The Effect Of Parecoxib On Burn Injury-induced M1/M2 Macrophage Polarization Through TLR4/MyD88/NF-κB Pathway

PART ONE Parecoxib regulates the polarization of M1/M2 macrophages through the TLR4/MyD88/NF-κB pathway to improve wound healing quality in mice with deep second-degree burnObjective Research to explore the role of parecoxib sodium on the quality of deep second-degree burn wound healing in mice and its possible molecular mechanism.Methods Male healthy ICR mice were randomly divided into five groups:sham group(Sham,n=6),burn group(B group,B,n=6),burn+parecoxib group(Burn+P Group,BP,n=6),burn+Neoseption 3 group(Burn+Neo group,BN,n=6),burn+parecoxib+Neoseption 3 group(Burn+P+Neo group,BPN,n=6).Neoseption 3 is an agonist of TLR4.Parecoxib sodium was administered by intraperitoneal injection at 0.75 mg/kg;Neoseption 3 was administered by intraperitoneal injection at a dose of 5 mg/kg,once a day for 3 weeks(3 days for some mice).Take the wound/heal tissue and serum at 100 hours and 3 weeks after the burn.Perform the following tests on the wound/healing tissue.HE for pathological analysis.Masson Trichrome staining to observe the degree of fibrosis.Realtime quantitative polymerase chain reaction(RT-qPCR)technology detects matrix metalloproteinase 1 mRNA(MMP-1 mRNA)and transforming growth factor-β1 mRNA(TGF-β1 mRNA),Cyclooxygenase-2 mRNA(COX2 mRNA),Tumor necrosis factor-a mRNA(TNF-α mRNA)expression.Immunohistochemical(IHC)method was used to detect the expression of CD86 and CD206,CD86 labeled M1 macrophages,CD206 labeled M2 macrophages,and the Image-Pro Plus image analysis system was used to quantitatively analyze the IHC experimental pictures.Immunofluorescence staining detects CD86(COX2),CD206(COX2)co-standard results;RT-qPCR and(or)Western Blot to detect the expression of Toll-like receptors 4(TLR4),Myeloid differentiation factor 88(MyD88)and nuclear factor-kappa beta(NF-κB).Enzyme-linked immunosorbent assay(ELISA)detects inducible nitric oxide synthase(iNOS),TNF-α,interleukin-10(IL-10)expression in serum.Results The wounds in the BP group were completely covered by epithelium 3 weeks after the burn,and the contraction was not obvious;the shape of collagen fibers is relatively uniform,while the wounds in the B,BN,and BPN groups were not completely healed.Compared with group B,BN and BPN group,the BP group reduced the infiltration of inflammatory cells in the wound/callal tissue 3 weeks after burn,reduced the area and degree of fibrosis,and reduced its COX2 mRNA,TNF-α mRNA,TGF-β1 mRNA,TLR4 mRNA,MyD88 mRNA,NF-κB mRNA and TLR4,NF-κB-P-P65 protein expression,reduce the expression of M1 macrophages(P<0.05).increase the expression of MMP-1 mRNA and M2 macrophages(P<0.05).Compared with the B,BP,and BPN groups,the BN group significantly increased its NF-κB-P65 protein expression(P<0.05),but there was no difference in the NF-κB-P65 protein expression between the B,BP and BPN groups(P>0.05).Compared with the B,BN and BPN groups,the BP group also reduced the expression of COX2 mRNA,TNF-α mRNA,TLR4 mRNA,MyD88 mRNA and NF-κB mRNA,decreased the expression of M1 macrophages,and increased the expression of M2 macrophages in 100H wound tissue after burns(P<0.05);At the same time,it reduced the expression of iNOS and TNF-α and increased the concentration of IL-10 in the serum(P<0.05).Conclusion Parecoxib sodium regulates the polarization of M1/M2 macrophages through the TLR4/MyD88/NF-KB pathway and inhibits inflammation,which is a possible mechanism for improving the quality of wound healing in mice with deep second-degree burns on the back of the mouse at 3 weeks.PART TWO Parecoxib regulates the polarization of M1/M2 macrophages through the TLR4/MyD88/NF-κB pathway to reduce lung inflammation after burnObjective To study the therapeutic effect of parecoxib sodium on pulmonary inflammation after deep second-degree burns in mice and its possible mechanism.Methods Male healthy ICR mice were randomly divided into five groups:sham group(Sham,n=6),burn group(B group,B,n=6),burn+parecoxib group(Burn+P Group,BP,n=6),burn+Neoseption 3 group(Burn+Neo group,BN,n=6),burn +parecoxib+Neoseption 3 group(Burn+P+Neo group,BPN,n=6).Neoseption 3 is an agonist of TLR4.Parecoxib sodium was administered by intraperitoneal injection at 0.75 mg/kg;Neoseption 3 was administered by intraperitoneal injection at a dose of 5 mg/kg,once a day for 3 days.Lung tissue and bronchoalveolar lavage fluid(BALF)were taken for testing at 100H after the burn.HE staining evaluates lung tissue pathological changes and counts lung injury scores.Detecting the wet/dry(W/D)ratio of lung tissue to evaluate pulmonary edema.Enzyme-linked immuno-soorbent assay(ELISA)was used to detect the concentration expression of pro-inflammatory factor-inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α)and anti-inflammatory factor interleukin 10(IL-10)in BALF.Immuno-histochemistry(IHC)method was used to detect the expression of CD86 and CD206 in lung tissues of each group,CD86 labeled Ml macrophages,CD206 labeled M2 macrophages,and the Image-Pro Plus image analysis system was used to quantitatively analyze the IHC experimental pictures.Immunofluorescence staining to detect lung tissue CD86 andCOX2,CD206 and COX2 co-standard results.RT-qPCR technology detects Tolllike receptors 4(TLR4)mRNA,Myeloid differentiation factor 88(MyD88)mRNA and nuclear transcription factor κB(NF-κB)mRNA expression in lung tissue.Results Compared with group B,BN and BPN group,BP group reduced lung tissue edema,hemorrhage and inflammatory cell infiltration;Significantly reduce the score of lung injury(P<0.05);Significantly reduce the ratio of wet weight to dry weight in lung tissue(P<0.05);significantly reduce the expression of iNOS and TNF-α in BALF and increase the concentration of IL-10(P<0.05);Reduce the expression of M1 macrophages and increase the expression of M2 macrophages(P<0.05);significantly reduce the expression of TLR4 mRNA,MyD88 mRNA and NF-κB mRNA(P<0.05).The TLR4 agonist Neoseption 3 reversed the above effects of parecoxib sodium.Conclusion Parecoxib sodium regulates the polarization of M1/M2 macrophages through the TLR4/MyD88/NF-κB pathway to reducein jury of lung tissue after burns.PART THREE In vitro study of parecoxib through the TLR4/MyD88/NF-KB pathway to regulate the polarization of M1/M2 macrophagesObjective To investigate the effect of each group of serum in animal experiments on the polarization of RAW264.7 macrophages and possible pathways in vitro.Methods The RAW264.7 macrophages were cultured with the post-burn 100H serum of each group in the mouse experiment and the serum of the control group to form six groups of in vitro studies(n=3).RAW264.7+fetal bovine serum group(Control group,Con),RAW264.7+sham burn group serogroup(R+Sham group,RS),RAW264.7+burn group serogroup(R+Burn group,RB),RAW264.7+(burn+parecoxib sodium group)serum group(R+Burn+P group,RBP),RAW264.7+(burn+Neoseption 3 group)serum group(R+Burn+Neo Group,RBN),RAW264.7+(burn+parecoxib sodium+Neoseption 3 groups)serum group(R+Burn+P+Neo group,RBPN).Neoseption 3 is an agonist of TLR4.CCK-8 detects the growth curve of 1-3 days and selects the best conditions for cell proliferation experiment;3 days later,flow cytometry detects the expression of CD86 and CD206,CD86 marks M1 macrophages,and CD206 marks M2 macrophages;Enzyme-linked immunosorbent assay(ELISA)detects the expression of pro-inflammatory factor-inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α)and the concentration of interleukin 10(IL-10)in the cell supernatant;Western Blot detects the protein expression of TLR4,MyD88,NF-κB-P-P65 and NF-κB-P65.Results Compared with the RB group,RBN group,and RBPN group,the RBP group significantly reduced the expression of iNOS and TNF-α,and increased the concentration of IL-10(P<0.05);significantly reduced the proportion of M1 macrophages expression and increased the proportion of M2 macrophages expression(P<0.05);Significantly reduce the expression of TLR4,MyD88,NF-κB-P-P65 protein(P<0.05).There was no significant difference in the protein expression of NF-κB-P65 in each group(P>0.05).Conclusion In vitro experiments confirmed that parecoxib sodium can regulate the polarization of M1/M2 macrophages through the TLR4/MyD88/NF-κB pathway.