大黄酸对小鼠肾脏的毒性机制
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摘要
目的:观察大黄酸长期给药对小鼠肾脏毒性的影响,探讨其可能的毒性机制,为临床合理用药及进一步研究提供一定依据。方法:昆明种小鼠30只,随机分为空白组和大黄酸低、高剂量组(0.175,0.35 g·kg~(-1)),每组10只,雌雄各半。连续灌胃给药60 d。给药期间,观察记录小鼠的一般情况;停止给药后检测动物血清尿素氮(BUN),肌酐(SCr),丙二醛(MDA),超氧化物歧化酶(SOD),肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)等指标。计算肾脏指数并检测肾脏中谷胱甘肽过氧化物酶(GSH-Px)和还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG),取肾脏,苏木精-伊红(HE)染色观察肾脏组织形态病理变化,免疫组化法检测肾脏转化生长因子-β_1(TGF-β_1)及半胱氨酸蛋白酶-3(Caspase-3)蛋白表达。结果:与同性别空白组比较,给药组小鼠BUN和SCr均升高(P<0.05,P<0.01),大黄酸高剂量组30 d后小鼠体质量下降明显,SOD活力明显降低(P<0.05,P<0.01),TNF-α含量增多(P<0.05),Caspase-3表达明显上升(P<0.05),雄性给药组肾脏指数下降明显(P<0.05,P<0.01),雄性大黄酸高剂量组GSH-Px含量显著下降(P<0.05),TGF-β_1表达增强(P<0.05)。肾脏组织形态病理变化,大黄酸高剂量组可见肾小管管腔中出现蛋白管型,肾小球和肾间质毛细血管充血,肾小管上皮细胞肿胀和淋巴细胞小灶性增生,且雄性小鼠病变更严重,大黄酸低剂量组以上表现较高剂量组不明显。结论:大黄酸长期大剂量给药对小鼠肾脏存在一定毒性,在0.35 g·kg~(-1)·d~(-1)剂量下毒性作用明显,且雄性机体毒性更加明显。其潜在毒性机制可能为引起谷胱甘肽抗氧化系统失衡,诱发过度氧化,触发炎症反应,激活Caspase-3的表达,进而诱导细胞凋亡。
Objective:To observe effect of long-term administration of rhein on the kidney toxicity of mice,and explore its possible toxic mechanism,in order to provide some basis for rational clinical drug use and further research.Method:The 30 Kunming mice(half male and half female)were randomly divided into 3groups:control group,low-dose rhein group and high-dose rhein group(0.175,0.35 g·kg~(-1)),with 10 mice in each group.The intragastric administration lasted for 60 days.During administration,general situations of the mice were observed and recorded.Serum urea nitrogen(BUN),creatinine(SCr),malondialdehyde(MDA),superoxide dismutase(SOD),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected after drug withdrawal.Kidney index was calculated,and glutathione peroxidase(GSH-Px)and reduced glutathione/oxidized glutathione(GSH/GSSG)ratio were measured.The kidneys were collected and histopathologically examined,and the protein expressions of transforming growth factor beta(TGF-β_1)and cysteine aspartic acid specific protease-3(Caspase-3)were detected by immunohistochemistry.Result:Compared with the control group of the same sex,BUN and SCr of the administration group increased significantly(P<0.05,P<0.01).In the high-dose group,the activity of SOD decreased significantly(P<0.05,P<0.01),while the expressions of TNF-αand Caspase-3increased significantly(P<0.05).In the male group,the renal index was significantly decreased(P<0.05,P<0.01),the GSH-Px content in the male high-dose group was significantly decreased(P<0.05),whereas the expression of TGF-β_1was increased(P<0.05).The histopathological changes of kidney were observed by hematoxylin-eosin(HE)staining.In the high-dose group,protein tubules were found in renal tubule lumen,glomerular and interstitial capillary congestion,tubule epithelial cell swelling and small focal proliferation of lymphocytes,and the pathological changes were more serious in male mice.The above performances in the lowdose group was less significant than those in the high-dose group.Conclusion:The toxicity of rhein in the kidney of mice was obvious at the dose of 0.35 g·kg~(-1)·d~(-1),and the toxicity in male organism is more obvious.The mechanism of its potential toxicity may cause the imbalance of glutathione antioxidant system,induce excessive oxidation,trigger inflammatory reaction,activate the expression of Caspase-3,and then induce apoptosis.
Objective:To observe effect of long-term administration of rhein on the kidney toxicity of mice,and explore its possible toxic mechanism,in order to provide some basis for rational clinical drug use and further research.Method:The 30 Kunming mice(half male and half female)were randomly divided into 3groups:control group,low-dose rhein group and high-dose rhein group(0.175,0.35 g·kg~(-1)),with 10 mice in each group.The intragastric administration lasted for 60 days.During administration,general situations of the mice were observed and recorded.Serum urea nitrogen(BUN),creatinine(SCr),malondialdehyde(MDA),superoxide dismutase(SOD),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected after drug withdrawal.Kidney index was calculated,and glutathione peroxidase(GSH-Px)and reduced glutathione/oxidized glutathione(GSH/GSSG)ratio were measured.The kidneys were collected and histopathologically examined,and the protein expressions of transforming growth factor beta(TGF-β_1)and cysteine aspartic acid specific protease-3(Caspase-3)were detected by immunohistochemistry.Result:Compared with the control group of the same sex,BUN and SCr of the administration group increased significantly(P<0.05,P<0.01).In the high-dose group,the activity of SOD decreased significantly(P<0.05,P<0.01),while the expressions of TNF-αand Caspase-3increased significantly(P<0.05).In the male group,the renal index was significantly decreased(P<0.05,P<0.01),the GSH-Px content in the male high-dose group was significantly decreased(P<0.05),whereas the expression of TGF-β_1was increased(P<0.05).The histopathological changes of kidney were observed by hematoxylin-eosin(HE)staining.In the high-dose group,protein tubules were found in renal tubule lumen,glomerular and interstitial capillary congestion,tubule epithelial cell swelling and small focal proliferation of lymphocytes,and the pathological changes were more serious in male mice.The above performances in the lowdose group was less significant than those in the high-dose group.Conclusion:The toxicity of rhein in the kidney of mice was obvious at the dose of 0.35 g·kg~(-1)·d~(-1),and the toxicity in male organism is more obvious.The mechanism of its potential toxicity may cause the imbalance of glutathione antioxidant system,induce excessive oxidation,trigger inflammatory reaction,activate the expression of Caspase-3,and then induce apoptosis.
引文
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[9]赵玲,胡昌江,潘新,等.长期服用生大黄、熟大黄对大鼠肝肾功能影响的比较[J].中国医院药学杂志,2015,35(15):1384-1387.
[10]WANG J B,ZHAO Y L,XIAO X H,et al. Assessment of the renal protection and hepatotoxicity of rhubarb extract in rats[J]. J Ethnopharmacol,2009,124(1):18-25.
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[12]谭汉全,尹雪梅. PTH、BUN及Cr联合检测在肾功能损伤早期诊断中的临床价值[J].海南医学院学报,2015,21(2):197-199.
[13]向春艳,何小燕,李逐波,等. TNF-α在癌症中的作用研究进展[J].生命科学,2012,24(3):250-254.
[14]XIONG J,SUN Q,JI K,et al. Epidermal growth factor promotes transforming growth factor-β1-induced epithelial-mesenchymal transition in HK-2 cells through a synergistic effect on Snail[J]. Mol Biol Rep,2013,41(1):241-250.
[2]王艳辉,赵海平,王伽伯,等.基于“有故无殒”思想的熟大黄对肝脏量-毒/效关系研究[J].中国中药杂志,2014,39(15):2918-2923.
[3]曾灵娜.基于“有故无殒”思想的大黄对肾脏保护与损伤双向作用的研究[D].昆明:昆明理工大学,2012.
[4]XUE J,CHEN F,WANG J,et al. Emodin protects against concanavalin a-induced hepatitis in mice through inhibiting activation of the p38 MAPK-NF-κB signaling pathway[J]. Cell Physiol Biochem,2015,35(4):1557-1570.
[5]邓诺,易艳,梁爱华,等.大黄肾脏毒性部分机制研究[J].中国中药杂志,2018,43(13):2777-2783.
[6]刘德明,周春燕,吴嘉思,等.大黄素通过线粒体通路诱导Hep G2细胞凋亡[J].中国实验方剂学杂志,2018,24(3):104-108.
[7]刘德明,周春燕,吴嘉思,等.大黄素通过激活线粒体Caspase-8通路诱导L02细胞凋亡[J].中药药理与临床,2017,33(5):23-26.
[8]王青秀.大黄及其主要成份的毒性毒理研究[D].北京:中国人民解放军军事医学科学院,2007.
[9]赵玲,胡昌江,潘新,等.长期服用生大黄、熟大黄对大鼠肝肾功能影响的比较[J].中国医院药学杂志,2015,35(15):1384-1387.
[10]WANG J B,ZHAO Y L,XIAO X H,et al. Assessment of the renal protection and hepatotoxicity of rhubarb extract in rats[J]. J Ethnopharmacol,2009,124(1):18-25.
[11]WANG J B,KONG W J,WANG H J,et al. Toxic effects caused by rhubarb(Rheum palmatum L.)are reversed on immature and aged rats[J]. J Ethnopharmacol,2011,134(2):216-220.
[12]谭汉全,尹雪梅. PTH、BUN及Cr联合检测在肾功能损伤早期诊断中的临床价值[J].海南医学院学报,2015,21(2):197-199.
[13]向春艳,何小燕,李逐波,等. TNF-α在癌症中的作用研究进展[J].生命科学,2012,24(3):250-254.
[14]XIONG J,SUN Q,JI K,et al. Epidermal growth factor promotes transforming growth factor-β1-induced epithelial-mesenchymal transition in HK-2 cells through a synergistic effect on Snail[J]. Mol Biol Rep,2013,41(1):241-250.