金雀异黄酮对大鼠视网膜缺血-再灌注损伤的保护作用

目的　探讨金雀异黄酮（GEN）对大鼠视网膜缺血-再灌注（I/R）损伤的保护作用。方法　选取30只健康SPF级大鼠，按照随机数字表法将其分为假手术组、I/R组和I/R+GEN组，每组各10只。I/R组和I/R+GEN组大鼠采用前房灌注生理盐水升高眼压法制备视网膜I/R损伤大鼠模型，I/R组大鼠术后每天给予注射生理盐水（1 mL·kg^-1·d^-1）；I/R+GEN组大鼠术后每天给予注射GEN （40 mL·kg^-1·d^-1）；假手术组大鼠行相应假手术后每天予以注射生理盐水（1 mL·kg^-1·d^-1），连续7 d后，颈椎脱臼法处死各组大鼠，取眼部组织。HE染色观察各组大鼠视网膜的形态以及内丛状层（IPL）、内核层（INL）和神经节细胞层（GCL）厚度；免疫荧光分析观察各组大鼠视网膜神经节细胞（RGC）的存活率；TUNEL染色检测各组大鼠视网膜细胞凋亡水平；检测各组大鼠视网膜组织中过氧化氢酶（CAT）、丙二醛(MDA)及超氧化物歧化酶(SOD)水平以反映视网膜氧化应激状态；Western blot分析三组大鼠视网膜中NLRP3、ASC、Caspase-1的蛋白表达。结果　I/R组、I/R+GEN组和假手术组大鼠视网膜总厚度分别为(114.37±7.32）μm、（155.31±6.83）μm和（178.98±13.65）μm（t=14.284，P<0.01），I/R组低于假手术组和I/R+GEN组，差异均有统计学意义（均为P<0.01）。I/R组大鼠视网膜IPL、INL和GCL厚度分别为（18.95±5.06）μm、（17.62±4.69）μm和（19.03±4.74）μm，I/R+GEN组大鼠视网膜IPL、INL和GCL厚度分别为（20.69±8.13）μm、（25.74±6.78）μm和（26.71±7.85）μm，假手术组大鼠视网膜IPL、INL和GCL厚度分别为（11.73±3.15）μm、（11.97±3.56）μm和（12.59±3.24）μm（均为P<0.05），I/R组与假手术组和I/R+GEN组三个指标比较差异均有统计学意义（均为P<0.01）。I/R组、I/R+GEN组和假手术组大鼠视网膜中RGC相对存活率分别为（26.87±3.12）%、（73.46±7.80）% 和（100.00±5.64）%（t=16.825，P<0.01），I/R组RGC相对存活率低于假手术组和I/R+GEN组，差异均有统计学意义（均为P<0.01）。I/R组、I/R+GEN组和假手术组大鼠视网膜中CAT、MDA、SOD含量总体比较差异均有统计学意义（均为P<0.01），I/R组与I/R+GEN组和假手术组三个指标比较差异均有统计学意义（均为P<0.05）。Western blot检测结果显示，三组大鼠视网膜中NLRP3、ASC、Caspase-1的蛋白表达总体比较差异均有统计学意义（均为P<0.01），组间两两比较差异均有统计学意义（均为P<0.05）。结论　GEN可通过提高视网膜细胞抗炎和抗氧化能力抑制视网膜细胞凋亡，从而起到对大鼠视网膜I/R损伤的保护作用。

Protective effect of genistein on retinal ischemia-reperfusion injury in rats

Objective　To explore the protective effect of genistein (GEN) on retinal ischemia-reperfusion (I/R) injury in rats.Methods　Thirty healthy SPF rats were selected and randomly divided into the sham operation group, I/R group, and I/R+GEN group, with 10 rats in each group. In the I/R group and I/R+GEN group, I/R rat models were prepared by anterior chamber infusion of normal saline to increase intraocular pressure. In the I/R group, rats were injected with 1 mL·kg^-1·d^-1 normal saline every day after operation. In the I/R+GEN group, rats were injected with 40 mL·kg^-1·d^-1 GEN every day after operation. In the sham operation group, rats were injected with 1 mL·kg^-1·d^-1 normal saline every day after the corresponding sham operation. Seven consecutive days later, all rats were dislocated and sacrificed, and their eye tissues were taken. The hematoxylin and eosin (HE) staining was used to observe the retinal morphology and thickness of inner plexiform layer (IPL), inner nuclear layer (INL) and ganglion cell layer (GCL). The immunofluorescence assay was used to determine the survival rate of retinal ganglion cells (RGC). The TUNEL staining was used to detect the apoptosis of retinal cells. The catalase (CAT), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured to reflect the oxidative stress state of rats’ retinas. Western blot was used to analyze the expression of NLRP3, ASC, and Caspase-1 proteins in the retina of all rats.Results　The total retinal thickness of rats in the I/R group, I/R+GEN group, and sham operation group were (114.37±7.32) μm, (155.31±6.83) μm, and (178.98±13.65) μm, respectively. The differences among the three groups were statistically significant (t=14.284, all P<0.01). The thickness of IPL, INL, and GCL of rats were (18.95±5.06) μm, (17.62±4.69) μm, and (19.03±4.74) μm in the I/R group, (20.69±8.13) μm, (25.74±6.78) μm, and (26.71±7.85) μm in the I/R+GEN group, (11.73±3.15) μm, (11.97±3.56) μm, and (12.59±3.24) μm in the sham operation group (all P<0.05). The differences among the three groups were statistically significant (all P<0.01). The survival rates of RGCs in the retina of rats in the I/R group, I/R+GEN group, and sham operation group were (26.87±3.12) %, (73.46±7.80) %, and (100.00±5.64) %, respectively. The differences among the three groups were statistically significant (t=16.825, all P<0.01). The content of CAT, MDA, and SOD in the retina of rats showed statistically significant differences among the three groups (all P<0.01). In the I/R group, the three indexes were statistically different from those in the I/R+GEN group and the sham operation group (all P<0.05). Western blot results showed that there were statistically significant differences in the expression levels of NLRP3, ASC, and Caspase-1 in the retina of rats among the three groups (all P<0.01), and these indexes also showed statistically significant differences between any two groups (all P<0.05).Conclusion　GEN can improve the anti-inflammatory and antioxidant capacity of retinal cells to inhibit apoptosis, thus enhancing the protective effect on retinal I/R injury in rats.