水飞蓟素灌胃对糖尿病肾病大鼠肾损伤的改善作用及其机制
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摘要
目的观察水飞蓟素(Sly)对糖尿病肾病(DN)大鼠肾损伤的改善作用,并探讨其作用机制。方法将40只SD大鼠随机分为4组(每组10只):糖尿病肾病组(DN组)、水飞蓟素低剂量治疗组(Sly-L组)、水飞蓟素高剂量治疗组(Sly-H组)、对照组。DN组、Sly-L组、Sly-H组均采用链脲佐菌素诱导制备DN大鼠模型。Sly-L组大鼠每24 h给予低剂量Sly(60 mg/kg)灌胃1次,Sly-H组大鼠每24 h给予高剂量Sly(120 mg/kg)灌胃1次,DN组、对照组给予等量生理盐水灌胃。各组大鼠灌胃8周后,采用代谢笼收集24 h尿液,取空腹鼠尾静脉血及眼眶血,然后处死大鼠,处死大鼠后迅速切取两侧肾脏,使用预冷生理盐水灌洗去除残留血液,滤纸吸干后备检。取鼠尾静脉血检测空腹血糖(FPG);取代谢笼收集的大鼠24 h尿液,采用双缩脲比色法测定24 h尿白蛋白和肌酐,计算尿白蛋白/肌酐比值(UACR);称取处死后大鼠体质量及肾脏质量,计算肾脏指数(肾脏重量/大鼠体质量,KI)。取大鼠眼眶血,采用ELISA法检测各组大鼠血清炎症细胞因子TNF-α、IL-1β、IL-6。取右肾组织,HE染色后光学显微镜下观察各组大鼠肾组织病理形态学变化,免疫组织化学法检测各组大鼠肾小管CD14。结果与对照组相比,DN组、Sly-L组及Sly-H组大鼠FPG、UACR、KI水平显著升高(P均<0. 01),体质量显著降低(P均<0. 01)。与DN组相比,Sly-L组、Sly-H组大鼠FPG、UACR、KI水平均显著降低(P均<0. 05)。DN组肾小球体积增大,部分肾小管管腔轻度或中度扩大,肾小管上皮细胞空泡样变性,可见炎性细胞浸润;与DN组相比,Sly-L组及Sly-H组肾小管和肾小球病理损伤减轻。与对照组相比,DN组、Sly-L组血清TNF-α、IL-1β、IL-6及肾小管CD14均显著升高(P均<0. 05),Sly-H组血清IL-6水平显著升高(P <0. 05)。与DN组相比,Sly-L组、Sly-H组血清TNF-α、IL-1β、IL-6及肾小管CD14均显著降低(P均<0. 05)。与Sly-L组相比,Sly-H组血清TNF-α、IL-1β、IL-6及肾小管CD14均显著降低(P均<0. 05)。结论 Sly可改善DN大鼠肾损伤,其作用机制与Sly可降低DN大鼠血清炎症细胞因子TNF-α、IL-1β、IL-6的水平并抑制肾小管CD14的表达有关。
Objective To explore the protective effect of silymarin( Sly) on the diabetic nephropathy( DN) rats and its possible mechanisms. Methods Forty Sprague-Dawley( SD) rats( 8 weeks old,weight 180-200 g) were randomly divided into four groups( 10 rats per group) : DN group,low-dose Sly treatment group( Sly-L group),high-dose Sly treatment group( Sly-H group),and normal control group. Rats in the DN group,Sly-L group,and Sly-H group were induced by streptozotocin( STZ,60 mg/kg). Rats in the Sly-L group and Sly-H group were given intragastric administration of lowdose Sly( 60 mg/Kg/d) and high-dose Sly( 120 mg/Kg/d) every 24 hours,respectively; rats in the DN group and the control group were treated with the same amount of normal saline. After 8 weeks treatment,tail vein blood was used for measuring the level of fasting blood glucose( FPG); metabolic cages were used for collecting 24 h urine,and urine albuminuria/creatinine( UACR) was calculated. Then all rats were sacrificed for measurement of body weight and kidney weight.The KI was calculated as follows: KI = kidney weight/body weight × 100%. The expression levels of tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β),and interleukin-6( IL-6) were detected by ELISA. HE staining was used to observe the pathological changes of the kidneys. The CD14 expression in the renal tubular was examined by immunohistochemistry. Results Compared with the control group,the body weight decreased in the DN group,and enlarged glomerular volume and inflammatory cell infiltration were observed,and FPG,KI and UACR levels also increased( all P < 0. 01).Compared with the DN group,morphological abnormality was significantly ameliorated in the Sly-L group and Sly-H group,and FPG,KI and UACR levels decreased in both of the Sly treatment groups( all P < 0. 05). Compared with the control group,the expression of CD14 in the glomerulus and the expression of TNF-α,1 L-1β,1 L-6 were up-regulated in the DN and Sly-L group( all P < 0. 05) and 1 L-6 expression increased in the Sly-H group( P < 0. 05). Compared with the DN group,CD14 expression and TNF-α,1 L-1β,and 1 L-6 levels all decreased significantly in the Sly-L group and Sly-H group( all P < 0. 05). Conclusion Sly can attenuate the renal injury of DN rats,which may be related to the inhibition of inflammatory cytokines TNF-α,IL-1β,IL-6 and CD14 expression.
Objective To explore the protective effect of silymarin( Sly) on the diabetic nephropathy( DN) rats and its possible mechanisms. Methods Forty Sprague-Dawley( SD) rats( 8 weeks old,weight 180-200 g) were randomly divided into four groups( 10 rats per group) : DN group,low-dose Sly treatment group( Sly-L group),high-dose Sly treatment group( Sly-H group),and normal control group. Rats in the DN group,Sly-L group,and Sly-H group were induced by streptozotocin( STZ,60 mg/kg). Rats in the Sly-L group and Sly-H group were given intragastric administration of lowdose Sly( 60 mg/Kg/d) and high-dose Sly( 120 mg/Kg/d) every 24 hours,respectively; rats in the DN group and the control group were treated with the same amount of normal saline. After 8 weeks treatment,tail vein blood was used for measuring the level of fasting blood glucose( FPG); metabolic cages were used for collecting 24 h urine,and urine albuminuria/creatinine( UACR) was calculated. Then all rats were sacrificed for measurement of body weight and kidney weight.The KI was calculated as follows: KI = kidney weight/body weight × 100%. The expression levels of tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β),and interleukin-6( IL-6) were detected by ELISA. HE staining was used to observe the pathological changes of the kidneys. The CD14 expression in the renal tubular was examined by immunohistochemistry. Results Compared with the control group,the body weight decreased in the DN group,and enlarged glomerular volume and inflammatory cell infiltration were observed,and FPG,KI and UACR levels also increased( all P < 0. 01).Compared with the DN group,morphological abnormality was significantly ameliorated in the Sly-L group and Sly-H group,and FPG,KI and UACR levels decreased in both of the Sly treatment groups( all P < 0. 05). Compared with the control group,the expression of CD14 in the glomerulus and the expression of TNF-α,1 L-1β,1 L-6 were up-regulated in the DN and Sly-L group( all P < 0. 05) and 1 L-6 expression increased in the Sly-H group( P < 0. 05). Compared with the DN group,CD14 expression and TNF-α,1 L-1β,and 1 L-6 levels all decreased significantly in the Sly-L group and Sly-H group( all P < 0. 05). Conclusion Sly can attenuate the renal injury of DN rats,which may be related to the inhibition of inflammatory cytokines TNF-α,IL-1β,IL-6 and CD14 expression.
引文
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[14]Sheela N,Jose MA,Sathyamurthy D,et al. Effect of silymarin on streptozotocin-nicotinamide-induced type 2 diabetic nephropathy in rats[J]. Iran J Kidney Dis,2013,7(2):117-123.
[15]张红,章向成,朱大龙.炎性反应与糖尿病肾病[J].国际内分泌代谢杂志,2015,35(1):49-52.
[16]Nguyen D,Ping F,Mu W,et al. Macrophage accumulation in human progressive diabetic nephropathy[J]. Nephrology,2010,11(3):226-231.
[17]郑寿焕,金光明,柳明洙. CD14启动子-159位点基因多态性与糖尿病肾病的相关性研究[J].中华内分泌代谢杂志,2009,25(4):409-411.
[18]Wu CH,Huang SM,Yen GC. Silymarin:a novel antioxidant with antiglycation and antiinflammatory properties in vitro and in vivo[J]. Antioxid Redox Signal,2011,14(3):353-366.
[19]Zhang H,Zhang R,Chen J,et al. High mobility group box1 inhibitor glycyrrhizic acid attenuates kidney injury in streptozotocininduced diabetic rats[J]. Kidney Blood Press Res,2017,42(5):894-904.
[20]Garcia-Garcia PM,Getino-Melian MA,Dominguez-Pimentel V,et al. Inflammation in diabetic kidney disease[J]. World J Diabetes,2014,5(4):431-443.
[21]Fallahzadeh MK,Dormanesh B,Sagheb MM,et al. Effect of addition of silymarin to renin-angiotensin system inhibitors on proteinuria in type 2 diabetic patients with overt nephropathy:a randomized,double-blind,placebo-controlled trial[J]. Am J Kidney Dis,2012,60(6):896-903.
[2]Li Z,Zou G,Li W,et al. Prevalence of diabetic nephropathy complicating non-diabetic renal disease among Chinese patients with type 2diabetes mellitus[J]. Eur J Med Res,2013,18(1):4.
[3]Sun YM,Su Y,Li J,et al. Recent advances in understanding the biochemical and molecular mechanism of diabetic nephropathy[J].Biochem Biophys Res Commun,2013,433(4):359-361.
[4]Kanasaki K,Taduri G,Koya D. Diabetic nephropathy:the role of inflammation in fibroblast activation and kidney fibrosis[J]. Front Endocrinol,2013,4(1):7.
[5]Chow F,Ozols E,Nikolic-Paterson DJ,et al. Macrophages in mouse type 2 diabetic nephropathy:Correlation with diabetic state and progressive renal injury[J]. Kidney Int,2004,65(1):116-128.
[6]Duran-Salgado,Montserrat B. Diabetic nephropathy and inflammation[J]. World J Diabetes,2014,5(3):393-398.
[7]Saller R,Meier R,Brignoli R. The use of silymarin in the treatment of liver diseases[J]. Drugs,2001,61(14):2035-2063.
[8]Shaker E,Mahmoud H,Mnaa S. Silymarin,the antioxidant component and Silybum marianum extracts prevent liver damage[J].Food Chem Toxicol,2010,48(3):803-806.
[9]Katiyar SK. Silymarin and skin cancer prevention:anti-inflammatory,antioxidant and immunomodulatory effects(Review)[J]. Int J Oncol,2005,26(1):169-176.
[10]Morishima C,Shuhart MC,Wang CC,et al. Silymarin inhibits in vitro T-cell proliferation and cytokine production in hepatitis C virus infection[J]. Gastroenterology,2010,138(2):671-681.
[11]Rafieian-Kopaie M,Nasri H. Silymarin and diabetic nephropathy[J]. J Renal Inj Prev,2012,1(1):3-5.
[12]Stolf AM,Cardoso CC,Acco A. Effects of silymarin on diabetes mellitus complications:a review[J]. Phytother Res,2017,31(3):366-374.
[13]Vessal G,Akmali M,Najafi P,et al. Silymarin and milk thistle extract may prevent the progression of diabetic nephropathy in streptozotocin-induced diabetic rats[J]. Ren Fail,2010,32(6):733-739.
[14]Sheela N,Jose MA,Sathyamurthy D,et al. Effect of silymarin on streptozotocin-nicotinamide-induced type 2 diabetic nephropathy in rats[J]. Iran J Kidney Dis,2013,7(2):117-123.
[15]张红,章向成,朱大龙.炎性反应与糖尿病肾病[J].国际内分泌代谢杂志,2015,35(1):49-52.
[16]Nguyen D,Ping F,Mu W,et al. Macrophage accumulation in human progressive diabetic nephropathy[J]. Nephrology,2010,11(3):226-231.
[17]郑寿焕,金光明,柳明洙. CD14启动子-159位点基因多态性与糖尿病肾病的相关性研究[J].中华内分泌代谢杂志,2009,25(4):409-411.
[18]Wu CH,Huang SM,Yen GC. Silymarin:a novel antioxidant with antiglycation and antiinflammatory properties in vitro and in vivo[J]. Antioxid Redox Signal,2011,14(3):353-366.
[19]Zhang H,Zhang R,Chen J,et al. High mobility group box1 inhibitor glycyrrhizic acid attenuates kidney injury in streptozotocininduced diabetic rats[J]. Kidney Blood Press Res,2017,42(5):894-904.
[20]Garcia-Garcia PM,Getino-Melian MA,Dominguez-Pimentel V,et al. Inflammation in diabetic kidney disease[J]. World J Diabetes,2014,5(4):431-443.
[21]Fallahzadeh MK,Dormanesh B,Sagheb MM,et al. Effect of addition of silymarin to renin-angiotensin system inhibitors on proteinuria in type 2 diabetic patients with overt nephropathy:a randomized,double-blind,placebo-controlled trial[J]. Am J Kidney Dis,2012,60(6):896-903.