白首乌C-21甾体总苷对肝损伤小鼠氧化应激通路的影响
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摘要
通过观察白首乌C-21甾体总苷对肝损伤小鼠氧化应激的影响来探讨白首乌的保肝作用机制。雄性ICR小鼠随机分为正常对照组、模型组、阳性药组及白首乌总苷高剂量(8 g·kg~(-1))和低剂量(4 g·kg~(-1))组,用四氯化碳(CCl4)复制小鼠急性肝损伤模型,采集各组动物血液和肝组织标本,测定血清中ALT和AST水平,HE染色观察肝脏病理学改变,比色法检测肝组织SOD活性和MDA含量,Western blot法检测肝脏NF-κB p65,p-IκBα,i NOS和COX-2的表达,RT-PCR法测定肝组织中TNF-α和IL-6 mRNA的表达。与模型组比较,白首乌C-21甾体总苷可明显降低模型动物血清中ALT,AST水平(P<0. 01),同时显著升高肝匀浆中SOD活性(P<0. 01),并降低MDA含量(P<0. 01); HE染色结果显示白首乌2个剂量组均不同程度地减轻肝细胞水肿、变性; Western blot和RT-PCR法结果显示,白首乌总苷组p-IκBα,i NOS,NF-κB p65和COX-2蛋白表达显著下降(P<0. 01),TNF-α和IL-6 mRNA表达明显下调(P<0. 05)。白首乌C-21甾体总苷对CCl4诱导的小鼠肝损伤有较好的预防作用,通过减轻氧化应激,从而发挥保肝作用,其机制与提高细胞SOD活性、降低MDA水平,抑制NF-κB通路激活,下调i NOS和COX-2蛋白表达有关。
The study aimed to investigate the mechanism of hepatoprotective effect of C-21 steroidal glucosides from Cynanchum auriculatum( Baishouwu) on oxidative stress in mice with liver injury. Mice were randomly divided into normal group,model group,positive control group,Baishouwu high group and Baishouwu low group. The liver injury model was induced by intraperitoneal injection of CCl4 peanut oil solution. All mice were sacrificed to collect blood and liver specimens. The activities of serum levels of ALT and AST were detected. The content of MDA and the activity of SOD in liver homogenate were examined by colorimetry method. Tissues were stained with hematoxylin-eosin for histological examination. The hepatic protein expressions of NF-κB p65,p-IκBα,i NOS and COX-2 were detected by Western blot. The mRNA expressions of TNF-α and IL-6 were determined by RT-PCR. It was found that treatment with C-21 steroidal glucosides from Baishouwu successfully attenuated liver injury induced by CCl4,as shown by decreased levels of serum biochemical indicators( AST,ALT)( P<0. 01). Administration of total C-21 steroidal glucosides enhanced the activity of SOD( P<0. 01) and decreased the content of MDA( P<0. 01) in liver homogenate. Microscopic features suggested that treatment with C-21 steroidal glucosides from Baishouwu was effective in inhibiting CCl4-induced hepatocyte edema and degeneration. Further studies showed that NF-κB p65 overexpression induced by CCl4 was decreased by C-21 steroidal glucosides,leading to the markedly down-regulated protein expression levels of p-IκBα,i NOS and COX-2,as well as the depression of TNF-α and IL-6 mRNA expressions. In conclusion,total C-21 steroidal glucosides from Baishouwu exhibited potent effect on oxidative stress pathway in mice with liver injury induced by CCl4,with enhanced activity of SOD,decreased content of MDA,and down-regulated levels of NF-κB p65,p-IκBα,i NOS and COX-2.
The study aimed to investigate the mechanism of hepatoprotective effect of C-21 steroidal glucosides from Cynanchum auriculatum( Baishouwu) on oxidative stress in mice with liver injury. Mice were randomly divided into normal group,model group,positive control group,Baishouwu high group and Baishouwu low group. The liver injury model was induced by intraperitoneal injection of CCl4 peanut oil solution. All mice were sacrificed to collect blood and liver specimens. The activities of serum levels of ALT and AST were detected. The content of MDA and the activity of SOD in liver homogenate were examined by colorimetry method. Tissues were stained with hematoxylin-eosin for histological examination. The hepatic protein expressions of NF-κB p65,p-IκBα,i NOS and COX-2 were detected by Western blot. The mRNA expressions of TNF-α and IL-6 were determined by RT-PCR. It was found that treatment with C-21 steroidal glucosides from Baishouwu successfully attenuated liver injury induced by CCl4,as shown by decreased levels of serum biochemical indicators( AST,ALT)( P<0. 01). Administration of total C-21 steroidal glucosides enhanced the activity of SOD( P<0. 01) and decreased the content of MDA( P<0. 01) in liver homogenate. Microscopic features suggested that treatment with C-21 steroidal glucosides from Baishouwu was effective in inhibiting CCl4-induced hepatocyte edema and degeneration. Further studies showed that NF-κB p65 overexpression induced by CCl4 was decreased by C-21 steroidal glucosides,leading to the markedly down-regulated protein expression levels of p-IκBα,i NOS and COX-2,as well as the depression of TNF-α and IL-6 mRNA expressions. In conclusion,total C-21 steroidal glucosides from Baishouwu exhibited potent effect on oxidative stress pathway in mice with liver injury induced by CCl4,with enhanced activity of SOD,decreased content of MDA,and down-regulated levels of NF-κB p65,p-IκBα,i NOS and COX-2.
引文
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[19]董永喜,王霞,董莉,等.辛芍组方对缺血再灌注损伤后神经细胞氧化应激及NF-κB信号通路的影响[J].山东医药,2016,56(42):5.
[20]曹婕,赵昕,刘明江,等.青蒿琥酯对ConA诱导小鼠自身免疫性肝损伤的保护作用研究[J].中国中药杂志,2018,43(10):2123.
[21] Lipton S A,Choi Y B,Pan Z H,et al. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds[J]. Nature(London),1993,364(6438):626.
[22]马琳,张玉玲,刘红梅,等.氧化槐定碱对四氯化碳致急性肝损伤小鼠TNF-α和COX-2表达的干预作用[J].宁夏医科大学学报,2011,33(7):622.
[23]张怡,王勇,孟慧,等.复方丹参片调控AT1介导花生四烯酸PLA2-COX2/5-LOX代谢途径防治心力衰竭的机制研究[J].中国中药杂志,2018,43(12):2593.
[2]王冬艳,华欣,叶记林,等.江苏地产白首乌C21甾体苷对高血脂大鼠血脂调节和肝脏保护作用研究[J].安徽医药,2015,19(8):1454.
[3]张士侠,李心,尹家乐,等.江苏地产白首乌C21甾苷抗衰老作用研究[J].实用老年医学,2007,21(2):104.
[4]饶雅琪,张丽,曾金祥,等.禾叶风毛菊醇提物对四氯化碳致小鼠肝损伤的保护作用[J].中国临床药理学杂志,2017,33(20):64.
[5]伍振辉,孟娴,丁永芳,等.白首乌C-21甾体总苷对人肝癌HepG2细胞增殖、侵袭与凋亡的影响[J].中国中药杂志,2018,43(9):1915.
[6]姚楠,顾晓洁,李友宾.白首乌中C21甾体皂苷类成分的抗肿瘤活性研究[J].中成药,2010,32(11):1975.
[7]曾郁敏,邱泽计,潘爱群,等.白首乌总苷与环磷酰胺合用对荷瘤小鼠免疫功能的影响[J].北京中医药大学学报,2009,32(3):167.
[8]张士侠,李心,尹家乐,等.江苏地产白首乌C21甾苷对D-半乳糖衰老模型小鼠的作用研究[J].中国中药杂志,2007,32(23):2511.
[9]郭肇铮,刘小青,王淑华,等.白首乌抗自由基损伤的研究[J].中国中西医结合杂志,1988(5):282.
[10]宋俊梅,丁霄霖.白首乌对超氧阴离子自由基清除作用的研究[J].中国野生植物资源,1997,18(9):61.
[11]王冬艳,华欣,叶记林,等.江苏地产白首乌C21甾体苷对高血脂大鼠的肝脏保护作用和抗氧化作用研究[J].河北医药,2016,38(3):335.
[12]段冷昕,高杨,胡举,等.二至丸对四氯化碳致小鼠急性肝损伤的防护作用及机制[J].中国药理学与毒理学杂志,2018,32(6):462.
[13] Aleksandar Ra2kovic',Neboj2a Pavlovic',Maja Kvrgic',et al.Effects of pharmaceutical formulations containing thyme on carbon tetrachloride-induced liver injury in rats[J]. BMC Complement Altern Med,2015,15(1):442.
[14]平键,陈红云,周扬,等.骨髓间充质干细胞移植对四氯化碳大鼠肝损伤的影响及一贯煎的干预作用[J].中国中药杂志,2018,43(19):3905.
[15]朱平生,孟玉,付双楠,等.CCl4致大鼠肝损伤24 h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):124.
[16]王睿,孙鹏,赵欣.虫茶粗多糖对四氯化碳诱导小鼠肝损伤预防效果研究[J].现代食品科技,2015,31(5):6.
[17] Hu B,Zhang H,Meng X,et al. Aloe-emodin from rhubarb(Rheum rhabarbarum)inhibits lipopolysaccharide-induced inflammatory responses in RAW264. 7 macrophages[J]. J Ethnopharmacol,2014,153(3):846.
[18] Steinebrunner N,Mogler C,Vittas S,et al. Pharmacologic cholinesterase inhibition improves survival in acetaminophen-induced acute liver failure in the mouse[J]. BMC Gastroenterol,2014,14(1):148.
[19]董永喜,王霞,董莉,等.辛芍组方对缺血再灌注损伤后神经细胞氧化应激及NF-κB信号通路的影响[J].山东医药,2016,56(42):5.
[20]曹婕,赵昕,刘明江,等.青蒿琥酯对ConA诱导小鼠自身免疫性肝损伤的保护作用研究[J].中国中药杂志,2018,43(10):2123.
[21] Lipton S A,Choi Y B,Pan Z H,et al. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds[J]. Nature(London),1993,364(6438):626.
[22]马琳,张玉玲,刘红梅,等.氧化槐定碱对四氯化碳致急性肝损伤小鼠TNF-α和COX-2表达的干预作用[J].宁夏医科大学学报,2011,33(7):622.
[23]张怡,王勇,孟慧,等.复方丹参片调控AT1介导花生四烯酸PLA2-COX2/5-LOX代谢途径防治心力衰竭的机制研究[J].中国中药杂志,2018,43(12):2593.