| Objective:To explore the molecular mechanism of G2 A in polarizing signal pathways that regulate macrophages to produce polarization in hypoxic environment in vitro.Methods:The cellculture method was used for research.The rat bone marrow-derived macrophages were divided into 4 groups:(1)normoxic group(NC): normoxic(21%O2)culture in cell incubator for 24 hours;(2)hypoxic group(H): Incubate in a hypoxic incubator(37 ℃,1% O2,5% CO2)for 24 hours;(3)Hypoxic LPC stimulation group(H + LPC): Add LPC(0.2μmol / L)stimulant to the medium,and Incubate in an oxygen incubator(37 ° C,1% O2,5% CO2)for 24 hours;(4)LPC stimulation plus G2 A interference group(H + LPC + G2A-)under hypoxia: use lentivirus interference technology to transfer to macrophage The cells were stained with G2 A si RNA lentivirus,LPC stimulant was added to the medium,and cultured in a hypoxia incubator(37 ° C,1% O2,5% CO2)for 24 hours.After the corresponding treatments in the above groups were completed,cell supernatants were collected for detection of inflammatory factors,and cellular RNA and proteins were extracted for related detection.Real-time quantitative PCR was used to detect the expression of i NOS and CD86 m RNA related to M1 macrophages and the expression of G2 A,STAT5,and IRF5 m RNA in the signaling pathway.Western-blot was used to detect the changes of G2 A,STAT5,IRF5 protein expression in cells.The expression of inflammatory factors IL-1β,IL-6,IL-23 and TNFα related to MI macrophages in the cell supernatant was detected by ELISA.Results:(1)m RNA measurement results of macrophages in each group: m RNAs of i NOS,CD86,G2 A,STAT5 / IRF5 signaling were higher in group H than in NC group,and the differences were statistically significant(t = 5.67,7.93,8.24,15.9,12.7,all P<0.05);the expression in the H + LPC group was significantly higher than that in the H group,and the differences were statistically significant(t = 5.40,4.51,4.66,3.77,4.49,all P <0.05);and The H + LPC + G2A-group was significantly lower than the H+ LPC group(t = 10.3,8.83,6.76,5.81,6.80,all P <0.05),but higher than the NC group(t = 5.67,7.93,3.63,8.55,12.7,all P <0.05);indicates that G2 A is highly expressed under hypoxia and that the expression of M1-type related surface markers(i NOS,CD86)is increased after macrophages are stimulated by LPC,indicating that LPC promotes macrophages under hypoxia Phages are transformed to M1 type.(2)Results of determination of macrophage protein in each group: The expression of G2 A,STAT5,IRF5 protein was higher in group H than in NC group(t = 9.40,3.34,20.6,all P <0.05);in H + LPC group The expressions were significantly higher than those in the H group(t = 2.41,3.24,2.78,all P <0.05);while the H + LPC + G2 Agroup was significantly lower than the H + LPC group,(t = 7.05,23.7,19.0,all P<0.05),but higher than those in the NC group(t = 6.35,37.0,9.37,all P <0.05);combined with the above results of STAT5 / IRF5 m RNA,it is suggested that LPC may activate G2 A / STAT5 / IRF5 signal under hypoxia The pathway regulates macrophage polarization.(3)Cytokine measurement results in macrophage culture fluid of each group: The expression of IL-1β,IL-6,IL-23,and TNFα inflammatory cytokines was higher in group H than in NC group(t = 17.5,12.5,9.86,14.7,all P<0.05);the expression in the H + LPC group was significantly higher than that in the H group(t = 12.9,7.60,10.8,11.1,all P <0.05);and the H + LPC + G2A-group was significantly higher than the H group.The + LPC group was significantly reduced(t =25.5,13.1,20.5,16.5,all P <0.05),but higher than the NC group(t = 7.72,6.82,3.75,5.80,all P <0.05).Conclusion:(1)LPC can promote the expression of IL-1β,IL-6,IL-23,TNFαinflammatory factors in macrophages under hypoxia,and it is significantly higher than that in the hypoxia group.Moreover,the surface markers(i NOS,CD86)of M1 macrophages were significantly increased by genetic and protein tests,suggesting that LPC can transform macrophages into a pro-inflammatory phenotype(M1).(2)Lentivirus interferes with the expression of G2 A in macrophages,which can significantly reduce the expression of M1 macrophage surface markers,reduce the polarization of macrophages to M1,and reduce the expression of inflammatory factors without being affected by LPC stimulation.(3)The expression levels of STAT5 and IRF5 were significantly reduced after interference with G2 A,suggesting that G2 A may regulate the polarization of macrophages to M1 type through the G2 A /STAT5 / IRF5 signal pathway in a hypoxic environment. |
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