金水缓纤方通过p53-KRT8信号通路减轻HPAEPICS细胞衰老和分化障碍改善肺纤维化的作用机制OA
Mechanism of Jinshui Huanxian Formula on improving pulmonary fibrosis by alleviating senescence and dysdifferentiation of HPAEPIC cells via p53-KRT8 signaling pathway
目的:探讨金水缓纤方通过调控p53-KRT8信号通路减轻人Ⅱ型肺泡上皮细胞株HPAEPIC细胞衰老和分化障碍以改善博来霉素(bleomycin,BLM)诱导的肺纤维化的作用机制.方法:采用0.63 µg/mL BLM诱导HPAEPIC细胞48 h构建细胞肺纤维化模型,分为空白组(CON组)、模型组(MOD组)、金水缓纤方组(JHF组,10%)、吡非尼酮组(PFD组,20 µg/mL)和p53抑制剂组(Pifithrin-α HBr抑制剂,PFT-α组,10 µmol/L).CCK-8检测细胞活力;RT-PCR检测细胞IL-6、IL-8、p21、p53、SPC、KRT8、IL-13 mRNA的表达水平;流式细胞术检测细胞周期;Western blot检测p53、KRT8、CLDN4、N-CAD、α-SMA蛋白表达水平;免疫荧光检测p53、p21、KRT8 表达水平.结果:与 CON组相比,MOD组细胞 G1 期百分比和 G1/G2 比值明显降低(P<0.01),G2期百分比明显升高(P<0.01);IL-6、IL-8、p21、p53、KRT8、CLDN4 mRNA表达水平明显上调(P<0.05),p53、KRT8、CLDN4、N-CAD、α-SMA蛋白表达水平明显增多(P<0.05),p53、p21、KRT8荧光表达水平明显增多(P<0.01);与MOD组相比,JHF组和PFT-α组G1期百分比明显升高(P<0.05),G2期百分比明显降低(P<0.01);IL-6、IL-8、p21、p53、KRT8、CLDN4 mRNA表达水平明显下调(P<0.05),p53、KRT8、CLDN4、N-CAD、α-SMA蛋白表达水平明显减少(P<0.05),p53、p21、KRT8荧光表达水平明显减少(P<0.01),各组KRT8 荧光与p21 荧光的皮尔森系数>0.5.结论:金水缓纤方可以改善BLM诱导的HPAEPIC细胞肺纤维化,其机制可能与抑制p53-KRT8通路,减轻HPAEPIC细胞衰老和分化障碍有关.
Objective:To explore the effects and mechanisms of Jinshui Huanxian Formula on improving bleomycin(BLM)-induced pulmonary fibrosis by alleviating type Ⅱ alveolar epithelial cells HPAEPIC senescence and dysdifferentiation via regulating the p53-KRT8 signaling pathway.Methods:HPAEPIC cells were induced by 0.63 µg/mL BLM for 48 h to establish a cellular pulmonary fibrosis model.The cells were divided into the blank group(CON group),the model group(MOD group),the Jinshui Huxian Formula group(JHF group,10%),the pirfenidone group(PFD group,20 µg/mL)and the p53 inhibitor group(pifithrin-α HBr inhibitor,PFT-α group,10 µmol/L).Cell viability was detected by CCK-8 assay.The mRNA expression levels of IL-6,IL-8,p21,p53,SPC,KRT8,and IL-13 were detected by RT-PCR.The cell cycle was detected by flow cytome-try.The expression levels of p53,KRT8,CLDN4,N-CAD,and α-SMA were detected by Western blot.The expression levels of p53,p21,and KRT8 was detected by immunofluorescence.Results:Compared to the CON group,the level of G1 phase per-centage and G1/G2 ratio in the MOD group were significantly decreased(P<0.01),the level of G2 phase percentage was signifi-cantly increased(P<0.01).The mRNA expression levels of IL-6,IL-8,p21,p53,KRT8,and CLDN4 were significantly up-regulated(P<0.05).The protein expression levels of p53,KRT8,CLDN4,N-CAD,and α-SMA were significantly in-creased(P<0.05).The fluorescence expression levels of p53,p21,and KRT8 were significantly increased(P<0.01).Com-pared to the MOD group,the levels of G1 phase percentage in the JHF and PFT-α groups were significantly increased(P<0.05),and the levels of G2 phase percentage was significantly decreased(P<0.01).The mRNA expression levels of IL-6,IL-8,p21,p53,KRT8,and CLDN4 were significantly down-regulated(P<0.05).The protein expression levels of p53,KRT8,CLDN4,N-CAD,and α-SMA were significantly reduced(P<0.05).The fluorescence expression levels of p53,p21,and KRT8 were significantly decreased(P<0.01).The Pearson's coefficient between KRT8 fluorescence and p21 fluorescence in all groups was>0.5.Conclusion:Jinshui Huanxian Formula can improve BLM-induced HPAEPIC cell pulmonary fibrosis,and its mecha-nism may be related to the alleviation of HPAEPIC cell senescence and dysdifferentiation via inhibiting the p53-KRT8 signaling pathway.
王宇;游梦月;余本嫚;陈媛媛;韩瑞婷;沈俊岭;余海滨
河南中医药大学第一附属医院呼吸科,河南 郑州 450000||河南中医药大学第一临床医学院,河南 郑州 450046||河南中医药大学呼吸疾病中医药防治省部共建协同创新中心,河南省中医药防治呼吸病重点实验室,河南 郑州 450046河南中医药大学第一附属医院呼吸科,河南 郑州 450000||河南中医药大学第一临床医学院,河南 郑州 450046||河南中医药大学呼吸疾病中医药防治省部共建协同创新中心,河南省中医药防治呼吸病重点实验室,河南 郑州 450046河南中医药大学第一附属医院呼吸科,河南 郑州 450000||河南中医药大学第一临床医学院,河南 郑州 450046河南中医药大学第一附属医院呼吸科,河南 郑州 450000||河南中医药大学第一临床医学院,河南 郑州 450046河南中医药大学第一附属医院呼吸科,河南 郑州 450000河南中医药大学第一附属医院呼吸科,河南 郑州 450000河南中医药大学第一附属医院呼吸科,河南 郑州 450000||河南中医药大学第一临床医学院,河南 郑州 450046||河南中医药大学呼吸疾病中医药防治省部共建协同创新中心,河南省中医药防治呼吸病重点实验室,河南 郑州 450046
医药卫生
肺纤维化衰老金水缓纤方KRT8Ⅱ型肺泡上皮细胞
Pulmonary fibrosisSenescenceJinshui Huanxian FormulaKRT8Type Ⅱ alveolar epithelial cells
《海南医科大学学报》 2026 (4)
258-268,11
This study was supported by the National Key Research Project for the Modernization of Traditional Chinese Medicine(2023YFC3502604)Natural Science Foundation Project of Henan Province(222300420221)Major Special Project of Traditional Chinese Medicine Research in Henan Province(2022ZYZD04)Scientific Research Program of Traditional Chinese Medicine in Henan Province(2021JDZY102,2023ZXZX1033)Special Project of Collaborative Innovation Center of Zhengzhou(2023XTCX043) 国家重点研究计划中医药现代化重点专项(2023YFC3502604)河南省自然科学基金(222300420221)河南省中医药研究重大专项(2022ZYZD04)河南省中医药科学研究专项(2021JDZY102,2023ZXZX1033)郑州市协同创新中心专项(2023XTCX043)
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