脂质纳米粒递送IL-35 mRNA对脂多糖诱导小鼠急性肺损伤的保护作用

Protective effect of IL-35 mRNA deliveried by lipid nanoparticles against lipopolysaccharide induced acute lung injury in mice

目的 探讨脂质纳米粒(LNP)递送白细胞介素35(IL-35)mRNA(IL-35 mRNA-LNP)对脂多糖(LPS)诱导小鼠急性肺损伤(ALI)的保护作用。方法 将56只小鼠随机分成7组,每组8只,分别为正常对照组、IL-35 mRNA-LNP(250μg·kg^(-1))组、LPS组、LPS+IL-35 mRNA-LNP(50,125和250μg·kg^(-1))组及LPS+地塞米松(DXM)组。IL-35 m RNA-LNP(250μg·kg^(-1))组、LPS+IL-35 m RNA-LNP(50,125和250μg·kg^(-1))组小鼠iv给予相应剂量的LNP包载IL-35 mRNA复合物,LPS+DXM组小鼠iv给予DXM(5 mg·kg^(-1)),随后除正常对照组和IL-35 mRNA-LNP(250μg·kg^(-1))组外,其他组均采用气管滴注LPS(5 mg·kg^(-1))建立ALI模型,造模24 h后处死小鼠,记录肺系数和肺组织湿/干重比(W/D);乳酸脱氢酶(LDH)试剂盒检测肺组织LDH活性;实时荧光定量PCR检测肺组织肿瘤坏死因子α(TNF-α)、IL-6和IL-1β mRNA水平;ELISA法检测肺组织IL-35,TNF-α,IL-6和IL-1β蛋白水平;苏木精-伊红(HE)染色观察肺组织病理变化;免疫荧光法检测肺组织淋巴细胞抗原6G(Ly6G)表达水平。结果 与正常对照组、LPS组和LPS+DXM组相比,LPS+IL-35mRNA-LNP(50,125和250μg·kg^(-1))组肺组织均显著表达IL-35蛋白(P<0.01)。与正常对照组相比,LPS组肺系数、肺组织W/D、LDH活性及TNF-α,IL-6和IL-1β mRNA水平以及蛋白水平均显著升高(P<0.01),出现肺泡出血、肺泡壁增厚和中性粒细胞浸润。与LPS组相比,LPS+IL-35 m RNA-LNP(50,125和250μg·kg^(-1))组肺系数、肺组织W/D、LDH活性及TNF-α,IL-6和IL-1β mRNA水平以及蛋白水平均显著降低(P<0.01),同时肺泡出血、肺泡壁增厚和中性粒细胞浸润明显改善。结论 IL-35 mRNA-LNP可在小鼠肺组织中表达IL-35蛋白,并通过抑制促炎因子表达,有效改善LPS诱导的小鼠ALI。

OBJECTIVE To investigate the protective effect of interleukin-35(IL-35)mRNA-lipid nanoparticles(LNP)against lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice.METHODS Fifity-six mice were randomly divided into 7 groups with 8 mice in each,including the normal control group,IL-35 mRNA-LNP(250μg·kg^(-1))group,LPS group,LPS+IL-35 mRNA-LNP(50,125 and 250μg·kg^(-1))group and LPS+Dexamethasone(DXM)group.Except for the normal control group and IL-35 mRNA-LNP(250μg·kg^(-1))group and ALI model was established by tracheal infusion of LPS in each of the other groups.IL-35 mRNA-LNP(250μg·kg^(-1))group and LPS+IL-35 mRNA-LNP(50,125 and 250μg·kg^(-1))group were injected with a corresponding dose of LNP encapsulated mRNA complex via the tail vein while the LPS+DXM group was injected with DXM via the tail vein.Lung coefficient and the wet to dry weight ratio(W/D)of lung tissue were recorded.The mRNA levels of inflammatory cytokines tumor necrosis factor-α(TNF-α),Interleukin-6(IL-6)and IL-1βof lung homogenates were detected by realtime fluorescence quantitative PCR(RT-qPCR).LDH activity of lung homogenates and the protein levels of IL-35,TNF-α,IL-6 and IL-1βin lung homogenate were detected by corresponding kits.Hematoxylineosin(HE)staining was used to observe and analyze the pathological injury to lung tissue.The expres⁃sion of Lymphocyte antigen 6G(Ly6G)was detected by Immunofluorescence to reflect the infiltration of neutrophils.RESULTS Compared with the normal control group,LPS group and LPS+DXM group,IL-35 protein expression levels in lung homogenates of the other groups were more significant(P<0.01).Compared with the normal control group,lung coefficient,W/D ratio of lung tissue,LDH activity,mRNA levels and the protein levels of TNF-α,IL-6 and IL-1βin lung homogenates were significantly increased in the LPS group(P<0.01),accompanied by alveolar hemorrhage,alveolar wall thickening and neutro⁃phils infiltration.After IL-35 mRNA-LNP administration,lung coefficient,W/D ratio of lung tissue,LDH activity,mRNA levels and the protein levels of TNF-α,IL-6 and IL-1βin lung homogenates were signifi⁃cantly decreased(P<0.01),and alveolar hemorrhage,alveolar wall thickening and neutrophil infiltration were obviously improved.CONCLUSION IL-35 mRNA-LNP can express IL-35 protein in lung tissue of mice,and effectively improve LPS-induced ALI in mice by inhibiting the expression of proinflammatory factors.