【作者】 孙龙；

【导师】 杨世忠； 迟宝荣；

【作者基本信息】 吉林大学 ， 内科学， 2007， 博士

【摘要】 作为一种多功能的核转录因子,PPAR-α可以在细胞水平抑制NF-κB的转录活性,调控炎性细胞因子的表达,从而抑制局部的炎症反应;可以调控脂肪酸氧化相关基因的表达,在肝脏脂肪细胞分化、脂质的储存、转运及脂肪酸氧化过程中发挥重要作用。本课题研究应用白酒灌胃制备酒精性肝损伤大鼠模型,动态观察了酒精性肝损伤进程中PPAR-α的基因表达变化及中药酒肝颗粒的干预作用,并对PPAR-α的基因表达与炎症因子、氧化应激指标之间进行了相关性分析,旨在研究探讨PPAR-α在酒精性肝损伤中可能的作用及酒肝颗粒的作用机理。研究结果提示:PPAR-α参与了酒精性肝损伤,并在其发生、发展进程中具有关键作用,有望成为治疗酒精性肝损伤新的靶点;中药酒肝颗粒可以上调PPAR-α的基因表达,抑制肝脏炎症反应,提高其抗氧化能力,对酒精性肝损伤的具有较好的防治作用。本课题研究应用Real-time PCR、RT-PCR及免疫组化等实验方法动态观察了PPAR-α在酒精性肝损伤形成过程中不同时间段的表达变化,结果初步证实PPAR-α在酒精性肝损伤中具有重要作用;同时,从基因及蛋白水平探讨了中药酒肝颗粒治疗酒精性肝损伤的作用机理,为其今后的临床推广应用提供了理论基础。更多还原

【Abstract】 Insobriety is becoming one severe social problem in the world. Long-term excessive drinking can lead to disorders of enzymatic activities and result in alcoholic liver injury (ALI). A series of pathologic physiology variations will be presented in the liver of ALI, including initial fatty degeneration, following alcoholic hepatitis, alcoholic hepatic fibrosis and final alcoholic liver cirrhosis. And some severe complications, such as ascitic fluid, upper gastrointestinal bleeding, hepatic encephalopathy and so on, might appear at last. The incidence of ALI is increasing in China during the recent years, which results in its second position just under virus hepatitis in liver diseases. ALI has already influenced people quality of life and health severely. Studies manifest that early ALI may be completely recovered if it could be diagnosed and treated in time. On the contrary, it could develop into alcoholic liver cirrhosis,liver cancer and even severe complications, where the pathological changes are too difficult to recover. Thus, it is very significant for the prognosis of patients with ALI to discover, stop drinking and treat positively at the initial stage.It is clear that excessive drinking is the causative agent of ALI. While its nosogenesis is very complicated, including toxic effects of alcohol and its metabolin, immuno-modulation, inflammatory factors, apoptosis, endotoxin, genetic polymorphism, additive effects of virus, etc. Moreover, ALI is also correlated with nutritional-state and genetic predisposition. Presently, it is generally accepted that inflammatory factors and oxidative stress play an important role in ALI. Alcohol intake can make some cell factors and mediators of inflammation increase massively, which may lead to inflammation, necrosis, apoptosis, fibrosis in liver and result in cell damage. Besides, long-term drinking can make prooxidants increase and antioxygens decrease, which may aggravate the oxidative stress and lead to liver cells death or apoptosis.Recently studies discovered that Peroxisome Proliferator Activated Receptor-α(PPAR-α), as a multifunctional nuclear factor, has many biological effects. PPAR-αcan inhibit the transcription and signal transduction of NF-κB (P65/ReLA subunit) and adjust the expression of inflammatory factors on cellular level, which might inhibit local inflammatory reaction. PPAR-αalso can control the expression of genes related with fatty acid oxidation in liver, which may play an important role in cytodifferentiation of adipose cells, storage and transport of lipids, fatty acid oxidation. Excitomotors of PPAR-αcan ameliorate fatty-degeneration of liver cells, reverse inflammatory reaction, and up-regulate the gene expression of fatty acid-binding proteins in liver. Studies hint that PPAR-αmight play a significant role in the pathologic mechanism of ALI, and might become one new therapeutic target for ALI. Hereto, there are less really therapeutical remedies for ALI yet. Chinese traditional medicine, with relatively less side effects, can regulate physiological reactions into balance by more targets. And it has a broad future of application and development in prevention and cure of ALI. Recently, there are numerous studies and reports on Chinese traditional medicine against ALI. The most are confined to observations of therapeutic effect, but less investigations on the mechanism of action. While, reports about studying effects of Chinese traditional medicine on expression of PPAR-αare rare. Jiugan Keli (JGKL) is a formula designed by Prof. Shi-zhong Yang on basis of his years of clinical experience. Prophase pharmacodynamics studies and clinical observations confirm that JGKL can alleviate hangover, decrease blood-fat level,protect liver function and lower activities of aminopherase efficiently. But its exact mechanism of action is still not clear now.In our study, we established ALI animal models successfully by lavage with distillate spirit. During the different stages of ALI (4w、8w、12w、16w), we observed the expression of PPAR-αand NF-κB in hepatic tissue of rats through molecular biology technique continuously. Besides, we also detected the level of TNF-α, IL-6 in serum, the content of MDA and the activity of SOD in hepatic tissue, ect. Then we analyzed the dependability between the expression of PPAR-αand other indexes, in order to identify the possible roles of PPAR-αin the development of ALI. Moreover, we treated ALI animals with JGKL and stop-drinking (spontaneous recovery) for 8w. Then we observed the expression of PPAR-αand NF-κB in hepatic tissue, the level of TNF-α, IL-6, FFA in serum, the content of MDA and the activity of SOD in hepatic tissue, ect. Moreover, In order to approach its mechanism of action against ALI further, we observed the effects of JGKL on the expression of PPAR-αand studied its mechanism of action initially. The following is the study outcomes:1) In the study, we established ALI animal models successfully by lavage with distillate spirit. A series of pathologic physiology variations were presented in hepatic tissue of rats with ALI, including adipose degeneration, inflammation, necrosis and slight fibrosis. And we found that the hepatic injury aggravated steadily with the increase of alcohol intake.2) During different stages of ALI, the gene expression of PPAR-αin hepatic tissue decreased obviously with the increase of alcohol intake.3) The expression of NF-κB in hepatic tissue of ALI rats increase steadily with the increase of alcohol intake.4) The level of TNF-α, IL-6, FFA in serum and the content of MDA in hepatic tissue of ALI increased with the increase of alcohol intake. While the activity of SOD in hepatic tissue decreased steadily with the increase of alcohol intake.5) Analysis of dependability indicates that the gene expression of PPAR-αwas correlated with the degree of liver injury, expression of inflammatory factors (NF-κB, TNF-α, IL-6) negatively. Meanwhile, there were certain inverse correlations between the gene expression of PPAR-αand the content of MDA, the level of FFA respectively. On the contrary, the gene expression of PPAR-αwas correlated with the activity of SOD in hepatic tissue positively.6) JGKL could against the further liver injury caused by alcohol efficiently. It might enhance the gene expression of PPAR-αand lower the expression of NF-κB, TNF-α, IL-6 in hepatic tissue. JGKL also could ameliorate the impaired hepatic function and lighten the oxidative stress. Besides, JGKL might protect liver cells and recover the impaired hepatic tissue. 7) The impaired hepatic function, the level of blood-fat, the indexes of oxidation/anti-oxidation and histopathological changes might improve gradually after stop-drinking. The expression of inflammatory factors (NF-κB, TNF-α, IL-6) decreased obviously, while the expression of PPAR-αincreased gradually.8) Combining stop-drinking and JGKL could treat ALI more significantly, which might enhance the expression of PPAR-α, weaken the expression of inflammatory factors (NF-κB, TNF-α, IL-6) in hepatic tissue, improve the indexes of oxidation/anti-oxidation and others obviously.The study results suggested that:1) PPAR-αmight play a significant role in the nosogenesis of ALI. The decreased expression of PPAR-αcan enhance the expression of inflammatory factors, degrade the power of removing free-radical and aggravate the oxidative stress. Thus, to increase the expression of PPAR-αmight be anticipated to become one new therapeutic target for ALI.2) JGKL has good preventive and therapeutic effects on ALI. PPAR-αshould be a target of its effects, and to increase the expression of PPAR-αmight be one of main mechanism of action of JGKL.3) Stop-drinking (spontaneous recovery) is a chief method for curing ALI. While, we found that combining stop-drinking and JGKL might be more efficiently to treat ALI.Significance and innovation of the study:1) The study is the first of its kind using real-time PCR and RT-PCR to observe the expression of PPAR-αin the different stages of ALI. The study demonstrates that the gene expression of PPAR-αdecreases obviously during different stages of ALI with the increase of alcohol intake. 2) The study is the first of its kind indicating through dependablity analysis that the gene expression of PPAR-αis correlated with the degree of liver injury, the expression of inflammatory factors (NF-κB, TNF-α, IL-6) negatively in ALI. Meanwhile, there are certain inverse correlations between the gene expression of PPAR-αand the indexes of oxidative stress (the content of MDA, the level of FFA) respectively. On the contrary, the gene expression of PPAR-αis correlated with the activity of SOD in hepatic tissue positively.3) The study is the first of its kind explaining mechanism of action agaist ALI of JGKL from gene and protein level. The study indicates that JGKL has good preventive and therapeutic effects on ALI. JGKL can enhance the expression of PPAR-α, which might decrease inflammatory reaction in hepatic tissue, improve the power of antioxygen and inhibit the lipid peroxidation efficiently.更多还原