MiR-124-5p Regulates LPS-induced Microglia Polarization Through CAMP/PKA/CREB Pathway

ObjectiveEstablish the inflammatory model by stimulating lipopolysaccharide(Lipopolysaccharide,LPS),and explore whether mi R-124-5p induced the polarization of microglia to M2 phenotype through c AMP pathway,thus inhibiting the inflammatory response.Methods1、Microglia cells were cultured in vitro,stimulated by LPS to cause inflammatory reactions,and Iba-1 protein expression was detected by immunofluorescence.2、Using the transfection technique,miR-124-5p mimics and miR-124-5p inhibitor were transfected into microglia.The experiment was divided into five groups: Con group,mimics NC group,mimics group,inhibitor NC group and inhibitor group,and mi R-124-5p was detected by RT-q PCR.3、miR-124-5p mimics and miR-124-5p inhibitor were transfected into microglia,and the cell culture supernatants were collected.The experiment was divided into six groups: Con group,LPS group,mimics NC + LPS group,mimics + LPS group,inhibitor NC + LPS group,and inhibitor + LPS group.The expression of IL-1 β(Interleukin-1 β,IL-1 β),tumor necrosis factor-α(tumor necrosis factor alpha,TNF-α),IL-10(Interleukin-10,IL-10),and IL-4(Interleukin-4,IL-4)were determined using ELISA.The protein expression of IL-1 β,i NOS,ARG-1,and CD206 was visualized by immunoblotting and immunofluorescence.4、Bioinformatics techniques to screen out possible pathways regulated by mi R-124-5p.5、mi R-124-5p mimics and mi R-124-5p inhibitor were transfected into microglia.The experiment was divided into six groups: Con group,LPS group,H89 + LPS group,H89 + LPS + mimics group,DB-c AMP + LPS group,and DB-c AMP + LPS group + mimics.The tein expression of c AMP/ p-PKA / p-CREB was determined by immunoblotting.Results1、Immunofluorescence identification showed that the microglia treated by LPS showed bright Iba-1 green fluorescence compared with the Con group.2、Real-time RT-q PCR showed enhanced expression of mi R-124-5p in the mimics group compared to the Con group and decreased in the inhibitor group compared to the Con group(P<0.05).3 、 ELISA results showed that compared with Con group,LPS group increased the expression levels of IL-1 β and TNF-α(P<0.05,P<0.01),the expression levels of IL-4 and IL-10 were decreased(P<0.05,P<0.01);Compared with the mimics NC+LPS group,the expression levels of IL-1β and TNF-α in the mimics +LPS group were decreased(P<0.05,P<0.01),the expression levels of IL-4 and IL-10 were increased(P<0.05,P<0.01).Compared with inhibitor NC+LPS group,the expression levels of IL-1β and TNF-α in inhibitor +LPS group were significantly increased(P<0.05,P<0.01),the expression levels of IL-4 and IL-10 in inhibitor +LPS group were significantly decreased(P<0.05,P<0.01).4、The results of immunofluorescence showed that the red fluorescence of i NOS and IL-1β was weak in Con group,and the red fluorescence of ARG-1and CD206 was obvious in Con group.After LPS treatment,i NOS and IL-1βshowed bright red fluorescence expression,while ARG-1 and CD206 showed weak red fluorescence expression.The red fluorescence expression of i NOS and IL-1β in mimics+LPS group was weaker,while the red fluorescence expression of ARG-1 and CD206 was stronger.i NOS and IL-1β showed obvious red fluorescence expression in inhibitor+LPS group,while ARG-1 and CD206 showed weak red fluorescence expression.5、The results of bioinformatics analysis showed that the c AMP pathway was the most strongly correlated inflammatory pathway of mi R-124-5p.6、Western blotting results showed that:(1)Compared with Con group,LPS group significantly increased the expression of i NOS and IL-1β,and significantly decreased the expression of ARG-1 and CD206(P<0.01);Compared with the mimics NC+LPS group,the expressions of i NOS and IL-1β in the mimics+LPS group were significantly decreased,while the expressions of ARG-1 and CD206 were significantly increased(P<0.01);Compared with inhibitor NC+LPS group,i NOS and IL-1β protein expressions were significantly increased,while ARG-1and CD206 protein expressions were significantly decreased in inhibitor+LPS group(P<0.01).(2)c AMP protein expression in LPS group was lower than that in Con group(P<0.05);After addition of H-89,c AMP protein expression in H-89+LPS group was significantly decreased(P<0.01);The expression level of c AMP protein in H-89+LPS+mimics group was higher than that in H-89+LPS group(P<0.05);After addition of DB-c AMP,the expression level of c AMP protein in DB-c AMP+LPS group was increased compared with LPS group(P<0.05);The expression level of c AMP protein in DB-c AMP+LPS+mimics group was higher than that in DB-c AMP+LPS group(P<0.05).p-PKA and p-CREB also showed the same trend.Conclusion1、Overexpression of miR-124-5p was promoted LPS-induced microglia cell polarization toward M2 phenotype.2、The miR-124-5p was regulated the cAMP / PKA / CREB pathway and promoted the polarization of microglia to the M2 phenotype and inhibited the inflammatory response.