The Effects Of BAMBI On Ox-LDL-induced Vascular Endothelial Cell Injury And Mechanism Research

Purpose:To investigate the effects of bmp and activin membrane-bound inhibitor(BAMBI)on the oxidized low density lipoprotein(ox-LDL)-induced injury in human umbilical vein endothelial cells(HUVECs)and its molecular mechanisms.Method:1.The role of BAMBI in ox-LDL-induced endothelial cell injuryHUVECs were divided into the following six groups:control group;ox-LDL group;BAMBI + ox-LDL group;pcDNA3.1 + ox-LDL group;si-BAMBI + ox-LDL group;si-control + ox-LDL group The MTT assay was used to detect cell proliferation.Flow cytometry was used to detect apoptosis.RT-PCR was used to detect mRNA expression.Western blot was used to detect the protein expression level.Flow cytometry was used to detect ROS levels.The kits were used to detect nitrite and cGMP levels.2.BAMBI plays a biological role by regulating TGF-?/Smad pathwayHUVECs were divided into the following six groups:control group;ox-LDL group;BAMBI + ox-LDL group;TGF-? + BAMBI + ox-LDL group;si-BAMBI +ox-LDL group;TGF--BAMBI + ox-LDL group.Detection methodswere the same with the method 1.3.Ox-LDL/TLR4/NF-?B signaling pathway regulates B AMBI/TGF-? pathway HUVECs were divided into the following eight groups:control group;ox-LDL group;BAMBI + ox-LDL group;CLI-095 + BAMBI + ox-LDL group;PDTC +BAMBI + ox-LDL group;si-BAMBI + LDL group;CLI-095 + si-BAMBI +ox-LDL group;PDTC + si-BAMBI + ox-LDL group.Detection methodswere the same with the method 1.4.MiR-373 regulates the expression of BAMBI in ox-LDL-induced HUVECsHUVECs were divided into the following six groups:control group,ox-LDL group,mimic + ox-LDL group,mimic + BAMBI + ox-LDL group,inhibitor +ox-LDL group,inhibitor + si-BAMBI + ox-LDL group The dual luciferase reporter assay kit was used to detect the targeting relationship between miR-373 and BAMBI.Detection methodswere the same with the method 1.Result:1.The role of BAMBI in ox-LDL-induced endothelial cell injuryThe results showed that BAMBI was down-regulated in ox-LDL-induced HUVECs.In ox-LDL-induced HUVECs,were,and of BAMBI overexpression promoted cell proliferation,reduced cell apoptosis,down-regulated the expression of inflammatory factors(IL-1?,IL-6 and TNF-a)and adhesion molecules(E-selectin,VCAM1 and ICAM1),inhibited oxidative stress by reducing ROS and MDA levels,while increasing the expression of SOD,and increasedthe production of NO and eNOS.The effectsofBAMBIinhibition in ox-LDL-induced HUVECs were opposite with that of BAMBI overexpression,including reducing cellproliferation,increasing cell apoptosis,upregulatingthe expression of IL-1?,IL-6,TNF-a,E-selectin,VC AM1 and IC AM1,promoting oxidative stress and reducing the production of NO and eNOS.2.BAMBI plays a biological role by regulating TGF-?/Smad pathwayThe results showed that the expression levels of TGF-?,p-Smad2 and p-Smad3 were up-regulated in ox-LDL-induced HUVECs.Overexpression of TGF-?partially reversed the protective effect of BAMBI overexpression aganist ox-LDL-induced endothelial cell injury,thereby inhibiting cell proliferation,increasing cell apoptosis,upregulating the expression of inflammatory factors and adhesion molecules,promoting oxidative stress and reducing the production of NO and eNOS.In contrast,overexpression of TGF-? aggravated the promoting effects of BAMBI inhibiton to ox-LDL-induced endothelial cell injury.3.Ox-LDL/TLR4/NF-?B signaling pathway regulates BAMBI/TGF-? pathwayThe results showed that the protein expression levels of TLR4,p-I?B-a and p-p65 were up-regulated in ox-LDL-induced HUVECs.The pretreatment of TLR4 inhibitor CLI-095 or NF-?B inhibitor PDTC up-regulated the expression of BAMBI,and enhanced the protective effect of BAMBI overexpression aganist ox-LDL-induced endothelial cell injury,partially reversedthe promoting effects of BAMBI inhibiton to ox-LDL-induced endothelial cell injury.4.MiR-373 regulates the expression of BAMBI in ox-LDL-induced HUVECsThe results showed that miR-373 was up-regulated in ox-LDL-induced HUVECs and targeted BAMBI mRNA 3'-UTR to regulate its expression.MiR-373 played a biological role by regulating BAMBI-mediated TGF-?/Smad signaling pathway.Inhibit the expression of miR-373 resisted ox-LDL-induced endothelial cell injury,including promoting cell proliferation,reducingcell apoptosis,down-regulating the expression of inflammatory factors and adhesion molecules,inhibiting oxidative stress and increasingthe production of NO and eNOS.Conclusion:In conclusion,the results of this study showed that in the ox-LDL-induced HUVECs,BAMBI expression was down-regulated and its downstream TGF-?/Smad pathway was activated.Overexpression of BAMBI inhibited TGF-?/Smad pathway,promoted cell proliferation,reduced apoptosis,down-regulated the expression of inflammatory factors and adhesion molecules,inhibited oxidative stress and increasedthe production ofNO and eNOS,and thus resistance to endothelial cell injury.Inhibition of TLR4/NF-?B signaling up-regulated the expression of BAMBI,suggesting that TLR4/NF-?B/BAMBI pathway plays an important role in endothelial cell injury.In addition,BAMBI was a target gene for miR-373,and miR-373 inhibitionprotected HUVECS againstendothelial cell injuryby upregulating BAMBI.Our study revealed the regulatory role of TLR4/NF-?kB(or miR-373)/BAMBI/TGF-?/Smad signaling in endothelial injury,further elucidating the mechanisms of endothelial injury,providing a theoretical basis for the prevention and treatment ofcardiovascular diseases that related with endothelial injury.