南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (12): 1815-1821.doi: 10.12122/j.issn.1673-4254.2022.12.09

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敲除S1PR3可缓解小鼠的急性肺损伤：基于抑制MAPK信号通路

房尚萍，袁 冉，孙任珂，马同军   

  1. 皖南医学院麻醉学院，麻醉学实验实训中心，法医学院，安徽 芜湖 241002
  • 出版日期:2022-12-20 发布日期:2023-01-12

Knockout of S1PR3 attenuates acute lung injury in mice by inhibiting the MAPK pathway

FANG Shangping, YUAN Ran, SUN Renke, MA Tongjun   

  1. School of Anesthesiology, Anesthesia Laboratory and Training Center, College of Forensic Medicine, Wannan Medical College, Wuhu 241002, China
  • Online:2022-12-20 Published:2023-01-12

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摘要： 目的 探讨敲除S1PR3是否通过抑制丝裂原活化蛋白激酶（MAPKs）途径改善脂多糖（LPS）诱导的小鼠急性肺损伤。方法 用LPS诱导小鼠急性肺损伤模型。雄性C57BL/6J和S1PR3敲除小鼠，随机分为4组：C57 NS组（C57，生理盐水处理）、C57 LPS组（C57，LPS诱导）、S1PR3-/- NS组（S1PR3敲除鼠，生理盐水处理）、S1PR3-/- LPS组（S1PR3敲除鼠，LPS诱导），8只/组。RT-qPCR检测S1PR3，IL-β和IL-18表达，HE染色检测肺部组织损伤，流式细胞术检测细胞凋亡水平，Western blot法检测caspase-1，GSDMD，p-JNK，p-ERK p-p38 蛋白表达水平。同时，Ⅱ型肺泡上皮细胞（MLE-12 细胞）铺板后分为 4 组：PBS 组、LPS 组、CYM5541组（仅加入S1PR3激动剂）、CYM5541+LPS组（加入S1PR3激动剂+LPS），检测各组细胞焦亡水平及MAPK信号通路分子的表达水平。结果 急性肺损伤小鼠肺组织S1PR3表达上调（P<0.001）；血清IL-1β和IL-18因急性肺损伤而明显升高（P<0.05）。急性肺损伤小鼠因敲除S1PR3肺出血、炎症渗出明显改善，肺湿干比重下降，细胞凋亡比例和焦亡相关蛋白如clv-caspase-1，GSDMD表达均降低（P<0.05）。MLE-12细胞因加入S1PR3激动剂，细胞焦亡相关蛋白表达均升高（P<0.05）。此外，MAPKs家族（JNK，ERK p38）的激活因S1PR3敲除后受到抑制，因加入S1PR3激动剂而表达明显升高（P<0.05）。结论 敲除S1PR3可通过抑制MAPK信号通路改善急性肺损伤。

关键词: S1PR3；急性肺损伤；MAPK通路；细胞焦亡；CYM5541

Abstract: Objective To investigate whether knockout of S1PR3 improves lipopolysaccharide (LPS)-induced acute lung injury in mice by inhibiting mitogen activated protein kinases (MAPKs). Methods Male C57BL/6J and S1PR3 knockout (S1PR3-/-) mice were both randomized into two groups (n=8) for intratracheal instillation of normal saline or LPS to induce acute lung injury. The expressions of S1PR3, IL-1β and IL-18 in the lung tissues were detected using RT-qPCR, lung tissue injury was observed with HE staining, and cell apoptosis was detected using flow cytometry. Western blotting was performed to detect the expression levels of caspase-1, GSDMD, p- JNK, p-ERK and p-p38 proteins. In the cell experiment, type II alveolar epithelial cells (MLE-12 cells) were treated with PBS, LPS, CYM5541 (a S1PR3 agonist), or CYM5541 + LPS, and the cell apoptosis and expression levels of MAPK signal pathway molecules were detected. Results The expression of S1PR3 was up-regulated and serum IL-1β and IL-18 levels were elevated significantly in the nontransgenic mice with acute lung injury (P<0.001). By comparison, the elevation of IL-1β and IL-18 levels was obviously reduced in S1PR3 knockout mice with acute lung injury, which also showed significant improvement of pulmonary hemorrhage, inflammation and exudation, lowered wet-to-dry ratio of the lungs, and decreased cell apoptosis and expressions of cleaved caspase-1 and GSDMD (P<0.05). In MLE-12 cells, treatment with the S1PR3 agonist significantly increased the expression of pyroptosis-associated proteins (P<0.05). S1PR3 knockout strongly inhibited the activation of MAPKs family (JNK and ERK p38; P<0.05), but their expressions were significantly increased following treatment with the S1PR3 agonist (P<0.05). Conclusion Inhibition of S1PR3 can improve LPS-induced acute lung injury in mice by inhibiting the activation of MAPK signaling.

Key words: S1PR3; acute lung injury; MAPK pathway; cell pyroptosis; CYM5541