解毒护肝汤对药物性肝损伤大鼠MIP-2和MIP-1β蛋白表达的影响
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摘要
目的:通过检测大鼠血清肝功能、血清生物标志物,炎性因子以及巨噬细胞炎性蛋白-2(MIP-2),巨噬细胞炎性蛋白-1β(MIP-1β)的表达,观察解毒护肝汤对药物性肝损伤大鼠的干预作用及机制。方法:采用灌服对乙酰氨基酚方法复制药物性肝损伤大鼠模型,将Wistar大鼠按体质量随机分为正常组,模型组,水飞蓟宾组(44. 1 mg·kg~(-1)),解毒护肝汤高、中、低剂量组(63,31. 5,15. 75 g·kg~(-1)),正常组和模型组给予生理盐水灌胃,其余各组给予相应药液灌胃,连续给药30 d。实验结束后,腹主动脉取血分离血清检测天门冬氨酸氨基转移酶(AST),丙氨酸氨基转移酶(ALT),总胆红素(TBIL),直接胆红素(DBIL),苹果酸脱氢酶(MDH),对氧磷酶1(PON1),谷氨酸脱氢酶(GLDH),精氨酸(ARG),嘌呤核苷酸磷酸化酶(PNP),肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6)的含量;苏木素-伊红(HE)染色法观察肝组织病理形态改变;免疫组化法观察MIP-1β的蛋白表达情况;用单荧光免疫组化观察MIP-2的蛋白表达情况;用酶联免疫吸附测定(ELISA)检测肝组织匀浆中TNF-α和IL-6的含量。结果:与正常组比较,模型组大鼠血清AST,ALT,DBIL,PON1,ARG,GLDH,MDH,PNP,TNF-α含量均明显升高(P <0. 05,P <0. 01),肝组织MIP-2,MIP-1β蛋白阳性表达增多;与模型组比较,解毒护肝汤中、低剂量组能明显降低转氨酶活性及胆红素含量(P <0. 05,P <0. 01),解毒护肝汤高剂量组能明显降低肝损伤大鼠血清PON1,MDH和TNF-α的含量(P <0. 05),解毒护肝汤中剂量组明显降低ARG,MDH的含量(P <0. 05),解毒护肝汤高、中剂量组均能明显降低肝组织MIP-2,MIP-1β蛋白的表达,解毒护肝汤中剂量组效果最优,各给药组对肝细胞病理形态均具有不同程度的保护作用。结论:解毒护肝汤中剂量组的护肝效果较优,且其作用机制可能通过降低TNF-α的含量,进而影响趋化因子MIP-2和MIP-1β对中性粒细胞的趋化,抑制炎性因子的释放,从而预防炎症发生。
Objective: To observe the effect and mechanism of Jiedu Hugan decoction on drug-induced liver injury in rats by detecting serum liver function, serum biomarkers, inflammatory factors, macrophage inflammatory protein-2( MIP-2) and macrophage inflammatory protein~(-1)β( MIP-1β). Method: The rat model of drug-induced liver injury was induced by acetaminophen( 1 g·kg~(-1)) orally once daily for 30 days. The sixty male adult Wistar rats were divided into five groups, control group, model group, administered silybin group( 44. 1 mg·kg~(-1)),Jiedu Hugan decoction high,medium and low dose groups( 63,31. 5,15. 75 g·kg~(-1)),normal group and model group were given normal saline gavage,and the other groups were given corresponding liquid gavage for 30 days. After the experiment,the abdominal aorta separation take blood serum aspartate amino transferase( AST),alanine aminotransferase( ALT),total bilirubin( TBIL),direct bilirubin( DBIL),malate dehydrogenase( MDH), enzyme for oxygen p1( PON1) and glutamate dehydrogenase( GLDH), arginine( ARG),purine nucleotide phosphorylase( PNP),tumor necrosis factor-α( TNF-α),interleukin-6( IL-6)content. Pathological morphological changes of liver tissues were observed by hematoxylin-eosin( HE) staining.The protein expression of MIP-1β was observed by immunohistochemistry. The protein expression of MIP-2 was observed by single fluorescence immunohistochemistry,and the contents of TNF-α and IL-6 in liver homogenate were detected by enzyme-linked immunosorbent assay( ELISA). Result: Compared with normal group,levels of AST,ALT,DBIL,PON1,ARG,GLDH,MDH,PNP and TNF-α in model group were significantly increased( P < 0. 05,P < 0. 01). Compared with model group,detoxification,Jiedu Hugan decoction low dose group can obviously decrease transaminase activity and content of bilirubin( P < 0. 05,P < 0. 01). Jiedu Hugan decoction high dose group can significantly reduce the levels of PON1,MDH and the content of TNF-α in liver injury rats( P < 0. 05),Jiedu Hugan decoction low dose group significantly reduce the levels of ARG,MDH( P < 0. 05),Jiedu Hugan decoction high,middle dose group can obviously reduce the liver tissue MIP-2,MIP-1β protein expression,detoxification protect liver soup effect of the optimal dose group,the pathological morphology of liver cell dosage group were with different degree of protection. Conclusion: The effect of Jiedu Hugan decoction in medium dose group is better,and its mechanism may affect the chemotaxis of neutrophils induced by MIP-2 and MIP-1β by reducing the content of TNF-α,thus inhibiting the release of inflammatory factors and preventing inflammation.
Objective: To observe the effect and mechanism of Jiedu Hugan decoction on drug-induced liver injury in rats by detecting serum liver function, serum biomarkers, inflammatory factors, macrophage inflammatory protein-2( MIP-2) and macrophage inflammatory protein~(-1)β( MIP-1β). Method: The rat model of drug-induced liver injury was induced by acetaminophen( 1 g·kg~(-1)) orally once daily for 30 days. The sixty male adult Wistar rats were divided into five groups, control group, model group, administered silybin group( 44. 1 mg·kg~(-1)),Jiedu Hugan decoction high,medium and low dose groups( 63,31. 5,15. 75 g·kg~(-1)),normal group and model group were given normal saline gavage,and the other groups were given corresponding liquid gavage for 30 days. After the experiment,the abdominal aorta separation take blood serum aspartate amino transferase( AST),alanine aminotransferase( ALT),total bilirubin( TBIL),direct bilirubin( DBIL),malate dehydrogenase( MDH), enzyme for oxygen p1( PON1) and glutamate dehydrogenase( GLDH), arginine( ARG),purine nucleotide phosphorylase( PNP),tumor necrosis factor-α( TNF-α),interleukin-6( IL-6)content. Pathological morphological changes of liver tissues were observed by hematoxylin-eosin( HE) staining.The protein expression of MIP-1β was observed by immunohistochemistry. The protein expression of MIP-2 was observed by single fluorescence immunohistochemistry,and the contents of TNF-α and IL-6 in liver homogenate were detected by enzyme-linked immunosorbent assay( ELISA). Result: Compared with normal group,levels of AST,ALT,DBIL,PON1,ARG,GLDH,MDH,PNP and TNF-α in model group were significantly increased( P < 0. 05,P < 0. 01). Compared with model group,detoxification,Jiedu Hugan decoction low dose group can obviously decrease transaminase activity and content of bilirubin( P < 0. 05,P < 0. 01). Jiedu Hugan decoction high dose group can significantly reduce the levels of PON1,MDH and the content of TNF-α in liver injury rats( P < 0. 05),Jiedu Hugan decoction low dose group significantly reduce the levels of ARG,MDH( P < 0. 05),Jiedu Hugan decoction high,middle dose group can obviously reduce the liver tissue MIP-2,MIP-1β protein expression,detoxification protect liver soup effect of the optimal dose group,the pathological morphology of liver cell dosage group were with different degree of protection. Conclusion: The effect of Jiedu Hugan decoction in medium dose group is better,and its mechanism may affect the chemotaxis of neutrophils induced by MIP-2 and MIP-1β by reducing the content of TNF-α,thus inhibiting the release of inflammatory factors and preventing inflammation.
引文
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[15]刘晓娜,彭双清,贾栗,等.对乙酰氨基酚致小鼠肝损伤早期血清苹果酸脱氢酶和嘌呤核苷磷酸化酶的变化[J].毒理学杂志,2015,29(1):54-56.
[16]乔靖怡,周璐,金若敏,等.四氯化碳致大鼠肝损伤早期血清总胆汁酸、α-谷胱甘肽S转移酶、嘌呤核苷磷酸化酶和鸟氨酸氨基甲酰转移酶的变化[J].中国药理学与毒理学杂志,2013,27(4):650-656.
[17] Meneses-Lorente G, Watt A, Sailim K, et al.Identification of early proteomic markers for hepatic steatosis[J]. Chem Res Toxicol,2006,19(8):986-998.
[18]朱平生,焦炎杰,付双楠,等.对乙酰氨基酚致大鼠肝损伤早期生物标志物水平的变化规律[J].中国实验方剂学杂志,2019,25(2):118-123.
[19]乔靖怡,金若敏,姚广涛,等.北豆根致大鼠肝损伤血清酶生物标志物的早期变化及联合检测[J].世界中医药,2014,9(2):166-170.
[20]陈晓宇,郭瑞娟,王菲,等.对氧磷酶1过表达对敌敌畏急性中毒小鼠肝损伤保护作用的研究[J].浙江医学,2016,38(15):1230-1233,1245.
[21]黄凯,李燕.对氧磷酶与慢性肝损伤[J].国际药学研究杂志,2011,38(2):112-117.
[22]张帆,韩新飞,张娜,等.重组人对氧磷酶1K192亚型对毒死蜱所致SD大鼠急性肝损伤的保护作用[J].中国医科大学学报,2016,45(2):101-105.
[23]周璐,徐婷婷,金若敏,等.吴茱萸致大鼠和小鼠肝损伤血清酶生物标志物变化的研究[J].中药新药与临床药理,2014,25(4):418-423.
[24]易婷婷,罗光成,胡帅,等.血浆谷氨酸脱氢酶活性测定在肝胆疾病中的临床应用评价[J].川北医学院学报,2016,31(4):532-534.
[25]赵元明.血清谷氨酸脱氢酶活性检测在肝胆疾病中的临床价值[J].检验医学,2013,28(2):163-165.
[26]逯青,木努热丁·艾则孜.早期手术对重症胆源性梗阻型胰腺炎的疗效及其对患者血清MIP-1α、MIP-1β和MCP-1的影响[J].中华普外科手术学杂志:电子版,2018,12(4):289-291.
[27] Heinrichs D,Berres M L,Nellen A,et al. The chemokine CCL3 promotes experimental liver fibrosis in mice[J].PLo S One,2013,8:e66106.
[28]桂坤,董亚琼,龙启忠,等.清解补肺汤辅助治疗支气管扩张对肺功能和BALF中MMP-9、MI-2及TIMP-1的影响[J].中药材,2018,41(10):2451-2453.
[29]梁法生,宋继昌,高英堂,等.趋化因子MIP-2在大鼠肝缺血/再灌注损伤后的表达[J].中国普通外科杂志,2004,13(2):104-106.
[2] Leise M D,Poterucha J J,Talwalkar J A. Drug-induced liver injury[J]. Mayo Clin Proc, 2014, 89(1):95-106.
[3] Chen J, Ahmad J. Cefdinir-induced hepatotoxicity:potential hazards of inappropriate antibiotic use[J]. J Gen Intern Med,2008,23(11):1914-1916.
[4]赵凤娥,石振东.中药相关性肝损伤的临床研究进展[J].世界华人消化杂志,2017,25(30):2689-2694.
[5]王君明,苗明三,屈凌波,等.蛋白质组学在肝损伤本质研究中的应用现状以策略[J].中国实验方剂学杂志,2013,19(9):358-362.
[6] Amacher D E. Use of proteomicmethods to identify serum biomarkers associated with ratliver toxicity or hypertrophy[J]. Clin Chem,2005,51(10):1796-1803.
[7]王海珍,李秀惠.药物性肝损伤发病机制研究进展[J].临床肝胆病杂志,2018,34(4):883-887.
[8]隗秀荣,朱文涛,杜庆鹏,等. 4种口服保肝药治疗药物性肝损伤的有效性、安全性和经济性分析[J].中国新药杂志,2017,26(8):965-968.
[9]庞连晶,李妮矫,符思.符思教授治疗药物性肝损害[J].长春中医药大学学报,2013,29(4):604-606.
[10]李水芹,李平,王飞,等.李平教授中医辨治严重药物性肝损害验案举隅[J].中华中医药杂志,2010,25(8):1236-1238.
[11]高晶,彭海燕,章永红.药物性肝损伤的中医药研究[J].长春中医药大学学报,2011,27(5):741-743.
[12]黄清杰,李茂星,刘雄.中医治疗药物性肝损伤研究进展[J].西部中医药,2012,25(7):124-126.
[13] Chomaker S,Warner R,Bock J,et al. Assessment of emerging biomarkers of liver injury in human subjects[J]. Toxicol Sci,2013,132(2):276-283.
[14] Bailey W J,Holder D,Patel H,et al. A performance evaluationof three drug-induced liver injury biomarkers in the rat:alpha-glutathione S-transferase,arginase 1,and 4-hydroxyphenyl-pyruvate dioxygenase[J]. Toxicol Sci,2012,130(2):229-244.
[15]刘晓娜,彭双清,贾栗,等.对乙酰氨基酚致小鼠肝损伤早期血清苹果酸脱氢酶和嘌呤核苷磷酸化酶的变化[J].毒理学杂志,2015,29(1):54-56.
[16]乔靖怡,周璐,金若敏,等.四氯化碳致大鼠肝损伤早期血清总胆汁酸、α-谷胱甘肽S转移酶、嘌呤核苷磷酸化酶和鸟氨酸氨基甲酰转移酶的变化[J].中国药理学与毒理学杂志,2013,27(4):650-656.
[17] Meneses-Lorente G, Watt A, Sailim K, et al.Identification of early proteomic markers for hepatic steatosis[J]. Chem Res Toxicol,2006,19(8):986-998.
[18]朱平生,焦炎杰,付双楠,等.对乙酰氨基酚致大鼠肝损伤早期生物标志物水平的变化规律[J].中国实验方剂学杂志,2019,25(2):118-123.
[19]乔靖怡,金若敏,姚广涛,等.北豆根致大鼠肝损伤血清酶生物标志物的早期变化及联合检测[J].世界中医药,2014,9(2):166-170.
[20]陈晓宇,郭瑞娟,王菲,等.对氧磷酶1过表达对敌敌畏急性中毒小鼠肝损伤保护作用的研究[J].浙江医学,2016,38(15):1230-1233,1245.
[21]黄凯,李燕.对氧磷酶与慢性肝损伤[J].国际药学研究杂志,2011,38(2):112-117.
[22]张帆,韩新飞,张娜,等.重组人对氧磷酶1K192亚型对毒死蜱所致SD大鼠急性肝损伤的保护作用[J].中国医科大学学报,2016,45(2):101-105.
[23]周璐,徐婷婷,金若敏,等.吴茱萸致大鼠和小鼠肝损伤血清酶生物标志物变化的研究[J].中药新药与临床药理,2014,25(4):418-423.
[24]易婷婷,罗光成,胡帅,等.血浆谷氨酸脱氢酶活性测定在肝胆疾病中的临床应用评价[J].川北医学院学报,2016,31(4):532-534.
[25]赵元明.血清谷氨酸脱氢酶活性检测在肝胆疾病中的临床价值[J].检验医学,2013,28(2):163-165.
[26]逯青,木努热丁·艾则孜.早期手术对重症胆源性梗阻型胰腺炎的疗效及其对患者血清MIP-1α、MIP-1β和MCP-1的影响[J].中华普外科手术学杂志:电子版,2018,12(4):289-291.
[27] Heinrichs D,Berres M L,Nellen A,et al. The chemokine CCL3 promotes experimental liver fibrosis in mice[J].PLo S One,2013,8:e66106.
[28]桂坤,董亚琼,龙启忠,等.清解补肺汤辅助治疗支气管扩张对肺功能和BALF中MMP-9、MI-2及TIMP-1的影响[J].中药材,2018,41(10):2451-2453.
[29]梁法生,宋继昌,高英堂,等.趋化因子MIP-2在大鼠肝缺血/再灌注损伤后的表达[J].中国普通外科杂志,2004,13(2):104-106.