六味地黄丸基于SIRT6/NF-κB信号通路对糖尿病伴肝损伤的保护作用
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摘要
目的:从炎症角度探讨六味地黄丸对糖尿病伴肝损伤的保护机制。方法:选取自发性2型糖尿病动物模型db/db雄性小鼠18只,按空腹血糖(FBG)随机分模型组、六味地黄丸组(9. 75 g·kg-1·d-1,1次/d)、白藜芦醇组(42. 6 mg·kg-1·d-1,1次/d); 12只同窝野生型db/m小鼠,按FBG随机分正常组和六味地黄丸对照组(9. 75 g·kg-1·d-1,1次/d),正常组和模型组等量蒸馏水灌胃,各组给药16周后内眦静脉取血后处死小鼠,检测FBG,甘油三酯(TG)和丙氨酸氨基转移酶(ALT)等变化,苏木素-伊红(HE)染色观察肝组织病理改变,蛋白免疫印迹法(Western blot)检测肝组织沉默信息调节因子6(SIRT6),核转录因子-κB p65(NF-κB p65),单核细胞趋化蛋白-1(MCP-1),血管细胞黏附因子-1(VCAM-1)和细胞间黏附因子-1(ICAM-1)蛋白表达。结果:与正常组比较,模型组小鼠FBG,TG和ALT显著升高(P <0. 01),NF-κB p65,MCP-1,VCAM-1和ICAM-1蛋白表达显著升高(P <0. 01),SIRT6蛋白表达显著降低(P <0. 01);与模型组比较,六味地黄丸组和白藜芦醇组血FBG,TG显著降低(P <0. 01),NF-κB p65,MCP-1和VCAM-1蛋白表达明显降低(P <0. 05,P <0. 01),SIRT6蛋白表达明显升高(P <0. 05),改善小鼠肝组织的肝细胞脂肪变性及炎细胞浸润。结论:六味地黄丸对糖尿病小鼠伴肝脏损伤有保护作用,其机制与调节血脂代谢,抑制炎症反应有关。
Objective: To investigate the protective mechanism of Liuwei Dihuangwan on diabetes mellitus with liver injury in terms of inflammation. Method: The 18 db/db mice were selected from animal model of spontaneous type 2 diabetes, mice were randomly divided into model group, Liuwei Dihuangwan group( 9. 75 g·kg-1·d-1,once a day),resveratrol group( 42. 6 mg·kg-1·d-1,once a day) based on fasting bloodglucose( FBG). The 12 litter wild db/m mice were randomly divided into normal group,Liuwei Dihuangwan group( 9. 75 g·kg-1·d-1,once a day). The normal group and the model group were given the same amount of distilled water by gavage. The mice in each group were treated for 16 weeks. FBG,triglyceride( TG) and alanine aminotransferase( ALT) were examined. Histopathological changes were observed by liver biopsy hematoxylin-eosin( HE) staining. Silent information regulator 6( SIRT6),nuclear factor-kappaB p65( NF-κB p65),monocyte chemoattractant protein-1( MCP-1),vascular cell adhesion molecule-1( VCAM-1) and intercellular adhesion molecule-1( ICAM-1) in the liver tissue were detected by Western blot. Result: Compared with normal group,FBG,TG and ALT in model group increased significantly( P < 0. 01),protein expression levels of NF-κB p65,MCP-1,VCAM-1 and ICAM-1 in model group were significantly up-regulated( P < 0. 01),the protein expression level of SIRT6 in model group was significantly down-regulated( P < 0. 01). Compared with model group,Liuwei Dihuangwan and resveratrol could significantly reduce the levels of serum FBG and TG( P < 0. 01),the protein expression of SIRT6 was significantly increased( P < 0. 01),NF-κB p65,MCP-1 and VCAM-1 were significantly decreased( P < 0. 05, P < 0. 01), and liver steatosis and inflammatory cells infiltration were alleviated.Conclusion: Liuwei Dihuangwan can protect the diabetes mice from liver injury,and its mechanism is related to the improvement of lipid metabolism and the inhibition of inflammatory response.
Objective: To investigate the protective mechanism of Liuwei Dihuangwan on diabetes mellitus with liver injury in terms of inflammation. Method: The 18 db/db mice were selected from animal model of spontaneous type 2 diabetes, mice were randomly divided into model group, Liuwei Dihuangwan group( 9. 75 g·kg-1·d-1,once a day),resveratrol group( 42. 6 mg·kg-1·d-1,once a day) based on fasting bloodglucose( FBG). The 12 litter wild db/m mice were randomly divided into normal group,Liuwei Dihuangwan group( 9. 75 g·kg-1·d-1,once a day). The normal group and the model group were given the same amount of distilled water by gavage. The mice in each group were treated for 16 weeks. FBG,triglyceride( TG) and alanine aminotransferase( ALT) were examined. Histopathological changes were observed by liver biopsy hematoxylin-eosin( HE) staining. Silent information regulator 6( SIRT6),nuclear factor-kappaB p65( NF-κB p65),monocyte chemoattractant protein-1( MCP-1),vascular cell adhesion molecule-1( VCAM-1) and intercellular adhesion molecule-1( ICAM-1) in the liver tissue were detected by Western blot. Result: Compared with normal group,FBG,TG and ALT in model group increased significantly( P < 0. 01),protein expression levels of NF-κB p65,MCP-1,VCAM-1 and ICAM-1 in model group were significantly up-regulated( P < 0. 01),the protein expression level of SIRT6 in model group was significantly down-regulated( P < 0. 01). Compared with model group,Liuwei Dihuangwan and resveratrol could significantly reduce the levels of serum FBG and TG( P < 0. 01),the protein expression of SIRT6 was significantly increased( P < 0. 01),NF-κB p65,MCP-1 and VCAM-1 were significantly decreased( P < 0. 05, P < 0. 01), and liver steatosis and inflammatory cells infiltration were alleviated.Conclusion: Liuwei Dihuangwan can protect the diabetes mice from liver injury,and its mechanism is related to the improvement of lipid metabolism and the inhibition of inflammatory response.
引文
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[21]易华,苏俊芳,李雪,等.基于Cx32探讨六味地黄丸增效自杀基因抗肝癌的缝隙连接机制[J].中国实验方剂学杂志,2019,25(1):76-81.
[22]张瑞义,舒适,李志杰,等.六味地黄丸对阿霉素肾病小鼠足细胞nephrin和podocin表达的影响[J].数理医药学杂志,2017,30(6):795-798.
[23]Ashraf N U,Altaf M. Epigenetics:an emergingfield in the pathogenesis of nonalcoholic fatty liver disease[J].Mutat Res,2018,778(10):1-12.
[24]Elhanati S,Kanfi Y,Varvak A,et al. Multiple regulatory layers of SREBP1/2 by SIRT6[J]. Cell Rep,2013,4(5):905-912.
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[26]XIAO C,WANG R,Lahusen T J,et al. Progression of chronic liver inflammation and fibrosis driven by activation of c-JUN signaling in sirt6 mutant mice[J].J Biol Chem,2012,287(50):41903-41913.
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[28]ZHENG X,ZHU S,CHANG S,et al. Protective effects of chronic resveratrol treatment on vascular inflammatory injury in streptozotocin-induced type 2 diabetic rats:role of NF-kappa B signaling[J]. Eur J pharmacol,2013,720(10):147-157.
[29]WEI M,LI Z,XIAO L,et al. Effects of ROS-relative NF-κB signaling on high glucose-induced TLR4 and MCP-1 expression in podocyte injury[J]. Mol Immunol,2015,doi:10. 1016/j. molimm. 2015. 09. 002.
[30]KANG L,HU J,WENG Y,et al. Sirtuin 6 prevents matrix degradation through inhibition of the NF-κB pathway in intervertebral disc degeneration[J]. Exp Cell Res,2017,352(2):322-332.
[31]Kim H S,XIAO C,WANG R H,et al. Hepatic-specific disruption of SIRT6 in mice results in fatty liver formation due to enhanced glycolysis and triglyceride synthesis[J]. Cell Metab,2010,12(3):224-236.
[32]邓小敏,唐丽丽,陆斌,等.生脉散合六味地黄丸治疗2型糖尿病气阴两虚证[J].中国实验方剂学杂志,2014,20(11):192-194.
[2]Cusi K. Treatment of patients with type 2 diabetes and non-alcoholic fatty liver disease:current approaches and future directions[J]. Diabetologia,2016,59(6):1112-1120.
[3]Prattichizzo F,Giuliani A,Ceka A,et al. Epigenetic mechanisms of endothelial dysfunction in type 2 diabetes[J]. Clin Epigenetics,2015,7(5):56.
[4]Roberto C. Metabolic actions of hypothalamic[J].Trends Endocrinol Metab,2012,23(4):179-185.
[5]Tasselli L,ZHENG W,Chua K F. SIRT6:novel mechanisms and links to aging and disease[J]. Trends Endocrinol Metab,2017,28(3):168-185.
[6]Vitielloa M,Zullo A,Servillo L,et al. Multiple pathways of SIRT6 at the crossroads in the control of longevity,cancer,and cardiovascular diseases[J]. Ageing Res Rev,2017,35(5):301-311.
[7]Matsushita T,Sasaki H,Takayama K,et al. The overexpression of sirt1 inhibited osteoarthritic gene expressiom changes induced by interleukin-1βin human chondrocytes[J]. J Orthop Res,2013,31(4):531-537.
[8]Kitada M,Kume S,Takeda-wetanabe A,et al. Sirtuins and renal diseases:relationship with aging and diabetic nephropathy[J]. Clin Sci,2013,124(3):153-164.
[9]李志杰,张悦,刘煜敏,等.六味地黄丸防治大鼠糖尿病肾病的实验研究[J].中华中医药学刊,2011,29(8):1728-1731,1929.
[10]何珂,朱丽华,陆西宛.六味地黄丸联合二甲双胍片治疗2型糖尿病临床疗效观察[J].中成药,2016,38(1):50-52.
[11]李志杰,张悦,陆海英,等.六味地黄丸对糖尿病肾病大鼠肾组织TGF-β1-smad通路的影响[J].时珍国医国药,2017,28(8):1811-1814.
[12]张大方,金若敏.药理与中药药理实验[M]. 3版.上海:科学技术出版社,2013:106.
[13]黄新忠,温东海,张敏,等.白藜芦醇对慢性肾衰竭大鼠肾脏的保护作用和相关机制[J].中华肾脏病杂志,2013,29(2):114-118.
[14]刘芳,杨华,周文江,等.诱发性2型糖尿病小鼠模型与自发性db/db小鼠特性的比较[J].中国实验动物学报,2014,22(6):54-59,74.
[15]陈曦阳光,吴君.中国糖尿病人群合并肝损伤相关危险因素Meta分析[J].肝脏,2017,22(9):800-805.
[16]Bazick J,Donithan M,Neuschwander-Tetri B A,et al.Clinical model for NASH and advanced fibrosis in adult patients with diabetes and NAFLD:guidelines for referral in NAFLD[J]. Diabetes Care,2015,38(7):1347-1355.
[17]Maximos M,Bril F,Portillo S P,et a1. The role of liver fat and insulin resistance as determinants of plasma aminotransferase elevation in nonalcoholic fatty liver disease[J]. Hepatology,2015,61(1):153-160.
[18]Hummel K P,Dickie M M,Colemen D L. Diabetes,a new mutation in the mouse[J]. Science,1966,153(3740):1127-1128.
[19]徐小倩,朱谊,王晓华.六味地黄丸(汤)研究进展[J].哈尔滨医药,2016,36(4):484-486.
[20]崔勇,王艳杰,赵丹玉,等.六味地黄丸对APP/PS1小鼠学习记忆能力及胆碱能系统的影响[J].中国实验方剂学杂志,2016,22(3):148-151.
[21]易华,苏俊芳,李雪,等.基于Cx32探讨六味地黄丸增效自杀基因抗肝癌的缝隙连接机制[J].中国实验方剂学杂志,2019,25(1):76-81.
[22]张瑞义,舒适,李志杰,等.六味地黄丸对阿霉素肾病小鼠足细胞nephrin和podocin表达的影响[J].数理医药学杂志,2017,30(6):795-798.
[23]Ashraf N U,Altaf M. Epigenetics:an emergingfield in the pathogenesis of nonalcoholic fatty liver disease[J].Mutat Res,2018,778(10):1-12.
[24]Elhanati S,Kanfi Y,Varvak A,et al. Multiple regulatory layers of SREBP1/2 by SIRT6[J]. Cell Rep,2013,4(5):905-912.
[25]韩在祺,崔佰吉,冯波,等. SIRT6对小鼠米色脂肪细胞适应性产热功能的影响[J].中国病理生理杂志,2018,34(10):1827-1833.
[26]XIAO C,WANG R,Lahusen T J,et al. Progression of chronic liver inflammation and fibrosis driven by activation of c-JUN signaling in sirt6 mutant mice[J].J Biol Chem,2012,287(50):41903-41913.
[27]Schonthaler H B,Guinea-Viniegra J,Wagner E F.Targeting inflammation by modulating the Jun/AP-1pathway[J]. Ann Rheum Dis,2011,doi:10. 1136/ard.2010. 140533.
[28]ZHENG X,ZHU S,CHANG S,et al. Protective effects of chronic resveratrol treatment on vascular inflammatory injury in streptozotocin-induced type 2 diabetic rats:role of NF-kappa B signaling[J]. Eur J pharmacol,2013,720(10):147-157.
[29]WEI M,LI Z,XIAO L,et al. Effects of ROS-relative NF-κB signaling on high glucose-induced TLR4 and MCP-1 expression in podocyte injury[J]. Mol Immunol,2015,doi:10. 1016/j. molimm. 2015. 09. 002.
[30]KANG L,HU J,WENG Y,et al. Sirtuin 6 prevents matrix degradation through inhibition of the NF-κB pathway in intervertebral disc degeneration[J]. Exp Cell Res,2017,352(2):322-332.
[31]Kim H S,XIAO C,WANG R H,et al. Hepatic-specific disruption of SIRT6 in mice results in fatty liver formation due to enhanced glycolysis and triglyceride synthesis[J]. Cell Metab,2010,12(3):224-236.
[32]邓小敏,唐丽丽,陆斌,等.生脉散合六味地黄丸治疗2型糖尿病气阴两虚证[J].中国实验方剂学杂志,2014,20(11):192-194.