| objective:To study the Effect of curcumin on cholesterol-related protein,cholesterol efflux and the secretion of tumor necrosis factor(TNF-?),monocyte chemotactic protein-1(MCP-1),interleukin-6(IL-6)in macrophages derived from human acute monocytic leukemia cell line(THP-1).To explore the possible mechanism and provide a reliable theoretical basis for the curcumin anti-atherosclerosis(AS)as well as some experimental basis for the prevention and treatment of cardiovascular diseases.Methods:In this study,THP-1 cells cultured in vitro was utlized as the research object.Phorbol-12-myristate-13-acetate(PMA)was applied to induced the formation of macrophages.Experiments were carried out as the followings manner.1 Test cell toxicity of curcuminTHP-1 derived macrophages were cultured with curcumin of different final concentrations(0-80 ?M).Cell viability was measured by Cell Counting Kit-8(CCK-8).2 Effect of curcumin on the expression of miR3 3a,TLR4,ABC A1 and intracellular lipid content,formation rate of foam cell,IL-6,TNF-? and MCP-1 secretion in THP-1 derived macrophagesExperimental group:(1)control group;(2)oxidized lowdensity lipoprotein(ox-LDL)(50 ?g/mL)group;(3)ox-LDL+ curcumin(40 pM)group;With Oil red O staining cells,cell morphology was observed with the light microscope,Foam cell formation rate was calculated.,Intracellular lipid content was detected by enzymatic measurement.and the ratio of cholesterol ester(CE)to total cholesterol(TC)was calculated.The expression of ATP binding cassette transporter A1(ABCA1),micro RNA(miRNA)33a and Toll like receptor(TLR)4 mRNA were detected by real-time fluorescence quantitative PCR(RT-qPCR).The protein content of ABCA1 and TLR4 were detected by Western Blot,.The secretion of IL-6,TNF-?,MCP-lwere detected by enzyme linked immunosorbent assay(ELISA)kit.3 Effect of curcumin with different concentration and time on cholesterol efflux rate of THP-1 derived macrophagesox-LDL induced the absorption of lipids in THP-1 derived macrophages,and then the cells were incubated with curcumin at different concentrations(0-40 ?M)or for varied time(6-24h)conditions.Cholesterol efflux rate was measued by cholesterol fluorescence-labeling method.4 Detection of liposome transfectionExperiment group:(1)control group;(2)foam cell model group;(3)ox-LDL+miR33a inhibitor group;(4)ox-LDL+control sequence group;miR33a inhibitor and control sequence were used to transfect into THP-1 derived macrophages by liposome transfection in vitro,and detection for the expression of miR33a was completed by RT-qPCR.5 Effect of curcumin on the expression of ABCA1,cholesterol efflux rate and the secretion of IL-6,TNF-?,MCP-1 by miR33a in THP-1 derived macrophagesExperimental groups:(1)control group;(2)foam cell model group;(3)ox-LDL+curcumin group;(4)ox-LDL+miR33a inhibitor group;(5)ox-LDL+curcumin +miR3 3 a inhibitor group;(6)ox-LDL+control group sequence group;(7)ox-LDL + curcumin +control sequence group.The expression of ABCA1 mRNA was detected by RT-qPCR,the content of ABCA1 protein was detected by Western Blot,the cholesterol efflux rate was detected by fluorescence-labeling method,and the contents of IL-6,TNF-and MCP-1 were detected by ELISA kit.6 Effect of curcumin on NF-KB/miR33a pathwayExperimental group:(1)control group;(2)foam cell model group;ox-LDL+ Curcumin;(4)ox-LDL+NF-?B inhibitor PDTC group;The expression of miR33a mRNA was detected by RT-qPCR.The expression of transcription factor kappa B(NF-?B)in nuclear,NF-?B inhibitor alpha(I?B?)and phosphorylation of I?B?(p-I?B?)in cytoplasm was examined by Western Blot.7 Effect of curcumin on the expression of ABCA1,cholesterol efflux rate and the secretion of IL-6,TNF-? and MCP-1 through the NF-?B/miR33a pathwayExperimental groups:(1)control group;(2)foam cell model group;(3)ox-LDL+ curcumin group;(4)ox-LDL+ miR33a inhibitor group;(5)ox-LDL+PDTC group;(6)ox-LDL+ curcumin +miR33a inhibitor group;(7)ox-LDL+curcumin +PDTC group;(8)ox-LDL+ control sequence group;The expression of ABCA1 mRNA was detected by RT-Qpcr.The content of ABCA1 protein was detected by Western Blot.The cholesterol efflux rate was detected by fluorescence-labeling method,and the contents of IL-6,TNF-a and MCP-1 were detected by ELISA kit.8 Effect of curcumin on the expression of ABCA1,cholesterol efflux rate and the secretion of IL-6,TNF-a,MCP-1 through the TLR4/NF-KB/miR33a pathwayExperimental groups:(1)control group;(2)foam cell model group;(3)ox-LDL+ curcumin group;(4)ox-LDL+TLR4 antibody group;(5)ox-LDL+curcumin +TLR4 antibody group;(6)ox-LDL+TLR4 non-related isotype antibody group;(7)ox-LDL+ curcumin +TLR4 non-related isotype antibody group;The expression of ABCA1 and miR33a mRNA was detected by RT-qPCR.The content of ABCA1,NF-?B p65 in nucleus,I?B? and p-I?B? intracellular were detected by Western Blot.The cholesterol efflux rate was detected by fluorescence-labeling method,and the contents of IL-6,TNF-? and MCP-1 were detected by ELISA kit.Results:1 Test cell toxicity of curcuminCompared with the control group,curcumin with a concentration range of had no significant toxicity on cell activity(P>0.05),but curcumin at the concentration of r80?M resulted in a decrease of cell activity.,the difference was statistically significant(P<0.05).2 Curcumin Promoted the expression of ABCA1,inhibited the expression of miR33a,TLR4,IL-6,TNF-? and MCP-1,decreased the content of intracellular lipid and the formation of foam cells in THP-1 macrophageCompared with the control group,the intracellular lipid accumulation and foam cell formation rate was increased,CE:TC increased up to 65.09±1.30,the expression of miR33a,TLR4 was increase,the secretion of IL-6,TNF-a and MCP-1 increased,ABCA1 expression decreased in the ox-LDL group.Compared with ox-LDL group,the content of intracellular lipid decreased,the expression of miR33a and TLR4 reduced,the secretion of IL-6,TNF-? and MCP-1 declined and the expression of ABCA1 increased in the ox-LDL+curcumin group,the difference was statistically significant(P<0.05).3 The cholesterol efflux rate of THP-1 derived macrophages was positively correlated with the concentration and time of curcuminCompared with the foam cell model group,curcumin with concentration of 10-40 ?M concentration and the treatment time of 6-24 h can significantly increase the cholesterol efflux rate,and the efflux rate was positively correlated with the concentration and time of curcumin,the difference was statistically significant(P<0.05).However there was no significant difference between the ox-LDL+5 ?M curcumin group and the model group(P>0.05).Cholesterol efflux rate of 48h group was no significantly different with that of 24 h group(P>0.05).4 Detection of liposome transfectionCompared with the control group,the expression of miR3 3 a mRNA in foam cell model group was significantly increased,the difference was statistically significant(P<0.05).Compared with the foam cell model group,the expression of miR33a mRNA in ox-LDL+miR33a inhibitor group was significantly decreased,the difference was statistically significant(P<0.05),while there was no significant difference between the ox-LDL+control sequence group and model group(P>0.05).5 Curcumin increase the expression of ABCA1,cholesterol efflux rate and decrease the secretion of IL-6,TNF-? and MCP-1 by inhibiting the miR33aCompared with the control group,the expression of ABCAI in foam cell model group was significantly decreased,the secretion of IL-6,TNF-a,MCP-1 increased,the difference was statistically significant(P<0.05).Compared with the foam cell model group,the expression of ABCA1 and cholesterol efflux rate increased significantly,the secretion of IL-6,TNF-?,MCP-1 were reduced in the ox-LDL+ curcumin group and the ox-LDL+miR33a inhibitor group,the difference was statistically significant(P<0.05),while there was no significant difference between ox-LDL+control group and foam cell model group(P>0.05).6 Curcumin inhibits NF-?B/miR3 3 a pathwayCompared with the control group,the expression of miR33a was increased significantly,the expression of NF-?B p65 in nuclei and the p-I?B?in cytoplasm were increased,the expression of I?B? in cytoplasmic was decreased in foam cell model group,the difference was statistically significant(P<0.05).Compared with the foam cell model group,the expression of miR33a was decreased significantly,the expression of NF-?B p65 in nuclei and the p-I?B? in cytoplasm were decreased,the expression of I?B? in cytoplasmic was up regulated in ox-LDL+ curcumin group and ox-LDL+PDTC group,the difference was statistically significant(P<0.05).7 Curcumin increase the expression of ABCA1,cholesterol efflux rate and decrease the secretion of IL-6,TNF-a and MCP-1 by inhibiting the NF-?B/miR33a pathwayCompared with the control group,the expression of ABCA1 was significantly decreased,the secretion of IL-6,TNF-a,MCP-1 was increased in foam cell model group,the difference was statistically significant(P<0.05).Compared with the foam cell model group,the expression of ABCA1 and cholesterol efflux rate was increased significantly,the secretion of IL-6,TNF-?,MCP-1 was decreased in ox-LDL+ curcumin group,ox-LDL+PDTC group,ox-LDL+miR33a inhibitor group,the difference was statistically significant(P<0.05),while there was no significant difference between ox-LDL+control sequence group and foam cell model group(P>0.05).8 Curcumin increase the expression of ABCA1,cholesterol efflux rate and decrease the secretion of IL-6,TNF-? and MCP-1 by inhibiting the TLR4/NF-KB/miR33a pathwayCompared with the control group,the expression of miR33a was increased,the expression of NF-?B p65 in nuclei and the p-I?B? in cytoplasm were increased,the expression of I?B? in cytoplasmic was decreased,the expression of ABCA1 was decreased,the secretion of IL-6,TNF-a and MCP-1 were increased in foam cell model group,the difference was statistical significance(P<0.05).Compared with the foam cell model group,the expression of miR33a was decreased significantly,the expression of NF-?B p65 in nuclei and the p-I?B? in cytoplasm were decreased,the expression of I?B? in cytoplasmic was increased,the expression of ABCA1 was increased,the secretion of IL-6,TNF-a and MCP-1 were decreased in ox-LDL+curcumin group,ox-LDL+TLR4 antibody group,the difference was statistically significant(P<0.05),while there was no significant difference between ox-LDL+TLR4 non-related isotype antibody group and foam cell model group(P>0.05).Conclusions:1 In the concentration range of 0-40 ?M,curcumin had no toxicity on THP-1 derived macrophages2 The results showed effect of curcumin on cholesterol efflux rate in an appropriate concentration range(10-40 ?M)and time range(6-24h)in a concentration-dependent manner and time-dependent manner.3 TLR4/NF-kappa B/miR33a signaling pathway mediates the expression of cholesterol reverse transfer related protein ABCA1 and cholesterol efflux in THP-1 macrophages,and is involved in regulating the secretion of IL-6,TNF-and MCP-1.4 Curcumin increase the expression of cholesterol reverse transport related protein ABCA1 and decrease the secretion of IL-6,TNF-? and MCP-1 by TLR4/NF-?B/miR33a pathway in THP-1 derived macrophages. |
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