| Background:Hypoxic pulmonary hypertension (HPH) is the main clinical type of pulmonary hypertension. HPH is an important pathophysiological stage in the pathogenesis and development of many cardiovascular diseases and pulmonary diseases. Hypoxia-induced pulmonary vasoconstriction and pulmonary vascular remodeling are two key pathophysiologic processes in HPH. However, the pathogenesis of HPH has not been elucidated thoroughly, and there is still no ideal therapeutic strategy for HPH. Therefore,to elucidate the underlying mechanism of HPH and look for effective prevention and treatment measures have been a major task faced by the medical field.Previous studies have indicated that endogenous opioid peptides (EOP) are synthesized and released by the heart during ischemia or hypoxia. EOP triggers cardioprotection in I/R myocardium. Our previous study also indicates that ischemic preconditioning promotes releasing of endogenous Dynorphin, which exerts protection against ischemia and reperfusion. However, whether Dynorphin elicits effect on HPH is unclear. In addition, our previous results show that exogenous K-opioid receptor agonist,U50,488H, plays the role of anti-HPH by dilating pulmonary artery, inhibiting remodeling of pulmonary vascular. Moreover, the expression of ?-OR is up-regulated during hypoxia.However, whether Dynorphin, an endogenous ?-OR agonist, can release from lung during hypoxia, and what is its role in HPH remains unclear.In recent years, studies have suggested that the calcium sensing receptor (CaSR) play an important role in the formation of HPH, and it may be a key target for prevention and treatment of HPH. It has been reported that CaSR is expressed in PASMCs, and the expression of CaSR increases during hypoxia. CaSR promotes vasoconstriction of pulmonary artery and proliferation of PASMCs, resulting in the occurrence of HPH. Our previous results showed that ?-OR activation significantly inhibits the remodeling of pulmonary vascular and proliferation of PASMCs induced by hypoxia, leading to the alleviation of HPH. It was also found unexpectedly that CaSR was negatively regulated by?-OR activation in our preliminary experiment. However, whether ?-OR activation exerts anti-HPH effect via inhibition of CaSR signaling remains unclear.In addition, inflammatory reaction is often involved in the process of hypoxia-induced pulmonary vasoconstriction and pulmonary vascular remodeling. Our previous study confirmed that ?-OR activation significantly inhibits the inflammatory reaction caused by myocardial ischemia. However, whether ?-OR activation can inhibit the inflammatory response induced by hypoxia in pulmonary artery has not been reported.Based on the above problems and our previous studies, the current study aims to investigate the following aspects (1) the role of Dynorphin A, an endogenous ?-OR agonist in anti-HPH; (2) whether ?-OR activation exhibits anti-HPH via regulating the function of CaSR; (3) preliminarily investigate the role of ?-OR activation in the pulmonary inflammatory response induced by hypoxia. The expected results will elucidate the effect of ?-OR mediated anti-HPH effect, and provide new experimental evidence for the clinical use of opioids in the prevention and treatment of HPH.Aims:1. To investigate the role of Dynorphin A,an endogenous ?-OR agonist in HPH.2. To investigate the effect of ?-OR activation on regulating the expression and function of CaSR and the role of CaSR in anti-HPH mediated by ?-OR activation.3. To investigate the role of ?-OR activation in the inflammatory response induced by hypoxia.Methods:1. SD male rats were exposed to chronic hypobaric and hypoxia condition for 1, 2 and 4 weeks, and hypoxia for 8 h per day. The hypobaric and hypoxia condition simulated pressure in altitude of about 5500 m high environment, pressure of atmosphere is about 50 kPa, the oxygen concentration is about 10%. In vivo, the effect of Dynorphin A and 50,488H (?-OR agonist) and nor-BNI (?-OR antagonist) on mPAP in HPH rats, and the effects of neomycin (CaSR agonist) and NPS2143 (CaSR antagonist) on hemodynamics of HPH rats were observed.2. Changes of Dynorphin A, ET-1 and Ang ? levels in blood and lung tissues of HPH rats were detected with ELISA kits.3. Effects of Dynorphin A 1-13, U50,488H, nor-BNI and Spermine (CaSR agonist) on pulmonary arterial tension, which were detected by perfusion technique in vitro, and the effects of U50,488H and NPS2143 pretreatment on the reaction of the pulmonary artery ring in HPH rats to ACh, which were also detected by perfusion technique.4. Proliferation of PASMCs was detected by MTT kit and BrdU kit. The effect of Spermine on the proliferation of PASMCs was analyzed by CCK-8 kit.5. Expression and localization of ?-OR and CaSR in PASMCs were detected with immunohistochemical staining.6. Interaction between ?-OR and CaSR in PASMCs was detected by immunoprecipitation.7. Effect of U50,488H on the expression of CaSR in the pulmonary artery of HPH rats was measured using Western blot. The effects of Spermine on the expression levels of PCNA,Erk, and p-Erk in PASMCs in normoxic and hypoxic rats were detected by Western blot.8. Ultrastructural changes of pulmonary vascular endothelium and right ventricle in HPH rats were detected by transmission electron microscopy to examine the effects of U50,488H and NPS2143 on the HPH.9. Level of IL-6, IL-10 and MCP-1 in serum during hypoxia were detected by liquid beads suspended chip.Results:Part ? Role of Dynorphin, an endogenous ?-OR agonist in HPH1. Mean pulmonary arterial pressure (mPAP) and right ventricular pressure (RVP)increased significantly, which were higher over diagnostic criteria in male SD rats after hypoxia for 2 and 4 weeks. The HPH rat model was successfully established after hypobaric hypoxia for 2 weeks.2. Level of Dynorphin increased in chronic hypoxia for 1 weeks and 2 weeks groups,whereas it decreased in the hypoxia for 4 weeks group. ET-1 and Ang? levels increased remarkably in both blood and lung tissues in the hypoxia for lweek, hypoxia for 2 weeks and also hypoxia for 4 weeks groups.3. Compared with the hypoxia for 2 weeks group, treatment with nor-BNI further increased mPAP during hypoxia. Dynorphin A 1-13 significantly decreased mPAP in hypoxia for 2 weeks and hypoxia for 4 weeks group. U50,488H also significantly inhibited the mPAP in hypoxia for 4 weeks group. The effect of of Dynorphin A was less than that of U50,488H.4. In vitro, Dynorphin A and U50,488H induced a significant relaxation in normoxia and HPH rat pulmonary artery rings, and both the effect of Dynorphin A and U50,488H were abolished by nor-BNI. In addition, the relaxation of Dynorphin A was less than that of U50,488H.5. At the cellular level, DynorphinA 1-13 significantly suppressed the proliferation of PASMCs, which was abolished by nor-BNI. The expression of ?-OR in PASMCs in hypoxia condition was increased and Dynorphin A1-13 further up-regulated the expression of ?-OR, which was abolished by nor-BNI.Part ? Effect of ?-OR activation on regulating the expression and function of CaSR and the role of CaSR in anti-HPH mediated by ?-OR activation1. ?-OR and CaSR were both expressed in pulmonary artery smooth muscle cells, and the two receptors were colocalized. The expression levels of the two receptors were up-regulated during hypoxia. Colocalization of the two receptors in hypoxic condition was more obvious compared with the normoxia group. Spermine, a CaSR agonist up-regulated the expression of two receptors, but expression of CaSR increased more significantly.However, U50,488H up-regulated the expression of ?-OR and down-regulated the expression of CaSR. Immunoprecipitation results demonstrated that there was interaction between two receptors.2. Spermine, a CaSR agonist, significantly increased vasoconstriction of pulmonary artery both in normoxia rats and HPH rats. The effect of Spermine on HPH rats was stronger than that on normoxia rats. The effect of Spermine on normoxia rats and HPH rats were inhibited by U50,488H, a ?-OR agonist, SB203580, a p38 inhibitor, SP600125, a JNK inhibitor, Sch772984, an ERK inhibitor, and U0126, a MER inhibitor, respectively. In addition, with NPS2143 treatment in advance, the relaxation of U50,488H on pulmonary artery was significantly cutoff.3. Neomycin, a CaSR agonist, raised RVP in normoxia rats. Neomycin also significantly up-regulated RVP in hypoxia for 3 weeks rats, and the effect and period of time of neomycin were obviously stronger and longer than those in normoxia group. The effect of neomycin was inhibited by U50,488H treatment. Both of U50,488H and NPS2143 significantly decreased the RVP and mPAP in hypoxia for 3 weeks group. And the effects of U50,488H were abolished by nor-BNI. In addition, the results of electron microscopy showed that U50,488H and NPS2143 significantly improved vascular endothelial dysfunction and cardiac structure in HPH rats.4. Compared with the normoxia group, vasorelaxation in response to ACh of the pulmonary artery of HPH rats was decreased. Treatment with U50488H and NPS2143 in advance significantly increased vasorelaxation of the pulmonary artery in response to ACh.5. At the cellular level, Spermine significantly promoted the OD value in normoxia PASMCs group, and activation of ?-OR inhibited the action of Spermine. In addition, the effect of Spermine was also inhibited by SB203580, a p38 inhibitor, SP600125, a JNK inhibitor, Sch772984, an ERK inhibitor, and U0126, a MER inhibitor, respectively.Compared with the normoxia group, the OD value in hypoxia for 24 h group increased significantly, and Spermine further increased the OD value. NPS2143 and U50,488H significantly reduced the OD value.6. At the cellular level, Spermine significantly promoted the expression of PCNA and p-Erk in normoxia PASMCs, which was inhibited by U50,488H, and the effect of U50,488H was abolished by nor-BNI. In addition, U50,488H and NPS2143 significantly reduced the expression of PCNA and p-Erk in hypoxia for 24 h group. Spermine further up-regulated the expression of PCNA and p-Erk in hypoxia for 24 h group.Part ? Role of ?-OR activation in the inflammatory response induced by hypoxiaSerum IL-6 and MCP-1 levels increased dramatically after 3 weeks hypoxia, but IL-10 level did not change significantly. ?-OR activation significantly inhibited the expression of IL-6 and MCP-1 induced by hypoxia, and significantly upregulated the expression of IL-10, which were abolished by nor-BNI.Conclusion:1. Dynorphin A, an endogenous ?-OR agonist, like U50,488H, a exogenous ?-OR agonist attenuates HPH through relaxation of the pulmonary artery and inhibition of proliferation of smooth muscle cells. Endogenous dynorphin A released in the early stage of hypoxia plays a compensatory effect in anti-HPH via stimulation of K-opioid receptor.2. ?-OR and CaSR are colocalized in PASMCs, and there is interaction between the two receptors. ?-OR negatively regulates the expression and function of CaSR. MAPKs signaling pathway mediated by CaSR is involved in the contraction of the pulmonary artery and proliferation of smooth muscle cell, thus promoting the occurrence of HPH.?-OR activation confers anti-HPH through inhibiting CaSR and the signaling pathway mediated by CaSR.3. ?-OR activation significantly inhibits the inflammatory response induced by hypoxia,and its underlying mechanism awaits further research. |
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