调肝理脾法对大鼠非酒精性脂肪性肝病脂质代谢和自噬相关蛋白-1表达的影响
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摘要
目的:观察调肝理脾法(MRLS)对实验性大鼠非酒精性脂肪肝病(NAFLD)的改善作用及对大鼠肝组织自噬相关蛋白Beclin1表达的影响,探讨调肝理脾法治疗非酒精性脂肪肝病的可能作用机制。方法:SD雄性大鼠(清洁级) 30只,随机分为对照组、模型组、调肝理脾组(各10只)。模型组、调肝理脾组大鼠皆予以高脂饮食共12周。调肝理脾法组大鼠造模同时给予调肝理脾方2ml/100g/d灌胃(相当于成人剂量的5倍),对照组大鼠给予正常饮食(普通饲料)及等量生理盐水灌胃。第12周末处死动物后,分别采集大鼠血液、肝脏标本,检测各组大鼠血清ALT、AST、TG、TC、HDL-C、LDL-C水平,通过HE染色观察肝脏脂肪变性情况,免疫组化法检测Beclin1在肝组织中表达的变化。结果:(1)HE染色提示模型组大鼠肝细胞脂肪变性程度和炎症活动度计分与正常对照组相比,差异有统计学意义(P <0. 001);调肝理脾法组脂肪变性程度和炎症活动度计分明显减轻,与模型组相比差异有统计学意义(P <0. 01)。(2)与对照组比较,模型组大鼠血清ALT、AST、TG、TC、HDL-C、LDL-C的水平均升高,差异有统计学意义(P <0.001)。与模型组比较,除HDL-C外,调肝理脾法组大鼠血清ALT、AST、TG、TC、LDL-C的水平均降低,差异有统计学意义(P <0.05)。(3)与对照组比较,模型组大鼠肝组织Beclin1蛋白表达水平略增高,差异无统计学意义(χ2=1. 250,P> 0. 05);与模型组比较,调肝理脾法组肝组织Beclin1蛋白表达水平明显增高,差异有统计学意义(χ2=6. 121,P <0. 05)。结论:调肝理脾法对实验性大鼠非酒精性脂肪肝病的肝功能、血脂等指标有很好的改善作用,其作用机制可能与升高大鼠肝组织内的自噬水平有关。
Objective: To observe the effect of method of regulating liver and spleen on lipid metabolism and the expression of Beclin1 in non-alcoholic fatty liver disease in rats,and to explore the impossible mechanism. Methods: Thirty male healthy SPF SD rats were randomized into the control group,high-fat group and MRLS group(10 mice in each group); the rats of high-fat group and MRLS group were fed with high fat diets for 12 weeks,and the MRLS group being fed with high-fat diet was applied by the method of regulating liver and spleen(2 ml/100 g/d) at the same time. The rats in control group was fed with standard diets and saline of the same volume was perfused intragastricly in the meantime. At the end of 12 th week,all the rats were sacrificed and we collected the blood samples and liver tissues from the rats immediately. The fatty degeneration,the serum levels of ALT,AST,TG,TC,HDL-C,LDL-C,,and the expression of Beclin1 were detected by HE stain,automatic biochemistry analyzers and immunohistochemistry respectively. Results:(1)The HE stain showed severe hepatocellular fatty degeneration in the high-fat group,and partly hepatocellular fatty degeneration in the MRLS group. The degree of fatty degeneration in the MRLS group was significantly lower than that in the high-fat group(P < 0. 05). There was no fatty degeneration in the control group.(2)Compared with those of the control group,beyond HDL-C increased slightly,the serum ALT,AST,TG,TC,LDL-C levels of the high-fat group increased significantly(P < 0. 05); Compared with those of the high-fat group,beyond HDL-C increased slightly,the serum ALT,AST,TG,TC,LDL-C levels of the MRLS group reduced significantly(P < 0. 05).(3)Compared with those of the control group,the expression of Beclin1 in high-fat group increased slightly,and there was no significant difference(P > 0. 05); Compared with those of the high-fat group,the expression of Beclin1 in the MRLS group increased significantly(P < 0. 05). Conclusion: The method of regulating liver and spleen had a good improvement on liver function and blood lipid in rats,and the mechanism was possibly correlated with the increased expression levels of autophagy-associated proteins in liver of rats.
Objective: To observe the effect of method of regulating liver and spleen on lipid metabolism and the expression of Beclin1 in non-alcoholic fatty liver disease in rats,and to explore the impossible mechanism. Methods: Thirty male healthy SPF SD rats were randomized into the control group,high-fat group and MRLS group(10 mice in each group); the rats of high-fat group and MRLS group were fed with high fat diets for 12 weeks,and the MRLS group being fed with high-fat diet was applied by the method of regulating liver and spleen(2 ml/100 g/d) at the same time. The rats in control group was fed with standard diets and saline of the same volume was perfused intragastricly in the meantime. At the end of 12 th week,all the rats were sacrificed and we collected the blood samples and liver tissues from the rats immediately. The fatty degeneration,the serum levels of ALT,AST,TG,TC,HDL-C,LDL-C,,and the expression of Beclin1 were detected by HE stain,automatic biochemistry analyzers and immunohistochemistry respectively. Results:(1)The HE stain showed severe hepatocellular fatty degeneration in the high-fat group,and partly hepatocellular fatty degeneration in the MRLS group. The degree of fatty degeneration in the MRLS group was significantly lower than that in the high-fat group(P < 0. 05). There was no fatty degeneration in the control group.(2)Compared with those of the control group,beyond HDL-C increased slightly,the serum ALT,AST,TG,TC,LDL-C levels of the high-fat group increased significantly(P < 0. 05); Compared with those of the high-fat group,beyond HDL-C increased slightly,the serum ALT,AST,TG,TC,LDL-C levels of the MRLS group reduced significantly(P < 0. 05).(3)Compared with those of the control group,the expression of Beclin1 in high-fat group increased slightly,and there was no significant difference(P > 0. 05); Compared with those of the high-fat group,the expression of Beclin1 in the MRLS group increased significantly(P < 0. 05). Conclusion: The method of regulating liver and spleen had a good improvement on liver function and blood lipid in rats,and the mechanism was possibly correlated with the increased expression levels of autophagy-associated proteins in liver of rats.
引文
[1]周滔,张声生,郁强.调肝理脾法治疗非酒精性脂肪肝的临床队列研究[J].北京中医药[J],2013,32(6):403-405.
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[8] KIM YC,GUAN KL. m TOR:a pharmacologic target for autophagy regulation[J]. J Clin Invest.,2015,125(1):25-32.
[9] WANG EY,BIALA AK,GORDON JW,et al. Autophagy in the heart:too much of a good thing?[J]. J Cardiovasc Pharmacol,2012,60(2):110-117.
[10] MIZUSHIMA N,KOMATSU M. Autophagy:renovation of cells and tissues[J]. Cell,2011,147(4):728-741.
[11] AITA VM,LIANG XH,MURTY VV,et al. Cloning and genomic organization of beclin 1,a candidate tumor suppressor gene on chromosome 17q21[J]. Genomics,1999,59(1):59-65.
[12] DJAVAHERI-MERGNY M,MAIURI MC,KROEMER G. Cross talk between apoptosis and autophagy by caspase-mediated cleavage of Beclin 1[J]. Oncogene,2010,29(12):1717-1719.
[13] ROBINET P,RITCHEY B,SMITH JD. Physiological difference in autophagic flux in macrophages from 2 mouse strains regulates cholesterol ester metabolism[J]. Arterioscler Thromb Vasc Biol,2013,33(5):903-910.
[14] LIU K,CZAJA MJ. Regulation of lipid stores and metabolism by lipophagy[J]. Cell Death Differ,2013,20(1):3-11.
[15] PapákováZ,DaňkováH,PáleníkováE,et al. Effect of short-andlong-term high-fat feeding on autophagy flux and lysosomal activity in rat liver[J]. Physiol Res,2012,61 Suppl 2:S67-76.,
[16] YANG L,LI P,FU S,et al. Defective hepatic antophagy in obesity promotes ER stress and causes insulin resistance[J]. Cell Metab,2010,11(6):467-478.
[17] KIM Kh,LEE MS. Autophagy as a crosstalk mediator of metabolic organs in regulation of energy metabolism[J]. Rev End ocr Metab Disord,2014,15(1):11-20.
[18] MARTINEZ-VICENTE M,TALLOCZY Z,WONG E,et al. Cargo recognition failure is responsible for inefficient autophagy in Huntington’s disease[J]. Nat Neurosci,2010,13(5):567-576.
[19]闫蓉,牛春燕,于璐,等.非酒精性脂肪性肝病细胞模型中自噬与脂质代谢的相互调节[J].临床肝胆病杂志,2017,33(10):1981-1986.
[20]黄丽琼,黄贵华,纪云西,等.细胞自噬与中医“阴阳”及“气”理论相关性探讨[J].辽宁中医杂志,2014,41(6):1147-1149.
[21]黎颖贤,敖海清.从微观角度探究细胞自噬与肝主疏泄功能相关性[J].辽宁中医药大学学报,2016,18(2):45-47.
[22]姜文秀,王英超,常诚.中医药调节细胞自噬刍议[J].中医杂志,2017,58(18):1566-1568.
[2]周滔,张声生,陈誩. 120例非酒精性脂肪肝单元证特点分析与健脾清肝方临床疗效[J].中西医结合肝病杂志,2009,19(4):209-210.
[3] SINGH R,KAUSHIK S,WANG Y,et al. Autophagy regulates lipid m etabolism[J]. Nature,2009,458(7242):1131-1135.
[4] WU X,ZHANG L,GURLEY E,et al. Prevention of free fatty acidinduced hepatic lipotoxicity by 18beta-glycytthetinic acid through lysosomal and mitochondrial pathways[J]. Hepatology,2008,47(6):1905-1915.
[5] DIEHL A,GOODMAN Z,ISHAK K. Alcohollike liver disease in nonalcoholics. A clinical and histologic comparison with alcohol-induced liver injury[J]. Gastroenterology,1998,95(4):1056-1062.
[6]王泰龄,刘霞,周元平等.慢性肝炎炎症活动度及纤维化程度计分方案[J].中华肝脏杂志,1998,6(4):195-197.
[7] CHOI J,JUNG W,KOO JS. Expression of autophagy-related markers Beclin1,light chain 3A,light chain 3B and P62 according to the molecular subtype of breast cancer[J]. Histopsthology,2013,62(2):275-286.
[8] KIM YC,GUAN KL. m TOR:a pharmacologic target for autophagy regulation[J]. J Clin Invest.,2015,125(1):25-32.
[9] WANG EY,BIALA AK,GORDON JW,et al. Autophagy in the heart:too much of a good thing?[J]. J Cardiovasc Pharmacol,2012,60(2):110-117.
[10] MIZUSHIMA N,KOMATSU M. Autophagy:renovation of cells and tissues[J]. Cell,2011,147(4):728-741.
[11] AITA VM,LIANG XH,MURTY VV,et al. Cloning and genomic organization of beclin 1,a candidate tumor suppressor gene on chromosome 17q21[J]. Genomics,1999,59(1):59-65.
[12] DJAVAHERI-MERGNY M,MAIURI MC,KROEMER G. Cross talk between apoptosis and autophagy by caspase-mediated cleavage of Beclin 1[J]. Oncogene,2010,29(12):1717-1719.
[13] ROBINET P,RITCHEY B,SMITH JD. Physiological difference in autophagic flux in macrophages from 2 mouse strains regulates cholesterol ester metabolism[J]. Arterioscler Thromb Vasc Biol,2013,33(5):903-910.
[14] LIU K,CZAJA MJ. Regulation of lipid stores and metabolism by lipophagy[J]. Cell Death Differ,2013,20(1):3-11.
[15] PapákováZ,DaňkováH,PáleníkováE,et al. Effect of short-andlong-term high-fat feeding on autophagy flux and lysosomal activity in rat liver[J]. Physiol Res,2012,61 Suppl 2:S67-76.,
[16] YANG L,LI P,FU S,et al. Defective hepatic antophagy in obesity promotes ER stress and causes insulin resistance[J]. Cell Metab,2010,11(6):467-478.
[17] KIM Kh,LEE MS. Autophagy as a crosstalk mediator of metabolic organs in regulation of energy metabolism[J]. Rev End ocr Metab Disord,2014,15(1):11-20.
[18] MARTINEZ-VICENTE M,TALLOCZY Z,WONG E,et al. Cargo recognition failure is responsible for inefficient autophagy in Huntington’s disease[J]. Nat Neurosci,2010,13(5):567-576.
[19]闫蓉,牛春燕,于璐,等.非酒精性脂肪性肝病细胞模型中自噬与脂质代谢的相互调节[J].临床肝胆病杂志,2017,33(10):1981-1986.
[20]黄丽琼,黄贵华,纪云西,等.细胞自噬与中医“阴阳”及“气”理论相关性探讨[J].辽宁中医杂志,2014,41(6):1147-1149.
[21]黎颖贤,敖海清.从微观角度探究细胞自噬与肝主疏泄功能相关性[J].辽宁中医药大学学报,2016,18(2):45-47.
[22]姜文秀,王英超,常诚.中医药调节细胞自噬刍议[J].中医杂志,2017,58(18):1566-1568.