| Background:2,4',5'-trihydroxyl-5,2'-dibromo diphenylmethanone(LM49)is a novel active polyphenol synthesized by our group in the process of structural optimization of seaweed polyphenols,LM49 exerted therapeutic effects against rat acute pyelonephritis in-vivo experiment,accompanied by increased IL-10 and decreased IL-1? and IL-6,indicated that LM49 had anti-inflammatory effect in bacterial inflammation.However,the anti-inflammatory mechanism is still unknown,RAW264.7 cells are the target cells for studying inflammation in vitro.Therefore,this paper mainly discusses the effect of LM49 on RAW264.7macrophages,and thus clarifies the molecular mechanism of LM49 exerting anti-inflammatory effects.Objective:In view of the role of macrophage polarization in the regulation of inflammatory response.The study aimed to investigate the relationship between the anti-inflammatory activity of LM49 and macrophage polarization,and to evaluate the anti-inflammatory activity of LM49 and its effect on macrophage polarization in vivo and in vitro,then to explore the potential mechanism of macrophage polarization.Method:Part ?: Effect of LM49 on LPS+INF-?-induced polarization of RAW264.7macrophages1.The cell viability of LM49 on RAW264.7 cell was detected by MTT assay.2.The RAW264.7 cells were exposed to LPS plus INF-? and treated with different concentrations of LM49,then the content of NO in the cell supernatant was detected by the Griess assay.3 The RAW264.7 cells were cultured with LPS plus INF-? and treated with different concentrations of LM49,the expression of CD16/32 and CD206 were analyzed by flow cytometry.4.The RAW264.7 cells were exposed to LPS plus INF-? and treated with different concentrations of LM49,the protein expression of i NOS and Arg-1 was analyzed by western-blot.5.The RAW264.7 cells were exposed to LPS plus INF-? and treated with different concentrations of LM49,the m RNA expression of Arg-1,i NOS,CD206,CD86,CD163,FIZZ,IL-6,TNF-?,IL-10,MCP-1 and IL-12 were examined by RT-PCR.Part ?: Potential mechanism of LM49-derived macrophage polarization in LPS/INF-?-induced RAW264.71.The RAW264.7 cells were exposed to LPS plus INF-? and treated with different concentrations of LM49,the m RNA expression of IRF-4,IRF-5,KLF4,SOCS1,SOCS3,NF-?B,STAT1,STAT3,STAT6,TLR4,Myd88,JAK1,JAK2 and JAK3 were tested by RT-PCR.2.The RAW264.7 cells were exposed to LPS plus INF-? and treated with different concentrations of LM49,the protein expression of TLR4,Myd88 and NF-?B were analyzed by western-blot.3.The RAW264.7 cells were exposed to LPS plus INF-? and treated with different concentrations of LM49,the protein expression of SOCS1,JAK2,p-JAK2,STAT1 and p-STAT1 were analyzed by western-blot.4.The RAW264.7 cells were exposed to LPS plus INF-? and treated with different concentrations of LM49,the protein expression of JAK1,p-JAK1,SOCS1,JAK3,p-JAK3,STAT6 and p-STAT6 were analyzed by western-blot.Part ?: Effect of LM49 on Kuffer cell polarization in LPS-induced acute liver injury1.The effects of LM49 on the activities of AST and ALT in serum of LPS-induced acute liver injury mice were detected by micropore method,and the levels of IL-6and TNF-? in serum were detected by ELISA.The effect of LM49 on liver histopathological examination in LPS-induced acute liver injury mice by H&E staining.2.The expression of Arg-1 in liver tissue in LPS-induced acute liver injury mice by immunohistochemical,the expression of CD16/32 and CD206 in KCs of LPS-induced acute liver injury mice were analyzed by flow cytometry.3.The effects of LM49 on the the expression of i NOS and Arg-1 in KCs of LPS-induced acute liver injury mice by RT-PCR and western blot.Result:1.LM49 has no obvious cytotoxicity on RAW264.7 cells at the concentration range of 0-30 ?mol·L-1.Subsequent experiments have selected doses of 5 ?mol·L-1,10?mol·L-1,20 ?mol·L-1.LM49 inhibits the expression of NO in the supernatant of RAW264.7 cells induced by LPS+INF-? in a dose-dependent manner.The protein expression of i NOS was inhibited and the protein expression of Arg-1 was induced by LM49 treatment.2.By using flow cytometry,LM49 treatment dose dependently reduced the expression of CD16/32 and elevated the expression of CD206 in RAW 264.7 cells induced by LPS+INF-?.And LM49 treatment significantly increased the m RNA expression of Arg-1,CD206,CD163,FIZZ and IL-10 in a concentration dependent manner,and strongly inhibited the m RNA expression of i NOS,IL-6,TNF-?,CD86,MCP-1 and IL-12 in RAW 264.7 cells3.By using RT-PCR,LM49 treatment reduced the m RNA expression of IRF-5,JAK2,SOCS3,STAT1,TLR4,Myd88 and NF-?B in RAW 264.7 cells induced by LPS+INF-?,and increased the m RNA expression of IRF-4,KLF4,SOCS1,JAK1,JAK3,STAT3 and STAT6 in RAW 264.7 cells,but had no effect on STAT3.4.LM49 treatment reduced the protein expression of TLR4,MyD88 and NF-kB in RAW 264.7 cells induced by LPS+INF-?.Meanwhile,especially high dose of LM49 significantly increased the protein expression of SOCS1,JAK1,p-JAK1,JAK3,p-JAK3,STAT6 and p-STAT6,and strongly reduced the protein expression of JAK2,p-JAK2,STAT1 and p-STAT1 in RAW 264.7 cells.5.In LPS-induced acute liver injury mice,LM49 declined the activities of ALT and AST in a concentration dependent manner,the concentrations of inflammatory cytokine IL-6 and TNF-? were inhibited in all LM49 groups.Pathological sections showed that LM49 could significantly improve the infiltration of inflammatory cells in the liver of LPS-induced acute liver injury mice.6.Immunohistochemical results showed that LM49 could significantly increase the expression of Arg-1 in liver,and flow cytometry showed that LM49 couldsignificantly increase the number of F4/80+CD206+ cells and decrease the number of F4/80+CD16/32+ cells.The results of RT-PCR and Western-blot showed that LM49 could significantly increase the expression of Arg-1 and decrease the expression of i NOS in KCs.Conclusion:1.LM49 significantly inhibited LPS/INF-?-induced M1 macrophages and drived to M2 macrophages polarization.2.LM49 could inhibit M1 polarization by down-regulating TLR4-Myd88-NF-?B and JAK2-STAT1 pathways,and promote M2 polarization via up-regulating JAK1/JAK3-STAT6 pathway.3.LM49 has hepatoprotective effect on LPS-induced acute liver injury in mice,and may play an anti-inflammatory role by polarizing KCs to M2-type. |
PDF Full Text Request