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 Pseudomonas 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 factors; 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), 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 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 serum 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 Pseudomonas aeruginosa keratitis, and its mechanism of action may be related to the inhibition of GSDMD-mediated activation of the atypical cellular pyroptosis pathway.
关键词
甘草酸 /
GSDMD介导的非典型细胞焦亡 /
铜绿假单胞菌 /
炎症 /
角膜损伤
Key words
glycyrrhizic acid /
gasdermin-D mediated atypical cellular pyroptosis /
Pseudomonas aeruginosa /
inflammation /
corneal damage
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基金
无锡市卫健委课题“甘草酸通过焦亡通路调控NLRP3影响角膜炎症的研究”（Q202348）
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