Expression of volume-activated chloride channel in rat PASMCs treated with hypoxia and hypercapnia and its relationship with MAPK pathway

AIM：To investigate the expression of volume-activated chloride channel (CLC3) in rat pulmonary artery smooth muscle cells (PASMCs) treated with hypoxia and hypercapnia and its relationship with MAPK pathway. METHODS：The method of enzyme digestion was used to isolate the PASMCs in male SD rat for cell primary culture. The cells were identified by immunofluorescence cytochemical method with mouse anti-rat α-smooth muscle actin antibody. The rat model of hypoxia and hypercapnia was established. The protein expression of CLC3 was detected by Western blotting. The mRNA expression of CLC3 was determined by RT-PCR. RESULTS：Compared with control group, the mRNA and protein expression of CLC3 in PASMCs was significantly raised in hypoxia and hypercapnia group. Compared with hypoxic and hypercapnic group, the expression of CLC3 was significantly reduced in ERK inhibitor U0126+ hypoxia and hypercapnia group, and was up-regulated in p38 inhibitor SB203580+ hypoxia and hypercapnia group. p38 activator anisomycin significantly decreased the expression of CLC3 at mRNA and protein levels in hypoxia and hypercapnia group. CONCLUSION：The expression of CLC3 at mRNA and protein levels in PASMCs increases under hypoxia and hypercapnia conditions. The ERK1/2 pathway mediates CLC3 expression in PASMCs induced by hypoxia and hypercapnia. Activation of p38 MAPK pathway down-regulates the expression of CLC3 at mRNA and protein levels induced by hypoxia and hypercapnia.     

Farrerol inhibits nicotine-induced proliferation of pulmonary vascular smooth muscle cells by enhancing Kv1.5 and Kv2.1 expression

AIM:To study the effect of farrerol (Far) on nicotine-induced proliferation of rat pulmonary smooth muscle cells (PASMCs), and further to explore its relationship with voltage-dependent potassium channels (Kv) 1.5 and Kv2.1. METHODS:Firstly, the effect of nicotine on the proliferation of PASMCs was detected by cell counting method, and the optimal concentration of nicotine was selected. Primary cultured PASMCs were randomly divided into 5 groups:normal control group, nicotine (1 μmol/L)group, nicotine (1 μmol/L) + Far (10^-6 mol/L, 10^-5 mol/L and 10^-4 mol/L) Far group. The activity of caspase-3 was measured by apoptosis kit, the cell viability was measured by CCK-8 assay, the apoptotic rate was analyzed by flow cytometry. The expression of Kv1.5 and Kv2.1, and apoptosis-related factors Bcl-2 and Bax at mRNA and protein levels was determined by RT-qPCR and Western blot respectively. RESULTS:Nicotine at 1 μmol/L increased the number of PASMCs to the maximum extent (P<0.01). Nicotine at 1 μmol/L significantly reduced the caspase-3 activity and enhanced the cell viability of the PASMCs (P<0.01). Farrerol at 10^-6~10^-4 mol/L eliminated the effect of PASMCs induced by nicotine in a concentration dependent manner. Compared with control group, nicotine at 1 μmol/L significantly increased the proliferation and inhibited the apoptotic rate of rat PASMCs (P<0.01). The apoptotic rate of PASMCs in farrerol intervention group was significantly higher than that in nicotine group (P<0.01). Nicotine at 1 μmol/L significantly inhibited the expression of Kv1.5, Kv2.1 and Bax but increased the expression of Bcl-2 in PASMCs (P<0.01). Farrerol at 10^-5 mol/L obviously inhibited the effect of PASMCs induced by nicotine. CONCLUSION:Farrerol eliminates nicotine-induced inhibition of caspase-3 and Bax, and enhancement of Bcl-2 in PASMCs by enhancing Kv1.5 and Kv2.1 expression.     

Role of SB203580 in enhancement of hypoxia hypercapnia-induced pulmonary vasoconstriction by voltage-dependent K+ channel blocker 4-aminopyridione in rats

AIM:To investigate the roles of voltage-dependent K+ channel (KV) and p38 mitogen-activated protein kinase (MAPK) signal pathway in the pathological process of hypoxia hypercapnia-induced pulmonary vasoconstriction (HHPV) in rats. METHODS:The second-order pulmonary artery rings isolated from SD rats in vitro was prepared, and randomly divided into control group (N group), hypoxia hypercapnia group (H group), hypoxia hypercapnia+DMSO incubation group (HD group), hypoxia hypercapnia+4-aminopyridione (4-AP) group (4-AP group), hypoxia hypercapnia+SB203580 incubation group (SB group) and hypoxia hypercapnia+4-AP+SB203580 incubation group (4-AP+SB group). Under acute hypoxia hypercapnia condition, the changes of the 3 stages of HHPV incubated by 4-AP or the combined application of 4-AP and SB203580 were observed. At the same time, the tension values of the rings were recorded. RESULTS:Under hypoxia hypercapnia condition, a biphasic pulmonary artery contractile response was observed. The phase II persistent vasoconstriction of the pulmonary artery rings incubated with 4-AP was enhanced. Under hypoxia hypercapnia condition, SB203580 significantly relieved the phase II persistent vasoconstriction induced by 4-AP. CONCLUSION: The KV blocker 4-AP enhances HHPV. SB203580 significantly relieves the phase II persistent vasoconstriction induced by 4-AP, indicating that the participation of p38 MAPK may be one of the important mechanisms for the regulation of HHPV by KV in the rats.     

Effect of bim silencing by siRNA on hypoxia-induced apoptosis of rat cardiomyocytes

AIM：To investigate the effect of BH3-only protein Bim (Bcl-2 interacting mediator of cell death) on apoptosis of rat cardiomyocytes induced by hypoxia. METHODS：Rat cardiomyocytes were isolated from infant rats aged 1~3 days and then primarily cultured. The antibody targeting α-actin of striated muscle was used to identify the cardiomyocytes. The siRNAs of bim were transfected into the cardiomyocytes with liposome, and the expression of Bim was determined by Western blotting. The cardiomyocytes were divided into blank control group, hypoxia group, hypoxia+liposome group, hypoxia+negative control siRNA group and hypoxia+bim-siRNA group.The frequency and rhythm of cardiomyocyte beating were observed and recorded under inverted microscope. The activity of lactate dehydrogenase (LDH) in the culture medium was assessed by automatic biochemical analyzer. The viability of the cells was analyzed by MTT assay. The cell apoptotic rate was measured by flow cytometry. The protein expression of Bim, Bax, Bcl-2, p-p38 MAPK and p38 MAPK was detected by Western blotting. RESULTS：Immunohistochemical identification confirmed that the rat cardiomyocytes were successfully cultured. The expression of Bim was obviously inhibited after transfected with bim-siRNAs and the silencing efficiency of bim-siRNA-2 was the highest (86.73%). The frequency of cardiomyocyte beating was slowed down after hypoxia and the rhythm was disordered, while the frequency of beating was obviously increased after silencting the expression of bim. Compared with control group, the LDH in the culture medium was increased (P<0.01), and the viability of the cardiomyocytes was reduced in hypoxia group (P<0.05). The apoptotic rate was increased (P<0.01). After transfection with bim-siRNA, the release of LDH was decreased, and the viability of the cardiomyocytes was increased. The apoptotic rate was decreased. The results of Western blotting showed that hypoxia increased the expression of Bax and p-p38 MAPK (P<0.05), and decreased the expression of Bcl-2 (P<0.01), while transfection with bim-siRNA reduced the effects caused by hypoxia (P<005). These were greatly related to the decrease of apoptosis. However, the expression of p38 MAPK was not changed. CONCLUSION：The apoptosis of cardiomyocytes induced by hypoxia can be inhibited by silencing the expression of bim gene by down-regulation of p-p38 MAPK and Bax expression and up-regulation of Bcl-2 expression.     

Role of MAPK inhibition in decrease of hypoxia hypercapnia-induced pulmonary vasoconstriction mediated by Panax notoginseng saponin R1 in rats

AIM：To investigate the role of Panax notoginseng saponin R1 in the pathological process of hypo-xia hypercapnia-induced pulmonary vasoconstriction (HHPV) and to observe the relationship with MAPK signal pathway in rats. METHODS：The model of pulmonary artery ring perfusion in vitro was used, and the rings were divided randomly into the following groups: normoxia group (N group); hypoxia hypercapnia group (H group); H+DMSO incubation group (HD group); H+R1 group, which was divided into 3 subgroups: low-concentration R1 group (RL group), middle-concentration R1 group (RM group) and high-concentration R1 group (RH group); H+SB203580 (p38 MAPK inhibitor) incubation group (S group); H+U0126 (ERK1/2 inhibitor) incubation group (U group); H+R1+SB203580 incubation group (RS group); H+R1+U0126 (RU group). Under acute hypoxia hypercapnia condition, the effects of different concentrations of R1 or R1 at the optimal concentration combined with U0126 or SB203580 on the 3 stages of HHPV were observed. At the same time, the changes of ring tension were recorded via the method of hypoxia hypercapnia condition reactivity. RESULTS：Under the hypoxia hypercapnia condition, a biphasic pulmonary artery contractile response (phase I acute vasoconstriction, phase I vasodilation and phase II persistent vasoconstriction) in the secondary pulmonary artery rings was observed. The treatments in HD group and RL group distinctly relieved the early phase I acute vasoconstriction of HHPV and reversed the phase II persistent vasoconstriction, but the effect in RM group was not obvious. The treatment in RH group enhanced both the early phase I acute vasoconstriction and the phase II persistent vasoconstriction of HHPV. RL and RH groups had significant differences compared with HD group. In contrast to HD group, the values of systolic peak in RS and RU groups decreased dramatically, and the phase II persistent vasoconstriction reversed to relaxation state. The HHPV in RS and RU groups was significantly relieved as compared with RL group. The HHPV in RS and RU groups was relieved as compared with S group and U group. CONCLUSION：R1 at concentration of 8 mg/L relieves acute HHPV in rats. The mechanism may be associated with p38 MAPK and ERK1/2 signaling pathway.     

Expression of voltage-gated potassium channel gene of pulmonary artery smooth muscle cells in COPD merge chronic hypoxic patients

AIM: To study the change of expressions of Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv3.1 genes in pulmonary artery smooth muscle cells (PASMCs) on COPD merge chronic hypoxic patients. METHODS: Human lung tissue was collected from surgical patients. RT-PCR technique was used to study the expression of Kv1.2, Kv1.3, Kv1.5, Kv2.1 and Kv3.1 genes. PASMCs were divided into two groups: ① PASMCs from normal human pulmonary artery, pure COPD patients and COPD merger chronic hypoxic patients pulmonary artery； ② Cultured PASMCs exposed to continual chronic hypoxia or normoxia. RESULTS: ① The expression of Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv3.1 encoding genes were found in human PASMCs exposed to either normixa or chronic hypoxia. ② The expression of Kv1.2, Kv1.5, Kv2.1 genes in PASMCs exposed to chronic hypoxia were significantly decreased compared with control groups (P<0.05). ③ The expression of Kv1.3, Kv3.1 genes in PASMCs exposed to chronic hypoxia showed no significant change compared with control groups (P>0.05). ④ The expression of Kv1.2, Kv1.5, Kv2.1, Kv3.1 genes in pure COPD patients were significantly increased compared with control groups (P<0.05). CONCLUSIONS: ①The results suggested that Kv1.2, Kv1.5, Kv2.1 genes may be oxygen sensitive gene. Their expressions are affected by chronic hypoxia, which probably play an important role in human pulmonary artery hypertension. ② Kv1.3, Kv3.1 genes may not be oxygen sensitive gene and their expression are not affected by chronic hypoxia, which might play a secondary role in human pulmonary artery hypertension.     

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.     

Effect of ginsenoside Rb1 on proliferation and serotonin transporter and 5-HT1B R expression in hypoxia-induced rat pulmonary artery smooth muscle cells via Rho/Rho-kinase pathway

AIM: To observe the effect of ginsenoside Rb1 on the proliferation and the expression of serotonin transporter (SERT), 5-hydroxytryptamine 1B receptor (5HT_1BR) in rat pulmonary artery smooth muscle cells (PASMCs) under hypoxia condition and the relationship with Rho/Rho-kinase signal pathway.METHODS: PASMCs were isolated from the adult male SD rats and primarily cultured. The subcultured cells from the 4th generation to the 6th generation were harvested and divided into normal group, and hypoxia group, different concentrations of Rb1 incubation groups treated with 50, 100 and 200 mg/L ginsenoside Rb1 under hypoxia (HR50, HR100 and HR200 groups, respectively). The viability of the PASMCs was measured by CCK-8 assay. BrdU positive cells were determined using flow cytometry. The expression of serotonin transporter and 5HT_1BR at mRNA and protein levels was detected by RT-PCR and Western blot, respectively. The PASMCs were randomly divided into normal group, hypoxia group, HR200 group and hypoxia+Y-27632 incubation group (HY group). The mRNA expression of Rho-kinase and phosphorylated myosin phosphatase target subunit 1 (p-MYPT1) protein level were investigated by RT-PCR and Western blot, respectively.RESULTS: Compared with normal group, the proliferation of PASMCs in hypoxia group was significantly increased (P<0.01). The cell viability and the expression of SERT and 5HT_1BR at mRNA and protein levels in all different concentrations of Rb1 groups were obviously decreased compared with hypoxia group (P<0.05). The mRNA expression of Rho-kinase and protein level of p-MYPT1 were markedly decreased in HR200 group, and no significant difference compared with HY group was observed (P<0.01).CONCLUSION: Treatment with ginsenoside Rb1 might prevent hypoxia-induced proliferation of PASMCs and over-expression of SERT and 5HT_1BR through inhibiting the Rho/Rho-kinase pathway.     

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.     

Emodin reduces hypoglycemia/hypoxia-induced injury of microglia by TLR4/NF-κB signaling pathway

AIM: To investigate the mechanism of emodin on the protection of glucose-deficient/anoxic microglia. METHODS: A microglia BV2 cell model induced by hypoglycemia/hypoxia (HH) was established. The glucose-deficient/anoxic cells treated with emodin were labeled as HH+emodin (20, 40 and 80 μmol/L) groups. The BV2 cells with TLR4 over-expression treated with emodin under hypoglycemia/hypoxia condition was labeled as HH+pcDNA-TLR4+ emodin (40 μmol/L) group. The cell viability was measured by MTT assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were detected by ELISA. The apoptosis was analyzed by flow cytometry. The protein levels of Bax, Bcl-2, TLR4, p-IκB and IκB were determined by Western blot. RESULTS: Compared with HH+DMSO group, the viability was significantly increased, the levels of LDH and TNF-α and apoptotic rate were significantly decreased, the protein levels of Bax, TLR4 and p-IκB were significantly decreased, the protein level of Bcl-2 was significantly increased in HH+emodin groups (P<0.05). Over-expression of TLR4 reversed the effect of emodin on promoting the viability and inhibiting apoptosis in the BV2 cells. CONCLUSION: Emodin has a protective effect on hypoglycemia/hypoxia induced microglia, and its mechanism may be related to the inactivation of TLR4/NF-κB signaling pathway.     

Effect of MAPK inhibition on the hypoxia hypercapnia-induced pulmonary vasoconstriction

AIM: To investigate isometric force displacement in isolated rat main pulmonary artery rings and right main branch pulmonary artery (second pulmonary artery) rings during hypoxia hypercapnia and the role of mitogen activated protein kinase (MAPK). METHODS: The main pulmonary artery rings were dissected from the male Sprague-Dawley rats and were randomly divided into control group and hypoxia hypercapnia group. The second pulmonary artery rings were also randomly divided into control group, hypoxia hypercapnia group, DMSO incubation group, U0126 incubation group and SB203580 incubation group. The tension changes of pulmonary artery rings were monitored in vitro. RESULTS: Under normoxia conditions, there was no statistically significant change between main pulmonary artery rings and second pulmonary artery rings. A biphasic pulmonary artery contractile response to hypoxia hypercapnia in the second pulmonary artery rings was observed instead of a sharp and transient increase in the main pulmonary artery tension. Both p38 MAPK inhibitor SB203580 and ERK1/2 inhibitor U0126 significantly attenuated the delayed, but not early, contractile phase of the biphasic pulmonary artery contraction. CONCLUSION: Acute hypoxia hypercapnia causes a biphasic pulmonary artery contractile response in the second pulmonary artery, and p38 MAPK and ERK1/2 may be two key mediators in the process.     

Effects of bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.     

Role of endoplasmic reticulum stress in Bim-mediated cardiomyocyte apoptosis induced by hypoxia

AIM:To investigate the role of endoplasmic reticulum stress (ERS) in the process of Bim-mediated cardiomyocyte apoptosis induced by hypoxia. METHODS:Cardiomyocytes were isolated from neonatal Sprague-Dawley rats aged 1~3 days, and primarily cultured in vitro. The antibody targeting α-striated muscle actin was used to identify the cardiomyocytes. The siRNAs targeting bim were transfected into cardiomyocytes with liposome, followed by detecting the expression of Bim by Western blotting. Cardiomyocytes were divided into five groups: blank control group, hypoxia group, hypoxia+liposome group, hypoxia+negative control siRNA group and hypoxia+Bim-siRNA group. The cell viability was determined by MTT assay, and the cell apoptotic rate and the intracellular calcium concentration were measured by flow cytometry. The protein expression of caspase-12 and inositol 1,4,5-triphosphate (IP3) was detected by Western blotting. RESULTS:Immunohistochemical identification confirmed that rat cardiomyocytes were successfully cultured. Green fluorescence was observed in the cells transfected with negative control siRNA under fluorescence microscope. The expression of Bim was obviously inhibited after transfected with Bim-siRNAs and the silencing efficiency of Bim-siRNA-2 was the highest (86.73%). Compared with blank control group, the viability of cardiomyocytes in hypoxia group was significantly reduced (P<005). Compared with hypoxia+negative control siRNA group, the viability of cardiomyocytes in hypoxia+Bim-siRNA group was significantly increased (P<005). The apoptotic rate and the intracellular calcium concentration of cardiomyocytes were obviously increased in hypoxia group (P<0.01), and were both decreased after bim silencing (P<005 or P<0.01). The expression of caspase-12 and IP3 was up-regulated in hypoxia group (P<005), and was down-regulated after bim silencing (P<005 or P<0.01). CONCLUSION: Cardiomyocyte apoptosis induced by hypoxia can be inhibited by silencing the expression of bim gene. Caspase-12 and IP3, as markers of ERS, may participate in the process of Bim-mediated cardiomyocyte apoptosis induced by hypoxia.     

Effects of CARHSP1 gene expression on viability,apoptosis and immune factors of vascular endothe-lial cells induced by hypoxia in atherosclerosis

AIM: To investigate the effect of calcium-regulated heat stable protein 1 (CARHSP1) gene expression on the viability, apoptosis and expression of interleukin-6 (IL-6) and C-reactive protein (CRP) in vascular endothe-lial cells induced by hypoxia.METHODS: The protein expression of CARHSP1 was detected by Western blot in atherosclerotic plaques. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia, and the cells were divided into normal culture group, hypoxia group, hypoxia+CARHSP1-siRNA group and hypoxia+pcDNA3.1-CARHSP1 group. The viability and apoptotic rate of the HUVECs were measured by CCK-8 assay and flow cytometry, respectively. The mRNA expression of IL-6 and CRP was detected by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot.RESULTS: The protein expression of CARHSP1 in atherosclerotic plaques was significantly higher than that in control group (P<0.05). Hypoxia significantly increased the expression of CARHSP1. The cell viability and the protein expression of Bcl-2 were significantly lower in hypoxia group than those in normal culture group (P<0.05). The apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were significantly higher than those in normal culture group (P<0.05). Compared with hypoxia group, the cell viability and protein expression of Bcl-2 were significantly increased in hypoxia+CARHSP1-siRNA group, while the apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were decreased significantly (P<0.05). The cell viability and protein expression of Bcl-2 were decreased significantly in hypoxia+pcDNA3.1-CARHSP1 group, while the apoptotic rate and the protein le-vels of IL-6, CRP, cleaved caspase-3 and Bax were increased significantly (P<0.05).CONCLUSION: The expression of CARHSP1 is increased in atherosclerotic plaques, and inhibition of CARHSP1 expression improves the viability, reduces the apoptosis, and down-regulates the expression of IL-6 and CRP in the HUVECs. Over-expression of CARHSP1 exerts the opposite effect.     

Role of thioredoxin nitration in doxorubicin-induced apoptosis of neonatal rat cardiomyocytes

AIM：To investigate the role of thioredoxin nitration in the apoptosis of neonatal rat cardiomyocytes (NRCMs) induced by doxorubicin (DOX). METHODS：Cardiomyocytes treated with DOX were isolated from newborn Sprague-Dawley rats and cultured in vitro. NRCMs were treated with DOX alone (DOX group), pretreated with Mn (III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP), a peroxynitrite (ONOO-) scavenger, and then treated with DOX (MnTMPyP+DOX group), or treated with MnTMPyP alone (MnTMPyP group). NRCMs without any treatment served as a normal control (control group). The viability of the cells was examined by MTT assay, and the apoptosis was measured by Hoechst 33258 nuclear staining kit. The activity of caspase-3 was detected by spectrophotometry. The expression of cleaved poly(ADP-ribose) polymerase 1 (PARP-1), apoptosis signal-regulating kinase 1 (ASK1), phosphorylated ASK1 (p-ASK1), p38 mitogen-activated protein kinase (p38 MAPK) and phosphorylated p38 MAPK (p-p38 MAPK) was measured by Western blotting. Immunoprecipitation and immunoblotting were performed to detect the formation of Trx-ASK1 and Trx-nitrotyrosine. RESULTS：DOX induced significant apoptosis of NRCMs. MnTMPyP could significantly attenuate the apoptosis induced by DOX. Compared with control group, Trx nitration in DOX group increased obviously. The increases in activity of caspase-3 and expression of cleaved PARP-1 and p-p38 MAPK were also observed, besides the expression of Trx-ASK1 compound and p-ASK1 decreased significantly (P<005). MnTMPyP could decrease the nitration of Trx. The decreases in activity of caspase-3 and expression of cleaved PARP-1 and p-p38 MAPK were detected in MnTMPyP+DOX group, while the expression of Trx-ASK1 compound and p-ASK1 increased significantly (P<005). CONCLUSION: DOX could induce significant apoptosis of NRCMs and increase Trx nitration. The process was significantly attenuated by pretreatment with MnTMPyP. Therefore, Trx nitration may play an important role in doxorubicin-induced apoptosis of cardiomyocytes.     

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.     

Role of calcium-sensing receptor in hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells

AIM: To explore the role of calcium-sensing receptor (CaSR) in rat pulmonary artery smooth muscle cells (PASMCs) and its effect on hypoxia-induced proliferation of PASMCs. METHODS: The expression and distribution of CaSRs were detected by Western blotting and immunofluorescence observation. The intracellular concentration of free calcium ([Ca]_i) was determined by confocal laser scanning microscopy. The cell viability was analyzed by MTT assay. The expression of PCNA and CaSRs was determined by Western blotting. RESULTS: CaSR protein was expressed in rat PASMCs. Hypoxia significantly increased the expression of CaSR and PCNA,[Ca]_i and the cell viability. GdCl₃ (an agonist of CaSR) or NPS2390 (an antagonist of CaSR) amplified or weakened the effect of hypoxia, respectively.CONCLUSION: CaSR is expressed in rat PASMCs. The activation of CaSR is involved in the proliferation of PASMCs induced by hypoxia.     

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.     

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.