非诺贝特对糖尿病视网膜病变小鼠视网膜神经细胞的保护作用及机制

Protective effect and mechanism of Fenofibrate on retinal neuro cells in mice with diabetic retinopathy

目的探讨嘌呤受体P2X7-NOD样受体蛋白-3(Nod-like receptor pyrin domain 3,NLRP3)炎性相关信号转导通路在糖尿病小鼠视网膜神经细胞中的表达情况,初步研究非诺贝特对糖尿病视网膜病变(diabetic retinopathy,DR)的保护作用。方法健康雄性小鼠60只,随机分为实验对照组(N组)、糖尿病组(D组)、非诺贝特治疗组(F组),D组及F组通过腹腔内注射链脲佐菌素制备DR模型,分别于造模后每天记录血糖情况,并于4周、8周、12周进行三组间血糖水平的比较。自造模成功后第2天起,F组每天口服非诺贝特100 mg·kg^-1连续喂养12周,4周后免疫荧光化学法观察视网膜神经胶质细胞及视网膜神经节细胞(retinal ganglion cell,RGC)的活化情况,Western blot检测视网膜组织中P2X7、NLRP3蛋白的表达情况。结果与N组相比,D组血糖水平在造模后4周、8周、12周随时间推移均有不同程度增长,差异均有统计学意义(均为P<0.05)。N组中,GFAP少量表达;D组4周时GFAP的表达主要分布于内界膜(inner limiting membrane,ILM)、RGC层和内丛状层(inner plexiform layer,IPL);非诺贝特治疗后,F组小鼠视网膜GFAP主要分布于ILM和RGC层,且密度与D组相比明显减弱。Western blot检测结果显示:N组中,视网膜P2X7和NLRP3蛋白仅有少量表达;D组中二者的表达水平在4周时明显升高,与N组相比,差异均有统计学意义(均为P<0.05);而此时F组经非诺贝特治疗后小鼠视网膜组织中P2X7和NLRP3蛋白的表达水平较D组明显降低,但仍高于N组,差异均有统计学意义(均为P<0.05)。结论非诺贝特可通过下调糖尿病小鼠视网膜组织中P2X7和NLRP3蛋白的表达从而发挥视网膜保护作用。

Objective To investigate the expression of purinergicreceptor P2 X7-Nod-like receptor pyrindomain 3(P2 X7-NLRP3) inflammatory signaling pathway in retinal neuro cells in mice and discuss the protective effect of Fenofibrate on diabetic retinopathy(DR).Methods Totally 60 healthy male mice were randomly divided into experimental control group(N group),diabetes group(D group) and Fenofibrate treatment group(F group).DR was induced by intraperitoneal injection of streptozotocin(STZ) in mice of D and F group.Serum glucose levels were measured everyday and compared among three groups at 4 weeks,8 weeks and 12 weeks after model establishment.Each mouse in F group was treated with Fenofibrate 100 mg·kg^-1 from 2 days to 12 weeks after DR successful establishment.Four weeks later,immunohistochemical staining was used to detect the activation of retina glial and retinal ganglion cell(RGC).Then,the expression of P2 X7 and NLRP3 was evaluated by Western blot.Results Compared with N group,serum glucose level in D group increased at 4 weeks,8 weeks and 12 weeks after model establishment to some extent,and significant difference was found between the two groups(all P<0.05).Low expression of GFAP in N group was detected,while it can be seen in inner limiting membrane(ILM),RGC and inner plexiform layer(IPL) of retina in D group at 4 weeks after model establishment.Compared to D group,GFAP in F group which was treated by Fenofibrate was found locating in ILM and RGC layers and the density decreased obviously.Western blot showed that tiny expression of P2 X7 and NLRP3 was found in N group,while their expression increased in D group at 4 weeks after model establishment and significant difference was found between D and N group(all P<0.05).After treatment with Fenofibrate,the expression of P2 X7 and NLRP3 in F group decreased significantly when compared with D group,but still significantly higher than N group(all P<0.05).Conclusion Fenofibrate may have neuroprotective effect on diabetic retinopathy by suppressing the activation of P2 X7 and NLRP3 in diabetic mice.