基于代谢组学的小檗皮对STZ诱导糖尿病大鼠肾病的保护机制研究
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摘要
目的:基于代谢组学方法,探索小檗皮对STZ诱导糖尿病肾病(DN)大鼠的保护机制,发现相关血清代谢标志物。方法:腹腔注射小剂量链脲佐菌素(STZ)复制DN大鼠模型,造模成功后,分别灌胃给予小檗皮0.7 g/kg水浸膏组、盐酸小檗碱+盐酸小檗胺171 mg/kg+48 mg/kg组和二甲双胍0.196 g/kg组,空白组和模型组给予等体积的生理盐水,每天1次,连续4周,同时检测空腹血糖水平(FBG)。采集大鼠血清检测血尿素氮(BUN)、肌酐(Scr)、尿酸(UA)、总蛋白(TP)、转换生长因子-β1(TGF-β1)和血管内皮生长因子(VEGF)的含量,电镜下观察肾脏病理形态学变化及测定肾小球基底膜厚度。同时,大鼠血清采用LC-MS代谢组学技术,主成分分析(PCA)、偏最小二乘判别分析(PLS-DA)等方法筛选并鉴定与DN相关的生物标志物,利用MetPa数据库分析相关代谢通路。结果:与空白组相比,模型组血清FBG、BUN、Scr、UA、TGF-β1和VEGF显著升高,TP显著降低;与模型组相比,给药组血清FBG、BUN、Scr、UA、TGF-β1和VEGF明显降低,TP显著升高;电镜结果表明给药组均能改善大鼠肾组织病理损伤,减少肾小球基底膜厚度。代谢组学结果表明给药组的代谢物能够显著区分,发现并初步鉴定了17个差异代谢物及6条相关通路。结论:小檗皮能够改善DN的病理变化和药效学指标,其作用机制可能为上调DN大鼠血清中的鸟氨酸含量,参与精氨酸与脯氨酸的代谢,而盐酸小檗碱、盐酸小檗胺可能是小檗皮发挥治疗DN作用的物质基础。
Objective: To study the protective effect and mechanism of Berberidis dictyophyllae Cortex on STZ induced diabetic nephropathy(DN) rat, and to discover the relative metabolic markers in serum. Methods: DN rat model was established by intraperitoneal injection of streptozotocin(STZ). Then Berberidis dictyophyllae Cortex extractum(0.7 g/kg), berberine hydrochloride + berbamine hydrochloride(171 mg/kg+48 mg/kg) and metformin(0.196 g/kg) were administered, rats in the control and the model group were given equal volume of normal saline once a day for 4 weeks. The level of FBG was determined. Rat serum was collected to detect the contents of blood urea nitrogen(BUN), creatinine(Scr), uric acid(UA), total protein(TP), transforming growth factor-beta 1(TGF-β1) and vascular endothelial growth factor(VEGF). The pathological morphological changes of the kidney and the thickness of the glomerular basement membrane were observed under electron microscope. Meanwhile, DN related biomarkers in rats serum were screened and identified by LC-MS metabolomics, principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA). And relevant metabolic pathways were analyzed by MetPa database. Results: Pharmacodynamic results showed that compared with the control group, the levels of blood glucose, BUN, Scr, UA, TGF-β1 and VEGF were significantly increased and TP was significantly decreased in the model group. Compared with the model group, the levels of blood glucose, BUN, Scr, UA, TGF-beta 1 and VEGF were significantly decreased, the level of TP was significantly in the drug administration groups. The electron microscopy results showed that the drug administration groups improved the renal histopathological injury and reduced the thickness of glomerular basement membrane. The metabolites in the drug administration groups were distinctly distinguished, 17 different metabolites and 6 related pathways were observed and preliminarily identified. Conclusion:Berberidis dictyophyllae Cortex treatment can ameliorat the pathological injury and pharmacodynamics of DN. Its mechanism may be related to up-regulating the content of ornithine in DN rat serum and participating in the metabolism of arginine and proline. Berberine hydrochloride and berbamine hydrochloride may be the material basis of Berberidis dictyophyllae Cortex in the treatment of DN.
Objective: To study the protective effect and mechanism of Berberidis dictyophyllae Cortex on STZ induced diabetic nephropathy(DN) rat, and to discover the relative metabolic markers in serum. Methods: DN rat model was established by intraperitoneal injection of streptozotocin(STZ). Then Berberidis dictyophyllae Cortex extractum(0.7 g/kg), berberine hydrochloride + berbamine hydrochloride(171 mg/kg+48 mg/kg) and metformin(0.196 g/kg) were administered, rats in the control and the model group were given equal volume of normal saline once a day for 4 weeks. The level of FBG was determined. Rat serum was collected to detect the contents of blood urea nitrogen(BUN), creatinine(Scr), uric acid(UA), total protein(TP), transforming growth factor-beta 1(TGF-β1) and vascular endothelial growth factor(VEGF). The pathological morphological changes of the kidney and the thickness of the glomerular basement membrane were observed under electron microscope. Meanwhile, DN related biomarkers in rats serum were screened and identified by LC-MS metabolomics, principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA). And relevant metabolic pathways were analyzed by MetPa database. Results: Pharmacodynamic results showed that compared with the control group, the levels of blood glucose, BUN, Scr, UA, TGF-β1 and VEGF were significantly increased and TP was significantly decreased in the model group. Compared with the model group, the levels of blood glucose, BUN, Scr, UA, TGF-beta 1 and VEGF were significantly decreased, the level of TP was significantly in the drug administration groups. The electron microscopy results showed that the drug administration groups improved the renal histopathological injury and reduced the thickness of glomerular basement membrane. The metabolites in the drug administration groups were distinctly distinguished, 17 different metabolites and 6 related pathways were observed and preliminarily identified. Conclusion:Berberidis dictyophyllae Cortex treatment can ameliorat the pathological injury and pharmacodynamics of DN. Its mechanism may be related to up-regulating the content of ornithine in DN rat serum and participating in the metabolism of arginine and proline. Berberine hydrochloride and berbamine hydrochloride may be the material basis of Berberidis dictyophyllae Cortex in the treatment of DN.
引文
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[3]Valencia Willy Marcos,Florez Hermes.How to prevent the microvascular complications of type 2 diabetes beyond glucose control.BMJ,2017,356∶i6505:1 ～ i6505:17.
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[7]李琪,杜欢,文焕松,等.藏药小檗皮中6种成分的含量测定及不同品种比较研究.中国中药杂志,2019,44(5)∶968～974.
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[9]向宇楠,王小艳,冯慧,等.外翻肠囊法考察盐酸小檗胺对盐酸小檗碱肠吸收特性的影响.中国实验方剂学杂志,DOI:10.13422/j.cnki.syfjx.20190749.
[10]叶凡,周邦华,王胜,等.藏药小檗膏提取物中3种生物碱含量的高效液相色谱法测定.时珍国医国药,2016,27(2)∶286 ～ 289.
[11]童东,王文倩,罗煜,等.藏药四味姜黄方对STZ诱导糖尿病肾病大鼠模型的剂量配比关系的初步研究.中成药,2018,40(3)∶516 ～ 524.
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[14]Goru Santosh Kumar,Gaikwad Anil Bhanudas.Novel reno-protective mechanism of Aspirin involves H2AK119 monoubiquitination and Set7 in preventing type 1 diabetic nephropathy.Pharmacol Rep,2018,70∶497 ～ 502.
[15]Chen Xia,Wang Hao,Jiang Minqian,et al.Huangqi (astragalus) decoction ameliorates diabetic nephropathy via IRS1-PI3K-GLUT signaling pathway.Am J Transl Res,2018,10∶2491 ～ 2501.
[16]Lenoir Olivia,Gaillard Fran?ois,Lazareth Hélène,et al.Deficiency sensitizes mice to peroxynitrite formation and diabetic glomerular microvascular injuries.J Diabetes Res,2017,1 ～ 7.
[17]Sutariya Brijesh,Saraf Madhusudan.Betanin,isolated from fruits of Opuntia elatior Mill attenuates renal fibrosis in diabetic rats through regulating oxidative stress and TGF-β pathway.J Ethnopharmacol,2017,198∶432 ～ 443.
[18]Ju Yinghui,Su Yong,Chen Qingqing,et al.Protective effects of Astragaloside IV on endoplasmic reticulum stress-induced renal tubular epithelial cells apoptosis in type 2 diabetic nephropathy rats.Biomed.Pharmacother.,2019,109∶84 ～ 92.
[19]郭晓玲,陈文军,檀金川,等.芪芍胶囊对糖尿病肾病大鼠的肾保护作用及对肾组织促血管生成素1表达的影响.中药药理与临床,2016,32(4)∶80 ～ 84.
[20]Sharma Dilip,Gondaliya Piyush,Tiwari Vinod,et al.Kaempferol attenuates diabetic nephropathy by inhibiting RhoA/Rho-kinase mediated inflammatory signalling.Biomed.Pharmacother.,2019,109∶1610 ～ 1619.
[21]Xiang Xiang,Cai Hong-Die,Su Shu-Lan,et al.Salvia miltiorrhiza protects against diabetic nephropathy through metabolome regulation and wnt/β-catenin and TGF-β signaling inhibition.Pharmacol.Res.,2018,139∶26 ～ 40.
[22]Tang Fengjuan,Hao Yarong,Zhang Xue,et al.Effect of echinacoside on kidney fibrosis by inhibition of TGF-β1/Smads signaling pathway in the db/db mice model of diabetic nephropathy.Drug Des Devel Ther.,2017,(11)∶2813 ～ 2826.
[23]Onions Karen L,Gamez Monica,Buckner Nicola R,et al.VEGFC reduces glomerular albumin permeability and protects against alterations in VEGF receptor expression in diabetic nephropathy.Diabetes,2019,68∶172 ～ 187.
[24]Zhao Guanhua,Hou Xilong,Li Xingyao,et al.Metabolomics analysis of alloxan-induced diabetes in mice using UPLC-Q-TOF-MS after Crassostrea gigas polysaccharide treatment.Int.J.Biol.Macromol.,2018,108∶550 ～ 557.
[25]Saleem Tahia,Dahpy Marwa,Ezzat Ghada,et al.The Profile of Plasma Free Amino Acids in Type 2 Diabetes Mellitus with Insulin Resistance:Association with Microalbuminuria and Macroalbuminuria.Appl.Biochem.Biotechnol,2019,DOI∶10.1007/s12010-019-02956-9.
[26]Du Yan,Xu Bing-Ju,Deng Xu,et al.Predictive metabolic signatures for the occurrence and development of diabetic nephropathy and the intervention of Ginkgo biloba leaves extract based on gas or liquid chromatography with mass spectrometry.J Pharm Biomed Anal,2018,166∶30 ～ 39.
[27]蔡稀,李蓉,曾粒,等.不同病因的慢性肾病患者血清氨基酸谱存在显著差异.中国生物化学与分子生物学报,2018,34(8)∶ 891 ～ 899.
[2]Wimmer Reiner A,Leopoldi Alexandra,Aichinger Martin,et al.Human blood vessel organoids as a model of diabetic vasculopathy.Nature,2019,565∶505 ～ 510.
[3]Valencia Willy Marcos,Florez Hermes.How to prevent the microvascular complications of type 2 diabetes beyond glucose control.BMJ,2017,356∶i6505:1 ～ i6505:17.
[4]帝玛尔·丹增彭措.晶珠本草.上海科学技术出版社.1,2012∶122.
[5]毛继祖,卡洛,毛韶玲.蓝琉璃.上海科学技术出版社.1,2012∶135.
[6]张静,吕秀梅,李艳,等.藏族药小檗皮不同药用部位中4种生物碱成分的HPLC含量测定.中国实验方剂学杂志,2017,23(2)∶37 ～ 42.
[7]李琪,杜欢,文焕松,等.藏药小檗皮中6种成分的含量测定及不同品种比较研究.中国中药杂志,2019,44(5)∶968～974.
[8]张燕,孟宪丽,岳丽珺,等.藏药小檗皮对糖尿病模型小鼠血糖水平影响的初步研究.现代生物医学进展,2013,13(19)∶3619 ～ 3622.
[9]向宇楠,王小艳,冯慧,等.外翻肠囊法考察盐酸小檗胺对盐酸小檗碱肠吸收特性的影响.中国实验方剂学杂志,DOI:10.13422/j.cnki.syfjx.20190749.
[10]叶凡,周邦华,王胜,等.藏药小檗膏提取物中3种生物碱含量的高效液相色谱法测定.时珍国医国药,2016,27(2)∶286 ～ 289.
[11]童东,王文倩,罗煜,等.藏药四味姜黄方对STZ诱导糖尿病肾病大鼠模型的剂量配比关系的初步研究.中成药,2018,40(3)∶516 ～ 524.
[12]Sangster T,Major H,Plumb R,et al.A pragmatic and readily implemented quality control strategy for HPLC-MS and GC-MS-based metabonomic analysis.Analyst,2006,131(10)∶1075 ～ 1078.
[13]Want E J,Masson P,Michopoulos F,et al.Global metabolic profiling of animal and human tissues via UPLC-MS.Nature Protocols,2012,8(1)∶17 ～ 32.
[14]Goru Santosh Kumar,Gaikwad Anil Bhanudas.Novel reno-protective mechanism of Aspirin involves H2AK119 monoubiquitination and Set7 in preventing type 1 diabetic nephropathy.Pharmacol Rep,2018,70∶497 ～ 502.
[15]Chen Xia,Wang Hao,Jiang Minqian,et al.Huangqi (astragalus) decoction ameliorates diabetic nephropathy via IRS1-PI3K-GLUT signaling pathway.Am J Transl Res,2018,10∶2491 ～ 2501.
[16]Lenoir Olivia,Gaillard Fran?ois,Lazareth Hélène,et al.Deficiency sensitizes mice to peroxynitrite formation and diabetic glomerular microvascular injuries.J Diabetes Res,2017,1 ～ 7.
[17]Sutariya Brijesh,Saraf Madhusudan.Betanin,isolated from fruits of Opuntia elatior Mill attenuates renal fibrosis in diabetic rats through regulating oxidative stress and TGF-β pathway.J Ethnopharmacol,2017,198∶432 ～ 443.
[18]Ju Yinghui,Su Yong,Chen Qingqing,et al.Protective effects of Astragaloside IV on endoplasmic reticulum stress-induced renal tubular epithelial cells apoptosis in type 2 diabetic nephropathy rats.Biomed.Pharmacother.,2019,109∶84 ～ 92.
[19]郭晓玲,陈文军,檀金川,等.芪芍胶囊对糖尿病肾病大鼠的肾保护作用及对肾组织促血管生成素1表达的影响.中药药理与临床,2016,32(4)∶80 ～ 84.
[20]Sharma Dilip,Gondaliya Piyush,Tiwari Vinod,et al.Kaempferol attenuates diabetic nephropathy by inhibiting RhoA/Rho-kinase mediated inflammatory signalling.Biomed.Pharmacother.,2019,109∶1610 ～ 1619.
[21]Xiang Xiang,Cai Hong-Die,Su Shu-Lan,et al.Salvia miltiorrhiza protects against diabetic nephropathy through metabolome regulation and wnt/β-catenin and TGF-β signaling inhibition.Pharmacol.Res.,2018,139∶26 ～ 40.
[22]Tang Fengjuan,Hao Yarong,Zhang Xue,et al.Effect of echinacoside on kidney fibrosis by inhibition of TGF-β1/Smads signaling pathway in the db/db mice model of diabetic nephropathy.Drug Des Devel Ther.,2017,(11)∶2813 ～ 2826.
[23]Onions Karen L,Gamez Monica,Buckner Nicola R,et al.VEGFC reduces glomerular albumin permeability and protects against alterations in VEGF receptor expression in diabetic nephropathy.Diabetes,2019,68∶172 ～ 187.
[24]Zhao Guanhua,Hou Xilong,Li Xingyao,et al.Metabolomics analysis of alloxan-induced diabetes in mice using UPLC-Q-TOF-MS after Crassostrea gigas polysaccharide treatment.Int.J.Biol.Macromol.,2018,108∶550 ～ 557.
[25]Saleem Tahia,Dahpy Marwa,Ezzat Ghada,et al.The Profile of Plasma Free Amino Acids in Type 2 Diabetes Mellitus with Insulin Resistance:Association with Microalbuminuria and Macroalbuminuria.Appl.Biochem.Biotechnol,2019,DOI∶10.1007/s12010-019-02956-9.
[26]Du Yan,Xu Bing-Ju,Deng Xu,et al.Predictive metabolic signatures for the occurrence and development of diabetic nephropathy and the intervention of Ginkgo biloba leaves extract based on gas or liquid chromatography with mass spectrometry.J Pharm Biomed Anal,2018,166∶30 ～ 39.
[27]蔡稀,李蓉,曾粒,等.不同病因的慢性肾病患者血清氨基酸谱存在显著差异.中国生物化学与分子生物学报,2018,34(8)∶ 891 ～ 899.