miR-210-3p对急性中耳炎小鼠炎症反应的影响及其机制实验研究OA

Objective:To investigate the effect of microRNA-210-3p(miR-210-3p)on inflammatory response in acute otitis media(AOM)mice and its potential underlying mechanism.Methods:A total of 60 male mice were en-rolled,10 mice were randomly assigned into normal control group(Ctrl group),and the remaining 50 mice were es-tablished into AOM models via intratympanic injection of Streptococcus pneumoniae through bullar puncture.Forty successfully modeled mice out of 45 qualified model animals were randomly divided into four groups:AOM model group,agomir NC group(AOM mice transfected with negative control agomir),miR-210-3p agomir group(AOM mice transfected with miR-210-3p agomir),and Ad-insulin-like growth factor 2(IGF2)group(AOM mice co-trans-fected with miR-210-3p agomir and AAV-oe IGF2),with 10 mice in each group.At 24 h after model establishment,mice in agomir NC group received intratympanic injection of 5 μg agomir NC;miR-210-3p agomir group was given 5 μg miR-210-3p agomir via tympanic membrane;Ad-IGF2 group was co-injected with 5 μg miR-210-3p agomir plus 1 × 107PFU Ad-IGF2;equal volume of 0.9％sodium chloride solution was intratympanically administrated in Ctrl and AOM groups synchronously.Interventions were performed once every other day for totally 3 times.At 24 h after the last intervention,auditory brainstem response(ABR)was detected;RT-qPCR was used to quantify the expres-sion levels of miR-210-3p,glucose transporter 1(GLUT1),hexokinase 3(HK3),6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3)and IGF2;HE staining was applied to observe histopathological changes of middle ear tissues;the counts of inflammatory cells and bacteria as well as concentrations of inflammatory factors in middle ear lavage fluid(MELF)were determined;dual-luciferase reporter assay was conducted to verify the targeted binding relationship between miR-210-3p and IGF2;Western blot was adopted to measure the expression of proteins related to IGF2/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway.Results:Compared with the Ctrl group,the ABR threshold was elevated in AOM group and agomir NC group;middle ear mucosal cells were swollen with obvious mucosal thickening,massive infiltration of neutrophils and lymphocytes,interstitial edema and vasodilation.Meanwhile,the counts of inflammatory cells and bacteria,concentrations of interleukin-8(IL-8),IL-1βand tumor necrosis factor-α(TNF-α)in MELF,mRNA levels of GLUT1,HK3,PFKFB3,IGF2 and protein levels of IGF2,as well as the ratios of p-PI3K/PI3K and p-AKT/AKT in middle ear tissues were significantly increased,whereas the expression of miR-210-3p was downregulated in the two groups(all P＜0.05).In contrast to AOM group and agomir NC group,miR-210-3p agomir group presented declined ABR threshold,basically regular polarity arrangement of middle ear mucosal epithelium,alleviated tissue swelling and dramatically reduced inflammatory cell infiltration;the counts of inflammatory cells,levels of IL-8,IL-1β and TNF-α in MELF,mRNA expressions of GLUT1,HK3,PFKFB3,IGF2,and protein expressions of IGF2,together with p-PI3K/PI3K and p-AKT/AKT ratios in middle ear tissues were downregulated,while bacterial count in MELF and tissue miR-210-3p expression were up-regulated(all P＜0.05).Compared with miR-210-3p agomir group,all above-mentioned index changes were reversed in Ad-IGF2 group(all P＜0.05).Conclusion:MiR-210-3p alleviates inflammatory response in AOM mice possibly by in-hibiting glycolysis,and its mechanism may be associated with the downregulation of IGF2/PI3K/AKT signaling pathway.