石斛乙醇提取物对2型糖尿病大鼠视网膜血管生成的影响

Effect of Ethanolic Extract of Dendrobium on Retinal Angiogenesis in Type 2 Diabetes Mellitus Rats

【目的】观察石斛乙醇提取物对2型糖尿病(T2DM)大鼠视网膜血管生成的作用及机制。【方法】随机选取85只大鼠,建立T2DM模型。将建模成功的80大鼠随机分为模型组,石斛乙醇提取物低、高剂量组与阳性药物组,每组20只,另设正常组(20只大鼠)。石斛乙醇提取物低、高剂量组分别给予石斛乙醇提取物100、300 mg·kg^(-1)灌胃,阳性药物组给予羟苯磺酸钙5.4 mg·kg^(-1)灌胃,正常组和模型组则给予等体积生理盐水灌胃。每日1次,持续12周。给药结束后,观察血浆空腹血糖(FPG)、血清空腹胰岛素(FINs)变化;苏木素-伊红(HE)染色法观察视网膜组织血管及病理学变化;酶联免疫吸附分析(ELISA)检测视网膜组织促炎因子白细胞介素6(IL-6)、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)含量;聚合酶链反应(PCR)法和蛋白免疫印迹(Western Blot)法分别检测视网膜组织缺氧诱导因子1α(HIF-1α)、血管内皮生长因子(VEGF)mRNA与蛋白表达情况。【结果】与正常组比较,模型组FPG、FINs、IL-6、IL-1β、TNF-α水平,HIF-1α、VEGF mRNA与蛋白表达量升高(P<0.05);与模型组比较,石斛乙醇提取物低、高剂量组和阳性药物组FPG、FINs、IL-6、IL-1β、TNF-α水平,HIF-1α、VEGF mRNA与蛋白表达量降低(P<0.05);石斛乙醇提取物高剂量组与阳性药物组FPG、FINs水平,HIF-1α、VEGF mRNA与蛋白表达量降低效果相当(P>0.05),阳性药物组IL-6、IL-1β、TNF-α水平低于石斛乙醇提取物高剂量组(P<0.05)。FFA结果显示,石斛乙醇提取物低、高剂量组和阳性药物组心血管数量、荧光素泄漏量低于模型组,高剂量组和阳性药物组疗效相当。HE结果显示,石斛乙醇提取物低、高剂量组和阳性药物组视网膜组织结构、内外膜层细胞排列以及细胞组织水肿均较模型组呈现不同程度的改善,高剂量组和阳性药物组疗效相当。【结论】石斛乙醇提取物可有效抑制T2DM大鼠视网膜血管生成,其机制可能与抑制HIF-1α/VEGF轴相关信号分子mRNA和蛋白表达及下游促炎因子有关。

Objective To observe the effect and mechanism of ethanolic extract of Dendrobium on retinal angiogenesis in type 2 diabetic mellitus(T2DM)rats.Methods Eighty-five rats were randomly selected to establish a T2DM model.The 80 successfully modeled rats were randomly divided into the model group,the Dendrobium ethanol extract low-and high-dose groups and positive drug group,with 20 rats in each group,and another normal group(20 rats)was set up.The low-and high-dose groups were given Dendrobium ethanol extract 100 and 300 mg·kg^(-1)by gavage respectively,the positive drug group was given calcium dobesilate 5.4 mg·kg^(-1)by gavage,and the normal group and model group were given equal volume of normal saline by gavage.The drug was administered once daily for 12 weeks.At the end of drug administration,changes in fasting plasma glucose(FPG)and serum fasting insulin(FINs)were observed;retinal tissue vascular and pathological changes were observed by hematoxylin-eosin(HE)staining;pro-inflammatory factors interleukin 6(IL-6),interleukin 1β(IL-1β)and tumour necrosis factorα(TNF-α)were measured by enzyme-linked immunosorbent assay(ELISA).The mRNA and protein expression of hypoxia-inducible factor 1α(HIF-1α)and vascular endothelial growth factor(VEGF)in retinal tissues were detected by polymerase chain reaction(PCR)and protein immunoblotting(Western Blot),respectively.Results Compared with the normal group,FPG,FINs,IL-6,IL-1β,TNF-αlevels,HIF-1α,VEGF mRNA and protein expression levels were increased in the model group(P<0.05);Compared with the model group,FPG,FINs,IL-6,IL-1β,TNF-αlevels,HIF-1α,VEGF mRNA and protein expression levels were decreased(P<0.05);there were no differences in the FPG,FINs levels,HIF-1α,VEGF mRNA and protein expression levels between the high-dose group and positive drug group(P>0.05),and IL-6,IL-1β,TNF-αlevels in the positive drug group were lower than those in the high-dose group(P<0.05).The FFA results showed that the cardiovascular number and fluorescein leakage in the Dendrobium ethanol extract low-and high-dose groups and the positive drug group were lower than those in the model group,and the high dose group and the positive drug group had therapeutic equivalence.HE results showed that the retinal tissue structure,inner and outer membrane layer cell arrangement and cell tissue edema in the Dendrobium ethanol extract low-and high-dose groups and the positive drug group showed different degrees of improvement compared with the model group,and the improvement was equivalent in the high-dose group and positive drug group.Conclusion Ethanolic extract of Dendrobium can effectively inhibit retinal angiogenesis in T2DM rats,and the mechanism may be related to the inhibition of HIF-1α/VEGF axis-related signaling molecules mRNA and protein expression.