Inhibitory Effect Of Gallic Acid On LPS-induced Inflammatory Response In Mouse J774A.1 Macrophages

Objective: To investigate the inhibitory effect of gallic acid(GA)on the inflammatory response of mouse J774 A.1 macrophage induced by lipopolysaccharide(LPS).Methods: 1.The cellular inflammation model was established by stimulating mouse J774 A.1 macrophages with LPS,and the cells were randomly divided into the normal group,LPS group,GA low,medium,and high dose groups(GA concentrations of 20,40,and 80 μmol/L,respectively).Cell survival rate was detected using the CCK-8 kit.The NO kit was used to detect the NO content in the cell culture.The TNF-α and IL-6 contents in the cell culture were determined with the ELISA kit.Intracellular COX-2,i NOS protein expressions were detected using western blot method.2.The macrophage inflammasome model was induced by LPS plus ATP in mouse J774 A.1 macrophages,and the cells were randomly divided into the normal group,LPS+ATP group,and GA low,medium,and high dose groups(GA concentrations of 20,40,and 80 μmol/L,respectively).Cell survival rate and LDH content were detected using the CCK-8 and LDH kits.The IL-1β contents in the cell culture were determined with the ELISA kit.The damages of mouse J774 A.1macrophages were observed using Hoechst/PI staining.The protein expressions of IL-1β,non-activated GSDMD was detected with the western blot method,and NLRP3 protein expression was observed using immunofluorescence staining.3.Intracellular TLR4,My D88,NF-κB,MAPK protein expressions were detected using western blot method.Results: 1.LPS had no significant effect on the survival rate of mouse J774 A.1macrophages at the concentration range of 100-1000 ng/m L(P>0.05),but significantly upregulated the secretion of NO and IL-6 of J774 A.1 cell,indicating the inflammatory model of mouse J774 A.1 macrophages was successfully constructed.GA had no significant effect on the survival rate of mouse J774 A.1 macrophages at the concentration range of 5-80 μmol/L(P>0.05).Compared with the normal group,the contents of NO,TNF-α,and IL-6 in the cell culture medium of the LPS group were significantly increased(P<0.05);compared with the LPS group,the contents of NO,TNF-α,and IL-6 in the cell culture medium of the GA groups were significantly reduced(P<0.05).Western blot results showed that COX-2,i NOS protein expressions were significantly up-regulated in the LPS group compared with the normal group(P<0.05);COX-2,i NOS protein expressions were significantly down-regulated in the GA groups compared with the LPS group(P<0.05).2.The cell survival rate of the LPS+ATP group was significantly reduced and the IL-1β,LDH content in cell culture medium was significantly increased compared with the normal group(P<0.05);but the cell survival rate of the GA groups significantly increased and the IL-1β,LDH content in cell culture medium significantly decreased compared with the LPS+ATP group(P<0.05).Hoechst/PI staining results showed that the cell damage was increased in the LPS+ATP group than the normal group(P<0.05),and the cell damage was significantly reduced in the GA groups than the LPS+ATP group(P<0.05).The western blot results showed that the expression of IL-1β protein was significantly upregulated in the LPS+ATP group compared with the normal group(P<0.05).However,the expression of IL-1β protein was significantly downregulated in the GA groups compared with the LPS+ATP group(P<0.05).The expression of GSDMD protein was significantly downregulated in the LPS+ATP group compared with the normal group(P<0.05).However,the expression of GSDMD protein was significantly upregulated in the GA groups compared with the LPS+ATP group(P<0.05).Immunofluorescence staining results showed that the expression of NLRP3 protein was significantly enhanced in the LPS+ATP group compared with the normal group;the expression of NLRP3 protein was significantly reduced in the GA group compared with the LPS+ATP group(P<0.05).3.Western blot results showed that TLR4,My D88,p-NF-κB,p-JNK,p-ERK,p-P38 protein expressions were significantly up-regulated in the LPS group compared with the normal group(P<0.05);TLR4,My D88,p-NF-κB,p-JNK,p-ERK,p-P38 protein expressions were significantly down-regulated in the GA groups compared with the LPS group(P<0.05).Conclusion: GA has an inhibitory effect on the LPS-induced inflammatory response of mouse J774 A.1 macrophages,and its anti-inflammatory effect may be related to the inhibition of TLR4/NF-κB and MAPK signaling pathways.