Studies On Inhibitory Effects And Molecular Mechanisms Of Garlic Organosulfur Compounds DATS On The Inflammation

Objective:In order to clarify scientific basis for garlic DATS in medical applications, and develop healthcare products, the anti-inflammatory action of garlic dially trisulfide （DATS） and the relationship between the anti-inflammatory and anti-inflammatory effects of DATS were investigated at the molecular level and animal model.Methods:RAW264.7 cells were treated with six kinds of GOSCs to screen their influence on cyclooxygenase-2 and inducible nitric oxide synthase expression by Western blotting. Prostaglandin E2 and nitrite were measured by ELISA and Griess Reagent System, respectively.Cytokines in culture medium were assayed by the multiplex technology. Proteins including inflammatory enzymes （COX-2 and iNOS）, protein kinases （ERK, p38, JNK, p65, AKT, TAK1） and antioxidant proteins （Nrf2, NQO1, HO-1, HSP70） were detected, using their respective antibodies by Western blotting. ROS level in RAW264.7 cells were mearured, using diclofenac sodium fluorescein （DCFH-DA） by flow cytometry. Mouse paw edema was induced by LPS and estimated by measuring the thickness of mouse paw. The serum levels of IL-6, MCP1 and TNF-a weremeasured by ELISA.Results 1:The inhibitory effects of GOSCs on the production of COX-2, iNOS, （PGE）₂ and NO in LPS-activated Raw264.7 cells. DATS is a strongest inhibitor among GOSCs. DATS inhibited the expression of COX-2 and iNOS in LPS-avtivated RAW264.7 cells in a dose dependent manner. As the same fansion, DATS also inhibited （PGE）₂ and NO production. These data suggest that DATS might extert the anti-inflammation activity by inhibiting the expression of COX-2 and iNOS, and the production （PGE）₂ and NO.Results 2:The inhibitory effects of DATS on the production of 23 cytokines in LPS-activated RAW264.7 cells.DATS significantly reduced six cytokines of which including IL-6, IL-10, IL-12 （p70）, TNF-a, KC and MCP-1 （P<0.01）, but the other 17 kinds of cytokines had no significant effect. These data suggest that DATS might extert the anti-inflammation activity also by inhibiting the production of six kinds of cytokines (IL-6, IL-10, IL-12 （p70）, TNF-a, KC and MCP-1.Results 3 Signal transduction pathway analysis showed that DATS not only inhibited the phosphorylation of LPS-induced ERK, p38, JNK and c-Jun protein to block MAPK signaling; but also inhibited the phosphorylation of LPS-induced I k B-a and p65 protein, and reduce I k B-a protein degradation and p65 nuclear translocation to block NF-k B pathway signaling. DATS further inhibited phosphorylation of TAK1 and AKT1 （upstream molecules of both NF-k B and MAPK）. The results revealed that DATS might suppress the expression of pro-inflammatory and foctors and cytokines by downregulating TAKl and AKT1-mediated NF-k B, MAPK and IF3 cascades.Results 4 Antioxidant assay results showed that DATS enhanced the expression of antioxidant proteins such as Nrf2, HO-1 and NQ01.DATS intervention significantly reduced ROS levels in LPA-activated RAW264.7 cells （P<001.）. The the levels of Nrf2, HO-1 and NQO1 protein were significantly increased （P<0.01） while HSP70 protein expression was significantly reduced （P<0.01）. Thus, DATS could enhance Nrf2, HO-1 and NQO1 antioxidant protein with decreased HSP70 protein to reduce the generation of LPS-induced ROS antagonizing oxidative stress.Results 5 In vivo data showed that DATS suppressed mouse paw edema induced by LPS. DATS decreased the edema by 31.8%, compared with LPS. Simultaneously, DATS could significantly reduce the expression of serum IL-6, MCP1 and TNF-a （p<0.05）. These data demonstrated the in vivo the anti-inflammatory effect of DATS that attenuated the production of LPS-induced inflammatory mediators to inhibit mouse paw edema.Conclusion DATS was a strongest inhibitor for cyclooxygenase and inducible nitric oxide synthase among GOSCs, and reduced the levels of LPS-induced IL-6, IL-10, IL-12（p70）, KC, MCP-1, and TNF-a. Cellular signaling analysis revealed that DATS downregulated AKT1/TAK-mediated mitogen-activated protein kinase （MAPK） and nuclear factor kappa-light-chain-enhancer of activated B cells （NF-k B） pathways. Furthermore, DATS activated Nrf2-mediated expression of HO-1 and NQO1 and reduced LPS-induced intracellular reactive oxygen species, whichmay contribute to suppress inflammatory mediator production. Finally, in vivo data demonstrated that DATS attenuated LPS-induced mouse paw edema. Thus, DATS as a potential inhibitor revealed antiinflammatory effect in both cell and animal models by downregulating AKTl/TAK-^ activated kinase-mediated NF-k B andMAPK signaling pathways.