藏红花素对视网膜缺血再灌注损伤小鼠视网膜神经节细胞的保护作用及其机制研究

目的　研究藏红花素对视网膜缺血再灌注损伤（RIRI）小鼠视网膜神经节细胞（RGC）的保护作用及其机制。方法　将144只C57BL/6小鼠随机分为3组:假手术组、模型组、藏红花素治疗组。模型组和藏红花素治疗组小鼠建立RIRI模型，藏红花素治疗组小鼠造模前30 min腹腔注射50 mg·kg^-1藏红花素。RIRI后14 d，视网膜铺片染色比较各组小鼠RGC密度差异。RIRI后24 h，HE染色比较各组小鼠视网膜内层厚度差异。于RIRI后不同时间点（0 h、3 h、6 h、9 h、12 h、15 h）取各组小鼠视网膜组织，通过多重基因定量分析系统检测NLRP3、ASC、Caspase-1、白细胞介素-1β（IL-1β） mRNA的表达变化。RIRI后6 h和12 h取各组小鼠视网膜组织，Western blot检测NLRP3、ASC、Caspase-1、IL-1β蛋白的表达，ELISA检测IL-1β蛋白的含量，并对比分析。结果　小鼠RIRI后14 d，视网膜铺片染色结果显示，藏红花素治疗组较模型组小鼠RGC密度增加约18.5%（P<0.05）。RIRI后24 h，HE染色结果显示，藏红花素治疗组小鼠视网膜内层厚度较模型组显著降低（P<0.01）。多重定量分析系统检测结果显示，RIRI后6 h、9 h及12 h，藏红花素治疗组小鼠视网膜组织中Caspase-1以及IL-1β mRNA表达较模型组均显著降低（均为P<0.05)。Western blot检测结果显示，藏红花素治疗组小鼠视网膜组织中Caspase-1以及IL-1β蛋白表达较模型组均显著降低（均为P<0.05)。RIRI后6 h、12 h，模型组小鼠视网膜组织中NLRP3、ASC mRNA和蛋白表达与假手术组相比无显著变化（均为P>0.05)。ELISA检测结果进一步证实，RIRI后6 h和12 h,藏红花素治疗组小鼠视网膜组织中IL-1β蛋白含量较模型组均显著降低（均为P<0.05)。结论　藏红花素通过抑制Caspase-1和IL-1β表达保护RIRI小鼠RGC。

Protective effect and mechanism of crocin on retinal ganglion cells of mice with retinal ischemia-reperfusion injury

Objective To explore the protective effect and mechanism of crocin on surviving retinal ganglion cells(RGC)of mice with retinal ischemia reperfusion injury(RIRI).Methods A total of 144 C57BL/6 mice were randomly divided into three groups sham operation group,model group,and crocin treatment group.RIRI model of mice in the model group and crocin treatment group was established.Mice in the crocin treatment group were intraperitoneally injected with 50 mg·kg^-1 crocin 30 min before modeling.RGC density was observed and compared by retinal flat-mount preparation and staining at 14 d after RIRI,while the internal retinal thickness was observed and compared by hematoxylin and eosin(HE)staining at 24 h after RIRI.The mRNA levels of NOD-like receptor protein 3(NLRP3),apoptotic speck-like protein containing a caspase recruitment domain(ASC),Caspase-1,and interleukin-1β(IL^-1β)in retinal tissues were detected and compared by multiple gene quantitative analysis system at 0 h,3 h,6 h,9 h,12 h,and 15 h respectively after RIRI,while the protein expression of NLRP3,ASC,Caspase-1,and IL^-1βin retinal tissues were detected and compared by Western blot at 6 h and 12 h respectively after RIRI.ELISA was used to analyze the variations in protein expression of IL^-1β.Results Retinal flat-mount staining results showed that on the 14th day after RIRI,the density of RGC in the crocin treatment group was 18.5%higher than that in the model group(P<0.05).HE staining results showed that 24 h after RIRI,the inner retinal thickness of mice in the crocin treatment group was significantly lower than that in the model group(P<0.01).Multiple gene quantitative analysis results showed that at 6 h,9 h and 12 h after RIRI,the mRNA levels of Caspase-1 and IL^-1βin the crocin treatment group were significantly lower than those in the model group(both P<0.05).Western blot results showed that the protein expression of Caspase-1 and IL^-1βin the crocin treatment group was significantly lower than that in the model group(both P<0.05).There were no significant differences in the mRNA and protein levels of NLRP3 and ASC in the retinal tissues of mice between the model group and the sham operation group at 6 h and 12 h respectively after RIRI(all P>0.05).ELISA results further confirmed that the IL^-1βprotein expression in the crocin treatment group was significantly lower than that in the model group at 6 and 12 h respectively after RIRI(both P<0.05).Conclusion Crocin protects RGC of mice with RIRI by inhibiting Caspase-1 and IL^-1βexpression.