ZLN005通过上调PGC-1α表达诱导巨噬细胞的M2型分化减轻脓毒症引起的急性肺损伤

doi:10.3969/j.issn.1006-6233.2022.02.02

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摘要 目的: 在脓毒症诱导的急性肺损伤(ALI)中探究小分子化合物ZLN005的作用及相关机制。方法: 利用盲肠结扎穿孔(CLP)术建立小鼠脓毒症诱导的急性肺损伤模型,并按处理方式的不同将小鼠分为四组,假手术(Sham)组,ZLN005组、CLP组及CLP+ZLN005组;应用RT-PCR、免疫荧光染色及Western blot实验检测各组小鼠肺组织中过氧化物酶体增殖物激活受体γ共激活因子-1α(PGC-1α)的mRNA及蛋白表达水平;ELISA检测肺泡支气管灌洗液(BALF)中炎症因子TNF-α、IL-1β、IL-6和IL-10的含量;HE染色观察各组小鼠肺组织病理组织学损伤;统计学方法分析CLP造模7d后各组小鼠的存活率;体外培养小鼠巨噬细胞系RAW264.7,转染靶向沉默PGC-1α的siRNA,RT-PCR及Western blot检测转染效率;利用LPS诱导脓毒症ALI的体外细胞模型,并将RAW264.7细胞分为五组,对照组(NC),ZLN005组,LPS组,LPS+ZLN005+si-NC和LPS+ZLN005+si-PGC-1α;RT-PCR检测各组细胞中M1型巨噬细胞标记物iNOS与MHC-Ⅱ与M2型巨噬细胞标记物Arg1与CD206的mRNA表达水平;Western blot检测各组细胞中AMPK-α的磷酸化水平(p-AMPK-α/AMPK-α)及线粒体生物发生中关键蛋白NRF1、NRF2和mtTFA的蛋白表达。结果: 成功建立小鼠脓毒症诱导的急性肺损伤模型;其中,与Sham组比较,ZLN005组与CLP+ZLN005组肺组织中PGC-1α的表达水平显著升高(P<0.05),CLP组中明显降低(P<0.05),但CLP+ZLN005组又较CLP组中PGC-1α的表达显著升高(P<0.05);各组小鼠的BALF、HE染色及术后7d的存活率结果显示,与CLP组和CLP+ZLN005组中BALF中促炎因子TNF-α、IL-1β、IL-6的表达与肺组织病理损伤水平较Sham组或ZLN005组明显升高(P<0.05),抑炎因子IL-10及术后7d的存活率却显著降低(P<0.05),而CLP+ZLN005组又较CLP组明显改善(P<0.05);在体外细胞实验中,与NC组相比,PGC-1α蛋白表达在ZLN005组与LPS+ZLN005组明显升高(P<0.05),LPS组显著降低(P<0.05);转染si-PGC-1α后RAW264.7巨噬细胞中PGC-1的表达被显著抑制(P<0.05);与NC组相比,LPS组、ZLN005+LPS+si-NC组与ZLN005+LPS+si-PGC-1α组中M1型巨噬细胞标记物的mRNA表达水平明显升高(P<0.05),同时ZLN005+LPS+si-NC组的M2型巨噬细胞标记物的mRNA表达也显著升高(P<0.05),但AMPK-α的磷酸化水平与线粒体生物发生中关键蛋白的表达均显著下降(P<0.05);而ZLN005+LPS+si-NC组又较LPS组与ZLN005+LPS+si-PGC-1α中M1型标记物表达显著升高(P<0.05),但M2型标记物与AMPK-α的磷酸化水平与线粒体生发蛋白的蛋白表达均显著降低(P<0.05)。结论: ZLN005能促进PGC-1α的表达以促进M2巨噬细胞的极化来保护脓毒症引起的肺损伤,而其相关作用机制可能与ZLN005通过AMPK信号通路促进线粒体生物发生相关。

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	张昊
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	杨璐瑜

关键词 ： ZLN005, 过氧化物酶体增殖物激活受体γ共激活因子-1α, M2型巨噬细胞, 脓毒症诱导的急性肺损伤

Abstract：Objective: To explore the role and mechanism of ZLN005 in sepsis induced acute lung injury (ALI). Methods: Sepsis induced acute lung injury model in mice was established by cecal ligation and puncture (CLP), and the mice were divided into four groups according to different treatment. Methods: sham group, ZLN005 group, CLP Group and CLP +ZLN005 group; RT-PCR, immunofluorescence staining and Western blot were used to detect peroxisome proliferator activated receptor (PPAR) in lung tissue of mice γ coactivator-1 α( PGC-1 α). The mRNA and protein expression levels were measured; ELISA was used to detect the contents of TNF-α,IL-1β,IL-6 and IL-10 in BALF; HE staining was used to observe the histopathological damage of lung tissue; the survival rate of mice in each group after 7 days of CLP modeling was analyzed by statistical method; RAW264.7 was cultured in vitro and transfected with si-PGC-1α siRNA. RT-PCR and Western blot were used to detect the transfection efficiency. RAW264.7 cells were divided into five groups: NC group, ZLN005 group, LPS group, LPS+ZLN005+si-NC and LPS+ZLN005+si-PGC-1 α. RT-PCR was used to detect the mRNA expression levels of M1 macrophage markers iNOS and MHC-Ⅱ, M2 macrophage markers Arg1 and CD206. The phosphorylation level of p-AMPK-α/AMPK-α and the key proteins in mitochondrial biogenesis of Nrf1, Nrf2 and mtTFA was detected by Western blot. Results: Compared with sham group, the mRNA and protein expression levels and fluorescence intensity of PGC-1α in lung tissues of ZLN005 group and CLP+ZLN005 group were significantly increased (P<0.05), while those in CLP group were significantly decreased (P<0.05). Compared with the CLP group, the above detection indexes in the CLP+ZLN005 group were significantly increased (P<0.05). BALF, HE staining and survival rate 7 days after operation of mice in each group showed that the expressions of pro-inflammatory factors TNF-α, IL-1β and IL-6 in BALF and the pathological injury level of lung tissue in CLP group and CLP+ZLN005 group were significantly higher than those in sham group or ZLN005 group (P<0.05). However, the survival rate of anti-inflammatory factor IL-10 and postoperative 7 days were significantly decreased (P<0.05). Compared with the CLP group, the change trends of the above indexes in the CLP+ZLN005 group were significantly decreased (P<0.05). Compared with NC group, mRNA expression levels of M1 iNOS and MHC-Ⅱ in LPS group, ZLN005+LPS+si-NC group and ZLN005+LPS+si-PGC-1α group were significantly increased (P<0.05), meanwhile, the mRNA expressions of Arg1 and CD206 in Zln005+LPS+Si-NC group were significantly increased (P<0.05), while the protein expressions of p-AMPK-α/AMPK-α and NRF1, NRF2 and mtTFA were decreased (P<0.05). Compared with ZLN005+LPS+si-NC group, the mRNA expressions of iNOS and MHC-Ⅱ in LPS group and ZLN005+LPS+si-PGC-1α were significantly increased (P<0.05). The mRNA expression of Arg1 and CD206 and protein expression of p-AMPK-α/AMPK-α and NRF1, NRF2 and mtTFA were significantly decreased (P<0.05). Conclusion: ZLN005 can promote the expression of PGC-1α and promote the polarization of M2 macrophages to protect sepsis induced lung injury, and the mechanism may be related to the promotion of mitochondrial biogenesis by ZLN005 through AMPK signaling pathway.

Key words： ZLN005 PGC-1α M2 macrophages Acute lung injury induced by sepsis

基金资助:湖北省卫生健康委科研项目,(编号:WJ2019M066)

引用本文:

张昊, 谭赟, 杨璐瑜. ZLN005通过上调PGC-1α表达诱导巨噬细胞的M2型分化减轻脓毒症引起的急性肺损伤[J]. 河北医学, 2022, 28(2): 182-189.
ZHANG Hao, TAN Yun, YANG Luyu. ZLN005 Alleviates Sepsis Induced Acute Lung Injury by Upregulating PGC-1α Expression to Induce M2-Type Differentiation of Macrophages. HeBei Med, 2022, 28(2): 182-189.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2022.02.02 或 http://www.hbyxzzs.cn/CN/Y2022/V28/I2/182

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