敲低NLRP12对高眼压大鼠RGCs的保护作用及其抑制细胞焦亡的机制  

Protective effect of NLRP12 knockdown on retinal damage in rats with ocular hypertension and its mechanism of inhibiting cell pyroptosis

作　　者：宋伟琼[1] 何芳[1] 杜玲芳[1] 谭华霞[1] 刘丹[2] Song Weiqiong;He Fang;Du Lingfang;Tan Huaxia;Liu Dan(Department of Ophthalmology,The First People's Hospital of Chenzhou,Chenzhou 423000,China;Department of Ophthalmology,Xiangya Hospital,Central South University,Changsha 410078,China)

机构地区：[1]湖南省郴州市第一人民医院眼科,郴州423000 [2]中南大学湘雅医院眼科,长沙410078

出　　处：《中华实验眼科杂志》2023年第2期110-118,共9页Chinese Journal Of Experimental Ophthalmology

基　　金：湖南省自然科学基金科卫联合项目(2019JJ80012)。

摘　　要：目的探讨敲低NOD样受体家族热蛋白结构域12(NLRP12)对高眼压大鼠视网膜神经节细胞(RGCs)炎症因子水平和视网膜损伤的影响及其机制。方法选取70只SPF级成年雄性SD大鼠,采用随机数字表法分为对照组、高眼压组、高眼压+小干扰RNA阴性对照(siNC)组、高眼压+siNLRP12组和高眼压+siNLRP12+重组大鼠caspase-1(rrcaspase-1)组,每组14只。其中对照组仅接受右眼结膜切口处理,其他各组均采用巩膜外静脉烧灼法建立大鼠右眼高眼压模型;高眼压+siNC组、高眼压+siNLRP12组和高眼压+siNLRP12+rrcaspase-1组建立高眼压模型后分别给予尾静脉注射siNC、siNLRP12和siNLRP12+rrcaspase-1试剂。巩膜外静脉烧灼术后1 d、1周、2周、3周,测量大鼠右眼眼压;巩膜外静脉烧灼术后3周,采用苏木精-伊红染色法观察各组大鼠视网膜结构,计数各组RGCs数量。将RGCs分为对照组、rrcaspase-1组、siNC+rrcaspase-1组、siNLRP12+rrcaspase-1组,其中rrcaspase-1组、siNC+rrcaspase-1组、siNLRP12+rrcaspase-1组分别采用rrcaspase-1、siNC+rrcaspase-1和siNLRP12+rrcaspase-1处理细胞24 h,对照组不予处理。采用Western blot法检测RGCs和大鼠视网膜组织中NLRP12、caspase-1、cleaved-caspase-1蛋白表达水平;采用酶联免疫吸附测定法检测大鼠血清或细胞培养物上清中肿瘤坏死因子α(TNF-α)及白细胞介素1β(IL-1β)浓度。结果与对照组比较,术后1、2、3周高眼压组眼压高于对照组,差异均有统计学意义(均P<0.05)。对照组视网膜各层组织清晰,RGCs呈单层排列,高眼压组和高眼压+siNC组RGCs层松散,视网膜内丛状层变薄。高眼压+siNLRP12组视网膜内丛状层较高眼压组增厚,高眼压+siNLRP12组和高眼压+siNLRP12+rrcaspase-1组RGCs层松散。对照组、高眼压组、高眼压+siNC组、高眼压+siNLRP12组和高眼压+siNLRP12+rrcaspase-1组RGCs数量分别为(119.31±23.25)、(89.19±16.98)、(88.87±13.92)、(109.33±10.25)和(92.89±12.58)个,总体比较Objective To investigate the effect and mechanism of NOD-like receptor family pyrin domain containing 12(NLRP12)knockdown on inflammatory factor levels and retinal injury in retinal ganglion cells(RGCs)of rats with high intraocular pressure.Methods Seventy SPF adult male SD rats were selected and randomized into control group,high intraocular pressure(IOP)group,high IOP+small interfering RNA negative control(siNC)group,high IOP+siNLRP12 group and high IOP+siNLRP12+recombinant rat caspase-1(rrcaspase-1)group,with 14 rats in each group.Rats in the control group were only treated with conjunctival incision in the right eye,and ocular hypertension model was established in the other four groups with external scleral vein cauterization.High IOP+siNC group,high IOP+siNLRP12 group and high IOP+siNLRP12+rrcaspase-1 group were injected with siNC,siNLRP12 and siNLRP12+rrcaspase-1 reagent via the tail vein,respectively.The IOP of the right eye was measured at 1 day,1,2 and 3 weeks after the operation.Three weeks after the operation,the retinal structure was observed by hematoxylin-eosin staining,and the number of RGCs in each group was counted.RGCs were divided into control group,rrcaspase-1 group,siNC+rrcaspase-1 group,siNLRP12+rrcaspase-1 group.The cells in rrcaspase-1 group,siNC+rrcaspase-1 group and siNLRP12+rrcaspase-1 group were treated with rrcaspase-1,siNC+rrcaspase-1 and siNLRP12+rrcaspase-1 reagent for 24 hours,respectively.No treatment was given to the control group.The expression levels of NLRP12,caspase-1 and cleaved-caspase-1 proteins in RGCs and retinal tissue were detected by Western blot.The concentrations of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in rat serum or cell culture supernatant were detected by enzyme-linked immunosorbent assay.The study protocol was approved by the Animal Ethics Committee of the First People's Hospital of Chenzhou(No.2020086).Results Compared with control group,the IOP was higher in high IOP group at 1,2 and 3 weeks after cauterization,and the differences were

关 键 词：青光眼 高眼压 视网膜神经节细胞 NOD样受体家族热蛋白结构域12 半胱天冬酶1 细胞焦亡 

分 类 号：R775[医药卫生—眼科]