Effect Of Photodynamic Therapy On Macrophage Polarization State,Phagocytic Activity And Secretion Of Inflammatory Factors And Its Mechanism

Macrophages are an important part of the innate immune effector cells of the human body,and play an important role in host defense.They are the most important phagocytes in the body,and have the functions of phagocytosis of pathogens,antigen presentation and secretion of a variety of cytokines.Since Elie Metchnikoff first reported macrophages in the literature in 1883,people have been studying macrophages.It has been found that macrophages have strong plasticity and adaptability to different environments.The functional state of macrophages is heterogeneous under different conditions.In normal tissues,macrophages are in a resting or inactive state,while the activation of macrophages is a state in which they play a powerful physiological function.Macrophages can polarize into different phenotypes under different tissue microenvironment and different cytokines.Rosenstreich found that cytokines secreted by lymphocytes promoted macrophages to phagocytosis of bacteria,and then found that interferon-γ secreted by lymphocytes transformed quiescent macrophages into macrophages with strong antibacterial and phagocytic abilities and secretion of inflammatory cytokines,namely classical activated macrophages.In 1989,with the discovery of Th1 and Th2 cells,researchers found that the secretion of interleukin-4 by Th2 cells can transform quiescent macrophages into alternately activated macrophages(M2).In 2010,the concept of macrophage polarization was modified again because of the founding of M2-like macrophages.These macrophages include M2 b type macrophages induced by immune complex,M2 c type macrophages induced by interleukin-10 and so on.The current mainstream view is that macrophages can be divided into two functional categories: macrophages classically activated(M1)and macrophages alternately activated(M2).M1 macrophages,also known as inflammatory macrophages,has the strong ability of antigen presenting and devouring,and can release a lot of proinflammatory factor,such as tumor necrosis factor and interleukin 1 beta,interleukin-12,interleukin-18,nitric oxide,etc.,participating Ⅰ type of immune response.M2 macrophages,known as anti inflammatory macrophages,can produce IL-10,transforming growth factor beta,arginase 1anti-inflammatory factorsetc,participating in Ⅱ type of immune response,plays a central role in the parasite,tissue remodeling,angiogenesis and allergic reaction.They can play different roles and trigger the transformation of macrophage phenotypes and functions to adapt to different vivo environmental factors,and chang dynamically between the two extremes of M1 and M2 phenotypes.Photodynamic therapy is a kind of method based on photosensitizer,oxygen and light interaction for the diagnosis and the treatment of diseases,PS after entering the human body,aggregat selectivily in proliferation faster cells such as tumor tissues and cells after infection and the lesion site.It can be stimulated by the specific wavelength light,generating a large number of oxide free radical which kill the cells to get the purpose of treatment.Due to its good therapeutic effect,no drug resistance and good cosmetic effect,PDT is becoming more and more popular among doctors and patients.PDT is widely used in the palliative treatment of condyloma acuminatum,acne,solar keratosis,basal cell carcinoma(BCC),Bowen’s disease and tumors.In 2007,China State Food and Drug Administration(CFDA)officially approved 5-aminolevulinic acid-photodynamic therapy for CA.Previous studies showed that PDT can kill the macrophages,especially infected by virus or pathogen,and increase in treatment of macrophage infiltration in some conditions.PDT in the regulation of macrophage polarization effect and its mechanism study is less,and prior research has shown that,different photosensitizer and light energy,under different conditions,PDT effect and mechanism of each are not identical.Therefore,the effect of ALA-PDT on macrophages and its mechanism deserve further study.In this study,C57 mice and mouse mononuclear macrophage RAW264.7 were studied to explore the regulation of PDT on the polarization state of macrophages and its mechanism,so as to explore the theoretical basis and provide data support for the regulation of PDT on the state of macrophages in the treatment of tumors and infectious diseasesMethods:1.The M1/M2 ratio of macrophages around the wound of mice treated with ALA-PDT was detected by immunofluorescence technique at 0,1 and 3 days after clean wound treatment.2.Quantitative polymerase chain reaction(q PCR)was used to detect the m RNA expressions of M1 and M2-labeled CD86,inducible nitric oxide synthase(i NOS),CD206 and Arg-1 in RAW264.7 cells treated with ALA-PDT.3.Western blot(WB)was used to detect the expression of M1 and M2 marker CD86,i NOS,CD206,Arg-1 and other proteins in RAW264.7 cells treated with ALA-PDT.4.NF-k B pathway expression in RAW264.7 cells treated with ALA-PDT was detected by using NF-k B activation-nuclear transport detection kit and WB assay.5.WB experiment was used to detect the expression of NF-k B pathway related proteins and M1 characteristic proteins CD86 and i NOS in RAW264.7 cells treated with ALA-PDT after the addition of NF-k B pathway inhibitor6.The m RNA expressions of inflammatory factors such as IL-1β,IL-6 and TNF-a in RAW264.7 cells treated with ALA-PDT were detected by Q-PCR assay.7.WB assay was used to detect the expression of inflammatory factors such as IL-1β,IL-6 and TNF-a in RAW264.7 cells treated with ALA-PDT8.Luminex liquid suspension chip technology was used to detect the protein concentrations of IL-1β,IL-6,TNF-a and other proteins in the supernatant of RAW264.7cells treated with ALA-PDT.9.WB experiment was used to detect the expression of ERK-MAPK pathway related proteins in RAW264.7 cells treated with ALA-PDT.10.WB experiment was used to detect the expression of ERK-MAPK pathway related proteins,inflammatory factors IL-1β,IL-6,TNF-a and other proteins in RAW264.7 cells treated with ALA-PDT after the addition of ERK inhibitor11.The phagocytic activity of RAW264.7 cells treated with ALA-PDT for 3 days was detected by HE staining and video recording method.Results:1.ALA-PDT increased the M1/M2 ratio of macrophages in C57 mice on the first day after treatment,and the difference was statistically significant(P<0.05),the difference disappeared on the third day.2.After the treatment of RAW264.7 cells with ALA-PDT,Q-PCR and WB experiments showed that the expression of CD86 and i NOS of M1 in PDT group was significantly increased compared with that in control group,and the expression of CD206 and Arg-1 of M2 were significantly decreased compared with that in control group,the difference was statistically significant(P<0.05).3.After the treatment of RAW264.7 cells with ALA-PDT,the results of Q-PCR,WB and Luminex liquid-phase suspension chip showed that the protein expressions of inflammatory factors IL-1β,IL-6 and TNF-a were enhanced in PDT group.4.After 3 days of treatment with ALA-PDT,RAW264.7 cells were co-cultured with Candida albicans.The phagocytic activity of the PDT group was significantly enhanced compared with the control group,and the difference was statistically significant(P<0.05).5.After the treatment of RAW264.7 cells with ALA-PDT,NF-k B activation and nuclear transport detection and WB test results showed that the NF-k B pathway P65 nuclear transfer was enhanced in the PDT group,and the expression of NF-k B pathway related proteins p-p65 and NF-k B 2 were enhanced,and the pathway was activated.6.After the addition of NF-k B pathway inhibitor,the expressions of NF-k B pathway related proteins p-p65 and NF-k B-2 in PDT group were enhanced and inhibited,while the expressions of M1 characteristic proteins CD86 and i NOS were enhanced and inhibited.7.After ALA-PDT was treated with RAW264.7 cells,WB test results showed that the proportion of ERK-MAPK pathway related proteins p-ERK/ERK and p-P38 /P38 increased significantly in PDT group.8.After the addition of ERK inhibitors,WB results showed that the increase in the proportion of ERK-MAPK pathway related proteins p-ERK/ERK,p-P38 /P38 was inhibited,and the expression of inflammation-related factors IL-1β,IL-6,TNF-a and other proteins were also inhibited.Conclusion:1.After the treatment of C57 mice,ALA-PDT could induce enhanced polarization of macrophages in local tissues toward M1.2.ALA-PDT can induce RAW264.7 cells to polarize to M1,enhance the inflammatory related proteins IL-1β,IL-6,TNF-a,and enhance the phagocytic activity of RAW264.7 cells.3.ALA-PDT induced macrophage polarization to M1 and increased secretion of inflammation-related proteins such as IL-1β,IL-6 and TNF-a may be related to ERK/MAPK signaling pathway.4.ALA-PDT induced macrophage polarization to M1 may be related to NF-k B signaling pathway and ERK/MAPK signaling pathway.