| Background:Photodynamic therapy(PDT)is a promising new approach to promote wound healing,and its effectiveness has been demonstrated in both clinical and animal studies.Macrophages are the key cells in wound healing and inflammatory response,but the mechanism of action of photodynamic therapy on macrophages in promoting wound healing is still unclear,and the research of photodynamic therapy on macrophages is mainly focused on its killing effect on macrophages,but the research on macrophage polarization is still less.Macrophages differentiate mainly toward two types,namely pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages.In a large number of clinical and experimental studies,it was found that the inflammatory response was significantly increased immediately after photodynamic therapy treatment,and the intensity of its early inflammatory response was positively related to the efficacy of the later treatment.Our group’s previous study also found that photodynamic can promote wound healing and increase M1-type macrophages in the traumatic tissue during the acute inflammatory phase,suggesting that ALA-PDT treatment may promote macrophage polarization to M1-type in the early stage and increase local pro-inflammatory factors,thus enhancing the local inflammatory response.Based on this,this study focuses on the effect of photodynamic therapy on macrophage polarization in the early stage of photodynamic therapy to investigate whether it affects macrophage polarization to pro-inflammatory M1 in the early stage of photodynamic therapy treatment and to explore the underlying mechanisms.ObjectiveIn vitro experiments were performed using RAW264.7 macrophages to investigate the effects of ALA-PDT on macrophage polarization,as well as on inflammatory factors and phagocytosis.These findings may lead us to note that the increased inflammatory response in the immediate trauma after photodynamic therapy,as well as the effectiveness may be related to the promotion of M1-type polarization of macrophages,which also provides a theoretical basis and data support for the clinical application of ALA-PDT.Methods and grouping1.Effect of ALA-PDT on the polarization of M0 macrophagesFirst,RAW264.7 macrophages were divided into four groups for treatment.(i)control group(untreated RAW264.7 cells)(ii)ALA group(cells were incubated in 1.0m M ALA for 4h)(iii)red light group(cells were irradiated with 1.5 J/cm2 red light)(iv)PDT treatment group(1.5J/cm2 red light irradiation after 1.0m M ALA incubation for 4h).After the above treatments,the protein and m RNA expression of M1/M2 macrophage markers,inflammatory factors(including Arg-1,CD86,CD206,TNF-α,i NOS,IL-1β,IL-6)were measured by Western blot and RT-PCR,and the phagocytic capacity of each group was recorded and compared using a live cell workstation.2.Effect of ALA-PDT on the polarization of M2 macrophagesFirstly,RAW264.7 cells were divided into blank group and treated group,the blank group was normal cultured RAW264.7 cells,the treated group was stimulated with IL-4 and IL-13(20 ng/ml)for 36-48 h.The changes of cell morphology were photographed and recorded by inverted microscope,and the expression of M1/M2 markers were detected by Western blot and RT-PCR.To verify whether the M2 macrophage polarization model was successfully established.After establishing the M2 macrophage model,the experiment was divided into four groups.(i)control group(M2 macrophages)(ii)ALA group(M2 macrophages were incubated with 1.0m M ALA for 4h)(iii)red light group(M2 macrophages were irradiated with 4J/cm2 red light)(iv)PDT group(M2 macrophages were incubated with 1.0m M ALA for 4h and then irradiated with 4J/cm2 red light).Western blot,RT-PCR and immunofluorescence were used to detect the changes of M1/M2 macrophage marker expression in each group,CD86,i NOS,TNF-α and IL-6 were used to label M1 macrophages,while Arg-1 and CD206 were used to label M2 macrophages.3.Mechanistic study of the effect of ALA-PDT on macrophage polarization3.1 Studies on the role of NLRP3 inflammatory inflammasomeThe cells were divided into four groups for treatment.(i)control group(untreated macrophages)(ii)NLRP3 inflammasome inhibitor treatment group(1u M NLRP3 inflammasome inhibitor MCC950 co-cultured for 1h)(iii)PDT treatment group(1.0m M ALA incubated for 4h with light avoidance and then 1.5J/cm2 red light irradiation)(iv)PDT and NLRP3 inflammasome inhibitor co-treatment(1u M NLRP3 inflammasome inhibitor MCC950 co-cultured for 1h and then 1.0m M ALA and then 1.5J/cm2 red light irradiation)inhibitor MCC950 was incubated for 1h,followed by 1.0m M ALA and 1.5J/cm2 red light irradiation).The expression of NLRP3,Caspase-1,ASC and CD86,i NOS,IL-1β,TNF-α and other proteins in each group after the addition of NLRP3 inhibitor was examined by Western blot.3.2 Exploring the role of ERK/MAPK pathway in.The cells were divided into four groups for treatment(i)control group(untreated macrophages)(ii)ERK inhibitor FR180204 treatment group(10u M ERK inhibitor incubated for 24h)(iii)PDT treatment group(1.0m M ALA incubated for 4h with light avoidance and then given 1.5J/cm2 red light irradiation)(iv)PDT and ERK inhibitor group(10u M ERK inhibitor incubated for 24 h and then given(1.0m M ALA and 1.5J/cm2 red light irradiation).After treatment,the expression of ERK,p-ERK,CD86,i NOS,NLRP3,Caspase-1,IL-1β and other proteins were detected in each group using WB assayResults:1.ALA-PDT promotes the polarization of M0 macrophages to M1Under the photodynamic conditions of 1 m M ALA concentration and 1.5 J/cm2 red light,both RT-PCR and WB experiments showed that the expression of M1 characteristic proteins CD86 and i NOS was significantly enhanced in macrophages in the PDT group compared with the control group,and the expression of M2 characteristic proteins CD206 and Arg-1 was significantly decreased compared with the control group,and the expression of inflammatory factors such as TNF-α,IL-1β and IL-6 The expression of inflammatory factors such as TNF-α,IL-1β and IL-6 was increased,while the phagocytosis of Candida albicans by macrophages was observed to be significantly enhanced in the ALA-PDT group compared with the control group.2.ALA-PDT promotes repolarization of M2 macrophages to M1The M2-related markers Arg-1 and CD206 increased in the treated group after IL-4 and IL-13 induction compared with the untreated group,and the M1-related markers CD86 and i NOS decreased compared with the untreated group,indicating the successful establishment of the M2 macrophage model.q-PCR and WB experiments showed that under the effect of 1 m M ALA concentration and 4 J/cm2 red light irradiation,the M1 characteristic proteins of macrophages CD86,i NOS,IL-6 and TNF-α were significantly enhanced and the expression of M2 characteristic proteins CD206 and Arg-1 were significantly decreased compared with the control group.3.Mechanism of action of ALA-PDT to promote the effect of M1 polarizationWB results showed that the expression of p-ERK protein and NLRP3 were enhanced after treatment with ALA-PDT,indicating that ALA-PDT activated ERK/MAPK and NLRP3 pathways.After adding NLRP3 inhibitor and ERK inhibitor treatment,macrophage M1 signature proteins i NOS and CD86 expression were attenuated,and IL-1β and TNF-α expression were reduced,indicating that photodynamic promotion of macrophage polarization and inflammatory factors were associated with ERK/MAPK and NLRP3 pathways.Meanwhile,when ERK pathway was blocked,NLRP3 inflammasome activation was inhibited and Caspase-1 protein expression was significantly reduced,indicating that NLRP3 is a downstream pathway of ERK.Conclusion:1.1m M ALA and 1.5J/cm2 red light of ALA-PDT promoted RAW264.7 macrophage polarization toward M1,along with inflammatory factor secretion and phagocytosis.2.1m M ALA and 4J/cm2 red light conditions of ALA-PDT can induce M2 type RAW264.7 repolarization toward M1.3.ALA-PDT-induced macrophage polarization toward M1 was associated with activation of ERK/MAPK--NLRP3 signaling pathway. |
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