柚皮素下调RhoA/ROCK信号通路改善糖尿病小鼠心肌纤维化机制探讨
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摘要
目的探讨柚皮素对糖尿病小鼠心肌纤维化的影响及其机制。方法将32只C57BL/6小鼠随机分为正常组(NS组)、糖尿病组(DM组)、柚皮素低剂量组(D+LN组)、柚皮素高剂量组(D+HN组)。采用链脲佐菌素(streptozotocin,STZ)连续腹腔注射建立1型糖尿病小鼠模型。给药8周后,处死前记录末次小鼠体质量及血糖,观察柚皮素对小鼠体质量及血糖的影响;HE及Masson染色观察心脏形态改变,并测量心肌胶原容积分数(collagen volume fraction,CVF);免疫组化法观察心脏组织RhoA、ROCK1、ROCK2蛋白的水平;Western Blot测定心脏组织RhoA、ROCK1、ROCK2、IV型胶原蛋白、纤连蛋白(Fibronectin,FN)的水平。结果与NS组比较,DM组小鼠成模后体质量下降,血糖升高(P <0.05);与DM组比较,各柚皮素处理组血糖、体质量差异无统计学意义(P> 0.05)。与NS组比较,DM组CVF增大,RhoA、ROCK1、ROCK2、IV型胶原蛋白、FN的水平升高(P <0.05);与DM组比较,各柚皮素处理组CVF减小,RhoA、ROCK1、ROCK2、IV型胶原蛋白、FN的水平下降(P <0.05)。结论柚皮素具有改善糖尿病小鼠心肌纤维化作用,其机制之一可能是下调了RhoA/ROCK信号通路。
Objective To investigate the effect of Naringenin on myocardial fibrosis and RhoA/ROCK signaling pathway in the diabetic mice. Methods C57 BL/6 mice(n = 32) were randomly divided into normal group(NS group),diabetes group(DM group),diabetes+low dose of Naringenin group(D+LN group),and diabetes+high dose of Naringenin group(D+HN group). Mice models of type 1 diabetes mellitus were established by intraperitoneal injection of streptozotocin(STZ). The mice in treatment group received different doses of Naringenin through intraperitoneal injection for 8 weeks. At the end of the study,the effects of Naringenin at different doses on the body weight and blood glucose were observed. The histopathological changes of the heart were observed with HE staining.The myocardial collagen volume fraction(CVF)was calculated by Masson staining. Immumohistochemical staining was used to test the protein expression of RhoA,ROCK1 and ROCK2. Western Blot was applied to determine the protein levels of RhoA, ROCK1, ROCK2, type IV collagen and fibronectin(FN). Results Compared with NS group,the body weight of the mice in DM group was decreased and the blood glucose was increased significantly(P < 0.05). However,no statistically difference in blood glucose and body weight between the treatment groups and DM group was observed(P > 0.05). As compared with NS group,CVF and the protein levels of RhoA,ROCK1,ROCK2, type IV collagen and FN were increased markedly in DM group(P < 0.05). Moreover, CVF and the protein levels of RhoA,ROCK1,ROCK2,type IV collagen and FN in DM group were higher than treatment groups(P < 0.05). Conclusion Naringenin significantly attenuates the myocardial fibrosis in the diabetic mice,which may act through down-regulating the RhoA/ROCK signaling pathway.
Objective To investigate the effect of Naringenin on myocardial fibrosis and RhoA/ROCK signaling pathway in the diabetic mice. Methods C57 BL/6 mice(n = 32) were randomly divided into normal group(NS group),diabetes group(DM group),diabetes+low dose of Naringenin group(D+LN group),and diabetes+high dose of Naringenin group(D+HN group). Mice models of type 1 diabetes mellitus were established by intraperitoneal injection of streptozotocin(STZ). The mice in treatment group received different doses of Naringenin through intraperitoneal injection for 8 weeks. At the end of the study,the effects of Naringenin at different doses on the body weight and blood glucose were observed. The histopathological changes of the heart were observed with HE staining.The myocardial collagen volume fraction(CVF)was calculated by Masson staining. Immumohistochemical staining was used to test the protein expression of RhoA,ROCK1 and ROCK2. Western Blot was applied to determine the protein levels of RhoA, ROCK1, ROCK2, type IV collagen and fibronectin(FN). Results Compared with NS group,the body weight of the mice in DM group was decreased and the blood glucose was increased significantly(P < 0.05). However,no statistically difference in blood glucose and body weight between the treatment groups and DM group was observed(P > 0.05). As compared with NS group,CVF and the protein levels of RhoA,ROCK1,ROCK2, type IV collagen and FN were increased markedly in DM group(P < 0.05). Moreover, CVF and the protein levels of RhoA,ROCK1,ROCK2,type IV collagen and FN in DM group were higher than treatment groups(P < 0.05). Conclusion Naringenin significantly attenuates the myocardial fibrosis in the diabetic mice,which may act through down-regulating the RhoA/ROCK signaling pathway.
引文
[1]JIA G,DEMARCO V G,SOWERS J R.Insulin resistance and hyperinsulinaemia in diabetic cardiomyopathy[J].Nature Reviews Endocrinology,2016,12(3):144-153.
[2]冯新星,陈燕燕.糖尿病心肌病的研究进展[J].中国循环杂志,2015,30(01):87-89.
[3]GUAN S J,MA Z H,WU Y L,et al.Long-term administration of fasudil improves cardiomyopathy in streptozotocin-induced diabetic rats[J].Food&Chemical Toxicology,2012,50(6):1874-1882.
[4]ZHOU H,LI Y.RhoA/Rho kinase:a novel therapeutic target in diabetic complications[J].Chin Med J(Engl),2010,123(17):2461-2466.
[5]ZHOU H,LI Y.Rho kinase inhibitors:potential treatments for diabetes and diabetic complications[J].Curr Pharm Des,2012,18(20):2964-2973.
[6]ZHOU H,LI Y,WANG M,et al.Involvement of RhoA/ROCK in myocardial fibrosis in a rat model of type 2 diabetes[J].Acta Pharmacol Sin,2011,32(8):999-1008.
[7]谢发江,冯健,李家富.RhoA/ROCK信号通路在糖尿病心肌病发病机制中的研究进展[J].临床心血管病杂志,2018,34(09):846-850.
[8]曾梦雅,王燕,李伍夫,等.柚皮素与代谢综合征的研究进展[J].中国动脉硬化杂志,2016,24(10):1055-1058.
[9]文利,陈蕾,孙一瑄,等.柚皮素通过抑制TGF-β1/smad信号传导通路缓解糖尿病肾病大鼠肾损伤[J].基础医学与临床,2016,36(7):896-901.
[10]刘付贞,潘德茂,陈景福,等.柚皮苷抗高糖诱导的心肌细胞损伤作用与抑制STAT3通路有关[J].中国动脉硬化杂志,2014,22(04):345-350.
[11]YOU Q,WU Z,WU B,et al.Naringin protects cardiomyocytes against hyperglycemia-induced injuries in vitro and in vivo[J].Journal of Endocrinology,2016,230(2):197.
[12]ZHANG J,QIU H,HUANG J,et al.Naringenin exhibits the protective effect on cardiac hypertrophy via EETs-PPARs activation in streptozocin-induced diabetic mice[J].Biochemical and biophysical research communications,2018,502(1):55-61.
[13]屈春梅,李红梅.柚皮素改善糖尿病肾病模型小鼠病情及机制研究[J].重庆医学,2018,47(23):3025-3027.
[14]URBINA P,SINGLA D K.BMP-7 attenuates adverse cardiac remodeling mediated through M2 macrophages in prediabetic cardiomyopathy[J].Am J Physiol Heart Circ Physiol,2014,307(5):H762-H772.
[15]JIA C,CHEN H,WEI M,et al.Gold nanoparticle-based miR155antagonist macrophage delivery restores the cardiac function in ovariectomized diabetic mouse model[J].Int J Nanomed,2017,12:4963.
[16]LIN G,BROWNSEY R W,MACLEOD K M.Complex regulation of PKCβ2 and PDK-1/AKT by ROCK2 in diabetic heart[J].Plos One,2014,9(1):e86520.
[17]KUETHE F,SIGUSCH H H,BORNSTEIN S R,et al.Apoptosis in patients with dilated cardiomyopathy and diabetes:a feature of diabetic cardiomyopathy?[J].Hormone and Metabolic Research,2007,39(09):672-676.
[18]GREEN J,CAO J,BANDARAGE U K,et al.Design,synthesis,and structure-activity relationships of pyridine-based rho kinase(ROCK)inhibitors[J].Journal of medicinal chemistry,2015,58(12):5028-5037.
[19]GAO H,HOU F,DONG R,et al.Rho-Kinase inhibitor fasudil suppresses high glucose-induced H9c2 cell apoptosis through activation of autophagy[J].Cardiovascular therapeutics,2016,34(5):352-359.
[20]SOLIMAN H,GADOR A,LU Y H,et al.Diabetes-induced increased oxidative stress in cardiomyocytes is sustained by a positive feedback loop involving Rho kinase and PKCβ2[J].American Journal of Physiology-Heart and Circulatory Physiology,2012,303(8):H989-H1000.
[21]HEGAB Z,MOHAMED T,STAFFORD N,et al.Advanced glycation end products reduce the calcium transient in cardiomyocytes by increasing production of reactive oxygen species and nitric oxide[J].FEBS open bio,2017,7(11):1672-1685.
[22]GUO R,LIU B,ZHOU S,et al.The protective effect of fasudil on the structure and function of cardiac mitochondria from rats with type 2 diabetes induced by streptozotocin with a high-fat diet is mediated by the attenuation of oxidative stress[J].BioMed research international,2013,2013(1):430791.
[23]WANG D,LUO P,WANG Y,et al.Glucagon-Like peptide-1protects against cardiac microvascular injury in diabetes via a c AMP/PKA/Rho-dependent mechanism[J].Diabetes,2013,62(5):1697-1708.
[24]FRATI G,SCHIRONE L,CHIMENTI I,et al.An overview of the inflammatory signalling mechanisms in the myocardium underlying the development of diabetic cardiomyopathy[J].Cardiovascular research,2017,113(4):378-388.
[25]YANG R,CHANG L,LIU S,et al.High glucose induces Rho/ROCK-dependent visfatin and type I procollagen expression in rat primary cardiac fibroblasts[J].Molecular medicine reports,2014,10(4):1992-1998.
[26]LAI D,GAO J,BI X,et al.The Rho kinase inhibitor,fasudil,ameliorates diabetes-induced cardiac dysfunction by improving calcium clearance and actin remodeling[J].Journal of Molecular Medicine,2017,95(2):155-165.
[27]DAI Q M,LU J,LIU N F.Fluvastatin attenuates myocardial interstitial fibrosis and cardiac dysfunction in diabetic rats by inhibiting over-expression of connective tissue growth factor[J].Chinese medical journal,2011,124(1):89-94.
[28]CHENG C I,CHEN P H,LIN Y C,et al.High glucose activates Raw264.7 macrophages through RhoA kinase-mediated signaling pathway[J].Cellular signalling,2015,27(2):283-292.
[2]冯新星,陈燕燕.糖尿病心肌病的研究进展[J].中国循环杂志,2015,30(01):87-89.
[3]GUAN S J,MA Z H,WU Y L,et al.Long-term administration of fasudil improves cardiomyopathy in streptozotocin-induced diabetic rats[J].Food&Chemical Toxicology,2012,50(6):1874-1882.
[4]ZHOU H,LI Y.RhoA/Rho kinase:a novel therapeutic target in diabetic complications[J].Chin Med J(Engl),2010,123(17):2461-2466.
[5]ZHOU H,LI Y.Rho kinase inhibitors:potential treatments for diabetes and diabetic complications[J].Curr Pharm Des,2012,18(20):2964-2973.
[6]ZHOU H,LI Y,WANG M,et al.Involvement of RhoA/ROCK in myocardial fibrosis in a rat model of type 2 diabetes[J].Acta Pharmacol Sin,2011,32(8):999-1008.
[7]谢发江,冯健,李家富.RhoA/ROCK信号通路在糖尿病心肌病发病机制中的研究进展[J].临床心血管病杂志,2018,34(09):846-850.
[8]曾梦雅,王燕,李伍夫,等.柚皮素与代谢综合征的研究进展[J].中国动脉硬化杂志,2016,24(10):1055-1058.
[9]文利,陈蕾,孙一瑄,等.柚皮素通过抑制TGF-β1/smad信号传导通路缓解糖尿病肾病大鼠肾损伤[J].基础医学与临床,2016,36(7):896-901.
[10]刘付贞,潘德茂,陈景福,等.柚皮苷抗高糖诱导的心肌细胞损伤作用与抑制STAT3通路有关[J].中国动脉硬化杂志,2014,22(04):345-350.
[11]YOU Q,WU Z,WU B,et al.Naringin protects cardiomyocytes against hyperglycemia-induced injuries in vitro and in vivo[J].Journal of Endocrinology,2016,230(2):197.
[12]ZHANG J,QIU H,HUANG J,et al.Naringenin exhibits the protective effect on cardiac hypertrophy via EETs-PPARs activation in streptozocin-induced diabetic mice[J].Biochemical and biophysical research communications,2018,502(1):55-61.
[13]屈春梅,李红梅.柚皮素改善糖尿病肾病模型小鼠病情及机制研究[J].重庆医学,2018,47(23):3025-3027.
[14]URBINA P,SINGLA D K.BMP-7 attenuates adverse cardiac remodeling mediated through M2 macrophages in prediabetic cardiomyopathy[J].Am J Physiol Heart Circ Physiol,2014,307(5):H762-H772.
[15]JIA C,CHEN H,WEI M,et al.Gold nanoparticle-based miR155antagonist macrophage delivery restores the cardiac function in ovariectomized diabetic mouse model[J].Int J Nanomed,2017,12:4963.
[16]LIN G,BROWNSEY R W,MACLEOD K M.Complex regulation of PKCβ2 and PDK-1/AKT by ROCK2 in diabetic heart[J].Plos One,2014,9(1):e86520.
[17]KUETHE F,SIGUSCH H H,BORNSTEIN S R,et al.Apoptosis in patients with dilated cardiomyopathy and diabetes:a feature of diabetic cardiomyopathy?[J].Hormone and Metabolic Research,2007,39(09):672-676.
[18]GREEN J,CAO J,BANDARAGE U K,et al.Design,synthesis,and structure-activity relationships of pyridine-based rho kinase(ROCK)inhibitors[J].Journal of medicinal chemistry,2015,58(12):5028-5037.
[19]GAO H,HOU F,DONG R,et al.Rho-Kinase inhibitor fasudil suppresses high glucose-induced H9c2 cell apoptosis through activation of autophagy[J].Cardiovascular therapeutics,2016,34(5):352-359.
[20]SOLIMAN H,GADOR A,LU Y H,et al.Diabetes-induced increased oxidative stress in cardiomyocytes is sustained by a positive feedback loop involving Rho kinase and PKCβ2[J].American Journal of Physiology-Heart and Circulatory Physiology,2012,303(8):H989-H1000.
[21]HEGAB Z,MOHAMED T,STAFFORD N,et al.Advanced glycation end products reduce the calcium transient in cardiomyocytes by increasing production of reactive oxygen species and nitric oxide[J].FEBS open bio,2017,7(11):1672-1685.
[22]GUO R,LIU B,ZHOU S,et al.The protective effect of fasudil on the structure and function of cardiac mitochondria from rats with type 2 diabetes induced by streptozotocin with a high-fat diet is mediated by the attenuation of oxidative stress[J].BioMed research international,2013,2013(1):430791.
[23]WANG D,LUO P,WANG Y,et al.Glucagon-Like peptide-1protects against cardiac microvascular injury in diabetes via a c AMP/PKA/Rho-dependent mechanism[J].Diabetes,2013,62(5):1697-1708.
[24]FRATI G,SCHIRONE L,CHIMENTI I,et al.An overview of the inflammatory signalling mechanisms in the myocardium underlying the development of diabetic cardiomyopathy[J].Cardiovascular research,2017,113(4):378-388.
[25]YANG R,CHANG L,LIU S,et al.High glucose induces Rho/ROCK-dependent visfatin and type I procollagen expression in rat primary cardiac fibroblasts[J].Molecular medicine reports,2014,10(4):1992-1998.
[26]LAI D,GAO J,BI X,et al.The Rho kinase inhibitor,fasudil,ameliorates diabetes-induced cardiac dysfunction by improving calcium clearance and actin remodeling[J].Journal of Molecular Medicine,2017,95(2):155-165.
[27]DAI Q M,LU J,LIU N F.Fluvastatin attenuates myocardial interstitial fibrosis and cardiac dysfunction in diabetic rats by inhibiting over-expression of connective tissue growth factor[J].Chinese medical journal,2011,124(1):89-94.
[28]CHENG C I,CHEN P H,LIN Y C,et al.High glucose activates Raw264.7 macrophages through RhoA kinase-mediated signaling pathway[J].Cellular signalling,2015,27(2):283-292.
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