姜黄素及其类似物J7对2型糖尿病大鼠睾丸氧化应激损伤的干预作用
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摘要
目的:研究姜黄素(CUR)及其类似物J7对糖尿病大鼠睾丸氧化应激损伤的干预作用。方法:60只SD大鼠随机分组,其中10只作为正常(NC)组,余50只通过高脂饮食和腹腔注射链脲佐菌素诱导建立糖尿病大鼠模型,造模成功后将其再分为4组:糖尿病(DM,n=12)组、姜黄素治疗(CUR,n=10)组、J7高剂量治疗(J+,n=10)组、J7低剂量治疗(J-,n=10)组。CUR组大鼠每天予以姜黄素20 mg/kg灌胃治疗,J+及J-组分别予以J7 20 mg/kg、10 mg/kg灌胃治疗,8周后处死大鼠,测量各组大鼠体重、空腹血糖,羟胺法和硫代巴比妥酸法分别检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,Western blot法检测t Nrf2、p Nrf2、CAT、NQO1蛋白表达水平,qRT-PCR检测CAT、NQO1、HO1 mRNA表达水平,光镜下观察大鼠睾丸形态学改变,免疫组化检测Nrf2及CAT蛋白表达情况。结果:DM组血糖、MDA水平升高(P<0.05),体重、SOD活性、p Nrf2/t Nrf2、CAT、NQO1蛋白及CAT、NQO1、HO1mRNA水平均有下降(P<0.05);光镜下见睾丸各级生精细胞减少、排列紊乱;免疫组化显示Nrf2蛋白核周表达量下降,CAT蛋白表达水平降低。经姜黄素及J7治疗后,三个治疗组的MDA水平均下降(P<0.05),SOD活性、p Nrf2/t Nrf2、CAT、NQO1蛋白及NQO1、HO1 mRNA水平均上升(P <0. 05),J+及J-组血糖显著下降(P <0. 05),J+组CAT mRNA水平显著上升(P<0.05); J+组p Nrf2/t Nrf2比值明显高于CUR组及J-组(P<0.05),J+组CAT蛋白水平也明显高于J-组(P<0.05),其余指标在三个治疗组间不具有显著性差异。光镜下见三个治疗组睾丸形态学病变减轻;免疫组化显示Nrf2蛋白核周表达量上升,CAT蛋白表达水平升高。提示高剂量J7抗糖尿病大鼠睾丸的氧化应激损伤的能力较强。结论:姜黄素及J7可在一定程度上对抗糖尿病大鼠睾丸的氧化应激损伤,其机制可能与Nrf2-ARE信号通路的激活相关。
Objective: To investigate the intervention of curcumin and its analogue J7 on oxidative stress injury in testis of type 2 diabetic rats. Methods: Sixty male SD rats,10 rats were chosen as normal control group( NC),the other 50 rats were assigned to experiment group. Experiment diabetic rats were induced by high-fat food and intraperitoneal injection of steptozotocin( STZ). After the model was established successfully,diabetic rats were divided into four groups randomly: diabetes mellitus group( DM,n = 12),curcumin treatment group( CUR,n = 10),high dose treatment group of J7( J+,n = 10),low dose treatment group of J7( J-,n = 10).The CUR group were intragastrically administered with curcumin 20 mg/kg daily,in addition,the J+ group and the J-group were intragastrically administered with J7 20 mg/kg and 10 mg/kg daily respectively. After 8 weeks,the fast blood glucose was detected biochemically. The activity of superoxide dismutase( SOD) and the level of malondialdehyde( MDA) were detected by hydroxylamine method and thiobarbituric acid method respectively. The protein expressions of the nuclear factor-erythroid 2-related factor 2( t Nrf2),phosphorylation of Nrf2( p Nrf2),catalase( CAT),NAD( P) H quinine oxidoreductase 1( NQO1) were measured by Western blot.The mRNA expressions of CAT,NQO1,hemeoxygenase-1( HO1) were measured by quantitative real-time PCR( qRT-PCR). Morphological structure of testis was observed by hematoxylin-eosin( HE) staining. The expressions of Nrf2 and CAT were also detected by immunohistochemical method. Results: The levels of fast blood glucose and MDA in DM group were increased significantly( P< 0.05),while the body weight,the activity of SOD,the protein expressions of p Nrf2/t Nrf2,CAT,NQO1 and the mRNA expressions of CAT,NQO1,HO1 were decreased( P<0.05). Under light microscope,the DM group showed disrupted histological appearance. Immunohistochemistry showed that the protein expressions of Nrf2 around the nucleus and CAT were decreased. With the treatment of curcumin and J7,the MDA levels in the three treatment groups were decreased( P < 0. 05). The activity of SOD,the protein expressions of p Nrf2/t Nrf2,CAT,NQO1 and the mRNA expressions of NQO1,HO1 were increased( P<0.05). the levels of fast blood glucose were decreased in the J+ and J-group( P<0.05),and the mRNA expression of CAT was increased in the J+ group( P<0.05). The ratio of p Nrf2/t Nrf2 in the J+ group was significantly higher than that in CUR and J-group( P< 0.05). The protein level of CAT in the J +group was also significantly higher than that in J-group( P< 0.05). There were no significant differences in other indexes among the three treatment groups. Under light microscope,the morphology was obviously improved in the three treatment groups. Immunohistochemistry showed that the protein expressions of Nrf2 around the nucleus and CAT were increased in the three treatment groups. It was suggested that high dose J7 had better antioxidant stress ability in testis of diabetic rats. Conclusion: Curcumin and J7 could inhibit the oxidative stress damage of testicular tissue in diabetic rats,which might be related with the activation of the Nrf2-ARE signaling pathway.
Objective: To investigate the intervention of curcumin and its analogue J7 on oxidative stress injury in testis of type 2 diabetic rats. Methods: Sixty male SD rats,10 rats were chosen as normal control group( NC),the other 50 rats were assigned to experiment group. Experiment diabetic rats were induced by high-fat food and intraperitoneal injection of steptozotocin( STZ). After the model was established successfully,diabetic rats were divided into four groups randomly: diabetes mellitus group( DM,n = 12),curcumin treatment group( CUR,n = 10),high dose treatment group of J7( J+,n = 10),low dose treatment group of J7( J-,n = 10).The CUR group were intragastrically administered with curcumin 20 mg/kg daily,in addition,the J+ group and the J-group were intragastrically administered with J7 20 mg/kg and 10 mg/kg daily respectively. After 8 weeks,the fast blood glucose was detected biochemically. The activity of superoxide dismutase( SOD) and the level of malondialdehyde( MDA) were detected by hydroxylamine method and thiobarbituric acid method respectively. The protein expressions of the nuclear factor-erythroid 2-related factor 2( t Nrf2),phosphorylation of Nrf2( p Nrf2),catalase( CAT),NAD( P) H quinine oxidoreductase 1( NQO1) were measured by Western blot.The mRNA expressions of CAT,NQO1,hemeoxygenase-1( HO1) were measured by quantitative real-time PCR( qRT-PCR). Morphological structure of testis was observed by hematoxylin-eosin( HE) staining. The expressions of Nrf2 and CAT were also detected by immunohistochemical method. Results: The levels of fast blood glucose and MDA in DM group were increased significantly( P< 0.05),while the body weight,the activity of SOD,the protein expressions of p Nrf2/t Nrf2,CAT,NQO1 and the mRNA expressions of CAT,NQO1,HO1 were decreased( P<0.05). Under light microscope,the DM group showed disrupted histological appearance. Immunohistochemistry showed that the protein expressions of Nrf2 around the nucleus and CAT were decreased. With the treatment of curcumin and J7,the MDA levels in the three treatment groups were decreased( P < 0. 05). The activity of SOD,the protein expressions of p Nrf2/t Nrf2,CAT,NQO1 and the mRNA expressions of NQO1,HO1 were increased( P<0.05). the levels of fast blood glucose were decreased in the J+ and J-group( P<0.05),and the mRNA expression of CAT was increased in the J+ group( P<0.05). The ratio of p Nrf2/t Nrf2 in the J+ group was significantly higher than that in CUR and J-group( P< 0.05). The protein level of CAT in the J +group was also significantly higher than that in J-group( P< 0.05). There were no significant differences in other indexes among the three treatment groups. Under light microscope,the morphology was obviously improved in the three treatment groups. Immunohistochemistry showed that the protein expressions of Nrf2 around the nucleus and CAT were increased in the three treatment groups. It was suggested that high dose J7 had better antioxidant stress ability in testis of diabetic rats. Conclusion: Curcumin and J7 could inhibit the oxidative stress damage of testicular tissue in diabetic rats,which might be related with the activation of the Nrf2-ARE signaling pathway.
引文
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[11]Guariguata L,Whiting DR,Hambleton I,et al.Global estimates of diabetes prevalence for 2013 and projections for 2035[J].Diabetes Res Clin Pract,2014,103(2):137-149.
[12]La Vignera S,Condorelli R,Vicari E,et al.Diabetes mellitus and sperm parameters[J].J Androl,2012,33(2):145-153.
[13]Agarwal A,Gupta S,Sikka S.The role of free radicals and antioxidants in reproduction[J].Curr Opin Obstet Gynecol,2006,18(3):325-332.
[14]Jaiswal AK.Nrf2 signaling in coordinated activation of antioxidant geneexpression[J].Free Radic Biol Med,2004,36(10):1199-1207.
[15]Nakamura BN,Lawson G,Chan JY,et al.Knockout of the transcription factor Nrf2 disrupts spermatogenesis in an age-dependent manner[J].Free Radic Biol Med,2010,49(9):1368-1379.
[16]Hegazy AM,El-Sayed EM,Ibrahim KS,et al.Dietary antioxidant for disease prevention corroborated by the Nrf2pathway[J].J Complement Integr Med,2019.
[17]Kuriakose MA,Ramdas K,Dey B,et al.A randomized double-blind placebo-controlled phase IIB trial of curcumin in oral leukoplakia[J].Cancer Prev Res(Phila),2016,9(8):683-691.
[18]Blanco-Garcia E,Otero-Espinar FJ,Blanco-Mendez J,et al.Development and characterization of anti-inflammatory activity of curcumin-loaded biodegradable microspheres with potential use in intestinal inflammatory disorders[J].Int J Pharm,2017,518(1-2):86-104.
[19]Anand P,Kunnumakkara AB,Newman RA,et al.Bioavailability of curcumin:problems and promises[J].Mol Pharm,2007,4(6):807-818.
[2]Mulholland J,Mallidis C,Agbaje I,et al.Male diabetes mellitus and assisted reproduction treatment outcome[J].Reprod Biomed Online,2011,22(2):215-219.
[3]Wu H,Kong L,Tan Y,et al.C66 ameliorates diabetic nephropathy in mice by both upregulating NRF2 function via increase in miR-200a and inhibiting miR-21[J].Diabetologia,2016,59(7):1558-1568.
[4]Wang Y,Zhang Z,Guo W,et al.Sulforaphane reduction of testicular apoptotic cell death in diabetic mice is associated with the upregulation of Nrf2 expression and function[J].Am J Physiol Endocrinol Metab,2014,307(1):E14-E23.
[5]Xu M,Dai DZ,Zhang Q,et al.Upregulated NADPH oxidase contributes to diabetic testicular complication and is relieved by strontium fructose 1,6-diphosphate[J].Exp Clin Endocrinol Diabetes,2010,118(7):459-465.
[6]Shehzad A,Ha T,Subhan F,et al.New mechanisms and the anti-inflammatory role of curcumin in obesity and obesity-related metabolic diseases[J].Eur J Nutr,2011,50(3):151-161.
[7]Hatcher H,Planalp R,Cho J,et al.Curcumin:from ancient medicine to current clinical trials[J].Cell Mol Life Sci,2008,65(11):1631-1652.
[8]林中民,焦立卓,郑怡,等.姜黄素衍生物B06对2型糖尿病大鼠心肌的保护作用及其机制探讨[J].中国应用生理学杂志,2014,30(1):38-42.
[9]曾聪聪,刘曦,刘网网,等.姜黄素衍生物B06对2型糖尿病大鼠肾脏的保护作用[J].中国应用生理学杂志,2015,31(1):38-42.
[10]林中民,王芳,张泉波,等.姜黄素衍生物B06对2型糖尿病大鼠睾酮合成的影响[J].中国病理生理杂志,2016,32(2):352-358.
[11]Guariguata L,Whiting DR,Hambleton I,et al.Global estimates of diabetes prevalence for 2013 and projections for 2035[J].Diabetes Res Clin Pract,2014,103(2):137-149.
[12]La Vignera S,Condorelli R,Vicari E,et al.Diabetes mellitus and sperm parameters[J].J Androl,2012,33(2):145-153.
[13]Agarwal A,Gupta S,Sikka S.The role of free radicals and antioxidants in reproduction[J].Curr Opin Obstet Gynecol,2006,18(3):325-332.
[14]Jaiswal AK.Nrf2 signaling in coordinated activation of antioxidant geneexpression[J].Free Radic Biol Med,2004,36(10):1199-1207.
[15]Nakamura BN,Lawson G,Chan JY,et al.Knockout of the transcription factor Nrf2 disrupts spermatogenesis in an age-dependent manner[J].Free Radic Biol Med,2010,49(9):1368-1379.
[16]Hegazy AM,El-Sayed EM,Ibrahim KS,et al.Dietary antioxidant for disease prevention corroborated by the Nrf2pathway[J].J Complement Integr Med,2019.
[17]Kuriakose MA,Ramdas K,Dey B,et al.A randomized double-blind placebo-controlled phase IIB trial of curcumin in oral leukoplakia[J].Cancer Prev Res(Phila),2016,9(8):683-691.
[18]Blanco-Garcia E,Otero-Espinar FJ,Blanco-Mendez J,et al.Development and characterization of anti-inflammatory activity of curcumin-loaded biodegradable microspheres with potential use in intestinal inflammatory disorders[J].Int J Pharm,2017,518(1-2):86-104.
[19]Anand P,Kunnumakkara AB,Newman RA,et al.Bioavailability of curcumin:problems and promises[J].Mol Pharm,2007,4(6):807-818.
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