积雪草酸对糖尿病大鼠血-视网膜屏障的保护作用

Protective effect of asiatic acid on blood-retinal barrier in diabetic rats

目的研究积雪草酸(AA)对糖尿病大鼠血—视网膜屏障(BRB)的保护作用及其可能机制。方法取健康8周龄雄性SD大鼠96只,按照随机数字表法分为正常对照组、糖尿病模型组、低剂量AA组和高剂量AA组,每组24只。取糖尿病模型组、低剂量AA组、高剂量AA组大鼠采用腹腔注射链脲佐菌素建立糖尿病模型,正常对照组注射等量枸橼酸盐缓冲液,模型建立后1个月,低剂量AA组和高剂量AA组分别给予37.5 mg/kg、75.0 mg/kg AA灌胃;正常对照组及糖尿病模型组给予等量0.5%羧甲基纤维素钠灌胃,每日1次。在给药第0、1、2、3、4周称量大鼠体质量并检测鼠尾静脉血糖值。给药后1个月取大鼠视网膜组织进行实验。采用苏木精—伊红染色法观察大鼠视网膜组织的病理变化;采用伊文思蓝定量法检测BRB的破坏情况;采用免疫荧光染色法检测视网膜组织中Occludin、Notch1、Jagged典型Notch配体1(JAG1)、Delta样典型Notch配体4(DLL4)蛋白的表达分布;采用实时荧光定量PCR法及Western blot法检测Occludin、Notch1、JAG1、DLL4 mRNA及蛋白的表达。结果给药第4周,高剂量AA组体质量明显高于糖尿病模型组,低剂量AA组和高剂量AA组血糖浓度均低于糖尿病模型组,差异均有统计学意义(均P<0.05)。正常对照组大鼠视网膜细胞排列整齐,结构层次清晰。糖尿病模型组大鼠视网膜明显增厚,外核层增厚,细胞排列紊乱,结构层次不清晰。低剂量AA组和高剂量AA组大鼠视网膜厚度和结构均较糖尿病模型组明显改善。正常对照组、糖尿病模型组、低剂量AA组和高剂量AA组视网膜中伊文思蓝含量依次为(3.07±1.30)、(13.73±3.88)、(9.57±2.69)和(6.55±1.61)ng/mg,总体差异有统计学意义(F=18.50,P<0.01),其中高剂量AA组视网膜中伊文思蓝含量明显低于糖尿病模型组,差异均有统计学意义(均P<0.01)。糖尿病模型组Occludin蛋白相对表达量明显低于其余3个组,Notch1、JAG1和DLL4蛋白相对表达量明显高于其余3个组,差异均有统计学意义(均P<0.05);高剂量AA组Occludin蛋白相对表达量明显高于低剂量AA组,Notch1、JAG1和DLL4蛋白相对表达量明显低于低剂量AA组,差异均有统计学意义(均P<0.05)。与正常对照组比较,糖尿病模型组及低剂量AA组Occludin mRNA相对表达量明显下调,Notch1、JAG1和DLL4 mRNA相对表达量明显上调,差异均有统计学意义(均P<0.01);高剂量AA组Occludin mRNA相对表达量明显高于糖尿病模型组和低剂量AA组,Notch1 mRNA相对表达量明显低于糖尿病模型组和低剂量AA组,JAG1和DLL4 mRNA相对表达量明显低于糖尿病模型组,差异均有统计学意义(均P<0.05)。结论AA可能通过抑制Notch1信号通路发挥对糖尿病大鼠BRB的保护作用。

Objective To investigate the protective effect of asiatic acid(AA)on blood-retinal barrier(BRB)in diabetic rats and its possible mechanism.Methods Ninety-six healthy 8-week-old male SD rats were randomly divided into normal control group,diabetes group,low-dose AA group and high-dose AA group,with 24 rats in each group.Intraperitoneal injection of streptozocin(STZ)was used to establish diabetes model.One month after the establishment of the model,the low-dose AA group and the high-dose AA group were given intragastrical administration of 37.5 mg/kg AA and 75.0 mg/kg AA,respectively,once a day according to grouping.The normal control group and the diabetes group were administrated with the same amount of 0.5%sodium carboxymethyl cellulose.The body weight of the rats were weighted at week 0,1,2,3,4 after intragastrical administration.Blood was taken from the tail vein and the blood glucose level was measured.The retina was obtained one month following the administration.Pathological changes of the rats retina were detected by hematoxylin-eosin(HE)staining.Evan's blue quantitative method was used to detect the damage of blood-retinal barrier(BRB).Immunofluorescence staining was performed to detect the distribution of Occludin,Notch1,Jagged canonical Notch ligand 1(JAG1)and Delta like canonical Notch ligand 4(DLL4)in retina.The mRNA and protein expressive levels of Occludin,Notch1,JAG1 and DLL4 were detected by Real-time PCR and Western blot.The study protocol was approved by a Scientific Research and Clinical Trial Ethics Committee of The First Affiliated Hospital of Zhengzhou University(No.2020-KY-228).The use and care of animals complied with the Guide for the Care and Use of Laboratory Animals of National Institutes of Health and the 3R rules.Results At 4 weeks after intragastrical administration,the body weight of the high-dose AA group was significantly higher than that of the diabetes group,and the blood glucose values were significantly lower in the high-dose AA group and the low-dose AA group in comparison with the diabetes group(all at P<0.05).The cells were arranged orderly with clear layered structure in the normal control group.In the diabetes group,the retina was thicker than that of the normal control group,with a thicker outer nuclear layer,disordered cell arrangement and unclear layered structure.Compared with the diabetes group,the total retinal thickness and structure were obviously improved in the low-dose AA group and the high-dose AA group.Evan's blue leakage in retina was(3.07±1.30),(13.73±3.88),(9.57±2.69)and(6.55±1.61)ng/mg in the normal control group,the diabetes group,the low-dose AA group and the high-dose AA group,respectively.There was a significant difference in leakage of Evan's blue among the four groups(F=18.50,P<0.01),among which the leakage of Evan's blue dye in the high-dose AA group was significantly lower than that of the diabetes group(P<0.01).Compared with the diabetes group,there was significantly higher relative expression level of Occludin protein and significantly lower relative expression levels of Notch1,JAG1 and DLL4 proteins in the other three groups(all at P<0.05).The relative expression level of Occludin protein was significantly higher and the relative expression levels of Notch1,JAG1 and DLL4 proteins were significantly lower in the high-dose AA group than those in the low-dose AA group(all at P<0.05).Compared with the normal control group,the Occludin mRNA expression level was significantly decreased and the expression levels of Notch1,JAG1 and DLL4 mRNA were significantly increased in the diabetes group and low-dose AA group(all at P<0.01).The Occludin mRNA expression level was higher and the Notch1 mRNA expression level was lower in the high-dose AA group than those in the diabetes group and the low-dose AA group,and the expression levels of JAG1 and DLL4 mRNA were lower in the high-dose AA group in comparison with the diabetes group,and the differences were statistically significant(all at P<0.05).Conclusions Asiatic acid might play a protective role on BRB in diabetic rats by inhibiting Notch1 signaling pathway.