槐果碱对酒精性肝病大鼠的保护作用及对细胞凋亡的影响
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摘要
目的观察槐果碱对酒精性肝病大鼠的保护作用及对细胞凋亡的影响。方法将40只健康雄性清洁级Wistar大鼠按照随机数字法分为正常组、模型组、槐果碱低剂量组和槐果碱高剂量组,每组10只。除正常组外,其余3组均给予酒精灌胃12周;槐果碱低剂量组和槐果碱高剂量组分别于灌胃第9周起同时给予槐果碱20 mg/(kg·d)和40 mg/(kg·d)背部皮下注射,共4周;正常组灌胃生理盐水及皮下注射生理盐水。实验结束后称取各组大鼠体质量、肝质量,计算肝指数,取血检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、三酰甘油(TG)和总胆固醇(TC)水平,HE染色进行肝脏组织病理学观察,用流式细胞技术检测肝脏细胞凋亡指数,分别用RT-PCR和Western blot法检测肝脏组织中半胱氨酸天冬氨酸蛋白酶(Caspase)-3、Caspase-8、Caspase-12 mRNA和蛋白表达水平。结果模型组大鼠体质量明显低于正常组(P <0. 05),肝指数和血清ALT、AST、TC、TG水平均明显高于正常组(P均<0. 05);槐果碱低剂量组和槐果碱高剂量组大鼠体质量均明显高于模型组(P均<0. 05),肝质量、肝指数和血清ALT、AST、TC水平均明显低于模型组(P均<0. 05),且槐果碱高剂量组升高及降低更为明显(P均<0. 05)。HE染色显示槐果碱低剂量组和槐果碱高剂量组肝脏细胞损伤明显减轻,并且呈剂量依赖性。模型组肝细胞凋亡指数和肝组织中Caspase-3、Caspase-8、Caspase-12 mRNA和蛋白表达水平均明显高于正常组(P均<0. 05),槐果碱低剂量组和槐果碱高剂量组均明显低于模型组(P均<0. 05),且槐果碱高剂量组均明显低于槐果碱低剂量组(P均<0. 05)。结论槐果碱具有明显保护酒精性肝病大鼠肝脏损伤的作用,可减少肝脏细胞凋亡,可能与抑制线粒体和内质网凋亡有关。
Objective It is to observe the protective effect of sophocarpine in rats with alcoholic liver disease and its impact on apoptosis. Methods Forty healthy male clean grade Wistar rats were randomly divided into normal group,model group,low dose of sophocarpine group and high dose group according to random number method,each group had 10 rats.Except the normal group,the other three groups were treated with alcohol by gavage for 12 weeks; the low dose and high dose of sophocarpine groups were treated with sophocarpine 20 mg/( kg·d) and 40 mg/( kg·d) from the 9th week of intragastric administration by subcutaneous injection for 4 weeks; the normal group was given normal saline and subcutaneous injection of normal saline. At the end of the experiment,the body weight and liver mass of the rats in each group were weighed,the liver index was calculated,and the levels of alanine aminotransferase( ALT),aspartate aminotransferase( AST),triglyceride( TG) and Total cholesterol( TC) were detected,the histopathological change of liver tissue was observed after HE staining,liver cell apoptosis index was detected by flow cytometry,the expression levels of mRNA and protein of Caspase-3,Caspase-8,Caspase-12 in liver tissue was detected by RT-PCR and Western blot respectively. Results The body weight of the model group was significantly lower while the liver index and serum levels of ALT,AST,TC,TG were significantly higher than those in the normal group( P < 0. 05). The body weight of sophocarpine groups was significantly higher while the liver index and serum levels of ALT,AST,TC,TG were significantly lower than those in the model group( P < 0. 05),the increase and decrease of the high-dose group were more obvious( P < 0. 05). HE staining showed that liver cell damage was significantly improved in the low-dose and high-dose groups of sophocarpine in a dose-dependent manner. The hepatocyte apoptosis index and the expression levels of Caspase-3,Caspase-8 and Caspase-12 mRNA and protein in the model group were significantly higher than those in the model group( all P < 0. 05),while these indexes in the low-dose and high-dose groups of sophocarpine were significantly lower than those in the model group( P < 0. 05),and the improvements in the high-dose group were the best P < 0. 05). Conclusion Sophocarpine has obvious protective effect in rats with alcoholic liver disease,which may alleviate the apoptosis of liver cells and its mechanism may be related to the inhibition of mitochondrial and endoplasmic reticulum apoptosis.
Objective It is to observe the protective effect of sophocarpine in rats with alcoholic liver disease and its impact on apoptosis. Methods Forty healthy male clean grade Wistar rats were randomly divided into normal group,model group,low dose of sophocarpine group and high dose group according to random number method,each group had 10 rats.Except the normal group,the other three groups were treated with alcohol by gavage for 12 weeks; the low dose and high dose of sophocarpine groups were treated with sophocarpine 20 mg/( kg·d) and 40 mg/( kg·d) from the 9th week of intragastric administration by subcutaneous injection for 4 weeks; the normal group was given normal saline and subcutaneous injection of normal saline. At the end of the experiment,the body weight and liver mass of the rats in each group were weighed,the liver index was calculated,and the levels of alanine aminotransferase( ALT),aspartate aminotransferase( AST),triglyceride( TG) and Total cholesterol( TC) were detected,the histopathological change of liver tissue was observed after HE staining,liver cell apoptosis index was detected by flow cytometry,the expression levels of mRNA and protein of Caspase-3,Caspase-8,Caspase-12 in liver tissue was detected by RT-PCR and Western blot respectively. Results The body weight of the model group was significantly lower while the liver index and serum levels of ALT,AST,TC,TG were significantly higher than those in the normal group( P < 0. 05). The body weight of sophocarpine groups was significantly higher while the liver index and serum levels of ALT,AST,TC,TG were significantly lower than those in the model group( P < 0. 05),the increase and decrease of the high-dose group were more obvious( P < 0. 05). HE staining showed that liver cell damage was significantly improved in the low-dose and high-dose groups of sophocarpine in a dose-dependent manner. The hepatocyte apoptosis index and the expression levels of Caspase-3,Caspase-8 and Caspase-12 mRNA and protein in the model group were significantly higher than those in the model group( all P < 0. 05),while these indexes in the low-dose and high-dose groups of sophocarpine were significantly lower than those in the model group( P < 0. 05),and the improvements in the high-dose group were the best P < 0. 05). Conclusion Sophocarpine has obvious protective effect in rats with alcoholic liver disease,which may alleviate the apoptosis of liver cells and its mechanism may be related to the inhibition of mitochondrial and endoplasmic reticulum apoptosis.
引文
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[11]Chen R,Liu S,Piao F,et al.2,5-hexanedione induced apoptosis in mesenchymal stem cells from rat bone marrow via mitochondria-dependent caspase-3 pathway[J].Ind Health,2015,53(3):222-235
[2]Huang A,Chang B,Sun Y,et al.Disease spectrum of alcoholic liver disease in Beijing 302 Hospital from 2002 to2013:A large tertiary referral hospital experience from7422 patients[J].Medicine(Baltimore),2017,96(7):e6163
[3]Peng Z,Chen L,Nussler AK,et al.Current sights for mechanisms of deoxynivalenol-induced hepatotoxicity and prospective views for future scientific research:A mini review[J].J Appl Toxicol,2017,37(5):518-529
[4]Chen H,Yang X,Feng Z,et al.Prognostic value of Caspase-3 expression in cancers of digestive tract:a meta-analysis and systematic review[J].Int J Clin Exp Med,2015,8(7):10225-10234
[5]Wang J,Li P,Jiang Z,et al.Diagnostic value of alcoholic liver disease(ALD)/nonalcoholic fatty liver disease(NAFLD)index combined with gamma-glutamyl transferase in differentiating ALD and NAFLD[J].Korean J Intern Med,2016,31(3):479-487
[6]黄银秋,周厚琴,张璐,等.槐果碱对小鼠免疫性肝损伤保护作用的研究[J].中国医院用药评价与分析,2017,17(3):297-299;303
[7]乔春萍,施建平,叶慧英,等.槐果碱对大鼠酒精性肝病的作用及机制[J].中华消化杂志,2012,32(8):543-548
[8]Petrasek J,Iracheta-Vellve A,Csak T,et al.STING-IRF3pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease[J].Proc Natl Acad Sci USA,2013,110(41):16544-16549
[9]Lim SW,Loh HS,Ting KN,et al.Antiproliferation and induction of caspase-8-dependent mitochondria-mediated apoptosis by beta-tocotrienol in human lung and brain cancer cell lines[J].Biomed Pharmacother,2014,68(8):1105-1115
[10]Zhang Q,Liu J,Chen S,et al.Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress[J].Apoptosis,2016,21(4):432-442
[11]Chen R,Liu S,Piao F,et al.2,5-hexanedione induced apoptosis in mesenchymal stem cells from rat bone marrow via mitochondria-dependent caspase-3 pathway[J].Ind Health,2015,53(3):222-235