高良姜素对青光眼大鼠视网膜神经节细胞的保护作用

Protective effect of galangin during the loss of retinal ganglion cells in glaucoma rats

目的研究高良姜素对青光眼大鼠视网膜神经节细胞(RGC)的保护作用。方法取48只SD大鼠随机分为对照组、对照+高良姜素组、高眼压组和高眼压+高良姜素组,每组12只(12眼)。对照+高良姜素组和高眼压+高良姜素组大鼠接受高良姜素滴眼液治疗,对照组和高眼压组大鼠则接受相同剂量二甲基亚砜溶剂滴眼。于造模后第0~28天监测大鼠眼压,并于造模后第28天处死大鼠,收集各组大鼠右眼球,并采用HE染色观察大鼠视网膜形态变化,采用免疫组织化学染色分析各组大鼠视网膜中GFAP、Toll样受体4(TLR4)和NOD样受体3(NLRP3)表达。从4只4 d龄大鼠视网膜中分离RGC,分为空白组、光气组和CoCl 2+高良姜素组。光气组和光气+高良姜素组RGC与200μmol·L-1 CoCl 2一起孵育48 h,光气+高良姜素组RGC中加入20μmol·L-1高良姜素干预48 h。收集各组细胞,采用流式细胞术和Western blot检测RGC的凋亡率及相关蛋白的相对表达情况,并采用ELISA检测空白组、光气组和光气+高良姜素组RGC中IL-18和IL-1β蛋白表达。结果造模后第3~28天,与对照组比较,高眼压组大鼠眼压均显著升高(均为P<0.001),高眼压+高良姜素组大鼠眼压差异均有统计学意义(均为P<0.05)。造模后第0~28天,对照组与对照+高良姜素组大鼠眼压差异均无统计学意义(均为P>0.05)。造模后第18~28天,与高眼压组比较,高眼压+高良姜素组大鼠眼压均持续显著降低(均为P<0.05)。对照组大鼠视网膜厚度、RGC生存率与对照+高良姜素组相比差异均无统计学意义(均为P>0.05);与对照组比较,高眼压组大鼠视网膜厚度、RGC生存率均显著减少(均为P<0.01),高眼压+高良姜素组大鼠视网膜厚度、RGC生存率差异均无统计学意义(均为P>0.05);与高眼压组比较,高眼压+高良姜素组大鼠视网膜厚度、RGC生存率均显著增加(均为P<0.05)。Western blot检测结果显示,与对照组比较,高眼压组大鼠视网膜组织中TLR4、NLRP3蛋白表达均显著上调(均为P<0.001),高眼压+高良姜素组大鼠视网膜组织中TLR4、NLRP3蛋白表达均显著降低(均为P<0.001),对照+高良姜素组大鼠视网膜组织中TLR4、NLRP3蛋白表达差异无统计学意义(均为P>0.05);与高眼压组比较,高眼压+高良姜素组大鼠视网膜组织中TLR4、NLRP3蛋白表达均显著降低(均为P<0.01)。在体外实验中,与空白组比较,光气组RGC中IL-18和IL-1β表达均显著增加(均为P<0.05);与光气组比较,光气+高良姜素组RGC中IL-18和IL-1β表达均显著减少(均为P<0.001);光气组RGC中TLR4、NLRP3蛋白表达较空白组均显著增加(均为P<0.001);且光气+高良姜素组RGC中TLR4、NLRP3蛋白表达较光气组均显著减少(均为P<0.01)。结论高良姜素在青光眼模型中可降低眼压并防止RGC凋亡,其保护机制可能与抑制TLR4/NLRP3炎症小体激活有关。

Objective To investigate the protective effect of galangin on glaucoma rats with retinal ganglion cell(RGC)loss.Methods Forty-eight Sprague\|Dawley rats were randomly divided into the control group,control+galangin group,high intraocular pressure(IOP)group,and high IOP+galangin group with 12 rats(12 eyes)in each group.The rats in the control+galangin group and high IOP+galangin group were treated with galangin eye drops,while rats in the control group and the high IOP group were treated with the same dose of dimethyl sulfoxide eye drops.The IOP of rats was monitored from 0 d to 28 d after modeling.The rats were killed on the 28 d,and the right eyeballs of rats in each group were collected.The retinal morphological changes were observed by Hematoxylin and Eosin staining,and expression levels of glial fibrillary acidic protein(GFAP),Toll-like receptor 4(TLR4)and NOD-like receptor protein 3(NLRP3)in the retina were analyzed by immunohistochemistry.The RGCs were isolated from the retina of 4 rats at 4 d and divided into three groups:blank group,CoCl 2 group,and CoCl 2+galangin group.The RGCs in the CoCl 2 group and the CoCl 2+galangin group were incubated with 200μmol·L-1 CoCl 2 for 48 h,and the CoCl 2+galangin group was additionally administered with 20μmol·L-1 galangin for 48 h.Cells from each group were collected,then the apoptosis rate and expression levels of related proteins of RGCs were detected by flow cytometry and Western blot,and the interleukin(IL)-18 and IL-1βproteins in the blank group,CoCl 2 group and CoCl 2+galangin group were detected by using enzyme-linked immunosorbent assay.Results From 3 d to 28 d after the modeling,compared with the control group,the IOP increased significantly in the high IOP group(all P<0.001),and that in the high IOP+galangin group showed statistically significant differences(all P<0.05).From 0 d to 28 d,there was no significant difference in the IOP between the control group and the control+galangin group(all P>0.05).From 18 d to 28 d,compared with the high IOP group,the IOP in the high IOP+galangin group steadily decreased(all P<0.05).The retinal thickness and RGC survival rate in the control group had no significant difference from the control+galangin group and high IOP+galangin group(all P>0.05).Compared with the control group,the retinal thickness and RGC survival rate in the high IOP group significantly declined(both P<0.01).Compared with the high IOP group,the retinal thickness and RGC survival rate in the high IOP+galangin group significantly increased(both P<0.05).The Western blot detection results showed that compared with the control group,the expression levels of TLR4 and NLRP3 in the retina of rats in the high IOP group significantly increased(both P<0.001),those in the high IOP+galangin group significantly decreased(both P<0.001),while those in the control+galangin group have no significant difference(both P>0.05).Compared with the high IOP group,the expression levels of TLR4 and NLRP3 in the retina of rats in the high IOP+galangin group significantly decreased(both P<0.01).These results were consistent with the immunofluorescence chemical staining results.In vitro,both IL-18 and IL-1βexpressions of RGCs significantly increased in the CoCl 2 group compared with the blank group(both P<0.05);those were significantly reduced in the CoCl 2+galangin group compared with the CoCl 2 group(both P<0.001).The TLR4 and NLRP3 protein expression levels in the RGCs increased in the CoCl 2 group compared with the blank group(both P<0.001),and those were significantly reduced in the CoCl 2+galangin group compared with the CoCl 2 group(both P<0.01).Conclusion Galangin reduces IOP and prevents RGC apoptosis in glaucoma models,and its protective mechanism may be related to the inhibition of TLR4/NLRP3 inflammasome activation.