The Study Of Micro RNA-146a Regulate Synthesis And Secretion Of Cytokines In Lipopolysaccharide-induced Distal Pulmonary Artery Smooth Muscle Cells By TLR4-IRAK-NF-?B Signaling Pathway

Objective: To test the dynamic changes of the microRNA-146 a expression level in distal pulmonary artery smooth muscle cells(PASMCs)of rat which induced by lipopolysacchride(LPS).And transfected plasmid to test whether it can through targeted TLR4 signaling pathways to adjust the synthesis and secretion of the LPS-induced pulmonary artery smooth muscle cells.Methods: The first part: 1.established the COPD model in rats by fumigation combining intratracheal instillation of LPS.The primary generation of PASMCs were isolated and cultured.The fourth or fifth generation cells were used in the experiment.2.PASMCs were divided into two groups: 1)the normal group: it didn't do any dispose;2)the LPS-induced group: it was induced by LPS(final concentration of 1?g/ml).The dynamic changes of the microRNA-146 a expression level were tested with Reverse transcriotionpolymerase chain reaction(RT-PCR)in culturing for 12 hours,24 hours,48 hours and 72 hours.The second part: we put exogenous microRNA-146 a import PASMCs by plasmid transfection method.And RT-PCR was used to detect the transfection rate,further to confirm the optimal transfection concentration and time.It was set up eight experimental groups:(1)the control group: without any intervention;(2)LPS group: stimulated with LPS(final concentration of 1?g/ml);(3)microRNA-146 a group: transfected microRNA-146 a plasmid and converted to complete medium after 6 hours;(4)LPS + scramble microRNA group: transfected microRNA-146 a missense chain plasmid,and converted to complete medium as well as stimulated with LPS after 6 hours;(5)LPS+ the lipofetamine2000 group: given the same dose of PBS and lipofectamine2000,and joined LPS;(6)LPS+ microRNA-146 a group: transfected microRNA-146 a plasmid,and converted to complete medium as well as stimulated with LPS after 6 hours;(7)the scramble group: transfected microRNA-146 a missense chain plasmid,and converted to complete medium after 6 hours;(8)the lipofetamine2000 group: given the same dose of PBS and lipofectamine2000.We collceted the cells and cell culture supernates after 48 hours.The level of IFN-?,PDGF-AB and IL-6 in cell culture supernates were analyzed by enzyme-linked immunesorbent assay(ELISA).The protein expression level of TLR4,IRAK-1,IKK,I?B and NF-?B in the PASMCs were analyzed by western blot analysis.The related proteins and cytokine secretion in LPS+ microRNA-146 a were used to correlation analysis.Results: The first part: compared with the normal group,the microRNA-146 a expressions in PASMCs have an increasing trend after LPS induced.When induced 12 hours,the amount of expression began to increase and hold a high level at 72 hours(P < 0.01).The second part: 1.through the plasmid transfection method,we have confirmed the optimal transfection concentration and time when the ratio of plasmid and lipofectamine2000 was 1:2 and the transfection was transfected to 48 hours.The expression of microRNA-146 a of about 18 times for the control group,and the difference was statistically significant.2.We transfected microRNA-146 a and induced LPS to the PASMCs,went on cultivating to 48 hours.The result was shown that:(1)Compared with the control group,the TLR4,phospho-IRAK-1,phospho-IKK,phospho-I?B and phosphor-NF-?B protein expression levels in the LPS group,LPS+scramble group and LPS + lipofectamine2000 obviously increased(P<0.01).When transfected exogenous microRNA-146 a to the PASMCs and made the microRNA-146 a overexpress,we can see that,compared with the LPS group the TLR4,phospho-IRAK-1,phospho-IKK,phospho-I?B,NF-?B protein expression levels significantly reduced(P<0.05).(2)Compared with the control group,the expression of IFN-?,PDGF-AB and IL-6 obviously rised in the LPS-induced groups(LPS group,LPS+scramble group and LPS + lipofectamine2000)and P<0.01.Compared with the LPS group,IFN-? and PDGF-AB expression in the LPS+ microRNA-146 a group reduced significantly,but IL-6 expression has no obvious down-regulation.(3)Put the related protein expression and PDGF-AB/IFN-?/IL-6 secretion in the supernatant fluit of the LPS+ microRNA-146 a group for correlation analysis.It shown a positive correlation between PDGF-AB with IFN-? and the difference was statistically significance(P<0.05).The secretion of IL-6 only has a negative correlation with the phospho-IKK protein expression(P<0.05),but no correlation with other protein expression.Conclusions: 1.Based on our results,the expression of microRNA-146 a up-regulated in PASMCs when induced by LPS and it hold a high level at 72 hours.2.Exogenous microRNA-146 a can negatively adjusted the TLR4 signaling pathways,futher to down-regulate the synthesis and secrete of the PDGF-AB and IFN-? in PASMCs in PASMCs.The synthesis and secrete of the IL-6 did not reduce but increase,and the reason would need further study.