The Preventive Effect And Mechanism Of Chronic Intermittent Hypoxia On Pulmonary Hypertension Induced By Monocrotaline In Rats

Pulmonary arterial hypertension(PAH) is a kind of disease characterized by pulmonary artery pressure increase mainly, and the basic pathological changes are pulmonary vasomotor dysfunction and vascular remodeling. In recent years, the researches on the pathogenesis of pulmonary hypertension and the development of treatment strategy have become the focus in the basic and clinical medicine.Epidemiological data showed that animals and people living in plateau have a stronger ability to adapt to hypoxia environment than the residents living in plain. One of the important reasons for this phenomenon is that the pulmonary vascular is weak response to hypoxic contraction, namely, the pulmonary vascular reactivity mechanism for high altitude hypoxia adaptation or acclimatization. Therefore, the adaptation to moderate hypoxia may have a certain curbing effect on pulmonary hypertension in which the change of pulmonary vasomotor function is a main pathophysiological characteristic.Chronic intermittent hypobaric hypoxia(CIHH) refers to a situation that people or animals are exposed to hypobaric hypoxia(simulated high altitude hypoxia environment) in a certain period of time discontinuously, and kept in normal oxygen environment for the rest of time. Previous studies have indicated that CIHH has protective effects on various tissues and organs such as the cardiovascular system. Also CIHH was found having anti-hypertension effect, but no research of CIHH on PAH is reported so far.In this study, the microvascular tension recording, immunohistochemistry, immunofluorescence, flow cytometry, ELISA, Western blot and q PCR methods were used to investigate the preventive effects of CIHH on PAH and the underlying mechanism in whole body, organ or tissue, cell or molecular, and gene levels in monocrotaline(MCT)-induced PAH rats to test our hypothesis that CIHH has a preventive effect against PAH through enhancing the relaxation of pulmonary arterioles.This study was divided into three parts: 1. To measure the pulmonary hemodynamic parameters to determine the preventive effect of CIHH on MCT-induced PAH by catheter method and observe the structural changes of lung tissue and pulmonary artery with light microscope. 2.To determine the effect of CIHH on vasomotion of rat pulmonary arteriola using microvascular tension recording method;To determine the effect of CIHH on endothelialdependent relaxation by measuring e NOS and CYP2J2 expression in pulmonary arterioles; To determine the effect of CIHH on endotheliumindependent relaxation by measuring the expression of BKCa channel in vascular smooth muscle of pulmonary arterioles; To determine the effect of CIHH on proliferation, phenotype transformation and contraction by detecting the expression of Ki-67 and ɑ-SM-actin in smooth muscle of pulmonary arterioles. 3. To determine the immuno-regulation mechanism of CIHH on MCT-induced PAH measure by measuring TNF-ɑ and IL-6 expression in serum with ELISA, TNF-ɑ and IL-6 expression in lung tissue with Western blot and q PCR methods, and ratio of CD4+/CD8+ T lymphocytes in peripheral blood and the apoptosis rate of spleen CD3+ T lymphocyte with flow cytometry; To determine the p38 MAPK and NF-KB signaling pathway in the preventive effect of CIHH on MCT-induced PAH by detecting the expression of P38MAPK/p-P38 and P65 / Ik Bɑ in pulmonary arterioles. Part One: The preventive effect of CIHH against pulmonary hypertension in MCT-induced PAH ratsObjectives: To clarify the preventive effect of CIHH against pulmonary hypertension in MCT-induced PAH rats.Methods: The rats were randomly divided into four groups: the normal control group(CON), pulmonary hypertension group(PAH), chronic intermittent hypobaric hypoxia group(CIHH), chronic intermittent hypobaric hypoxia+pulmonary hypertension group(CIHH+MCT). The CIHH rats were treated with a hypobaric hypoxia simulating 5000 m altitude for 28 days, 6 hours per day in a hypobaric chamber. The PAH rats accepted a single dose injection of MCT(60 mg/kg) intraperitoneally to induce PAH. The CIHH+MCT rats accepted 28-days CIHH before PAH generation and the experiments were performed 14 days after MCT injection. The body weight and blood pressure were measured once weekly, the pulmonary hemodynamics were determined with catheterization, and the structural changes of lung tissue and pulmonary artery were observed under light microscope.Results:1.CIHH treatment had no effect on blood pressure, heart rate and body weight in rats under the normal basic condition(P>0.05), but antagonized the increase of heart rate and the decrease of body weight in PAH rats(P<0.05).2.The mean pulmonary artery pressure(m PAP) was higher in PAH rats than in CON rats(P<0.01); but there was no significant difference of m PAP among CIHH, CIHH+MCT and CON rats(P>0.05).3.The right ventricle hypertrophy index(RVHI) was higher in PAH rats than in other three groups rats(P<0.01), but there was no significant difference of RVHI among CON, CIHH, and CIHH+MCT rats(P>0.05).4.The HE stain under light microscopy showed normal structure and normal wall thickness of pulmonary artery in CIHH and CON rats. The wall thickness was increased obviously and the lumen was narrowed or even closed in pulmonary arterioles in PAH rats. In CIHH+MCT rats, only mild thickness of arterial wall was observed and there was no significant stenosis in vascular lumen.There was no infiltration of inflammatory cells in the lung tissue and around pulmonary vessels in CON and CIHH rats. The macrophages was increased in alveoli, the mononuclear cells were infiltrated around the pulmonary arterioles, and the perivascular inflammation scale was increased significantly in PAH rats. All those changes of PAH were improved significantly in CIHH+MCT rats.The result of pulmonary vascular parameters showed that the medial wall thickness(MT%) was increased and vessel lumen transverse area(VA%) was decreased in PAH rats compared with CON rats(P<0.01). The MT% was decreased and VA% was increased in CIHH+MCT rats compared with PAH rats(P<0.01).Summary:1. CIHH pretreatment can effectively prevent the generation of MCT-induced pulmonary hypertension in rats and improve right ventricular hypertrophy and pulmonary artery remodeling;2.The prevention effect of CIHH pretreatment on MCT-induced PAH may be related to the inhibition of pulmonary vascular remodeling and immune regulation of CIHH.Part Two:Effects of CIHH on relaxation and contraction of pulmonary arterioles in MCT-induced PAH ratsObjective: To determine the effect of CIHH on the contraction and relaxation of pulmonary arterioles and the underlying mechanism in MCT-induced PAH rats.Methods: The CIHH rats were treated with a hypobaric hypoxia treatment simulating 5000 m altitude for 28 days, 6 hours per day in a hypobaric chamber. The PAH rats accepted a single dose injection of MCT(60 mg/kg) intraperitoneally to induce PAH. The CIHH+MCT rats accepted 28-days CIHH before PAH formation and experiments were performed 14 days after MCT injection. The body weight and blood pressure were measured once weekly, the pulmonary hemodynamics were determined with catheterization, and the structural changes of lung tissue and pulmonary artery were observed under light microscope.The e NOS and CYP2J2 expression of pulmonary arterioles were measured by using Western blot and real time polymerase chain reaction(q PCR) methods to determine the effect of CIHH on endothelial-dependent relaxation in pulmonary arterioles;The BKCa channel protein expression of vascular smooth muscle was measured by Western blot and q PCR to determine the effect of CIHH on endothelium-independent relaxation in pulmonary arterioles;The Ki-67 and ɑ-SM-actin expression of pulmonary arterioles were assayed by immunofluorescence, Western blot and q PCR methods respectively to determine the effect of CIHH on proliferation, phenotype transformation and the contraction in smooth muscle of pulmonary arterioles.Result:1.Compared with CON rats, the endothelial-dependent relaxation in pulmonary artery rings was decreased in PAH rats(P<0.05) and increased in CIHH rats(P<0.05). The decrease of pulmonary artery rings in PAH rats was prevented effectively by CIHH pretreatment(P<0.05).2.Compared with CON rats, the endothelium-independent relaxation in pulmonary artery rings was reduced in PAH rats(P<0.05) increased in CIHH rats(P<0.05). The decrease of pulmonary artery rings in PAH rats was prevented effectively by CIHH pretreatment(P<0.05).3.Compared with CON rats, the reactivity of pulmonary artery ring to PE was decreased and the contraction was diminished in PAH rats(P<0.05). CIHH pretreatment can increase the contractile responses to PE and prevent the decrease of contractile responses to PE in CIHH+MCT rats(P<0.05).4.Compared with CON rats, e NOS and CYP2J2 expression of pulmonary arterioles was down-regulated in PAH rats(P<0.05) and slightly up-regulated in CIHH rats(P>0.05). The down-regulation of e NOS and CYP2J2 induced by MCT in pulmonary arterioles was effectively prevented in CIHH+MCT rats(P<0.05).5. Compared with CON rats, ɑ-SM-actin and BKCa expression of pulmonary arterioles was down-regulated in PAH rats(P<0.05) and was up-regulated in CIHH rats(P <0.05). The down-regulation of ɑ-SM-actin and BKCa induced by MCT in the pulmonary arterioles was effectively prevented in CIHH+MCT rats(P<0.05).6.Compared with CON rats, Ki-67 expression of pulmonary arterioles was up-regulated in PAH rats(P<0.01). The up-regulation of Ki-67 expression induced by MCT in pulmonary arterioles was effectively prevented in CIHH+MCT rats(P < 0.05).Summary:1. The preventive effect of CIHH pretreatment on MCT-induced PAH in rats may be realized by enhancing the endothelium-dependent and endothelium-independent relaxation, and regulating the contraction in pulmonary arterioles.2. CIHH pretreatment could enhance the endothelium-dependent relaxation in pulmonary arterioles through up-regulating the expression of e NOS and CYP2J2 in pulmonary arterioles.3. CIHH pretreatment could enhance the endothelium-independent relaxation in pulmonary arterioles through up-regulating the expression of BKCa in smooth muscle cells of pulmonary arterioles.4. CIHH pretreatment could inhibit the pathological proliferation and phenotype transformation of vascular smooth muscle,regulate the contractile function of pulmonary arterioles through up-regulating ɑ-SM-actin expression and down-regulating Ki-67 expression in pulmonary arterioles. Part Three Immune regulation mechanism of CIHH preventive effect on pulmonary hypertension in MCT-induced PAH ratsObjective: To investigate the immune regulation mechanism for the prevention of CIHH on pulmonary hypertension induced by MCT in rats.Methods: The rats were randomly divided into four groups: the normal control group(CON), pulmonary hypertension group(PAH), chronic intermittent hypobaric hypoxia treated group(CIHH), chronic intermittent hypobaric hypoxia + pulmonary hypertension group(CIHH+MCT). The PAH rats accepted a single dose injection of MCT(60 mg/kg)intraperitoneally to induce PAH. The CIHH+MCT rats accepted 28-days CIHH before PAH formation and experiments were performed 14 days after MCT injection. The body weight and blood pressure were measured once weekly. The expression of TNF-ɑ and IL-6 in serum and lung tissue was assayed by ELISA and immunofluorescence methods; The ratio of CD4+/CD8+ T lymphocyte in peripheral blood and the apoptosis rate of splenic CD3+ T lymphocyte were determined by flow cytometry.The protein and gene expression of P38MAPK/p-P38 and P65/Ik Bɑwere detected by Western blot and Qpcr. And the signal pathways for the PAH prevention of CIHH were measured in MCT-induced PAH rats.Result:1. Compared with CON rats, TNF-ɑ and IL-6 expression in serum and lung tissue were up-regulated in PAH rats(P<0.01). The up-regualtion of TNF-ɑ and IL-6 induced by MCT was prevented effectively in CIHH+MCT rats(P<0.01).2. Compared with CON rats, CD4 T lymphocyte was increased and CD8 T lymphocyte was decreased in peripheral blood(P<0.01), and the apoptosis rate of splenic CD3+ T lymphocytes was decreased in PAH rats(P<0.01). CD4 T lymphocyte was decreased and CD8 T lymphocyte was increased in peripheral blood(P<0.01), and the apoptosis rate of splenic CD3+ T lymphocytes was increased(P<0.01) in CIHH rats compared with CON rats. The increased CD4 T lymphocyte, the decreased CD8 T lymphocyte, and the decreased apoptosis rate of splenic CD3+ T lymphocyte induced by MCT were prevented effectively in CIHH+MCT rats(P<0.01).3. Compared with CON rats, the expression of p-P38 and P38 MAPK were up-regulated in lung tissue of PAH rats(P<0.01), the expression of p-P38 and P38MAPK(P<0.01) were down-regulated in lung tissue of CIHH rats. The up-regulation of p-P38 induced by MCT were prevented effectively in CIHH+MCT rats(P<0.01).4. Compared with CON rats, the expression of P65 was up-regulated and Ik Bɑ was down-regulated(P<0.01) in lung tissue of PAH rats, the expression of P65 was down-regulated and Ik Bɑ was up-regulated(P<0.01) in lung tissue of CIHH rats. The up-regulation of P65 and down-regulation of Ik Bɑ induced by MCT were prevented effectively in CIHH+MCT rats(P<0.01).Summary:1. The preventive effect of CIHH on MCT-induced PAH in rats may be related to the inhibition of pro-inflammatory cytokines TNF-ɑ and IL-6 expression.2. The preventive effect of CIHH on MCT-induced PAH in rats may be related to the maintenance of T lymphocyte proliferation/apoptosis and the balance of CD4+/CD8+ subsets.3. The immune-regulation of CIHH may be performed through the P38 MAPK and NF-кB signal pathways in MCT-induced PAH rats.Conclusion1.CIHH pretreatment has a preventive effect against MCT-induced pulmonary hypertension in rats, which might be related to the inhibition of pulmonary vascular remodeling and immune regulation of CIHH.2. CIHH pretreatment enhances the endothelium-dependent and endothelium-independent relaxation in pulmonary arterioles through up-regulating the expression of e NOS, CYP2J2 and BKCa channel in pulmonary arterioles, which contributes to the prevention of PAH.3. CIHH pretreatment inhibits the pathological proliferation and phenotype transformation of vascular smooth muscle through up-regulating ɑ-SM-actin expression and down-regulating Ki-67 expression in pulmonary arterioles, which contributes to the prevention of PAH.4. CIHH pretreatment inhibits production of pro-inflammatory, maintains the balance of T lymphocyte proliferation/apoptosis and of CD4+/CD8+ subsets through p38 MAPK and NF-кB signal pathways, which contributes to the prevention of PAH.