| Objective: The purpose of this study was to: 1.Analyze lipopolysaccharide(LPS)-induced acute respiratory distress Syndrome(ARDS)rat bronchoalveolar lavage fluid(BALF),spleen and periphery.The primary extraction method of macrophages from three different sources of blood and the better selection of culture.2.To investigate the effect of Mild Hypothermia(MHT)on LPS-induced alveolar macrophage(AM)Toll-like Receptor 4 protein and downstream proinflamatory mediators.Methods: 1.Density gradient centrifugation was used to extract primary macrophages from BALF,spleen and peripheral blood of LPS-induced ARDS SD(Sprague-Dawley)rats.The extracted cells were identified and counted by immunochemical staining.And the purity calculation,and the Cell Counting Kit-8(CCK-8)kit was used to detect the primary macrophage activity extracted by the three methods.2.To analyze the effect of hypothermia treatment on AM TLR4 protein and downstream roinflamatory mediators in LPS-induced ARDS rats.The changes of TLR4,My D88,Tumor Necrosis Factors-?(TNF-?)and inducible Nitric Oxide Synthase(i NOS)were analyzed by establishing a rat ARDS model.Each group was randomly divided into a LPS intervention group and a Phosphate Buffered Saline(PBS)control group;the concentration in the primary macrophages was 0 ug/ml,1ug/ml,5ug/ml,8ug/ml,10ug/ml,15ug/ml LPS,the optimal concentration gradient was determined by LPS intervention of primary macrophages through CCK-8 kit,After the primary macrophages were cultured in a medium with a temperature of 37?,a volume fraction of 5% CO 2 and a humidity of 95 % for 24 h,the cells were randomly divided into a sub-hypothermia group and a normal temperature(Normothermia,NT)group.The cells were cultured in a CO2 incubator at 32? and 37?,and LPS intervention group was added Stimulation was performed with 5ug/ml LPS,and an equal amount of PBS was added to the control group.Macrophage RNA was extracted at 0h,1h,4h,8h,16 h and 24 h,and macrophage TNF-? and i NOS were detected by real-time polymerase chain reaction(RT-PCR).m RNA expression changes;macrophage culture supernatant was extracted,and the expression of TNF-? and i NOS in macrophage culture supernatant was detected by enzyme-linked immunosorbent assay(Elisa).After culture for 1 h,The total protein of macrophages was extracted at 4h,8h,16 h and 24 h.The expression of TLR4 and My D88 protein in macrophages was detected by Western blotting(WB).Results: 1.The mean number of primary macrophages extracted by alveolar lavage fluid,spleen and peripheral blood were(1.14±0.14)×106,(9.29±0.19)×106 and(3.17±0.13)×106,the difference between the three groups was significant(P < 0.05);the activity of the primary macrophages extracted by the three methods was compared,and the macrophages extracted from the alveolar lavage fluid were significantly higher than the other ones.There were no significant differences between the spleen and the primary macrophages extracted from the peripheral blood(P > 0.05).The primary macrophages extracted by the three methods were immunochemically detected to obtain the cell purity.They were(95.779±1.170)%,(94.132±1.412)%,(94.012±0.954)%,and the difference was not statistically significant(P > 0.05).2.The optimum concentration gradient of LPS intervention on primary macrophages was determined by CCK-8 kit,and the optimal LPS concentration was 5 ug/ml.3.The expression of TLR4 and My D88 protein in macrophages: Compared with the normal temperature group,the expression of TLR4 protein decreased significantly at 4h,8h,16 h and 24 h after sub-hypothermia intervention(4h: 0.376± 0.011 vs.0.262±0.032,8h: 0.588±0.040 vs.0.390±0.029;16h: 0.767±0.013 vs.0.555±0.050,24h: 0.493±0.026 vs.0.317±0.031,all P<0.05);after hypothermia intervention,at 1h,4h,8h,16 h,My D88 protein is significantly decreased(1h: 0.433 ± 0.016 vs.0.240 ± 0.034,4h: 0.524 ± 0.026 vs.0.351 ± 0.020,8h: 0.636 ± 0.034 vs.0.395 ± 0.011;16h: 0.726 ± 0.008 vs.0.568 ± 0.023,all P<0.05).4.Expression of TNF-? m RNA and i NOS m RNA in macrophages(2-? ?Ct): Compared with normal temperature group,TNF-? m RNA and i NOS m RNA expression in hypothermia group were 1h,4h,8h,16 h after intervention.And 24 h decreased significantly(TNF-? m RNA: 1h: 0.802 ± 0.244 ng / L vs.0.366 ± 0.074 ng / L,4h: 1.644 ± 0.157 ng / L vs.0.771 ± 0.121 ng / L,8 h: 3.213 ± 0.857 ng / L vs.2.007 ± 0.690 ng / L,16 h: 7.916 ± 2.292 ng / L vs.3.944 ± 0.447 ng / L,24 h: 4.116 ± 1.340 ng / L vs.2.517 ± 1.165 ng / L;i NOS m RNA: 1h: 0.679 ± 0.147 ng / L vs.0.366 ± 0.129 ng / L,4h: 1.170 ± 0.148 ng / L vs.0.797 ± 0.101 ng / L,8 h: 2.403 ± 0.307 ng / L vs.1.193 ± 0.097 ng / L,16 h: 4.986 ± 1.131 ng / L vs.3.252±0.768 ng / L,24h: 8.867±1.395 ng / L vs.4.529±1.395 ng / L,all P<0.05).5.Expression of TNF-? and i NOS inflammatory mediators in macrophage culture supernatant: Compared with normal temperature group,TNF-? expression in hypothermia group decreased significantly at 4 h,8 h,16 h and 24 h after intervention,i NOS expression There was a significant decrease at 1h,4h,8h,16 h and 24 h after intervention(TNF-?: 4h: 128.859±23.667 ng / L vs.48.784±7.627 ng / L,8h: 214.136±14.460 ng / L vs.110.594 ±5.957 ng / L,16 h: 390.949 ± 5.789 ng / L vs.216.305 ± 11.229 ng / L,24 h: 600.659 ± 9.415 ng / L vs.348.981 ± 15.844 ng / L;i NOS m RNA: 1 h: 84.297 ± 0.582 ng / L vs.64.798±5.479 ng / L,4h: 134.043±6.205 ng / L vs.71.458±3.866 ng / L,8h: 285.510±69.423 ng / L vs.133.124±34.114 ng / L,16 h: 737.387±160.174 ng / L vs.304.097±51.948 ng / L,24h: 431.907±42.335 ng / L vs.237.965±8.985 ng / L,all P<0.05).Conclusion: 1.Compared with the primary macrophages extracted by the three methods,the purity of macrophages was not statistically significant.The primary macrophages extracted from alveolar lavage fluid had the strongest activity,and the primary macrophages extracted from the spleen.The highest number of primary macrophages extracted from peripheral blood is between the two.2.Hypothermia down-regulates LPS-induced TLR4/My D88 signaling pathway in alveolar lavage fluid macrophages of ARDS rats,thereby inhibiting transcription and expression of inflammatory mediators. |
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