microRNA-1247通过靶向CCR16抑制脂多糖诱导的小鼠急性肺炎的机制
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摘要
目的探讨microRNA-1247通过趋化因子CC配体16(CCR16)抑制脂多糖诱导的急性肺炎的作用机制。方法选取8周龄雌性BABL/c小鼠30只,随机分为3组,即急性肺炎模型组(6 h)、急性肺炎模型组(12 h)和正常组,每组10只小鼠。模型组小鼠采取气管滴加LPS(8 mg/kg),正常组给予与LPS含量相同的生理盐水。分别在LPS造模6 h和12 h后对小鼠进行麻醉解剖,取腹腔动脉中的血液检测各组的血气值;检测肺组织干湿重含量;应用qRT-PCR以及ELISA检测TNF-α,IL-1β及microRNA-1247表达量;应用蛋白质免疫印迹实验检测CCR16、TLR4、IRAK6、TAK、IKK与NF-κB p52含量。结果模型组小鼠与正常组相比较,模型组小鼠的PaO_2,氧和指数(PaO_2/FiO_2)降低,肺叶组织干湿重比增加,差异有统计学意义(P<0.05)。而PaCO_2值,炎症因子TNF-α、IL-1β的mRNA表达水平差异无统计学意义(P>0.05)。模型组在造模后6 h和12 h肺叶组织中的microRNA-1247含量与CCR16蛋白表达量增高均有统计学意义(P<0.05)。细胞表面受体蛋白TLR4表达量与正常组相比较,TLR4表达量降低,差异有统计学意义(P<0.05)。IRAK6,TAK,IKK以及转位入核蛋白NF-κB与正常组相比较,在造模后6 h和12 h组织中NF-κB蛋白表达量降低,差异有统计学意义(P<0.05)。结论在急性肺炎模型中,microRNA-1247是通过趋化因子配体CCR16抑制因LPS诱导的急性肺炎而导致的各种细胞因子及蛋白表达升高。
Objective To explore the mechanism of the inhibitory effect of microRNA(miR)-1247 on lipopolysaccharide(LPS)-induced acute pneumonia. Methods Thirty 8-week-old female BABL/c mice were randomly divided into three groups: acute pneumonia model group(6 h), acute pneumonia model group(12 h) and normal group, with 10 mice in each group. LPS solution(8 mg/kg) was instilled into the mouse trachea of the model group, and the normal group was instilled an equal volume of physiological saline. Autopsy was performed after anesthesia at 6 h and 12 h after LPS modeling. The blood gas from the celiac artery was measured. The dry and wet weight of lung tissue was measured. qRT-PCR and ELISA were used to detect the expression levels of TNF-α, IL-1β and miR-1247 in the pulmonary tissues. The levels of CCR16, TLR4, IRAK6, TAK, IKK and NF-κB p52 proteins in the lung tissues of each group were detected by western blot. Results Compared with the normal group, the PaO_2, oxygen index(PaO_2/FiO_2) of the model group mice was decreased, and the dry-wet weight ratio of the lung was significantly increased(P < 0.05). However, the differences in PaCO_2 and mRNA expression levels of TNF-α and IL-1β between the two groups were not statistically significant(P > 0.05). The levels of miR-1247 and CCR16 protein expression in the lung tissues of the model group were significantly increased at 6 h and 12 h after modeling(P < 0.05). Compared with the normal group, the expression of TLR4 on the cell surface was significantly lower(P < 0.05). Compared with the normal group, the expressions of IRAK6, TAK, IKK and transfected nuclear protein NF-κB in the tissues at 6 h and 12 h after modeling were decreased significantly(P < 0.05).Conclusions The results of this study show that miR-1247 inhibites the increase of various cytokines and proteins in the LPS-induced acute pneumonia model via chemokine ligand CCR16.
Objective To explore the mechanism of the inhibitory effect of microRNA(miR)-1247 on lipopolysaccharide(LPS)-induced acute pneumonia. Methods Thirty 8-week-old female BABL/c mice were randomly divided into three groups: acute pneumonia model group(6 h), acute pneumonia model group(12 h) and normal group, with 10 mice in each group. LPS solution(8 mg/kg) was instilled into the mouse trachea of the model group, and the normal group was instilled an equal volume of physiological saline. Autopsy was performed after anesthesia at 6 h and 12 h after LPS modeling. The blood gas from the celiac artery was measured. The dry and wet weight of lung tissue was measured. qRT-PCR and ELISA were used to detect the expression levels of TNF-α, IL-1β and miR-1247 in the pulmonary tissues. The levels of CCR16, TLR4, IRAK6, TAK, IKK and NF-κB p52 proteins in the lung tissues of each group were detected by western blot. Results Compared with the normal group, the PaO_2, oxygen index(PaO_2/FiO_2) of the model group mice was decreased, and the dry-wet weight ratio of the lung was significantly increased(P < 0.05). However, the differences in PaCO_2 and mRNA expression levels of TNF-α and IL-1β between the two groups were not statistically significant(P > 0.05). The levels of miR-1247 and CCR16 protein expression in the lung tissues of the model group were significantly increased at 6 h and 12 h after modeling(P < 0.05). Compared with the normal group, the expression of TLR4 on the cell surface was significantly lower(P < 0.05). Compared with the normal group, the expressions of IRAK6, TAK, IKK and transfected nuclear protein NF-κB in the tissues at 6 h and 12 h after modeling were decreased significantly(P < 0.05).Conclusions The results of this study show that miR-1247 inhibites the increase of various cytokines and proteins in the LPS-induced acute pneumonia model via chemokine ligand CCR16.
引文
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[3] Gaur S,Agnihotri R,Costantini,et al.Alzheimer’s disease and chronic periodontitis:is there an association?[J].Geriatr Gerontol Int,2015,15(4):391-404.
[4] Latta CH,Brothers HM,Wilcock DM,et al.Neuroinflammation in Alzheimer’s disease;A source of heterogeneity and target for personalized therapy[J].Neuroscience,2015,302(8):103-111.
[5] Han JE,Choi JW.Control of JNK for an activation of NADPH oxidase in LPS stimulated BV2 microglia[J].Arch Pharm Res,2012,35(4):709-715.
[6] Cai Z,Hussain MD,Yan LJ.Microglia,neuroinflammation,and β-amyloid protein in Alzheimer’s disease [J].Int J Neurosci,2014,124(5):307-321.
[7] Chéret C,Gervais A,Lelli A,et al.Neurotoxic activation of microglia is promoted by a nox1dependent NADPH oxidase[J].J Neurosci,2008,28(46):12039-12051.
[8] Lin GH,Lee YJ,Choi DY,et al.Antiamyloidogenic e?ect of thiacremonone through anti-in?amation in vitro and in vivo models[J].J Alzheimers Dis,2012,29(21):659-676.
[9] Owens R,Grabert K,Davies CL,et al.Divergent neuroin?ammatory regulation of microglial TREM expression and involvement of NF-κB [J].Front Cell Neurosci,2017,11(15):56.
[10] Badshah H,Ali T,Kim MO.Osmotin attenuates LPS-induced neuroin?ammation and memory impairments via the TLR4/NFκB signaling pathway [J].Sci Rep,2016,6(13):24493.
[11] Gomez-Cambronero J.Lack of effective translational regulation of PLD expression and exosome biogenesis in triple-negative breast cancer cells [J].Cancer Metastasis Rev,2018,37(2-3):491-507.
[12] Stoeck K,Schmitz M,Ebert E,et al.Immune responses in rapidly progressive dementia:a comparative study of neuroinflammatory markers in Creutzfeldt-Jakob disease,Alzheimer’s disease and multiple sclerosis [J].J Neuroinflamm,2014,11(10):170.
[13] Kaminska B,Mota M,Pizzi M.Signal transduction and epigenetic mechanisms in the control of microglia activation during neuroinflammation [J].Biochim Biophys Acta,2016,1862(3):339-351.
[14] 程东良,梁媛,陈衍晨.MiRNA-1247-3P 在急性呼吸窘迫综合征细胞模型中的表达及功能分析[J].实用医学杂志,2015,31(17):2802-2804.
[15] Xu Y,Ito T,Fushimi S,et al.Spred-2 deficiency exacerbates lipopolysaccharide-induced acute lung inflammation in mice [J].PLoS One,2014,9(9):e108914.
[16] Yi JH,Hye Jin Park,Beak SJ,et al.Danggui-Jakyak-San enhances hippocampal long-term potentiation through the ERK/CREB/BDNF cascade [J].J Ethnopharmacol,2015,175(4):481-489.
[17] Dang Y,Mu Y,Wang K,et al.Papaverine inhibits lipopolysaccharide-induced microglial activation by suppressing NF-κB signaling pathway [J].Drug Des Dev Ther,2016,10(1):851-859.
[18] Schulte LN,Westermann AJ,Vogel J,et al.Differential activation and functional specialization of miR-146 and miR-155 in innate immune sensing [J].Nucleic Acids Res,2013,41(1):542-553.