| Background Rheumatoid arthritis(RA)is a autoimmune disease characterized by inflammation and proliferation of synovial tissue resulting in bone and cartilage destruction.The pathogenesis of RA has not been fully elucidated.The role of mast cells and their associated cytokines in the pathogenesis of RA is one of the hot-spots recent years.Mast cells account for about 3% of the total number of normal joint synovial cells,and their role may be involved in repairing tissue damage or clearing pathogens.However,the number of MCs in the synovium of RA patients may be 6~25 times that of healthy people.Normally,too many mast cells are detrimental to the development of RA.Existing experimental studies have confirmed that acupuncture at "Zusanli" has anti-inflammatory,analgesic,and immune-regulating effects,and has good application prospects in anti-RA,but the molecular mechanism by which it exerts anti-inflammatory and immune-regulation is not clear.It is unclear whether its anti-RA mechanism is related to regulating mast cell function.To this end,we have conducted a series of studies on this issue,established a collagen-induced arthritis(CIA)rat model,and investigated the effect of electroacupuncture at "Zusanli" on the inflammatory responses in CIA rats and explored its potential molecular mechanisms.Objective To enunciate the electroacupuncture possible molecular mechanisms of the anti-inflammatory and immuneregulative effects from the foot swelling,arthritis index and pathologic histological changes,bovine type Ⅱcollagen induced rats model were prepared.Observing the effects of electroacupuncture on the number and function of mast cells in synovial tissue of CIA rats,and evaluating the effects of electroacupuncture on IL-33-induced mast cell inflammation through in vitro experiments,combining IL-33 stimulation after transfection with mi R-155 mimic mast cells,to elucidate the possible molecular mechanism of electroacupuncture in anti-collagen-induced arthritis.Methods1.SPF male SD rats were divided into four groups(n = 30 per group),according to the digital random method namely blank control group,model(CIA)group,EA+CIA group,Sham+CIA group.Normal 7 days later,according to the following method to dispose: blank control group wound not be any processing;in addition to the blank control group,in the rest of the rats using Ⅱ collagen type and incomplete freund’s adjuvant mixed emulsifying subcutaneous injection mold,namely collagen-induced arthritis(CIA)rat model.After the modeling was completed,the arthritis index was used to evaluate the degree of joint damage in rats every two days,until the rats were sacrificed.2.After the modeling was completed,the blank control group and the model control group did not give any interference.The CIA model +electroacupuncture treatment group selected "Zusanli" acupoint to give electroacupuncture stimulation.The following stimulus parameters were selected: a continuous wave at 2 Hz and 1 m A for 5 min,2 Hz and 1.5 m A for5 min,and2 Hz and 2 m A for 20 min.EA stimulation was repeated rom day 1to day 35.The rats in the CIA model + sham acupuncture group were given electroacupuncture stimulation next to the "Zusanli" acupoint 5mm.Before each treatment,the body weight and foot swelling of the rats in each group were measured until the rats were sacrificed.3.Twenty-four hours after the last electroacupuncture treatment,the rats were anesthetized with chloral hydrate via intraperitoneal injection and fixed on a rat plate.Blood was collected from the abdominal aorta and centrifuged to collect serum from each group.Part of the rats were taken from the left knee joint tissue,and the other part was taken from the left synovial tissue.The tissue was fixed,decalcified,and embedded into sections;the rat spleen was isolated and the spleen mononuclear cells were extracted;gathered sterile peritoneal fluid.Experiment Ⅰ1 The arthritis index was used to evaluate the degree of joint damage of rat arthritis,the weight of rats were measured,foot swelling of CIA rats was measured with foot volume method.2 Histopathological changes of joints were observed by HE staining.3 ELISA method was used for detecting the content of total anti-CII Ig G,anti-CII Ig G1,anti-CII Ig G2 a,TNF-α,IL-1β,IL-6,IL-17 in serum.4 The level of RORγt and IL-17 in splenic lymphocytes were detected with flow cytometry.Experiment(47)1 IL-33 expression in synovial tissues were detected by immunohistochemical assay.2 ELISA method was used for detecting the content of IL-33 in serum and the content of IL-33,TNF-α,IL-1β,IL-6,IL-17 in synovial tissues.3 The gene expression level of IL-33 m RNA was detected by RT-PCR assay.4 Toluidine blue staining showed the number of mast cells and degranulation in synovial tissue.Experiment Ⅲ1 Isolation of rat abdominal mast cells and identification with toluidine blue staining.2 ELISA was used to detect the release of TNF-α,IL-1β,IL-6,MCP-1,IL-6,IL-13 and histamine from mast cells stimulated by IL-33.3 Spectrophotometric analysis measured the release of β-hexosaminidase release in vitro.4 Real-Time RT-PCR was used to detecte the gene expression of ST2 and mi R-155 m RNA in mast cells stimulated by IL-33.5 Western blot assay was used to detecte the protein expression related to NF-κB p65 transcription and P38 MAPK signaling pathway in mast cells stimulated by IL-33.Experiment Ⅳ1 Real-Time RT-PCR was used to detecte the gene expression of mi R-155 in mast cells transfected with mi R-155 mimic.2 ELISA was used to detect the release of TNF-α,IL-1β,IL-6,MCP-1,IL-6,IL-13 of mast cells transfected with mi R-155 mimic.3 Western blot assay was used to detecte the protein expression related to NF-κB p65 transcription and P38 MAPK signaling pathway in mast cells transfected with mi R-155 mimic.Results1.Electroacupuncture at “Zusanli” alleviated collagen-induced arthritis in rats(CIA).In the model control group,the food of the rats in the model group gradually swelled,and the degree of swelling reached its peak on the 21 st day,and then gradually subsided;local skin congestion,redness and swelling,and the arthritis score reached its peak on the 24 th day;the weight of CIA rats gradually decreased.The state of rats in the Sham+CIA group were basically the same as those in the model control group.The EA+CIA group had basically the same performance before treatment as the model control group.After electroacupuncture treatment,the posterior condyle swelling degree of CIA rats was reduced(P<0.05),the arthritis score was reduced(P<0.05),and the weight loss was slowed(P<0.05).The HE staining results showed that compared with the model control group,the knee joint synovial tissue proliferation of the EA+CIA group was reduced,the inflammatory cell infiltration was significantly reduced,(P < 0.05)and the histopathological score was significantly reduced(P < 0.05).ELISA test results showed that electroacupuncture could reduce the type II collagen antibody(total anti-CII Ig G antibody concentration,anti-CII Ig G1 antibody concentration,and anti-CII Ig G2 a antibody)concentration in the serum to varying degrees(P<0.01、P<0.05、P<0.01),and could inhibit the release of inflammatory factor(TNF-α,IL-1β,IL-6 and IL-17)in serum(P<0.01、P<0.01、P<0.01、P< 0.05).Flow cytometry results showed that electroacupuncture can significantly reduce the proportion of CD4+RORγt+ cells and the proportion of CD4+IL-17+ T cells in CD3+CD4+T cells of CIA rats(P<0.01、P<0.05).2.Electroacupuncture inhibited the production of IL-33 and infiltration of mast cells in CIA rats.Immunohistochemical results showed that compared with the blank control group,the content of IL-33 in the synovial tissue of the model control group and the EA+CIA group were significantly increased,and,compared with the model control group,the content of IL-33 in the EA+CIA group decreased significantly(P < 0.01).ELISA detected the levels of IL-33 in serum and synovial tissue of rats,the results trend was consistent with the results of immunohistochemistry(P<0.01),and the content of IL-33 in serum and the content of IL-33 in synovial tissue showed a positive correlation(r =0.9196,p = 0.0012).Further RT-PCR test results showed that compared with the control group,electro-acupuncture could significantly reduce the expression of IL-33 m RNA in serum(P<0.01).Toluidine blue staining results showed that electroacupuncture can significantly reduce the hypertrophy and infiltration of synovial tissue in CIA rats compared with the control group(P<0.05、P<0.01).ELISA test results showed that compared with the model control group,electroacupuncture could significantly reduce the release of inflammatory factors TNF-α,IL-1β,IL-6 and IL-17 in synovial tissue(P <0.01、P<0.01、P<0.05、P<0.05),and there was a positive correlation between the number of cells and the release of inflammatory factors in the synovial tissue.3.Electroacupuncture restrained the inflammation of mast cell induced by IL-33.IL-33 co-cultured with peritoneal mast cells isolated from each group of rats,mast cells were observed with toluidine blue staining,and inflammatory factors were detected by ELISA.The results showed that compared with the model control group,the secretion of TNF-α,IL-5,IL-1β,IL-6,MCP-1 and IL-13 and the release of histamine from mast cells in the EA+CIA group were all decreased significantly(P<0.05、P<0.01、P<0.01、P<0.01、P<0.05、P < 0.01 、 P < 0.05).Spectrophotometric results showed that electroacupuncture can significantly inhibit the release of β-hexosaminidase from mast cells in vitro compared with the model control group(P<0.05).ELISA detected the levels of related antibodies in the cultured mast cell supernatants.The results showed that the levels of IL-5 and IL-13 in the supernatants were positively correlated with the levels of anti-CⅡ Ig G1 and anti-CⅡ Ig G2 a,respectively.The results of flow cytometry showed that the contents of IL-1β and IL-6 were positively correlated with the proportion of CD4+IL-17+cells in CD3+CD4+T cells.The results of Real-Time PCR showed that electroacupuncture significantly reduced the expression of mi R-155 mRNA in mast cells but had no effect on ST2,which is a IL-33 receptor(P<0.01).Western blot results showed that electro-acupuncture can significantly reduce the expression level of p-IKKα/β in cytosolic proteins(P<0.001)、the expression level of NF-κB p65 in nucleoproteins,and decrease the value of p-P38/P38 in cytosolic protein(P<0.005),and increase IκBα expression in cytosolic proteins(P<0.005).4.Electroacupuncture inhibits mast cell inflammation regulated by IL-33 via targeting mi R-155 thereby alleviating collagen-induced arthritis in rats.Peritoneal mast cells of rats were isolated,and mi R-155 mimic transfection of mast cells in the EA+CIA group resulted in overexpression of mi R-155 m RNA(P<0.005).ELISA test results showed that compared with the EA+CIA group,the release of mast cell inflammatory factors TNF-α,IL-1β,IL-6,MCP-1,IL-5 and IL-13 increased significantly stimulated by IL-33(P<0.01、P<0.01、P<0.01、P<0.05、P<0.05、P<0.05).Western blot analysis showed that compared with the EA+CIA group,the value of p-P38/P38 and the protein expression of p-IKKα/β were significantly up-regulated in the cytoplasmic protein of mast cells after mi R-155 mimic transfection under IL-33 stimulation(P<0.005、P<0.005),while the protein expression of IκBα was significantly decreased;simultaneously(P<0.01),the protein expression of NF-κB p65 in nuclear proteins was significantly increased(P<0.005).Conclusion1.Electroacupuncture has a marked improvement on collagen-induced arthritis.Electro-acupuncture can reduce the infiltration of inflammatory cells in synovial tissue of CIA rats,inhibit the content of type Ⅱ collagen antibodies and the secretion of inflammatory cytokines TNF-α,IL-1β,IL-6 and IL-17,and restrain the activation of Th17 cell in spleen,they are the key to their effect on regulating the immune function of CIA rats.2.Electroacupuncture exerts its therapeutic effect by reducing the infiltration of mast cells in synovial tissue of CIA rats,reducing the production of IL-33 in local synovial tissue and in general blood circulation,and suppressing the inflammation response of mast cells induced by IL-33.3.Mi R-155 is involved the effect of electroacupuncture on inhibiting IL-33-induced mast cell inflammation in CIA rats.In summary,we speculate that electroacupuncture may target mi R-155 to negatively regulate the release of IL-33 from mast cells,thereby exerting an anti-collagen-induced arthritis effect. |
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