Expression of NO and H2S in hypoxic pulmonary hypertension and intervention of sodium nitrite in rats

AIM: To observe the change of nitric oxide(NO)and hydrogen sulfide(H2S)in blood and lung homogenate of hypoxic pulmonary hypertension(HPH)rat model, and to discuss the meaning of inhalation sodium nitrite and these factors in the treatment of HPH. METHODS: Fifty healthy male Wistar rats were assigned randomly into 5 groups(10 rats each): normoxia control group(NC), normoxia sodium nitrite group(NNI), hypoxic control group(HC), hypoxic normal saline group(HNS)and hypoxic sodium nitrite group(HNI). The mean pulmonary arterial pressure(mPAP), weight of right ventricle, weight of left ventricle plus septum, and the ratio of the weight of right ventricle to that of left ventricle plus septum(right ventricle hypertrophy index, RVHI)were also determined. The serum level of NO and plasma level of H2S were measured, and at the same time the levels of NO in the lung homogenate were detected. The structures in pulmonary arteries were examined using optical microscope. RESULTS: After model established, compared to that in the normoxia groups, the body weight decreased significantly in hypoxia groups(P<0.05), although no difference of body weight in five groups before producing model was observed. Compared to that in normoxia groups, the levels of mPAP and RVHI increased significantly in hypoxia groups, and compared to that in hypoxia control groups and hypoxia normal saline group, mPAP and RVHI levels decreased significantly in hypoxia sodium nitrite group(P<0.05). Compared to that in normoxia groups, the serum level of NO decreased significantly in hypoxia groups(P<0.05). NO level in lung homogenate decreased significantly in hypoxia control group and hypoxia normal saline group as compared to that in normoxia groups(P<0.05), and no obvious difference between hypoxic sodium nitrite group and normoxia groups was found. The plasma level of H2S was decreased significantly in hypoxia groups(P<0.05)as compared to that in normoxia groups. H2S level increased significantly in hypoxia sodium nitrite group as compared to that in hypoxia control groups and hypoxia normal saline group(P<0.05). Observation under optical microscope, the lumen structure of lung in normoxia control group was normal. No significant change in normoxia sodium nitrite group was found. The proliferation of smooth muscle cells(SMCs), the collagen fiber deposition in the vessel wall and every caliber thickening was observed in hypoxic control group. The same changes were also observed in hypoxic normal saline group. The thickened caliber was relieved significantly in hypoxic nitrite group. CONCLUSION: Pulmonary hypertension and right ventricle reconstitution can be relieved by inhalation of sodium nitrite, and can be regulated by the level of NO and H2S in rats. Above all, inhalation of sodium nitrite may degrade HPH directly or by affecting the externalization and synthesizing of gas signaling molecule indirectly.     

Effect of diltiazem on heme oxygenase-1 and nitric oxide synthase in rats with pulmonary hypertension

AIM: To investigate the action of diltiazem (a calcium antagonist) on the expression of heme oxygenase (HO) -1 and nitric oxide synthase (NOS) in the small pulmonary arteries (SPA) of rat in chronic hypoxia. METHODS: Chronic pulmonary arterial hypertension models were established by treating the rats in hypoxic environment for 6 weeks. After 2 weeks of hypoxia, rats were treated with diltiazem (15 mg/kg/day). Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were measured. Pathological changes in the lungs were observed under the light microscope and transmission electron microscope. The expression and distribution of heme oxygenase (HO) -1, endothelial NOS (eNOS) and inducible NOS (iNOS) were tested by immunohistochemistry and Western blot. Guanosine-3', 5'-cyclic monophosphate (cGMP) of lung tissues were detected with radioimmunoassay. RESULTS: Diltiazem significantly decreased abnormal RVSP, and RVHI in model rats, attenuated the SPA media thickeness, and recovered abnormal eNOS and iNOS expression in SPA. Whereas diltiazem had little effect on the increased HO-1 expression in SPA caused by hypoxia and ultrastructure injury in endothelium. cGMP levels were corresponded with HO-1. CONCLUSION: Diltiazem has a significant effect on inhibiting hypoxic pulmonary hypertension structural remodeling. These effects might be partly attributed to the suppression of iNOS, promotion of eNOS, and not attenuation HO-1 expression in the lung of hypoxic rats.     

Effects of different β-adrenergic receptors on cardiac systolic and diastolic functions in hypoxic stress rats

AIM:To explore the effects of different β-adrenergic receptors (β-AR) on the left and right ventricular systolic and diastolic functions in rats under acute hypoxic stress. METHODS:The healthy male SD rats were randomly divided into 4 groups (n=7):control group, non-selected β-AR blocker propranolol group, selected β₁-AR blocker atenolol group and selected β₂-AR blocker ICI 118,551 group, and then the rats were exposed to normoxia (20.9% O₂, 79.1% N₂) and hypoxia (15.0% O₂, 85.0% N₂) condition respectively at the altitude of 2 260 m (Xining, China). The heart rate (HR), the left ventricular systolic pressure (LVSP), the right ventricular systolic pressure (RVSP), and the maximum raise/decline rate of left and right ventricular pressure (±dp/dt_max) were monitored, and the arterial blood gas in normoxia and hypoxia condition were compared to explore the effect of β-AR on the left and right ventricular systolic and diastolic functions in acute hypoxic stress rats. RESULTS:Under normoxia condition, the LVSP, ±dp/dt_max of left ventricular were decreased in propranolol group, atenolol group and ICI 118,551 group, the RVSP and ±dp/dt_max of right ventricle were decreased in propranolol group and atenolol group (P<0.05). Under hypoxia condition, the PaO₂, LVSP, ±dp/dt_max of left ventricle were decreased in all groups compared with the normoxia group, and the ±dp/dt_max of right ventricle was increased in all groups (P<0.05), also the degree of index change in control group was more obvious than that in propranolol group and atenolol group. CONCLUSION:The activation of β₁-AR is an important compensatory regulation for heart function during hypoxic stress. However, the compensatory enhancement of right heart function under acute hypoxia condition which through tonogenic dilation is more significant for maintaining the normal circulating blood flow.     

Levels of CGRP and ET-1 in plasma of pulmonary artery and thoracic aorta and in the extractives of lung and ventricular tissues of the chronic hypoxic rats

AIM AND METHODS: To explore the effects calcitonin gene-related peptide (CGRP) and endothelin-1(ET-1) on the mechanisms of hypoxic pulmonary hypertension (HPH), the contents of CGRP and ET-1 in plasma of pulmonary artery and thoracic aorta and in extractives of lung and ventricular tissues of the chronic hypoxic rats were determined by radioimmunoassay. The changes of their hemodynamic indices and right heart hypertrophy index were monitored simultaneously. RESULTS: The level of pulmonary artery plasma CGRP was significantly higher than that of thoracic aorta plasma, but just the reverse was ET-1 or the ratio of ET-1 and CGRP in control rats( P<0.01). Compared with controls, the level of pulmonary artery plasma CGRP was gradually reduced in all hypoxic rats, but ET-1 was enhanced after 7 and 14 days of hypoxia and was decreased after 21 days of hypoxia. With prolonging time exposed to hypoxia, the level of thoracic aorta plasma CGRP was markedly increased in hypoxic animals compared with controls ( P<0.05), the positive correlation significantly with increased pulmonary arterial pressure( r = -0.896, P =0.05), but the lower level of thoracic aorta plasma ET-1 showed or negative correlation with pulmonary arterial pressure. CONCLUSIONS: These data suggest that the unbalance of effects of CGRP and ET may plays an important role in regulating the resistance of pulmonary circulation and has close relation with the formation of HPH.     

Asiaticoside attenuates hypoxic pulmonary hypertension in mice by inhibiting NF-κB/p38 signaling pathway

AIM: To investigate whether asiaticoside attenuates hypoxic pulmonary hypertension by inhibiting p38/NF-κB signaling pathway. METHODS: BALB/c mice (n=30) were randomly divided into normoxia (N) group, hypoxia (H) group, and hypoxia+asiaticoside group. Right ventricular systolic pressure (RVSP), mean carotid artery pressure (mCAP), the weight ratio of right ventricle/(left ventricle+ventricular septum)[RV/(LV+S)], the ratio of right ventricle/body weight (RV/BW), vessel wall area/vessel total area (WA/TA) and vessel wall diameter/vessel wall total diameter (WT/TT) were determined after the model was established. The protein levels of p38, p-p38, NF-κB and p-NF-κB in the lung tissues were detected by Western blot. The fluorescence intensity of p-p38 and p-NF-κB were measured by immunofluorescence method. The serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA. RESULTS: Compared with N group, the levels of RVSP, RV/(LV+S), RV/BW, WA/TA and WT/TT were significantly increased in H group, while administration of asiaticoside decreased the levels of RVSP, RV/(LV+S), RV/BW, WA/TA and WT/TT (P<0.05). Compared with N group, the relative protein levels of p-p38 and p-NF-κB in H group were significantly increased (P<0.05), and the concentrations of IL-6 and TNF-α were significantly increased, which were apparently attenuated by asiaticoside injection. CONCLUSION: Inhibition of p38/NF-κB signaling pathway and reduction of inflammatory responses may be the important mechanisms of asiaticoside in the prevention and treatment of hypoxic pulmonary hypertension.     

Effect of hypercapnia on lysyl oxidase-dependent collagen cross-linking and hypoxia-induced pulmonary hypertension in rat

AIM: To investigate the effect of hypercapnia on hypoxia-induced pulmonary hypertension and the changes of lysyl oxidase (LOX) and extracellular matrix collagen cross-links in the rat. METHODS: Sprague-Dawley rats were randomly divided into 4 groups:normoxia group, hypoxia group, hypercapnia group and hypoxia+hypercapnia group. LOX activity was detected by fluorescence spectrophotometry. LOX protein expression was detected by immunohistochemistry and Western blot. The mRNA expression of LOX in the pulmonary artery was detected by real-time PCR. RESULTS: The levels of mean pulmonary artery pressure (mPAP), RV/(LV+S) and WA/TA in hypoxia group were significantly higher than those in normoxia group (P<0.01). Moreover, the levels of mPAP and RV/(LV+S) in hypoxia+hypercapnia group were significantly lower than those in hypoxia group (P<0.01). However, no significant difference of mPAP and RV/(LV+S) between hypercapnia group and normoxia group was observed. In hypoxia group, the collagen cross-links in the lung tissue was significantly higher than that in normoxia group and hypercapnia group (P<0.01). Importantly, collagen cross-links in the lung tissue of hypoxia+hypercapnia group was significantly lower than that in hypoxia group (P<0.01). There was no significant difference in collagen cross-links between hypercapnia group and normoxia group. The expression of LOX at mRNA and protein levels and its activity in the pulmonary arteries of hypoxia group were significantly increased as compared with normoxia group (P<0.01). Furthermore, the expression of LOX at mRNA and protein levels and its activity in the pulmonary arteries in hypoxia+hypercapnia group were lower than those in hypoxia group (P<0.01). CONCLUSION: Hypoxia not only up-regulates LOX but also promotes collagen cross-linking in the rat lung, which contributes to the development of pulmonary hypertension. Hypercapnia inhibits hypoxia-induced LOX expression and collagen cross-linking, therefore impairing the progress in hypoxia-induced pulmonary hypertension.     

The expression of adrenomedullin mRNA in the right ventricle in chronic hypoxic rats

AIM: To study the role of adrenomedullin(AM) in the pathogenesis of hypoxic pulmonary hypertension. METHODS: Rats were exposed to chronic hypoxia for 14 days. After the measurement of the right ventricular systolic pressure (RVSP), the rats were executed. The weight of the right ventricle (RV), the left ventricle(LV) and the ventricular septum(SP) were determined. The ration RV/(LV+SP) was used to express the thickness of RV. In situ hybridization was used for the detection of the expression of AM mRNA in the lung and RV. RESULTS: The RVSP in the hypoxic group was (63.63±3.42) mmHg,which was significantly higher than that in control group [(34.13±3.40) mmHg]. The RV/(LV+SP) in hypoxic group was 0.439±0.039,which was increased obviously when compared with that of control (0.230±0.025). The level of AM mRNA expressed in the RV in the hypoxia group was significantly higher than that in the control group. CONCLUSION: The expression of AM mRNA in RV increased in the hypoxic condition, which suggests that AM may attenuate the inappropriate increase in pulmonary artery pressure.     

Effects of Tongxinluo on expression of apoptotic factors in cerebral tissues of hypoxic preconditioned mice

AIM: To observe the effects of Tongxinluo (TXL), a Chinese medicine, on hypoxic tolerance and expression of Bcl-associated X (Bax) and B-cell leukemia/lymphoma 2 (Bcl-2) in hypoxia-preconditioned mice. METHODS: The mice were randomly divided into groups of hypoxia preconditioning without (control group) or with TXL treatment (TXL group). The mice in TXL group were administered with TXL at dose of 1.52 g·kg^-1·d^-1 crude drug for 5 days. The mouse was exposed to normoxia (0 run, H₀) and acute repetitive hypoxia for 1-5 runs (H₁-H₅) by placing the animal in an air-sealed jar. The hypoxic environment was established in the jar through consumption of the oxygen by the respiration of the mouse. A gasp breath was regarded as the hypoxic tolerant limit of the mouse and the animal was then transferred to another new jar. The mouse was exposed to hypoxia in this way for 5 times. In each run of hypoxic exposure, the time of hypoxic tolerance was measured. Western blotting was used to measure the protein levels of hypoxia inducible factor-1α (HIF-1α), Bax and Bcl-2 in the cerebral cortex. RESULTS: The hypoxic tolerance time in control and TXL groups was gradually increased run by run (P<0.01 or P<0.05). The protein levels of HIF-1α and Bcl-2 in the two groups were gradually increased (P<0.01 or P<0.05). Bax in control and TXL groups was significantly increased in H₁. After H₁, Bax was decreased run by run (P<0.01 or P<0.05). Compared with control group, the tolerance time, the expression of HIF-1α and Bcl-2 in the cerebral cortex in TXL group were increased in H₁,H₃ and H₅. However, the expression of Bax was lower than that in control group in every run. CONCLUSION: Hypoxia preconditioning makes the organism produce a strong adaptive response. The increase in Bcl-2 and the decrease in Bax may be involved in the mechanism of adaptation. TXL obviously increases the adaptive ability of the mice to hypoxia.     

Effects of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells

AIM: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1α) and stromal cell-derived factor 1 (SDF-1) in pulmonary artery endothelial cells (PAECs) of SD rats and to investigate the role of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells to PAECs. METHODS: Immunomagnetic beads were used to separate and purify the CD34⁺/CXCR4⁺ progenitor cells derived from the peripheral circulation of SD rats. The expression of HIF-1α and SDF-1 in PAECs exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) was detected by immunofluorescence, Western blotting and ELISA. The migration index and adhesion rate were measured in the progenitor cells, which were subjected to the following different treatments: (1) normoxia (21% O₂); (2) hypoxia 12 h; (3) hypoxia 12 h +HIF-1α inhibitor (2ME2); (4) hypoxia 12 h+SDF-1 neutralizing antibody; (5) hypoxia 12 h+2ME2+SDF-1 neutralizing antibody.RESULTS: The expression of HIF-1α and SDF-1 in PAECs was effectively induced by the hypoxic exposure, and both of them reached the peak levels after 12 h of hypoxic treatment (P<0.01), while administration of 2ME2 decreased the hypoxia-induced SDF-1 expression (P<0.05). Treatment of the PAECs with 2ME2 or SDF-1 neutralizing antibody attenuated the migration index and adhesion rate of progenitor cells to the PAECs (P<0.05). CONCLUSION: There is a HIF-1α/SDF-1 signaling axis in hypoxia-exposed PAECs, which may play a crucial role in the migration and adhesion of progenitor cells to PAECs.     

Expression and distribution of osteopontin in the development of pulmonary hypertension induced by hypoxia-hypercapnia

AIM: To study the expression and distribution of osteopontin (OPN) in lungs and pulmonary arteries in pulmonary hypertensive rats induced by hypoxia-hypercapnia, and to explore the role of OPN in pathogenesis of pulmonary hypertension. METHODS: Forty-eight male Sprague-Dawley rats (Weight 180 g-220 g) were randomly divided into four groups: normal control group (NC), hypoxic hypercapnia 1-week，2-week and 4-week group (1HH, 2HH and 4HH). The expressions of OPN mRNA and protein in lungs and pulmonary arteries were detected by RT-PCR and immunohistochemistry. ELISA was used to detect the concentration of OPN in lung homogenates. The content of OPN in pulmonary arteries was detected by Western blotting. RESULTS: ① The mean pulmonary artery pressure (mPAP) and weight ratio of right ventricle to left ventricle and septum ［RV/(LV+S)］ in all hypoxic hypercapniac groups were higher than those in normal control group (P<0.01), respectively. Differences of mean carotid artery pressure (mCAP) among these four groups were not significant (P>0.05). ② The expression of OPN mRNA was significantly increased in pulmonary arteries and lung tissues in hypoxic hypercapnic groups compared with normal control group (P<0.01). ③ The result of immunohistochemistry showed that OPN was only detected in bronchus and alveolar epithelium, but not detected in pulmonary arterioles of normal control group. In contrast，OPN expression was evident in pulmonary arterioles of 1HH rats，especially in media. Moreover, the expression of OPN was markedly increased in group 2HH and 4HH. ④ OPN levels in lung homogenates in 1HH, 2HH and 4HH were increased by 69%, 128% and 187% (P<0.01), respectively, compared with control rats. ⑤ Western blotting analysis showed that the contents of OPN were significantly higher in all hypoxic hypercapnic groups than those in NC group (P<0.01).CONCLUSION: The expressions of OPN in pulmonary arteioles and lung are increased in rats with pulmonary hypertension. OPN might play an important role in the pathogenesis of pulmonary hypertension induced by chronic hypoxia and hypercapnia.     

Expression of Jagged2/Notch3 signaling molecules in pulmonary hypertensive rats induced by monocrotaline

AIM: To study the expression of Jagged2/Notch3 signaling molecules in pulmonary vascular wall of pulmonary hypertensive rats induced by monocrotaline. METHODS: SD rats were randomly divided into normal control group (C group,n=15), solvent control group (S group,n=15) and monocrotaline model groups (M group,n=15). The model of pulmonary hypertension was established by a single subcutaneous injection of monocrotaline (50 mg/kg). The rats in S group were given a single subcutaneous injection of the same dose of solvent. After 4 weeks, the pulmonary vascular remodeling was assessed by HE staining, and the mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) were determined by right heart catheterization. The expression of Jagged2/Notch3/Hes5 molecules in the pulmonary vascular wall was detected by immunohistochemical method and real-time PCR. RESULTS: Compared with S group and C group, the percentage of medial wall thickness of smaller arteries in model group increased significantly (P<0.01). The levels of mPAP and RVSP in M group were significantly higher than those in S group and C groups (P<0.01). The results of real-time PCR showed that the expression of Jagged2, Notch3 and Hes5 was significantly increased in M group compared with S group and C group. The data from immunohistochemical detection indicated that Jagged2 mainly expressed in the intima of small lung artery, Notch3 and Hes5 mainly expressed in the medial smooth muscle cells. Compared with S group and C group, the expression of Jagged2 and Notch3 was significantly increased in the lung small arteries of M group. CONCLUSION: The activation of Jagged2/Notch3 signaling pathway might play an important role in the formation of pulmonary hypertension.     

Protective effect of salidroside on rats with hypoxic pulmonary hypertension by suppressing oxidative stress

AIM: To study whether salidroside plays a protective role in hypoxia-induced pulmonary hypertension by suppressing oxidative stress. METHODS: Sprague-Dawley rats were randomly divided into 4 groups:normoxia (N) group, hypoxia for 4 weeks (H₄) group, low-dose salidroside (hypoxia for 4 weeks and treatment with salidroside at 16 mg/kg, H₄S16) group and high-dose salidroside (hypoxia for 4 weeks and treatment with salidroside at 32 mg/kg, H₄S32) group. The mean pulmonary arterial pressure (mPAP), the weight ratio of right ventricle/(left ventricle+septum)[RV/(LV+S)] and vessel wall area/vessel total area (WA/TA) were evaluated. The levels of malondialdehyde (MDA) in the serum and lung tissues were detected by colorimetric method. The levels of 8-iso-prostaglandin F_2α (8-iso-PGF_2α) in the serum and lung tissues were measured by ELISA. The activity of superoxide dismutase (SOD) in the serum was analyzed by hydroxylamine method. The expression of NAPDH oxidase 4 (NOX4) and SOD1 in the lung tissues was determined by Western blot. RESULTS: Compared with N group, the levels of mPAP, RV/(LV+S) and WA/TA in H₄ group were significantly increased, which were apparently attenuated by salidroside injection in a dose-dependent manner. Meanwhile, salidroside administration apparently decreased the levels of MDA and 8-iso-PGF_2α in the serum and lung tissues, as well as the expression of NOX4 in the lung tissues. Besides, compared with N group, the activity of SOD in the serum and the expression of SOD1 in the lung tissues in H₄ group were significantly decreased, while administration of salidroside increased the activity of SOD in the serum and the expression of SOD1 in the lung tissues in a dose-dependent manner. CONCLUSION: Salidroside protects the pulmonary vessels from remodeling and attenuates hypoxia-induced pulmonary hypertension by inhibiting oxidative stress.     

Effect of diazoxide on change of H2O2 in rat pulmonary artery smooth muscle cells and proliferation of hypoxic rat pulmonary artery smooth muscle cells

AIM: To investigate the contribution of diazoxide,an opener of mitochondrial ATP-sensitive K+ channel (MitoK_ATP),and mitochondrial membrane potential (ΔΨm) to change of H₂O₂ in rat pulmonary artery smooth muscle cells (PASMCs) and to unbalance between cell proliferation and apoptosis of PASMCs induced by hypoxia.METHODS: The rat PASMCs were isolated from fresh normal lung tissues and cultured,which were divided into 6 groups,as follows: ① control group;② diazoxide group;③ 5-HD group;④chronic hypoxia group;⑤ chronic hypoxia+diazoxide group;⑥ chronic hypoxia +5-HD group.The relative change in mitochondrial potential was detected with rhodamine fluorescence (R-123) technique.The level of H₂O₂ in rat PASMCs was detected with chemiluminescence method.The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.RESULTS: After exposed to diazoxide for 24 h,the intensity of R-123 fluorescence,the level of H₂O₂ and the A value in normoxic rat PASMCs were significantly increased,and the apoptosis of rat PASMCs was significantly decreased as compared with control group (P<0.05).However,there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24 h.Chronic hypoxia or chronic hypoxia+diazoxide markedly increased the intensity of R-123 fluorescence,the level of H₂O₂ and the A value in rat PASMCs,and also markedly decreased the apoptosis of rat PASMCs as compared with control group (P<0.05),and these changes were more significant in chronic hypoxia +diazoxide group than those in chronic hypoxia group (P<0.05).5-HD partly weakened the effect of hypoxia on the intensity of R-123 fluorescence,the level of H₂O₂,the A value and the apoptosis of rat PASMCs (P<0.05).Significant and positive correlations were found between the intracellular H₂O₂ and the R-123 fluorescence or the A value.Significant and negative correlation was found between the intracellular H₂O₂ and the apoptosis of rat PASMCs.CONCLUSION: The results suggest that the opening of MitoK_ATP followed by a depolarization of ΔΨm can contribute to the increase in the level of H₂O₂ in rat PASMCs and to the proliferation of rat PASMCs induced by hypoxia.This might be a mechanism of the development of hypoxic pulmonary hypertension.     

Effect of Panax notoginoside on pulmonary arterial pressure and expression of p38 MAPK in lung tissues of hypoxic rats

AIM: To observe the effect of Panax notoginoside (PNS) on the pulmonary artery pressure and the p38 mitogen-activated protein kinase(p38 MAPK) in lung tissues of rats treated with hypoxia. METHODS: Thirty adult male SD rats were randomly divided into 3 groups. The rats in normal control group were exposed to normal conditions, the rats in hypoxia group were exposed to isobaric hypoxia, and the rats in hypoxia+PNS group were treated with PNS under the condition of hypoxia. After 4 weeks of treatment, the mean pulmonary arterial pressure (mPAP) and the mean carotid arterial pressure (mCAP) were measured by cardiac catheterization. The heart was isolated, and the right ventricle (RV), left ventricle plus ventricular septum (LV+S) were weighed to calculate the ratio of RV/(LV+S).The quantity of phospho-p38 MAPK(p-p38 MAPK) in rat pulmonary arterioles was determined by the method of immunohistochemistry and the mRNA content of p38 MAPK was tested by RT-PCR. RESULTS: The mPAP and RV/(LV+S) in hypoxia group were higher than those in normal control group. The expression of p-p38 MAPK in rat pulmonary arterioles and p38 MAPK mRNA in the lung tissues were higher than those in normal control group (P<0.05). The mPAP, RV/(LV+S), the expression of p-p38 MAPK in rat pulmonary arterioles and p38 MAPK mRNA in the lung tissues in hypoxia+PNS group were significantly lower than those in hypoxia group (P<0.05).CONCLUSION: PNS possesses the preventive and therapeutic effect on hypoxic pulmonary hypertension by decreasing p-p38 MAPK and down-regulation of p38 MAPK mRNA in the lungs.     

Roles of ERK pathway and Panax notoginoside treatment in hypoxic hypercapnia pulmonary hypertension in rats

AIM: To investigate the roles of Panax notoginoside (PNS) and ERK1/2 signaling pathway in the pathological process of chronic hypoxic hypercapnia pulmonary hypertension in rats.METHODS: The animal model of chronic hypoxic hypercapnia pulmonary hypertension was set up in 72 male Sprague-Dawley rats and the animals were randomly divided into 6 groups: normal (N) group, hypoxic hypercapnia for 3-day (H_3d) group, hypoxic hypercapnia for 1-week (H_1w) group, hypoxic hypercapnia for 2-week (H_2w) group, hypoxic hypercapnia for 4-week (H_4w) group and PNS treatment (H_p) group.The rats in H_p group were injected with PNS (50 mg·kg^-1·d^-1, ip) before placing the animals into the hypoxic hypercapnia chamber.The rats in other groups were injected with normal saline (2 mL/kg, ip).The morphological changes of the pulmonary artery were observed under microscope with HE staining.Western blotting was used to detect the protein expression of p-ERK.The protein levels of p-ERK in the lung tissues and pulmonary blood vessels were determined by immunohistochemistry.RESULTS: The ratios of WA/TA in H_1w, H_2w, H_4w and H_p groups were higher than that in N group (P<0.05).The ratio of WA/TA in H_p group was obviously lower than that in H_4w group (P<0.05).The protein expression of p-ERK was barely positive in N group, but was up-regulated in the pulmonary tissues in all hypoxic rats.Compared with N group, the protein level of p-ERK was markedly up-regulated in H_3d group, reached its peak in H_2w group, and tended to decline in H_4w group (P<0.05).In pulmonary arterial tunica intima and tunica media, p-ERK protein was dramatically expressed in all hypoxic rats compared with the control animals (P<0.05).In the lung tissues, the protein level of p-ERK in H_p group was lower than that in H_4w group (P<0.05).In pulmonary arterial tunica intima and tunica media, the protein level of p-ERK in H_p group was lower by 84.86% than that in H_4w group (P<0.05).CONCLUSION: ERK1/2 as a signal transducer may play an important role in the development of hypoxia and hypercapnia induced pulmonary hypertension.PNS inhibits the expression of ERK1/2, thus attenuating the development of pulmonary hypertension and improving pulmonary vascular remodeling.     

Effects of monocrotaline-induced pulmonary hypertension on expression of Hippo signaling pathway-related molecules in rat lung

AIM:To investigate the expression of Hippo signaling pathway-related molecules in the lung tissues of the rats with pulmonary hypertension induced by monocrotaline for exploring the significance of Hippo signaling pathway in the development of pulmonary hypertension. METHODS:SD rats (n=45) were randomly divided into control group (n=15) and model group (n=30). The rats in model group was given neck subcutaneous injection of monocrota-line at 60 mg/kg to establish pulmonary hypertension model, and the rats in control group was injected with the same volume of normal saline. Four weeks later, right ventricular systolic pressure (RVSP) was measured by right cardiac catheterization, and right ventricular hypertrophy index (RVHI) and right ventricular mass index (RVMI) were calculated. The remodeling of the pulmonary arterioles was observed by HE staining, and medial thickness/external diameter (M/E%) was evaluated. The fibrosis of lung tissues was detected by Masson staining. The protein expression of Yes-associated protein (YAP), tafazzin (TAZ) and TEAD was detected by immunohistochemistry, and the protein and mRNA levels of YAP, TAZ and TEAD in lung tissues were determined by Western blot and RT-qPCR. RESULTS:Compared with control group, the vascular wall in model group was thickened significantly, the M/E% was increased (P<0.01), the pulmonary fibrosis was obvious, and the RVSP and RVHI in model group were significantly higher than those in control group (P<0.01). The immunohistochemical staining showed that the protein expression of YAP, TAZ and TEAD in the pulmonary arterioles in model group was significantly higher than that in control group. The YAP, TAZ and TEAD protein and mRNA levels in the lung tissues were also higher than those in control group (P<0.05). CONCLUSION:The activation of Hippo signaling molecules may promote the remodeling of pulmonary arterioles and further regulate the development of monocrotaline-induced pulmonary hypertension.     

Effect of chronic hypoxia on L-Arginine/nitric oxide pathway in rat pulmonary artery

AIM: To study the effect of chronic hypoxia on L-Arginine/NO pathway in rat pulmonary artery. METHODS: Changes in pulmonary artery L-Arginine(L-Arg) transport, nitric oxide synthase (NOS) activity, plasma nitrite level and L-Arg level in HPH rats were investigated. RESULTS: (1) The mean pulmonary arterial pressure (mPAP) and weight ratio of right ventricle to left ventricle and septum (RV/LV+S) of HPH group were higher than those in control group (P＜0.01). (2) Plasma L-Arg level in HPH group was not significantly changed. (3) At low (0.2 mmol/L)or high(5.0 mmol/L)concentration of L-Arg, the velocity of L-Arg transport in HPH group was lower than that in control group (P＜0.05 or P＜0.01). (4) The activity of pulmonary artery tNOS, iNOS and cNOS in HPH group were increased by 38.0%, 32.8% and 53.0%, respectively (P＜0.01), compared with control group. (5) Plasma NO level of HPH group was decreased, which was negative correlation to mPAP and RV/LV+S (P＜0.01). CONCLUSION: The decrease of nitric oxide generation might result from L-Arg transport injury, while pulmonary artery tNOS, iNOS and cNOS activity were enhanced during chronic hypoxia.     

Effect of polidatin on the activity of phospholipase A2and pulmonary structural remodeling in rats with hypoxia-induced pulmonary hypertension

AIM: To study the relationship between the activity of phospholipase A₂ (PLA₂) and pulmonary structural remodeling with the model of chronic isobaric hypoxic pulmonary hypertension. METHODS: 29 healthy Sprague-Dawley rats were randomly divided into normal control group, chronic hypoxic group and hypoxia plus Polidatin (PD) group. By diameter, the arteries were divided into two groups: arteries of group I (30 μm-100 μm) and group II (101 μm-200μm). The mean pulmonary arterial pressure (mPAP) was measured by inserting a microcatheter into the pulmonary artery. The PLA₂ activity was measured with modified microtitrimetic method. The pulmonary tissue and arterioles morphology changes were examined under light microscope. RESULTS: It was found that after 21 days hypoxia, the mean pulmonary arterial pressure (mPAP), the PLA₂ activity in blood and lung homogenate increased significantly. The media thickness of group I arteries increased (P<0.01) while that of group II arteries had no significant changes. The ratio of media area and adventitia of both groups was raised. Under light microscope, it was observed that pulmonary vascular endothelium proliferated, media became thickening and adventitia matrix increased. Pretreatment with PD could attenuate the changes mentioned above. CONCLUSION:PLA₂ plays an important inducing role through promotion of the pulmonary vascular structural remodeling in the formation of chronic hypoxic pulmonary hypertension.     

Hypoxia simulated to high altitude promotes right ventricular ACE2 activity in adult SD rats independent of pulmonary artery hypertension

AIM: To determine the right ventricular angiotensin converting enzyme2 (ACE2) activity in adult SD rats under normoxia and hypoxia environment, and to detect the relationship between ACE2 and high altitude heart disease initially. METHODS: Adult male Sprague Dawley (SD) rats were raised under hypoxia environment simulated to high altitude (5 000 m, 23 h/d), then divided into hypoxia 1 d, 15 d and 30 d groups randomly. The control group was set up under normoxia environment. The right ventricular function, ponderal index, pulmonary artery pressure and right ventricular ACE2 activity were determined. The effect of captopril or nitrendpine on cardiac ACE2 activity in hypoxia 30 d group was also observed. RESULTS: The dramatic up-regulation of cardiac ACE2 expression of mRNA and protein and its activity in hypoxia 30 d group was observed, together with obvious increase in right ventricular function, ponderal index and pulmonary artery pressure. Although captopril or nitrendipine depressed pulmonary artery pressure and cardiac function dramatically, no significant alteration of right ventricular ACE2 activity was detected. CONCLUSION: Chronic hypoxic exposure promotes the ACE2 expression and activity in right ventricular, indicating that ACE2 may be correlated to the changes of cardiac architecture and function induced by hypoxia. Up-regulation of ACE2 expression maybe contribute to the increase in right ventricular ACE2 activity, and pulmonary artery hypertension may not be the main reason for the changes of ACE2 activity under hypoxia environment.