Inhibitory effect of notoginsenoside monomer R1 on activation of p38 MAPK signaling pathway induced by hypoxic hypercapnia in PASMCs

AIM: To explore the mechanism of notoginsenoside monomer R₁ (R₁) against hypoxic hypercapnia-induced pulmonary vasoconstriction (HHPV) by investigating the effect of R₁ on p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in pulmonary arterial smooth muscle cells (PASMCs) under the condition of hypoxia and hypercapnia. METHODS: Primary cultured PASMCs, which were isolated from Sprague-Dawley rats, were incubated in logarithmic growth phase from the 2nd to 5th generation with different concentrations (8, 40 and 100 mg/L) of R₁ under the condition of 6% CO₂ plus 1% O₂ for 24 h. The expression of p38 at mRNA and protein levels was detected by RT-PCR and Western blotting,respectively. RESULTS: The results of Western blotting and RT-PCR analysis indicated that the protein and mRNA expression levels of p-p38 MAPK were significantly higher in hypoxic hypercapnia group with DMSO control than those in normoxia control group (P<0.01). In R₁ treatment groups, the levels of p-p38 MAPK protein and p38 MAPK mRNA were markedly decreased (P<0.01) in a dose-dependent manner. CONCLUSION: p38 MAPK signaling pathway may mediate hypoxic hypercapnia pulmonary vasoconstriction in rats. Notoginsenoside monomer R₁ attenuates HHPV, which may be related to blockage of p38 MAPK signal pathway.     

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.     

Effect of hydroxylamine on pulmonary arterial hypertension induced by chronic hypoxic hypercapnia in rats

AIM: To explore the effects of hydroxylamine on the pulmonary arterial pressure in chronic hypoxic hypercapnic rats. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into 3 groups (8 rats in each group): the normal control group (NC), hypoxic hypercapnia+normal saline group (NS), hypoxic hypercapnia+hydroxylamine group (HA). The animals in NS and HA groups were kept in the O₂ (9%-11%) and CO₂ (5%-6%) cabin, 8 h a day and 6 days a week for 4 weeks. Before entering the cabin, the rats in HA group were administered with 1 mL hydroxylamine (12.5 mg/kg) by intraperitoneal injection, while the rats in NS group were given intraperitoneal injection of 1 mL saline solution. The mean pulmonary arterial pressure (mPAP) was measured by external jugular vein cannulation. The heart was removed, and the right ventricle (RV) and the left ventricle plus the septum (LV+S) were dissected. The ratio of the wet weight of the RV to that of the LV+S was calculated. The changes of the pulmonary vascular construction were observed under optical microscope. The concentration of H₂S in the plasma was measured with a spectrometer. The expression of cystathionine-γ-lyase (CSE) in the pulmonary arterioles and bronchi was measured by immunohistochemistry and RT-PCR. RESULTS: The values of mPAP, RV/(LV+S),vessel wall area/total area (WA/TA) and media thickness of pulmonary arterioles (PAMT) in NS group and HA group were significantly higher than those in NC group (P<0.05). The level of H₂S in the plasma, the content of CSE protein and the expression of CSE mRNA in NC group were significantly lower than those in NS group (P<0.05). The values of mPAP, RV/(LV+S), WA/TA and PAMT in HA group were significantly lower than those in NS group (P<0.05). The level of H₂S in the plasma, the content of CSE protein and the expression of CSE mRNA in HA group were significantly higher than those in NS group (P<0.05). CONCLUSION: Hydroxylamine may decrease the pulmonary arterial hypertension induced by chronic hypoxic hypercapnia in rats by increasing the level of H₂S in the plasma, the content of CSE protein and the mRNA expression of CSE, thus improving the pulmonary vascular structural remodeling.     

Effects of hypoxia and hypercapnia on expression of soluble guanylate cyclase in rat pulmonary artery

AIM:To investigate the expression of soluble guanylate cyclase protein and its mRNA in rat pulmonary artery after exposure to hypoxia and hypercapnia.METHODS:Male Sprague-Dawley rats were randomly split into 4 group, which were hypoxic hypercapnic (HH 1 week, HH 2 weeks, HH 4 weeks) group and control group, to copy pulmonary hypertensive animal model. The expression of sGCα₁ and β₁ subunits protein of medial and small pulmonary artery was performed by immunohistochemistry with a polycolonal antibody. In situ hybridization was performed on the rat lung tissue using sGC oligonuclear probe to assay the expression of sGCα₁subunit mRNA.RESULTS:The sGCα₁ and β₁ subunits protein and sGCα₁ subunit mRNA were faint staining in the pulmonary small and medium artery in HH1 week, HH 2 weeks and HH 4 weeks groups compared with control group (all P＜0.01).CONCLUSION:sGC subunit mRNA and protein expression in pulmonary small and medium artery were decreased after exposure to hypoxia and hypercapnia, which took part in the development of the pulmonary hypertension.     

Relationship between expression of heme oxygenase-1 mRNA and pulmonary hypertention induced by hypoxic hypercapnia

AIM: To study the effect of chronic hypoxic hypercapnia on expression of heme oxygenase-1 (HO-1). METHODS: Sprague-Dawley rats were randomly divided into three groups: control group(A),hypoxic hypercapnic group(B), hypoxic hypercapnia+hemin group(C). HO-1 and HO-1 mRNA were observed in pulmonary arterioles by the technique of immunohistochemistry and in situ hybridization. RESULTS: ① mPAP and weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) were significantly higher in rats of B group than those of A and C group (P<0.01). Differences of mCAP were not significant in three groups(P>0.05). ② Blood CO concentration was significantly higher in rats of B group than that of A group (P<0.01), it was much higher in C group than that of B group(P<0.01). ③ Light microscopy showed that vessel well area/total area (WA/TA), density of medial smooth muscle cell (SMC) and media thickness of pulmonary arterioles were much higher in rats of B group than those of A and C group (P<0.01). ④ The observation by electron microscopy showed proliferation of medial smooth muscle cells and collageous fibers of pulmonary arterioles in rats of B group, hemin could reverse the changes mentioned above. ⑤ HO-1 and HO-1 mRNA in pulmonary arterioles was significantly higher in rats of B group than those of A group(P<0.01), and they were significantly higher in rats of C group than those of B group (P<0.01). CONCLUSION: Expression of HO-1 mRNA and HO-1 in pulmonary arterioles was enhanced by hypoxic hypercapnia. Hemin partly inhibited pulmonary hypertension and pulmonary vessel remodeling by enhancing the expression of HO-1 mRNA and HO-1.     

USP14 regulates H2O2 induced oxidative stress in H9c2 cells

AIM:To evaluate the effect of inhibiting ubiquitin-specific protease 14(USPl4) activity on oxidative stress induced by H₂O₂ of H9c2 cells.METHODS:The H9c2 cells were incubated with H₂O₂ at 25 μmol/L for 2 h to establish the oxidative stress injury model.The cells were divided into control group,H₂O₂ group,IU1 group (25 μmol/L or 50 μmol/L) and IU1+H₂O₂ group.The H9c2 cells activity was measured by MTS assay.The level of intracellular reactive oxygen species (ROS) and cell survival rate were analyzed by flow cytometry assay.The changes of the mitogen-activated protein kinase (MAPK) family related proteins were detected by Western blot.RESULTS:Compared with control group,the cell activity and the viability rate in H₂O₂ group were decreased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were increased (P<0.05).Compared with H₂O₂ group,the cell activity and the viability rate of the H9c2 cells in IU1+H₂O₂ group were increased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were decreased (P<0.05).CONCLUSION:Inhibition of USPl4 activity reduces the oxidative stress injury of the H9c2 cells.The mechanism may be related to inhibition of the MAPK signaling and down-regulation of apoptosis related proteins.     

Relationship between thromboxane synthase gene and prostacyclin synthase gene expression and pulmonary hypertension induced by hypoxic hypercapnia

AIM: To study the effect of chronic hypoxic hypercapnia on gene expression of thromboxane synthase and prostacyclin synthase in pulmonary arterioles. METHODS: Sprague-Dawley rats were randomly divided into two groups: control group and hypoxic hypercapnic group. TXS mRNA and PGI₂-SmRNA were observed in pulmonary arterioles by in situ hybridization. RESULTS: mPAP, weight ratio of right ventricle (RV) to left ventricle plus septum(LV+S), contents of TXB₂ and 6-keto-PGF1_α in plasma and lung and TXS mRNAin pulmonary arterioles were much higher in rats of hypoxic hypercapnic group than those of control group. Differences of PGI₂-SmRNA in pulmonary arterioles were not significant in two groups. Light microscopy showed hypertrophy of vessel smooth muscle cells and vessel cavity straitness were found in hypoxic hypercapnic group. CONCLUSION: Changes of gene expressions of thromboxane synthase and prostacyclin synthase and imbalance of TXA₂/PGI₂ may play an important role in hypoxic hypercapnic pulmonary hypertension.     

Changes of interleukin-6 in pulmonary hypertension mice induced by chronic hypoxic-hypercapnia

AIM: To investigated the changes of interleukin-6 (IL-6) in the pulmonary hypertension mice induced by chronic hypoxic hypercapnia. METHODS: Sixteen male C57BL/6 mice were randomly divided into 2 groups (8 mice in each group): normal control group and chronic hypoxic hypercapnia group. The mice in chronic hypoxic hypercapnia group were placed in a sealed chamber where O₂ concentration was kept at 9%~11%, and the CO₂ concentration at 5% ~6%, 8 h a day, 6 days a week for 4 weeks. The right ventricular (RV) weight, the weight of left ventricle plus ventricular septum (LV+S) were measured and right ventricular hypertrophy index was calculated. The structural changes of the pulmonary arteries were assessed by the method of histology with HE staining. The vessel wall diameter/total diameter (WT%) and the vessel wall area/total area (WA%) were analyzed by Image-Pro Plus 6.0 software. The protein expression of IL-6 in the lungs of the mice was determined by immunohistochemistry and ELISA, and the mRNA expression of IL-6 in the lungs was determined by RT-PCR. RESULTS: Compared with control group, RV/(LV+S), MT%, MA% and the expression of IL-6 at mRNA and protein levels were significantly increased in chronic hypoxic hypercapnia group. CONCLUSION: In the environment of chronic hypoxia and hypercapnia, the expression of interleukin-6 was elevated in mouse lungs, which may closely related to the development of pulmonary hypertension.     

Phosphatidylinositol 3-kinase regulates hypoxia-inducible factor 1α in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension

AIM:To investigate relationship among phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in lung of rats with hypoxia-inducible pulmonary hypertension. METHODS:Forty male adult Wistar rats were randomly divided into five groups (eight rats in each group):control group (C group) and groups with hypoxia for 3, 7, 14 and 21 days (H3, H7, H14 and H21 group). Mean pulmonary arterial pressure (mPAP), right ventric hypertrophy index (RVHI) and vessel morphometry were measured. The levels of HIF-1α mRNA expression in lung tissue was measured by in siteu hybridization (ISH). The protein expression of HIF-1α，VEGF and phosphorylated protein kinase β (P-AKT) were observed by immunohistochemistry or Western blotting. RESULTS:mPAP increased significantly 7 days after hypoxia ［(23.53±1.78) mmHg］, peaked 14 days after hypoxia, then remained on the high level. Pulmonary artery remodeling index (extern diameter 100 μm) and RVIH became evident 14 days after hypoxia. Expression of P-AKT protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima and tunica media in all hypoxia rats. HIF-1α mRNA staining was poorly positive in control，hypoxia for 3 days and hypoxia for 7 days, but began to increase significantly 14 days after hypoxia (0.305±0.104, P<0.05), then remained stable. Expression of HIF-1α protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima in all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1α protein was markedly up-regulated after 3 days (0.029±0.019, P<0.05 ), reached its peak 7 days after hypoxia (0.232±0.008, P<0.05), then tended to decline 14 days and 21 days after hypoxia. Expression of VEGF protein began to increase 7 days after hypoxia (0.188±0.018, P<0.05), reached its peak 14 days after hypoxia (0.238±0.017, P<0.05), then remained on the high level in pulmonary arterial tunica intima. Linear correlation analysis showed that P-AKT, HIF-1α mRNA, VEGF and mPAP were correlated with vessel the morphometry and RVHI (P<0.01). P-AKT was positively correlated with HIF-1α and VEGF (tunica intima). CONCLUSION:P-AKT, HIF-1α and VEGF are all involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats.     

Effect of ligustrazine on pulmonary arterial pressure and pulmonary arterial wall collagen of chronic hypoxic hypercapnic rats

AIM:To study the effect of ligustrazine on pulmonary hypertensive rats induced by hypoxic hypercapnia. METHODS:Thirty rats were randomly divided into three groups:control group(A),hypoxic hypercapnic group(B), hypoxic hypercapnia+ligustrazine(lig.) group(C). RESULTS: (1) Mean pulmonary arterial pressure(mPAP)of group B was significantly higher than that of group A and mPAP of group C was significantly lower than that of group B(P＜0.01),differences of mean carotid pressure(mCAP) were not significant among three groups (P＞0.05); (2)Electron microscopy and immunohistochemistry showed ligustrazine could inhibit the diposition of collagenous fiber(collagen typeⅠ)in pulmonary arterioles induced by hypoxic hypercapnia; (3) Plasma endothelin level of group C was significantly lower than that of group B (P＜0.01), serum (NO ₂^-/NO₃^-) of group C was significantly higher than that of group B (P＜0.01). CONCLUSION:Ligustrazine can inhibit pulmonary hypertension and the diposition of collagen type Ⅰ in pulmonary arterial wall induced by hypoxic hypercapnia.     

Effect of diltiazem on pulmonary arterial pressure and ceNOS mRNA expression in pulmonary arteries in chronic hypoxic hypercapnic rats

AIM: To investigate the effect of diltiazem on mean pulmonary arterial pressure (mPAP) and nitric oxide synthase (NOS) in arterioles in chronic hypoxic hypercapnic rats. METHODS: Twenty-four rats were randomly divided into three groups: control group (A), hypoxic hypercapnic group (B), hypoxic hypercapnia+ diltiazem group (C), constitutive endothelial NOS (ceNOS) were observed in arterioles of rats using the technique of immunohistochemistry, ceNOS mRNA were observed by the technique of in situ hybridization. RESULTS: (1) mPAP was significantly higher in rats of B group than that of A and C group(P<0.01). Differences of mCAP were not significant between A group and B groups (P>0.05), but mCAP was lower in rats of C group than that in B group. (2) Light microscopy showed WA/TA (vessel wall area/total area) was significantly lower in rats of C group than that of B group (P<0.01), electron microscopy showed that diltiazem inhibited the proliferation of smooth muscle cells and collageous fibers of pulmonary arterioles in chronic hypoxic hypercapnic rats. (3) Immunohistochemistry showed the average value of integral light density (LD) of ceNOS in pulmonary arterioles was significantly higher in rats of C group than that of B group (P<0.01), in situ hybridization showed LD of ceNOS mRNA in pulmonary arterioles was significantly higher in rats of C group than that of B group (P<0.01). CONCLUSION: Diltiazem inhibited pulmonary hypertension, the proliferation of smooth muscle cells and collagenous fibers of pulmonary arterioles in chronic hypoxic hypercapnic rats by incresing the expression of ceNOS in pulmonary arterioles.     

Changes of NO- and H2O2-dependent soluble guanylate cyclase pathway in the hypoxic hypercapnic pulmonary hypertension rats

AIM: To study the effect of hypoxia and hypercapnia on nitric oxide (NO) in plasma and superoxide dismutase (SOD), catalase (CAT), soluble guanylate cyclase (sGC), cyclic guanosine monophospholate (cGMP) in lung tissue in rats, and to explore the effect of NO- and H2O2-sGC pathway on the development of the pulmonary hypertension. METHODS: The model of hypoxic and hypercapnic 1, 2, 4-week group (HH 1 week, HH 2 weeks, HH 4 weeks) and control group was set up. NO content in plasma, CAT and SOD in rat lung were determined by spectrophotometry. The sGC activity in lung tissue was detected by enzyme kinetic analysis. cGMP content in lung tissue was examined with ［125I］-radioimmunoassay. RESULTS: The mean pulmonary artery pressure (mPAP) showed significantly higher in HH 1 week, HH 2 weeks and HH 4 weeks groups compared with control group (all P<0.05). NO concentration in plasma, CAT, SOD, basal or nitroprusside-or H2O2- stimulated sGC activity and cGMP concentration in lung homogenates were significantly lower (P<0.05, P<0.01, P<0.01, respectively) in HH 1 week, HH 2 weeks and HH 4 weeks groups compared with control group. CONCLUSION: The inhibition of NO- and H2O2-sGC pathway by hypoxia and hypercapnia plays an important role in the development of 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.     

新书推荐：《分子克隆实验指南》（第三版）

AIM: To study the effect of panax notoginseng sponins (PNS) on L-type Ca²⁺ current in isolated right ventricle myocytes from chronic hypoxic rats. METHODS: Using whole cell patch clamp recording technique,we measured I_Ca-L in isolated right ventricle myocytes which were divided into three group:control group, chronic hy-poxic group and chronic hypoxic group with PNS(100 mg·kg^-1·d^-1). RESULTS: The result showed I_Ca-L of cells from chronic hypoxic group were significantly larger than the other two groups(P＜0.05). CONCLUSION: PNS decreases L-type Ca²⁺ current of the right ventricle myocytes from chronic hypoxic rats.     

Effect of ligustrazine on hydrogen sulfide system in lung tissue of rats with pulmonary hypertension induced by hypoxic hypercapnia

AIM: To investigate the effect of ligustrazine on hydrogen sulfide (H2S) system in pulmonary hypertension induced by hypoxic hypercapnia in rats. METHODS: Thirty Sprague-Dawley rats were randomly divided into 3 groups: control group (C), hypoxic hypercapnia group (HH), and hypoxic hypercapnia+ligustrazine group (HH+L). The change of hemodynamics was measured. The ratio of vessel wall area and total area of arteriae pulmonalis were observed under light microscope. The apoptosis of arteriae pulmonalis was tested with TdT-mediated dUTP nick end labeling (TUNEL), and the apoptosis index was calculated. Plasma level of hydrogen sulfide and activity of hydrogen sulfide generating enzymes in homogenates of rat lung tissue were evaluated by sensitive modified sulfide electrode method. Cystathionine-γ-lyase (CSE) mRNA in lung tissues was determined by RT-PCR. RESULTS: The level of mean pulmonary arterial pressure, the ratio of vessel wall area/total area and the right ventricle/left ventricle+septum were significantly higher in HH group than those in C group, and the value was obviously lower in HH+LTZ group than that in HH group (all P<0.01). The mean carotid arterial pressure of 3 groups had no significant difference (P>0.05). The apoptotic index of arteriae pulmonalis in HH group and HH+LTZ group was significantly lower than that in C group, and that in HH+LTZ group was significantly higher than that in HH group (P<0.05 or P<0.01). Plasma level of H2S, the activity of H2S generating enzymes in homogenates of rat lung tissue, cystathionine-γ-lyase (CSE) mRNA in lung tissues in HH group were significantly lower than those in C group (all P<0.01), and those in HH+LTZ group were significantly lower than those in HH group (all P<0.01). CONCLUSION: Ligustrazine up-regulates the expression of cystanthionine-γ-splitting enzyme (CSE), enhances the activity of CSE and increases the level of H2S to prevent pulmonary hypertension induced by hypoxic hypercapnia.     

Role of reactive oxygen species and ERK1/2 signaling pathway in proliferation and apoptosis of hypoxic rat pulmonary arterial smooth muscle cells

AIM:To investigate the change of reactive oxygen species (ROS) production in hypoxic pulmonary arterial smooth muscle cells (PASMCs) of rats, the effect of ROS on the expression of extracellular signal-regulated kinase (ERK)1/2 protein, and the role of ROS and ERK1/2 in the imbalance between proliferation and apoptosis of PASMCs.METHODS: Primary cultures of PASMCs were established and cells between passages 2 to 3 were used for experiments. PASMCs were treated with tiron, a membrane permeable ROS scavenger, and PD98059, an ERK1/2 inhibitor, under normoxia or hypoxia condition. The ROS production was measured by DCFH-DA and NBT reduction. The expression of phosphorylated-ERK1/2 (p-ERK1/2) protein was detected by immunofluorescence. Cell proliferation was examined by MTT colorimetric assay and the expression of PCNA. Cell apoptosis was detected by TUNEL.RESULTS: (1)Compared with control group, the ROS levels in hypoxia group were significantly increased (P<0.01). (2) In hypoxia group, the proliferative capacity was higher and the apoptosis index was lower than those in control group (P<0.01). Tiron significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). (3) The expression of p-ERK1/2 in hypoxia group were higher than that in control group (P<0.01), which were significantly suppressed by tiron (P<0.01)．(4) PD98059 significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). The proliferative capacity and apoptosis index was similar in hypoxia+tiron+PD98059 group to those in hypoxia+tiron group (P>0.05).CONCLUSION:The hypoxia-mediated increase in PASMCs proliferation and the decrease in PASMCs apoptosis are related to the overproduction of intracellular ROS through downstream activation of ERK1/2. ROS and ERK1/2 play important roles in the hypoxic remodeling of pulmonary artery.     

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 relationship between the activity of phospholipase A2 and chronic hypoxic pulmonary hypertension

AIM:To study the relationship between the activity of phospholipase A₂ (PLA₂) and chronic hypoxic pulmonary hypertension. METHODS:29 healthy SD rats were randomly divided into normal control group, chronic hypoxic group and hypoxia plus Polidatin (PD) group. The model of rat chronic hypoxic pulmonary hypertension was made by method of intermittent isobaric hypoxia for 21 days. The mean pulmonary arterial pressure (mPAP) was measured by inserting a microcatheter into the pulmonary artery. RESULTS:After exposing hypoxia for 21 days, the mPAP, R/L+S, the PLA₂ activity, TXB₂, MDA in plasma and lung homogenate increased significantly, while 6-k-PGF_1α, SOD decreased significantly. Pretreatment with PD could relieve the changes mentioned above.CONCLUSION:PLA₂ plays an important inducing role through its metabolic products and the interactional radicals in the formation of chronic hypoxic pulmonary hypertension.     

Role of EndoMT in HHPH based on TGF-β/Smads signaling pathway and regulated by Yiqi-Wenyang-Huoxue-Huatan formula

AIM: To investigate the relationship between transforming growth factor-β (TGF-β)/Smads signaling pathway and pulmonary arterial endothelial-mesenchymal transition (EndoMT) in hypoxia-hypercapnia pulmonary hypertension (HHPH) process and the regulatory effect of Yiqi-Wenyang-Huoxue-Huatan formula (YWHHF). METHODS: Healthy male SD rats were randomly divided into 5 groups:normal control (N) group, hypoxia-hypercapnia (HH) group, high-dose YWHHF (YH) group, middle-dose YWHHF (YM) group and low-dose YWHHF (YL) group. The rats in N group was housed in normoxic environment, and the rats in the other 4 groups were housed in hypoxia-hypercapnia environment (9%~11% O₂ and 5%~6% CO₂) for 4 weeks, 8 h/d, 6 d/week. The excess water vapor was absorbed by anhydrous CaCl₂, and CO₂ was absorbed by sodium hydroxide. The rats in YWHHF groups were put into the oxygen chamber before the same volume of YWHHF at different concentrations were given (200 g/L for YH group, 100 g/L for YM group and 50 g/L for YL group). The average pulmonary artery pressure and the average carotid artery pressure were measured during the operation. After operation, the right ventricular free wall and left ventricle plus interventricular septum were collected for determining the right ventricular hypertrophy index. Moreover, the morphological changes of the lung tissues were observed under light microscope. The mRNA and protein levels of α-smooth muscle actin (α-SMA), CD31, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were detected by RT-PCR and Western blot. RESULTS: Compared with N group, the pulmonary artery mean pressure, the mRNA and protein expression of α-SMA, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were increased, the levels of CD31 were decreased (P<0.05), and the lung tissue damage was observed in the other 4 groups. Compared with HH group, the pulmonary artery mean pressure, the mRNA and protein expression of α-SMA, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were decreased, while the mRNA and protein levels of CD31 were increased. Moreover, the lung tissue damage was reduced in YH, YM and YL groups. CONCLUSION: TGF-β/Smads pathway may be involved in the process of EndoMT under hypoxia and hypercapnia condition, and YWHHF may reduce EndoMT by inhibiting the expression of TGF-β/Smads pathway-related molecules.