS的失衡,促进异常合成的细胞外基质的降解,减少细胞外基质的过度沉积。
Hepatic fibrosis is a pathologic process that every kind of disease cause liver injure and inflammation.It is the hallmark of most chronic liver diseases. Its essense is excessive deposition of extracellular matrix (ECM) components in liver, which occurs due to an imbalance between the production and degradation of matrix. Collagen is the most important part of ECM.
In previous studies, Hepatic fibrosis is limited to a passive stationary state. With the development of molecular biological technology, People have made large progress in many ways,such as hepatic stellate cell activation, Establishment of Animal Model , cytokine signaling ,the molecular targeted therapyin Hepatic fibrosis and so on . transforming growth factor-β1 ,tissue inhibit of metallo Proteinase and extracellular matrix components have become a hotspot for research.
In current research work, anti-hepatic fibrosis drug main contents include the type of cytoprotection, hepatic stellate cell activation, Cytokines activity, suppressed extracellular matrix production and secretion, Promote degradation of extracellular matrix, traditional medicine monomer and Chinese Herbal Compound. But HF is a complex process there is no established therapy for hepatic fibrosis。
The characteristics of Traditional Chinese Medicine (TCM) treatment are multiple positions, multiple links and multiple targets. There are much superiority in the prevention and treatment on Diabetes Mellitus. And now, to search the traditional medicine drugs or the extract of these drugs is important in the prevention and treatment of HF. If we can explore the possible mechanisms of these drugs, it will benefit the TCM modernization, the treatment of HF.
To study the effect of Huazhuo Jiedu Yiqi’formula on the effect of immunity hepatic fibrosis rats induced by pig’s serum. Investigate on Pathological Mechanism of Hepatic fibrosis,so that provide theoretical and experimental basis for treating liver fibrosis.
Objectives
The aim was to study the effect of The ZhuoDu theory in the position of Therapy for Hepatic Fibrosis, and explore its possible mechanism.. The research included preliminary Study of The ZhuoDu theory on Therapy for Hepatic Fibrosis, effect of Huazhuo Jiedu Yiqi’formula on the levels of serum ALT,AST,ALB,TP and Pathomorphology in hepatic fibrosis rats;effect of Huazhuo Jiedu Yiqi’formula on the levels of serum HA,LN,PCⅢ,CIV and Pathomorphology in hepatic fibrosis rats;Huazhuo Jiedu Yiqi’formula on the expression of TGF-β1 in liver tissue of hepatic fibrosis rats; Huazhuo Yiqi’formula on the expression of TIMP-1 in liver tissue of hepatic fibrosis rats.
Methods
Total 60 clean male Wistar rats weighed 200～220 grams were randomly divided into six groups:namely,the normal group, the model group, colchicine prevention group,(C Prevention group for short),Huazhuo Jiedu Yiqi’formula Prevention group(HJY Prevention group for short), colchicine treatment group(C treatment group), Huazhuo Jiedu Yiqi’formula treatment group(HJY reatment group)with 10 rats for each group .In addition to the normal group,pig’s serum( 0.5ml per mouse, twice a week) was established in rats by intraperitoneal injection for eight weeks. The drugs were given through stomach-perfusion to C Prevention group and HJY Prevention group at the beginning of experiment.The drugs are given at the same time every day.The treatment groups were given drug at the end of the eighth week. The rats in the normal control and model groups received intragastric administration of saline(2ml each time).
Five rats were killed in each group at the 8th and 12th weekend. We draw blood from abdominal aorta, ALT、AST level were measured with Reit′s method; The contents of TP andAlb were measured using biurea method and bromocresol green method ;HA、LN、PCⅢ、CⅣlevel were measured with radioimmunoassay(RIA). The specimens were taken and the degree of hepatic fibrosis was judged by routine haematoxylin-eosin staining and Masson staining.TGF-β1 protein and mRNA expression were determined using immunohistochemical technique and RT-PCR.TIMP-1 protein and mRNA expression were determined using immunohistochemical technique and RT-PCR .
Data was collected and analyzed through t test or analysis of variance. The statistic software was SPSS. Differences with P values <0.05 were considered significant.
Results
1 Effect of Huazhuo JieDu Yiqi’formula on the levels of serum ALT,AST,ALB,TP and pathomorphology in hepatic fibrosis rats.
1.1 Effect of Huazhuo JieDu Yiqi’formula on the levels of serum ALT,AST,ALB,TP in hepatic fibrosis rats.
Compared with the normal group, the content of ALT and AST was significantly increased with liver injure in the model group(P<0.05). TP、ALB of serum can be increased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05). Compared with the model group, All medicine intervention group decreased the level of serum ALT and AST and increased the concentration of serum TP,ALB at the end of 12 weeks ,there were statistic differences among the groups(P<0.05) .The effect of Chinese Herbal Compound can reduce the evels of serum ALT,AST,ALB,TP in hepatic fibrosis rats and the effect is better than Colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
1.2 Effect of Huazhuo JieDu Yiqi’formula to pathomorphology in hepatic fibrosis rats.
The structure of hepatic lobule were integrated hepatic cord radially ranged as the center of navel vein.There was not degeneration and necrosis in liver tissue. There was not collagen proliferation.Compared with normal group,the model control rats had obvious liver inflammation including degeneration,necrosis,fibrosis and collagen proliferation.Pseudolobule proliferation could be seen in individual animal.At the end of 8th weeks, Hepatic lobule in colchicine prevention group and HJY Prevention group lessed injured than model group. Hepatocellular degeneration and necrosis and inflammatory cell infiltration could be seen in liver tissue.Collagen proliferation in the two groups was less than in model group,it were mainly scattered in hepatic sinusoid and disse clearance. There was not pseudolobule proliferation. Pathological examination in HJY Prevention group was better than C prevention group. At the end of 12th weeks, Pathological examination in the treatment groups less injured than in model group. Pathological examination in C treatment group was similar to that of C prevention group ,the result was same between HJY Prevention group and treatment group.And The effect of Chinese Herbal Compound is better than colchicine.
2 Effect of Huazhuo Yiqi’formula on the levels of serum HA,LN,PCⅢ,CIV and pathomorphology in hepatic fibrosis rats
2.1 Effect of Huazhuo Yiqi’formula on the levels of serum HA,LN,PCⅢ,CIV and pathomorphology in hepatic fibrosis rats
Compared with the normal group, the levels of serum HA,LN,PCⅢ,CIV could be increased from treament group with the significant difference(P<0.05) . The levels of serum HA,LN,PCⅢ,CIV could be decreased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05).Compared with the model group, All medicine intervention group decreased the level of serum HA,LN,PCⅢ,CIV at the end of 12 weeks,There were statistic differences among the groups(P<0.05).The Effect of Chinese Herbal Compound can reduce the levels of serum HA,LN,PCⅢ,CIV in hepatic fibrosis rats and the effect is better than colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
2.2 Effect of Huazhuo Yiqi’formula to pathomorphology in hepatic fibrosis rats.
The structure of hepatic lobule were integrated hepatic cord radially ranged as the center of navel vein.There was not degeneration and necrosis in liver tissue. There was not collagen proliferation.Compared with normal rats,the model control rats had obvious liver inflammation including degeneration,necrosis,fibrosis and collagen proliferation .Pseudolobule proliferation could be seen in individual animal.At the end of 8th weeks, Hepatic lobule in C prevention group and HJY Prevention group lessed injured than model group. Hepatocellular degeneration and necrosis and inflammatory cell infiltration could be seen in liver tissue. Collagen proliferation in the two groups was less than in model group,it were mainly scattered in hepatic sinusoid and disse clearance. There was not pseudolobule proliferation. Pathological examination in HJY Prevention group was better than C prevention group. At the end of 12th weeks, Pathological examination in the treatment groups less injured than in model group. Pathological examination in C treatment group was similar to that of C prevention group ,the result was same between HJY Prevention group and treatment group.And The effect of Chinese Herbal Compound is better than colchicine.
3 Experimental study on Huazhuo Jiedu Yiqi’formula effect on TGF-β1 of the immunity hepatic fibrosis rats
3.1 Experimental study on Huazhuo Jiedu Yiqi’Formula effect on TGF-β1 pretine of the immunity hepatic fibrosis rats
The positive expression cells of normal group was a few. the expression of TGF-β1 was strongly positive in model group.The level was higher than normal group. It could be manifested as heterogeneously vinegar particles.Less positive expression of TGF-β1 cell can be seen in the liver tissue of rats form normal group,the cell proportion of TGF-β1 cell expression in the liver tissue of rats form model group were significantly higer than that of the normal group,which were manifested by the unsymmetric brown yellow particulates(P<0.05). The protein level of TGF-β1 of immunity hepatic fibrosis rats could be decreased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05).Compared with the model group, All medicine intervention group decreased the protein level of TGF-β1 of immunity hepatic fibrosis rats at the end of 12 weeks,There were statistic differences among the groups(P<0.05). The Effect of Chinese Herbal Compound can reduce the expression of TGF-β1 protein of immunity hepatic fibrosis rats and the effect is better than Colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
3.2 Experimental study on Huazhuo Jiedu Yiqi’formula effect on TGF-β1 mRNA of the immunity hepatic fibrosis rats
The results showed that the expressions of TGF-β1 mRNA in the model groups were increased significantly, respectively compared with normal control group and the difference was significant (P<0.05). The expressions of TGF-β1 mRNA of immunity hepatic fibrosis rats could be decreased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05).Compared with the model group, All medicine intervention group decreased the expressions of TGF-β1 mRNA of immunity hepatic fibrosis rats at the end of 12 weeks,There were statistic differences among the groups(P<0.05).The Effect of Chinese Herbal Compound could reduce the expression of TGF-β1 mRNA of hepatic fibrosis rats and the effect is better than Colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
4 Experimental study on Huazhuo Jiedu Yiqi’formula effect on TIMP-β1 of the immunityhepatic fibrosis rats
4.1 Experimental study on Huazhuo Jiedu Yiqi’formula effect on TIMP-1 pretine of the immunity hepatic fibrosis rats
The positive expression cells of normal group was a few. the expression of TIMP-1 was strongly positive in model group.The level was higher than normal group. It could be manifested as heterogeneously vinegar particles.Less positive expression of TIMP-1cell can be seen in the liver tissue of rats form normal group,the cell proportion of TIMP-1 cell expression in the liver tissue of rats form model group were significantly higer than that of the normal group,which were manifested by the unsymmetric brown yellow particulates(P<0.05). The protein level of TIMP-1 of immunity hepatic fibrosis rats could be decreased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05).Compared with the model group, All medicine intervention group decreased the protein level of TIMP-1 of immunity hepatic fibrosis rats at the end of 12 weeks,There were statistic differences among the groups(P<0.05). The Effect of Chinese Herbal Compound can reduce the expression of TIMP-1 protein of immunity hepatic fibrosis rats and the effect is better than colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
4.2 Experimental study on Huazhuo Jiedu Yiqi’formula effect on TIMP-1 mRNA of the immunity hepatic fibrosis rats
The results showed that the expressions of TIMP-1 mRNA in the model groups were increased significantly, respectively compared with normal control group and the difference was significant (P<0.05). The expressions of TIMP-1 mRNA of immunity hepatic fibrosis rats could be decreased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05).Compared with the model group, All medicine intervention group decreased the expressions of TIMP-1 mRNA of immunity hepatic fibrosis rats at the end of 12 weeks,There were statistic differences among the groups(P<0.05).The Effect of Chinese Herbal Compound could reduce the expression of TIMP-1 mRNA of hepatic fibrosis rats and the effect is better than Colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
Conclusions
1 ZhuoDu theory has an important effect on etiology and pathogenesis in tcm analysis ,it play an important role in diagnosis and treatment of hepatic fibrosis.
2 Huazhuo Jiedu Yiqi’formula can improve the liver pathologic injur and ameliorate hepatocyte injury in hepatic fibrosis rats , exert an anti-hepatic fibrosis effect.
3 Huazhuo Jiedu Yiqi’formula decreased the level of serum ALT and AST and increased the concentration of serum TP,ALB, it may play a role in liver-protection,Anti-inflammatory effect of Huazhuo Jiedu Yiqi’formula lead the resistance against oxidant-induced injury anti-hepatic fibrosis,which is better than colchicines.
4 Huazhuo Jiedu Yiqi’formula could significantly reduce serum HA, LN,PCⅢ, CⅣand collagen hyperplasia in liver of hepatic fibrosis rats, exert an anti-hepatic fibrosis effect.
5 Jiedu Huazhuo Yiqi’formula could reduce the expression of TGF-β1 protein and mRNA of immunity hepatic fibrosis rats, suppresse theaetivation of HSCs, decrease the deposition of ECM.
6 Jiedu Huazhuo Yiqi’formula could significantly reduce the expression of TIMP-1 protein and mRNA of immunity pig serum-induced hepatic fibrosis of rat, which might play a role in regulating the disequilibrium between MMPS and TIMPS, promote the degrading of ECM, decrease the deposition of ECM.
以往的研究局限于将肝纤维化归为一种被动的静止状态,随着分子生物学研究方法和技术的日新月异,人们在肝星状细胞(hepatic stellate cells, HSC)活化、多种动物模型的建立、细胞因子信号传导通路、基因靶向治疗等方面取得了较大的进展。转化生长因子-β1(transforming growth factor- beta1-1,TGF-β1)、基质金属蛋白酶抑制因子-1(tissue inhibit of metallo -Proteinase,TIMP-1)、ECM成分等已经成为研究的热点。
目前研究的抗纤维化药物主要包括细胞保护类、HSC活化抑制类、细胞因子活性类、抑制ECM合成及分泌类、促进ECM降解类、中药单体和复方等。但肝纤维化是一个复杂的病理过程,临床上至今缺乏确切特效的抗纤维化药物与方法。
中医药治疗疾病具有多方位、多环节、多靶点的特点,在肝纤维化的预防和治疗方面有其独特的优势。在当前形势下,寻找能够改善HF状态的中药或者中药提取物,并对其相关机制进行探索,不仅有利于中药现代化的研究,也将对HF治疗产生深远的影响。
我们通过观察化浊解毒益气方对猪血清诱导的HF大鼠的干预研究,探讨肝纤维化的病理机制,为肝纤维化的治疗提供理论及实验依据。
目的:以“浊毒”理论为依据探讨其在肝纤维化治疗中的地位,探讨化浊解毒益气方对肝纤维化大鼠的影响及其可能的作用机制。主要内容包括以“浊毒”立论论治肝纤维化的理论初探;化浊解毒益气方对肝纤维化大鼠血清AST、ALT、ALB、TP和病理形态学的影响;化浊解毒益气方对肝纤维化大鼠血清HA、LN、PCⅢ、Ⅳ型胶原和病理形态学的影响;化浊解毒益气方对肝纤维化大鼠肝组织TGF-β1表达的影响;化浊解毒益气方对肝纤维化大鼠肝组织TIMP-1表达的影响。
方法:清洁级Wistar大鼠60只,雄性,体重200~220g。适应性喂养一周后随机分为正常对照组、模型对照组、秋水仙碱预防组(简称西药预防组)、化浊解毒益气方预防组(简称中药预防组)、秋水仙碱治疗组(简称西药治疗组)、化浊解毒益气方治疗组(简称中药治疗组)六组,每组10只。除正常对照组外,其余各组均采用未灭活的猪血清腹腔注射,每次0.5ml/只,每周两次,连续8周。秋水仙碱预防组和中药预防组于造模的同时给药,每天上午灌胃一次,每次2ml,连续12周。秋水仙碱治疗组和中药治疗组于造模的第8周末给药,每天上午灌胃一次,每次2ml,连续4周。正常对照组给予等量生理盐水。
各组分别于造模的第8周末、第12周末处死5只大鼠,腹主动脉取血,分离血清,测定各组大鼠干预后血清中丙氨酸氨基转移酶(ALT)、天门冬氨基转移酶(AST)、白蛋白(ALB)、总蛋白(TP)、透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(CⅣ)水平。其中ALT、AST用赖氏法测定;ALB用溴甲酚绿比色法测定;TP用双缩脲法测定;HA、LN、PCⅢ、CⅣ用放射免疫分析法测定。留取大鼠肝组织,采用苏木精-伊红染色和Masson染色(胶原纤维特殊染色法)观察肝组织病理形态学的变化。采用免疫组化和RT-PCR技术测定肝组织TGF-β1、TIMP-1蛋白和mRNA表达情况。所得数据经过整理,采用SPSS统计软件进行t检验或方差分析。
结果:
1化浊解毒益气方对肝纤维化大鼠血清AST、ALT、ALB、TP和病理形态学的影响
1.1化浊解毒益气方对肝纤维化大鼠血清AST、ALT、ALB、TP的影响
与正常对照组相比,模型组大鼠血清ALT、AST升高,ALB、TP降低,差异有统计学意义(P<0.05)。与模型对照组相比,第8周末,西药预防组和中药预防组各组大鼠血清ALT、AST下降,TP、ALB升高,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物处理组大鼠血清ALT、AST下降,TP、ALB升高,差异有统计学意义(P<0.05),且中药预防组优于西药预防组,中药治疗组优于西药治疗组(P<0.05)。但是西药处理组之间、中药处理组之间各组大鼠血清指标差异均无统计学意义(P>0.05)。
1.2化浊解毒益气方对肝纤维化大鼠肝脏病理形态学的影响
正常组肝小叶结构完整,肝细胞围绕中央静脉呈放射状排列,无变性坏死,无纤维组织增生。模型组肝小叶结构严重破坏,肝细胞广泛脂肪变性、坏死伴炎性细胞浸润,纤维组织大量增生,个别动物可见假小叶形成。第8周末,各药物预防组肝小叶结构受损轻,可见肝细胞脂肪变性及炎性细胞浸润,但较模型组显著减轻。胶原纤维增生较模型组减轻,主要散在于肝血窦和disse间隙内,无假小叶形成,中药预防组优于西药预防组。第12周末,各药物干预组肝脏损伤程度均较模型组轻,西药治疗组病理改变与西药预防组相似,中药治疗组病理改变与中药预防组相似。且中药预防组优于西药预防组,中药治疗组优于西药治疗组。
2化浊解毒益气方对肝纤维化大鼠血清HA、LN、PCⅢ、Ⅳ型胶原和病理形态学的影响
2.1化浊解毒益气方对肝纤维化大鼠血清HA、LN、PCⅢ、Ⅳ型胶原的影响
与正常对照组相比,模型组大鼠血清HA、LN、PCⅢ、Ⅳ型胶原升高,差异有统计学意义(P<0.05)。与模型对照组相比,第8周末,西药预防组和中药预防组各组大鼠血清HA、LN、PCⅢ、Ⅳ型胶原下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物处理组大鼠血清HA、LN、PCⅢ、Ⅳ型胶原下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组,中药治疗组优于西药治疗组(P<0.05)。但是西药处理组之间、中药处理组之间各组大鼠血清指标差异均无统计学意义(P>0.05)。
2.2化浊解毒益气方对肝纤维化大鼠病理形态学的影响
正常组肝小叶结构完整,肝细胞围绕中央静脉呈放射状排列,无变性坏死,无纤维组织增生。模型组肝小叶结构严重破坏,肝细胞广泛脂肪变性、坏死伴炎性细胞浸润,绿色纤维组织大量增生,个别动物可见假小叶形成。8周末,各预防组肝小叶结构受损轻,可见肝细胞脂肪变性及炎性细胞浸润,但较模型组显著减轻。胶原纤维增生较模型组减轻,主要散在于肝血窦和disse间隙,无假小叶形成。且中药预防组优于西药预防组。12周末,各药物干预组肝脏损伤程度均较模型组显著减轻,且中药预防组优于西药预防组。中药治疗组优于西药治疗组。
3化浊解毒益气方对肝纤维化大鼠肝组织TGF-β1表达的影响
3.1化浊解毒益气方对肝纤维化大鼠肝组织TGF-β1蛋白表达的影响。
正常组大鼠肝组织见少许TGF-β1阳性表达细胞,模型组大鼠肝组织中表达的阳性细胞比例显著高于正常组,表现为不均匀的黄褐色颗粒状物。与正常对照组相比,模型组大鼠肝组织TGF-β1蛋白表达增高,差异有统计学意义(P<0.05)。8周末,与模型对照组相比,西药预防组和中药预防组两组大鼠肝组织TGF-β1蛋白表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物干预组大鼠肝组织TGF-β1蛋白表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05),中药治疗组优于西药治疗组。但是西药预防组与治疗组之间、中药预防组和治疗组之间各组大鼠肝组织TGF-β1蛋白表达差异均无统计学意义(P>0.05)。
3.2化浊解毒益气方对肝纤维化大鼠肝组织TGF-β1mRNA表达的影响
与正常对照组相比,模型组大鼠肝组织TGF-β1mRNA表达增高,差异有统计学意义(P<0.05)。8周末,与模型对照组相比,西药预防组和中药预防组两组大鼠肝组织TGF-β1mRNA表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物干预组大鼠肝组织TGF-β1mRNA表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05),中药治疗组优于西药治疗组。但是西药预防组与治疗组之间、中药预防组和治疗组之间各组大鼠肝组织TGF-β1mRNA表达差异均无统计学意义(P>0.05)。
4化浊解毒益气方对肝纤维化大鼠肝组织TIMP-1表达的影响
4.1化浊解毒益气方对肝纤维化大鼠肝组织TIMP-1蛋白表达的影响
正常组大鼠肝组织见少许TIMP-1阳性表达细胞,模型组大鼠肝组织中表达的阳性细胞比例显著高于正常组,表现为不均匀的黄褐色颗粒状物。与正常对照组相比,模型组大鼠肝组织TIMP-1蛋白表达增高,差异有统计学意义(P<0.05)。8周末,与模型对照组相比,西药预防组和中药预防组两组大鼠肝组织TIMP-1蛋白表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物干预组大鼠肝组织TIMP-1蛋白表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05),中药治疗组优于西药治疗组。但是西药预防组与治疗组之间、中药预防组和治疗组之间各组大鼠肝组织TIMP-1蛋白表达差异均无统计学意义(P>0.05)。
4.2化浊解毒益气方对肝纤维化大鼠肝组织TIMP-1mRNA表达的影响
与正常对照组相比,模型组大鼠肝组织TIMP-1mRNA表达增高,差异有统计学意义(P<0.05)。8周末,与模型对照组相比,西药预防组和中药预防组大鼠肝组织TIMP-1mRNA表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05)。12周末,各药物干预组大鼠肝组织TIMP-1mRNA表达下降,差异有统计学意义(P<0.05),且中药预防组优于西药预防组(P<0.05),中药治疗组优于西药治疗组。但是西药预防组与治疗组之间、中药预防组和治疗组之间各组大鼠肝组织TIMP-1mRNA表达差异均无统计学意义(P>0.05)。
结论:
1“浊毒”理论的提出对肝纤维化的中医病因病机分析具有重要意义,在肝纤维化的诊断和治疗中,占有重要的地位。
2化浊解毒益气方能有效改善肝纤维化大鼠肝组织病理改变、减轻肝细胞的损伤,减少胶原纤维的生成,从而发挥其抗纤维化的作用。
3化浊解毒益气方能显著降低大鼠血清AST、ALT含量,升高TP、ALB含量,减轻肝细胞的损伤,保护肝功能,通过保肝、抗炎而发挥其抗纤维化的作用。
4化浊解毒益气方能显著降低大鼠血清HA、LN、PCⅢ型胶原、Ⅳ型胶原含量,减少细胞外基质的生成,从而发挥其抗纤维化的作用。
5化浊解毒益气方能显著降低大鼠肝组织TGF-β1蛋白和mRNA的表达,抑制肝星状细胞的活化,通过调控相关细胞因子生物作用,促进细胞外基质的降解。
6化浊解毒益气方能显著降低大鼠肝组织TIMP-1蛋白和mRNA的表达,通过调节MMPS和TIMPS的失衡,促进异常合成的细胞外基质的降解,减少细胞外基质的过度沉积。
Hepatic fibrosis is a pathologic process that every kind of disease cause liver injure and inflammation.It is the hallmark of most chronic liver diseases. Its essense is excessive deposition of extracellular matrix (ECM) components in liver, which occurs due to an imbalance between the production and degradation of matrix. Collagen is the most important part of ECM.
In previous studies, Hepatic fibrosis is limited to a passive stationary state. With the development of molecular biological technology, People have made large progress in many ways,such as hepatic stellate cell activation, Establishment of Animal Model , cytokine signaling ,the molecular targeted therapyin Hepatic fibrosis and so on . transforming growth factor-β1 ,tissue inhibit of metallo Proteinase and extracellular matrix components have become a hotspot for research.
In current research work, anti-hepatic fibrosis drug main contents include the type of cytoprotection, hepatic stellate cell activation, Cytokines activity, suppressed extracellular matrix production and secretion, Promote degradation of extracellular matrix, traditional medicine monomer and Chinese Herbal Compound. But HF is a complex process there is no established therapy for hepatic fibrosis。
The characteristics of Traditional Chinese Medicine (TCM) treatment are multiple positions, multiple links and multiple targets. There are much superiority in the prevention and treatment on Diabetes Mellitus. And now, to search the traditional medicine drugs or the extract of these drugs is important in the prevention and treatment of HF. If we can explore the possible mechanisms of these drugs, it will benefit the TCM modernization, the treatment of HF.
To study the effect of Huazhuo Jiedu Yiqi’formula on the effect of immunity hepatic fibrosis rats induced by pig’s serum. Investigate on Pathological Mechanism of Hepatic fibrosis,so that provide theoretical and experimental basis for treating liver fibrosis.
Objectives
The aim was to study the effect of The ZhuoDu theory in the position of Therapy for Hepatic Fibrosis, and explore its possible mechanism.. The research included preliminary Study of The ZhuoDu theory on Therapy for Hepatic Fibrosis, effect of Huazhuo Jiedu Yiqi’formula on the levels of serum ALT,AST,ALB,TP and Pathomorphology in hepatic fibrosis rats;effect of Huazhuo Jiedu Yiqi’formula on the levels of serum HA,LN,PCⅢ,CIV and Pathomorphology in hepatic fibrosis rats;Huazhuo Jiedu Yiqi’formula on the expression of TGF-β1 in liver tissue of hepatic fibrosis rats; Huazhuo Yiqi’formula on the expression of TIMP-1 in liver tissue of hepatic fibrosis rats.
Methods
Total 60 clean male Wistar rats weighed 200～220 grams were randomly divided into six groups:namely,the normal group, the model group, colchicine prevention group,(C Prevention group for short),Huazhuo Jiedu Yiqi’formula Prevention group(HJY Prevention group for short), colchicine treatment group(C treatment group), Huazhuo Jiedu Yiqi’formula treatment group(HJY reatment group)with 10 rats for each group .In addition to the normal group,pig’s serum( 0.5ml per mouse, twice a week) was established in rats by intraperitoneal injection for eight weeks. The drugs were given through stomach-perfusion to C Prevention group and HJY Prevention group at the beginning of experiment.The drugs are given at the same time every day.The treatment groups were given drug at the end of the eighth week. The rats in the normal control and model groups received intragastric administration of saline(2ml each time).
Five rats were killed in each group at the 8th and 12th weekend. We draw blood from abdominal aorta, ALT、AST level were measured with Reit′s method; The contents of TP andAlb were measured using biurea method and bromocresol green method ;HA、LN、PCⅢ、CⅣlevel were measured with radioimmunoassay(RIA). The specimens were taken and the degree of hepatic fibrosis was judged by routine haematoxylin-eosin staining and Masson staining.TGF-β1 protein and mRNA expression were determined using immunohistochemical technique and RT-PCR.TIMP-1 protein and mRNA expression were determined using immunohistochemical technique and RT-PCR .
Data was collected and analyzed through t test or analysis of variance. The statistic software was SPSS. Differences with P values <0.05 were considered significant.
Results
1 Effect of Huazhuo JieDu Yiqi’formula on the levels of serum ALT,AST,ALB,TP and pathomorphology in hepatic fibrosis rats.
1.1 Effect of Huazhuo JieDu Yiqi’formula on the levels of serum ALT,AST,ALB,TP in hepatic fibrosis rats.
Compared with the normal group, the content of ALT and AST was significantly increased with liver injure in the model group(P<0.05). TP、ALB of serum can be increased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05). Compared with the model group, All medicine intervention group decreased the level of serum ALT and AST and increased the concentration of serum TP,ALB at the end of 12 weeks ,there were statistic differences among the groups(P<0.05) .The effect of Chinese Herbal Compound can reduce the evels of serum ALT,AST,ALB,TP in hepatic fibrosis rats and the effect is better than Colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
1.2 Effect of Huazhuo JieDu Yiqi’formula to pathomorphology in hepatic fibrosis rats.
The structure of hepatic lobule were integrated hepatic cord radially ranged as the center of navel vein.There was not degeneration and necrosis in liver tissue. There was not collagen proliferation.Compared with normal group,the model control rats had obvious liver inflammation including degeneration,necrosis,fibrosis and collagen proliferation.Pseudolobule proliferation could be seen in individual animal.At the end of 8th weeks, Hepatic lobule in colchicine prevention group and HJY Prevention group lessed injured than model group. Hepatocellular degeneration and necrosis and inflammatory cell infiltration could be seen in liver tissue.Collagen proliferation in the two groups was less than in model group,it were mainly scattered in hepatic sinusoid and disse clearance. There was not pseudolobule proliferation. Pathological examination in HJY Prevention group was better than C prevention group. At the end of 12th weeks, Pathological examination in the treatment groups less injured than in model group. Pathological examination in C treatment group was similar to that of C prevention group ,the result was same between HJY Prevention group and treatment group.And The effect of Chinese Herbal Compound is better than colchicine.
2 Effect of Huazhuo Yiqi’formula on the levels of serum HA,LN,PCⅢ,CIV and pathomorphology in hepatic fibrosis rats
2.1 Effect of Huazhuo Yiqi’formula on the levels of serum HA,LN,PCⅢ,CIV and pathomorphology in hepatic fibrosis rats
Compared with the normal group, the levels of serum HA,LN,PCⅢ,CIV could be increased from treament group with the significant difference(P<0.05) . The levels of serum HA,LN,PCⅢ,CIV could be decreased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05).Compared with the model group, All medicine intervention group decreased the level of serum HA,LN,PCⅢ,CIV at the end of 12 weeks,There were statistic differences among the groups(P<0.05).The Effect of Chinese Herbal Compound can reduce the levels of serum HA,LN,PCⅢ,CIV in hepatic fibrosis rats and the effect is better than colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
2.2 Effect of Huazhuo Yiqi’formula to pathomorphology in hepatic fibrosis rats.
The structure of hepatic lobule were integrated hepatic cord radially ranged as the center of navel vein.There was not degeneration and necrosis in liver tissue. There was not collagen proliferation.Compared with normal rats,the model control rats had obvious liver inflammation including degeneration,necrosis,fibrosis and collagen proliferation .Pseudolobule proliferation could be seen in individual animal.At the end of 8th weeks, Hepatic lobule in C prevention group and HJY Prevention group lessed injured than model group. Hepatocellular degeneration and necrosis and inflammatory cell infiltration could be seen in liver tissue. Collagen proliferation in the two groups was less than in model group,it were mainly scattered in hepatic sinusoid and disse clearance. There was not pseudolobule proliferation. Pathological examination in HJY Prevention group was better than C prevention group. At the end of 12th weeks, Pathological examination in the treatment groups less injured than in model group. Pathological examination in C treatment group was similar to that of C prevention group ,the result was same between HJY Prevention group and treatment group.And The effect of Chinese Herbal Compound is better than colchicine.
3 Experimental study on Huazhuo Jiedu Yiqi’formula effect on TGF-β1 of the immunity hepatic fibrosis rats
3.1 Experimental study on Huazhuo Jiedu Yiqi’Formula effect on TGF-β1 pretine of the immunity hepatic fibrosis rats
The positive expression cells of normal group was a few. the expression of TGF-β1 was strongly positive in model group.The level was higher than normal group. It could be manifested as heterogeneously vinegar particles.Less positive expression of TGF-β1 cell can be seen in the liver tissue of rats form normal group,the cell proportion of TGF-β1 cell expression in the liver tissue of rats form model group were significantly higer than that of the normal group,which were manifested by the unsymmetric brown yellow particulates(P<0.05). The protein level of TGF-β1 of immunity hepatic fibrosis rats could be decreased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05).Compared with the model group, All medicine intervention group decreased the protein level of TGF-β1 of immunity hepatic fibrosis rats at the end of 12 weeks,There were statistic differences among the groups(P<0.05). The Effect of Chinese Herbal Compound can reduce the expression of TGF-β1 protein of immunity hepatic fibrosis rats and the effect is better than Colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
3.2 Experimental study on Huazhuo Jiedu Yiqi’formula effect on TGF-β1 mRNA of the immunity hepatic fibrosis rats
The results showed that the expressions of TGF-β1 mRNA in the model groups were increased significantly, respectively compared with normal control group and the difference was significant (P<0.05). The expressions of TGF-β1 mRNA of immunity hepatic fibrosis rats could be decreased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05).Compared with the model group, All medicine intervention group decreased the expressions of TGF-β1 mRNA of immunity hepatic fibrosis rats at the end of 12 weeks,There were statistic differences among the groups(P<0.05).The Effect of Chinese Herbal Compound could reduce the expression of TGF-β1 mRNA of hepatic fibrosis rats and the effect is better than Colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
4 Experimental study on Huazhuo Jiedu Yiqi’formula effect on TIMP-β1 of the immunityhepatic fibrosis rats
4.1 Experimental study on Huazhuo Jiedu Yiqi’formula effect on TIMP-1 pretine of the immunity hepatic fibrosis rats
The positive expression cells of normal group was a few. the expression of TIMP-1 was strongly positive in model group.The level was higher than normal group. It could be manifested as heterogeneously vinegar particles.Less positive expression of TIMP-1cell can be seen in the liver tissue of rats form normal group,the cell proportion of TIMP-1 cell expression in the liver tissue of rats form model group were significantly higer than that of the normal group,which were manifested by the unsymmetric brown yellow particulates(P<0.05). The protein level of TIMP-1 of immunity hepatic fibrosis rats could be decreased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05).Compared with the model group, All medicine intervention group decreased the protein level of TIMP-1 of immunity hepatic fibrosis rats at the end of 12 weeks,There were statistic differences among the groups(P<0.05). The Effect of Chinese Herbal Compound can reduce the expression of TIMP-1 protein of immunity hepatic fibrosis rats and the effect is better than colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
4.2 Experimental study on Huazhuo Jiedu Yiqi’formula effect on TIMP-1 mRNA of the immunity hepatic fibrosis rats
The results showed that the expressions of TIMP-1 mRNA in the model groups were increased significantly, respectively compared with normal control group and the difference was significant (P<0.05). The expressions of TIMP-1 mRNA of immunity hepatic fibrosis rats could be decreased from C Prevention group and HJY Prevention group significantly at the end of 8 weeks(P<0.05). The effect of HJY Prevention group was better than C prevention group(P<0.05).Compared with the model group, All medicine intervention group decreased the expressions of TIMP-1 mRNA of immunity hepatic fibrosis rats at the end of 12 weeks,There were statistic differences among the groups(P<0.05).The Effect of Chinese Herbal Compound could reduce the expression of TIMP-1 mRNA of hepatic fibrosis rats and the effect is better than Colchicine (P<0.05) . But there was no significant difference between the effect of prevention and treatment with using two medicines(P>0.05).
Conclusions
1 ZhuoDu theory has an important effect on etiology and pathogenesis in tcm analysis ,it play an important role in diagnosis and treatment of hepatic fibrosis.
2 Huazhuo Jiedu Yiqi’formula can improve the liver pathologic injur and ameliorate hepatocyte injury in hepatic fibrosis rats , exert an anti-hepatic fibrosis effect.
3 Huazhuo Jiedu Yiqi’formula decreased the level of serum ALT and AST and increased the concentration of serum TP,ALB, it may play a role in liver-protection,Anti-inflammatory effect of Huazhuo Jiedu Yiqi’formula lead the resistance against oxidant-induced injury anti-hepatic fibrosis,which is better than colchicines.
4 Huazhuo Jiedu Yiqi’formula could significantly reduce serum HA, LN,PCⅢ, CⅣand collagen hyperplasia in liver of hepatic fibrosis rats, exert an anti-hepatic fibrosis effect.
5 Jiedu Huazhuo Yiqi’formula could reduce the expression of TGF-β1 protein and mRNA of immunity hepatic fibrosis rats, suppresse theaetivation of HSCs, decrease the deposition of ECM.
6 Jiedu Huazhuo Yiqi’formula could significantly reduce the expression of TIMP-1 protein and mRNA of immunity pig serum-induced hepatic fibrosis of rat, which might play a role in regulating the disequilibrium between MMPS and TIMPS, promote the degrading of ECM, decrease the deposition of ECM.
引文
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