地黄合剂介导p38 MAPK通路对糖尿病大鼠主动脉的影响

Effect of Rehmannia glutinosa Mixture on aortas in diabetic rats via p38 MAPK pathway

目的研究地黄合剂抑制p38有丝分裂原活化蛋白激酶(p38 mitogen activated protein kinase,p38 MAPK)对糖尿病大鼠主动脉蛋白羰基和炎症因子的影响。方法50只SPF大鼠均分为5组,分别为正常组、糖尿病组、氯沙坦组、低剂量地黄(合剂)组、高剂量地黄(合剂)组,除正常组外,其他组大鼠建立糖尿病大鼠模型,对其用药,麻醉抽血,开腹取出主动脉,采用放免法、苏木素-伊红染色法(HE染色法)、蛋白印迹法(Western blot)等检测大鼠基本指标、主动脉病理学变化情况、p38 MAPK通路相关蛋白情况、蛋白羰基相关指标水平、炎性因子情况并进行对比分析。结果与正常组相比,糖尿病组大鼠体质量下降,血糖和血管紧张素2升高(P<0.05);氯沙坦组和高剂量地黄组体质量明显升高,血糖和血管紧张素2降低(P<0.05);低、高剂量地黄组组织形态均有不同程度的变化,高剂量地黄组和氯沙坦组大鼠细胞变化减轻程度最为明显(P<0.001),低剂量地黄组次之(P<0.05)。与糖尿病组相比,正常组大鼠的p38 MAPK、cAMP反应元件结合蛋白1(cAMP response element-binding protein 1,CREB1)、MAPK激酶3/6(MAPK kinase3/6,MKK3/6)、环氧化酶2(cyclooxygenase 2,COX2)水平明显升高,丝裂原活化蛋白激酶磷酸酶1(MAP kinase phosphatase-1,MKP-1)降低(P<0.05),低、高剂量地黄组p38 MAPK、COX2明显降低(P<0.05),氯沙坦组和高剂量地黄组p38 MAPK、CREB1、MKK3/6、COX2水平显著降低,MKP-1明显升高(P<0.05);丙二醛(malondialdehyde,MDA)明显降低(P<0.05);超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽(glutathione,GSH)、谷胱甘肽过氧化物酶(gbutathione peroxidase,GSH-px)显著升高(P<0.05)。与糖尿病组相比,氯沙坦组炎性因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、单核细胞趋化蛋白-1(monocyte chemoattractant protein,MCP-1)、C反应蛋白(C-reactive protein,CRP)、白细胞介素6(interleukin 10,IL-10)明显降低(P<0.001);低剂量地黄组TNF-α、MCP-1、IL-10有所降低(P<0.05),高剂量地黄组TNF-α、MCP-1、IL-10显著降低(P<0.001)。结论高剂量地黄合剂的药效与氯沙坦相当,能够有效阻断p38 MAPK信号通路,改善糖尿病大鼠主动脉的病理组织结构,降低主动脉蛋白羰基水平,抑制氧化还原反应的发生,降低炎性因子水平。

Objective To study the effect of Rehmannia glutinosa Mixture(RGM)on p38 mitogen activated protein kinase(p38MAPK)on aortic protein carbonyl and inflammatory factors in diabetes rats.Methods 50 SPF rats were divided into 5 groups:normal group,diabetic group,losartan group and low and high dose RGM group.Except for the normal group,the other groups were used to establish diabetic rat models,Except for the normal group,the aortas of the other groups were taken out after anesthesia,and the blood samples were collected.Radioimmunoassay,hematoxylin and eosin staining,Western blot and other methods were employed to detect the basic indicators of the rats,the pathological changes of the aortas,the p38 MAPK pathway related proteins,and the level of protein carbonyl related indicators.The inflammatory factors were analyzed and compared.Results Compared with the normal group,the weight of the rats in the diabetic group decreased,and the blood sugar and angiotensin 2 increased(P<0.05);the weight of the losartan group and the high-dose RGM group increased significantly,and the blood sugar and angiotensin 2 decreased(P<0.05).The tissue morphology of the low-dose and high-dose RGM groups showed different degrees of changes.The high-dose RGM group and the losartan group had the most significant reduction in cell changes(P<0.001).The mixture group followed(P<0.05).Compared with the diabetic group,the levels of p38 MAPK,cAMP response element-binding protein 1(CREB1),MAPK kinase 3/6(MKK3/6),and cyclooxygenase 2(COX2)in the normal group were significantly increased,the MKP-1 decreased(P<0.05),and the p38 MAPK and COX2 in the low-dose and high-dose RGM groups were significantly decreased(P<0.05).The levels of p38 MAPK,CREB1,MKK3/6,COX2 in the losartan group and the high-dose RGM group were significantly reduced,and the MKP-1 was significantly increased(P<0.05).Malondialdehyde(MDA)decreased significantly(P<0.05);Superoxide dismutase(SOD),glutathione(GSH)and glutathione peroxidase(GSH-px)increased significantly(P<0.05).Compared with diabetic group,the losartan group has inflammatory factors,tumor necrosis factor-α(TNF-α),monocyte chemoattractant protein(MCP-1),C-reactive protein(CRP)and interleukin-10(IL-10)decreased significantly(P<0.001),the TNF-α,MCP-1,IL-10 in the high-dose RGM group was significantly reduced(P<0.001).Conclusion The efficacy of RGM is equivalent to that of losartan.RGM can effectively block the p38 MAPK signaling pathway,improve the pathological structure of the aorta of diabetic rats,reduce the level of aortic protein carbonyls,inhibit the occurrence of redox reactions,and reduce the inflammatory factors level.