Investigating The Therapeutic Effects Of Cyanidin-3-O-Glucoside On Rheumatoid Arthritis

Background:Rheumatoid arthritis?RA?is a common autoimmune disease.At present,the pathological mechanisms of RA are not fully understood,and the drugs available for treating this disease are limited.CD38 is a glycoprotein on the cell membrane that functions as a cyclic ADP-ribose hydrolase?cADPRH?.CD38 catalyzes the conversion of nicotinamide adenine dinucleotide?Coenzyme I,NAD+?to cyclic ADP-ribose?cADPR?to regulate calcium ion?Ca2+?homeostasis.Through transcriptome analysis,we found that CD38 was highly expressed in RA synovial tissue.Postigo et al.found that knockout of CD38 in mice significantly alleviated the occurrence and development of collagen-induced arthritis?CIA?.We also detected a high proportion of CD38+CD56+cells in the peripheral blood of RA patients.CD56 is an important marker of natural killer?NK?cells.Studies have shown that NK cells contribute to the progression of RA by regulating the secretion of tumor necrosis factor?TNF?-a,interferon?IFN?-y,and other cytokines to modulate the functions of immune cells,such as B cells,T cells,macrophages,and fibroblasts.Thus,we hypothesized that CD38 and CD38+ NK cells play important roles in RA and might be therapeutic targets.Cyanidin-3-O-glucoside,which is usually found in the form of chlorinated cyanidin-3-O-glucoside?kuromaninchloride,cyanidin-3-O-glucoside chloride,1-benzopyrylium,7-dihydroxy-2-?3,4-dihydroxyphenyl?-3-??--D-glucopyranosyloxy?-chloride?,is referred to as C3G.The molecular formula of C3G is C21H21ClO11.The structural core of C3G is a 2-phenylbenzopyran cation,which belongs to a family of flavonoid compounds.C3G has antitumor,anti-inflammatory and antioxidant effects Studies also found that C3G is an inhibitor of CD38 and competitively inhibits the function of CD38 by binding to the active site.This binding prevents CD38 from catalyzing the synthesis of cADPR and leads to accumulation of intracellular NAD?+?in dendritic cells,HL-60 cells,and chronic lymphocytic leukemia.Therefore,we postulated that C3G might be used to treat RA by inhibiting CD38 activity.Although increasing evidence shows that C3G,which is naturally derived from many plants,may provide protection against neurodegenerative diseases,no other molecular target except CD38 has been reported.Sirtuin 6?Sirt6?is a member of the sirtuin protein family and alleviates inflammatory responses in CIA mice.This study also explored the effects of C3G on Sirt6 expression and its downstream pathways in CD38+NK cellsMethods:We aimed to determine the pathogenic role of CD38 and the effect of C3G on RA in this study,which may provide a basis for developing C3G as a therapeutic agent for RA.In the present study,C3G was used to treat rats with bovine type II collagen-induced arthritis?CIA?model rats,and it was cultured with RA synovial fibroblast?RASF?-like cells,cultured mononuclear cells?MNCs?,and CD38+NK cells derived from RA peripheral blood or synovial fluid.The changes in lymphocyte subsets and proinflammatory cytokine levels were measured.Additionally,CD38+ NK cells with C3G treatment were cocultured with MNCs depleted of CD38+NK,and the mechanism of MNC differentiation into T regulatory?Treg?cells was investigatedResults:C3G injection significantly alleviated CIA.C3G also significantly increased the level of interleukin?IL?-10 and the regulatory T?Treg?cell proportion,and it decreased the interleukin?IL?-6 and interferon?IFN?-? levels and CD38+NK cell proportion in rat peripheral blood and synovial fluid.Additionally,C3G significantly increased RASF apoptosis and decreased RASF proliferation and IL-6 production in the culture medium.Furthermore,C3G stimulated MNCs to increase IL-2 and IL-10 production and the Treg cell proportion,and it caused MNCS to decrease IL-6 and IFN-y production and the CD38+ NK cell proportion.Although CD38+NK cells significantly decreased the Treg cell proportion and IL-10 level in MNCs,CD38+NK cells that had been pretreated with C3G increased the proportion of Treg cells and IL-10 levels and decreased the IL-6 and IFN-y levels in the coculture In CD38+NK cells,C3G significantly increased Sirtuin 6?Sirt6?expression and the tumor necrosis factor?TNF?-? level,and it decreased natural killer group 2D?NKG2D?expression and the IFN-y level.However,when CD38+NK cells were treated with Sirt6 siRNA,C3G didn't change the NKG2D expression,the TNF-? level sharply decreased,and the IFN-y level increased.When MNCs were cocultured with C3G-pretreated CD38+NK cells in the presence of TNF-? and an anti-IFN-y antibody,the IL-10+Treg cell proportion significantly increased.When MNCs were cocultured with C3 G-pretreated CD38+NK cells in the presence of IFN-? and an anti-TNF-?antibody,the IL-10+Treg cell proportion sharply decreased.When CIA rats were injected with both C3G and the Sirt6 inhibitor OSS₁28167,the rats exhibited joint inflammation and a low Treg cell proportion,but the CD38+NK proportion was still low.Conclusion:C3G has therapeutic effects on CIA and RA.C3G decreased the proportion of CD38+cells,RASF proliferation,and pro-inflammatory cytokine secretion,and it increased the Treg cell proportion.C3G also elevated Sirt6 expression to suppress NKG2D expression,increase TNF-? secretion,and decrease IFN-y secretion in CD38+ NK cells,which stimulates MNCs to differentiate into Treg cells.This study also demonstrates that the inhibition of Treg cell differentiation in MNCs by CD38+NK cells is a potential cause of the immune imbalance in RA and CIA.