Silent TUFM regulates mitochondrial autophagy through the AMPK/mTOR signaling pathway and its effect on pul-monary arterial hypertension in a pulmonary heart disease model rat
Cui Benke
Wang Yan
Lu Yunfeng
Du Juan
Zhai Yuhan
Abstract：Objective To explore the mechanism by which mitochondrial translation elongation factor Tu(TUFM)promotes vascular remodeling in pulmonary arterial hypertension(PAH)through mitochondrial autophagy.Methods The experiment was conducted in the Central Laboratory of Liaoning Provincial People's Hospital from January 2022 to June 2023.Thirty-six healthy male Sprague Dawley rats were randomly divided into a blank control(Ctrl)group,a model(PAH)group,a TUFM overexpression(OE)group,an OE negative control(OE-NC)group,a short hairpin RNA(Sh)knockout TUFM(Sh)group,and a Sh-NC negative control(Sh-NC)group,with 6 rats in each group.Except for the Ctrl group,all other rats were given a one-time intraperitoneal injection of 1%monocrotaline(60 mg/kg)to induce cardiogenic pulmonary edema(PAH)in a rat model.Rat pulmonary artery smooth muscle cells(PASMC)were cultured under low oxygen(3%O2)conditions for24 hours to simulate the in vivo pulmonary arterial hypertension microenvironment.They were divided into normoxic(Norm)group,hypoxic(Hyp)group,small interfering RNA(SiRNA-1)group,SiRNA-2 group,Si-NC group,OE-NC group,and OE group.Right heart catheterization and pulsed Doppler ultrasound were used to detect pulmonary hemodynamics in rats.He-matoxylin-eosin staining was used to detect the pathological structure of pulmonary arterioles.Immunofluorescence co stai-ning was used to detect tissue localization of TUFM.Cell counting method is used to detect cell proliferation.Observation of mitochondrial structure and autophagosomes using transmission electron microscopy.Protein immunoblotting was used to detect the expression of TUFM,autophagy,apoptosis,and adenosine phosphate activated protein kinase(AMPK)/mam-malian rapamycin target protein(mTOR)pathway related proteins.Results Compared with the Ctrl group,the expression of TUFM protein in the PAH group rats increased and was mainly associated with PASMC markers α smooth muscle actin(α-SMA)is co localized in the intima of pulmonary arterioles,but not with endothelial cell marker CD31.The pulmonary artery systolic pressure(PASP)increases,the pulmonary artery acceleration time(PAAT)shortens,the distal pulmonary arteriole wall shows concentric thickening,and the lumen is almost blocked.The expression of TUFM,benzyl chloride 1 recombinant protein(BECN1),human microtubule associated protein light chain 3(LC3)II/I,and B lymphocyte tumor 2(Bcl2)proteins in-creases,while the expression of P62,Bcl2 related X protein(Bax),and apoptosis activating factor(Apaf)proteins decreases(P＜0.05).Compared with the PAH group,the OEgroup showed an increase in PASP,a decrease in PAAT,an increase in pulmonary artery wall thickness,an increase in pulmonary artery TUFM,BECN1,LC3II/I,and Bcl2 expression,and a de-crease in P62,Bax,and Apaf expression(P＜0.05).Compared with the PAH group,the Sh group showed a decrease in PASP,an increase in PAAT,an improvement in pulmonary artery wall thickness and luminal stenosis,a decrease in TUFM,BECN1,LC3II/I,and Bcl2 expression,and an increase in P62,Bax,and Apaf expression(P＜0.05).Compared with the Norm group,the Hyp group showed an increase in TUFM protein expression in PASMC cells.Compared with the Si-NC group cells,the SiRNA-1 and SiRNA-2 groups showed increased expression of P62 and Bax proteins,decreased expression of BECN1,LC3II/I,Bcl2,and TUFM,intact mitochondrial structure,decreased proliferation activity of PASMC cells,decreased expression of p-AMPK,and increased expression of p-mTOR(P＜0.05).Compared with the OE-NC group,the expression of P62 and Baxproteins was reduced in the OE group,while the expression of BECN1,LC3II/I,Bcl2,and TUFM was increased.Some mitochondria were damaged and collapsed,and cristae rupture disappeared.The proliferation activity of PASMC cells was significantly increased,and the expression of p-AMPK was increased and p-mTOR was decreased in cells(P＜0.05).Conclusion Silencing TUFM can promote mitochondrial autophagy and accelerate apoptosis of PAH pulmonary artery smooth muscle cells by activating the AMPK/mTOR signaling pathway.
Keywords：Pulmonary arterial hypertensionMitochondrial translation elongation factor TuSmooth muscle cellsMitochondrial autophagyAMPK/mTOR pathwayRats
Publication Date：2024-04-18
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：9( 478-486 )