Preliminary Study On The Activation Of Mouse Macrophage And The Anti-inflammatory Mechanisms Of Punicalagin In Vivo

Objective: To investigate the anti-inflammatory activity of punicalagin(PUN)on mouse macrophages and their anti-inflammatory mechanisms.Methods:(1)RAW264.7 cells or mouse primary peritoneal macrophages(PMs)were randomly divided into normal control group,LPS(lipopolysaccharide,10 ?g/mL)group,PUN(50 ?mol/L)group and LPS+PUN group.After 4 h,8 h and 12 h of drug treatment,to intervene to detect the mRNA expression of M1/M2 macrophage-associated inflammatory cytokines,such as tumor necrosis factor alpha(TNF-?),interleukin-1?(IL-1?),and interleukin-10(IL-10)by qRTPCR,and The biomarkers iNOS,Arg-1 were tested by Dynamic changes of mRNA expression.Meanwhile iNOS,Arg-1 were tested by Western blot.The cytokines TNF-?,IL-6,IL-1? and IL-10 were tested by ELISA assay.(2)To investigate the effects of PUN on MAPK and JAK/STAT signaling pathways in activated macrophages by Western blot.(3)To investigate the protective influence of PUN high(50 mg/kg),medium(25 mg/kg)and low(12.5 mg/kg)dose on acute paw edema.The expression of IL-1?,IL-6,CCL-2 and other related inflammatory factors and chemokines in mouse paw tissue homogenate was detected by ELISA.Results:(1)Compared with the LPS group,after being treated with PUN,the expressions of iNOS,TNF-? and IL-6 were obviously decreased,and the difference was statistically significant(P<0.05).While,the expression of Arg-1and IL-10,which represented the M2 macrophage,increased and the difference was statistically significant(P<0.05),However,there was no significant difference in the expression of Arg-1 protein(P>0.05).(2)The results showed that PUN(50 ?mol/L)inhibited p-ERK,p-JAK1,p-STAT1 and p-STAT3,the difference was statistically significant(P<0.05),and JAK could be up-regulated.The p-p38 and p-JNK had no significant effect and there was no statistical difference(P>0.05).(3)Compared with the normal control group,acute paw edema in the high-dose PUN group(50 mg/kg)significantly resolved at 12 h after drug treatment,and inhibited the expression of inflammatory cytokines or chemokines such as IL-6,CCL-2 and CCL-4.The expression of the factors was statistically significant(P<0.05).Conclusion:(1)PUN has anti-inflammatory effects on macrophages in vitro:inhibits the secretion of inflammatory factors and chemokines;promotes the expression of anti-inflammatory factors;promotes macrophage transformation from M1 to M2.(2)PUN has strong antiinflammatory activity,and its mechanism can be achieved not only by regulating ERK/MAPK pathway,but also by inhibiting JAK/STAT signaling pathway.(3)PUN has a certain anti-inflammatory protective effect on acute paw edema.