红毛五加多糖对大鼠肝细胞免疫损伤的保护作用及其机制
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摘要
红毛五加属于五加科植物,而五加科植物大多具有调节免疫力的双向调节作用,红毛五加多糖是从中药红毛五加中提取的单一组分,目前关于红毛五加多糖的抑炎作用还少有报道。为了探究红毛五加多糖AHP-Ⅱ对LPS导致的大鼠肝细胞免疫损伤的保护作用及其作用机制,该研究将实验分为空白组、模型组(LPS,40μg·mL~(-1))和AHP-Ⅱ低、中、高剂量组(25,50,100μg·mL~(-1))五组,以LPS(40μg·mL~(-1))来制备大鼠肝细胞免疫损伤模型,先用ELISA方法检测细胞分泌TNF-α的水平及流式检测ROS的含量,来探究AHP-Ⅱ不同剂量对炎症因子的抑制作用,再用western方法检测P-JNK2的蛋白水平来进一步探究AHP-Ⅱ的抑制作用机制。结果表明:低、中、高剂量组的AHP-Ⅱ均可使肝细胞损伤后的TNF-α含量下降。同时,AHP-Ⅱ中、高剂量组均可使损伤后的肝细胞ROS的分泌量下降,且AHP-Ⅱ高剂量组抑制ROS的作用最强。在加入AHP-Ⅱ后,JNK2蛋白的磷酸化水平呈剂量依赖性降低,AHP-Ⅱ高剂量组抑制作用最强。这说明红毛五加多糖AHP-Ⅱ可以通过降低P-JNK2的蛋白含量来抑制炎症因子TNF-α及ROS的水平,以发挥免疫保护作用。
Acanthopanax giraldii belongs to the Araliaceae plant,and most of the Araliaceae plants have a two-way regulation effect in regulating immunity. A. giraldii polysaccharide is a single component extracted from the traditional Chinese medicine A. giraldii. The anti-inflammatory effect of polysaccharides has rarely been reported. This study aimed to explore the protective effect and mechanism of polysaccharide AHP-Ⅱon LPS-induced hepatocyte injury in rats. This experiment was divided into five groups: blank group,model group( LPS,40 μg·m L~(-1)),AHP-Ⅱlow,moderate,highdose group( 25,50,100 μg ·m L~(-1)). LPS( 40 μg·m L~(-1)) was used to prepare the rat hepatocyte immune injury model. The cytotoxic effect of AHP-Ⅱon hepatocytes was first examined by CCK-8 method to eliminate interference,and the secreted level of TNF-α by the cells and the level of ROS were measured by ELISA and flow cytometry respectively to establish the hepatocyte immune injury model. Then the inhibitory effect of different doses of AHP-Ⅱ on inflammatory factors was explored,and the level of JNK2 protein and its phosphorylation were detected by western method to further explore the inhibitory mechanism of AHP-Ⅱ. The results showed that the experimental dose of AHP-Ⅱhad no damage to cells by CCK-8 method,which indicates that the reduction of inflammatory factors secreted by cells treated by AHP-Ⅱis not due to its cytotoxic effect. AHP-Ⅱ groups( 25,50,100 μg ·m L~(-1)) decreased TNF-α content durig hepatocyte injury. At the same time,the high-dose AHP-Ⅱgroup can reduce the secretion of ROS durig hepatocytes injury,and the high-dose AHP-Ⅱgroup has the strongest effect in inhibiting ehe conrent of ROS. After the addition of AHP-Ⅱ,the phosphorylation level of JNK2 protein decreased in a dose-dependent manner,and the AHP-Ⅱhigh-dose group had the strongest inhibitory effect. This indicate that the polysaccharide AHP-Ⅱ can play an immunoprotective role by reducing the protein content of P-JNK2 and inhibiting the levels of TNF-α and ROS. The results of this study provide a scientific reference for the hepatoprotective effect of A. giraldii,and promote the further development and utilization of the resources of A. giraldii.
Acanthopanax giraldii belongs to the Araliaceae plant,and most of the Araliaceae plants have a two-way regulation effect in regulating immunity. A. giraldii polysaccharide is a single component extracted from the traditional Chinese medicine A. giraldii. The anti-inflammatory effect of polysaccharides has rarely been reported. This study aimed to explore the protective effect and mechanism of polysaccharide AHP-Ⅱon LPS-induced hepatocyte injury in rats. This experiment was divided into five groups: blank group,model group( LPS,40 μg·m L~(-1)),AHP-Ⅱlow,moderate,highdose group( 25,50,100 μg ·m L~(-1)). LPS( 40 μg·m L~(-1)) was used to prepare the rat hepatocyte immune injury model. The cytotoxic effect of AHP-Ⅱon hepatocytes was first examined by CCK-8 method to eliminate interference,and the secreted level of TNF-α by the cells and the level of ROS were measured by ELISA and flow cytometry respectively to establish the hepatocyte immune injury model. Then the inhibitory effect of different doses of AHP-Ⅱ on inflammatory factors was explored,and the level of JNK2 protein and its phosphorylation were detected by western method to further explore the inhibitory mechanism of AHP-Ⅱ. The results showed that the experimental dose of AHP-Ⅱhad no damage to cells by CCK-8 method,which indicates that the reduction of inflammatory factors secreted by cells treated by AHP-Ⅱis not due to its cytotoxic effect. AHP-Ⅱ groups( 25,50,100 μg ·m L~(-1)) decreased TNF-α content durig hepatocyte injury. At the same time,the high-dose AHP-Ⅱgroup can reduce the secretion of ROS durig hepatocytes injury,and the high-dose AHP-Ⅱgroup has the strongest effect in inhibiting ehe conrent of ROS. After the addition of AHP-Ⅱ,the phosphorylation level of JNK2 protein decreased in a dose-dependent manner,and the AHP-Ⅱhigh-dose group had the strongest inhibitory effect. This indicate that the polysaccharide AHP-Ⅱ can play an immunoprotective role by reducing the protein content of P-JNK2 and inhibiting the levels of TNF-α and ROS. The results of this study provide a scientific reference for the hepatoprotective effect of A. giraldii,and promote the further development and utilization of the resources of A. giraldii.
引文
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WANG L,2016.Protective effect and mechanism of andrographolide on anit-induced liver injury in rats[D].Dalian:Dalian Medicine University.[王雷,2016.穿心莲内酯对ANIT诱导的大鼠肝损伤的保护作用及其机制研究[D].大连:大连医科大学.]
WANG FH,HAN JC,LI DF,et al.2017.Dendrobium holoshan extract can reduce alcoholic liver injury in mice by mediating NF-B/p65 and p38 MAPK[J].Res Dev Nat Prod,29(4):569-574.[王凤华,韩吉春,李德芳,等,2017.霍山石斛水提取物通过介导NF-κB/p65和p38MAPK减轻小鼠酒精性肝损伤[J].天然产物研究与开发,29(4):569-574.]
XU LX,YAN LJ,2017.Protective effect and mechanism of betulinic acid on acute liver injury induced by CCl4in mice[J].J Nanjing Univ Trad Chin Med,33(6):618-622.[徐立新,严丽军,2017.白桦脂酸对CCl4诱导小鼠急性肝损伤的保护作用及机制研究[J].南京中医药大学学报,33(6):618-622.]
ZHANG Q,ZHANG YT,LI SM,2014.Research progress of baicalin protecting liver and protecting liver[J].Strair Pharmacol,26(1):8-10.[张强,张燕堂,李素梅,2014.黄芩苷保肝护肝作用的研究进展[J].海峡药学,26(1):8-10.]
ZHANG LH,CI H,2012.Pharmacological effect and clin application of silymarin on liver protection[J].Capital Med,(8):47-48.[张立海,慈慧,2012.水飞蓟素保肝的药理作用及临床应用[J].首都医药,(8):47-48.]
ZHAO X,LI GJ,HU YY,et al.,2017.Study on the improvement effect and mechanism of kuding tea polyphenol extract on the liver injury induced by carbon tetrachloride in mice[J].Food Ind Technol,39(4):289-295.[赵欣,李贵节,胡园园,等,2017.苦丁茶多酚提取物对四氯化碳诱导小鼠肝损伤的改善作用及机制研究[J].食品工业科技,39(4):289-295.]
ZHANG PJ,XING XY,LIU BY,et al.,2011.Study on the protective effect of goji barbarum polysaccharide on experimental liver injury[J].Chin Med Clin Pract,11(11):1286-1287.[章培军,邢雁霞,刘斌钰,等,2011.枸杞多糖对实验性肝损伤保护作用的研究[J].中国药物与临床,11(11):1286-1287.]
ZHANG ZG,QIAN SH,HUANG HC,2011.Protective effects of fruit extracts from piniculus chinensis on acute liver injury induced by carbon tetrachloride in mice[J].Ginseng Res,23(3):11-14.[张正光,钱士辉,黄厚才,2011.细柱五加果实提取物对四氯化碳致小鼠急性肝损伤的保护作用[J].人参研究,23(3):11-14.]
ZHU RF,LE S,MA ZF,et al.,2006.Protective effect of p38MAPK inhibitor on hepatic injury in sepsis rats[J].J Clin Exp Med,(7):855-856.[朱瑞芳,乐胜,马中富,等,2006.P38 MAPK抑制剂对脓毒症大鼠肝损伤的保护作用[J].临床和实验医学杂志,(7):855-856.]
GAO Y,ZHENG P,YAN L,et al.,2013.Effects and preliminary mechanism of matrine on chronic alcoholic liver injury in rats[J].Chin Psychopharm Bull,29(7):1012-1016.[高艳,郑萍,闫琳,等,2013.苦参碱对大鼠慢性酒精性肝损伤的作用及初步机制研究[J].中国药理学通报,29(7):1012-1016.]
HAO CY,BAI T,NAN JX,et al.,2012.Effects of acanthopanhydric acid on alcoholic liver injury[J].Chin J Exp Trad Med Form,18(24):198-200.[郝乘仪,白婷,南极星,等,2012.刺五加酸对酒精性肝损伤的影响[J].中国实验方剂学杂志,18(24):198-200.]
JIN CL,LIN D,LEI Y,2017.Hepatoprotective effects of kaemprerol-3-O-α-l-arabinopyranosyl-7-O-α-l-rhamnopyranoside ond-galactosamine and lipopolysaccharide caused hepatic failure in mice[J].Molecules,22:1755.
JU Y,LUO Q,DANG YL,2005.Protective effects of five plus polysaccharides with red hair on tetrachloride in rats with liver injury[J].Phamacol Clin Pract Trad Chin Med,(3):25-26.[鞠洋,骆勤,党月兰,2005.红毛五加多糖对四氯化碳肝损伤大鼠的保护作用[J].中药药理与临床,(3):25-26.]
LIN QY,2007.Anti-inflammation effect of acanthopanax senticosus hydrochloride extract and its mechanism[D].Dalian:Dalian University Technology.[林秋叶,2007.刺五加水提物抗炎作用及其机制研究[D].大连:大连理工大学.]
LU F,YANG XD,ZHANG Y,et al.,2016.Based on metabonomics method acanthopanax polysaccharides on immunological liver injury in mice protection[J].J Trad Chin Med New Drug Clin Pharmacol,27(6):823-829.[卢芳,杨晓丹,张颖,等,2016.基于代谢组学方法探讨刺五加多糖对免疫性肝损伤小鼠的保护作用[J].中药新药与临床药理,27(6):823-829.]
SUN YJ,2012.Extraction of total flavonoids from acanthopanax senticosus stems and leaves and their antioxidant effects[D].Changchun:Jilin Agriculture University.[孙永杰,2012.刺五加茎叶中总黄酮的提取及其抗氧化功效研究[D].长春:吉林农业大学.]
SULTAN M,BEN-ARI Z,MASOUD R,et al.,2017.Interleukin-1αand Interleukin-1βplay a central role in the pathogenesis of fulminant hepatic failure in mice[J].PLoSONE,12(9):e0184084.
SONG ZZ,2016.Study on target action of angelica polysaccharide liver and mechanism of protecting acute liver injury[D].Wuhan:Huazhong University Science Technology.[宋之臻,2016.当归多糖肝靶向作用及保护急性肝损伤机理的研究[D].武汉:华中科技大学.]
TIAN J,JIN QF,LI ZY,et al.,2016.Effects of compound yinbai granule on MAPK signaling pathway in rats with acute liver injury caused by carbon tetrachloride[J].J Chin Med,34(1):111-113.[田静,金巧菲,李兆翌,等,2016.复方茵柏颗粒对四氯化碳致急性肝损伤大鼠MAPK信号通路的影响[J].中华中医药学刊,34(1):111-113.]
WANG RQ,CHANG XQ,WEN ZY,2013.Anti-acute inflammation and immune-enhancing effects of acanthopanax senticosus hydrochloride extract[J].Tianjin Trad Chin Med,30(2):109-111.[王瑞强,常晓强,温占勇,2013.刺五加水提物的抗急性炎症及免疫增强作用[J].天津中医药,30(2):109-111.]
WENG SQ,YAO QY,LI X,et al.,2015.Protective effects of quercetin on acute liver injury induced by edamame A in mice[J].J Gastroenterol Hepatol,(1):40-44.[翁书强,姚群燕,李希,等,2015.槲皮素对刀豆蛋白A诱导小鼠急性肝损伤的保护作用[J].胃肠病学和肝病学杂志,(1):40-44.]
WANG L,2016.Protective effect and mechanism of andrographolide on anit-induced liver injury in rats[D].Dalian:Dalian Medicine University.[王雷,2016.穿心莲内酯对ANIT诱导的大鼠肝损伤的保护作用及其机制研究[D].大连:大连医科大学.]
WANG FH,HAN JC,LI DF,et al.2017.Dendrobium holoshan extract can reduce alcoholic liver injury in mice by mediating NF-B/p65 and p38 MAPK[J].Res Dev Nat Prod,29(4):569-574.[王凤华,韩吉春,李德芳,等,2017.霍山石斛水提取物通过介导NF-κB/p65和p38MAPK减轻小鼠酒精性肝损伤[J].天然产物研究与开发,29(4):569-574.]
XU LX,YAN LJ,2017.Protective effect and mechanism of betulinic acid on acute liver injury induced by CCl4in mice[J].J Nanjing Univ Trad Chin Med,33(6):618-622.[徐立新,严丽军,2017.白桦脂酸对CCl4诱导小鼠急性肝损伤的保护作用及机制研究[J].南京中医药大学学报,33(6):618-622.]
ZHANG Q,ZHANG YT,LI SM,2014.Research progress of baicalin protecting liver and protecting liver[J].Strair Pharmacol,26(1):8-10.[张强,张燕堂,李素梅,2014.黄芩苷保肝护肝作用的研究进展[J].海峡药学,26(1):8-10.]
ZHANG LH,CI H,2012.Pharmacological effect and clin application of silymarin on liver protection[J].Capital Med,(8):47-48.[张立海,慈慧,2012.水飞蓟素保肝的药理作用及临床应用[J].首都医药,(8):47-48.]
ZHAO X,LI GJ,HU YY,et al.,2017.Study on the improvement effect and mechanism of kuding tea polyphenol extract on the liver injury induced by carbon tetrachloride in mice[J].Food Ind Technol,39(4):289-295.[赵欣,李贵节,胡园园,等,2017.苦丁茶多酚提取物对四氯化碳诱导小鼠肝损伤的改善作用及机制研究[J].食品工业科技,39(4):289-295.]
ZHANG PJ,XING XY,LIU BY,et al.,2011.Study on the protective effect of goji barbarum polysaccharide on experimental liver injury[J].Chin Med Clin Pract,11(11):1286-1287.[章培军,邢雁霞,刘斌钰,等,2011.枸杞多糖对实验性肝损伤保护作用的研究[J].中国药物与临床,11(11):1286-1287.]
ZHANG ZG,QIAN SH,HUANG HC,2011.Protective effects of fruit extracts from piniculus chinensis on acute liver injury induced by carbon tetrachloride in mice[J].Ginseng Res,23(3):11-14.[张正光,钱士辉,黄厚才,2011.细柱五加果实提取物对四氯化碳致小鼠急性肝损伤的保护作用[J].人参研究,23(3):11-14.]
ZHU RF,LE S,MA ZF,et al.,2006.Protective effect of p38MAPK inhibitor on hepatic injury in sepsis rats[J].J Clin Exp Med,(7):855-856.[朱瑞芳,乐胜,马中富,等,2006.P38 MAPK抑制剂对脓毒症大鼠肝损伤的保护作用[J].临床和实验医学杂志,(7):855-856.]
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