依那普利联合姜黄素对防治小鼠肝纤维化效果及机制研究
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摘要
目的:肝纤维化(hepatic fibrosis,HF)是各种原因所致的肝脏慢性疾病的共同病理过程,是慢性肝病发展为肝硬化的早期和必经阶段,抑制甚或逆转肝纤维化向前发展极为重要。肝纤维化的形成涉及多种细胞、细胞因子及肝组织内细胞外基质的变化。核因子-κB(nuclear factor-kappaB,NF-κB)是一种广泛存在于体内多种细胞的核转录因子,与机体的免疫和炎症反应及凋亡调控密切相关,Bcl-2家族是它的下游基因,在细胞凋亡的基因调控过程中起着至关重要的作用,Bcl-2家族成员构成比例决定细胞存亡。姜黄素(curcumin)是从姜黄属中药分离出的主要药理活性成分,具有抗炎、抗脂质过氧化、抗肿瘤、保肝等多种药理作用。近来研究发现,血管紧张素转化酶抑制剂(angiotensin-converting enzyme inhibitors,ACEI)对肝纤维化具有明显抑制作用。本实验通过检测CCL4诱导的小鼠肝纤维化模型肝组织中NF-KB及相关凋亡蛋白Bcl-2、Bax的表达,研究姜黄素和依那普利单独应用和联合应用对肝纤维化的影响,探讨这些指标在肝纤维化发生中的机制,并为肝纤维化的治疗提供新的靶点。
方法:8周龄雄性昆明种小鼠45只随机分为5组,正常对照组(5只)、模型组(10只)、姜黄素治疗组(10只)、依那普利治疗组(10只)和姜黄素联合依那普利治疗组(10只)。除正常对照组外,各组小鼠均给予四氯化碳5ul/g体重腹腔注射建立肝纤维化模型,每周2次,共10周。姜黄素治疗组、依那普利治疗组和姜黄素联合依那普利治疗组(以后简称联合治疗组)小鼠分别从第二周到第十周给予姜黄素100ug/g、依那普利10mg/kg、及姜黄素100ug/g联合依那普利10mg/kg体重,每周3次灌胃处理,正常对照组给予蒸馏水灌胃,每周3次。所有小鼠均于10周处死。留取血清检测肝功能ALT、AST和血清肝纤维化指标HA;留取部分肝组织,10%中性福尔马林固定,石蜡包埋,苏木精-伊红(HE)染色及Masson染色观察肝组织病理学改变,免疫组织化学染色方法检测IKB、NF-kBP65、Bcl-2、Bax的含量,逆转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)法检测肝组织Bcl-2、Bax mRNA的表达。
结果:
1小鼠一般情况:正常组小鼠精神好,活动自如,毛发有光泽,每周体重增长正常。模型组小鼠随着四氯化碳注射时间的延长,变得易激惹,精神萎靡,食欲下降,体重增长慢或不增,各治疗组小鼠的情况优于模型组。模型组、姜黄素治疗组、依那普利治疗组、联合治疗组、对照组小鼠体重依次为:34.43±4.08、43.38±4.75、43.56±2.88、49.75±3.62、53.60±3.51。10周末,与正常对照组相比,其他四组小鼠体重下降,差异有统计学意义(P值均<0.05)。与模型组比较,其他四组小鼠体重增加明显,差异有统计学意义(P值<0.05);对照组及联合治疗组小鼠体重高于姜黄素治疗组和依那普利治疗组(P<0.05);姜黄素治疗组与依那普利治疗组间、对照组与联合治疗组间小鼠体重比较无显著差异(P >0.05)。
2小鼠血清学指标检测:实验动物血清ALT水平,模型组、依那普利治疗组、姜黄素治疗组、联合治疗组、正常对照组水平,依次为:155.29±21.56U/L,92.11±16.89U/L,83.75±14.54U/L,47.50±9.17U/L,26.60±5.22U/L。对照组及各治疗组血清ALT水平均低于模型组(P<0.05),联合治疗组效果优于依那普利及姜黄素单药治疗组(P<0.05),依那普利治疗组与姜黄素治疗组间血清ALT水平无显著差异(P >0.05);血清AST水平,模型组、依那普利治疗组、姜黄素治疗组、联合治疗组、正常对照组,依次为:205.29±15.86U/L,123.22±10.47U/L,98.38±12.41U/L,79.88±9.83U/L,65.20±16.41 U/L,各组之间AST水平的两两比较均有统计学意义,P<0.05;模型组、依那普利治疗组、姜黄素治疗组、联合治疗组、正常对照组,实验动物血清HA水平依次为:1680.22±94.19ng/ml,869.89±110.53ng/ml , 608.71±120.74ng/ml , 396.13±78.17ng/ml ,144.20±37.09ng/ml,各组之间HA水平的两两比较均有统计学意义,P <0.05。
3肝组织病理学检测
3.1肝组织大体标本形态:正常对照组小鼠肝脏为红褐色,柔软,明亮有光泽;模型组小鼠肝脏大体色泽暗淡,表面欠光滑,体积较正常对照组减小,肝脏边缘变钝,质地较实,肝脏表面可见散在黄色脂肪颗粒;依那普利治疗组、姜黄素治疗组小鼠肝脏大体色泽偏暗,体积较正常对照组略有减小,联合治疗组小鼠肝脏红褐色,有光泽,体积未见明显缩小。
3.2肝组织病理HE染色:正常对照(G0)组小鼠肝组织结构清晰,肝小叶结构正常,肝细胞索排列规则有序,肝细胞大小均匀,无变性、坏死;模型组(G3-G4)小鼠多数正常小叶结构破坏或消失,由汇管区和中央静脉伸出粗大胶原纤维条索分割,包绕肝小叶,肝细胞索排列紊乱,肝细胞浊肿明显,坏死出血较多,纤维间隔内有大量炎性细胞浸润,部分可见假小叶形成;依那普利治疗组和姜黄素治疗组(G2-G3)可见汇管区有少量的纤维组织增生,未见假小叶形成,可见炎症细胞浸润,肝细胞水肿较模型组减轻;联合治疗组(G1-G2)优于依那普利治疗组和姜黄素治疗组。
3.3肝组织病理Masson染色:显微镜下观察绿色胶原纤维分布情况,正常对照组(S0)仅于血管周围可见少量绿色纤维,细且短,肝小叶结构正常,未见纤维增生;模型组(S3-S4)肝脏组织中纤维明显增多,分布广泛,可相互连接形成较粗大的纤维间隔,多位于汇管区和血管周围;依那普利治疗组及姜黄素治疗组(S2-S3)可见较细的绿色纤维走行于血管周围;联合治疗组(S1-S2)纤维化程度较依那普利治疗组及姜黄素治疗组轻。
4肝组织免疫组织化学染色
4.1肝组织bcl-2蛋白免疫组织化学染色:与空白阴性对照比较,以细胞和组织棕黄色染色或深棕黄色为阳性表达。在正常小鼠肝窦、肝细胞浆和中央静脉呈低水平表达,在模型组小鼠肝组织中分布广泛,主要表达在汇管区、纤维间隔、肝窦、肝细胞膜、中央静脉和肝细胞浆也有一定表达。五组bcl-2表达量:联合治疗组(1.31±0.51)%>姜黄素治疗组(1.17±0.41)%>依那普利治疗组(0.97±0.25)%>模型组(0.70±0.36) %﹥正常对照组(0.58±0.34)%,两两比较,P <0.05,差异具有统计学意义。
4.2肝组织Bax蛋白免疫组织化学染色:与空白阴性对照比较,以细胞和组织染为棕黄色染色视为阳性表达。Bax在正常小鼠中央静脉及其周围的肝窦呈低水平表达。在肝纤维化模型小鼠肝组织中主要表达在肝细胞浆,主要为变性的肝细胞浆,严重时可呈弥漫性分布,其次在肝窦、纤维间隔和中央静脉。五组bax表达量:模型组(1.52±0.20)%>依那普利治疗组(0.88±0.27)%>姜黄素治疗组(0.85±0.31)%>联合治疗组(0.63±0.22)%>正常对照组(0.25±0.20)%,两两比较,P <0.05,差异具有统计学意义。
4.3肝组织NF-κBp65蛋白免疫组织化学染色:NF-κBp65阳性物质呈棕黄色颗粒,弥漫分布于细胞质中,激活后位于细胞核中。在对照组小鼠肝组织中较少表达NF-κB p65,模型组则有较强的表达,主要分布于肝细胞、HSC细胞,胞浆内亦有表达,各治疗组NF-κB p65表达较对照组有所增加,且阳性表达主要位于肝细胞浆中,各组水平依次为:模型组(2.20±0.10)%>依那普利治疗组(1.18±0.13)%>姜黄素治疗组(0.95±0.07)%>联合治疗组(0.62±0.06) %>正常对照组(0.42±0.06)%。两两比较,P <0.05,差异具有统计学意义。
4.4肝组织IKB蛋白免疫组织化学染色:IκB在肝组织中的表达与NF-κB p65相似,在对照组小鼠肝组织中肝细胞浆内有较弱的表达,细胞核内无表达;在模型组及各治疗组中肝细胞浆及胞核中可见弥漫性棕黄色颗粒表达,联合治疗组以胞浆表达增多为主,胞核及HSC中也见阳性表达,而模型组胞浆及胞核中表达均较联合组明显减少,各组水平依次为联合治疗组(2.20±0.14)%>姜黄素治疗组(1.35±0.12)%>依那普利治疗组(1.08±0.07)%>模型组(0.94±0.13)%>对照组(0.50±0.06)%。两两比较,P <0.05,差异具有统计学意义。
5肝组织RT-PCR检测
5.1肝组织bcl-2mRNA表达:联合治疗组、姜黄素治疗组、依那普利治疗组、模型组、对照组,bcl-2mRNA表达水平明显下降:联合治疗组(1.40±0.09)%>姜黄素治疗组(1.19±0.15)%>依那普利治疗组(0.87±0.12)%>模型组(0.56±0.07)%>正常对照组(0.30±0.09)%,五组间两两比较,P<0.05,差异具有统计学意义。
5.2肝组织bax mRNA表达:模型组、依那普利治疗、姜黄素治疗组、联合治疗组、正常对照组bax mRNA表达水平明显下降:模型组(1.06±0.14)%>依那普利治疗组(0.92±0.07)%>姜黄素治疗组(0.79±0.08)%>联合治疗组(0.67±0.09)%>正常对照组(0.51±0.10)%,五组间两两比较,P <0.05,差异具有统计学意义。
6肝组织纤维化程度与肝组织Bcl-2和Bax蛋白表达的相关关系:Bax与纤维化程度呈正相关,纤维化越重,Bax表达越多;Bcl-2/Bax的比值与纤维化程度呈负相关,纤维化越重,比值越小。
结论:
1应用10%CC14橄榄油溶液腹腔注射10周,可成功建立符合血清生化学和病理特征的小鼠肝纤维化模型。
2肝细胞凋亡是肝纤维化发病的重要机制之一,核因子NF-κB与Bcl-2基因家族与肝细胞凋亡的关系最为密切。Bcl-2是最重要的抗凋亡因子,又被称为“生存基因”。Bax基因是Bcl-2的同源基因,是最重要的促凋亡因子,两者之间的比值决定细胞的生存或凋亡,Bcl-2/Bax比值增加,细胞凋亡减少,Bcl-2/Bax比值降低,细胞凋亡增加。Bax与纤维化程度呈正相关,Bcl-2/Bax的比值与纤维化程度呈负相关。
3单用姜黄素及依那普利可以预防四氯化碳所致的小鼠肝纤维化的形成,姜黄素联合依那普利应用时,对预防四氯化碳所致的小鼠肝纤维化效果最好,机理是通过抑制NF-κB活性进而了抑制Bax的表达,促进Bcl-2的表达,抑制了肝细胞凋亡,而减轻肝了纤维化。
Objective: Hepatic fibrosis (hepatic fibrosis, HF) is a chronic liver disease due to various reasons. Hepatitic fibrosis is a common pathological process for chronic liver disease progression to cirrosis. Reverse liver fibrosis in early stage is extremely important for blocking liver cirrosis. Hepatic fibrosis related to a variety of cells, cytokines and extracellular matrix changes of liver tissue. Nuclear factor-κB (nuclear factor-kappaB, NF-κB) is a broad variety of cells in the body of nuclear transcription factor, the body's immune and inflammatory responses is closely related to apoptosis regulation, Bcl-2 family is its downstream gene, in the process of gene regulation of apoptosis plays a crucial role. Bcl-2 family members decided to constitute a percentage of cell survival. Curcumin (curcumin) the main pharmacologically active ingredient is isolated from Curcuma medicine, has anti-inflammatory,anti-lipid peroxidation, anti-tumor, nourish the liver and a variety of pharmacological effects. Recent studies found that angiotensin-converting enzyme inhibitors (ACEI) inhibit hepatic fibrosis effectivly. In this study, by detecting NF-KB and related apoptotic protein Bcl-2, Bax expression in the CCL4-induced model of liver fibrosis, to explore the effect of combination therapy of curcumin and enalapril therapy, determine the molecule mechanism. Provide a new target for treatment hepatic fibrossis.
Methods: Forty-five 8-week-old male Kunming mice were randomly divided into five groups, the normal control group(n=10), model group(n=10), curcumin treatment group(n=10), enalapril treatment group (n=10), and curcumin combined with enalapril treatment group(n=10). In addition to the normal control group, mice in each group were treated with carbon tetrachloride 5ul/g body weight intraperitoneal injection to establish hepatic fibrosis model, two times a week, a total of 10 weeks. Curcumin treatment group, enalapril treatment group and the curcumin combined with enalapril treatment group (after here referred to as combination therapy group) were from the second week to 10th week to give a gavage treatment curcumin 100ug/g, enalapril 10mg/kg, and curcumin 100ug/g combined with enalapril 10mg/kg, 3 times per week, the normal control group was given distilled water, 3 times a week. All mice were killed at end of 10 weeks. Blood and liver tissue were collected for test serum ALT, AST and HA levels. Part of liver tissue werefixed in 10% neutral formalin, paraffin-embedded. Hematoxy lin-eosin (HE) staining and Masson staining to observe histopathological changes,immunohistochemical staining analyze IKB, NF-kBP65, Bcl-2, Bax protein expression. Reverse transcription polymerase chain reaction (RT-PCR) detect liver tissue Bcl-2, Bax mRNA expression.
Results
l General situations of mice:The mice in normal control group are in good spirit,hair shiny,weekly weight gain gradually.Model group of mice injected with carbon tetrachloride,become irritability, apathetic,loss of appetite, weight gain is slow or no increase. The treated mice were better than the model group mice. The body weight of mice in model group, enalapril treatment group, curcumin-treated group, combination therapy group and the normal control group were followed: 34.43±4.08,43.56±2.88,43.38±4.75,49.75±3.62,53.60±3.51. Compared with the normal control group, the body weight in other four groups were lower. The difference was statistically significant (P﹤0.05). Compared with the model group, the other four groups of mice gained weight significantly, the difference was statistically significant (P﹤0.05). The control group and combined treatment group body weight were higher than curcumin treatment group and the enalapril treatment group (P﹤0.05); The difference between curcumin treatment group and enalapril treatment group, normal control group and combined treatment group have no significant difference (P﹥0.05).
2 The serum levels of ALT, AST and HA: serum ALT levels of experimental mice, model control group, enalapril treatment group, curcumin treatment group, combination therapy group and normal control group, followed by:155.29±21.56U/L, 92.11±16.89U/L, 83.75±14.54U/L, 47.50±9.17U/L, 26.60±5.22U/L. The treatment group and the control group, serum ALT were lower than model group (P﹤0.05), combination therapy more effective than enalapril and curcumin treatment group (P﹤0.05), the serum ALT levels between the enalapril treatment group and the curcumin treatment was no significant difference (P﹥0.05). Serum AST levels, model control group, enalapril treatment group, curcumin treatment group, combination therapy group and normal control group, followed by:205.29±15.86U/L, 123.22±10.47U/L, 98.38±12.41U/L, 79.88±9.83U/L, 65.20±16.41U/L, the AST levels of between each groups are statistically significant, P﹤0.05. Experimental mice serum HA levels, model group, enalapril treatment group, curcumin-treated group, combination therapy group, normal control group, serum HA levels followed:1680.22±94.19ng/ml, 869.89±110.53ng/ml,608.71±120.74ng/ml, 396.13±78.17ng/ml, 144.20±37.09ng/ml, the HA levels of between two groups are statistically significant,P﹤0.05.
3 Liver tissue Pathological detect
3.1 Pattern of liver tissue: In normal control group, the liver of mice is reddish-brown, soft, bright and shiny. In model group, the liver of mice is darker then the normal control group, smooth surface, due to the size decrease compared with the normal control group, the edge of liver become blunt and texture is solid, it can be seen yellow fat particle scattered at the surface of liver. Enalapril-treated group and curcumin treatment group livers of mice in general liver’s color was a little darker, liver size was slight decrease compared with the normal control group, the combination therapy group of mice liver reddish-brown, shiny, size no significantly reduced compared with the normal control group.
3.2 liver pathology HE staining: in normal control group(G0), the liver structure is organizational, hepatic lobule structure was normal, liver cells cord orderly arranged, the size of liver cells were orderly and uniform, no degeneration and necrosis. In model group of mice(G3-G4), the normal lobule structure was damaged or disappeared, the portal area and central vein out of thick collagen fiber cable divided and surrounded the hepatic lobule, the liver cell cord disarrangement, hepatocellular obvious necrosis, a large number of inflammatory cell infiltration in fiber interval and can see the false lobules formationing. Enalapril treatment group and curcumin treatment group(G2-G3) shows a small number of fibrous tissue, no pseudolobule formation and see infiltration of inflammatory cells, compared with model group, the liver cell’s edema reduced; combined treatment group(G1-G2) was better than enalapril treatment group and the curcumin treatment group.
3.3 Liver pathology Masson staining:we focuse on the distribution of green collagen fibers under microscope, in normal control group(S0), only a small amount of green perivascular fibers, thin and short, the structure of liver unit is normal, no fibrosis was seen; In the model group(S3-S4), liver fibers can be seen increased significantly, widely distributed and interconnected to form larger coarse fiber spacing, most of which located in the portal areas and perivascular tube; In enalapril treatment group and the curcumin treatment group(S2-S3) smaller green fibers can be seen around the blood vessels. Compared with enalapril treatment group and the curcumin treated group fibrosis in combined treatment group(S1-S2) was slightly.
4 Liver tissue immunohistochemical staining study
4.1 Bcl-2 immunohistochemical staining: The positive expression of Bcl-2 protein was stained brown in cells and tissues compared with the blank negative control. In normal control group, there was a low level expression in Sinusoids, liver cytoplasm and the central vein. In the model group Bcl-2 protein are widely distributed in liver tissue, mainly in periportal areas, fiber spacing, sinusoids, liver cell membrane, the central vein and Liver cytoplasm. The sequence of Bcl-2 expression in five groups are : Combined treatment group(1.31±0.51)%﹥curcumin treatment group(1.17±0.41)%﹥enalapril treatment group(0.97±0.25)%﹥Model group(0.70±0.36) %﹥normal control group(0.58±0.34)%. two two compared among the groups,P <0.05, differences with statistical significance.
4.2 Bax immunohistochemical staining: The positive expression of bax protein was stained brown in cells and tissues compared with the blank negative control. In normal mice liver tissue, the central vein and its surrounding sinusoid was low-level expression. In the model group mainly expressed in liver cytoplasm, mainly in degeneration of liver cytoplasm, in severe cases can diffuse distribution, followed by the sinusoid, fiber spacing and central venous. Bax expression in five groups are: model control group(1.52±0.20)%﹥enalapril treatment group ( 0.88±0.27 ) %﹥curcumin treatment group(0.85±0.31)%﹥combination therapy(0.63±0.22)%﹥normal control group(0.25±0.20)%. two two compared among the groups, P<0.05, differences with statistical significance.
4.3 NF-κBp65 immunohistochemical staining: NF-κB p65 positive substance showed brown particles, expression in the cytoplasm and was activated in the nucleus. In normal mice liver tissue, less expression of NF-κBp65. In the model group, there are strong expression, Mainly distributed in liver cells, HSC cells, the cytoplasm was also expressed. Compared with the control group, the expression in treatment groups were increased, and the positive expression mainly located in the cytoplasm of liver cells. NF-κBp65 expression in five groups:model group(2.20±0.10)%﹥enalapril treatment group(1.18±0.13)%﹥curcumin treatment group(0.95±0.07)%﹥combination therapy(0.62±0.06) %﹥normal control group(0.42±0.06)%. two two compared among the groups, P<0.05, differences with statistical significance.
4.4 IκB immunohistochemical staining: IκB in liver tissue expression was similar to NF-κBp65, In normal mice liver tissue, there was a weak expressin in liver cells cytoplasm, no expression within the nucleus. In the model group and the treatment groups, liver cytoplasm and nucleus were expressed brown-yellow granules, Combination therapy group mainly expression in cytoplasmic, the nucleus and HSC also see the expression. Compare to the Combined treatment group, the expression in model group expressed in cytoplasm and nucleus significantly reduced. IκB expression of five groups:Combined treatment group(2.20±0.14)% >curcumin treatment group(1.35±0.12)%>enalapril treatment group(1.08±0.07)%>model group(0.94±0.13)%> control group(0.50±0.06)%. two two compared among the groups, P <0.05, differences with statistical significance.
5 Liver tissue RT-PCR detect
5.1 The expression of Bcl-2 mRNA in liver tissue: Combination therapy group, curcumin treatment group, enalapril treatment group, model group, normal control group, Bcl-2mRNA expression was significantly decreased. The expression of liver tissue Bcl-2mRNA level order: combined treatment group(1.40±0.09)%>curcumin treatment group(1.19±0.15)%>enalapril treatment group(0.87±0.12)%>model group(0.56±0.07)%> control group(0.30±0.09)%. two two compared among the groups, P <0.05, differences with statistical significance.
5.2 The expression of BaxmRNA in liver tissue: Model control group, enalapril treatment, curcumin treated group, combination therapy group, normal control group, Bax mRNA expression was significantly decreased, the expression of liver tissue Bax mRNA level order: model group(1.06±0.14)%>enalapril treatment group(0.92±0.07)%>curcumin treatment group(0.79±0.08)%>combined treatment group (0.67±0.09)%>normal control group(0.51±0.10)%, two two compared among the groups, P <0.05, differences with statistical significance.
6 corelationship analysis of liver firosis stage and liver tissue Bcl-2 and Bax protein levels, Bax was positively correlated with the degree of fibrosis, fibrosis was heavier and Bax expression more; Bcl-2/Bax ratio was negatively correlated with the degree of fibrosis, fibrosis was heavier the ratio was smaller.
Conclusion
1 The hepatic fibrosis models could be established successfully by intraperitoneal injection with the mixture of 10% CCL4 and olive oil for 10 weeks. The Serum biochemical and pathological features are consistent with hepatic fibrosis in human.
2 Apoptosis is one of the important mechanism for the pathogenesis of liver fibrosis, Nuclear factor NF-κB and Bcl-2 gene family is most closely related to hepatocyte apoptosis, Bcl-2 is the most important anti-apoptotic factor, also known as the "survival genes", Bax was homologous gene of Bcl-2, it was the most important anti-apoptotic factor, The ratio of Bcl-2/Bax decided cell's survival or apoptosis,Bcl-2/Bax ratio increased and apoptosis reduced, Bcl-2/Bax ratio decreased and apoptosis increased. Bax was positively correlated with the degree of fibrosis, Bcl-2/Bax ratio was negatively correlated with the degree of fibrosis.
3 Curcumin and enalapril alone can prevent carbon tetrachloride-induced liver fibrosis in mice, Curcumin combined with enalapril prevent carbon tetrachloride-induced liver fibrosis is the best. The mechanism may be related with inhibiting NF-κB activity and promoting the expression of Bcl-2, reducing the expression of bax, thus inhibit liver cell apoptosis and reduce liver fibrosis.
方法:8周龄雄性昆明种小鼠45只随机分为5组,正常对照组(5只)、模型组(10只)、姜黄素治疗组(10只)、依那普利治疗组(10只)和姜黄素联合依那普利治疗组(10只)。除正常对照组外,各组小鼠均给予四氯化碳5ul/g体重腹腔注射建立肝纤维化模型,每周2次,共10周。姜黄素治疗组、依那普利治疗组和姜黄素联合依那普利治疗组(以后简称联合治疗组)小鼠分别从第二周到第十周给予姜黄素100ug/g、依那普利10mg/kg、及姜黄素100ug/g联合依那普利10mg/kg体重,每周3次灌胃处理,正常对照组给予蒸馏水灌胃,每周3次。所有小鼠均于10周处死。留取血清检测肝功能ALT、AST和血清肝纤维化指标HA;留取部分肝组织,10%中性福尔马林固定,石蜡包埋,苏木精-伊红(HE)染色及Masson染色观察肝组织病理学改变,免疫组织化学染色方法检测IKB、NF-kBP65、Bcl-2、Bax的含量,逆转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)法检测肝组织Bcl-2、Bax mRNA的表达。
结果:
1小鼠一般情况:正常组小鼠精神好,活动自如,毛发有光泽,每周体重增长正常。模型组小鼠随着四氯化碳注射时间的延长,变得易激惹,精神萎靡,食欲下降,体重增长慢或不增,各治疗组小鼠的情况优于模型组。模型组、姜黄素治疗组、依那普利治疗组、联合治疗组、对照组小鼠体重依次为:34.43±4.08、43.38±4.75、43.56±2.88、49.75±3.62、53.60±3.51。10周末,与正常对照组相比,其他四组小鼠体重下降,差异有统计学意义(P值均<0.05)。与模型组比较,其他四组小鼠体重增加明显,差异有统计学意义(P值<0.05);对照组及联合治疗组小鼠体重高于姜黄素治疗组和依那普利治疗组(P<0.05);姜黄素治疗组与依那普利治疗组间、对照组与联合治疗组间小鼠体重比较无显著差异(P >0.05)。
2小鼠血清学指标检测:实验动物血清ALT水平,模型组、依那普利治疗组、姜黄素治疗组、联合治疗组、正常对照组水平,依次为:155.29±21.56U/L,92.11±16.89U/L,83.75±14.54U/L,47.50±9.17U/L,26.60±5.22U/L。对照组及各治疗组血清ALT水平均低于模型组(P<0.05),联合治疗组效果优于依那普利及姜黄素单药治疗组(P<0.05),依那普利治疗组与姜黄素治疗组间血清ALT水平无显著差异(P >0.05);血清AST水平,模型组、依那普利治疗组、姜黄素治疗组、联合治疗组、正常对照组,依次为:205.29±15.86U/L,123.22±10.47U/L,98.38±12.41U/L,79.88±9.83U/L,65.20±16.41 U/L,各组之间AST水平的两两比较均有统计学意义,P<0.05;模型组、依那普利治疗组、姜黄素治疗组、联合治疗组、正常对照组,实验动物血清HA水平依次为:1680.22±94.19ng/ml,869.89±110.53ng/ml , 608.71±120.74ng/ml , 396.13±78.17ng/ml ,144.20±37.09ng/ml,各组之间HA水平的两两比较均有统计学意义,P <0.05。
3肝组织病理学检测
3.1肝组织大体标本形态:正常对照组小鼠肝脏为红褐色,柔软,明亮有光泽;模型组小鼠肝脏大体色泽暗淡,表面欠光滑,体积较正常对照组减小,肝脏边缘变钝,质地较实,肝脏表面可见散在黄色脂肪颗粒;依那普利治疗组、姜黄素治疗组小鼠肝脏大体色泽偏暗,体积较正常对照组略有减小,联合治疗组小鼠肝脏红褐色,有光泽,体积未见明显缩小。
3.2肝组织病理HE染色:正常对照(G0)组小鼠肝组织结构清晰,肝小叶结构正常,肝细胞索排列规则有序,肝细胞大小均匀,无变性、坏死;模型组(G3-G4)小鼠多数正常小叶结构破坏或消失,由汇管区和中央静脉伸出粗大胶原纤维条索分割,包绕肝小叶,肝细胞索排列紊乱,肝细胞浊肿明显,坏死出血较多,纤维间隔内有大量炎性细胞浸润,部分可见假小叶形成;依那普利治疗组和姜黄素治疗组(G2-G3)可见汇管区有少量的纤维组织增生,未见假小叶形成,可见炎症细胞浸润,肝细胞水肿较模型组减轻;联合治疗组(G1-G2)优于依那普利治疗组和姜黄素治疗组。
3.3肝组织病理Masson染色:显微镜下观察绿色胶原纤维分布情况,正常对照组(S0)仅于血管周围可见少量绿色纤维,细且短,肝小叶结构正常,未见纤维增生;模型组(S3-S4)肝脏组织中纤维明显增多,分布广泛,可相互连接形成较粗大的纤维间隔,多位于汇管区和血管周围;依那普利治疗组及姜黄素治疗组(S2-S3)可见较细的绿色纤维走行于血管周围;联合治疗组(S1-S2)纤维化程度较依那普利治疗组及姜黄素治疗组轻。
4肝组织免疫组织化学染色
4.1肝组织bcl-2蛋白免疫组织化学染色:与空白阴性对照比较,以细胞和组织棕黄色染色或深棕黄色为阳性表达。在正常小鼠肝窦、肝细胞浆和中央静脉呈低水平表达,在模型组小鼠肝组织中分布广泛,主要表达在汇管区、纤维间隔、肝窦、肝细胞膜、中央静脉和肝细胞浆也有一定表达。五组bcl-2表达量:联合治疗组(1.31±0.51)%>姜黄素治疗组(1.17±0.41)%>依那普利治疗组(0.97±0.25)%>模型组(0.70±0.36) %﹥正常对照组(0.58±0.34)%,两两比较,P <0.05,差异具有统计学意义。
4.2肝组织Bax蛋白免疫组织化学染色:与空白阴性对照比较,以细胞和组织染为棕黄色染色视为阳性表达。Bax在正常小鼠中央静脉及其周围的肝窦呈低水平表达。在肝纤维化模型小鼠肝组织中主要表达在肝细胞浆,主要为变性的肝细胞浆,严重时可呈弥漫性分布,其次在肝窦、纤维间隔和中央静脉。五组bax表达量:模型组(1.52±0.20)%>依那普利治疗组(0.88±0.27)%>姜黄素治疗组(0.85±0.31)%>联合治疗组(0.63±0.22)%>正常对照组(0.25±0.20)%,两两比较,P <0.05,差异具有统计学意义。
4.3肝组织NF-κBp65蛋白免疫组织化学染色:NF-κBp65阳性物质呈棕黄色颗粒,弥漫分布于细胞质中,激活后位于细胞核中。在对照组小鼠肝组织中较少表达NF-κB p65,模型组则有较强的表达,主要分布于肝细胞、HSC细胞,胞浆内亦有表达,各治疗组NF-κB p65表达较对照组有所增加,且阳性表达主要位于肝细胞浆中,各组水平依次为:模型组(2.20±0.10)%>依那普利治疗组(1.18±0.13)%>姜黄素治疗组(0.95±0.07)%>联合治疗组(0.62±0.06) %>正常对照组(0.42±0.06)%。两两比较,P <0.05,差异具有统计学意义。
4.4肝组织IKB蛋白免疫组织化学染色:IκB在肝组织中的表达与NF-κB p65相似,在对照组小鼠肝组织中肝细胞浆内有较弱的表达,细胞核内无表达;在模型组及各治疗组中肝细胞浆及胞核中可见弥漫性棕黄色颗粒表达,联合治疗组以胞浆表达增多为主,胞核及HSC中也见阳性表达,而模型组胞浆及胞核中表达均较联合组明显减少,各组水平依次为联合治疗组(2.20±0.14)%>姜黄素治疗组(1.35±0.12)%>依那普利治疗组(1.08±0.07)%>模型组(0.94±0.13)%>对照组(0.50±0.06)%。两两比较,P <0.05,差异具有统计学意义。
5肝组织RT-PCR检测
5.1肝组织bcl-2mRNA表达:联合治疗组、姜黄素治疗组、依那普利治疗组、模型组、对照组,bcl-2mRNA表达水平明显下降:联合治疗组(1.40±0.09)%>姜黄素治疗组(1.19±0.15)%>依那普利治疗组(0.87±0.12)%>模型组(0.56±0.07)%>正常对照组(0.30±0.09)%,五组间两两比较,P<0.05,差异具有统计学意义。
5.2肝组织bax mRNA表达:模型组、依那普利治疗、姜黄素治疗组、联合治疗组、正常对照组bax mRNA表达水平明显下降:模型组(1.06±0.14)%>依那普利治疗组(0.92±0.07)%>姜黄素治疗组(0.79±0.08)%>联合治疗组(0.67±0.09)%>正常对照组(0.51±0.10)%,五组间两两比较,P <0.05,差异具有统计学意义。
6肝组织纤维化程度与肝组织Bcl-2和Bax蛋白表达的相关关系:Bax与纤维化程度呈正相关,纤维化越重,Bax表达越多;Bcl-2/Bax的比值与纤维化程度呈负相关,纤维化越重,比值越小。
结论:
1应用10%CC14橄榄油溶液腹腔注射10周,可成功建立符合血清生化学和病理特征的小鼠肝纤维化模型。
2肝细胞凋亡是肝纤维化发病的重要机制之一,核因子NF-κB与Bcl-2基因家族与肝细胞凋亡的关系最为密切。Bcl-2是最重要的抗凋亡因子,又被称为“生存基因”。Bax基因是Bcl-2的同源基因,是最重要的促凋亡因子,两者之间的比值决定细胞的生存或凋亡,Bcl-2/Bax比值增加,细胞凋亡减少,Bcl-2/Bax比值降低,细胞凋亡增加。Bax与纤维化程度呈正相关,Bcl-2/Bax的比值与纤维化程度呈负相关。
3单用姜黄素及依那普利可以预防四氯化碳所致的小鼠肝纤维化的形成,姜黄素联合依那普利应用时,对预防四氯化碳所致的小鼠肝纤维化效果最好,机理是通过抑制NF-κB活性进而了抑制Bax的表达,促进Bcl-2的表达,抑制了肝细胞凋亡,而减轻肝了纤维化。
Objective: Hepatic fibrosis (hepatic fibrosis, HF) is a chronic liver disease due to various reasons. Hepatitic fibrosis is a common pathological process for chronic liver disease progression to cirrosis. Reverse liver fibrosis in early stage is extremely important for blocking liver cirrosis. Hepatic fibrosis related to a variety of cells, cytokines and extracellular matrix changes of liver tissue. Nuclear factor-κB (nuclear factor-kappaB, NF-κB) is a broad variety of cells in the body of nuclear transcription factor, the body's immune and inflammatory responses is closely related to apoptosis regulation, Bcl-2 family is its downstream gene, in the process of gene regulation of apoptosis plays a crucial role. Bcl-2 family members decided to constitute a percentage of cell survival. Curcumin (curcumin) the main pharmacologically active ingredient is isolated from Curcuma medicine, has anti-inflammatory,anti-lipid peroxidation, anti-tumor, nourish the liver and a variety of pharmacological effects. Recent studies found that angiotensin-converting enzyme inhibitors (ACEI) inhibit hepatic fibrosis effectivly. In this study, by detecting NF-KB and related apoptotic protein Bcl-2, Bax expression in the CCL4-induced model of liver fibrosis, to explore the effect of combination therapy of curcumin and enalapril therapy, determine the molecule mechanism. Provide a new target for treatment hepatic fibrossis.
Methods: Forty-five 8-week-old male Kunming mice were randomly divided into five groups, the normal control group(n=10), model group(n=10), curcumin treatment group(n=10), enalapril treatment group (n=10), and curcumin combined with enalapril treatment group(n=10). In addition to the normal control group, mice in each group were treated with carbon tetrachloride 5ul/g body weight intraperitoneal injection to establish hepatic fibrosis model, two times a week, a total of 10 weeks. Curcumin treatment group, enalapril treatment group and the curcumin combined with enalapril treatment group (after here referred to as combination therapy group) were from the second week to 10th week to give a gavage treatment curcumin 100ug/g, enalapril 10mg/kg, and curcumin 100ug/g combined with enalapril 10mg/kg, 3 times per week, the normal control group was given distilled water, 3 times a week. All mice were killed at end of 10 weeks. Blood and liver tissue were collected for test serum ALT, AST and HA levels. Part of liver tissue werefixed in 10% neutral formalin, paraffin-embedded. Hematoxy lin-eosin (HE) staining and Masson staining to observe histopathological changes,immunohistochemical staining analyze IKB, NF-kBP65, Bcl-2, Bax protein expression. Reverse transcription polymerase chain reaction (RT-PCR) detect liver tissue Bcl-2, Bax mRNA expression.
Results
l General situations of mice:The mice in normal control group are in good spirit,hair shiny,weekly weight gain gradually.Model group of mice injected with carbon tetrachloride,become irritability, apathetic,loss of appetite, weight gain is slow or no increase. The treated mice were better than the model group mice. The body weight of mice in model group, enalapril treatment group, curcumin-treated group, combination therapy group and the normal control group were followed: 34.43±4.08,43.56±2.88,43.38±4.75,49.75±3.62,53.60±3.51. Compared with the normal control group, the body weight in other four groups were lower. The difference was statistically significant (P﹤0.05). Compared with the model group, the other four groups of mice gained weight significantly, the difference was statistically significant (P﹤0.05). The control group and combined treatment group body weight were higher than curcumin treatment group and the enalapril treatment group (P﹤0.05); The difference between curcumin treatment group and enalapril treatment group, normal control group and combined treatment group have no significant difference (P﹥0.05).
2 The serum levels of ALT, AST and HA: serum ALT levels of experimental mice, model control group, enalapril treatment group, curcumin treatment group, combination therapy group and normal control group, followed by:155.29±21.56U/L, 92.11±16.89U/L, 83.75±14.54U/L, 47.50±9.17U/L, 26.60±5.22U/L. The treatment group and the control group, serum ALT were lower than model group (P﹤0.05), combination therapy more effective than enalapril and curcumin treatment group (P﹤0.05), the serum ALT levels between the enalapril treatment group and the curcumin treatment was no significant difference (P﹥0.05). Serum AST levels, model control group, enalapril treatment group, curcumin treatment group, combination therapy group and normal control group, followed by:205.29±15.86U/L, 123.22±10.47U/L, 98.38±12.41U/L, 79.88±9.83U/L, 65.20±16.41U/L, the AST levels of between each groups are statistically significant, P﹤0.05. Experimental mice serum HA levels, model group, enalapril treatment group, curcumin-treated group, combination therapy group, normal control group, serum HA levels followed:1680.22±94.19ng/ml, 869.89±110.53ng/ml,608.71±120.74ng/ml, 396.13±78.17ng/ml, 144.20±37.09ng/ml, the HA levels of between two groups are statistically significant,P﹤0.05.
3 Liver tissue Pathological detect
3.1 Pattern of liver tissue: In normal control group, the liver of mice is reddish-brown, soft, bright and shiny. In model group, the liver of mice is darker then the normal control group, smooth surface, due to the size decrease compared with the normal control group, the edge of liver become blunt and texture is solid, it can be seen yellow fat particle scattered at the surface of liver. Enalapril-treated group and curcumin treatment group livers of mice in general liver’s color was a little darker, liver size was slight decrease compared with the normal control group, the combination therapy group of mice liver reddish-brown, shiny, size no significantly reduced compared with the normal control group.
3.2 liver pathology HE staining: in normal control group(G0), the liver structure is organizational, hepatic lobule structure was normal, liver cells cord orderly arranged, the size of liver cells were orderly and uniform, no degeneration and necrosis. In model group of mice(G3-G4), the normal lobule structure was damaged or disappeared, the portal area and central vein out of thick collagen fiber cable divided and surrounded the hepatic lobule, the liver cell cord disarrangement, hepatocellular obvious necrosis, a large number of inflammatory cell infiltration in fiber interval and can see the false lobules formationing. Enalapril treatment group and curcumin treatment group(G2-G3) shows a small number of fibrous tissue, no pseudolobule formation and see infiltration of inflammatory cells, compared with model group, the liver cell’s edema reduced; combined treatment group(G1-G2) was better than enalapril treatment group and the curcumin treatment group.
3.3 Liver pathology Masson staining:we focuse on the distribution of green collagen fibers under microscope, in normal control group(S0), only a small amount of green perivascular fibers, thin and short, the structure of liver unit is normal, no fibrosis was seen; In the model group(S3-S4), liver fibers can be seen increased significantly, widely distributed and interconnected to form larger coarse fiber spacing, most of which located in the portal areas and perivascular tube; In enalapril treatment group and the curcumin treatment group(S2-S3) smaller green fibers can be seen around the blood vessels. Compared with enalapril treatment group and the curcumin treated group fibrosis in combined treatment group(S1-S2) was slightly.
4 Liver tissue immunohistochemical staining study
4.1 Bcl-2 immunohistochemical staining: The positive expression of Bcl-2 protein was stained brown in cells and tissues compared with the blank negative control. In normal control group, there was a low level expression in Sinusoids, liver cytoplasm and the central vein. In the model group Bcl-2 protein are widely distributed in liver tissue, mainly in periportal areas, fiber spacing, sinusoids, liver cell membrane, the central vein and Liver cytoplasm. The sequence of Bcl-2 expression in five groups are : Combined treatment group(1.31±0.51)%﹥curcumin treatment group(1.17±0.41)%﹥enalapril treatment group(0.97±0.25)%﹥Model group(0.70±0.36) %﹥normal control group(0.58±0.34)%. two two compared among the groups,P <0.05, differences with statistical significance.
4.2 Bax immunohistochemical staining: The positive expression of bax protein was stained brown in cells and tissues compared with the blank negative control. In normal mice liver tissue, the central vein and its surrounding sinusoid was low-level expression. In the model group mainly expressed in liver cytoplasm, mainly in degeneration of liver cytoplasm, in severe cases can diffuse distribution, followed by the sinusoid, fiber spacing and central venous. Bax expression in five groups are: model control group(1.52±0.20)%﹥enalapril treatment group ( 0.88±0.27 ) %﹥curcumin treatment group(0.85±0.31)%﹥combination therapy(0.63±0.22)%﹥normal control group(0.25±0.20)%. two two compared among the groups, P<0.05, differences with statistical significance.
4.3 NF-κBp65 immunohistochemical staining: NF-κB p65 positive substance showed brown particles, expression in the cytoplasm and was activated in the nucleus. In normal mice liver tissue, less expression of NF-κBp65. In the model group, there are strong expression, Mainly distributed in liver cells, HSC cells, the cytoplasm was also expressed. Compared with the control group, the expression in treatment groups were increased, and the positive expression mainly located in the cytoplasm of liver cells. NF-κBp65 expression in five groups:model group(2.20±0.10)%﹥enalapril treatment group(1.18±0.13)%﹥curcumin treatment group(0.95±0.07)%﹥combination therapy(0.62±0.06) %﹥normal control group(0.42±0.06)%. two two compared among the groups, P<0.05, differences with statistical significance.
4.4 IκB immunohistochemical staining: IκB in liver tissue expression was similar to NF-κBp65, In normal mice liver tissue, there was a weak expressin in liver cells cytoplasm, no expression within the nucleus. In the model group and the treatment groups, liver cytoplasm and nucleus were expressed brown-yellow granules, Combination therapy group mainly expression in cytoplasmic, the nucleus and HSC also see the expression. Compare to the Combined treatment group, the expression in model group expressed in cytoplasm and nucleus significantly reduced. IκB expression of five groups:Combined treatment group(2.20±0.14)% >curcumin treatment group(1.35±0.12)%>enalapril treatment group(1.08±0.07)%>model group(0.94±0.13)%> control group(0.50±0.06)%. two two compared among the groups, P <0.05, differences with statistical significance.
5 Liver tissue RT-PCR detect
5.1 The expression of Bcl-2 mRNA in liver tissue: Combination therapy group, curcumin treatment group, enalapril treatment group, model group, normal control group, Bcl-2mRNA expression was significantly decreased. The expression of liver tissue Bcl-2mRNA level order: combined treatment group(1.40±0.09)%>curcumin treatment group(1.19±0.15)%>enalapril treatment group(0.87±0.12)%>model group(0.56±0.07)%> control group(0.30±0.09)%. two two compared among the groups, P <0.05, differences with statistical significance.
5.2 The expression of BaxmRNA in liver tissue: Model control group, enalapril treatment, curcumin treated group, combination therapy group, normal control group, Bax mRNA expression was significantly decreased, the expression of liver tissue Bax mRNA level order: model group(1.06±0.14)%>enalapril treatment group(0.92±0.07)%>curcumin treatment group(0.79±0.08)%>combined treatment group (0.67±0.09)%>normal control group(0.51±0.10)%, two two compared among the groups, P <0.05, differences with statistical significance.
6 corelationship analysis of liver firosis stage and liver tissue Bcl-2 and Bax protein levels, Bax was positively correlated with the degree of fibrosis, fibrosis was heavier and Bax expression more; Bcl-2/Bax ratio was negatively correlated with the degree of fibrosis, fibrosis was heavier the ratio was smaller.
Conclusion
1 The hepatic fibrosis models could be established successfully by intraperitoneal injection with the mixture of 10% CCL4 and olive oil for 10 weeks. The Serum biochemical and pathological features are consistent with hepatic fibrosis in human.
2 Apoptosis is one of the important mechanism for the pathogenesis of liver fibrosis, Nuclear factor NF-κB and Bcl-2 gene family is most closely related to hepatocyte apoptosis, Bcl-2 is the most important anti-apoptotic factor, also known as the "survival genes", Bax was homologous gene of Bcl-2, it was the most important anti-apoptotic factor, The ratio of Bcl-2/Bax decided cell's survival or apoptosis,Bcl-2/Bax ratio increased and apoptosis reduced, Bcl-2/Bax ratio decreased and apoptosis increased. Bax was positively correlated with the degree of fibrosis, Bcl-2/Bax ratio was negatively correlated with the degree of fibrosis.
3 Curcumin and enalapril alone can prevent carbon tetrachloride-induced liver fibrosis in mice, Curcumin combined with enalapril prevent carbon tetrachloride-induced liver fibrosis is the best. The mechanism may be related with inhibiting NF-κB activity and promoting the expression of Bcl-2, reducing the expression of bax, thus inhibit liver cell apoptosis and reduce liver fibrosis.
引文
1聂青和.肝纤维化的逆转策略及研究现状(述评).世界华人消化杂志,2005,13(10):1165-1174
2 ELSHARKAWY AM, MANN DA. Nuclear factor-kappaB andthe hepatic inflammation-fibrosis-cancer axis Hepatology, 2007, 4(62): 590-597
3 SEN R, BALTIMORE D. Multiple nuclear factors interactwith the immunoglobulin enhancer sequences . Cell, 1986, 4(65): 705-716
4 Okamoto T, Sanda T, Asamitsu K.NF-kappa B signaling andcarcinogenesis. CurrPharm Des, 2007, 13(5): 447-462
5 Heyninck K, Wullaert A, Beyaert R. Nuclear factor-kappa B plays a central role in tumour necrosis factor-mediated liver disease . Biochem Pharmacol, 2003, 66(8): 1409
6 Bruck R, AshkenaziM, WeissS, eta.l Prevention of liver cirrhosis in rats by curcumin . Liver Int, 2007, 27: 373-383
7 Liu YG, Chen HC, Jiang YP. Efects of cureumin on priary cucturo rat hatocyte injuried by CCh . Chinese Traditional Patent Medicine, 2003, 25(3): 522-525
8 BARRETO R, KAWAKITA S, TSUCHIYA J, et al.Metal-induced oxidative damage in cultured hepatocytes and hepatic lysosomal fraction: beneficial effectof a curcumin/absinthium compound . Chin J DigDis, 2005, 6: 31-36
9 BAVA S V, PULIAPPADAMBA V T, DEEPTI A, etal. Sensitization of taxol-induced apoptosis by curcumin involves down-regulation of nuclear Factor-κB and the Serine. Threonine Kinase Akt and is independent of tu-bulin polymerization . J Biol Chem, 2005, 280: 6301-6308
10 REYES-GORDILLO K, SEGOVIA J, SHIBAYAMA M, et al. Curcumin protects against acute liver damagein the rat by inhibiting NF-KappaB, proinflammatory cytokines production and oxidative stress. BiochiBiophys Acta, 2007, 22: 989-996
11舒建昌,叶国荣,吕霞,等.姜黄素治疗肝纤维化及其作用机制的初步研究.中华肝脏病杂志,2007,15(10):753-757
12成扬,平键,刘成,等.姜黄素激活过氧化物酶体增殖因子活化受体γ信号对大鼠肝星状细胞基质金属蛋白酶2,9活性和胞核核因子-κBp65表达的影响.中国中西医结合杂志,2007,27(5):439-442
13 CHOUDHURI T, PAL S, AGWARL M L, et al. Curcumin induces apoptosis in human breast cancer cells through P53-dependent bax induction. FEBS Lett, 2002, 512: 334-340
14 SuzukiY, Ruiz-OrtegaM, LorenzoO, et al Inflammation and angiotensinⅡ. Int J Biochem CellBio, 2003, 35(6): 881-900
15 Olaso E, Friedman SL. Molecular regulation of hepatic fibrogenesis. JHepato, 1998, 29(5): 836-47
16 LiD, Friedman SL. Liver fibrogenesis and the role of hepatic stellate cells: new insights and prospects for therapy. Jgastroenterol Hepato, 1999, 14(7): 618-33
17 Joish VN, Oderda GM. Cost-utility analysis and quality adjusted life years. J Pain Palliat Care Pharmacother, 2005, 19(1): 57-61
18 Yoshiji H, Kuriyama S, Fukui H.Blockade of renin-angiotensin system in antifibrotic therapy. J Gastroenterol Hepatol, 2007, 22(Suppl 1): S93-S95.
19 Wagatsuma Y, Naritaka Y, Shimakawa T, et al. Clinical usefulness of the angiotensinⅡreceptor antagonist losartan in patients with portal hypertensive gastropathy. Hepatogastroenterology, 2006, 53(68): 171-174
20 Baldwin AS. The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol, 1996, 14: 649-81
21 Barnes PJ, Karim M. Nuclear factor-KB-a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med, 1997, 336(15): 1066-71
22 Kubota T, Fujisakik, Itoh Y, et al, Apoptotic injury in Culturecl human hepatocytes induced by HMG-COA redutase inhibitors. Biochem Pharmacol, 2004, 07: 2175-2186
23 Adachi M, Hignchi H, Miuras, et al. Bax interacts with the voltage-dependent action channel and mediates ethanol-induced apoptosis in rat hepatocytes Am J physial Gastrointest. Liver Physial, 2004, 287: G695-G705
24 Tsamandas AC, Thomopoulos K, zolota V, et al Potential role of bcl-2 and bax mRNA and protein expression in chronic hepatitis type Band c: a clinicopathologic Study. Mod pathol, 2003, 16: 1273-1288
25 Knaravias DD, Tsamandas AC, Tepetes K, et al, Bcl-2 and BAX expressin and cell proliferation after partial hepatectorny with and without is chemia, on cholestatic liver in rats, an experimental study, J surg Res, 2003, 110;399-408
1高春芳,陆伦根.纤维化疾病的基础和临床.上海科学技术出版社,2004,:265
2韩硬海,李树桐.临床肝脏病学.山东科学技术出版社,2005,:174
3 Zhau XY, Murphy FR, Gehdu N, et al. Engagement of aintegrin regulates proliferation and apoptosis of hepatic stellate cells. J Biol Chem, 2004, 279(23): 23996-24006
4段雪辉,杨东华.血管紧张素Ⅱ与肝星状细胞的研究进展.临床荟萃, 2006,3:380
5王玉珍,娄慧卿,扈彩霞,等. RhoA和肌球蛋白轻链在肝纤维化大鼠肝组织中的表达.基础医学与临床,2007,27(3):275-278
6 IredaleJP. Models of liver fibrosis : exploring the dynamic nature of inflammation and repair In a solid organ. J Clin Invest. 2007, 117: 539-548
7 ProsserCC, YenRD. Wu.J. Molecular therapy for hepatic injury and fibrosis:where are we? World J Gastroenterol, 2006, 12: 509-515
8 GonzaloT, Beljaars L, van de BovenkampM, et al. Local inhibition of liver fibrosis by specific delivery of a platelet-derived growth factor kinase inhibitor to hepatic stellate cells. J Pharmacol Exp Ther, 2007, 321(3): 856-865
9 Ruddell RG, OakleyF, HussainZ, er al. A role for serotonin (5-HT) in hepatic stellate cell function and liver fibrosis; Am J Pathol. 2006, 169: 861-876
10 LiG, XieQ, ShiY, et al I nhibition of connective tissuegrowth factorby siRNA prevents liver fibrosis in rats. JGeneMed, 2006, 8(7): 889-900
11 Yeom SY, Jeoung D, Ha KS, et al Small interfering RNA(siRNA) targetted to Smad3 inhibits transforming growth factor-beta signaling. Biotechnol Lett, 2004 , 26(9): 699-703
12 Williams EJ, Gaca MD, Brigstock DR,et al. Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells. J Hepatol, 2000, 32: 754-761
13 Saile B, Nlatthes N, Knittel T, et al.Transforming growth factor beta and tumor necrosis factor alpha inhibit both apoptosis and proliferation of activated rat hepatic stellate cells. Hepatology, 1999, 30: 196-202
14 Pinzani M, Marra F, Carloni V. Signal transduction in hepatic stellate cells. Liver, 1998, 18: 2-3
15刘小菁,吴红斌,刘芳,等.肿瘤坏死因子α对肝星状细胞表达结缔组织生长因子的影响.中华肝脏病杂志,2001年,9:15-17
16姜慧卿,赵东强.肿瘤坏死因子-α对肝星状细胞增殖与凋亡的影响.中国病理生理杂志,2003,19:1353-1356
17陈治新,张莉娟,陈运新,等.胰岛素样生长因子-1受体在实验性肝纤维化中的表达及IL-10的干预作用。福建医科大学学报,2002,36:134-136
18 Everett H, McFadden G. Viruses and apoptosis: meddling with itochondria. Virology, 2001, 288: 1-7
19 Novo E, Marra F, Zamara E. Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans . Gut, 2006, 55(8): 1174-82
20 MalhiH, GoresG J, Lemasters J J. Apoptosis and necrosis in the liver: a tale of two deaths? . Hepatology, 2006, 43 (2Suppl1) : S31-44
21 Taimr P, HiguchiH, Kocova E, et al Activated stellate cells express the TRAIL receptor-2 /death receptor-5 and undergo TRAIL-mediated apoptosis . Hepatology, 2003, 37(1): 87-95
22 GuoY T, LengX S, LiT, et al. Effect of ligand of peroxisome pro-liferator-activated receptor gamma on the biological characters of hepatic stellate cells . World J Gastroenterol, 2005, 11(30): 4735-9
23陈新,王玉珍,修贺明,等.神经生长因子在肝纤维化大鼠肝星状细胞凋亡中的作用。中华医学杂志。2006,86:1985- 1988
24 Shen H, Fan J, Minuk G, et al. Apoptotic and survival signals in hepatic stellate cells. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2007, 32: 726- 734
25张永进,方步武.蛋白酶体抑制剂诱导肝星状细胞凋亡的研究进展。中华肝脏病杂志。2007,15:878- 880
26 Anan A, Baskin- Bey ES, Bronk SF, et al. Proteasome inhibition induces hepatic stellate cell apoptosis. Hepatology 2006, 43: 335- 344
27 Gressner AM. Liver fibrosis: perspectives in pathobiochemical research and clinical outlook. Eur J Clin Chem Clin Biochem, 199129:293-311
2 ELSHARKAWY AM, MANN DA. Nuclear factor-kappaB andthe hepatic inflammation-fibrosis-cancer axis Hepatology, 2007, 4(62): 590-597
3 SEN R, BALTIMORE D. Multiple nuclear factors interactwith the immunoglobulin enhancer sequences . Cell, 1986, 4(65): 705-716
4 Okamoto T, Sanda T, Asamitsu K.NF-kappa B signaling andcarcinogenesis. CurrPharm Des, 2007, 13(5): 447-462
5 Heyninck K, Wullaert A, Beyaert R. Nuclear factor-kappa B plays a central role in tumour necrosis factor-mediated liver disease . Biochem Pharmacol, 2003, 66(8): 1409
6 Bruck R, AshkenaziM, WeissS, eta.l Prevention of liver cirrhosis in rats by curcumin . Liver Int, 2007, 27: 373-383
7 Liu YG, Chen HC, Jiang YP. Efects of cureumin on priary cucturo rat hatocyte injuried by CCh . Chinese Traditional Patent Medicine, 2003, 25(3): 522-525
8 BARRETO R, KAWAKITA S, TSUCHIYA J, et al.Metal-induced oxidative damage in cultured hepatocytes and hepatic lysosomal fraction: beneficial effectof a curcumin/absinthium compound . Chin J DigDis, 2005, 6: 31-36
9 BAVA S V, PULIAPPADAMBA V T, DEEPTI A, etal. Sensitization of taxol-induced apoptosis by curcumin involves down-regulation of nuclear Factor-κB and the Serine. Threonine Kinase Akt and is independent of tu-bulin polymerization . J Biol Chem, 2005, 280: 6301-6308
10 REYES-GORDILLO K, SEGOVIA J, SHIBAYAMA M, et al. Curcumin protects against acute liver damagein the rat by inhibiting NF-KappaB, proinflammatory cytokines production and oxidative stress. BiochiBiophys Acta, 2007, 22: 989-996
11舒建昌,叶国荣,吕霞,等.姜黄素治疗肝纤维化及其作用机制的初步研究.中华肝脏病杂志,2007,15(10):753-757
12成扬,平键,刘成,等.姜黄素激活过氧化物酶体增殖因子活化受体γ信号对大鼠肝星状细胞基质金属蛋白酶2,9活性和胞核核因子-κBp65表达的影响.中国中西医结合杂志,2007,27(5):439-442
13 CHOUDHURI T, PAL S, AGWARL M L, et al. Curcumin induces apoptosis in human breast cancer cells through P53-dependent bax induction. FEBS Lett, 2002, 512: 334-340
14 SuzukiY, Ruiz-OrtegaM, LorenzoO, et al Inflammation and angiotensinⅡ. Int J Biochem CellBio, 2003, 35(6): 881-900
15 Olaso E, Friedman SL. Molecular regulation of hepatic fibrogenesis. JHepato, 1998, 29(5): 836-47
16 LiD, Friedman SL. Liver fibrogenesis and the role of hepatic stellate cells: new insights and prospects for therapy. Jgastroenterol Hepato, 1999, 14(7): 618-33
17 Joish VN, Oderda GM. Cost-utility analysis and quality adjusted life years. J Pain Palliat Care Pharmacother, 2005, 19(1): 57-61
18 Yoshiji H, Kuriyama S, Fukui H.Blockade of renin-angiotensin system in antifibrotic therapy. J Gastroenterol Hepatol, 2007, 22(Suppl 1): S93-S95.
19 Wagatsuma Y, Naritaka Y, Shimakawa T, et al. Clinical usefulness of the angiotensinⅡreceptor antagonist losartan in patients with portal hypertensive gastropathy. Hepatogastroenterology, 2006, 53(68): 171-174
20 Baldwin AS. The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol, 1996, 14: 649-81
21 Barnes PJ, Karim M. Nuclear factor-KB-a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med, 1997, 336(15): 1066-71
22 Kubota T, Fujisakik, Itoh Y, et al, Apoptotic injury in Culturecl human hepatocytes induced by HMG-COA redutase inhibitors. Biochem Pharmacol, 2004, 07: 2175-2186
23 Adachi M, Hignchi H, Miuras, et al. Bax interacts with the voltage-dependent action channel and mediates ethanol-induced apoptosis in rat hepatocytes Am J physial Gastrointest. Liver Physial, 2004, 287: G695-G705
24 Tsamandas AC, Thomopoulos K, zolota V, et al Potential role of bcl-2 and bax mRNA and protein expression in chronic hepatitis type Band c: a clinicopathologic Study. Mod pathol, 2003, 16: 1273-1288
25 Knaravias DD, Tsamandas AC, Tepetes K, et al, Bcl-2 and BAX expressin and cell proliferation after partial hepatectorny with and without is chemia, on cholestatic liver in rats, an experimental study, J surg Res, 2003, 110;399-408
1高春芳,陆伦根.纤维化疾病的基础和临床.上海科学技术出版社,2004,:265
2韩硬海,李树桐.临床肝脏病学.山东科学技术出版社,2005,:174
3 Zhau XY, Murphy FR, Gehdu N, et al. Engagement of aintegrin regulates proliferation and apoptosis of hepatic stellate cells. J Biol Chem, 2004, 279(23): 23996-24006
4段雪辉,杨东华.血管紧张素Ⅱ与肝星状细胞的研究进展.临床荟萃, 2006,3:380
5王玉珍,娄慧卿,扈彩霞,等. RhoA和肌球蛋白轻链在肝纤维化大鼠肝组织中的表达.基础医学与临床,2007,27(3):275-278
6 IredaleJP. Models of liver fibrosis : exploring the dynamic nature of inflammation and repair In a solid organ. J Clin Invest. 2007, 117: 539-548
7 ProsserCC, YenRD. Wu.J. Molecular therapy for hepatic injury and fibrosis:where are we? World J Gastroenterol, 2006, 12: 509-515
8 GonzaloT, Beljaars L, van de BovenkampM, et al. Local inhibition of liver fibrosis by specific delivery of a platelet-derived growth factor kinase inhibitor to hepatic stellate cells. J Pharmacol Exp Ther, 2007, 321(3): 856-865
9 Ruddell RG, OakleyF, HussainZ, er al. A role for serotonin (5-HT) in hepatic stellate cell function and liver fibrosis; Am J Pathol. 2006, 169: 861-876
10 LiG, XieQ, ShiY, et al I nhibition of connective tissuegrowth factorby siRNA prevents liver fibrosis in rats. JGeneMed, 2006, 8(7): 889-900
11 Yeom SY, Jeoung D, Ha KS, et al Small interfering RNA(siRNA) targetted to Smad3 inhibits transforming growth factor-beta signaling. Biotechnol Lett, 2004 , 26(9): 699-703
12 Williams EJ, Gaca MD, Brigstock DR,et al. Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells. J Hepatol, 2000, 32: 754-761
13 Saile B, Nlatthes N, Knittel T, et al.Transforming growth factor beta and tumor necrosis factor alpha inhibit both apoptosis and proliferation of activated rat hepatic stellate cells. Hepatology, 1999, 30: 196-202
14 Pinzani M, Marra F, Carloni V. Signal transduction in hepatic stellate cells. Liver, 1998, 18: 2-3
15刘小菁,吴红斌,刘芳,等.肿瘤坏死因子α对肝星状细胞表达结缔组织生长因子的影响.中华肝脏病杂志,2001年,9:15-17
16姜慧卿,赵东强.肿瘤坏死因子-α对肝星状细胞增殖与凋亡的影响.中国病理生理杂志,2003,19:1353-1356
17陈治新,张莉娟,陈运新,等.胰岛素样生长因子-1受体在实验性肝纤维化中的表达及IL-10的干预作用。福建医科大学学报,2002,36:134-136
18 Everett H, McFadden G. Viruses and apoptosis: meddling with itochondria. Virology, 2001, 288: 1-7
19 Novo E, Marra F, Zamara E. Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans . Gut, 2006, 55(8): 1174-82
20 MalhiH, GoresG J, Lemasters J J. Apoptosis and necrosis in the liver: a tale of two deaths? . Hepatology, 2006, 43 (2Suppl1) : S31-44
21 Taimr P, HiguchiH, Kocova E, et al Activated stellate cells express the TRAIL receptor-2 /death receptor-5 and undergo TRAIL-mediated apoptosis . Hepatology, 2003, 37(1): 87-95
22 GuoY T, LengX S, LiT, et al. Effect of ligand of peroxisome pro-liferator-activated receptor gamma on the biological characters of hepatic stellate cells . World J Gastroenterol, 2005, 11(30): 4735-9
23陈新,王玉珍,修贺明,等.神经生长因子在肝纤维化大鼠肝星状细胞凋亡中的作用。中华医学杂志。2006,86:1985- 1988
24 Shen H, Fan J, Minuk G, et al. Apoptotic and survival signals in hepatic stellate cells. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2007, 32: 726- 734
25张永进,方步武.蛋白酶体抑制剂诱导肝星状细胞凋亡的研究进展。中华肝脏病杂志。2007,15:878- 880
26 Anan A, Baskin- Bey ES, Bronk SF, et al. Proteasome inhibition induces hepatic stellate cell apoptosis. Hepatology 2006, 43: 335- 344
27 Gressner AM. Liver fibrosis: perspectives in pathobiochemical research and clinical outlook. Eur J Clin Chem Clin Biochem, 199129:293-311