生淫羊藿与炙淫羊藿对肾阳虚证水肿模型大鼠的影响
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摘要
目的:研究生品与炮制后的淫羊藿对肾阳虚水肿模型大鼠的作用,为阐明炮制前后淫羊藿治疗肾阳虚水肿的作用机制。方法:用氢化可的松联合盐酸多柔比星复制肾阳虚水肿大鼠模型,即造模第1,8天分别尾静脉注射盐酸多柔比星(3. 5 mg·kg-1),与此同时,连续14 d腹腔注射强化可的松注射液(3. 75 mg·kg-1·d-1)进行造模,造模结束后将造模成功的大鼠分为模型组,生淫羊藿组(204. 86 mg·kg-1),炙淫羊藿组(204. 86 mg·kg-1),同时设立正常组,各组大鼠分别给予相应药物(10 m L·kg-1),正常组和模型组给予同体积蒸馏水,连续灌胃14 d。给药结束后,大鼠代谢笼法测24 h尿量,采用紫外分光光度法测定尿液中尿蛋白的含量;血清中指标的测定,采用全自动生化分析仪测血浆白蛋白(ALB),血清总蛋白(TP),血清肌酐(SCr),血清尿素氮(BUN)含量;采用酶联免疫测定(ELISA)检测环磷酸腺苷(c AMP),环磷酸鸟苷(c GMP),三碘甲状腺原氨酸(T3),甲状腺素(T4),雌二醇(E2),睾酮(T)指标含量,取肾脏适量做苏木素-伊红(HE)染色病理切片。结果:与正常组比较,模型组大鼠从外观指标,生化指标,病理切片指标上均证明模型组大鼠处于肾阳虚水肿状态(P <0. 05,P <0. 01);与模型组比较,生淫羊藿给药组与炙淫羊藿给药组对肾阳虚水肿的外观指标与病理切片指标均有回调作用,且在生化指标上,生、炙淫羊藿给药组均能回调尿量,尿蛋白,SCr,BUN,c AMP/c GMP,E2,T指标(P <0. 05,P <0. 01),但是回调强度有所不同。生淫羊藿给药组极显著性回调尿量,尿蛋白,SCr,BUN(P <0. 01);炙淫羊藿给药组极显著性回调c AMP/c GMP,E2,T(P <0. 01)。此外,生淫羊藿给药组还可以回调ALB,TP(P <0. 05,P <0. 01),而炙淫羊藿给药组对这2个指标没有回调作用,炙淫羊藿给药组可以回调T3,T4,肛温(P <0. 05,P <0. 01),而生淫羊藿给药组对这2个指标没有回调作用。结论:生淫羊藿与炙淫羊藿给药组均可以治疗肾阳虚水肿,机制可能与改善阿霉素所致的肾小球足细胞损伤有关。生淫羊藿与炙淫羊藿治疗肾阳虚水肿侧重点有所不同,生淫羊藿性寒,更侧重于加强肾阳虚水肿大鼠的肾脏排泄功能来治疗肾阳虚水肿。炙淫羊藿经羊油炮制后产生了淫羊藿苷等物质,药性由寒转温,偏重改善肾阳虚水肿大鼠肾阳虚状态来治疗肾阳虚水肿。
Objective: To compare the effects between crude and processed Epimedii Folium on the rats of kidney-Yang deficiency and edema,in order to discuss the mechanism. Method: The rat model of kidney-Yang edema was duplicated with hydrocortisone and doxorubicin hydrochloride. At day 1 and day 8 of modeling,the rats were injected with doxorubicin hydrochloride(3. 5 mg·kg-1) via tail vein,while being intraperitoneally injected with hydrocortisone(3. 75 mg·kg-1·d-1) for 15 days. After modeling,the rats were divided into model group,crude Epimedii Folium group(204. 86 mg·kg-1) and processed Epimedii Folium group(204. 86 mg·kg-1),and a normal control group was set up additionally. Rats in each treatment group were given the corresponding drugs,and those in normal and model groups were given the same volume of normal saline by gavage for continuous 14 days. At the end of the administration,24 h urine was measured by rat metabolic cage method; 24 h urinary protein was detected by ultraviolet spectrophotometry; the content of plasma albumin(ALB) and total serum protein(TP) were measured by an automatic biochemical analyzer; and the content of adenosine monophosphate was measured by enzyme-linked immunosorbent assay(ELISA). Cyclic adenosine monophosphate(cAMP),cyclic guanosine monophosphate(cGMP),triiodothyronine(T3),thyroxine(T4),serum creatinine(SCr),serum urea(BUN),estradiol(E2),testosterone(T) indicator content were determined by enzyme-linked immunosorbent assay(ELISA). Kidney tissues were collected to make pathological section by htoxylin eosin(HE) staining. Result: Compared with the normal group,all the indicators of appearance,biochemistry and pathological section in the model group indicated kidney Yang deficiency edema in the rats(P < 0. 05,P < 0. 01).Compared with the model group,both crude Epimedii Folium group and processed Epimedii Folium group showed a regulatory effect on indicators of appearance and pathological section kidney Yang deficiency edema; and in biochemical indicators,both crude Epimedii Folium group and processed Epimedii Folium group can regulate urine,urine protein,SCr,BUN,cAMP/cGMP,E2,T indicators to varying degrees(P < 0. 05,P < 0. 01).Crude Epimedii Folium group showed a significantly regulatory effect on urine,urine protein,SCr,BUN(P <0. 01); whereas processed Epimedii Folium group showed a significantly regulatory effect on cAMP/cGMP,E2,T(P < 0. 01). In addition,crude Epimedii Folium group can also regulate ALB,TP(P < 0. 05,P < 0. 01),while processed Epimedii Folium group had no regulatory effect on these two indicators; however,processed Epimedii Folium group could regulate T3,T4 and anal temperature(P < 0. 05,P < 0. 01),while crude Epimedii Folium group had no regulatory effect on these two indicators. Conclusion: Both crude Epimedii Folium group and processed Epimedii Folium group have certain effect in treating kidney Yang deficiency edema,and the possible mechanism is to alleviate glomerular podocyte injury induced by doxorubicin. Their emphasis in the treatment of kidney Yang deficiency edema is different. Crude Epimedii Folium has a cold property,and can treat kidney Yang deficiency edema by strengthening renal excretion function of rats with kidney Yang deficiency edema. Processed Epimedii Folium is processed with sheep oil to produce icariin and other substances,and its property changed from cold to warm,with a focus on alleviating the kidney Yang deficiency status of rats with kidney Yang deficiency edema.
Objective: To compare the effects between crude and processed Epimedii Folium on the rats of kidney-Yang deficiency and edema,in order to discuss the mechanism. Method: The rat model of kidney-Yang edema was duplicated with hydrocortisone and doxorubicin hydrochloride. At day 1 and day 8 of modeling,the rats were injected with doxorubicin hydrochloride(3. 5 mg·kg-1) via tail vein,while being intraperitoneally injected with hydrocortisone(3. 75 mg·kg-1·d-1) for 15 days. After modeling,the rats were divided into model group,crude Epimedii Folium group(204. 86 mg·kg-1) and processed Epimedii Folium group(204. 86 mg·kg-1),and a normal control group was set up additionally. Rats in each treatment group were given the corresponding drugs,and those in normal and model groups were given the same volume of normal saline by gavage for continuous 14 days. At the end of the administration,24 h urine was measured by rat metabolic cage method; 24 h urinary protein was detected by ultraviolet spectrophotometry; the content of plasma albumin(ALB) and total serum protein(TP) were measured by an automatic biochemical analyzer; and the content of adenosine monophosphate was measured by enzyme-linked immunosorbent assay(ELISA). Cyclic adenosine monophosphate(cAMP),cyclic guanosine monophosphate(cGMP),triiodothyronine(T3),thyroxine(T4),serum creatinine(SCr),serum urea(BUN),estradiol(E2),testosterone(T) indicator content were determined by enzyme-linked immunosorbent assay(ELISA). Kidney tissues were collected to make pathological section by htoxylin eosin(HE) staining. Result: Compared with the normal group,all the indicators of appearance,biochemistry and pathological section in the model group indicated kidney Yang deficiency edema in the rats(P < 0. 05,P < 0. 01).Compared with the model group,both crude Epimedii Folium group and processed Epimedii Folium group showed a regulatory effect on indicators of appearance and pathological section kidney Yang deficiency edema; and in biochemical indicators,both crude Epimedii Folium group and processed Epimedii Folium group can regulate urine,urine protein,SCr,BUN,cAMP/cGMP,E2,T indicators to varying degrees(P < 0. 05,P < 0. 01).Crude Epimedii Folium group showed a significantly regulatory effect on urine,urine protein,SCr,BUN(P <0. 01); whereas processed Epimedii Folium group showed a significantly regulatory effect on cAMP/cGMP,E2,T(P < 0. 01). In addition,crude Epimedii Folium group can also regulate ALB,TP(P < 0. 05,P < 0. 01),while processed Epimedii Folium group had no regulatory effect on these two indicators; however,processed Epimedii Folium group could regulate T3,T4 and anal temperature(P < 0. 05,P < 0. 01),while crude Epimedii Folium group had no regulatory effect on these two indicators. Conclusion: Both crude Epimedii Folium group and processed Epimedii Folium group have certain effect in treating kidney Yang deficiency edema,and the possible mechanism is to alleviate glomerular podocyte injury induced by doxorubicin. Their emphasis in the treatment of kidney Yang deficiency edema is different. Crude Epimedii Folium has a cold property,and can treat kidney Yang deficiency edema by strengthening renal excretion function of rats with kidney Yang deficiency edema. Processed Epimedii Folium is processed with sheep oil to produce icariin and other substances,and its property changed from cold to warm,with a focus on alleviating the kidney Yang deficiency status of rats with kidney Yang deficiency edema.
引文
[1]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2015:320.
[2]牛锐等.淫羊藿炮制前后对小鼠血浆睾酮及附性器官的影响[J].中国中药杂志,1989,14(8):14-20.
[3]李苗,曾梦楠,张贝贝,等.麻黄水煎液及拆分组分对肾阳虚水肿大鼠的影响[J].中国实验方剂学杂志,2017,23(22):218-223.
[4]王洪玉,陈平平,董婉茹,等.阿霉素诱发肾性水肿病症大鼠模型的建立[J].中国比较医学杂志,2016,26(12):6-9.
[5]吴珊珊,龚晓猛,张美等.缩泉丸中益智仁盐炙前后对肾阳虚大鼠肾脏功能的改善作用[J].中国实验方剂学杂志,2016,22(5):1-4.
[6]谭成,冉小库,窦德强.等.白术对大鼠肾阳虚型下焦水肿影响的实验研究[J].中华中医药学刊,2017,35(2):352-354.
[7]刘嘉琪,王秋红,匡海学.防己与茯苓配伍对肾性水肿模型大鼠的影响[J].中华中医药杂志,2017,32(9):4248-4251.
[8]郑筱萸.中药新药临床研究指导原则[M].北京:中国医药科技出版社,2002:385-388.
[9] Quinn C J,Burns P D,Gibson N M,et al. Effects of chronic endurance exercise on doxorubicin-induced thymic damage[J]. Integr Cancer Ther,2016,15(4):535-541.
[10] Park E J,Kwon H K,Choi Y M,et al. Doxorubicin induces cytotoxicity through upregulation of p ERKdependent ATF3[J]. PLo S One,2012,7(9):44-50.
[11]陈英华,欧阳轶强,孙琪,等.肾阳虚证动物模型规范化研究中诊断指标选择的初步探讨[J].中国中医基础医学杂志,2003,9(10):26-30.
[12]崔凯恒,嵇冰.肾阳虚证动物模型现代评价指标研究概述[J].甘肃中医药大学学报,2016,33(1):75-78.
[13]吴文辉,胡麟,冯健,等.淫羊藿炮制前后对正常和肾阳虚小鼠肾上腺VC水平的影响[J].中成药,2014,34(11):2397-2399.
[14]周一帆,星点设计-效应面法优选淫羊藿油炙工艺[J].中国实验方剂学杂志,2012,18(16):16-18.
[15]卢文丽,方肇勤,潘志强,等.小鼠气、血、阴、阳、虚等八种模型四诊的比较和评价[J].中国中医基础医学杂志,2006,12(6):433-438.
[16]张悦,魏民,王谦,等.阿霉素复制大鼠微小病变肾病模型的研究[J].临床与实验病理学杂志,2001,4(17):178-179.
[17]熊瑞,胡昌江,张美,等.生、炙淫羊藿对肾阳虚小鼠耐寒能力和交配能力研究[J].亚太传统医药,2015,11(3):5-7.
[18]李寅超,何永侠,孙曼,等.比较以不同品质的羊脂油炙淫羊藿的温肾阳作用[J].中国实验方剂学杂志,2013,19(19):197-202.
[2]牛锐等.淫羊藿炮制前后对小鼠血浆睾酮及附性器官的影响[J].中国中药杂志,1989,14(8):14-20.
[3]李苗,曾梦楠,张贝贝,等.麻黄水煎液及拆分组分对肾阳虚水肿大鼠的影响[J].中国实验方剂学杂志,2017,23(22):218-223.
[4]王洪玉,陈平平,董婉茹,等.阿霉素诱发肾性水肿病症大鼠模型的建立[J].中国比较医学杂志,2016,26(12):6-9.
[5]吴珊珊,龚晓猛,张美等.缩泉丸中益智仁盐炙前后对肾阳虚大鼠肾脏功能的改善作用[J].中国实验方剂学杂志,2016,22(5):1-4.
[6]谭成,冉小库,窦德强.等.白术对大鼠肾阳虚型下焦水肿影响的实验研究[J].中华中医药学刊,2017,35(2):352-354.
[7]刘嘉琪,王秋红,匡海学.防己与茯苓配伍对肾性水肿模型大鼠的影响[J].中华中医药杂志,2017,32(9):4248-4251.
[8]郑筱萸.中药新药临床研究指导原则[M].北京:中国医药科技出版社,2002:385-388.
[9] Quinn C J,Burns P D,Gibson N M,et al. Effects of chronic endurance exercise on doxorubicin-induced thymic damage[J]. Integr Cancer Ther,2016,15(4):535-541.
[10] Park E J,Kwon H K,Choi Y M,et al. Doxorubicin induces cytotoxicity through upregulation of p ERKdependent ATF3[J]. PLo S One,2012,7(9):44-50.
[11]陈英华,欧阳轶强,孙琪,等.肾阳虚证动物模型规范化研究中诊断指标选择的初步探讨[J].中国中医基础医学杂志,2003,9(10):26-30.
[12]崔凯恒,嵇冰.肾阳虚证动物模型现代评价指标研究概述[J].甘肃中医药大学学报,2016,33(1):75-78.
[13]吴文辉,胡麟,冯健,等.淫羊藿炮制前后对正常和肾阳虚小鼠肾上腺VC水平的影响[J].中成药,2014,34(11):2397-2399.
[14]周一帆,星点设计-效应面法优选淫羊藿油炙工艺[J].中国实验方剂学杂志,2012,18(16):16-18.
[15]卢文丽,方肇勤,潘志强,等.小鼠气、血、阴、阳、虚等八种模型四诊的比较和评价[J].中国中医基础医学杂志,2006,12(6):433-438.
[16]张悦,魏民,王谦,等.阿霉素复制大鼠微小病变肾病模型的研究[J].临床与实验病理学杂志,2001,4(17):178-179.
[17]熊瑞,胡昌江,张美,等.生、炙淫羊藿对肾阳虚小鼠耐寒能力和交配能力研究[J].亚太传统医药,2015,11(3):5-7.
[18]李寅超,何永侠,孙曼,等.比较以不同品质的羊脂油炙淫羊藿的温肾阳作用[J].中国实验方剂学杂志,2013,19(19):197-202.