| Objective:Monocytes play a multifaceted role in rheumatoid arthritis(RA),and previous studies have shown that long-term upregulation of NAMPT can aggravate inflammatory arthritis.Therefore,this article mainly discusses the effect of NAMPT intervention of QLY on the rat model of rheumatoid arthritis.Methods:Eighteen male healthy SD rats were randomly and equally divided into normal group(Normal),model group(AIA)and Qingluoyu group(QLY),and each group was divided into 6 groups.The model group(AIA)and Qingluoyu group(QLY)were used to induce arthritis in SD rats with Freund’s adjuvants to establish a rheumatoid arthritis model.After successful modelling,the three groups of rats were gavaged,the QLY group received the appropriate dose of therapeutic drugs,and the Normal and AIA groups re-ceived the same dose of normal saline for 30 consecutive days.During this period,the general condition of the rats was monitored;after the rats were anaesthetised and sacri-ficed,the histomorphology of the rat ankle joint was observed by haematoxylin and eosin staining(HE),and the expression of i NOS and Arg-1 in rat spleen tissue was observed by immunohistochemistry(IHC).Expression levels of IL-1βand TNF-αin rat serum,GM-CSF and Arg-1 in spleen tissue were monitored by enzyme-linked immunosorbent assay(ELISA).The expression of CD4~+IFN-γ~+and CD68~+CD86~+in rat peripheral blood was analysed by flow cytometry.In vitro experiments:1.In vitro isolation and culture of peripheral blood mononuclear cells(PBMC),verification of the inflammatory effect of QLY on PBMC by ELISA and WB detection methods,and detection of the effect of NAD~+expression on monocytes.2.Normal rat spleen lymphocytes were isolated and cultured in vitro,stimulated with LPS(1μg/m L),10%QLY-containing serum and FK866(100 nmol/L),CD4~+IFN-γ~+and CD68~+CD86~+were detected by flow cytometry,and IL-1β,TNF-α,IFN-γexpression and NAD~+expression in the supernatant were detected by ELISA method;3.Cultured monocytes in vitro,treated with NAMPT inhibitor FK866(100 nmol/L)and agonist NMN(5 mg/m L)to stimulate downregulation and upregulation of NAMPT,respectively,and verified that QLY intervened in monocyte polarisation via NAMPT to alleviate inflammation;4.Isolate and culture normal fibroblast-like synovial cells and co-culture with normal monocytes and monocytes stimulated with 10%AIA rat serum and detect the effect of monocytes on FLS in inflammatory pathology by ELISA and WB methods;5.On the basis of 4,superimpose 10%QLY drug-containing serum stimulation,observe the therapeutic effect of QLY on FLS cells by WB method,and ver-ify whether QLY reverses the effect of monocytes on FLS cells;6.Monocytes were co-cultured with FLS cells and stimulated with LPS(1μg/m L),FK866(100 nmol/L),QLY and QLY+NMN(5 mg/m L),respectively,and QLY+NMN were detected by ELISA and WB methods to determine the state of QLY mediating FLS by regulating monocyte in-flammation.Results:1.In vivo experiments showed that compared with the normal group,SD rats in the AIA model group lost weight,the foot and claw joints were hyperemic and swollen,the thymus and spleen tissues of immune organs were enlarged,and the inflammation score was increased;The results of HE staining of rat joints showed that,compared with the normal group,the joints of rats in the AIA group were extensively infiltrated with inflammation,and the cartilage and subchondral bone were severely eroded,and the phe-nomenon of angiopan appeared.The pathological histochemical results showed that the spleen tissue of rats in the AIA group had obvious positive expression of i NOS and a low positive expression rate of Arg-1.ELISA results showed that the serum expression levels of IL-1βand TNF-αwere higher in the AIA model group than in the normal group.The expression level of GM-CSF in the spleen of rats in the AIA group was also increased,while the expression of Arg-1 was decreased.The proportion of M1 and Th1 cells in the peripheral blood of rats in the AIA group was increased;Compared with the AIA group,after QLY treatment,the weight,thymus and spleen tissue morphology of the rats gradu-ally returned to normal,the swelling of the foot and claw joints decreased,the arthritis score decreased,HE staining showed that there was no large synovial invasion in the bone and joint,The expression of i NOS in the spleen tissue was low,the opposite trend of Arg-1,the expression of IL-1β,TNF-αin serum and GM-CSF in the spleen were downregu-lated,the expression of Arg-1 in the spleen increased,and CD4~+IFN-γ~+and CD68~+CD86~+in the peripheral blood decreased.2.In vitro experiments:ELISA results showed that compared with normal PBMC,IL-1β,IL-6,GM-CSF and NAD+were significantly in-creased in LPS-stimulated PBMC,IL-10 and Arg-1 were significantly decreased,inflam-matory cytokines and NAD+were decreased and inflammatory cytokines were increased after treatment with 10%QLY-containing serum.The WB method shows the same result;After isolation of splenic lymphocytes in vitro,compared with the normal group,the pro-portion of CD4~+IFN-γ~+and CD68~+CD86~+cells in splenic lymphocytes increased after LPS stimulation,and after stimulation with 10%QLY-containing serum and NAMPT in-hibitor FK866,the proportion of CD4~+IFN-γ~+and CD68~+CD86~+cells decreased signifi-cantly,and the ELISA results were the same as those of flow cytometry.NAMPT was downregulated,and when monocytes were stimulated with LPS,FK866 and QLY drug-containing serum,the results of the WB method showed that compared with normal mon-ocytes,the expression of NAMPT and i NOS protein increased and the expression of Arg-1 protein decreased in monocytes after LPS stimulation,and the protein expression of NAMPT and i NOS decreased after stimulation with 10%QLY-containing serum and the NAMPT inhibitor FK866,and the expression of Arg-1 protein was upregulated,and the ELISA results showed the same trend.The results of the WB method showed that com-pared with monocyte cells stimulated with LPS,the expression of NAMPT and i NOS protein increased and the expression of Arg-1 protein decreased after stimulation with the NAMPT agonist NMN,and after stimulation with 10%QLY drug-containing serum,the expression of NAMPT and i NOS protein decreased and the expression of Arg-1 protein increased.The ELISA results also showed the same trend as the WB;The results of the WB method showed that the expression of MMP3 and p-p65 proteins was significantly increased in FLS cells cocultured with monocytes stimulated with 10%AIA serum com-pared to FLS cells cocultured with normal monocytes.The expression of proteins such as NAMPT decreased after the superimposition of 10%drug-containing serum QLY;Mon-ocytes were co-cultured with FLS,monocytes were treated with LPS,LPS+NMN,LPS+FK866,LPS+QLY and LPS+NMN+QLY respectively,FLS cells were harvested,compared with the LPS group,the expression of NAMPT,MMP3,p-JNK and p-p65 pro-teins in FLS was increased in the LPS+NMN group,whereas the expression of NAMPT and MMP3 proteins was decreased in the LPS+FK866 and LPS+QLY groups.Phosphor-ylation of JNK and p65 was inhibited,and protein levels were also reduced in the LPS+QLY+NMN group,but not as much as in the LPS+FK866 and LPS+QLY groups.It was found that,compared to the LPS group,the M1 phenotype and cytokine IL-1βwere significantly increased in the NMN group,while the M2 phenotype and anti-inflamma-tory factor IL-10 were reduced,and the LPS+FK866 and LPS+QLY groups showed the opposite trend to NMN.Conclusions:1.QLY can alleviate the swelling of the joints of rats in the AIA model group,inhibit the inflammation of rats in the AIA model group,and reduce the ratio of M1 macrophages and Th1 cells in peripheral blood;2.QLY can inhibit the inflammation of PBMC and affect inflammatory arthritis through NAMPT;3.QLY can intervene in monocytes polarization through NAMPT to reverse inflammation to a certain extent;4.QLY regulates FLS inflammation by regulating monocytes and restores and regulates joint immune homeostasis. |
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