| Objective: To investigate the effects of icariin(ICA)on pulmonary artery remodeling of pulmonary arterial hypertension in monocrotaline-treated CX3CL1-/-mice and wild-type(WT)mice,and to analyze the relationship between anti-PAH effects of ICA and CX3CL1.Methods: There were 28 male WT and CX3CL1-/-mice,they were randomly divided into control group,model group,ICA treatment group(60,120 mg/kg).All mice were injected with MCT(70 mg/kg)or equel volume dd H2 O subcutaneously to establish PAH model.After 7 days MCT treatment,ICA or dd H2 O were given by intragastric administration according to groups for 21 days.FS,EF,RVID;d,LV Mass,LV Vol;d,LV Vol;s and Stroke Volume of CX3CL1-/-and WT mice were measured by B-ultrasound.Mortality was calculated.Right ventricular weight,Left ventricular + ventricular septal weight and calculate the right ventricular hypertrophy index(RVHI=RV/LV+S).The morphological change of the pulmonary artery was observed by HE staining.Immunofluorescence method was used to observe the infiltration of pulmonary arterial by inflammatory cells.Results: CX3CL1-/-mice: Compared with the control group,FS,EF,Stroke Volume decreased(P<0.01 or P<0.05).RVID;d,LV Mass,LV Vol;s significantly increased(P<0.01 or P<0.05),LV Vol;d did't been significantly changed.RVHI increased(P<0.05),and the mortality rate was 12.5% in the model group.Pulmonary artery thickened and inflammatory cells infiltrated in pulmonary arterial in the model group.Compared with the model group,EF was significantly increased(P<0.01),RVID;d,LV Mass and LV Vol;s decreased significantly(P<0.01 or P<0.05),LV Vol;d,Stroke Volume and RVHI had no significant changes in ICA treatment group.The mortality rate was 12.5% and 0 in ICA(60,120 mg/kg)respectively.Pulmonary arterythickening was reduced and there are few inflammatory cells infiltrated in pulmonary artery in ICA treatment group.WT mice: Compared with the control group,FS,EF were significantly decreased(P<0.01),RVID;d,LV Mass,LV Vol;d,LV Vol;s and Stroke Volume significantly increased(P<0.01);RVHI significantly increased(P<0.01),the mortality rate was42.82%,Pulmonary arterial wall was thickened,luminal stenosis and a large number of inflammatory cell infiltration in the model group.Compared with the model group,FS,EF,Stroke volume were significantly increased(P<0.01 or P<0.05),RVID;d,LV Mass,LV Vol;s decreased significantly(P<0.01 or P<0.05),LV Vol;d had no significant changes in ICA treatment group.RVHI was decreased(P<0.05).The mortality rate was 28.57% in ICA 60 mg/kg and 14.29% in ICA 120 mg/kg.Pulmonary artery thickening was reduced and there are few inflammatory cells infiltrated in pulmonary artery in ICA treatment group.Comparison of CX3CL1-/-mice with WT mice:The levels of FS,EF,LV Vol;d,Stroke Volume in CX3CL1-/-mice were higher than those in WT mice.The levels of RVID;d,LV Mass,LV Vol;s in CX3CL1-/-mice were lower than WT mice,of which FS?EF?LV Vol;d?Stroke Volume(P<0.01 or P<0.05).Pulmonary arterial wall thickness,lumen size and inflammatory cells in CX3CL1-/-mice were lower than WT mice.In the model group,the levels of FS,EF,LV Vol;d,LV Vol;s and Stroke Volume were higher than those of WT mice.RVID;d and LV Mass were lower than those of WT mice,of which Stroke Volume(P<0.05)in ICA 60 mg/kg and LV Mass(P<0.05)in ICA 120 mg/kg.Pulmonary arterial wall thickness,lumen size and inflammatory cells in CX3CL1-/-mice were lower than WT mice.The effects of ICA120 mg/kg was higher than that of 60 mg/kg.Conclusion: ICA can attenuate the hemodynamics and pulmonary vascular remodeling of MCT-induced PAH model mice.The effect to improve the PAH model of CX3CL1-/-mice better than WT mice.Objective: To investigate the effects of icariin(ICA)on migration and adhesion of human acute monocytic leukemia cell line(THP-1)to human umbilical vein endothelial cells(HUVECs)induced by tumor necrosis factor(TNF-?),and explore its mechanisms.Methods:(1)Immunofluorescence labeling technology was used to evaluate the protein expression of CX3CL1/CX3CR1 in THP-1 or HUVECs.(2)THP-1 and HUVECs were planted respectively in upper chamber and lower chamber of transwells in vitro.The cells were divided into six groups: control group,model group(TNF-? 10 ng/m L),ICA(10-8 mol/L ? 10-7 mol/L ?10-6 mol/L)and solvent group(DMSO,0.1%).The number of THP-1 migrate and adhere to HUVECs induced by TNF-? and observe the effect of ICA(10-8,10-7,10-6 mol/L)on the model.After 4 h of ICA administration,TNF-?(10 ng/m L)was added to co-culture system for 24 h.The upper champer of transwells was moved and medium were collected for cell count by cell counter.The adherent cells were captured using immunofluorescence double labeling.The concentrations of secreted soluble CX3CL1(s CX3CL1)in the supernatants and membrane CX3CL1(m CX3CL1)in HUVECs were measured by ELISA kit.Western blot technology was used to evaluate the protein level of CX3CL1 in HUVECs?CX3CR1 in THP-1?I?B-? and P-NF-?B(p65).(3)We select the NF-?B inhibitor pyrrolidine dithiocarbamate(PDTC)as a positive control to analysis the effect of ICA on the inhibition of THP-1 migration adhesion and NF-?B /CX3CL1 signaling pathway.The cells cultured by transwell were divided into 4groups: control group,model group(TNF-? 10 ng/m L),PDTC(50 ?mol/L)group,ICA(10-6 mol/L)group.After 4 h of ICA and PDTC administration,TNF-?(10ng/m L)was added to co-culture system for 24 h.The number of migration and adhesion of THP-1,s CX3CL1 and m CX3CL1 protein detection method as above.Immunofluorescence labeling technology was used to detect NF-?B nuclear transcription.Western blot technology was used to evaluate the protein level of I?B-??P-NF-?B(p65).(4)To further confirm the relationship between ICA inhibits THP-1 migration adhesion and CX3CL1.THP-1 and HUVECs were planted by transwells.The number of THP-1 migrate and adhere to HUVECs induced by CX3CL1(50 ng/m L)and observe the effect of ICA(10-8,10-7,10-6 mol/L)on the model.After 4 h of ICA administration,CX3CL1(50 ng/m L)was added to co-culture system for 24 h.The detection methods of THP-1 migrate and adhere to HUVECs as above.Western blot technology was used to evaluate the protein level of CX3CR1?p-NF-?B p65 and TNF-?.Result: CX3CL1 and CX3CR1 protein expression in HUVECs and THP-1.Compared with the control group,the number of migration and adhesion of THP-1 to HUVECs was significantly increased induced by TNF-?(P < 0.01),and increased protein levels of s CX3CL1,m CX3CL1,CX3CL1 and CX3CR1,as well as activating NF-?B.ICA(10-8?10-7?10-6 mol/L)could cut down the number of migration and adhesion of THP-1 to HUVECs(P<0.05 or P<0.01),and inhibite the protein levels of s CX3CL1,m CX3CL1,CX3CL1 and CX3CR1(P<0.01 or P<0.05),and inhibite the phosphorylation of NF-?B p65 and blocking the degradation of I?B protein(P<0.01 or P<0.05).We found that PDTC has the same function with ICA,the effect of ICA slightly higher than PDTC.Compared with the control group,the number of migration and adhesion of THP-1 to HUVECs induced by CX3CL1 was significantly increased(P < 0.01),and increased protein levels of CX3CR1,p-NF-?B p65 and TNF-?(P<0.05).Pretreatment with ICA(10-8?10-7?10-6 mol/L)could cut down the number of migration and adhesion of THP-1 to HUVECs(P<0.05 or P<0.01),and inhibite the protein levels of CX3CR1,p-NF-?B p65 and TNF-?(P<0.05 or P<0.01).Conclusions:ICA(10-8 ~ 10-6 mol/L)inhibit the migration and adhesion of THP-1 to HUVECs induced by TNF-?/CX3CL1 through inhibition of NF-?B/CX3CL1 signaling pathway. |
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