加味四逆汤防治病毒性肝炎的临床及实验研究
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摘要
目的：
乙型病毒性肝炎(HBV)是由乙肝病毒感染而引起的一种传染病,我国是高发流行区,每年约有30万人因肝病而死亡,其中多数与HBV感染有关,并且该病有10%-20%可变成肝硬化或肝癌,严重威胁着我们的生活和健康。在慢性乙型肝炎时,由于自身免疫功能失调,机体产生的抗体不足以清除体内的乙肝病毒,导致病毒大量复制,持续不断的造成肝细胞损伤,最终形成肝硬化甚至肝癌。寻找确切有效且简单的方法治疗本病越来越引起人们的关注。我们根据清代名医黄元御“一气周流,土枢四象”以及医圣“见肝之病,知肝传脾”的理论,利用《伤寒论》名方四逆汤加上山茱萸而形成调节人体免疫为主的加味四逆汤。原方四逆汤主要治疗阳虚阴寒内盛之少阴寒化证,具有回阳救逆之功效。现代药理研究表明：四逆汤有抗休克、强心、改善微循环、抗血脂以及对病理状态下的机体有抗炎、免疫调节等多种药理作用。山茱萸已被证实具有抗氧化、免疫调节、抗炎等方面的功效。我们通过临床观察了加味四逆汤治疗慢性乙型肝炎的疗效。
现代研究认为,乙型肝炎是由乙型肝炎病毒引起的一种免疫相关性疾病,机体感染HBV后的组织损伤并非其在肝细胞内复制繁殖直接作用的结果,而是一系列宿主免疫反应造成肝细胞的病理性免疫反应造成的免疫损害。刀豆蛋白复制形成的肝损伤模型与临床病毒性肝炎的表现十分相似,在现代研究中多作为病毒性肝炎的常用模型。在肯定临床疗效的基础上,通过复制刀豆蛋白急性免疫性肝损伤模型,采用流式细胞、ELISA等方法观察加味四逆汤对免疫性肝损伤的保护作用,并初步探讨其保护作用机制。
方法：
临床研究：共观察了慢性乙型肝炎患者32例,中药用加味四逆汤治疗,每日1剂,对照组用西药拉米夫定治疗,每日100mg,3个月为1疗程,共治疗2个疗程,然后分析治疗前后症状体征、肝功能及肝纤维化指标的变化。
实验研究：雌性NIH小鼠随机分成5组：正常对照组、模型对照组、阳性药物组、加味四逆汤大剂量组、加味四逆汤小剂量组,采用Con A尾静脉注射制作急性免疫性肝损伤模型。12h后处死动物取肝脏及血清检测各组小鼠肝脏指数,血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)活性,以及肝组织超氧化物歧化酶(SOD)活力和脂质过氧化产物丙二醛(MDA)的水甲,并观察肝组织病理学形态。通过FACS Cali bur流式细胞术测定全血中CD4+、 CD8+T细胞亚群,并以ELISA方法测定血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)的活性和细胞因子小鼠肿瘤坏死因子(TNF-a)、小鼠r干扰素(IFN-r)和白介素-10的水平。所有数据均以均数±标准差表示,以SPSS17.0软件进行统计分析,计量资料两组间比较用t检验,组间比较采用单因素方差分析,组间两两比较采用LSD法检验,统计前均对各组数据进行正态性检验和方差齐性检验,取a=0.05为显著性检验水准。
结果：
临床研究：
两组病例在年龄、性别及病情分布上无明显差异。经过治疗,治疗组病例在症状及体征的改善上明显好转,与对照组比较有统计学意义；但在肝功能及肝纤维化指标的检测上,两组比较没有明显差异。
实验研究：
1、肝组织大体形态改变
肉眼观察：正常组肝脏形态正常,被膜光整,色泽红润。模型组肝脏体积增大,被膜肿胀,边缘变钝,表面可见点状及片状的出血坏死点,用药组接近于正常。镜下观察：正常组肝组织结构完整、清晰,肝细胞无变性、坏死现象,无炎症细胞浸润。模型组肝小叶结构破坏,模糊不清,可见多数肝细胞出现点状、小片状或弥漫性坏死,坏死区域内有大量淋巴细胞和单核细胞浸润,有多数胞浆内吞噬细胞碎片和脂褐素的枯否细胞增生；此外大多数肝细胞胞浆肿胀、疏松,有的呈气球样变,并可见许多圆形类圆形嗜伊红的凋亡肝细胞。用药组肝小叶结构接近于正常,肝细胞坏死和炎症细胞浸润减少。
2、加味四逆汤对免疫性肝损伤小鼠ALT、 AST水甲的影响
小鼠肝功能检测结果显示,模型对照组小鼠ALT、 AST水平明显高于空白对照组(P均<0.05),与正常组比较差异具有统计学意义(P<0.01),表明实验造模成功；与模型组比较,联苯双酯组和加味四逆汤组能不同水平减低转氨酶水平(P<0.05),说明加味四逆汤组能降低免疫性肝损伤小鼠ALT和AST活性。
3、加味四逆汤对小鼠肝脏匀浆SOD与MDA的影响
用Con A造模后,小鼠MDA水甲明显高于空白对照组,SOD活性显著低于空白对照组(P<0.01)。给予药物治疗后,与模型对照组比较,加味四逆汤组SOD水平不同程度提高(P<0.01),MDA水平显著减低(P<0.05)。
4、加昧四逆汤对IFN-r、 TNF-a和IL-10分泌的影响
结果表明：模型对照组IFN-r、 TNF-a明显高于空白对照组(P<0.01)；西药治疗组和加味四逆汤组治疗后,与模型对照组比较,IFN-r、TNF-a分泌明显降低(P<0.01)；治疗组之间IFN-r、 TNF-a的含量无统计学意义(P>0.05)。而血清IL-10含量模型对照组较正常组明显减低(P<0.01)；西药治疗组与模型对照组比较IL-10活性提高(P<0.01),但仍然低于空白对照组；加味四逆汤干预后,IL-10的含量也得到了一定提高,与模型对照组比较有差异(P<0.05)。
5、加味四逆汤对外周血CD3、 CD4、 CD8的影响
实验结果表明模型组小鼠全血中CD4+、CD8+比率较正常对照组明显下降,与正常组比较差异有显著性(P<0.05)；与模型组比较,加味四逆汤各剂量组能引起小鼠全血CD4+、 CD8+比率明显升高(P<0.05),说明加味四逆汤能明显上调在免疫性肝损伤中已经明显降低的小鼠全血中CD4+、 CD8+比率,即提高外周血T淋巴细胞亚群的比率,减少其向肝脏的浸润,从而减小其细胞毒性作用对肝细胞的损伤。
结论：
临床研究：
加味四逆汤在治疗慢性乙型肝炎的方面,有比较好的疗效,对改善患者的生活质量尤为明显,在改善肝功能及防止肝纤维化方面也有疗效,且无明显毒副作用。
实验研究：
1、小鼠经尾静脉注射ConA12h后,ALT、 AST水平均明显升高,其血清学指标改变与文献报道一致,说明肝损伤造模成功。
2、加味四逆汤能明显降低模型小鼠血清转氨酶ALT、 AST活性,说明加味四逆汤对Con A造成的免疫性肝损伤具有保护作用。并且可明显减轻免疫性肝损伤小鼠肝组织小叶结构的破坏,减少肝组织炎性细胞的浸润,降低肝细胞变性、坏死和凋亡的形成,达到保护肝组织的目的。
3、加味四逆汤能明显升高肝损伤模型小鼠肝组织SOD活性,同时降低其MDA的含量,表明其对小鼠免疫性肝损伤具有一定抗氧化,减少自由基产生,减轻肝组织过氧化损伤的作用。其机制可能是直接清除自由基,阻断或终止自由基连锁反应链,从而阻止或抑制氧自由基反应和脂质过氧化反应,最终抑制脂质过氧化产物MDA的生成。因此,加味四逆汤可能是通过稳定细胞膜、抗脂质过氧化和阻断细胞凋亡的发生达到抗肝损伤目的,而其机体作用机制还有待进一步研究。
4、加味四逆汤能明显下降模型小鼠血清IFN-r、 TNF-a水平,提高IL-10水甲,因此我们推测加味四逆汤通过下调过度免疫炎性因子和增加抗炎性细胞因子的释放起到保护免疫性肝损伤的作用。
5、加味四逆汤能明显上调在模型小鼠外周血中明显降低的CD4+、 CD8+比率,说明加味四逆汤可提高模型小鼠外周血T淋巴细胞亚群的比例,减少其向肝脏的浸润,从而减小对肝细胞的细胞毒性作用,因此我们推测加味四逆汤保护肝损伤的作用与调节T细胞亚群间相互甲衡和小鼠肝损伤过程中的免疫应答水甲作用有关。
Objective
Hepatitis B virus (HBV) is an infectious disease caused by hepatitis B virus infection, China is a high prevalence area, about300000people died due to liver disease each year, most of which are related with HBV infection, and the disease of10%-20%can be turned into liver cirrhosis or liver cancer, is a serious threat to our life and health. In chronic hepatitis patients, due to autoimmune disorders, antibodies produced by the body are not enough to clear the body of the hepatitis B virus, resulting in a large number of virus replication, continue to cause liver cell damage, eventually cirrhosis or liver cancer. Method for finding the exact effective and simple treatment of the disease more and more attention. According to the Qing Dynasty famous doctor Huang Yuanyu "circular flow of Qi, soil and pivot Sasang" medicine "see liver disease, liver Chuan spleen" theory, the "Treatise on the" side of Sini Decoction with hill dogwood and regulating the body's immune based Modified Sini Decoction. The original Sini Decoction mainly treating deficiency of Yang Yin Han Sheng little cold syndrome, has the functions of Yang deficiency. Modern pharmacological research show that:Sini Decoction has shock resistance, strong heart, improve microcirculation, blood lipid and anti inflammatory, immune regulation and other pharmacological effects on the pathological state of the body. Cornus officinalis have been shown to have antioxidant, immunomodulatory, anti-inflammatory and other aspects. We observed the effect of Modified Sini Decoction in the treatment of chronic hepatitis B by clinical. Modern research thinks, hepatitis B is a kind of immune related diseases caused by the hepatitis B virus, tissue injury after HBV replication is not the direct role in the liver cell of the result of infection, but a series of host immune response caused by immune damage caused by pathological immune response in liver cells. Liver injury model and clinical hepatitis concanavalin copy form performance is very similar, as many commonly used models of viral hepatitis in the modern study of. Based on affirming the clinical curative effect on acute immune liver, by copying con damage model, to observe the protective effect of Modified Sini Decoction on immunological liver injury using flow cytometry, ELISA and other methods, and to explore its protective mechanism.
Methods
Clinical study:32cases of patients with chronic hepatitis B were observed by Modified Sini Decoction, traditional Chinese medicine treatment, daily1agent, the control group was treated with lamivudine treatment, daily100mg,3months to1course of treatment, a total of2courses of treatment, then analyze the changes before and after treatment, symptoms and signs, liver function and indexes of hepatic fibrosis.
Experimental study:Female NIH mice were randomly divided into5groups: normal control group, model control group, positive drug group, Modified Sini Decoction Modified Sini Decoction in high dose group, low dose group, acute immune liver injury model by tail vein injection of Con A.12h after the death of animal liver and serum of mice were detected the liver index, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, and liver tissue superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels, and to observe the pathological morphology. Determination of blood CD4+, CD8+T lymphocyte subsets by FACS Cali bur flow cytometry, and the ELISA method for the determination of serum glutamic oxaloacetic transaminase (AST), alanine aminotransferase (ALT) activity and cytokines in mice tumor necrosis factor (TNF-a), mouse interferon R (IFN-r) and interleukin-10. All the data are mean+standard deviation said, statistical analysis was performed by SPSS17.0software, the measurement data between two groups were compared using t test, compared with single factor analysis of variance, between the two two groups were compared with LSD test statistics, all the data in each group were test of normality and homogeneity of variance test, take a=0.05is a significant test level.
Results
Clinical study:two cases had no significant difference in age, gender and disease distribution. After treatment, the treatment group in the symptoms and signs were improved markedly, with statistical significance compared with the control group; but in the detection of liver function and hepatic fibrosis index, the two groups have no significant difference. Experimental study:
1、 liver morphological changes.
Macroscopic observation:normal liver morphology was normal, smooth, ruddy color. Model group liver volume increased, membrane swelling, blunt edge, surface punctate and patchy necrosis and hemorrhage, treatment group was close to normal. Microscopy:normal liver tissue of the group structure is complete, clear, liver cell degeneration, no necrosis, no inflammatory cell infiltration. Fuzzy damage model group, hepatic lobules, the most visible liver cells appeared punctate, patchy or diffuse necrosis, necrosis region has a large number of lymphocytes and mononuclear cells infiltration, the majority of intracellular phagocytosis of cellular debris and lipofuscin Kupffer cell hyperplasia; in addition, the majority of liver cell cytoplasmic swelling, osteoporosis, some are ballooning degeneration, and there were many round in eosinophil apoptosis eosin liver cells. Use structure of hepatic lobule were close to normal, reduce liver cell necrosis and inflammatory cell infiltration.
2、 Effect of Modified Sini Decoction on immunological liver injury in mice, the levels of AST ALT
Results liver function of mice showed, model mice of control group ALT, AST levels were significantly higher than that of control group (all P<0.05), compared with the normal group had statistical significance (P<0.01), showed that the experimental modeling success; compared with the model group, bifendate group and Modified Sini Decoction group four different levels reduced transaminase level (P<0.05), showed that Modified Sini Decoction group four can reduce the immunological liver injury in mice ALT and AST activity.
3、 Effect of Modified Sini Decoction on mice liver homogenate SOD and MDA
Using Con A model, MDA levels in mice were significantly higher than that of control group, SOD activity was significantly lower than that in control group (P<0.01). After treatment, compared with model group, Modified Sini group Decoction SOD levels increased in different degrees (P<0.01), the level of MDA was significantly decreased (P<0.05).
4、 Effect of Modified Sini Decoction on IFN-r, TNF-a and IL-10secretion
The results show that:the model control group IFN-r, TNF-a were significantly higher than that of control group (P<0.01); western medicine treatment group and Modified Sini group Decoction after treatment, compared with model group, IFN-r, TNF-a secretion decreased significantly (P<0.01); the content of IFN-r, TNF-a between treatment groups was not statistically significant (P>0.05). The content of serum IL-10and the model control group decreased significantly than that of control group (P<0.01); western medicine treatment group and model control group increased IL-10activity (P<0.01), but still lower than the control group; Modified Sini Decoction intervention, the content of IL-10was also improved, compared with the model group differences (P<0.05).
5、 Effect of Modified Sini Decoction on peripheral blood CD3, CD4, CD8
The experimental results show that the model group mice whole blood CD4+, CD8+ratio compared to the normal control group decreased significantly, there was significant difference compared with the normal group (P<0.05); compared with the model group, Modified Sini Decoction each dose group can cause mouse whole blood CD4+, CD8+ratio was significantly increased (P<0.05), Modified Sini Decoction CD4+, CD8+the ratio of blood mice have increased markedly lower in immunological liver injury in the higher ratio, namely peripheral blood T lymphocyte subsets, reduce its to liver infiltration, thereby reducing its cytotoxic effect on liver cell injury.
Conclusion
Clinical research:Modified Sini Decoction in the treatment of chronic hepatitis B, have a better effect, especially for improving the quality of life of patients, in the improvement of liver function and prevent liver fibrosis also has curative effect, without obvious side effect. Experimental study:
1、 Mice via tail vein injection of ConA12h, ALT, AST levels were significantly elevated, the serological indexes changes consistent with the reported literature, that liver injury rats.
2、 Modified Sini Decoction can significantly decrease the serum transaminase mice model ALT, AST activity, that has protective effects on liver immune Modified Sini Decoction for Con A damage. And can obviously reduce the lobules liver injury mouse immunological liver damage, reduce the infiltration of inflammatory cells in the liver tissue, reduce the formation of necrosis and apoptosis of liver cell degeneration,, to protect the liver tissue objective.
3、 Modified Sini Decoction could significantly increase the activity of SOD of liver injury model of mouse liver tissue, while reducing the content of MDA, show that it has certain antioxidant on immunological liver injury in mice, reduce the production of free radicals, reducing liver peroxidation. The mechanism may be directly scavenging free radicals, free radical chain reaction chain blocked or terminated, so as to prevent or inhibit the reaction of oxygen free radical and lipid peroxidation, generate the final inhibition of lipid peroxidation product MDA. Therefore, Modified Sini Decoction may be through the cell membrane stability, resisting lipid peroxidation and blocking apoptosis reached against liver injury, and its mechanism of action remains to be further studied body.
4、 Modified Sini Decoction can significantly decrease the levels of serum IFN-r, TNF-a model in mice, increase the level of IL-10, so we speculated that Modified Sini Decoction by downregulating the excessive proinflammatory cytokines and anti-inflammatory cytokines release to protect the immune liver injury.
5、 Modified Sini Decoction upregulated significantly decreased in peripheral blood of the mouse model of CD4+, CD8+ratio, that proportion of T lymphocyte subsets in peripheral blood of Modified Sini Decoction can improve the model mice, reduce the liver infiltration, thereby reducing the cell toxicity to liver cells, so we speculated that the immune response level effect of Modified Sini Decoction to protect the liver injury and regulation of T cell subsets in mutual equilibrium and in the process of liver injury in mice.
乙型病毒性肝炎(HBV)是由乙肝病毒感染而引起的一种传染病,我国是高发流行区,每年约有30万人因肝病而死亡,其中多数与HBV感染有关,并且该病有10%-20%可变成肝硬化或肝癌,严重威胁着我们的生活和健康。在慢性乙型肝炎时,由于自身免疫功能失调,机体产生的抗体不足以清除体内的乙肝病毒,导致病毒大量复制,持续不断的造成肝细胞损伤,最终形成肝硬化甚至肝癌。寻找确切有效且简单的方法治疗本病越来越引起人们的关注。我们根据清代名医黄元御“一气周流,土枢四象”以及医圣“见肝之病,知肝传脾”的理论,利用《伤寒论》名方四逆汤加上山茱萸而形成调节人体免疫为主的加味四逆汤。原方四逆汤主要治疗阳虚阴寒内盛之少阴寒化证,具有回阳救逆之功效。现代药理研究表明：四逆汤有抗休克、强心、改善微循环、抗血脂以及对病理状态下的机体有抗炎、免疫调节等多种药理作用。山茱萸已被证实具有抗氧化、免疫调节、抗炎等方面的功效。我们通过临床观察了加味四逆汤治疗慢性乙型肝炎的疗效。
现代研究认为,乙型肝炎是由乙型肝炎病毒引起的一种免疫相关性疾病,机体感染HBV后的组织损伤并非其在肝细胞内复制繁殖直接作用的结果,而是一系列宿主免疫反应造成肝细胞的病理性免疫反应造成的免疫损害。刀豆蛋白复制形成的肝损伤模型与临床病毒性肝炎的表现十分相似,在现代研究中多作为病毒性肝炎的常用模型。在肯定临床疗效的基础上,通过复制刀豆蛋白急性免疫性肝损伤模型,采用流式细胞、ELISA等方法观察加味四逆汤对免疫性肝损伤的保护作用,并初步探讨其保护作用机制。
方法：
临床研究：共观察了慢性乙型肝炎患者32例,中药用加味四逆汤治疗,每日1剂,对照组用西药拉米夫定治疗,每日100mg,3个月为1疗程,共治疗2个疗程,然后分析治疗前后症状体征、肝功能及肝纤维化指标的变化。
实验研究：雌性NIH小鼠随机分成5组：正常对照组、模型对照组、阳性药物组、加味四逆汤大剂量组、加味四逆汤小剂量组,采用Con A尾静脉注射制作急性免疫性肝损伤模型。12h后处死动物取肝脏及血清检测各组小鼠肝脏指数,血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)活性,以及肝组织超氧化物歧化酶(SOD)活力和脂质过氧化产物丙二醛(MDA)的水甲,并观察肝组织病理学形态。通过FACS Cali bur流式细胞术测定全血中CD4+、 CD8+T细胞亚群,并以ELISA方法测定血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)的活性和细胞因子小鼠肿瘤坏死因子(TNF-a)、小鼠r干扰素(IFN-r)和白介素-10的水平。所有数据均以均数±标准差表示,以SPSS17.0软件进行统计分析,计量资料两组间比较用t检验,组间比较采用单因素方差分析,组间两两比较采用LSD法检验,统计前均对各组数据进行正态性检验和方差齐性检验,取a=0.05为显著性检验水准。
结果：
临床研究：
两组病例在年龄、性别及病情分布上无明显差异。经过治疗,治疗组病例在症状及体征的改善上明显好转,与对照组比较有统计学意义；但在肝功能及肝纤维化指标的检测上,两组比较没有明显差异。
实验研究：
1、肝组织大体形态改变
肉眼观察：正常组肝脏形态正常,被膜光整,色泽红润。模型组肝脏体积增大,被膜肿胀,边缘变钝,表面可见点状及片状的出血坏死点,用药组接近于正常。镜下观察：正常组肝组织结构完整、清晰,肝细胞无变性、坏死现象,无炎症细胞浸润。模型组肝小叶结构破坏,模糊不清,可见多数肝细胞出现点状、小片状或弥漫性坏死,坏死区域内有大量淋巴细胞和单核细胞浸润,有多数胞浆内吞噬细胞碎片和脂褐素的枯否细胞增生；此外大多数肝细胞胞浆肿胀、疏松,有的呈气球样变,并可见许多圆形类圆形嗜伊红的凋亡肝细胞。用药组肝小叶结构接近于正常,肝细胞坏死和炎症细胞浸润减少。
2、加味四逆汤对免疫性肝损伤小鼠ALT、 AST水甲的影响
小鼠肝功能检测结果显示,模型对照组小鼠ALT、 AST水平明显高于空白对照组(P均<0.05),与正常组比较差异具有统计学意义(P<0.01),表明实验造模成功；与模型组比较,联苯双酯组和加味四逆汤组能不同水平减低转氨酶水平(P<0.05),说明加味四逆汤组能降低免疫性肝损伤小鼠ALT和AST活性。
3、加味四逆汤对小鼠肝脏匀浆SOD与MDA的影响
用Con A造模后,小鼠MDA水甲明显高于空白对照组,SOD活性显著低于空白对照组(P<0.01)。给予药物治疗后,与模型对照组比较,加味四逆汤组SOD水平不同程度提高(P<0.01),MDA水平显著减低(P<0.05)。
4、加昧四逆汤对IFN-r、 TNF-a和IL-10分泌的影响
结果表明：模型对照组IFN-r、 TNF-a明显高于空白对照组(P<0.01)；西药治疗组和加味四逆汤组治疗后,与模型对照组比较,IFN-r、TNF-a分泌明显降低(P<0.01)；治疗组之间IFN-r、 TNF-a的含量无统计学意义(P>0.05)。而血清IL-10含量模型对照组较正常组明显减低(P<0.01)；西药治疗组与模型对照组比较IL-10活性提高(P<0.01),但仍然低于空白对照组；加味四逆汤干预后,IL-10的含量也得到了一定提高,与模型对照组比较有差异(P<0.05)。
5、加味四逆汤对外周血CD3、 CD4、 CD8的影响
实验结果表明模型组小鼠全血中CD4+、CD8+比率较正常对照组明显下降,与正常组比较差异有显著性(P<0.05)；与模型组比较,加味四逆汤各剂量组能引起小鼠全血CD4+、 CD8+比率明显升高(P<0.05),说明加味四逆汤能明显上调在免疫性肝损伤中已经明显降低的小鼠全血中CD4+、 CD8+比率,即提高外周血T淋巴细胞亚群的比率,减少其向肝脏的浸润,从而减小其细胞毒性作用对肝细胞的损伤。
结论：
临床研究：
加味四逆汤在治疗慢性乙型肝炎的方面,有比较好的疗效,对改善患者的生活质量尤为明显,在改善肝功能及防止肝纤维化方面也有疗效,且无明显毒副作用。
实验研究：
1、小鼠经尾静脉注射ConA12h后,ALT、 AST水平均明显升高,其血清学指标改变与文献报道一致,说明肝损伤造模成功。
2、加味四逆汤能明显降低模型小鼠血清转氨酶ALT、 AST活性,说明加味四逆汤对Con A造成的免疫性肝损伤具有保护作用。并且可明显减轻免疫性肝损伤小鼠肝组织小叶结构的破坏,减少肝组织炎性细胞的浸润,降低肝细胞变性、坏死和凋亡的形成,达到保护肝组织的目的。
3、加味四逆汤能明显升高肝损伤模型小鼠肝组织SOD活性,同时降低其MDA的含量,表明其对小鼠免疫性肝损伤具有一定抗氧化,减少自由基产生,减轻肝组织过氧化损伤的作用。其机制可能是直接清除自由基,阻断或终止自由基连锁反应链,从而阻止或抑制氧自由基反应和脂质过氧化反应,最终抑制脂质过氧化产物MDA的生成。因此,加味四逆汤可能是通过稳定细胞膜、抗脂质过氧化和阻断细胞凋亡的发生达到抗肝损伤目的,而其机体作用机制还有待进一步研究。
4、加味四逆汤能明显下降模型小鼠血清IFN-r、 TNF-a水平,提高IL-10水甲,因此我们推测加味四逆汤通过下调过度免疫炎性因子和增加抗炎性细胞因子的释放起到保护免疫性肝损伤的作用。
5、加味四逆汤能明显上调在模型小鼠外周血中明显降低的CD4+、 CD8+比率,说明加味四逆汤可提高模型小鼠外周血T淋巴细胞亚群的比例,减少其向肝脏的浸润,从而减小对肝细胞的细胞毒性作用,因此我们推测加味四逆汤保护肝损伤的作用与调节T细胞亚群间相互甲衡和小鼠肝损伤过程中的免疫应答水甲作用有关。
Objective
Hepatitis B virus (HBV) is an infectious disease caused by hepatitis B virus infection, China is a high prevalence area, about300000people died due to liver disease each year, most of which are related with HBV infection, and the disease of10%-20%can be turned into liver cirrhosis or liver cancer, is a serious threat to our life and health. In chronic hepatitis patients, due to autoimmune disorders, antibodies produced by the body are not enough to clear the body of the hepatitis B virus, resulting in a large number of virus replication, continue to cause liver cell damage, eventually cirrhosis or liver cancer. Method for finding the exact effective and simple treatment of the disease more and more attention. According to the Qing Dynasty famous doctor Huang Yuanyu "circular flow of Qi, soil and pivot Sasang" medicine "see liver disease, liver Chuan spleen" theory, the "Treatise on the" side of Sini Decoction with hill dogwood and regulating the body's immune based Modified Sini Decoction. The original Sini Decoction mainly treating deficiency of Yang Yin Han Sheng little cold syndrome, has the functions of Yang deficiency. Modern pharmacological research show that:Sini Decoction has shock resistance, strong heart, improve microcirculation, blood lipid and anti inflammatory, immune regulation and other pharmacological effects on the pathological state of the body. Cornus officinalis have been shown to have antioxidant, immunomodulatory, anti-inflammatory and other aspects. We observed the effect of Modified Sini Decoction in the treatment of chronic hepatitis B by clinical. Modern research thinks, hepatitis B is a kind of immune related diseases caused by the hepatitis B virus, tissue injury after HBV replication is not the direct role in the liver cell of the result of infection, but a series of host immune response caused by immune damage caused by pathological immune response in liver cells. Liver injury model and clinical hepatitis concanavalin copy form performance is very similar, as many commonly used models of viral hepatitis in the modern study of. Based on affirming the clinical curative effect on acute immune liver, by copying con damage model, to observe the protective effect of Modified Sini Decoction on immunological liver injury using flow cytometry, ELISA and other methods, and to explore its protective mechanism.
Methods
Clinical study:32cases of patients with chronic hepatitis B were observed by Modified Sini Decoction, traditional Chinese medicine treatment, daily1agent, the control group was treated with lamivudine treatment, daily100mg,3months to1course of treatment, a total of2courses of treatment, then analyze the changes before and after treatment, symptoms and signs, liver function and indexes of hepatic fibrosis.
Experimental study:Female NIH mice were randomly divided into5groups: normal control group, model control group, positive drug group, Modified Sini Decoction Modified Sini Decoction in high dose group, low dose group, acute immune liver injury model by tail vein injection of Con A.12h after the death of animal liver and serum of mice were detected the liver index, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, and liver tissue superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels, and to observe the pathological morphology. Determination of blood CD4+, CD8+T lymphocyte subsets by FACS Cali bur flow cytometry, and the ELISA method for the determination of serum glutamic oxaloacetic transaminase (AST), alanine aminotransferase (ALT) activity and cytokines in mice tumor necrosis factor (TNF-a), mouse interferon R (IFN-r) and interleukin-10. All the data are mean+standard deviation said, statistical analysis was performed by SPSS17.0software, the measurement data between two groups were compared using t test, compared with single factor analysis of variance, between the two two groups were compared with LSD test statistics, all the data in each group were test of normality and homogeneity of variance test, take a=0.05is a significant test level.
Results
Clinical study:two cases had no significant difference in age, gender and disease distribution. After treatment, the treatment group in the symptoms and signs were improved markedly, with statistical significance compared with the control group; but in the detection of liver function and hepatic fibrosis index, the two groups have no significant difference. Experimental study:
1、 liver morphological changes.
Macroscopic observation:normal liver morphology was normal, smooth, ruddy color. Model group liver volume increased, membrane swelling, blunt edge, surface punctate and patchy necrosis and hemorrhage, treatment group was close to normal. Microscopy:normal liver tissue of the group structure is complete, clear, liver cell degeneration, no necrosis, no inflammatory cell infiltration. Fuzzy damage model group, hepatic lobules, the most visible liver cells appeared punctate, patchy or diffuse necrosis, necrosis region has a large number of lymphocytes and mononuclear cells infiltration, the majority of intracellular phagocytosis of cellular debris and lipofuscin Kupffer cell hyperplasia; in addition, the majority of liver cell cytoplasmic swelling, osteoporosis, some are ballooning degeneration, and there were many round in eosinophil apoptosis eosin liver cells. Use structure of hepatic lobule were close to normal, reduce liver cell necrosis and inflammatory cell infiltration.
2、 Effect of Modified Sini Decoction on immunological liver injury in mice, the levels of AST ALT
Results liver function of mice showed, model mice of control group ALT, AST levels were significantly higher than that of control group (all P<0.05), compared with the normal group had statistical significance (P<0.01), showed that the experimental modeling success; compared with the model group, bifendate group and Modified Sini Decoction group four different levels reduced transaminase level (P<0.05), showed that Modified Sini Decoction group four can reduce the immunological liver injury in mice ALT and AST activity.
3、 Effect of Modified Sini Decoction on mice liver homogenate SOD and MDA
Using Con A model, MDA levels in mice were significantly higher than that of control group, SOD activity was significantly lower than that in control group (P<0.01). After treatment, compared with model group, Modified Sini group Decoction SOD levels increased in different degrees (P<0.01), the level of MDA was significantly decreased (P<0.05).
4、 Effect of Modified Sini Decoction on IFN-r, TNF-a and IL-10secretion
The results show that:the model control group IFN-r, TNF-a were significantly higher than that of control group (P<0.01); western medicine treatment group and Modified Sini group Decoction after treatment, compared with model group, IFN-r, TNF-a secretion decreased significantly (P<0.01); the content of IFN-r, TNF-a between treatment groups was not statistically significant (P>0.05). The content of serum IL-10and the model control group decreased significantly than that of control group (P<0.01); western medicine treatment group and model control group increased IL-10activity (P<0.01), but still lower than the control group; Modified Sini Decoction intervention, the content of IL-10was also improved, compared with the model group differences (P<0.05).
5、 Effect of Modified Sini Decoction on peripheral blood CD3, CD4, CD8
The experimental results show that the model group mice whole blood CD4+, CD8+ratio compared to the normal control group decreased significantly, there was significant difference compared with the normal group (P<0.05); compared with the model group, Modified Sini Decoction each dose group can cause mouse whole blood CD4+, CD8+ratio was significantly increased (P<0.05), Modified Sini Decoction CD4+, CD8+the ratio of blood mice have increased markedly lower in immunological liver injury in the higher ratio, namely peripheral blood T lymphocyte subsets, reduce its to liver infiltration, thereby reducing its cytotoxic effect on liver cell injury.
Conclusion
Clinical research:Modified Sini Decoction in the treatment of chronic hepatitis B, have a better effect, especially for improving the quality of life of patients, in the improvement of liver function and prevent liver fibrosis also has curative effect, without obvious side effect. Experimental study:
1、 Mice via tail vein injection of ConA12h, ALT, AST levels were significantly elevated, the serological indexes changes consistent with the reported literature, that liver injury rats.
2、 Modified Sini Decoction can significantly decrease the serum transaminase mice model ALT, AST activity, that has protective effects on liver immune Modified Sini Decoction for Con A damage. And can obviously reduce the lobules liver injury mouse immunological liver damage, reduce the infiltration of inflammatory cells in the liver tissue, reduce the formation of necrosis and apoptosis of liver cell degeneration,, to protect the liver tissue objective.
3、 Modified Sini Decoction could significantly increase the activity of SOD of liver injury model of mouse liver tissue, while reducing the content of MDA, show that it has certain antioxidant on immunological liver injury in mice, reduce the production of free radicals, reducing liver peroxidation. The mechanism may be directly scavenging free radicals, free radical chain reaction chain blocked or terminated, so as to prevent or inhibit the reaction of oxygen free radical and lipid peroxidation, generate the final inhibition of lipid peroxidation product MDA. Therefore, Modified Sini Decoction may be through the cell membrane stability, resisting lipid peroxidation and blocking apoptosis reached against liver injury, and its mechanism of action remains to be further studied body.
4、 Modified Sini Decoction can significantly decrease the levels of serum IFN-r, TNF-a model in mice, increase the level of IL-10, so we speculated that Modified Sini Decoction by downregulating the excessive proinflammatory cytokines and anti-inflammatory cytokines release to protect the immune liver injury.
5、 Modified Sini Decoction upregulated significantly decreased in peripheral blood of the mouse model of CD4+, CD8+ratio, that proportion of T lymphocyte subsets in peripheral blood of Modified Sini Decoction can improve the model mice, reduce the liver infiltration, thereby reducing the cell toxicity to liver cells, so we speculated that the immune response level effect of Modified Sini Decoction to protect the liver injury and regulation of T cell subsets in mutual equilibrium and in the process of liver injury in mice.
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