Role of transcription factor Bmal1-mediated mitochondrial membrane permeability disturbance in the oxidative stress response of smooth muscle cells mediated by acute hypoxia

HE Siyuan; GE Yiling; XU Jin; WANG Yumeng; BU Yingrui; LI Peijie; XIE Manjiang

doi:10.13276/j.issn.2097-1656.2026.01.016

Abstract：Objective To investigate the effects of brain and muscle arnt-like 1(Bmal1)on mitochondrial membrane permeability and oxidative stress in vascular smooth muscle cells under acute hypoxic conditions.Methods In this study,an acute hypoxic environment was simulated by exposing pulmonary artery smooth muscle cells(PASMCs)to 10 mL/L low oxygen conditions.The cells were divided into control group,hypoxia group for 6 h,hypoxia group for 12 h,and hypoxia group for 18 h to observe intracellular mitochondrial oxidative stress[expression levels of reactive oxygen species(ROS),malondialdehyde(MDA),and glutathione(GSH)],mitochondrial membrane permeability[mitochondrial membrane potential(MMP)and the degree of mitochondrial permeability transition pore(mPTP)opening],and the protein expression of transcription factor Bmal1.In addition,lentiviral vectors were used to overexpress and knockdown Bmal1,and PASMCs were divided into normoxic control group,normoxic overexpression group,normoxic knockdown group,hypoxic control group,hypoxic overexpression group,and hypoxic knockdown group to observe the changes in mitochondrial membrane permeability and oxidative stress under acute hypoxic conditions.To investigate whether Bmal1 modulates oxidative stress levels through the regulation of mPTP,the mPTP inhibitor cyclosporin A(CsA)was used to divide PASMCs into normoxic group,normoxic+CsA group,hypoxic group,hypoxic+CsA group,hypoxic+overexpression group,and hypoxic+overexpression+CsA group to observe the changes of mPTP and ROS.Results Compared with the control group,acute hypoxia induced a time-dependent enhancement of oxidative stress in PASMCs,as evidenced by ROS accumulation and a continuous increase in MDA levels observed at 6,12,and 18 h of hypoxia(P<0.01).Concurrently,the GSH levels decreased progressively(P<0.01).Both the reduction in MMP and the opening of mPTP also exhibited a clear time-dependent pattern(P<0.01).In comparison to the control group,Bmal1 protein expression was elevated at 6,12,and 18 h of hypoxia(P<0.01).Furthermore,overexpression of Bmal1 in PASMCs under hypoxic conditions using adenovirus resulted in enhanced ROS accumulation and mPTP opening(P<0.01).However,treatment with CsA mitigated the increased ROS accumulation(P<0.01)and reduced mPTP opening(P<0.01)induced by Bmal1 overexpression.Conclusion Bmal1 is capable of sensing hypoxic signals and,in turn,of mediating hypoxia-induced oxidative stress in vascular smooth muscle by modulating mitochondrial membrane permeability.