The Effect And Mechanism Of High Mobility Group Protein 1 (HMGB1) And Curcumin On Endothelial-mesenchymal Transition

Background Heart failure(HF)is a major public health issue,cardiac fibrosis is the common pathology of end-stage heart failure.Myocardial fibrosis has become an important target for treatment of heart failure.Abnormal Endothelial-mesenchymal transition(EndMT)is one of the key mechanisms of myocardial fibrosis.Therefore,it is necessary to study the mechanism of EndMT and explore the effective drugs that can delay and even reverse the development of EndMT,and to provide potential treatment of heart failure.Lipopolysaccharides(LPS)can contribute to EndMT-induced fibrosis through activation of TLR4.High mobility group protein 1(HMGB1)is also an inflammatory ligand of TLR4,and HMGB1 has been shown to induce pulmonary fibrosis through TGF-?1/Smad2/3 pathway.Although there is no direct evidence that whether HMGB1 can induce EndMT through TGF-?1/Smad2/3 pathway,EndMT is a special type of EMT.Therefore,HMGB1,similar to LPS,which is also a TLR4 inflammatory ligand,may induce EndMT through TLR4/NF-?B/TGF-?1/Smad2/3 signaling to lead to myocardial fibrosis.Studies have shown that Curcumin has anti-fibrosis effects.However,whether Curcumin inhibits myocardial fibrosis by inhibiting EndMT,and the mechanism of Curcumin inhibiting EndMT is unclear.Methods This study is divided into there parts:(1)to determine the available stimulating concentration of major drugs,such as Curcumin,Ang ?,HMGB1.(2)To investigate the effects of Curcumin(Cur)on angiotensin(Ang)?-induced EndMT and the mechanisms underlying the anti-fibrotic effect of Cur in Human umbilical vein endothelial cells:four groups:a normal control group,Ang ? group,Cur-treated group,and Losartan-treated group.The morphological changes of each group were recorded by microscope;Cell viability of each group was measured by CCK8 assay;the migration ability of groups was tested by Scratch-Wound assay;Western blot method was used to test the expression of VE-cadherin,a-SMA,and TGF-?1.(3)To investigate whether HMGB1 can induce EndMT in endothelial cells,and whether HMGB1 induce EndMT by activating TLR4/TGF-?1 signaling pathway,and whether Curcumin can inhibit HMGB1-induced EndMT and its mechanism;divided into following groups:control group,HMGB1 group,CLI-095 +HMGB1 group,CLI-095 group,Curcumin + HMGB1 group.The morphological changes of the cells were observed under microscope.Scratch test was used to detect the migration of each group.The mRNA expression of VE-cadherin,a-SMA,TGF-?1 and collagen ? were detected by RT-QPCR.The expression of VE-cadherin,a-SMA,TLR4,total NF-?B,Phospho-NF-?B p65,TGF-?1,Smad2/3,Phospho-Smad2/3 and collagen ? were detected by Western blot.VE-cadherin and a-SMA were detected in each group by immunofluorescence.Results Part 1:The final stimulating concentration of Curcumin is12.50?M,Ang ? is 1?M and HMGB1 is 800ng/ml.Part 2:Cellular morphology was significantly different after Ang? treatment,showing a more spindle-shaped fibroblast phenotype.However,Cur and Losartan blocked the effect of Ang?treatment and the morphology of the cells was similar to that of untreated control cells.CCK8 showed that Ang? dose-dependently inhibited HUVECs viability,The viability of each group were greater than 50%(P<0.05).Scratch-Wound assay suggested that Ang? significantly promoted migration ability(P=0.044 for 0.1?mol/L Ang ? group,P<0.01 for 1?mol/L Ang II group),and the effects were reversed by Cur and Losartan(both P<0.05).Pretreatment of HUVECs with Cur and Losartan inhibited Ang?-induced expression of a-SMA,TGF-?1,and increased the expression of VE-cadherin,and the effects of Cur and Losartan on expression of a-SMA,TGF-?1,and VE-cadherin had no significant difference(P>0.05).Part 3:Cellular morphology was different after HMGB1 treatment,showing a more spindle-shaped fibroblast phenotype.However,Cur and CLI-095 blocked the effect of HMGB1 treatment and the morphology of the cells was similar to that of untreated control cells,showing "cobblestone" morphology.Scratch-Wound assay suggested that HMGB1 significantly promoted migration ability(P<0.05),and the effects were reversed by Cur and CLI-095(P<0.05).Compared with the control group,HMGB1 down-regulated the expression both of VE-cadherin mRNA and protein(P<0.001),the expression of interstitial cell marker a-SMA mRNA and protein was significantly increased induced by HMGB1(P<0.001),while the expression of Phospho-NF-?B p65,TGF-?1,Phospho-Smad2/3 and collagen ? mRNA and protein all was significantly up-regulated(P<0.05).However,pretreatment of HUVECs with Cur and CLI-095 inhibited HMGB1-induced expression of both mRNA and protein ofa-SMA,TGF-?1,collagen ?,and increased the mRNA and protein expression of VE-cadherin(P<0.05),and down-regulated the protein expression of the effects of Phospho-NF-?B p65,TGF-?1,and Phospho-Smad2/3.Immunofluorescence showed that the expression of VE-cadherin protein(red)decreased and the expression of a-SMA protein(green)increased in HMGB 1 group compared with control group.VE-cadherin(red)fluorescence in Curcumin and CLI-095 group increased,while the a-SMA green fluorescence was not obvious.Conclusions1.Curcumin inhibit Ang ?-induced aberrant EndMT by down-regulating TGF-?1 expression,and the anti-EndMT effect of Cur and losartan has no significant difference.2.HMGB 1 can induce aberrant EndMT through activation of TLR4/NF-?B/TGF-?1/Smad2/3 signaling pathway,and heighten the secretion of collagen in endothelial cells.3.Curcumin and CLI-095 can inhibit HMGB1-induced EndMT by down-regulating TLR4/NF-?B/TGF-?1/Smad2/3 signaling pathway and reduce the secretion of collagen.