摘要
目的探讨垂体腺苷酸环化酶激活肽-38(pituitary adenylate cyclase-activating peptide-38,PACAP38)在糖尿病视网膜病变(diabetic retinopathy,DR)过程中的视网膜保护是否由瞬时受体电位通道6(TRPC6)介导。方法将30只SD大鼠随机分为空白对照组和STZ组,每组15只。经单次腹腔注射链脲佐菌素(streptozotocin,STZ)诱发大鼠糖尿病动物模型。根据不同的干预方式,在注射STZ 2周后,一次性向大鼠一眼玻璃体内注射100μmol·L-1 PACAP38(STZ+PACAP38组),另一眼注射等量的PBS作为对照(STZ对照组)。采用Western blot和免疫组织化学分析视网膜组织中瞬时受体电位离子通道蛋白6(transient receptor potential channel 6,TRPC6)的表达情况。利用光学相干断层扫描检测各组大鼠的视网膜厚度。将视网膜神经节细胞系RGC-5细胞暴露于高血糖和低氧环境,分别给予PACAP38、TRPC6通道激动剂(OAG)、PAC1受体拮抗剂(PACAP6-38)、TRPC6通道阻滞剂(SKF96365)处理,对照组用完全培养基常规培养,采用MTT法分析不同药物处理对RGC-5细胞活力的影响。结果与空白对照组大鼠视网膜厚度相比较,STZ对照组视网膜厚度显著增加(P<0.05),STZ+PACAP38组大鼠视网膜厚度较STZ对照组明显减少(P<0.05)。免疫组织化学结果显示与空白对照组相比较,STZ对照组及STZ+PACAP38组大鼠神经节细胞层、内丛状层、内核层和外丛状层中均检测到了TRPC6免疫阳性信号,且STZ对照组TRPC6免疫阳性表达显著高于STZ+PACAP38组(P<0.05)。Western blot分析显示,STZ组大鼠视网膜TRPC6蛋白表达显著高于STZ+PACAP38组(P<0.01)。与HG+DFO+OAG组相比,PACAP38与OAG联合处理可显著提高RGC-5细胞活力(均为P<0.01)。结论PACAP38可抑制STZ诱导的DR大鼠视网膜增厚,提高体外暴露于高血糖/低氧环境中RGC-5细胞活力,其可能机制是通过调控TRPC6过表达有效逆转了糖尿病大鼠视网膜病变。
Objective To investigate whether the effect of pituitary adenylate cyclase activating peptide(PACAP38)on retina in diabetic retinopathy(DR)was mediated by transient receptor potential channel 6(TRPC6).Methods Thirty SD rats were randomly divided into a normal control group and a streptozotocin(STZ)model group,with 15 in each group.A single intraperitoneal injection of STZ induced an animal model of diabetes in rats.According to different intervention methods,two weeks after STZ injection,100μmol·L-1 PACAP38(STZ+PACAP38 group)was injected into the vitreous cavity of one eye of rat at one time,and the same amount of PBS was used as a control(STZ group)in the other eye.Western blot and immunohistochemistry were used to analyze the expression of TRPC6 in retinal tissue.Retinal thickness of rats was measured by means of optical coherence tomography.Retinal ganglion cell line(RGC-5 cells)were exposed to hyperglycemia and hypoxia,and were treated with PACAP38,TRPC6 channel agonist(OAG),PAC1 receptor antagonist(PACAP6-38),and TRPC6 channel blocker(SKF96365)respectively,while the normal control group was treat normally in complete medium.The cell viability of each group was analyzed by MTT methods.Results Compared with the normal control group,the thickness of the retina in the STZ group increased significantly(P<0.05),while the thickness of the retina in the STZ+PACAP38 group was thinner than that in the STZ group(P<0.05).From the results of immunohistochemistry,compared with the control group,TRPC6 immunopositive signals were detected in the ganglion cell layer,inner plexiform layer,inner core layer and outer plexiform layer of the STZ group and STZ+PACAP38 group.The expression of TRPC6 immunopositive signal in STZ group was significantly higher than that in STZ+PACAP38 group(P<0.05).Western blot analysis showed that the expression of TRPC6 protein in STZ group was significantly higher than that in STZ+PACAP38 group(P<0.01).Compared with the HG+DFO+OAG group,the combined treatment of PACAP38 and OAG significantly increased the viability of RGC-5 cells(P<0.01).Conclusions PACAP38 can inhibit retinal thickening of DR rats induced by STZ,and increase the viability of RGC-5 cells exposed to hyperglycemia/hypoxia in vitro.The possible mechanism is that it can effectively reverse the retinopathy of diabetic rats by regulating TRPC6 overexpression.
作者
皮百木
宋玫侠
PI Baimu;SONG Meixia(Department of Ophthalmology,Kaifeng Eye Hospital,Kaifeng Central Hospital,Kaifeng 475000,Henan Province,China;Department of Ophthalmology,Joint Logistics Support Force 988 Hospital,Zhengzhou 450007,Henan Province,China)
出处
《眼科新进展》
CAS
北大核心
2020年第7期625-630,共6页
Recent Advances in Ophthalmology
