急性高眼压致大鼠视网膜节细胞焦亡及抗Caspase-1处理的神经保护作用

Acute ocular hypertension induced pyropotosis in rat retinal ganglion cells and protective effect of inhibiting Caspase-1 in rat model

目的 探讨在SD大鼠急性高眼压模型中,细胞焦亡及抗Caspase-1处理对视网膜神经节细胞(retinal ganglion cell, RGCs)的保护作用。方法 42只SPF级雄性SD大鼠采用随机数字表法分为正常对照组、急性高眼压模型组和VX-765(Caspase-1抑制剂)干预组,均以右眼为实验眼。模型组和干预组用生理盐水前房灌法建立急性高眼压模型,干预组在建模当日及次日玻璃体腔注入VX-765(200μmol/L)溶液4μL,模型组于相同时间玻璃体腔注入等容量生理盐水。正常对照组不处理。造模后7 d处死大鼠,HE染色观察视网膜形态学变化及RGCs密度;免疫组化观察RGCs并检测视网膜平均光密度;实时荧光定量PCR检测视网膜Caspase-1、gsdmd、IL-1b、IL-18分子mRNA表达;Western blot法检测Caspase1-p20、gsdmd-n、IL-1b、IL-18蛋白表达。结果 模型组大鼠视网膜厚度为(164.53±1.04)μm,低于正常对照组[(181.44±5.13)μm]和干预组[(173.15±2.15)μm],模型组RGCs密度为(332.87±16.37)个/mm^(2),低于正常对照组[(520.39±18.62)个/mm^(2)]和干预组[(373.97±20.32)个/mm^(2)],模型组大鼠视网膜光密度为(0.443±0.014),低于正常对照组(0.517±0.015)和干预组(0.492±0.006),差异均有统计学意义(P<0.05)。模型组大鼠视网膜IL-1b、IL-18 mRNA相对表达量高于正常对照组和干预组,Caspase 1-p20、gsdmd-n、IL-1b、IL-18蛋白相对表达量同样高于正常对照组和干预组,差异均有统计学意义(P<0.05)。结论 急性高眼压暴露后,依赖Caspase-1的细胞焦亡通路分子表达明显增多,抑制Caspase-1活性能减轻视网膜组织损伤,有效保护RGCs,减少RGCs死亡数量。

Objective To investigate the protective effect of pyroptosis and anti-Caspase-1 treatment on retinal ganglion cells(RGCs) in rats with acute ocular hypertension. Methods Forty-two SPF male SD rats were randomly divided into normal control group, acute ocular hypertension model group and VX-765(Caspase-1 inhibitor) intervention group, with the right eye as the experimental eye. Acute ocular hypertension was inflicted by anterior chamber irrigation with normal saline in the model group and intervention group. The rats in the intervention group were further given 4 μL VX-765(200 μmol/L) solution by intravitreal injection on the modeling day and the next day, and those of the model group were injected with equal volume of normal saline in vitreous cavity at the same time. No such treatment was performed on the rats from the control group. All rats were killed at the seventh day after modeling. Retinal morphology and the density of RGCs were observed by HE staining. The survival of RGCs and average absorbance of retina were observed by immunohistochemical assay. The mRNA levels of Caspase-1, gsdmd, IL-1 b and IL-18 were detected by real-time fluorescence quantitative PCR. The protein expression of Caspase 1-p20, gsdmd-n, IL-1 b and IL-18 were detected by Western blotting. Results The retinal thickness was 164.53±1.04 μm in the model group, which was significantly thinner than that in the normal control group(181.44±5.13 μm) and the intervention group(173.15±2.15 μm). The density of RGCs was(332.87±16.37)/mm^(2) in the model group, obviously lower than that in the normal control group [(520.39±18.62)/mm^(2)] and the intervention group [(373.97±20.32)/mm^(2)]. The average absorbance of retina was 0.443±0.014 in the model group, statistically lower than in the normal control group(0.517±0.015) and the intervention group(0.492±0.006). The differences were statistically significant in above values(P<0.05). The mRNA relative expression levels of IL-1 b and IL-18 were significantly higher in the model group than the control group and intervention group(P<0.05). The protein levels of Caspase1-p20, gsdmd-n, IL-1 b and IL-18 were remarkably higher in the model group than the other 2 groups(P<0.05). Conclusion Acute ocular hypertendion exposure enhances the expression of the molecules in Caspase-1-dependent pyropotosis pathway. So, inhibiting Caspase-1 activity can reduce the severity of retinal injury, protect RGCs effectively, and reduce RGCs death.