摘要
目的:探究甘草酸通过抑制Gasdermin-D(GSDMD)介导的非典型细胞焦亡改善铜绿假单胞菌诱导的炎症和角膜损伤.方法:制备铜绿假单胞菌角膜炎大鼠模型,将大鼠随机分为对照组、PA组和GLY+PA组.比较各组角膜炎症指数;酶联免疫吸附试验(ELISA)检测血清炎症因子;苏木素-伊红(HE)染色检测角膜组织病理学变化;免疫荧光检测角膜组织中半胱氨酸的天冬氨酸蛋白水解酶11(caspase-11)和GSDMD阳性表达;分别用蛋白质印迹法和qRT-PCR检测检测角膜组织中半胱氨酸的天冬氨酸蛋白水解酶4(caspase-4)、半胱氨酸的天冬氨酸蛋白水解酶5(caspase-5)、caspase-11、GSDMD蛋白和mRNA表达.结果:PA组大鼠角膜炎症指数和血清中白介素1β(IL-1β)、白介素18(IL-18)水平、炎症细胞密度、caspase-11和GSDMD阳性表达面积百分比及角膜组织中caspase-4、caspase-5、caspase-11、GSDMD蛋白和mRNA表达均明显高于对照组;和PA组相比,GLY+PA组大鼠角膜炎症指数和和血清中IL-1β、IL-18水平、炎症细胞密度、caspase-11和GSDMD阳性表达面积百分比及角膜组织中caspase-4、caspase-5、caspase-11、GSDMD蛋白和mRNA表达均明显降低.结论:甘草酸可减轻铜绿假单胞菌角膜炎大鼠角膜炎症反应和组织损伤,其作用机制可能和抑制GSDMD介导的非典型细胞焦亡途径激活有关.
Abstract
Objective To investigate how glycyrrhetinic acid improves inflammation and corneal injury induced by Pseu-domonas aeruginosa by inhibiting Gasdermin-D(GSDMD)mediated atypical cell pyroptosis.Methods Prepare a rat model of Pseudomonas aeruginosa keratitis and randomly divide the rats into a control group,PA group,and GLY+PA group.Compare the keratitis index of each group;Enzyme linked immunosorbent assay(ELISA)was used to detect serum inflammatory fac-tors;Hematoxylin eosin(HE)staining was used to detect pathological changes in corneal tissue;Immunofluorescence detection showed positive expression of caspase-11 and GSDMD in corneal tissue for cysteine;Protein blotting and qRT PCR were used to detect the expression of caspase-4,caspase-5,caspase-11,GSDMD protein,and mRNA in corneal tissue.Results The keratitis index and serum levels of interleukin-1β(IL-1 β)(78.54±8.03)and interleukin-18(IL-18)(26.31±2.74),inflam-matory cell density,percentage of positive expression areas of caspase-11 and GSDMD,as well as the expression of caspase-4,caspase-5,caspase-11,GSDMD protein and mRNA in corneal tissue of rats in the PA group were significantly higher than those in the control group;Compared with the PA group,the GLY+PA group significantly reduced the keratitis index and se-rum levels of IL-1β(46.27±5.11)and IL-18(13.52±1.46),inflammatory cell density,percentage of positive expression areas of caspase-11 and GSDMD,as well as the expression of caspase-4,caspase-5,caspase-11,GSDMD protein and mRNA in corneal tissue.Conclusion Glycyrrhizic acid inhibits the corneal inflammatory response and tissue damage in rats with Pseu-domonas aeruginosa keratitis,and its mechanism of action may be related to the inhibition of GSDMD-mediated activation of the atypical cellular pyroptosis pathway.
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