Role Of Autophagy In κ-opioid Receptor Activation Against Hypoxic Pulmonary Hypertension And Its Underlying Mechanisms

Background:Pulmonary hypertension（PH）is a common clinical pathological process and a clinical complication of many heart and lung diseases.Hypoxic pulmonary hypertension（HPH）is an important type of PH.The basic features of HPH are the vasocontraction and remodeling of the pulmonary artery,resulting in right heart failure and death^[1-3],but its pathogenesis remains unclear.Previous studies have demonstrated that the proliferation of pulmonary artery smooth muscle cells（PASMCs）is the key factor for the occurrence of pulmonary vascular remodeling^[4-6].Therefore,inhibition of PASMC proliferation and vascular remodeling under hypoxia is one of the most important measures to prevent HPH.κ-opioid receptor（κ-OR）is widely present in the cardiovascular system^[7].In our previous studiy,we have demonstrated that the selectiveκ-OR agonist U50,488H protects against myocardial ischemia-reperfusion injury by activatingκ-OR.In addition,κ-OR is also expressed on pulmonary artery and its expression is increased under hypoxia^[8].We further showed thatκ-OR stimulation plays a role in anti-HPH,but its specific mechanisms are not clear.Autophagy is a cell-adapted degradation process that plays an important role in both physiological and pathological conditions.On the one hand,autophagy eliminates toxic substances,pathogens,and damaged organelles inside cells,thereby maintaining the normal metabolic balance of cells and the homeostasis of the internal environment;on the other hand,external stimuli may cause excessive autophagy and cell damage,which in turn leads to the occurrence of certain diseases.Recent studies showed that the expression of LC3B,an autophagy protein marker,is elevated in the lungs of PH patients and HPH mice,indicating that autophagy may be closely related to the occurrence of HPH.Subsequent studies further demonstrated that chloroquine,an autophagy inhibitor,may have anti-HPH effect.Is the mechanism of anti-HPH afterκ-OR stimulation related to autophagy?We further searched the literature and it is showed thatμ-OR stimulation could inhibit autophagy induced by lipopolysaccharide in bone marrow macrophages^[9],but it is still unclear whetherκ-OR stimulation modulates autophagy in PASMCs.To this end,we used in vivo and in vitro models of HPH to investigate the role and mechanism of PASMC autophagy in the activation ofκ-OR against HPH.Objectives:（1）To investigate the role ofκ-OR activation in anti-HPH.（2）To investigate the role of autophagy in HPH.（3）To investigate the role of autophagy inκ-OR activation against HPH and its underlying mechanisms.Methods:（1）SD male rats were placed in a hypobaric hypoxia chamber（air pressure 50 kpa,oxygen concentration 10%）,and the hypoxia time was 8 hours per day for 3 consecutive weeks.After hypoxia,the effects of U50,488H,a selectiveκ-OR agonist,nor-BNI,a selectiveκ-OR antagonist,and chloroquine,an autophagy inhibitor on the mean pulmonary arterial pressure（mPAP）and the right ventricular pressure（RVP）on hemodynamics of HPH rats were measured.（2）HE staining was used to detect the effects of U50,488H,nor-BNI and chloroquine on pulmonary vascular remodeling in HPH rats.（3）Primary and subculture of PASMCs were used.HPH cell model was established in a hypoxic incubator（93%N₂,5%CO₂,2%O₂,37°C）for 24 h.（4）CCK-8 was used to detect the effects of U50,488H,nor-BNI and chloroquine on PASMC proliferation under hypoxia.（5）The effects of U50,488H,nor-BNI and chloroquine on the apoptosis of PASMCs under hypoxia were detected by flow cytometry.（6）Western blotting analysis detected the expression ofβ-actin,LC3B,p62,Beclin-1,caspase 3,p-mTOR,mTOR,p-AMPK,and AMPK proteins in different tissues and cells.（7）Autophagic process of PASMCs was detected by mRFP-GFP-LC3 double-labeled adenovirus transfection.Results:I.Role ofκ-OR activation in anti-HPH（1）After 3 weeks of chronic hypoxia,the mPAP and RVP in rats were increased significantly.The thickness of pulmonary artery wall was increased,and the lumen was decreased.At the cellular level,hypoxia for 24 h significantly induced the proliferation of PASMCs,indicating that the HPH models of animal and cell are established successfully.（2）U50,488H,a selectiveκ-OR agonist,reversed the increase in mPAP and RVP,and inhibited the pulmonary artery remodeling under hypoxia by activatingκ-OR.The effects of U50,488H were abolished by nor-BNI,a selectiveκ-OR antagonist.（3）U50,488H inhibited PASMC proliferation and promoted its apoptosis in a dose-dependent manner under hypoxic conditions.These effects were blocked by nor-BNI.II.Role of autophagy in HPH（1）Rats developed severe HPH after chronic hypoxia for 3 weeks,and chloroquine,an autophagy inhibitor reduced the mPAP and RVP of HPH rats and inhibited the pulmonary vascular remodeling in HPH rats.（2）At the cellular level,chloroquine,an autophagy inhibitor not only inhibited PASMC proliferation,but also promoted its apoptosis induced by hypoxia.III.Role of autophagy inκ-OR activation against HPH and its underlying mechanisms（1）After 3 weeks of hypoxia,the expressions of LC3B,Beclin-1 and p62 were monitored.Compared with the normal group,the ratio of LC3B-II/-I and Beclin-1 expression were significantly increased and p62 expression was decreased in the pulmonary arteries of hypoxic rats,indicating that hypoxia could induce an increase in autophagy in HPH rats.（2）Compared with the hypoxic group,pre-administration of U50,488H significantly decreased the ratio of LC3B-II/-I and Beclin-1 expression and increased the p62expression of pulmonary arteries of HPH rats,suggesting that U50,488H inhibits autophagy in HPH rats.（3）At the cellular level,hypoxia induced autophagy in PASMCs.Western blotting and mRFP-GFP-LC3 puncta formation assays indicated that U50,488H inhibits autophagy in a dose-dependent manner in PASMCs under hypoxia,and this effect is blocked by nor-BNI.（4）U50,488H inhibited PASMC proliferation and induced PASMC apoptosis by inhibiting autophagy.（5）Hypoxia increased AMPK phosphorylation and mTOR dephosphorylation in PASMCs,whereas U50,488H reversed hypoxia-induced changes,and these effects were abolished by nor-BNI.（6）AICAR,an AMPK selective agonist and rapamycin,a mTOR selective blocker attenuated the inhibitory effect of U50,488H on autophagy in PASMCs under hypoxia,respectively.Conclusion:（1）κ-OR activation elicits an anti-HPH effect.（2）Hypoxia induces autophagy in PASMCs,and the latter leads to PASMC proliferation and reduce the apoptosis of PASMCs,resulting in the occurrence of HPH.（3）κ-OR activation inhibits autophagy by inhibiting the AMPK-mTOR signaling pathway,thereby reducing PASMC proliferation and increasing PASMC apoptosis under hypoxia to achieve the effects of inhibiting pulmonary vascular remodeling and anti-HPH.