Abstract: 【Objective】 The aim of the study was to explore the effect of dimethyl alpha-ketoglutarate (DMKG) on immunomodulatory effect of canine adipose-derived mesenchymal stem cells (cAD-MSCs).【Method】 In this experiment,DMKG medium with different concentrations was used to explore the effects of DMKG on the proliferation of cAD-MSCs.After cAD-MSCs were pretreated with 1 mmol/L DMKG,the levels of inflammatory cytokines of tumor nerosis factor-α (TNF-α),interleakin-1β (IL-1β),IL-6,IL-10 and transforming growth factor-β (TGF-β) in culture supernatant were determined by ELISA.RAW264.7 cells were cultured in medium containing 100,250,500,1 000 and 2 000 ng/mL lipopolysaccharide (LPS),respectively.After 24 h,the survival rate of cells was detected by CCK-8 assay to screen the optimal concentration of LPS was selected to establish RAW264.7 inflammation model.The cells were divided into blank control group,LPS group,MSCs group and DMKG-MSCs group,the cAD-MSCs pretreated with DMKG were co-cultured with RAW264.7 cells under 250 ng/mL LPS condition for 24 h,and the proliferation activity of the cells was detected by CCK-8 assay,the cell migration capacity was detected by Transwell assay,the content of NO in culture supernatant was determined by Griess assay,the mRNA expression levels of iNOs,IL-1β,IL-6,IL-10 and Arg-1 genes were detected by Real-time quantitative PCR,the surface markers CD80⁺ and CD86⁺ of M1 macrophages were detected by flow cytometry.【Result】 Compared with MSCs group,the secretion of TGF-β and IL-10 in culture supernatant after DMKG pretreatment of MSCs was significantly increased (P＜0.05),while the secretion of IL-1β and IL-6 were significantly inhibited (P＜0.05),and the secretion of TNF-α was not significantly different(P＞0.05).The proliferation activity of RAW264.7 cells reached the highest when LPS concentration was 250 ng/mL.Compared with LPS group,MSCs and DMKG pretreated cAD-MSCs extremely significantly inhibited the proliferation activity and migration ability of RAW264.7 cells (P＜0.01),inhibited NO release and down-regulated the mRNA expression level of iNOs (P＜0.01).In addition,mRNA expression levels of IL-1β (P＜0.01) and IL-6 in RAW264.7 cells were down-regulated,IL-10 was up-regulated (P＜0.05 or P＜0.01),the mRNA expression level of M2 macrophage surface marker Arg-1 was extremely significantly up-regulated (P＜0.01),and the expressions of surface markers CD80⁺ and CD86⁺ in M1 macrophages were extremely significantly decreased (P＜0.01).Compared with MSCs group,DMKG pretreated cAD-MSCs inhibited macrophage activation better.【Conclusion】 DMKG could effectively enhance the immunomodulatory effect of cAD-MSCs and increased expression of trophic factor TGF-β and anti-inflammatory cytokine IL-10,and the decreased expression of proinflammatory cytokines IL-1β and IL-6,inhibit the activation of RAW264.7 cells under the condition of LPS stimulation,and reduce the cellular inflammatory response.

Key words: dimethyl alpha-ketoglutarate (DMKG); canine adipose-derived mesenchymal stem cells (cAD-MSCs); immunomodulatory