Abstract：
Objective: Septic patients are especially vulnerable to acute lung injury (ALI). Calycosin (CAL) has various promising pharmacological activities. This paper aims to expound on the role of CAL in mice with sepsis-induced ALI and the associated mechanisms.Methods: Mouse models of sepsis-induced ALI were established using lipopolysaccharide (LPS). Pulmonary histopathological changes were observed by HE staining. Cell apoptosis was assessed by TUNEL staining. Pulmonary edema was evaluated by measuring wet/dry weight. Bronchoalveolar lavage fluid (BALF) was collected to count inflammatory cells. In vitro LPS models were established using MLE-12 cells. miR-375-3p expression was determined by RT-qPCR. Cell viability and apoptosis were assessed by MTT assay and flow cytometry. Levels of inflammatory cytokines were determined by ELISA. The target relationship between miR-375-3p and ROCK2 was analyzed by the dual-luciferase assay. ROCK2 protein level was determined by Western blot.Results: miR-375-3p was weakly-expressed in mice with sepsis-induced ALI, and CAL treatment elevated miR-375-3p expression. CAL treatment mitigated pulmonary tissue damage and edema, decreased apoptosis and inflammatory cells, downregulated levels of pro-inflammatory cytokines, and upregulated levels of anti-inflammatory cytokines in mice with sepsis-induced ALI. CAL treatment increased MLE-12 cell viability and decreased apoptosis and inflammation in MLE-12 cells. Inhibition of miR-375-3p partially abrogated CAL-mediated protective action on MLE-12 cells. miR-375-3p attenuated LPS-induced MLE-12 cell injury by targeting ROCK2.Conclusion: CAL upregulates miR-375-3p to target ROCK2, thus protecting against sepsis-induced ALI in mice.
摘要：
目的:脓毒症患者尤其容易发生急性肺损伤(ALI)。毛蒜素（CAL）具有多种有前途的药理活性。本文旨在阐述CAL在脓毒症诱导的ALI小鼠中的作用及相关机制。方法:采用脂多糖(LPS)建立脓毒症小鼠ALI模型。HE染色观察肺组织病理学改变。通过TUNEL染色评估细胞凋亡。通过测量湿/干重评估肺水肿。收集支气管肺泡灌洗液(BALF)计数炎性细胞。使用MLE-12细胞建立体外LPS模型。通过RT-qPCR测定miR-375-3p表达。通过MTT分析和流式细胞术评估细胞活力和凋亡。通过ELISA测定炎性细胞因子的水平。通过双荧光素酶实验分析miR-375-3p与ROCK2的靶关系。通过蛋白质印迹测定ROCK2蛋白水平。结果：miR-375-3p在脓毒症诱导的ALI小鼠中弱表达，和CAL治疗升高miR-375-3p表达。CAL治疗减轻肺组织损伤和水肿，细胞凋亡和炎症细胞减少，促炎细胞因子水平下调，并上调脓毒症诱导的ALI小鼠的抗炎细胞因子水平。CAL处理增加了MLE-12细胞活力并减少了MLE-12细胞的凋亡和炎症。miR-375-3p的抑制部分消除了CAL介导的对MLE-12细胞的保护作用。miR-375-3p通过靶向ROCK2减轻LPS诱导的MLE-12细胞损伤。结论：CAL上调miR-375-3p靶向ROCK2，从而保护脓毒症诱导的小鼠ALI。
