Kaempferol Alleviates Corneal Transplantation Rejection By Inhibiting Macrophage M1 Polarization Via Autophagy-NLRP3 Inflammasome Pathway

BackgroundWhen there is inflammation and vascularization in the implantation bed,the"immune privilege" ability of the cornea is destroyed,and it was called high-risk corneal transplantation.The immune rejection after keratoplasty is still the biggest threat to the success rate of high-risk keratoplasty patients.Classical immunosuppressants have short duration,easy to cause infection,metabolic disorders and other side effects,which can not be ignored.It is necessary to explore the mechanism of corneal transplantation rejection and develop new therapies.Corneal transplantation rejection is a complex immune reaction involving many kinds of immune cells.The uptake of graft antigen by antigen presenting cells headed by macrophages is the first step of corneal transplantation rejection.In recent years,macrophages have been found to have different polarization directions,which correspond to the outcome of inflammatory response or anti-inflammatory and pro repair.However,macrophage polarization has not been deeply studied in the field of corneal transplantation.In this study,we investigated the effect of macrophage polarization on corneal allograft rejection in vivo,and the molecular mechanism of kaempferol regulating macrophage polarization through autophagy NLRP3 inflammasome pathway in vitro.Method1.Inhibitory effect of kaempferol on M1 polarization of macrophages after corneal transplantation.(1)Objective to explore the relationship between macrophage polarization and corneal transplantation rejection:to establish penetrating keratoplasty model in rats,which were divided into autograft group and allograft group.The RI values of the grafts were recorded daily and the survival analysis was performed.On the 5th,9th and 14th day after operation,the corneas of each group were taken,and the mRNA expressions of M1 macrophage pro-inflammatory cytokines IL-6,iNOS,TNF-? and cxcl-10 were detected by real-time fluorescent quantitative PCR.(2)Objective to explore the effect of kaempferol in vivo and the relationship between kaempferol and macrophage polarization:the rat penetrating keratoplasty model was established and divided into autograft group,allograft group,kaempferol injection group and kaempferol+3-MA group:each group was treated with auto transplantation,allogeneic transplantation,allogeneic transplantation+intraperitoneal injection of 50 mg/kg kaempferol,allogeneic transplantation+intraperitoneal injection of kaempferol+intraperitoneal injection 10 mg/kg autophagy inhibitor 3-MA was injected.The corneal morphology was observed by microscope every day,and the graft rejection time was observed by survival analysis.The expression of IL-6,iNOS,TNF-? and CXCL-10 mRNA was detected by real-time fluorescence quantitative PCR;the rate of CD11b and CD86 positive cells was detected by immunofluorescence CO staining.2.Mechanism of kaempferol inhibiting macrophage M1 polarization through autophagy-NLRP3 inflammasome pathway(1)Effect of kaempferol on macrophage polarization in vitro:in order to clarify the molecular mechanism of kaempferol regulating macrophage polarization,THP-1derived macrophages were divided into control group,LPS group and kaempferol group:each group was treated with no special treatment,LPS treatment,LPS and kaempferol treatment respectively.CCK-8 kit was used to detect the toxicity of kaempferol on macrophages;real-time quantitative PCR was used to detect the mRNA expression of M1 type pro-inflammatory cytokines;flow cytometry was used to detect the proportion of M1 and M2 macrophages.(2)Objective to explore the effect of kaempferol on NLRP3 inflammasome:control group,LPS group,kaempferol group.The mRNA expressions of NLRP3,ASC and Caspase-1 were detected by real-time quantitative PCR,and the protein expressions of NLRP3 and its downstream pro-IL-1?,IL-1?,pro-Caspase-1 and Caspase-1 were detected by Western blot.(3)To explore the effect of kaempferol on autophagy:control group,LPS group,kaempferol group.The mRNA expressions of LC3 and p62 were detected by real-time quantitative PCR,and the autolysosome formation was observed by CO staining with small molecule autolysosome dyes DALGreen and LC3b.(4)Objective to explore whether kaempferol can affect macrophage polarization through autophagy-NLRP3 inflammasome pathway:control group,LPS group,kaempferol group,kaempferol+3-MA group.The protein expression of LC3II/I,p62 and the protein expression of NLRP3,pro-IL-1?,IL-1?,pro-caspase-1 and caspase-1 were detected by Western blot.The fluorescence intensity of NLRP3 and the proportion of CD86+cells were detected by CO immunofluorescence staining.The ratio of M1/M2 was detected by flow cytometry.Result1.Compared with the autograft group,the M1 polarization of macrophages in the allograft group was significantly increased.2.After in vivo injection of kaempferol,survival analysis showed that kaempferol could effectively delay the occurrence of corneal transplantation rejection in rats,and kaempferol could significantly reduce the infiltration of M1 macrophages after transplantation.3.After CO administration of kaempferol and autophagy inhibitor 3-MA in vivo,the inhibitory effect of kaempferol on M1 macrophage polarization was reversed.4.Kaempferol directly inhibited the polarization of M1 macrophages and the activation of NLRP3 inflammasome in vitro.5.Kaempferol can inhibit the polarization of M1 macrophages through autophagyNLRP3 inflammasome pathway,and exert anti-inflammatory effect in vitro.ConclusionWe found that kaempferol can inhibit the polarization of M1 macrophages through autophagy-NLRP3 inflammasome pathway,thus delaying the corneal transplantation rejection.