白头翁皂苷B4对实验性急性肾功能损伤的保护作用
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摘要
目的:研究探讨白头翁皂苷B4对急性肾功能损伤的保护作用,以及对其进行小鼠急性毒性评价。方法:通过体外细胞实验,以LPS诱导人胚肾细胞HEK293损伤模型,用白头翁皂苷B4进行干预24、48 h,MTT法检测细胞活性及炎性因子的表达,观察白头翁皂苷B4对肾细胞损伤的作用。整体动物实验,分别以甘油、顺铂和脂多糖(LPS)造成大鼠或小鼠急性肾功能损伤,尾静脉注射不同剂量的白头翁皂苷B4,给药一定时间后行眼眶采血,分离血清,检测反映肾功能指标的尿素氮(BUN)、肌酐(Cre)、总蛋白(TP)及尿液中蛋白含量,来观察白头翁皂苷B4对急性肾功能损伤的作用;给小鼠尾静脉注射不同剂量的白头翁皂苷B4,连续观察2周,根据死亡率计算半数致死量(LD50),初步评价白头翁皂苷B4的用药安全性。结果:白头翁皂苷B4能提高损伤的肾细胞活性,下调炎性因子TNF-α的表达。白头翁皂苷B4静脉给药对甘油,顺铂和LPS所导致的血清BUN,Cre升高均有明显的抑制作用,同时白头翁皂苷B4高剂量(2. 5 mg·kg-1)还可以明显降低甘油致急性肾损伤大鼠的尿蛋白浓度,其在大鼠有效剂量为1. 25～2. 5 mg·kg-1,小鼠有效剂量为5～10 mg·kg-1。白头翁皂苷B4对小鼠静脉给药的LD50为3. 36 g·kg-1,95%可信限为3. 34～3. 37 g·kg-1,根据小鼠最大给药剂量20 mg·kg-1,计算出其临床安全指数为168(3. 36/0. 02),具有较高的临床安全范围。结论:白头翁皂苷B4对于实验性急性动物肾功能损伤具有明确的保护作用,同时以其较大的临床安全指数提示该化合物具有较好的新药开发前景。
Objective: To investigate the protective effect of Pulsatilla saponin B4 on acute renal injury and acute toxicity in mice. Methods: In vitro,the cell experiments were performed to induce HEK293 injury model by LPS,and B4 was used for 24 and 48 hours. MTT assay was used to detect the activity of cells and the expression of inflammatory factors by q PCR to observe the effect of B4 on renal cell injury. The animal experiments were conducted in rats and mice. In pharmacological study,rats or mice were given glycerol,cisplatin,and lipopolysaccharide( LPS) respectively for acute renal injury. B4 was injected into the caudal vein at different doses. After given for a certain period of time,orbital blood was collected and the serum was separated. Serum BUN,creatinine( Cre),total protein( TP) and rat's urinary protein in rats were determined to evaluate the effect of B4 on the renal damage. In the acute toxicity,mice were injected intravenously with different doses of B4 for2 weeks. According to the death,the half-lethal dose( LD50) was calculated. Results: B4 could increase the activity of injured renal cells and down-regulate the expression of inflammatory factor TNF-α. It also inhibit the increase of serum BUN,Cre induced by cisplatin and LPS caused by increased significantly. At the same time,B4 with high dose( 2. 5 mg·kg-1) could also significantly reduce rat urine protein concentration. Its effective dose were 1. 25-2. 5 mg·kg-1in rats,and 5-10 mg·kg-1in mice. The LD50 for intravenous administration of B4 to mice was 3. 36 g·kg-1and the 95% confidence limit was 3. 34 to 3. 37 g·kg-1. And therefore,the clinical safety index of B4 was 168( 3. 36/0. 02) based on the maximum dose of 20 mg·kg-1in mice,showing B4 has a high clinical safety range. Conclusion: B4 has definite protective effect on experimental acute renal impairment,and meanwhile its large clinical safety index suggests that this compound has a good prospects for the development of new drugs.
Objective: To investigate the protective effect of Pulsatilla saponin B4 on acute renal injury and acute toxicity in mice. Methods: In vitro,the cell experiments were performed to induce HEK293 injury model by LPS,and B4 was used for 24 and 48 hours. MTT assay was used to detect the activity of cells and the expression of inflammatory factors by q PCR to observe the effect of B4 on renal cell injury. The animal experiments were conducted in rats and mice. In pharmacological study,rats or mice were given glycerol,cisplatin,and lipopolysaccharide( LPS) respectively for acute renal injury. B4 was injected into the caudal vein at different doses. After given for a certain period of time,orbital blood was collected and the serum was separated. Serum BUN,creatinine( Cre),total protein( TP) and rat's urinary protein in rats were determined to evaluate the effect of B4 on the renal damage. In the acute toxicity,mice were injected intravenously with different doses of B4 for2 weeks. According to the death,the half-lethal dose( LD50) was calculated. Results: B4 could increase the activity of injured renal cells and down-regulate the expression of inflammatory factor TNF-α. It also inhibit the increase of serum BUN,Cre induced by cisplatin and LPS caused by increased significantly. At the same time,B4 with high dose( 2. 5 mg·kg-1) could also significantly reduce rat urine protein concentration. Its effective dose were 1. 25-2. 5 mg·kg-1in rats,and 5-10 mg·kg-1in mice. The LD50 for intravenous administration of B4 to mice was 3. 36 g·kg-1and the 95% confidence limit was 3. 34 to 3. 37 g·kg-1. And therefore,the clinical safety index of B4 was 168( 3. 36/0. 02) based on the maximum dose of 20 mg·kg-1in mice,showing B4 has a high clinical safety range. Conclusion: B4 has definite protective effect on experimental acute renal impairment,and meanwhile its large clinical safety index suggests that this compound has a good prospects for the development of new drugs.
引文
[1]梁勇满,赵容,许亮,等.中药白头翁本草考证与中国白头翁属植物分类[J].中国实验方剂学杂志,2017,23(5):203-209.
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[3]管咏梅,孙振,张文秀,等.白头翁总皂苷提取物的基本理化性质考察[J].中国实验方剂学杂志,2014,20(14):41-44.
[4]俞冰荟,饶毅,杨世林,等.白头翁提取物中总皂苷及皂苷类成分的测定[J].中国实验方剂学杂志,2014,20(15):65-68.
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[8]陈凯,张文明,喻凯,等.白头翁中抗肿瘤物质的筛选及其作用机制研究[J].天然产物研究与开发,2016,28(12):1875-1879.
[9]罗颖颖,陈兰英,简晖,等.白头翁皂苷调节Bel-7402人肝癌异体移植瘤裸鼠能量代谢的研究[J].中草药,2014,4(7):973-977.
[10]罗颖颖,陈兰英,崔亚茹,等.白头翁皂苷抑制人HT29结肠癌细胞增殖及诱导凋亡作用研究[J].中药药理与临床,2013,29(5):52-56.
[11] LIU M,ZHAO X,XIAO L,et al. Cytotoxicity of the compounds isolated from Pulsatilla chinensis saponins and apoptosis induced by 23-hydroxybetulinic acid[J]. Pharm Biol,2015,53(1):1-9.
[12] CHEN Z,GUAN Y,ZHOU L,et al. Preparation and Characterization of Colon-Targeted Particles of Pulsatilla chinensis Saponins[J]. Nat Prod Commun,2015,10(2):237-238.
[13]罗颖颖,严新,谢欣序,等.白头翁总皂苷及其主要成分抗肿瘤药效及其构效关系的研究[J].中国现代中药,2018,20(7):791-796.
[14]杨浩强,黄向阳,孟晓燕,等. IL-18、TNF-α、CRP在甘油致大鼠急性肾损伤中的变化及意义[J].海南医学,2015,26(17):2506-2509.
[15]赵晓琴,刘唐威,邝晓聪.甘油致大鼠急性肾功能衰竭肾小管细胞凋亡的动态实验观察[J].中华急诊医学杂志,2003,12(3):167-170.
[16]董思敏,王海璐,王全凯,等.鹿茸水提物减轻顺铂所致的小鼠肾损伤[J].中国病理生理杂志,2016,32(8):1466-1470.
[17]朱方强,江军,黄宏.姜黄素抑制内毒素所致小鼠急性肾功能损害的作用[J].第三军医大学学报,2005,27(11):1074-1077.
[18]杨树勤.中国医学百科全书:医学统计学[M].上海:上海科学技术出版社,1992:196-204.
[19]赵晓琴,刘唐威,邝晓聪,等.甘油致大鼠急性肾功能衰竭肾小管细胞凋亡的动态实验观察[J].中华急诊医学杂志,2003,12(3):167-170.
[20]郭德玉,李斌,张丽,等.甘油致急性肾功能衰竭大鼠模型[J].中国实验动物学杂志,1997,7(4):212-215.
[21]梁正林,邝晓聪,温冠媚,等.大鼠急性肾功能衰竭中细胞凋亡的实验观察及其意义[J].广西医科大学学报,2008,25(1):25-26.
[22]钱超.癌症患者顺铂化疗相关肾毒性的临床进展[J].实用癌症杂志,2014,29(5):607-609.
[23]张静,周文.顺铂肾毒性相关机制及其防护研究现状[J].中国药学杂志,2012,47(22):1785-1789.
[24]孟晓燕.顺铂致急性肾损伤的研究进展[J].医学综述,2014,20(21):3949-3951.
[25]朱晓玲,Li Ke,王永钧,等. LPS对BALB/C小鼠肾小管上皮细胞TLR4和TNFα、IL-6表达的影响[J].医学研究杂志,2008,37(1):40-43.
[26]袁玉荣. Toll样受体4在急性肾功能衰竭中的作用[J].黑龙江医药,2010,23(6):907-910.
[27]孙佳,夏海鸣.内毒素休克大鼠血清中TNF-α、皮质醇、LPS的测定及肾上腺的病理改变[J].东南大学学报(医学版),2011,30(5):728-732.
[28] KELLUM A J,PROWLE J R. Paradigms of acute kidney injury in the intensive care setting[J]. Nat Rev Nephrol,2018,14(4):217-230.
[2] JIN M M,ZHANG W D,XU Y M,et al. Simultaneous determination of 12 active components in the roots of Pulsatilla chinensis using tissue-smashing extraction with liquid chromatography and mass spectrometry[J]. J Sep Sci,2017,40(6):1283-1292.
[3]管咏梅,孙振,张文秀,等.白头翁总皂苷提取物的基本理化性质考察[J].中国实验方剂学杂志,2014,20(14):41-44.
[4]俞冰荟,饶毅,杨世林,等.白头翁提取物中总皂苷及皂苷类成分的测定[J].中国实验方剂学杂志,2014,20(15):65-68.
[5]杨连荣,张丽杰,姚姣姣,等.白头翁皂苷B4在正常大鼠和溃疡性结肠炎大鼠体内组织分布情况的比较[J].中国实验方剂学杂志,2017,23(21):93-97.
[6]缪成贵,周国梁,秦梅颂,等.白头翁皂苷对佐剂性关节炎大鼠FZD8表达的影响[J].中国中药杂志,2015,40(20):4063-4067.
[7] XU Q M,SHU Z,HE W J,et al. Antitumor activity of Pulsatilla chinensis(Bunge)Regel saponins in human liver tumor 7402 cells in vitro and in vivo[J]. Phytomedicine,2012,19(3/4):293-300.
[8]陈凯,张文明,喻凯,等.白头翁中抗肿瘤物质的筛选及其作用机制研究[J].天然产物研究与开发,2016,28(12):1875-1879.
[9]罗颖颖,陈兰英,简晖,等.白头翁皂苷调节Bel-7402人肝癌异体移植瘤裸鼠能量代谢的研究[J].中草药,2014,4(7):973-977.
[10]罗颖颖,陈兰英,崔亚茹,等.白头翁皂苷抑制人HT29结肠癌细胞增殖及诱导凋亡作用研究[J].中药药理与临床,2013,29(5):52-56.
[11] LIU M,ZHAO X,XIAO L,et al. Cytotoxicity of the compounds isolated from Pulsatilla chinensis saponins and apoptosis induced by 23-hydroxybetulinic acid[J]. Pharm Biol,2015,53(1):1-9.
[12] CHEN Z,GUAN Y,ZHOU L,et al. Preparation and Characterization of Colon-Targeted Particles of Pulsatilla chinensis Saponins[J]. Nat Prod Commun,2015,10(2):237-238.
[13]罗颖颖,严新,谢欣序,等.白头翁总皂苷及其主要成分抗肿瘤药效及其构效关系的研究[J].中国现代中药,2018,20(7):791-796.
[14]杨浩强,黄向阳,孟晓燕,等. IL-18、TNF-α、CRP在甘油致大鼠急性肾损伤中的变化及意义[J].海南医学,2015,26(17):2506-2509.
[15]赵晓琴,刘唐威,邝晓聪.甘油致大鼠急性肾功能衰竭肾小管细胞凋亡的动态实验观察[J].中华急诊医学杂志,2003,12(3):167-170.
[16]董思敏,王海璐,王全凯,等.鹿茸水提物减轻顺铂所致的小鼠肾损伤[J].中国病理生理杂志,2016,32(8):1466-1470.
[17]朱方强,江军,黄宏.姜黄素抑制内毒素所致小鼠急性肾功能损害的作用[J].第三军医大学学报,2005,27(11):1074-1077.
[18]杨树勤.中国医学百科全书:医学统计学[M].上海:上海科学技术出版社,1992:196-204.
[19]赵晓琴,刘唐威,邝晓聪,等.甘油致大鼠急性肾功能衰竭肾小管细胞凋亡的动态实验观察[J].中华急诊医学杂志,2003,12(3):167-170.
[20]郭德玉,李斌,张丽,等.甘油致急性肾功能衰竭大鼠模型[J].中国实验动物学杂志,1997,7(4):212-215.
[21]梁正林,邝晓聪,温冠媚,等.大鼠急性肾功能衰竭中细胞凋亡的实验观察及其意义[J].广西医科大学学报,2008,25(1):25-26.
[22]钱超.癌症患者顺铂化疗相关肾毒性的临床进展[J].实用癌症杂志,2014,29(5):607-609.
[23]张静,周文.顺铂肾毒性相关机制及其防护研究现状[J].中国药学杂志,2012,47(22):1785-1789.
[24]孟晓燕.顺铂致急性肾损伤的研究进展[J].医学综述,2014,20(21):3949-3951.
[25]朱晓玲,Li Ke,王永钧,等. LPS对BALB/C小鼠肾小管上皮细胞TLR4和TNFα、IL-6表达的影响[J].医学研究杂志,2008,37(1):40-43.
[26]袁玉荣. Toll样受体4在急性肾功能衰竭中的作用[J].黑龙江医药,2010,23(6):907-910.
[27]孙佳,夏海鸣.内毒素休克大鼠血清中TNF-α、皮质醇、LPS的测定及肾上腺的病理改变[J].东南大学学报(医学版),2011,30(5):728-732.
[28] KELLUM A J,PROWLE J R. Paradigms of acute kidney injury in the intensive care setting[J]. Nat Rev Nephrol,2018,14(4):217-230.