楤木总皂苷对2型糖尿病肾病小鼠VEGF和MMP-9蛋白表达的影响
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摘要
目的观察楤木总皂苷对2型糖尿病小鼠肾脏组织血管内皮生长因子(VEGF)和基质金属蛋白酶-9(MMP-9)表达的影响。方法 90只清洁级昆明小鼠适应性喂养10 d后,分为正常组(n=10)、5个造模组80只小鼠(模型组、阳性药物贝那普利干预组以及楤木总皂苷低、中和高剂量治疗组)。除正常组外所有小鼠高脂高糖饮食一个月+一次性链脲佐菌素(STZ)诱导2型糖尿病肾脏损害小鼠模型,模型构建成功共25只小鼠死亡(55只剩余小鼠随机分为每组11只),55只小鼠治疗6周后处死,检测各组BUN、SCr、血糖、胰岛素、炎性因子IL-1α、IL-6和TNF-α水平的变化,同时免疫组化和Western blot检测肾组织VEGF和MMP-9蛋白表达位置及表达水平。结果与正常组相比,模型组血糖、胰岛素、BUN、SCr、IL-1α、IL-6和TNF-α含量均明显升高,且差异均有统计学意义(P <0. 05),阳性药物组和楤木总皂苷三个剂量组上述指标均降低(P <0. 05),楤木总皂苷高剂量组血清BUN、SCr含量明显低于阳性药物组(P <0. 05),而楤木总皂苷三个剂量组血糖、IL-1α、IL-6和TNF-α水平明显低于阳性药物组(P <0. 05);与正常组相比,模型组VEGF蛋白表达水平明显升高(P <0. 05),MMP-9蛋白表达水平明显降低(P <0. 05);与模型组相比,阳性药物组和楤木总皂苷三个剂量组VEGF水平均降低(P <0. 05),MMP-9表达均升高(P <0. 05),此外楤木总皂苷高剂量组与阳性药物组VEGF、MMP-9水平差异无统计学意义(P>0. 05)。结论楤木总皂苷可明显降低2型糖尿病小鼠血糖、血清胰岛素和血清炎症因子,还可以下调VEGF蛋白、升高MMP-9蛋白水平,进而保护肾脏。
Objective To observe the effect of aralia saponins on the expression of vascular endothelial growth factor( VEGF) and matrix metalloproteinase-9( MMP-9) in the kidney of diabetic nephropathy mice. Methods After 10 days of adaptive feeding,90 clean Kunming mice were randomly divided into the normal group( n = 10) and 5 model groups( model group,positive drug benazepril intervention group,aralia saponins low,middle and high doses treatment groups). Excepted the normal group,the kidney damage model of type 2 diabetes mellitus in mice was induced by high-fat and high-sugar diet for one month plus disposable streptozotocin( STZ). The model was successfully constructed and killed after 6 weeks of treatment. A total of 25 mice failed to establish the model. And totally 55 mice were randomly divided into 5 groups with 11 mice in each group. The serum changes of blood urea nitrogen( BUN),serum creatinine( SCr),insulin,inflammatory factors interleukin-1α( IL-1α),interleukin-6( IL-6) and tumor necrosis factor-α( TNF-α) in each group were detected. The expression of vascular endothelial growth factor and MMP-9 protein in renal tissue were detected by immunohistochemistry and Western blot. Results Compared with the normal group,the levels of blood glucose,insulin,BUN,SCr,IL-1α,IL-6 and TNF-α in the model group were significantly increased( P < 0. 05). The above indexes were decreased in positive drug group and aralia saponins treatment groups. The contents of insulin,BUN and SCr in the high dose of aralia saponins group were significantly lower than those parameters in benazepril group( P < 0. 05). In addition,the contents of blood glucose,IL-1α,IL-6 and TNF-α in the three dose aralia saponins groups were significantly lower than those parameters in the benazepril group( P < 0. 05). Compared with the normal group,the expression level of VEGF protein in the model group was significantly higher( P < 0. 05),and the expression level of MMP-9 protein was significantly lower( P < 0. 05). Compared with the model group,both benazepril and aralia saponins can reduce VEGF( P < 0. 05),increase MMP-9( P < 0. 05). In addition for VEGF and MMP-9,the high dose of aralia saponins group and benazepril group was basically same.Conclusions Aralia saponins can significantly reduce blood glucose,insulin and serum inflammatory factors,while downregulate VEGF and increase MMP-9 protein levels,thereby protecting the kidneys of diabetic nephropathy mice.
Objective To observe the effect of aralia saponins on the expression of vascular endothelial growth factor( VEGF) and matrix metalloproteinase-9( MMP-9) in the kidney of diabetic nephropathy mice. Methods After 10 days of adaptive feeding,90 clean Kunming mice were randomly divided into the normal group( n = 10) and 5 model groups( model group,positive drug benazepril intervention group,aralia saponins low,middle and high doses treatment groups). Excepted the normal group,the kidney damage model of type 2 diabetes mellitus in mice was induced by high-fat and high-sugar diet for one month plus disposable streptozotocin( STZ). The model was successfully constructed and killed after 6 weeks of treatment. A total of 25 mice failed to establish the model. And totally 55 mice were randomly divided into 5 groups with 11 mice in each group. The serum changes of blood urea nitrogen( BUN),serum creatinine( SCr),insulin,inflammatory factors interleukin-1α( IL-1α),interleukin-6( IL-6) and tumor necrosis factor-α( TNF-α) in each group were detected. The expression of vascular endothelial growth factor and MMP-9 protein in renal tissue were detected by immunohistochemistry and Western blot. Results Compared with the normal group,the levels of blood glucose,insulin,BUN,SCr,IL-1α,IL-6 and TNF-α in the model group were significantly increased( P < 0. 05). The above indexes were decreased in positive drug group and aralia saponins treatment groups. The contents of insulin,BUN and SCr in the high dose of aralia saponins group were significantly lower than those parameters in benazepril group( P < 0. 05). In addition,the contents of blood glucose,IL-1α,IL-6 and TNF-α in the three dose aralia saponins groups were significantly lower than those parameters in the benazepril group( P < 0. 05). Compared with the normal group,the expression level of VEGF protein in the model group was significantly higher( P < 0. 05),and the expression level of MMP-9 protein was significantly lower( P < 0. 05). Compared with the model group,both benazepril and aralia saponins can reduce VEGF( P < 0. 05),increase MMP-9( P < 0. 05). In addition for VEGF and MMP-9,the high dose of aralia saponins group and benazepril group was basically same.Conclusions Aralia saponins can significantly reduce blood glucose,insulin and serum inflammatory factors,while downregulate VEGF and increase MMP-9 protein levels,thereby protecting the kidneys of diabetic nephropathy mice.
引文
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[19]邹淑君,许树军,李靖,等.辽东楤木叶皂苷成分及其药学研究进展[J].中医药学报,2017,45(4):111-114. DOI:10. 3969/j.issn. 1002-2392. 2017. 04. 031.
[20]谭玉婷,鲁卫星,赵俊男.龙牙楤木总皂苷对缺血再灌注损伤大鼠心肌细胞线粒体细胞色素C和膜电位的影响及其作用机制[J].西部中医药,2016,29(12):9-12. DOI:10. 3969/j. issn.1004-6852. 2016. 12. 003.
[2]熊燕,林瑗.糖尿病并发症的研究进展[J].中国医师杂志,2017,19(10):1441-1449. DOI:10. 3760/cma. j. issn. 1008-1372. 2017. 10. 001.
[3]李庆,宋学君,李志军.糖尿病肾病细胞因子的研究进展[J].中国中西医结合急救杂志,2017,24(1):99-100. DOI:10.3969/j. issn. 1008-9691. 2017. 01. 030.
[4] Rossi GP,Seccia TM,Barton M,et al. Endothelial factors in the pathogenesis and treatment of chronic kidney disease Part I:General mechanisms:a joint consensus statement from the European Society of Hypertension Working Group on Endothelin and Endothelial Factors and The Japanese Society of Hypertension[J]. J Hypertens,2018,36(3):451-461. DOI:10. 1097/HJH. 0000000000001599.
[5] Hanefeld M,Engelmann K,Appelt D,et al. Intra-individual variability and circadian rhythm of vascular endothelial growth factors in subjects with normal glucose tolerance and type 2 diabetes[J]. PLo S One,2017,12(10):e0184234. DOI:10. 1371/journal. pone. 0184234.
[6]田鲁,杨椹,王琦,等.楤木总皂苷对2型糖尿病模型小鼠肾组织Toll样受体的影响[J].国际中医中药杂志,2018,40(3):236-241. DOI:10. 3760/cma. j. issn. 1673-4246. 2018. 03. 011.
[7] Dabbs DJ. Diagnostic Immunohistochemistry E-Book:Theranostic and Genomic Applications[M]. Elsevier Health Sciences,2018.
[8]席秋江,韩莉,楼小亮.糖尿病大鼠模型的实验室研究[J].江西医药,2017,52(12):1295-1296,1299. DOI:10. 3969/j. issn.1006-2238. 2017. 12. 011.
[9]闫永恒,李渐鹏,刘战伟,等.不同2型糖尿病肾病模型建立方法及成模特点比较[J].中国食物与营养,2016,22(1):65-69.DOI:10. 3969/j. issn. 1006-9577. 2016. 01. 015.
[10]陈薇宇,李双蕾,谢婧,等. 2型糖尿病KKAy小鼠动物模型的研究进展[J].临床医药文献电子杂志,2017,4(54):10681-10682. DOI:10. 3877/j. issn. 2095-8242. 2017. 54. 149.
[11]蒋朝晖,吕玉晶,赵芳,等.高脂高糖饮食结合链脲佐菌素建立2型糖尿病大鼠模型的改良[J].中国比较医学杂志,2011,21(1):33-35,44,后插1. DOI:10. 3969/j. issn. 1671. 7856. 2011. 01. 007.
[12]陆少君,曾伟斌,臧林泉. 2型糖尿病大鼠模型制备实验研究[J].广东药科大学学报,2017,33(5):624-628. DOI:10.16809/j. cnki. 2096-3653. 2017062304.
[13]徐云,陈雪辉,尹清风. TGF-β1/SGK1信号通路在糖尿病肾病中的作用[J].中国医师杂志,2017,19(10):1483-1487. DOI:10. 3760/cma. j. issn. 1008-1372. 2017. 10. 008.
[14] Li SY,Huang PH,Yang AH,et al. Matrix metalloproteinase-9deficiency attenuates diabetic nephropathy by modulation of podocyte functions and dedifferentiation[J]. Kidney Int,2014,86(2):358-369. DOI:10. 1038/ki. 2014. 67.
[15] Kolseth IB,Reine TM,Parker K,et al. Increased levels of inflammatory mediators and proinflammatory monocytes in patients with type I diabetes mellitus and nephropathy[J]. J Diabetes Complications,2017,31(1):245-252. DOI:10. 1016/j. jdiacomp. 2016. 06. 029.
[16] Hsu YH,Li HH,Sung JM,et al. Interleukin-20 targets podocytes and is upregulated in experimental murine diabetic nephropathy[J].Exp Mol Med,2017,49(3):e310. DOI:10. 1038/emm. 2016. 169.
[17] Wallner C,Schira J,Wagner JM,et al. Application of VEGFA and FGF-9 enhances angiogenesis,osteogenesis and bone remodeling in type 2 diabetic long bone regeneration[J]. PLo S One,2015,10(3):e0118823. DOI:10. 1371/journal. pone. 0118823.
[18]刘军民,林励,丁平,等.广东产四种楤木根中齐墩果酸含量[J].中药材,2000,23(1):8-9. DOI:10. 3321/j. issn:1001-4454. 2000. 01. 004.
[19]邹淑君,许树军,李靖,等.辽东楤木叶皂苷成分及其药学研究进展[J].中医药学报,2017,45(4):111-114. DOI:10. 3969/j.issn. 1002-2392. 2017. 04. 031.
[20]谭玉婷,鲁卫星,赵俊男.龙牙楤木总皂苷对缺血再灌注损伤大鼠心肌细胞线粒体细胞色素C和膜电位的影响及其作用机制[J].西部中医药,2016,29(12):9-12. DOI:10. 3969/j. issn.1004-6852. 2016. 12. 003.