| Background and objectiveCadmium(Cd)is a heavy metal contaminan which accumulates in the body to cause liver,kidney,lung,bone,and digestive tract damage.In addition,studies have shown that Cd exposure increased the risk of cardiovascular disease by promoting the occurrence of atherosclerosis(AS).However,the mechanism of Cd-promoted AS is still unclear.Macrophage polarization to M1 phenotype plays an important role in the development of AS.And the activation of Janus protein tyrosine kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signal pathway can promote macrophage polarization to M1 phenotype.Cd-promote the development of AS may through activating JAK2/STAT3 signal pathway to promote macrophage polarization to the M1 phenotype.Curcumin,as a plant-active substance,has a protective effect on cardiovascular diseases,but no research has explored the role of curcumin in the occurrence of AS promoted by Cd.In this study,in vivo and in vitro experiments were used to establish the role of macrophage polarization in the progress of Cd-promoted AS,in which whether if Cd can promote macrophage polarization to M1 phenotype through activated JAK2/STAT3 pathway.What’s more,the protective effect and the mechanism of curcumin on Cd-promoted AS were also detected.Methods1.Study on the role of macrophage polarization in Cd induced AS(1)Dose-response relationship of Cd to ApoE-/-mouse ASApoE-/-mice were exposed to 0,50,100 and 200 mg/L cadmium chloride(CdCl2)through drinking water for 12 weeks.The cardiovascular tissues of mice were collected to observe the formation of aortic plaque by oil red O staining.Plasma was collected to detect TG,T-CHO,LDL and HDL.Enzyme-linked immunosorbent assay(ELISA)was used to measure the concentration of AS molecular marker VCAM-1.Determine the optimal concentration of Cd to promote the construction of AS model.(2)Regulation of Cd on macrophage polarization in ApoE-/-mouseApoE-/-mice were exposed with or without 100 mg/L CdCl2 through drinking water for 12 weeks.The aortic tissue,blood and urine were collected.The concentration of Cd in blood,urine and tissue was measured by ICP-MS.QRT-PCR was used to analysis the mRNA expression of F4/80,CD86,CD206,TNF-α,IL-6 and IL-4.The expression of CD86 and CD206 in aorta was analyzed by immunofluorescence.ELISA was used to detect the expression of TNF-α,IL-6 and IL-4 in plasma.To clarify the regulatory role of Cd exposure on macrophage polarization in the process of promoting AS.(3)Regulation of Cd on the polarization of RAW264.7RAW264.7 cells were treated with different concentrations(0,0.38,0.75,1.5,3.0,6.0,12.0,and 24.0 μmol/L)CdCl2 for 24 hours.The cell viability was determined by CCK-8 to determine the maximum concentration value of the pair without affecting the cell activity.RAW264.7 cells treated with 0,0.75,1.5,and 3.0 μmol/L CdCl2 for 24 hours.QRT-PCR was used to analyze the mRNA expression of CD86,CD206,TNF-α,IL-6,and IL-4.The fluorescence intensity of CD86 and CD206 was measured by flow cytometry.And the concentration of TNF-α,IL-6,and IL-4 in culture supernatant was measured by ELISA.2.Study on the mechanism of JAK2/STAT3 signal pathway in Cd induced macrophage polarization(1)Regulation of Cd on JAK2/STAT3 signal pathway in ApoE-/-mouseApoE-/-mice were exposed with or without 100 mg/L CdCl2 by drinking water for 12 weeks.The cardiovascular tissues were collected.The mRNA expression of JAK2 and STAT3 in aortic tissue was analyzed by QRT-PCR.The expression of JAK2/STAT3 in aortic tissue was analyzed by immunohistochemistry.To observe the regulatory effect of Cd exposure on the JAK2/STAT3 signal pathway expression in ApoE-/-mice.(2)Regulation of Cd on JAK2/STAT3 signal pathway in RAW264.7 cellsRAW264.7 cells were treated with 0,0.75,1.5,and 3.0 μmol/L CdCl2 for 24 hours.The mRNA expression of JAK2 and STAT3 were analyzed by QRT-PCR.Western-blot was used to detect the expression of JAK2,STAT3,p-JAK2,and p-STAT3.To clarify the regulatory effect of Cd exposure on the JAK2/STAT3 signal pathway expression in RAW264.7 cells.(3)Mechanism of JAK2/STAT3 signal pathway in Cd-induced polarization of RAW264.7 cellsRAW264.7 with or without pacritinib pretreatment for 30 minutes,then treated with or without 3.0 μmol/L CdCl2 for 24 hours.The expression of JAK2,STAT3,p-JAK2,and p-STAT3 were detected by western-blot.QRT-PCR was used to analyze the mRNA expression of CD86,CD206,JAK2,STAT3,TNF-α,IL-6,and IL-4.The fluorescence intensity of CD86 and CD206 was measured by flow cytometry.The concentration of TNF-α,IL-6 and IL-4 in culture supernatant were detected by ELISA.To clarify the role of JAK2/STAT3 signal pathway in macrophage polarization induced by Cd.3.The protective effect of curcumin on Cd-promoted AS(1)Protective effect of curcumin on Cd-promoted AS in ApoE-/-mouseApoE-/-mice were exposed with or without100 mg/L CdCl2 in drinking water,and gavage with 100 mg/kg curcumin.After 12 weeks,the cardiovascular tissue and plasma of mice were collected.The formation of aortic plaque was observed by oil red O staining and pathological examination.TG,T-CHO,LDL,and HDL in plasma were detected.The expression of AS molecular marker VCAM-1 was detected by ELISA.To clarify the protective effect of curcumin on AS promoted by Cd exposure.(2)Regulation of curcumin on macrophage polarization induced by CdApoE-/-mice were exposed with or without 100 mg/L CdCl2 in drinking water,and gavage with 100 mg/kg curcumin.After 12 weeks,the cardiovascular tissue,plasma,and urine of mice were collected.The concentration of Cd in blood,urine,and aorta tissue was measured by ICP-MS.QRT-PCR was used to analyze the mRNA expression of F4/80,CD86,CD206,TNF-α,IL-6,and IL-4.The expression of CD86 and CD206 was analyzed by immunofluorescence.The concentration of TNF-α,IL-6,and IL-4 in plasma was detected by ELISA.RAW264.7 cells were pretreated with curcumin for 30 minutes,then treated with or without 3.0 μmol/L CdCl2 for 24 hours.QRT-PCR was used to analyze the mRNA expression of CD86,CD206,TNF-α,IL-6,and IL-4.The fluorescence intensity of CD86 and CD206 was measured by flow cytometry.The concentration of TNF-α,IL-6,and IL-4 in culture supernatant was detected by ELISA.To clarify the regulatory effect of curcumin on macrophage polarization induced by Cd exposure.(3)Regulatory effect of curcumin on Cd-activated JAK2/STAT3 signal pathwayApoE-/-mice were exposed with or without 100 mg/L CdCl2 in drinking water,and gavage with 100 mg/kg curcumin for 12 weeks.The cardiovascular tissues of mice were collected.The mRNA expression of JAK2 and STAT3 was analyzed by QRT-PCR.The expression of JAK2/STAT3 was analyzed by immunohistochemistry.To observe the regulatory effect of curcumin on JAK2/STAT3 signal pathway activated by Cd exposure in ApoE-/-mice.RAW264.7 cells were pretreated with curcumin for 30 minutes,then treated with or without 3.0 μmol/L CdCl2 for 24 hours.The mRNA expression of JAK2 and STAT3 was analyzed by QRT-PCR.Western-blot was used to detect the expression of JAK2,STAT3,p-JAK2,and p-STAT3.To clarify the regulatory effect of curcumin on the JAK2/STAT3 signal pathway activated by Cd in RAW264.7 cells.Results1.Study on the role of macrophage polarization in Cd promoted AS(1)Cd promoted the AS of ApoE-/-mouse with a dose-response relationship50,100,and 200 mg/L CdCl2 can promote the formation of aortic root plaque in ApoE-/-mice,increase plasma TG,T-CHO,LDL,and VCAM-1 level,and inhibit HDL.100 mg/L CdCl2 was the most obvious dose.(2)Cd promotes macrophage polarization to M1 phenotype in ApoE-/-mouseThe Cd concentration in blood,urine,and aorta in 100 mg/L CdCl2 group was higher than control group.The mRNA and protein expression of CD86,TNF-α,and IL-6 in ApoE-/-mice was higher than control group.But there was no significant difference in the mRNA and protein expression of CD206 and IL-4.(3)Cd promoted the polarization of the RAW264.7 cell to M1 phenotype 3.0 μmol/L CdCl2 was the maximum concentration that had not affected RAW264.7 cell viability.0.75,1.5,and 3.0 μmol/L CdCl2 promoted the fluorescence intensity of CD86,the expression of TNF-α and IL-6.There was no significant effect on the expression of CD206 and IL-4.2.Study on the mechanism of JAK2/STAT3 in Cd-induced macrophage polarization(1)Cd promoted the expression of JAK2/STAT3 signal pathway in the aortic root of ApoE-/-miceThe expression of JAK2/STAT3 signal pathway in the aorta of ApoE-/-mice was more significant in 100 mg/L CdCl2 than control group,especially in plaques.(2)Cd activated JAK2/STAT3 signal pathway in RAW264.7 cellCompared with 0 μmol/L CdCl2 group,0.75,1.5,and 3.0 μmol/L CdCl2 groups significantly activate JAK2/STAT3 signal pathway in RAW264.7 cells.(3)Inhibition of JAK2/STAT3 signal pathway activation can antagonize Cd-induced macrophage polarization to M1 phenotypePretreatment with pacritinib decreased the expression of CD86,TNF-α,and IL-6 up-regulated by CdCl2.There was no significant differential expression of CD206 and IL-4.3.Protective effect of curcumin on cadmium induced atherosclerosis(1)Curcumin accelerated Cd-promoted ASCompared with CdCl2 group the formation of plaques in ApoE-/-mice was lower in the control group,the curcumin group,and the CdCl2+curcumin group.And the plasma VCAM-1 level was also lower in the control group,the curcumin group,and the CdCl2+curcumin group than CdCl2 group,but the plasma HDL was higher.The plasma TG,T-CHO,and LDL in the CdCl2 group and the CdCl2+curcumin group had no significant difference.(2)Curcumin inhibited Cd-induced macrophage polarization to M1 phenotypeThere was no significant difference in Cd concentration in blood,urine,and aorta of 100 mg/L CdCl2 group and CdCl2+curcumin group.But the expression of CD86,TNF-α,and IL-6 in ApoE-/-mice was reduced in control group,curcumin group,and CdCl2+curcumin group.There was no significant difference between CD206 and IL-4 in all groups.And in RAW264.7 cells,the expression of CD86,TNF-α,and IL-6 were reduced in control group,curcumin group,and CdCl2+curcumin group compared with CdCl2 group,while the expression of CD206 was up-regulated.There was no significant difference in IL-4 in all groups.(3)curcumin inhibited the Cd-activated JAK2/STAT3 pathwayCompared with 100 mg/L CdCl2 group,the expression of JAK2/STAT3 signal pathway in the control group,the curcumin group,and the CdCl2+curcumin group was reduced.And the expression of JAK2/STAT3 signal pathway in RAW264.7 cells in control group,curcumin group,and CdCl2+curcumin group was down-regulated which compared with CdCl2 group.Conclusions1.Cd exposure can promote the occurrence of AS in ApoE-/-mice with a dose-response relationship.100 mg/L CdCl2 is the most significant dose.2.Cd can activate macrophage JAK2/STAT3 pathway to promote macrophage polarization to M1 phenotype,which finally promotes the development AS of ApoE-/mouse.3.Curcumin inhibits the macrophage polarization to M1 phenotype which is induced by JAK2/STAT3 pathway to alleviate the Cd-promoted AS in ApoE-/-mouse. |
PDF Full Text Request