黄连素对糖尿病大鼠血管钙化的影响及其机制研究
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摘要
目的探讨黄连素对糖尿病大鼠血管钙化的影响及其可能机制。方法将90只雄性SD大鼠随机分为空白组、单纯钙化组、单纯糖尿病组、糖尿病+钙化组、糖尿病+钙化+低浓度黄连素组、糖尿病+钙化+中浓度黄连素组、糖尿病+钙化+高浓度黄连素组、糖尿病+钙化+ERK抑制剂组、糖尿病+钙化+p38抑制剂组,每组10只。各糖尿病组采用高糖、高脂饲料喂养加链尿佐菌素腹腔注射建立2型糖尿病大鼠模型,各钙化组采用维生素D_3注射+尼古丁灌胃建立血管钙化模型。各黄连素组在糖尿病+血管钙化的基础上给予100 mg/kg、200 mg/kg、400 mg/kg黄连素灌胃,糖尿病+钙化+ERK抑制剂组、糖尿病+钙化+p38抑制剂组在糖尿病+血管钙化基础上给予1 mg/kg PD98059、2 mg/kg SB203580腹腔注射,均连续4周。实验结束后用钙离子检测试剂盒检测主动脉组织中钙含量,Von Kossa染色检测主动脉组织中钙盐沉积情况,免疫组化法检测主动脉组织中骨形态发生蛋白-2(BMP-2)蛋白表达情况,蛋白印记法检测主动脉组织中p-ERK1/2、ERK1/2、p-p38、p38蛋白表达情况。结果糖尿病模型建立成功后,糖尿病各组大鼠体质量逐渐降低,但各黄连素组大鼠体质量下降速度慢于糖尿病+钙化组(P均<0.05);药物干预4周中,糖尿病各组大鼠血糖水平比较差异均无统计学意义(P均>0.05)。与空白组比较,单纯钙化组大鼠主动脉组织中钙含量明显增加(P<0.05),黑色钙盐银染颗粒沉积,BMP-2表达明显增多(P<0.05);与单纯钙化组比较,糖尿病+钙化组大鼠主动脉组织中钙含量明显增加(P<0.05),黑色钙盐银染颗粒沉积明显增加,BMP-2表达明显增多(P<0.05);与糖尿病+钙化组比较,各黄连素组及糖尿病+钙化+ERK抑制剂组、糖尿病+钙化+p38抑制剂组大鼠主动脉组织中钙含量明显减少(P均<0.05),黑色钙盐银染颗粒沉积减少,BMP-2表达明显减少(P均<0.05),且随黄连素浓度增加作用越明显。与糖尿病+钙化组比较,各黄连素组主动脉组织中p-ERK1/2、p-p38蛋白相对表达量明显降低(P均<0.05),且随黄连素浓度增加降低越明显。结论黄连素可抑制糖尿病大鼠的血管钙化,其机制可能是通过抑制ERK1/2、p38 MAPK信号通路实现的。
Objective It is to investigate the effect and its possible mechanism of berberine(BBR) on vascular calcification in diabetic rats. Methods Ninety male SD rats were randomly divided into 9 groups: control group(group A), simple calcification group(group B), simple diabetes group(group C), diabetes+calcification group(group D), diabetes+calcification+low dose of BBR group(group E), diabetes+calcification+medium dose of BBR group(group F), diabetes+calcification+high dose of BBR group(group G), diabetes+calcification+ERK1/2 inhibitor group(group H), diabetes+calcification+p38 inhibitor group(group J), 10 rats in each group. Each diabetic group was treated with high-sugar and high-fat diet and intraperitoneal injection of streptozotocin to establish model of type 2 diabetes,every calcification group was treated with vitamin D3 injection+nicotine by gavage to establish model of vascular calcification. Group E,group F, group G were treated with different concentrations of BBR(100 mg/kg,200 mg/kg, 400 mg/kg) for 4 weeks on the basis of diabetes+vascular calcification.Group H and group J were respectively treated with PD98059 1 mg/(kg·d), SB203580 2 mg/(kg·d) by intraperitoneal injection for 4 weeks on the basis of diabetes+vascular calcification. Calcium content in aortic tissue was detected by calcium ion detection kit, detection of calcium salt deposition in aorta by Von Kossa staining, immunohistochemical detection of BMP2 protein expression in aortic tissues, western blotting was used to detect the expression of p-ERK1/2, ERK1/2, p-p38, p38 proteins in the aorta. Results After the establishment of the diabetes model, the body weight of the rats in the diabetic group gradually decreased, but the decrease speed of body weight of the BBR groups was slower than that of the diabetic + calcification group(P<0.05). During the 4 weeks of drug intervention, there was no significant difference in blood glucose levels among every diabetic group(P>0.05). Compared with group A, the calcium content in aorta tissue of rats in group B increased significantly(P<0.05), black calcium salt silver staining particle deposition, the expression of BMP2 was significantly increased(P<0.05); Compared with group B, the calcium content in aorta tissue in group D increased significantly(P<0.05), the deposition of black calcium salt silver staining particles increased obviously, the expression of BMP2 was significantly increased; Compared with group D, the calcium content in in aorta tissue of ERK1/2 inhibitor group, p38 inhibitor group and different concentrations of BBR group was decreased(P<0.05), the deposition of black calcium salt silver staining particles reduced, the expression of BMP2 was decreased,and with the increase of BBR concentration, the effect was more obvious; Compared with group D, the expression of p-ERK1/2 and p-p38 protein in aorta tissue decreased significantly in different concentrations of BBR groups(P<0.05),and with the increase of berberine concentration, the effect ws more obvious. Conclusion Berberine can inhibit vascular calcification in diabetic rats, and its mechanism may be achieved by inhibiting ERK1/2 and p38 MAPK signaling pathway.
Objective It is to investigate the effect and its possible mechanism of berberine(BBR) on vascular calcification in diabetic rats. Methods Ninety male SD rats were randomly divided into 9 groups: control group(group A), simple calcification group(group B), simple diabetes group(group C), diabetes+calcification group(group D), diabetes+calcification+low dose of BBR group(group E), diabetes+calcification+medium dose of BBR group(group F), diabetes+calcification+high dose of BBR group(group G), diabetes+calcification+ERK1/2 inhibitor group(group H), diabetes+calcification+p38 inhibitor group(group J), 10 rats in each group. Each diabetic group was treated with high-sugar and high-fat diet and intraperitoneal injection of streptozotocin to establish model of type 2 diabetes,every calcification group was treated with vitamin D3 injection+nicotine by gavage to establish model of vascular calcification. Group E,group F, group G were treated with different concentrations of BBR(100 mg/kg,200 mg/kg, 400 mg/kg) for 4 weeks on the basis of diabetes+vascular calcification.Group H and group J were respectively treated with PD98059 1 mg/(kg·d), SB203580 2 mg/(kg·d) by intraperitoneal injection for 4 weeks on the basis of diabetes+vascular calcification. Calcium content in aortic tissue was detected by calcium ion detection kit, detection of calcium salt deposition in aorta by Von Kossa staining, immunohistochemical detection of BMP2 protein expression in aortic tissues, western blotting was used to detect the expression of p-ERK1/2, ERK1/2, p-p38, p38 proteins in the aorta. Results After the establishment of the diabetes model, the body weight of the rats in the diabetic group gradually decreased, but the decrease speed of body weight of the BBR groups was slower than that of the diabetic + calcification group(P<0.05). During the 4 weeks of drug intervention, there was no significant difference in blood glucose levels among every diabetic group(P>0.05). Compared with group A, the calcium content in aorta tissue of rats in group B increased significantly(P<0.05), black calcium salt silver staining particle deposition, the expression of BMP2 was significantly increased(P<0.05); Compared with group B, the calcium content in aorta tissue in group D increased significantly(P<0.05), the deposition of black calcium salt silver staining particles increased obviously, the expression of BMP2 was significantly increased; Compared with group D, the calcium content in in aorta tissue of ERK1/2 inhibitor group, p38 inhibitor group and different concentrations of BBR group was decreased(P<0.05), the deposition of black calcium salt silver staining particles reduced, the expression of BMP2 was decreased,and with the increase of BBR concentration, the effect was more obvious; Compared with group D, the expression of p-ERK1/2 and p-p38 protein in aorta tissue decreased significantly in different concentrations of BBR groups(P<0.05),and with the increase of berberine concentration, the effect ws more obvious. Conclusion Berberine can inhibit vascular calcification in diabetic rats, and its mechanism may be achieved by inhibiting ERK1/2 and p38 MAPK signaling pathway.
引文
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[7] Yang R,Teng X,Li H,et al. Hydrogen Sulfide Improves Vascular Calcification in Rats by Inhibiting Endoplasmic Reticulum Stress[J]. Oxid Med Cell Longev,2016,2016:9095242
[8] Byon CH,Chen Y. Molecular Mechanisms of Vascular Calcification in Chronic Kidney Disease:The Link between Bone and the Vasculature[J]. Curr Osteoporos Rep,2015,13(4):206-215
[9] Yao Y,Bennett BJ,Wang X,et al. Inhibition of bone morphogenetic proteins protects against atherosclerosis and vascular calcification[J]. Circ Res,2010,107(4):485-494
[10] Bostrom KI,Jumabay M,Matveyenko A,et al. Activation of vascular bone morphogenetic protein signaling in diabetes mellitus[J]. Circ Res,2011,108(4):446-457
[11] Nakagami H,Osako MK,Morishita R. New concept of vascular calcification and metabolism[J]. Curr Vasc Pharmacol,2011,9(1):124-127
[12] Lacolley P,Regnault V,Segers P,et al. Vascular Smooth Muscle Cells and Arterial Stiffening:Relevance in Development,Aging,and Disease[J]. Physiol Rev,2017,97(4):1555-1617
[13] Brodeur MR,Bouvet C,Bouchard S,et al. Reduction of advanced-glycation end products levels and inhibition of RAGE signaling decreases rat vascular calcification induced by diabetes[J]. PLoS One,2014,9(1):e85922
[14] Li Z,Geng YN,Jiang JD,et al. Antioxidant and anti-inflammatory activities of berberine in the treatment of diabetes mellitus[J]. Evid Based Complement Alternat Med,2014,2014:289264
[15] Cui G,Qin X,Zhang Y,et al. Berberine differentially modulates the activities of ERK,p38 MAPK,and JNK to suppress Th17 and Th1 T cell differentiation in type 1 diabetic mice[J]. J Biol Chem,2009,284(41):28420-28429
[16] 刘慰华,刘世明,林双峰,等. 黄连素通过S1P2-MAPK信号通路抗糖尿病肾纤维化作用机制研究[J]. 中国药理学通报,2013,29(5):723-728
[17] 李晓明,王青竹,石婧,等. 小檗碱改善动脉粥样硬化小鼠的血管炎性反应和钙化[J]. 基础医学与临床,2018,38(2):163-168
[18] Rodríguez-Carballo E,Gámez B,Ventura F. p38 MAPK Signaling in Osteoblast Differentiation[J]. Front Cell Dev Biol,2016,4:40
[2] Zhou L,Yang Y,Wang X,et al. Berberine stimulates glucose transport through a mechanism distinct from insulin[J]. Metabolism,2007,56(3):405-412
[3] Kwon DH,Kim YK,Kook H. New Aspects of Vascular Calcification:Histone Deacetylases and Beyond[J]. J Korean Med Sci,2017,32(11):1738-1748
[4] Simmons CA,Nikolovski J,Thornton AJ,et al. Mechanical stimulation and mitogen-activated protein kinase signaling independently regulate osteogenic differentiation and mineralization by calcifying vascular cells[J]. J Biomech,2004,37(10):1531-1541
[5] 张青,李琰,陈磊. 黄连素对2型糖尿病及其并发症的治疗作用及相关机制研究进展[J]. 中国中药杂志,2015,40(9):1660-1665
[6] Zhang M,Lv XY,Li J,et al. The characterization of high-fat diet and multiple low-dose streptozotocin induced type 2 diabetes rat model[J]. Exp DiabetesRes,2008,2008:704045
[7] Yang R,Teng X,Li H,et al. Hydrogen Sulfide Improves Vascular Calcification in Rats by Inhibiting Endoplasmic Reticulum Stress[J]. Oxid Med Cell Longev,2016,2016:9095242
[8] Byon CH,Chen Y. Molecular Mechanisms of Vascular Calcification in Chronic Kidney Disease:The Link between Bone and the Vasculature[J]. Curr Osteoporos Rep,2015,13(4):206-215
[9] Yao Y,Bennett BJ,Wang X,et al. Inhibition of bone morphogenetic proteins protects against atherosclerosis and vascular calcification[J]. Circ Res,2010,107(4):485-494
[10] Bostrom KI,Jumabay M,Matveyenko A,et al. Activation of vascular bone morphogenetic protein signaling in diabetes mellitus[J]. Circ Res,2011,108(4):446-457
[11] Nakagami H,Osako MK,Morishita R. New concept of vascular calcification and metabolism[J]. Curr Vasc Pharmacol,2011,9(1):124-127
[12] Lacolley P,Regnault V,Segers P,et al. Vascular Smooth Muscle Cells and Arterial Stiffening:Relevance in Development,Aging,and Disease[J]. Physiol Rev,2017,97(4):1555-1617
[13] Brodeur MR,Bouvet C,Bouchard S,et al. Reduction of advanced-glycation end products levels and inhibition of RAGE signaling decreases rat vascular calcification induced by diabetes[J]. PLoS One,2014,9(1):e85922
[14] Li Z,Geng YN,Jiang JD,et al. Antioxidant and anti-inflammatory activities of berberine in the treatment of diabetes mellitus[J]. Evid Based Complement Alternat Med,2014,2014:289264
[15] Cui G,Qin X,Zhang Y,et al. Berberine differentially modulates the activities of ERK,p38 MAPK,and JNK to suppress Th17 and Th1 T cell differentiation in type 1 diabetic mice[J]. J Biol Chem,2009,284(41):28420-28429
[16] 刘慰华,刘世明,林双峰,等. 黄连素通过S1P2-MAPK信号通路抗糖尿病肾纤维化作用机制研究[J]. 中国药理学通报,2013,29(5):723-728
[17] 李晓明,王青竹,石婧,等. 小檗碱改善动脉粥样硬化小鼠的血管炎性反应和钙化[J]. 基础医学与临床,2018,38(2):163-168
[18] Rodríguez-Carballo E,Gámez B,Ventura F. p38 MAPK Signaling in Osteoblast Differentiation[J]. Front Cell Dev Biol,2016,4:40