Study On The Role And Mechanism Of Efferocytosis In The Suppression Of Synovitis By Pulsed Electromagnetic Field

Synovium is a layer of connective tissue membrane attached to the inner surface of the synovial joint capsule.Synovium is of great significance for maintaining the normal structure and function of joints.Synovial lesions represented by synovitis are closely involved in the pathological process of various joint diseases including osteoarthritis(OA)and rheumatoid arthritis(RA).Also,synovitis is closely related to joint swelling and pain in patients.Pulsed electromagnetic field(PEMF)as a non-invasive physical therapy has been used clinically to treat various diseases including non-union fractures,postoperative pain and edema,OA,plantar fasciitis and so on.Researchers have explored the mechanism of PEMF treatment effect.Existing research suggests that PEMF treatment can reduce the expression and secretion of multiple inflammatory factors including interleukin-1 beta(IL-1?)and tumor necrosis factor-alpha(TNF-?).In addition,PEMF can also play a role by increasing calcium ion influx and then increasing the binding of calcium ion to calmodulin(Ca M)and further increasing the nitric oxide synthase to increase the release of nitric oxide(NO).Other studies have shown that PEMF can also exert biological therapeutic effects by increasing the expression of heat shock protein 70(HSP70)and up-regulating adenosine receptors.At present,clinical studies have shown that PEMF treatment can improve pain in patients with OA.However,whether this treatment effect is related to its regulation of OA synovial inflammation is still not well known.This study intends to explore the regulation effect and possible mechanism of PEMF on synovitis through the mouse synovial inflammation model and in vitro cell experiments.Methods:1.Male C57BL/6J mice aged 6-8 weeks were used to establish the destabilization of the medial meniscus(DMM)model and air pouch model.2.The parameters of PEMF were: pulse waveform,1.5m T intensity,75 Hz frequency,and 10% duty cycle.3.After DMM surgery,the mice were treated with PEMF(2 hours/day,5 days/week,2 weeks in total).After all mice were sacrificed,the knee joints were removed and frozen sections were performed after fixed and embedded.Sections of the knee joints were stained with hematoxylin and eosin(H&E).The degree of synovitis of the knee joint was observed and quantified by histological score of synovitis.4.The air pouch was injected with LPS to induce inflammation and treated with PEMF(for a total of 4 hours).Mice were sacrificed at 8 hours after LPS injection,and lavage fluid was collected.The contents of IL-1? and TNF-? in the lavage fluid were measured by ELISA.5.Frozen sections of the skin were stained with H&E,and the number of nucleated cells in the synovial-like layer was counted.Ly-6G and F4/80 were used to label neutrophils and macrophages respectively.The proportion of neutrophils and macrophages was calculated.6.Mouse primary neutrophils were isolated and labeled with carboxyfluorescein diacetate,succinimidyl ester(CFDA SE)for 24 hours to induce apoptosis.RAW264.7 cells treated with PEMF for 1 hour were co-cultured with CFDA SE-labelled apoptotic neutrophils for 4 hours.The efferocytosis effect of RAW264.7 was observed by a confocal microscope,and the efferocytosis ability of RAW264.7 cells was quantified by flow cytometry.7.The effects of PEMF on the expression of total and phosphorylation levels of JNK,ERK,and P38 proteins in RAW264.7 cells and bone marrow derived macrophages(BMDM)were detected by western blot(WB).8.The effect of PEMF on LPS-induced IL-1? and TNF-? m RNA expression in RAW264.7 cells was detected by reverse transcription-polymerase chain reaction(RT-PCR).Results:1.PEMF could reduce the severity of knee synovitis in the DMM model.2.PEMF could reduce the secretion of LPS induced IL-1? and TNF-? in the air pouch model.3.PEMF did not change the total number of nucleated cells in the synovial-like layer of the air pouch model,but it decreased the proportion of neutrophils and increased the proportion of macrophages.4.PEMF could increase phagocytosis of RAW264.7 cells to apoptotic mouse primary neutrophils(efferocytosis).5.PEMF could inhibit the phosphorylation of P38 in RAW264.7 cells and BMDM cells.6.PEMF could inhibit mRNA levels of LPS-induced IL-1? and TNF-? in RAW264.7 cells.Conclusion:PEMF can inhibit the severity of synovitis in the DMM model and air pouch model.This inhibitory effect may be achieved by enhancing the efferocytosis effect of macrophages.In terms of mechanism,PEMF can inhibit the phosphorylation level of P38 in macrophages and reduce the expression and secretion of TNF-?,which in turn may lead to an enhanced efferocytosis of macrophages.