Abstract：
Acute lung injury (ALI) is a common disease with complex pathogenesis. However, the treatment is mainly symptomatic with limited clinical options. Asiaticoside (AS), a Chinese herbal extract, has protective effects against LPS-induced ALI in mice and inhibits nitric oxide and prostaglandin E2 synthesis; however, the specific mechanism of AS in the prevention and treatment of LPS-induced ALI needs further study. Sema4D/CD72 pathway, mitochondrial dysfunction, and miRNA-21 are closely associated with inflammation. Therefore, the present study aimed to explore whether AS exerts its therapeutic effect on ALI by influencing Sema4D/CD72 pathway and mitochondrial dysfunction, restoring the balance of inflammatory factors, and influencing miRNA-21 expression. Cell and animal experiments were performed to investigate the effect of AS on ALI. Lipopolysaccharide (LPS) was used to establish the ALI model. CCK8 and flow cytometry were used to detect the cell viability and apoptosis rate. HE staining and wet-to-dry weight ratio (W/D) of lung tissue were determined. The expressions of Sema4D, CD72, NF-κB p65, Bax, Bcl2, and caspase 3 in RAW264.7 cells and lung tissues were detected by western blot, and the levels of IL-10 and IL-1β induced by LPS in supernatant of RAW264.7 cells and BALF were measured by ELISA. And the expression of miRNA-21 in cells and lung tissues was detected by fluorescence quantitative PCR. The result shows that AS treatment suppressed LPS-induced cell damage and lung injury in mice. AS treatment could alleviate the pathological changes such as inflammatory infiltration and histopathological changes in the lungs caused by LPS, and reduce the ratio of W/D. AS significantly alleviated the decrease of mitochondrial membrane potential induced by LPS, inhibited the increase of ROS production, and reduced the expression of mitochondrial fission proteins Drp1 and Fis1. The high-dose AS group significantly downregulated the expression of Sema4D, CD72, phosphorylated NF-κB p65, and apoptosis-related proteins, decreased the pro-inflammatory factor IL-1β, and enhanced the level of anti-inflammatory factor IL-10. In addition, AS promoted miRNA-21 expression. These effects inhibited apoptosis and restored the balance between anti- and pro-inflammatory factors. This represents the inaugural report elucidating the mechanism by which AS inhibits the Sema4D/CD72 signaling pathway. These findings offer novel insights into the potential application of AS in both preventing and treating ALI.
摘要：
急性肺损伤(ALI)是一种常见病,发病机制复杂。然而,治疗主要是对症治疗，临床选择有限。Asiaticoside（AS），一种中草药提取物，对LPS诱导的小鼠ALI具有保护作用，并抑制一氧化氮和前列腺素E2的合成；然而，AS防治LPS诱导的ALI的具体机制有待进一步研究。Sema4D/CD72通路,线粒体功能障碍，miRNA-21与炎症密切相关。因此,本研究旨在探讨AS是否通过影响Sema4D/CD72通路和线粒体功能障碍发挥对ALI的治疗作用,恢复炎症因子的平衡，并影响miRNA-21的表达。进行细胞和动物实验以研究AS对ALI的影响。采用脂多糖(LPS)建立ALI模型。CCK8和流式细胞术检测细胞活力和凋亡率。测定HE染色和肺组织湿干重比(W/D)。Sema4D的表达，CD72,NF-κBp65,Bax,Westernblot检测RAW264.7细胞和肺组织中的Bcl2和caspase3,ELISA法检测LPS诱导的RAW264.7细胞上清液和BALF中IL-10和IL-1β的水平。并用荧光定量PCR检测细胞和肺组织中miRNA-21的表达。结果表明，AS治疗可抑制LPS诱导的小鼠细胞损伤和肺损伤。AS治疗可减轻LPS引起的肺部炎症浸润和组织病理学改变,并降低W/D的比值。AS显著缓解LPS诱导的线粒体膜电位降低,抑制了ROS产生的增加，并降低线粒体裂变蛋白Drp1和Fis1的表达。高剂量AS组显着下调Sema4D的表达，CD72、磷酸化NF-κBp65和凋亡相关蛋白，降低促炎因子IL-1β，并增强抗炎因子IL-10的水平。此外,AS促进miRNA-21表达。这些作用抑制细胞凋亡并恢复抗炎因子和促炎因子之间的平衡。这代表了阐明AS抑制Sema4D/CD72信号通路的机制的首次报道。这些发现为AS在预防和治疗ALI方面的潜在应用提供了新的见解。
