丹参抑制NLRP3炎症小体减轻胆汁淤积性肝损伤
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摘要
目的:探讨丹参抑制肝脏核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体活化、减轻胆汁淤积性肝损伤的作用机制。方法:40只雄性C57BL/6小鼠随机分为正常组、模型组及丹参低、高剂量组,每组10只。模型组和丹参组小鼠采用0.1%DDC饲料连续喂养3周的方法诱导胆汁淤积性肝损伤,同时正常组小鼠给予不含DDC饲料喂养。造模1周后,丹参低、高剂量组小鼠每日分别灌胃给予1.5 g/kg和3.0 g/kg丹参浸膏粉溶液,正常组与模型组灌胃给予等体积双蒸水,连续14 d。造模3周末收集各组小鼠血清和肝组织。试剂盒检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)和总胆红素(TBIL)水平,计算肝脏指数; HE染色观察肝组织病理学及炎症分级的变化; ELISA检测肝组织白介素-1β(IL-1β)含量;免疫组化观察肝脏NLRP3蛋白表达。结果:与正常组比较,模型组小鼠血清ALT、AST、TBIL水平和肝脏指数明显升高(P<0.01),HE染色结果显示,肝内汇管区和胆管周围出现大量炎症细胞浸润,肝组织促炎因子IL-1β含量和NLRP3阳性表达显著上调(P<0.01)。丹参干预2周后可降低模型小鼠血清ALT、AST、TBIL水平和肝脏指数(P<0.05,P<0.01),明显减轻肝组织病变,改善炎症细胞浸润,降低肝组织IL-1β含量,并减少NLRP3阳性表达(P<0.01)。与丹参低剂量组比较,丹参高剂量组改善肝功能及肝组织病变、抑制IL-1β表达及NLRP3炎症小体活化的作用效果更为明显(P<0.05)。结论:丹参可有效减轻DDC诱导的小鼠胆汁淤积性肝损伤,其机制与抑制NLRP3炎症小体活化有关。
Objective: To investigate the effects of Salvia Miltiorrhiza( SM) on ameliorating cholestatic liver injury by inhibiting nucleotide binding oligonucleotide domain like receptor protein 3( NLRP3) inflammasome activation. Methods: Forty male C57BL/6mice were randomly divided into the normal group,model group,SM groups with low-and high-dose,10 mice in each group. The mice in the model group and SM groups were fed with 0. 1% diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate( DDC) diet for continuous 3 weeks to induce cholestatic liver injury,meanwhile,the mice in the normal group were fed with diet without DDC. After modeling for one week,the mice in SM groups were treated with SM extract powder solution at daily dose of 1.5 g/kg and 3.0 g/kg by intragastric administration,respectively; the mice in the normal and the model group were treated with distilled water at equivalent volume,with a course of 14 days. 3 weeks after modeling,the serum and the liver tissues were collected. The serum levels of ALT,AST and TBIL were determined using commercial kits. The liver index was calculated. The changes of liver histopathology and inflammation grading were observed by HE staining.The level of IL-1β in liver tissue was measured by ELISA.The expression of NLRP3 inflammasome in liver was detected by immunohistochemistry. Results: Compared with the normal group,the serum levels of ALT,AST and TBIL and the liver index were significantly increased in the model group( P<0.01). HE staining showed a large number of inflammatory cell infiltration in the portal tract and around the bile duct,and the content of IL-1β and the positive expression of NLRP3 in liver tissue were significantly up-regulated( P<0.01). After treatment for 2 weeks,SM could decrease the serum levels of ALT,AST and TBIL and the liver index in model mice( P<0.05,P<0.01),significantly alleviate the pathological changes of liver tissue,improve the inflammatory cellinfiltration,decrease the content of IL-1β and reduce the positive expression of NLRP3( P<0.01).Compared with the low-dose group,the effects of improving liver function and liver pathological changes,inhibiting the expression of IL-1β and activation of NLRP3 inflammasome were more significant in the SM group with high-dose( P<0.05). Conclusion: Salvia Miltiorrhiza can effectively alleviate DDC induced cholestatic liver injury in mice,which is associated with inhibition on NLRP3 inflammasome activation.
Objective: To investigate the effects of Salvia Miltiorrhiza( SM) on ameliorating cholestatic liver injury by inhibiting nucleotide binding oligonucleotide domain like receptor protein 3( NLRP3) inflammasome activation. Methods: Forty male C57BL/6mice were randomly divided into the normal group,model group,SM groups with low-and high-dose,10 mice in each group. The mice in the model group and SM groups were fed with 0. 1% diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate( DDC) diet for continuous 3 weeks to induce cholestatic liver injury,meanwhile,the mice in the normal group were fed with diet without DDC. After modeling for one week,the mice in SM groups were treated with SM extract powder solution at daily dose of 1.5 g/kg and 3.0 g/kg by intragastric administration,respectively; the mice in the normal and the model group were treated with distilled water at equivalent volume,with a course of 14 days. 3 weeks after modeling,the serum and the liver tissues were collected. The serum levels of ALT,AST and TBIL were determined using commercial kits. The liver index was calculated. The changes of liver histopathology and inflammation grading were observed by HE staining.The level of IL-1β in liver tissue was measured by ELISA.The expression of NLRP3 inflammasome in liver was detected by immunohistochemistry. Results: Compared with the normal group,the serum levels of ALT,AST and TBIL and the liver index were significantly increased in the model group( P<0.01). HE staining showed a large number of inflammatory cell infiltration in the portal tract and around the bile duct,and the content of IL-1β and the positive expression of NLRP3 in liver tissue were significantly up-regulated( P<0.01). After treatment for 2 weeks,SM could decrease the serum levels of ALT,AST and TBIL and the liver index in model mice( P<0.05,P<0.01),significantly alleviate the pathological changes of liver tissue,improve the inflammatory cellinfiltration,decrease the content of IL-1β and reduce the positive expression of NLRP3( P<0.01).Compared with the low-dose group,the effects of improving liver function and liver pathological changes,inhibiting the expression of IL-1β and activation of NLRP3 inflammasome were more significant in the SM group with high-dose( P<0.05). Conclusion: Salvia Miltiorrhiza can effectively alleviate DDC induced cholestatic liver injury in mice,which is associated with inhibition on NLRP3 inflammasome activation.
引文
[1]中华医学会肝病学分会,中华医学会消化病学分会,中华医学会感染病学分会.胆汁淤积性肝病诊断和治疗共识(2015)[J].胃肠病学,2016,21(1):39-51.
[2] MARONI L,AGOSTINELLI L,SACCOMANNO S,et al. Nlrp3Activation Induces Il-18 Synthesis and Affects the Epithelial Barrier Function in Reactive Cholangiocytes[J]. Am J Pathol,2017,187(2):366-376.
[3]HANEKLAUS M,O’Neill L A. NLRP3 at the interface of metabolism and inflammation[J]. Immunol Rev,2015,265(1):53-62.
[4]齐艳艳,史永照.丹参对缺血再灌注损伤的保护作用及机制研究进展[J].上海中医药大学学报,2010,24(2):89-91.
[5]PENG Y, YANG T, HUANG K,et al. Salvia Miltiorrhiza Ameliorates Liver Fibrosis by Activating Hepatic Natural Killer Cells in Vivo and in Vitro[J]. Front Pharmacol,2018(9):762.
[6]LOK A S,LINDSAY I, SCHEUER P J, et al. Clinical and histological features of delta infection in chronic hepatitis B virus carriers[J]. J Clin Pathol,1985,38(5):530-533.
[7]SCHEUER P J. Classification of chronic viral hepatitis:a need for reassessment[J]. J Hepatol,1991,13(3):372-374.
[8]KIM J Y,AN H J,KIM W H,et al. A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis[J]. Am J Pathol,2007,171(2):525-536.
[9]AFONINA I S,ZHONG Z,KARIN M,et al.Limiting inflammationthe negative regulation of NF-κB and the NLRP3 inflammasome[J].Nat Immunol,2017,18(8):861-869.
[2] MARONI L,AGOSTINELLI L,SACCOMANNO S,et al. Nlrp3Activation Induces Il-18 Synthesis and Affects the Epithelial Barrier Function in Reactive Cholangiocytes[J]. Am J Pathol,2017,187(2):366-376.
[3]HANEKLAUS M,O’Neill L A. NLRP3 at the interface of metabolism and inflammation[J]. Immunol Rev,2015,265(1):53-62.
[4]齐艳艳,史永照.丹参对缺血再灌注损伤的保护作用及机制研究进展[J].上海中医药大学学报,2010,24(2):89-91.
[5]PENG Y, YANG T, HUANG K,et al. Salvia Miltiorrhiza Ameliorates Liver Fibrosis by Activating Hepatic Natural Killer Cells in Vivo and in Vitro[J]. Front Pharmacol,2018(9):762.
[6]LOK A S,LINDSAY I, SCHEUER P J, et al. Clinical and histological features of delta infection in chronic hepatitis B virus carriers[J]. J Clin Pathol,1985,38(5):530-533.
[7]SCHEUER P J. Classification of chronic viral hepatitis:a need for reassessment[J]. J Hepatol,1991,13(3):372-374.
[8]KIM J Y,AN H J,KIM W H,et al. A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis[J]. Am J Pathol,2007,171(2):525-536.
[9]AFONINA I S,ZHONG Z,KARIN M,et al.Limiting inflammationthe negative regulation of NF-κB and the NLRP3 inflammasome[J].Nat Immunol,2017,18(8):861-869.