摘要
目的探讨荆防颗粒对db/db小鼠血糖代谢的调控作用及对其视网膜病变(diabetic retinopaphy,DR)的保护作用。方法将60只雄性db/db小鼠随机分为模型组、二甲双胍(120 mg/kg)组及荆防颗粒(1、2 g/kg)组,每组15只,另取15只雄性db/m小鼠作为对照组,每周检测小鼠体质量、空腹血糖、摄食量及饮水量等基础指标,并在实验结束后进行口服葡萄糖耐量试验(oral glucose tolerance test,OGTT);采用ELISA检测小鼠视网膜组织白细胞介素-1β(interleukin-1β,IL1β)、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、丙二醛(malondialdehyde,MDA)水平和过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)活性;采用PAS染色检测视网膜组织病理变化;采用Western blotting检测视网膜组织中血管内皮生长因子(vascular endothelial growth factor,VEGF)、紧密连接跨膜蛋白(Claudin-1)、胰岛素受体底物1(insulin receptor substrate 1,IRS1)、磷酸化IRS1(phosphorylated IRS1,p-IRS1)、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、磷酸化PI3K(phosphorylated PI3K,p-PI3K)、蛋白激酶B(protein kinase B,Akt)、磷酸化Akt(phosphorylated Akt,p-Akt)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、剪切型半胱氨酸天冬氨酸蛋白酶-3(cleaved cystein-asparate protease-3,cleaved Caspase-3)蛋白表达。结果与对照组比较,模型组小鼠空腹血糖、摄食量、饮水量明显升高(P<0.01),OGTT中曲线下面积(AUC)显著升高(P<0.01);视网膜组织中IL-1β、IL-6、TNF-α和MDA含量显著升高(P<0.01),CAT、SOD活性显著下降(P<0.01);视网膜组织周细胞降低,新生血管增多(P<0.01),并且视网膜组织中Claudin-1、p-IRS1/IRS1、p-PI3K/PI3K、p-Akt/Akt、Bcl-2蛋白表达下调(P<0.01),VEGF、Bax、cleaved Caspase-3蛋白表达上调(P<0.01)。与模型组比较,荆防颗粒组小鼠体质量明显升高(P<0.05、0.01),空腹血糖、摄食量、饮水量均有不同程度下降(P<0.05、0.01);视网膜组织中IL-1β、IL-6、TNF-α、MDA含量显著下降(P<0.05、0.01),CAT、SOD活性显著上升(P<0.05、0.01);视网膜组织病理形态有所改善,视网膜组织中Claudin-1、p-IRS1/IRS1、p-PI3K/PI3K、p-Akt/Akt、Bcl-2蛋白表达显著上调(P<0.05、0.01),VEGF、Bax、cleaved Caspase-3蛋白表达显著下调(P<0.05、0.01)。结论荆防颗粒对db/db小鼠视网膜病变具有一定的改善作用,其作用机制可能为调节小鼠视网膜组织中VEGF及Claudin-1蛋白表达,进而激活胰岛素受体IRS-1/PI3K/Akt信号通路,启动Caspase级联反应,抑制视网膜上皮细胞的凋亡,从而对视网膜病变起到一定的保护作用。
Objective To investigate the regulation of Jingfang Granules(荆防颗粒)on blood glucose metabolism and protective effect of diabetic retinopaphy(DR)in db/db mice.Methods A total of 60 male db/db mice were randomly divided into model group,metformin(120 mg/kg)group,Jingfang Granules(10,20 mg/kg)groups,with 15 mice in each group,and 15 male db/m mice were selected as control group.The basic indexes such as body weight,fasting blood glucose,food intake and water intake of mice were detected weekly.After the experiment,oral glucose tolerance test(OGTT)was detected;The levels of interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α),malondialdehyde(MDA)and the activities of catalase(CAT)and superoxide dismutase(SOD)in mouse retinal tissue were detected by ELISA;PAS staining was used to detect the pathological changes of retinal tissue;Western blotting was used to detect the protein expressions of vascular endothelial growth factor(VEGF),tight junction transmembrane protein(Claudin-1),insulin receptor substrate 1(IRS1),phosphorylated IRS1(p-IRS1),phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),phosphorylated Akt(p-Akt),B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),and cleaved cystein-asparate protease-3(cleaved Caspase-3)in retinal tissue.Results Compared with control group,fasting blood glucose,food intake and water intake of mice in model group were significantly increased(P<0.01),AUC in OGTT was significantly increased(P<0.01);The levels of IL-1β,IL-6,TNF-α,MDA in retinal tissue were increased(P<0.01).CAT and SOD activities were decreased(P<0.01);The number of peripheral cells in retinal tissue was decreased and the number of new blood vessels was increased(P<0.01),protein expressions of Claudin-1,p-IRS1/IRS1,p-PI3K/PI3K,p-Akt/Akt and Bcl-2 were down-regulated(P<0.01),VEGF,Bax and cleaved Caspase-3 protein expressions were up-regulated(P<0.01).Compared with model group,body weight of mice in Jingfang Granules group was significantly increased(P<0.05,0.01),fasting blood glucose,food intake and water intake were decreased to varying degrees(P<0.05,0.01);The levels of IL-1β,IL-6,TNF-αand MDA in retinal tissue were decreased(P<0.05,0.01).CAT and SOD activities were increased(P<0.05,0.01);The histopathologic morphology of retina was improved,and the protein expressions of Claudin-1,p-IRS1/IRS1,p-PI3K/PI3K,p-Akt/Akt,Bcl-2 in retinal tissue were up-regulated(P<0.05,0.01),VEGF,Bax and cleaved Caspase-3 protein expressions were down-regulated(P<0.05,0.01).Conclusion Jingfang Granules can improve retinopathy in db/db mice to a certain extent,and its mechanism may be related to regulating the expressions of VEGF and Claudin-1 protein in retinal tissue of mice,thus activating the insulin receptor IRS-1/PI3K/Akt signaling pathway,initiating the Caspase cascade reaction,and inhibiting the apoptosis of retinal epithelial cells.Thus,it has a certain protective effect on retinopathy.
作者
刘冬光
赵文学
徐燕
姚茹
孟雪
姚景春
刘忠
LIU Dongguang;ZHAO Wenxue;XU Yan;YAO Ru;MENG Xue;YAO Jingchun;LIU Zhong(New Drug Pharmacology Center of Lunan Pharmaceutical Group Co.,Ltd.,Linyi 276006,China;State Key Laboratory of Integration and Innovation of Classic Formula and Modern Chinese Medicine,Linyi 276006,China;Linyi Key Laboratory of Immunopharmacology and Toxicology of Natural Drugs,Linyi 276006,China)
出处
《中草药》
CAS
CSCD
北大核心
2024年第9期2996-3005,共10页
Chinese Traditional and Herbal Drugs
基金
山东省重大科技创新工程(2020CXGC010505,2021CXG010508)。
