白桦酯酸通过阻断TLR4/NF-κB信号通路抑制肝纤维化及酒精性脂肪肝的实验研究
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摘要
肝纤维化是发生在所有慢性肝病患者上的一种可逆的创伤性反应。肝纤维化是发展到肝硬化的必经阶段,并与肝癌有关,是所有慢性肝病晚期并发症的共同病理基础。在肝损伤的过程中,活化的肝星状细胞(HSC)是肝纤维化形成过程中产生胶原的主要细胞,关于HSC细胞增殖及ECM产生中的分子机制研究是近来关注的热点。抑制炎症反应,对抗氧化应激,调节相关细胞因子的活性,干扰细胞因子信号转导等途径能够有效地抑制HSC的增殖、活化,从而有效对抗肝纤维化。肝细胞是各种肝毒性介质作用的主要靶细胞,包括各种肝炎病毒,酒精的代谢物和胆汁酸等,破坏肝细胞并释放大量活性氧簇(ROS)和炎性介质,造成炎症细胞的活化并进一步激活HSC,加剧肝纤维化的发生。因此,发现肝保护药物是发展抗肝纤维化治疗的迫切需要。
本实验采用硫代乙酰胺和酒精建立肝纤维化模型,通过体内、体外实验研究白桦酯酸的抗肝纤维化作用及潜在的作用机制。
(1)使用硫代乙酰胺(200mg/kg)建立大鼠肝纤维化模型,腹腔注射硫代乙酰胺每周两次连续六周。白桦酯酸(20mg/kg or50mg/kg)与硫代乙酰胺同时给药或在其之后给药直至第六周或第八周,考察白桦酯酸的肝脏预防作用和保护作用。实验结果显示白桦酯酸能够有效对抗和治疗肝纤维化,并减少了由硫代乙酰胺刺激后造成的肝组织中羟脯氨酸和α-SMA的增多。体外实验表明,白桦酯酸能够有效减少由TNF-α激活的HSC的活性,同时在正常人张氏肝细胞中没有毒性作用。此外,白桦酯酸也能够明显降低α-SMA和TIMP-1的水平,增加MMP-13的表达,抑制了TLR4、MyD88的水平并激发了NF-κB的核转导活性,伴有时间依赖性。
(2)使用乙醇建立酒精肝模型。小鼠灌胃给药乙醇(5g/kg)3次间隔12h。同时给予白桦酯醇或白桦酯酸(50mg/kg或20mg/kg)。体外实验,使用乙醇(50mM)激活HSC,并处理25μM和12.5μM的白桦酯醇和白桦酯酸,观察24小时。病理组织学结果显示在体内白桦酯醇和白桦酯酸能够抑制酒精诱导的肝脏脂肪变性和坏死,同时,白桦酯醇和白桦酯酸能够有效抑制CYP2E1和SREBP-1的表达。体外实验结果表明,酒精浓度在20-200mM没有影响细胞的增殖,而且能够有效抑制ECM的聚集减少collagen-1的水平和α-SMA的表达。同时增加了调控SREBP-1基因的主要蛋白AMPK和STAT3的表达,白桦酯醇和白桦酯酸的保护机制可能与抑制TLR4、MyD88的信号通道,抑制NF-κB的活性有关。且白桦酯酸对抗乙醇诱导的肝纤维化具有更强的保护作用。
根据上述实验结果可推断白桦酯酸对抑制肝纤维化具有潜在的药理学治疗特性。但是仍需要进一步深入研究、确定其确切的保护机制。
Fibrosis is the progressive pathological process in which the body's wound healing and tissue remodeling mechanisms respond to liver injury in all chronic hepatopath. Hepatic fibrosis is the necessary stage in the development of cirrhosis and plays an important part in liver cancer. It is the common pathological foundation in all advanced stage complications of chronic liver disease. The main cell type responsible for the development of fibrosis is the activated hepatic stellate cell (HSC). The molecule mechanism of activated hepatic stellate cells proliferate and synthesize extracellular matrix proteins to produce the fibrous scar is the research point recently. It has been suggested that the use of anti-inflammatory, anti-oxidative stress agents might be useful in preventing the activation of HSCs, consequently, in the development of liver fibrosis. HSC is the target cell that suffer from various kinds of hepatic toxic agents including hepatitis virus, alcohol metabolites and bile acid. The hepatic toxic agents destroy the hepatocyte, release reactive oxygen species (ROS) and inflammatory mediators causing that activate inflammatory cells and contributes to activate HSC to aggravate the development of liver fibrosis.
This study select TAA and alcohol to make animal liver fibrosis and acute ethanol fatty liver models, research on the hepato-protective effect and the underlying mechanism of BA in vivo and in vitro.
(1) Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA,200mg/kg) twice weekly for6weeks in Wistar rats. The administration of BA (20mg/kg or50mg/kg) was started following TAA injections and was continued for6or8weeks to evaluate both the preventive and the protective effects. BA demonstrated great efficacy in preventing and curing hepatic fibrosis via attenuating the TAA-mediated increases in liver'tissue hydroxyproline and α-smooth muscle actin (a-SMA). In vitro, BA effectively decreased the HSC-T6cell viability induced by TNF-a and showed low toxicity in normal human chang liver cells. Moreover, BA significantly attenuated the expression of a-SMA and tissue inhibitor of metalloproteinase-1(TIMP-1) and increased the levels of matrix metalloprotease (MMP)-13. BA also inhibited the expression of toll-like receptor4(TLR4), myeloid differentiation factor88(MyD88) and the activation of nuclear factor-κB (NF-κB) in a time-dependent manner.
(2) Mice were treated with ethanol (5g/kg, body weight) by gavage every12h for a total of3doses to induce acute fatty liver. BA and BT (50or20mg/kg) were gavaged simultaneously with ethanol for3doses. Hepatic stellate cells was activated by ethanol (50mM) for24h and simultaneous treated with BA or BT25μM and12.5μM to observe the protective effect against ethanol in vitro. BA or BT administration significantly reduced the increases in serum ALT, AST and triglyceride levels at4h after the last ethanol administration. BA and BT were also found to prevent ethanol-induced hepatic steatosis and necrosis, as indicated by liver histopathological studies. Meanwhile, both of them showed a powerful inhibitory effect on CYP2E1and SREBP-1expression. In vitro, ethanol concentrations among20-200mM exhibited hardly any effect on cell viability and treated with B A and BT did not influence the cell proliferation. Moreover, BA and BT effectively reduced the ECM accumulation via decreasing collagen-1levels and attenuating the expression of a-SMA. However, BA and BT also excellently decreased AMPK and STAT3levels that controlled SREBP-1expression in ethanol induced hepatic steatosis. The hepato-protective effect of BA and BT may be related with the inhibition of TLR4/MyD88signal pathway and the activation of NF-κB. Among that, BA showed a more powerful hepato-protective effect on ethanol induced liver steatosis.
Thus, BA may be useful as a potential pharmacological therapy for the prevention of liver fibrosis and acute fatty liver. Further investigation is required to determine the exact protective mechanism and to discover the active candidates.
本实验采用硫代乙酰胺和酒精建立肝纤维化模型,通过体内、体外实验研究白桦酯酸的抗肝纤维化作用及潜在的作用机制。
(1)使用硫代乙酰胺(200mg/kg)建立大鼠肝纤维化模型,腹腔注射硫代乙酰胺每周两次连续六周。白桦酯酸(20mg/kg or50mg/kg)与硫代乙酰胺同时给药或在其之后给药直至第六周或第八周,考察白桦酯酸的肝脏预防作用和保护作用。实验结果显示白桦酯酸能够有效对抗和治疗肝纤维化,并减少了由硫代乙酰胺刺激后造成的肝组织中羟脯氨酸和α-SMA的增多。体外实验表明,白桦酯酸能够有效减少由TNF-α激活的HSC的活性,同时在正常人张氏肝细胞中没有毒性作用。此外,白桦酯酸也能够明显降低α-SMA和TIMP-1的水平,增加MMP-13的表达,抑制了TLR4、MyD88的水平并激发了NF-κB的核转导活性,伴有时间依赖性。
(2)使用乙醇建立酒精肝模型。小鼠灌胃给药乙醇(5g/kg)3次间隔12h。同时给予白桦酯醇或白桦酯酸(50mg/kg或20mg/kg)。体外实验,使用乙醇(50mM)激活HSC,并处理25μM和12.5μM的白桦酯醇和白桦酯酸,观察24小时。病理组织学结果显示在体内白桦酯醇和白桦酯酸能够抑制酒精诱导的肝脏脂肪变性和坏死,同时,白桦酯醇和白桦酯酸能够有效抑制CYP2E1和SREBP-1的表达。体外实验结果表明,酒精浓度在20-200mM没有影响细胞的增殖,而且能够有效抑制ECM的聚集减少collagen-1的水平和α-SMA的表达。同时增加了调控SREBP-1基因的主要蛋白AMPK和STAT3的表达,白桦酯醇和白桦酯酸的保护机制可能与抑制TLR4、MyD88的信号通道,抑制NF-κB的活性有关。且白桦酯酸对抗乙醇诱导的肝纤维化具有更强的保护作用。
根据上述实验结果可推断白桦酯酸对抑制肝纤维化具有潜在的药理学治疗特性。但是仍需要进一步深入研究、确定其确切的保护机制。
Fibrosis is the progressive pathological process in which the body's wound healing and tissue remodeling mechanisms respond to liver injury in all chronic hepatopath. Hepatic fibrosis is the necessary stage in the development of cirrhosis and plays an important part in liver cancer. It is the common pathological foundation in all advanced stage complications of chronic liver disease. The main cell type responsible for the development of fibrosis is the activated hepatic stellate cell (HSC). The molecule mechanism of activated hepatic stellate cells proliferate and synthesize extracellular matrix proteins to produce the fibrous scar is the research point recently. It has been suggested that the use of anti-inflammatory, anti-oxidative stress agents might be useful in preventing the activation of HSCs, consequently, in the development of liver fibrosis. HSC is the target cell that suffer from various kinds of hepatic toxic agents including hepatitis virus, alcohol metabolites and bile acid. The hepatic toxic agents destroy the hepatocyte, release reactive oxygen species (ROS) and inflammatory mediators causing that activate inflammatory cells and contributes to activate HSC to aggravate the development of liver fibrosis.
This study select TAA and alcohol to make animal liver fibrosis and acute ethanol fatty liver models, research on the hepato-protective effect and the underlying mechanism of BA in vivo and in vitro.
(1) Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA,200mg/kg) twice weekly for6weeks in Wistar rats. The administration of BA (20mg/kg or50mg/kg) was started following TAA injections and was continued for6or8weeks to evaluate both the preventive and the protective effects. BA demonstrated great efficacy in preventing and curing hepatic fibrosis via attenuating the TAA-mediated increases in liver'tissue hydroxyproline and α-smooth muscle actin (a-SMA). In vitro, BA effectively decreased the HSC-T6cell viability induced by TNF-a and showed low toxicity in normal human chang liver cells. Moreover, BA significantly attenuated the expression of a-SMA and tissue inhibitor of metalloproteinase-1(TIMP-1) and increased the levels of matrix metalloprotease (MMP)-13. BA also inhibited the expression of toll-like receptor4(TLR4), myeloid differentiation factor88(MyD88) and the activation of nuclear factor-κB (NF-κB) in a time-dependent manner.
(2) Mice were treated with ethanol (5g/kg, body weight) by gavage every12h for a total of3doses to induce acute fatty liver. BA and BT (50or20mg/kg) were gavaged simultaneously with ethanol for3doses. Hepatic stellate cells was activated by ethanol (50mM) for24h and simultaneous treated with BA or BT25μM and12.5μM to observe the protective effect against ethanol in vitro. BA or BT administration significantly reduced the increases in serum ALT, AST and triglyceride levels at4h after the last ethanol administration. BA and BT were also found to prevent ethanol-induced hepatic steatosis and necrosis, as indicated by liver histopathological studies. Meanwhile, both of them showed a powerful inhibitory effect on CYP2E1and SREBP-1expression. In vitro, ethanol concentrations among20-200mM exhibited hardly any effect on cell viability and treated with B A and BT did not influence the cell proliferation. Moreover, BA and BT effectively reduced the ECM accumulation via decreasing collagen-1levels and attenuating the expression of a-SMA. However, BA and BT also excellently decreased AMPK and STAT3levels that controlled SREBP-1expression in ethanol induced hepatic steatosis. The hepato-protective effect of BA and BT may be related with the inhibition of TLR4/MyD88signal pathway and the activation of NF-κB. Among that, BA showed a more powerful hepato-protective effect on ethanol induced liver steatosis.
Thus, BA may be useful as a potential pharmacological therapy for the prevention of liver fibrosis and acute fatty liver. Further investigation is required to determine the exact protective mechanism and to discover the active candidates.
引文
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