五味子苷B调控miR-370-3p/CCL3轴对幼年肺炎小鼠免疫炎症因子水平的影响

Impacts of Schisandrin B on Immune Inflammatory Factors in Juvenile Mice with Pneumonia by Regulating miR-370-3p/CCL3 Axis

目的:探讨五味子苷B(Sch B)在幼年肺炎小鼠的作用机制。方法:将90只幼年雄性小鼠随机分为对照组、模型组、Sch B低剂量组(Sch BL组)、Sch B高剂量组(Sch BH组)、Sch BH+antagomir-NC组、Sch BH+miR-370-3p antagomir组各15只。Sch BL组和Sch BH组小鼠分别灌胃20、60 mg/kg的Sch B;Sch BH+antagomir-NC组和Sch BH+miR-370-3p antagomir组,先将1 nmol的miR-370-3p antagomir、antagomir-NC用20μL的磷酸盐缓冲液(PBS)溶解,在Sch B灌胃后将miR-370-3p antagomir、antagomir-NC质粒分别经尾静脉注射到小鼠体内;对照组和模型组给予等量生理盐水。每天1次,持续给药7 d。测定各组小鼠肺组织的湿干质量比;采用苏木精-伊红(HE)染色观察各组小鼠肺组织的病理形态;检测各组小鼠肺组织中炎症因子水平;流式细胞术检测外周血T淋巴细胞亚群;实时定量反转录聚合酶链式反应(qRT-PCR)检测肺组织中miR-370-3p和CCL3的mRNA水平;双荧光素酶实验验证miR-370-3p与CCL3的靶向关系。结果:与对照组相比,模型组幼鼠肺组织结构紊乱,肺泡壁变厚,出现大量炎性细胞浸润,组织受损严重,肺组织湿干质量比、CD8^(+)、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、CCL3 mRNA水平均升高,CD4^(+)、CD4^(+)/CD8^(+)、miR-370-3p水平均降低(P均<0.05)。与模型组相比,Sch BL组和Sch BH组幼鼠肺组织中肺泡壁变薄,炎性细胞浸润明显减少,损伤减轻,肺组织湿干质量比、CD8^(+)、TNF-α、IL-6、CCL3 mRNA水平均降低,CD4^(+)、CD4^(+)/CD8^(+)、miR-370-3p水平均升高(P均<0.05)。加入miR-370-3p antagomir进行回补实验,结果显示Sch B对肺炎幼鼠免疫功能和炎症保护作用被逆转,且CCL3 mRNA水平升高(P<0.05);双荧光素酶报告基因实验验证了miR-370-3p与CCL3存在靶向关系。结论:Sch B能够通过靶向调节miR-370-3p/CCL3轴来增强幼年肺炎小鼠免疫功能,并抑制炎症反应。

Objective:To investigate the mechanism of Schisandrin B(Sch B)in juvenile mice with pneumonia.Methods:Ninety juvenile male mice were randomly divided into the control group,model group,Sch B low dose group(Sch BL group),Sch B high dose group(Sch BH group),Sch BH+antagomir-NC group and Sch BH+miR-370-3p antagomir group,with 15 mice in each group.Sch BL group and Sch BH group were given 20 and 60 mg/kg Sch B,respectively.For the Sch BH+antagomir-NC group and Sch BH+miR-370-3p antagomir group,1 nmol of miR-370-3p antagomir and antagomir-NC were first dissolved in 20μL of phosphate buffer solution(PBS),and Sch B was gavaged,miR-370-3p antagomir,antagomir-NC plasmid were injected into mice by tail vein,respectively.The control group and model group were given the same amount of normal saline.The drug was administered once a day for 7 d.The wet to dry weight ratio of pulmonary tissue of mice in each group was measured.Hematoxylin Eosin(HE)staining was applied to observe the pathological morphology of pulmonary tissue in each group of mice.Levels of inflammatory factors in pulmonary tissues of mice were detected.Flow cytometry was applied to detect T lymphocyte subsets in peripheral blood.Quantitative real time polymerase chain reaction(qRT-PCR)was used to detect the levels of miR-370-3p and CCL3 mRNA in the pulmonary tissues.Double Luciferase experiment was applied to verify the targeting relationship between miR-370-3p and CCL3.Results:Compared with the control group,the pulmonary tissue structure of juvenile mice in the model group was disordered,with thickened alveolar walls,a large number of inflammatory cell infiltration,and severe tissue damage,the value of wet to dry weight,percentage of CD8+,levels of tumour necrosises factor(TNF)-α,interleukin(IL)-6 and CCL3 mRNA increased,CD4+,CD4+/CD8+,and miR-370-3p decreased(P<0.05).Compared with the model group,the alveolar wall in pulmonary tissue of juvenile mice in Sch BL group and Sch BH group became thinner,inflammatory cell infiltration reduced significantly,and damage was alleviated,the value of wet to dry weight,CD8+,levels of TNF-α,IL-6 and CCL3 mRNA decreased,CD4+,CD4+/CD8+,and miR-370-3p increased(P<0.05).The addition of miR-370-3p antagomir for compensatory experiments showed that the protective effects of Sch B on immune function and inflammation in juvenile mice with pneumonia were reversed,and the level of CCL3 mRNA increased(P<0.05).Double Luciferase reporter gene experiment verified that there was a targeting relationship between miR-370-3p and CCL3.Conclusion:Sch B can enhance the immune function of juvenilea mice with pneumonia and inhibit the inflammatory response by targeting the miR-370-3p/CCL3 axis.