双环醇对实验性非酒精性脂肪肝的保护作用及机制研究
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摘要
非酒精性脂肪性肝病(Nonalcoholic Fatty Liver Disease, NAFLD)是无过量饮酒史(<140g乙醇／周),但肝脏组织学病变与酒精性肝病(Alcoholic Liver disease,ALD)相似的临床综合症,包括单纯性脂肪肝(simple steatosis)和非酒精性脂肪性肝炎(Nonalcoholic Steatohepatitis, NASH),后者与肝硬化和肝癌关系密切。近年来随着生活水平的提高,NAFLD的发病率逐年攀升,普通人群中约有14%至24%患有NAFLD,已成为最常见的肝脏疾患之一。目前,限制热量摄取是治疗NAFLD唯一有效的手段,但对多数患者而言常难以坚持,因此,寻找有效的药物治疗NAFLD药物十分必要。
双环醇(bicyclol)是我所研制的治疗慢性肝炎的国家一类新药。临床资料显示,双环醇在改善慢性乙肝病人临床症状和肝功能的同时,对乙肝病毒复制也有一定抑制作用,且停药后反跳率低,未发现明显不良反应。以往药理研究表明,双环醇对多种化学毒物、酒精等引起的急慢性肝损伤模型均具有较好的肝保护作用,其作用机制与其清除自由基、保护线粒体功能等密切相关。
本实验室既往研究表明,双环醇对实验性非酒精性脂肪肝有显著的保护作用,但对相关机制尚未进行深入研究。因此,本研究选用四环素和高脂饮食引起的两种非酒精性脂肪肝模型观察双环醇的保护作用,并对相关机制进行深入探讨,为其临床应用提供科学可靠的实验依据。
1.双环醇对四环素引起小鼠脂肪肝的保护作用及机制研究
单次腹腔注射四环素(tetracycline,200mg/kg)可引起小鼠急性脂肪肝,表现为血清丙氨酸氨基转移酶(alanine transaminase, ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase, AST)升高,肝脏甘油三酯(TG)及胆固醇(CHO)聚积,肝细胞小泡性脂肪变及水样变性等病理改变。双环醇预防给药(300mg/kg)可降低血清ALT及AST水平的升高、抑制甘油三酯及胆固醇的蓄积,同时改善上述肝脏组织病理学改变。
体内脂肪酸氧化主要发生在三个细胞器,即线粒体和过氧化酶体的β氧化以及微粒体ω氧化。四环素注射24h后小鼠肝脏线粒体β氧化速率显著下降,过氧化酶体(acetyl-coenzyme A oxidase, AOX)及微粒体(CYP2E1及CYP4A)脂肪酸氧化也受到显著抑制,表现为上述三个细胞器中四个限速酶(ACD、AOX、CYP2E1以及CYP4A)的氧化速率与对照组相比分别降低了34%、40%、58%和30%。线粒体长链脂酰辅酶A脱氢酶(long-chain acyl-CoA dehydrogenase, LCAD)、微粒体脂肪酸代谢酶CYP4A10及CYP4A12的mRNA表达也受到显著抑制。双环醇预防给药(300mg/kg)可显著提高线粒体乙酰辅酶A脱氢酶(acetyl CoA dehydrogenase,ACD)、过氧化酶体AOX、微粒体CYP2E1以及CYP4A的氧化速率,同时增加LCAD以及CYP4A10、CYP4A12的mRNA表达。过氧化酶体增殖活化受体(peroxisome proliferator-activated receptor a, PPARα)为调节脂肪酸代谢过程所需多种酶表达的转录因子。四环素注射24h后,小鼠肝脏PPARa mRNA表达显著下降,双环醇预防给药(300mg/kg)可在一定程度上提高该基因的表达。
线粒体损伤尤其是线粒体呼吸链(mitochondrial respiratory chain, MRC)功能异常在NAFLD的发病过程中发挥关键作用。四环素注射24h后,肝脏MRC I和Ⅳ活性与对照组相比分别降低42%、31%。双环醇预防给药(300mg/kg)可显著增加MRC I和MRC IV活性,使之恢复到接近正常水平。此外,注射四环素24h后,小鼠肝脏线粒体对罗丹明123(rhodamine123,R123)的摄取量明显少于对照组,对高钙浓度诱发肿胀的敏感性显著降低,提示线粒体发生了膜渗透性转换。双环醇预防给药(300mg/kg)可显著改善受损的线粒体功能,表现为线粒体对R123的摄取量以及高钙浓度引发的吸光度下降幅度均明显增加。ATPase是反映线粒体能量代谢状况的重要指标,注射四环素24h后,小鼠肝脏ATPase活性显著下降。双环醇预防给药(300mg/kg)能够显著提高受损的ATPase活性,使之接近正常水平。
线粒体损伤能够引起ROS生成增加,造成肝组织氧化损伤。氧化应激是启动及维持NASH的重要病理因素。注射四环素24h后,小鼠肝脏MDA显著升高,而GSH含量及SOD活性也出现代偿性升高。双环醇预防给药(300mg/kg)能够改善MDA升高,使GSH含量及SOD活性恢复至接近正常水平。固醇调控元件结合蛋白(Sterol Regulatory Element-Binding Protein, SREBP)-lc是调控肝脏脂质合成代谢的重要转录因子。但四环素腹腔注射24h后,小鼠肝脏SREBP-1c及其靶基因脂肪酸合成酶(Fatty acid synthesis, FAS)及固醇辅酶A脱氢酶(stearoyl-CoA desaturase, SCD)的mRNA表达未见显著变化。由此可见,单次注射四环素能引起小鼠出现明显脂肪肝,损伤机制与抑制PPARα及其靶基因、氧化损伤有关。双环醇对四环素引起脂肪肝的保护作用与其调控PPARa转导通路紊乱及抑制氧化损伤密切相关。
2.双环醇对高脂饮食引起大鼠脂肪肝的保护作用及机制研究
高脂饮食喂养四周后,大鼠出现明显脂肪肝,表现为肝脏脂质聚积(肝脏TG、CHO以及FFA含量明显增加),血脂异常(出现高胆固醇和低甘油三酯血症),肝细胞损伤及小泡性脂肪变、水样变性以及炎性细胞浸润等病理改变。双环醇预防给药或治疗给药(150,300mg/kg)可有效地抑制HFD引起的肝脏脂质堆积、血中TG水平降低,并且改善上述肝脏病理学损伤。
SREBP-1c是调控肝脏脂肪酸及甘油三酯合成过程中诸多代谢酶表达的重要转录因子。高脂饮食喂养4周后,大鼠肝脏SREBP-1c前体蛋白及成熟型nSREBP-1蛋白表达显著升高,分别为对照组的3.5倍及2倍,双环醇预防及治疗给药(300mg／kg)均能明显降低SREBP-1c前体蛋白及成熟型nSREBP-1c蛋白表达,使之接近正常水平。
乙酰辅酶A羧化酶(acetyl-CoA carboxylase, ACC)、FAS以及SCD是脂肪酸代谢过程中的关键酶,其转录过程受SREBP-1c的调控。高脂饮食喂养4周后,大鼠肝脏ACC、FAS以及SCD的mRNA表达均显著升高,ACC及FAS的蛋白表达也显著增加,分别为对照组的1.84倍和3.1倍。双环醇对ACC、FAS以及SCD mRNA表达均有不同程度的抑制作用,并能显著抑制ACC及FAS的蛋白表达。
胰岛素诱导基因(Insulin induced genes, Insigs)是调控SREBP-1活化的重要因子,能造成SREBP-SCAP(SREBP裂解活化蛋白,SREBP cleavage-activating protein, SCAP)在内质网(ER)的滞留。高脂饮食喂养4周后,大鼠肝脏Insig-1 mRNA表达显著下降,Insig-2a mRNA表达无显著变化,双环醇预防及治疗给药(300mg/kg)对Insig-1和Insig-2a的mRNA的表达均无显著影响。
由此可见,高脂饮食喂养4周能够引起大鼠脂肪肝发生,其损伤机制与高脂饮食增加SREBP-1c的翻译及活化,从而增加脂质合成基因的转录有关。双环醇对高脂饮食引起大鼠脂肪肝的保护作用与其从翻译水平抑制SREBP-1c的表达进而调控脂质合成基因的转录相关。
综上所述,双环醇对四环素及高脂饮食引起的急慢性实验性脂肪肝均有显著的保护作用,不仅可以降低血清转氨酶的升高、抑制肝脏甘油三酯及胆固醇蓄积,还可显著改善肝脏小泡性脂肪变、水样变性及炎性损伤等病理改变。其保护作用机制可归纳为：
1)抑制氧化应激：减轻脂质过氧化,使GSH含量及SOD活性的代偿性升高恢复至接近正常水平。
2)减轻线粒体损伤：维持线粒体膜完整性和正常膜电位,恢复线粒体ATPase活性,增加MRCI和Ⅳ活性。
3)调控PPARa及其靶基因的表达：增加降低的PPARa表达,增加ACD、AOX、CYP2E1以及CYP4A的mRNA表达及活性。
4)调控SREBP-1c转导通路：抑制SREBP-1c的前体蛋白及成熟型蛋白的表达,抑制负责脂肪酸合成的靶基因ACC、FAS以及SCD的mRNA及蛋白表达。
上述研究为进一步了解双环醇的肝保护作用特点以及临床治疗NAFLD提供了有参考价值的实验依据。
Nonalcoholic Fatty Liver Disease (NAFLD) is a syndrome with liver histopathologic findings, which resembles alcoholic hepatitis in patients who lack a history of significant alcohol comsumption (<140 g ethanol/week). The spectrum of histologic abnormalities of NAFLD ranges over simple steatosis, nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and even hepatocellular carcinoma or liver failure. The prevalence of NAFLD in the general population is estimated to be 14% and 24% and has markedly increased recently. To date, the only effective treatment of NAFLD is caloric restriction, which is difficult to achieve for most NAFLD patients, it is of great necessity to find the effective treatment of NAFLD, including drug therapy.
Bicyclol is a novel anti-hepatitis drug for the treatment of chronic viral hepatitis. It has been shown to improve the clinical symptoms and inhibit virus replication in HBV patients, featured by low rebounding rate, few adverse effects and convenient usage. Previous pharmacological studies indicated that bicyclol had hepatoprotective action against acute and chronic experimental liver injury caused by certain toxins and alcohol. The hepatoprotective mechanisms of bicyclol were related with the clearance of reactive oxygen species, regulation of mitochondrial dysfunction, etc.
Previous study in our laboratory has also demostrated its role in protecting experimental animals from steatosis (induced by high fat diet, tetracycline, carbon tetrachloride and ethionine), however, in-depth investigation on related mechanisms are still needed. Therefore, acute and chronic experimental steatosis models were established in the present study to observe the protective effect of bicyclol and related mechanisms, so as to provide experimental evidences for its clinical application.
Part I:Protective effect of bicyclol on tetracycline-induced fatty liver in mice and related mechanisms
Single dose of tetracycline injection caused hepatic steatosis as evidenced by the elevation of serum ALT and AST, accumulation of hepatic TG and CHO, and morphologic changes (small lipid droplets and hydropic degeneration of hepatocytes), which reflected early alterations of NAFLD in mice. Bicyclol treatment significantly protected against tetracycline-induced fatty liver by reducing elevated serum ALT&AST levels and alleviating hepatic lipid accumulation biochemically and pathologically.
The oxidation of fatty acids occurs in three subcellular organelles. Mitochondria and peroxisome degrade fatty acid byβ-oxidation, while smooth endoplasmic reticulum metabolizes fatty acid viaω-oxidation by CYP2E1 and CYP4A subfamily. The mitochondrialβ-oxidation was found to be inhibited by 34% after tetracycline injection. In addition, tetracycline also reduced the activities of enzymes responsible for fatty acid oxidation in peroxisome (AOX) and microsome (CYP2E1, CYP4A) were also inhibited by 40%,58% and 30%, respectively in mice. Furthermore, mRNA levels of LCAD, CYP4A10 and CYP4A12 also decreased. Administration with bicyclol ameliorated the decrease of fatty acid oxidation at both enzyme activity and mRNA levels.
Peroxisome proliferators-activated receptor a (PPARa) is a kind of transcription factor regulating a serial of enzymes responsible for fatty acid oxidation. The mRNA expression of hepatic PPARa was inhibited after tetracycline injection. Bicyclol (300mg/kg) showed protective effect against tetracycline induced decrease of PPARa mRNA expression.
Mitochondrial dysfunction, particularly mitochondrial respiratory chain (MRC) deficiency, plays a key role in the physiopathology of NAFLD. Hepatic MRC I andⅣwere significantly inhibited by 42% and 31% respectively after tetracycline injection. Pretreatment of bicyclol (300mg/kg) reversed the hepatic MRC complexI&IV activity to normal levels. Besides, the isolated mitochondria had a rapid onset of MPT (membrane permeability transition, MPT) by decreased uptake of cationic dye Rodamine 123 and lowered sensitivity to Ca2+after tetracycline injection. Bicyclol remarkably improved tetracycline-induced alteration mentioned above. ATPase is a kind of marker reflecting the condition of mitochondrial energy metabolism. Pretreatment with bicyclol (300mg/kg) significantly reversed the injured activity of ATPase to normal level.
Mitochondrial dysfunction accelerates the production of ROS and induced hepatic oxidative injury. Oxidative stress was another pathogenic factor in initiating and maintaining of NASH. Hepatic MDA content was elevated 24h after tetracycline injection. GSH content and SOD activity were also found increased compensably. Pretreatment with bicyclol (300mg/kg) significantly inhibited the elevation of MDA, decreased GSH content and SOD activity.
Sterol regulatory element-binding protein (SREBP)-lc is a key transcription factor regulating lipid synthesis. No obvious alteration of mRNA expression of SREBP-1c and its target genes SCD, FAS was observed after tetracycline injection.
In conclusion, obvious hepatic steatosis was observed after a single dose of tetracycline in mice. The mechanism was found to be partly due to the inhibition of PPARa and its regulated genes, and mitochondrial dysfunction. Bicyclol showed significant protective effect on hepatic steatosis induced by tetracycline through the modulation of PPARa pathway and protection of mitochondrial injury.
Part II:Effect of bicyclol on high fat diet (HFD) induced fatty liver in rat and related mechanisms
After 4 week feeding with HFD, rat had severe fatty liver as evidenced by hepatic TG and CHO accumulation, abnormal serum lipids and pathological changes (including microvesicualr steatosis, hepatocyte hydropic degeneration and inflammatory cell infiltration). Oral administration of bicyclol (150,300mg/kg/day×2 to 4 weeks) could significantly inhibit the increase of hepatic lipid contents, regulate the decreasing of serum TG level, and alleviated hepatic pathological damage mentioned above.
SREBP-lc is an important transcription factor that regulating fatty acid and TG synthesis. After feeding with HFD for 4 weeks, both precursor and matured protein of SREBP-1c were increased to 3.5 and 2 fold of control respectively. There was no alteration in mRNA expression of SREBP-lc. Pretreatment and treatment with bicyclol (150,300mg/kg) significantly reversed the elevation of precursor and matured SREBP-1c protein to normal level.
Acetyl-CoA carboxylase (ACC), fatty acid synthesis (FAS) and stearoyl-CoA desaturase (SCD) are key enzymes responsible for fatty acid synthesis and are regulated by SREBP-lc. The mRNA expression of hepatic ACC, FAS and SCD were elevated significantly after feeding with HFD for 4 weeks. The protein of ACC and FAS were also increased to 1.84 and 3.1 fold of control. Bicyclol showed protective effect on the indexes mentioned above to different extent.
Insulin induced genes (Insigs) are important factors participating in the activation process of SREBP-1c. There expression could result in the detention of SREBP-SCAP (SREBP cleavage-activating protein, SCAP) in Endoplasmic Reticulum (ER). The mRNA expression of rat hepatic Insig-1 was reduced significantly after feeding with HFD for 4 weeks, whereas Insig-2a mRNA expression showed no alteration. Pretreatment and treatment with bicyclol showed no effect on mRNA expression of Insig-1 and Insig-2a.
In summary, bicyclol had significant protective effect on HFD-induced fatty liver. Its mechanisms were related to the inhibition on SREBP-lc expression in protein level, thereby suppressing the transcription of enzymes responsible for fatty acid synthesis.
In conclusion, bicyclol had a notable protective effect on tetracycline-induced acute fatty liver in mice and HFD-induced chronic fatty liver in rat. It can not only significantly inhibit the elevation of serum transaminase and the accumulation of hepatic triglyceride and cholesterol, but also improve pathological changes, including microvesicualr steatosis, hepatocyte hydropic degeneration and inflammatory cell. The possible mechanisms included:
1) Inhibition of oxidative stress:attenuate lipid peroxidation, decrease the conpensable elevation of GSH content and SOD activity.
2) Attenuation on mitochondrial dysfunction:maintain mitochondrial membrane integrity and normal membrane potential, restore mitochondrial ATPase activity, increase MRC I and MRC IV activity.
3) Regulation on the expression of PPARa and its target genes:inhibit the decrease expression of PPARa, increase ACD, AOX, CYP2E1 and CYP4A at mRNA and enzyme activity level.
4) Inhibition SREBP-lc regulatory pathway:inhibit the precursor and matured protein of SREBP-1c, reduce the mRNA and protein expression of SREBP-lc target genes responsible for fatty acid synthesis, including ACC, FAS and SCD.
With the results mentioned above, the present study will provide valuable experimental evidences for further investigation on the hepatoprotective effect of bicyclol and the possibility of clinical application in the treatment of NAFLD.
双环醇(bicyclol)是我所研制的治疗慢性肝炎的国家一类新药。临床资料显示,双环醇在改善慢性乙肝病人临床症状和肝功能的同时,对乙肝病毒复制也有一定抑制作用,且停药后反跳率低,未发现明显不良反应。以往药理研究表明,双环醇对多种化学毒物、酒精等引起的急慢性肝损伤模型均具有较好的肝保护作用,其作用机制与其清除自由基、保护线粒体功能等密切相关。
本实验室既往研究表明,双环醇对实验性非酒精性脂肪肝有显著的保护作用,但对相关机制尚未进行深入研究。因此,本研究选用四环素和高脂饮食引起的两种非酒精性脂肪肝模型观察双环醇的保护作用,并对相关机制进行深入探讨,为其临床应用提供科学可靠的实验依据。
1.双环醇对四环素引起小鼠脂肪肝的保护作用及机制研究
单次腹腔注射四环素(tetracycline,200mg/kg)可引起小鼠急性脂肪肝,表现为血清丙氨酸氨基转移酶(alanine transaminase, ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase, AST)升高,肝脏甘油三酯(TG)及胆固醇(CHO)聚积,肝细胞小泡性脂肪变及水样变性等病理改变。双环醇预防给药(300mg/kg)可降低血清ALT及AST水平的升高、抑制甘油三酯及胆固醇的蓄积,同时改善上述肝脏组织病理学改变。
体内脂肪酸氧化主要发生在三个细胞器,即线粒体和过氧化酶体的β氧化以及微粒体ω氧化。四环素注射24h后小鼠肝脏线粒体β氧化速率显著下降,过氧化酶体(acetyl-coenzyme A oxidase, AOX)及微粒体(CYP2E1及CYP4A)脂肪酸氧化也受到显著抑制,表现为上述三个细胞器中四个限速酶(ACD、AOX、CYP2E1以及CYP4A)的氧化速率与对照组相比分别降低了34%、40%、58%和30%。线粒体长链脂酰辅酶A脱氢酶(long-chain acyl-CoA dehydrogenase, LCAD)、微粒体脂肪酸代谢酶CYP4A10及CYP4A12的mRNA表达也受到显著抑制。双环醇预防给药(300mg/kg)可显著提高线粒体乙酰辅酶A脱氢酶(acetyl CoA dehydrogenase,ACD)、过氧化酶体AOX、微粒体CYP2E1以及CYP4A的氧化速率,同时增加LCAD以及CYP4A10、CYP4A12的mRNA表达。过氧化酶体增殖活化受体(peroxisome proliferator-activated receptor a, PPARα)为调节脂肪酸代谢过程所需多种酶表达的转录因子。四环素注射24h后,小鼠肝脏PPARa mRNA表达显著下降,双环醇预防给药(300mg/kg)可在一定程度上提高该基因的表达。
线粒体损伤尤其是线粒体呼吸链(mitochondrial respiratory chain, MRC)功能异常在NAFLD的发病过程中发挥关键作用。四环素注射24h后,肝脏MRC I和Ⅳ活性与对照组相比分别降低42%、31%。双环醇预防给药(300mg/kg)可显著增加MRC I和MRC IV活性,使之恢复到接近正常水平。此外,注射四环素24h后,小鼠肝脏线粒体对罗丹明123(rhodamine123,R123)的摄取量明显少于对照组,对高钙浓度诱发肿胀的敏感性显著降低,提示线粒体发生了膜渗透性转换。双环醇预防给药(300mg/kg)可显著改善受损的线粒体功能,表现为线粒体对R123的摄取量以及高钙浓度引发的吸光度下降幅度均明显增加。ATPase是反映线粒体能量代谢状况的重要指标,注射四环素24h后,小鼠肝脏ATPase活性显著下降。双环醇预防给药(300mg/kg)能够显著提高受损的ATPase活性,使之接近正常水平。
线粒体损伤能够引起ROS生成增加,造成肝组织氧化损伤。氧化应激是启动及维持NASH的重要病理因素。注射四环素24h后,小鼠肝脏MDA显著升高,而GSH含量及SOD活性也出现代偿性升高。双环醇预防给药(300mg/kg)能够改善MDA升高,使GSH含量及SOD活性恢复至接近正常水平。固醇调控元件结合蛋白(Sterol Regulatory Element-Binding Protein, SREBP)-lc是调控肝脏脂质合成代谢的重要转录因子。但四环素腹腔注射24h后,小鼠肝脏SREBP-1c及其靶基因脂肪酸合成酶(Fatty acid synthesis, FAS)及固醇辅酶A脱氢酶(stearoyl-CoA desaturase, SCD)的mRNA表达未见显著变化。由此可见,单次注射四环素能引起小鼠出现明显脂肪肝,损伤机制与抑制PPARα及其靶基因、氧化损伤有关。双环醇对四环素引起脂肪肝的保护作用与其调控PPARa转导通路紊乱及抑制氧化损伤密切相关。
2.双环醇对高脂饮食引起大鼠脂肪肝的保护作用及机制研究
高脂饮食喂养四周后,大鼠出现明显脂肪肝,表现为肝脏脂质聚积(肝脏TG、CHO以及FFA含量明显增加),血脂异常(出现高胆固醇和低甘油三酯血症),肝细胞损伤及小泡性脂肪变、水样变性以及炎性细胞浸润等病理改变。双环醇预防给药或治疗给药(150,300mg/kg)可有效地抑制HFD引起的肝脏脂质堆积、血中TG水平降低,并且改善上述肝脏病理学损伤。
SREBP-1c是调控肝脏脂肪酸及甘油三酯合成过程中诸多代谢酶表达的重要转录因子。高脂饮食喂养4周后,大鼠肝脏SREBP-1c前体蛋白及成熟型nSREBP-1蛋白表达显著升高,分别为对照组的3.5倍及2倍,双环醇预防及治疗给药(300mg／kg)均能明显降低SREBP-1c前体蛋白及成熟型nSREBP-1c蛋白表达,使之接近正常水平。
乙酰辅酶A羧化酶(acetyl-CoA carboxylase, ACC)、FAS以及SCD是脂肪酸代谢过程中的关键酶,其转录过程受SREBP-1c的调控。高脂饮食喂养4周后,大鼠肝脏ACC、FAS以及SCD的mRNA表达均显著升高,ACC及FAS的蛋白表达也显著增加,分别为对照组的1.84倍和3.1倍。双环醇对ACC、FAS以及SCD mRNA表达均有不同程度的抑制作用,并能显著抑制ACC及FAS的蛋白表达。
胰岛素诱导基因(Insulin induced genes, Insigs)是调控SREBP-1活化的重要因子,能造成SREBP-SCAP(SREBP裂解活化蛋白,SREBP cleavage-activating protein, SCAP)在内质网(ER)的滞留。高脂饮食喂养4周后,大鼠肝脏Insig-1 mRNA表达显著下降,Insig-2a mRNA表达无显著变化,双环醇预防及治疗给药(300mg/kg)对Insig-1和Insig-2a的mRNA的表达均无显著影响。
由此可见,高脂饮食喂养4周能够引起大鼠脂肪肝发生,其损伤机制与高脂饮食增加SREBP-1c的翻译及活化,从而增加脂质合成基因的转录有关。双环醇对高脂饮食引起大鼠脂肪肝的保护作用与其从翻译水平抑制SREBP-1c的表达进而调控脂质合成基因的转录相关。
综上所述,双环醇对四环素及高脂饮食引起的急慢性实验性脂肪肝均有显著的保护作用,不仅可以降低血清转氨酶的升高、抑制肝脏甘油三酯及胆固醇蓄积,还可显著改善肝脏小泡性脂肪变、水样变性及炎性损伤等病理改变。其保护作用机制可归纳为：
1)抑制氧化应激：减轻脂质过氧化,使GSH含量及SOD活性的代偿性升高恢复至接近正常水平。
2)减轻线粒体损伤：维持线粒体膜完整性和正常膜电位,恢复线粒体ATPase活性,增加MRCI和Ⅳ活性。
3)调控PPARa及其靶基因的表达：增加降低的PPARa表达,增加ACD、AOX、CYP2E1以及CYP4A的mRNA表达及活性。
4)调控SREBP-1c转导通路：抑制SREBP-1c的前体蛋白及成熟型蛋白的表达,抑制负责脂肪酸合成的靶基因ACC、FAS以及SCD的mRNA及蛋白表达。
上述研究为进一步了解双环醇的肝保护作用特点以及临床治疗NAFLD提供了有参考价值的实验依据。
Nonalcoholic Fatty Liver Disease (NAFLD) is a syndrome with liver histopathologic findings, which resembles alcoholic hepatitis in patients who lack a history of significant alcohol comsumption (<140 g ethanol/week). The spectrum of histologic abnormalities of NAFLD ranges over simple steatosis, nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and even hepatocellular carcinoma or liver failure. The prevalence of NAFLD in the general population is estimated to be 14% and 24% and has markedly increased recently. To date, the only effective treatment of NAFLD is caloric restriction, which is difficult to achieve for most NAFLD patients, it is of great necessity to find the effective treatment of NAFLD, including drug therapy.
Bicyclol is a novel anti-hepatitis drug for the treatment of chronic viral hepatitis. It has been shown to improve the clinical symptoms and inhibit virus replication in HBV patients, featured by low rebounding rate, few adverse effects and convenient usage. Previous pharmacological studies indicated that bicyclol had hepatoprotective action against acute and chronic experimental liver injury caused by certain toxins and alcohol. The hepatoprotective mechanisms of bicyclol were related with the clearance of reactive oxygen species, regulation of mitochondrial dysfunction, etc.
Previous study in our laboratory has also demostrated its role in protecting experimental animals from steatosis (induced by high fat diet, tetracycline, carbon tetrachloride and ethionine), however, in-depth investigation on related mechanisms are still needed. Therefore, acute and chronic experimental steatosis models were established in the present study to observe the protective effect of bicyclol and related mechanisms, so as to provide experimental evidences for its clinical application.
Part I:Protective effect of bicyclol on tetracycline-induced fatty liver in mice and related mechanisms
Single dose of tetracycline injection caused hepatic steatosis as evidenced by the elevation of serum ALT and AST, accumulation of hepatic TG and CHO, and morphologic changes (small lipid droplets and hydropic degeneration of hepatocytes), which reflected early alterations of NAFLD in mice. Bicyclol treatment significantly protected against tetracycline-induced fatty liver by reducing elevated serum ALT&AST levels and alleviating hepatic lipid accumulation biochemically and pathologically.
The oxidation of fatty acids occurs in three subcellular organelles. Mitochondria and peroxisome degrade fatty acid byβ-oxidation, while smooth endoplasmic reticulum metabolizes fatty acid viaω-oxidation by CYP2E1 and CYP4A subfamily. The mitochondrialβ-oxidation was found to be inhibited by 34% after tetracycline injection. In addition, tetracycline also reduced the activities of enzymes responsible for fatty acid oxidation in peroxisome (AOX) and microsome (CYP2E1, CYP4A) were also inhibited by 40%,58% and 30%, respectively in mice. Furthermore, mRNA levels of LCAD, CYP4A10 and CYP4A12 also decreased. Administration with bicyclol ameliorated the decrease of fatty acid oxidation at both enzyme activity and mRNA levels.
Peroxisome proliferators-activated receptor a (PPARa) is a kind of transcription factor regulating a serial of enzymes responsible for fatty acid oxidation. The mRNA expression of hepatic PPARa was inhibited after tetracycline injection. Bicyclol (300mg/kg) showed protective effect against tetracycline induced decrease of PPARa mRNA expression.
Mitochondrial dysfunction, particularly mitochondrial respiratory chain (MRC) deficiency, plays a key role in the physiopathology of NAFLD. Hepatic MRC I andⅣwere significantly inhibited by 42% and 31% respectively after tetracycline injection. Pretreatment of bicyclol (300mg/kg) reversed the hepatic MRC complexI&IV activity to normal levels. Besides, the isolated mitochondria had a rapid onset of MPT (membrane permeability transition, MPT) by decreased uptake of cationic dye Rodamine 123 and lowered sensitivity to Ca2+after tetracycline injection. Bicyclol remarkably improved tetracycline-induced alteration mentioned above. ATPase is a kind of marker reflecting the condition of mitochondrial energy metabolism. Pretreatment with bicyclol (300mg/kg) significantly reversed the injured activity of ATPase to normal level.
Mitochondrial dysfunction accelerates the production of ROS and induced hepatic oxidative injury. Oxidative stress was another pathogenic factor in initiating and maintaining of NASH. Hepatic MDA content was elevated 24h after tetracycline injection. GSH content and SOD activity were also found increased compensably. Pretreatment with bicyclol (300mg/kg) significantly inhibited the elevation of MDA, decreased GSH content and SOD activity.
Sterol regulatory element-binding protein (SREBP)-lc is a key transcription factor regulating lipid synthesis. No obvious alteration of mRNA expression of SREBP-1c and its target genes SCD, FAS was observed after tetracycline injection.
In conclusion, obvious hepatic steatosis was observed after a single dose of tetracycline in mice. The mechanism was found to be partly due to the inhibition of PPARa and its regulated genes, and mitochondrial dysfunction. Bicyclol showed significant protective effect on hepatic steatosis induced by tetracycline through the modulation of PPARa pathway and protection of mitochondrial injury.
Part II:Effect of bicyclol on high fat diet (HFD) induced fatty liver in rat and related mechanisms
After 4 week feeding with HFD, rat had severe fatty liver as evidenced by hepatic TG and CHO accumulation, abnormal serum lipids and pathological changes (including microvesicualr steatosis, hepatocyte hydropic degeneration and inflammatory cell infiltration). Oral administration of bicyclol (150,300mg/kg/day×2 to 4 weeks) could significantly inhibit the increase of hepatic lipid contents, regulate the decreasing of serum TG level, and alleviated hepatic pathological damage mentioned above.
SREBP-lc is an important transcription factor that regulating fatty acid and TG synthesis. After feeding with HFD for 4 weeks, both precursor and matured protein of SREBP-1c were increased to 3.5 and 2 fold of control respectively. There was no alteration in mRNA expression of SREBP-lc. Pretreatment and treatment with bicyclol (150,300mg/kg) significantly reversed the elevation of precursor and matured SREBP-1c protein to normal level.
Acetyl-CoA carboxylase (ACC), fatty acid synthesis (FAS) and stearoyl-CoA desaturase (SCD) are key enzymes responsible for fatty acid synthesis and are regulated by SREBP-lc. The mRNA expression of hepatic ACC, FAS and SCD were elevated significantly after feeding with HFD for 4 weeks. The protein of ACC and FAS were also increased to 1.84 and 3.1 fold of control. Bicyclol showed protective effect on the indexes mentioned above to different extent.
Insulin induced genes (Insigs) are important factors participating in the activation process of SREBP-1c. There expression could result in the detention of SREBP-SCAP (SREBP cleavage-activating protein, SCAP) in Endoplasmic Reticulum (ER). The mRNA expression of rat hepatic Insig-1 was reduced significantly after feeding with HFD for 4 weeks, whereas Insig-2a mRNA expression showed no alteration. Pretreatment and treatment with bicyclol showed no effect on mRNA expression of Insig-1 and Insig-2a.
In summary, bicyclol had significant protective effect on HFD-induced fatty liver. Its mechanisms were related to the inhibition on SREBP-lc expression in protein level, thereby suppressing the transcription of enzymes responsible for fatty acid synthesis.
In conclusion, bicyclol had a notable protective effect on tetracycline-induced acute fatty liver in mice and HFD-induced chronic fatty liver in rat. It can not only significantly inhibit the elevation of serum transaminase and the accumulation of hepatic triglyceride and cholesterol, but also improve pathological changes, including microvesicualr steatosis, hepatocyte hydropic degeneration and inflammatory cell. The possible mechanisms included:
1) Inhibition of oxidative stress:attenuate lipid peroxidation, decrease the conpensable elevation of GSH content and SOD activity.
2) Attenuation on mitochondrial dysfunction:maintain mitochondrial membrane integrity and normal membrane potential, restore mitochondrial ATPase activity, increase MRC I and MRC IV activity.
3) Regulation on the expression of PPARa and its target genes:inhibit the decrease expression of PPARa, increase ACD, AOX, CYP2E1 and CYP4A at mRNA and enzyme activity level.
4) Inhibition SREBP-lc regulatory pathway:inhibit the precursor and matured protein of SREBP-1c, reduce the mRNA and protein expression of SREBP-lc target genes responsible for fatty acid synthesis, including ACC, FAS and SCD.
With the results mentioned above, the present study will provide valuable experimental evidences for further investigation on the hepatoprotective effect of bicyclol and the possibility of clinical application in the treatment of NAFLD.
引文
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