RESUMEN
The present study was to investigate the role of TRPC6 in pulmonary artery smooth muscle cells (PASMCs) proliferation and apoptosis under hypoxia and hypercapnia. PASMCs were isolated from chloral hydrate-anesthetized male Sprague-Dawley (SD) rats. Cellular purity was assessed by immunofluorescence staining for smooth muscle α-actin under fluorescence microscopy. Passage 4-6 PASMCs were starved for 24 h in serum-free DMEM and divided into 5 groups randomly: normoxia, hypoxia and hypercapnia, DMSO, TRPC6 inhibitor SKF-96365 and TRPC6 activator OAG groups. The normoxic group was incubated under normoxia (5% CO2, 21% O2, 37 °C) for 24 h, and the others were incubated with corresponding drugs under hypoxic and hypercapnic (6% CO2, 5% O2, 37 °C) atmosphere for 24 h. TRPC6 mRNA was detected by reverse transcription-PCR. TRPC6 protein was detected by Western blotting. The proliferation of PASMCs was performed by CCK-8 kit. Apoptosis of the PASMCs was detected using TUNEL assay. The [Ca2+]i in the PASMCs was measured using Fura 2-AM fluorescence. The results showed that the expressions of TRPC6 mRNA and protein, and [Ca2+]i were upregulated under hypoxic and hypercapnic conditions. Hypoxia and hypercapnia promoted cellular proliferation and inhibited apoptosis in the PASMCs. OAG enhanced the above-mentioned effects of hypoxia and hypercapnia, whereas SKF-96365 reversed these effects. These results suggest that TRPC6 may play a role in PASMCs proliferation and apoptosis under hypoxia and hypercapnia by regulating [Ca2+]i.
Asunto(s)
Apoptosis , Hipercapnia/fisiopatología , Miocitos del Músculo Liso/metabolismo , Canales Catiónicos TRPC/metabolismo , Actinas , Animales , Calcio/metabolismo , Hipoxia de la Célula , Proliferación Celular , Células Cultivadas , Imidazoles , Masculino , Músculo Liso Vascular/citología , Arteria Pulmonar/citología , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To investigate the expression of mRNA and protein of Calcium activated chloride channel (CLCA2) in hypoxic pulmonary artery smooth muscle cell (PASMCs) of rat and it's relationship with ERK1/2 signal pathway. METHODS: PASMCs were randomly divided into 5 groups including normal group(N group), hypoxia group(H group), DMSO group(D group), U0126 group (U group) and Staurosporine aglycone group(SA group). The protein expression of CLCA2 in PASMCs was detected by Western blot.The mRNA expression of CLCA2 was detected by half quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The mRNA and protein expressions of CLCA2 in H group were significantly higher than N group (P<0.01). Comparing with D group,the mRNA and protein expressions of CLCA2 were significantly increased in U group (P<0.01),the mRNA expression of CLCA2 in SA group was obviously decreased (P<0.01) with slightly decreasing of its protein expression. CONCLUSIONS: Hypoxia promotes the expressions of mRNA and protein of CLCA2 in rat PASMCs. The ERK1/2 pathway activator Staurosporine aglycone reduces the mRNA and protein expression of CLCA2 in rats PASMCs and the ERK1/2 pathway inhibitor U0126 induces the upregulation of the mRNA and protein expressiosn of CLCA2 in rats PASMCs.
Asunto(s)
Canales de Cloruro/metabolismo , Sistema de Señalización de MAP Quinasas , Miocitos del Músculo Liso/metabolismo , Animales , Carbazoles/farmacología , Hipoxia de la Célula , Células Cultivadas , Alcaloides Indólicos/farmacología , Músculo Liso Vascular/citología , Arteria Pulmonar/citología , RatasRESUMEN
OBJECTIVE: To explore the relationship between hypoxic pulmonary arterial smooth muscle cells(PASMCs)proliferation, apop-tosis and mitogen-activated protein kinases(MAPK) signal pathway in rats. METHODS: PASMCs were obtained from male SD rats by the enzyme digestion method and primarily cultured; PASMCs were identified through two methods:immunofluorescence staining and light microscopy; the 4~6th generation PASMCs of logarithmic growth state of good growth period were selected, and randomly divided into 7 groups:normoxic con-trol group (N), hypoxia group (H), DMSO group (D), extracellular signal-regulated kinase1/2(ERK1/2) inhibitor-U0126 group (U) and p38MAPK inhibitor-SB203580 group (S), the p38MAPK activator-Anisomycin group (A), the ERK1/2 activator-Staurosporine Aglycone group (SA). When all the models were completed, the all groups joined the CCK-8 to measure cell proliferation; cell apoptosis of each group was detected by TUNEL kit after the modeling. RESULTS: Compared with N group, the expression of OD value in H group was up-regulated (0.990 ±0.041 vs 1.143 ±0.033,P < 0.01). There was no statistical significance on PASMCs apoptosis index(AI) in H group (4.913 ±0.451 vs 5.452 ±0.557, P > 0.05); Compared With H group, there were no statistical significance on the expression of PASMCs OD value and apoptosis index(AI)in D group (1.143 ±0.033 vs 1.142 ±0.049,5.452 ±0.557 vs 5.402 ±0.651,P > 0.05); the expression of OD value in U group was down-regulated, and the expression of AI was up-regulated (1.143 ±0.033 vs 0.985 ±0.078, 5.452 ±0.557 vs 10.145 ±2.545, P < 0.01); the expression of OD value in S group was up-regulated, and the expression of AI was down-regulated (1.143 ±0.033 vs 1.295 ±0.039, 5.452 ±0.557 vs 3.093 ±0.409, P < 0.01); the expression of OD value in A group was down-regulated, and the expres-sion of AI was up-regulated (1.143 ±0.033 vs 0.347 ±0.067, 5.452 ±0.557 vs 25.753 ±1.262, P < 0.01); the expression of OD value in SA group was up-regulated, and the expression of AI was down-regulated (1.143 ±0.033 vs 1.685 ±0.100, 5.452 ±0.557 vs 1.700 ±0.095, P < 0.01). CONCLUSIONS: The regulation of PASMCs' proliferation and apoptosis under hypoxia condition have a relationship with the participation of MAPK signal pathway.
Asunto(s)
Apoptosis , Proliferación Celular , Sistema de Señalización de MAP Quinasas , Miocitos del Músculo Liso/citología , Animales , Hipoxia de la Célula , Células Cultivadas , Masculino , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/enzimología , Arteria Pulmonar/citología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by increased pulmonary arteriolar resistance. Pulmonary vasoconstriction has been proved to play a significant role in PAH. We previously reported that Panax notoginseng saponins (PNS) might attenuate hypoxia and hypercapnia-induced pulmonary vasoconstriction (HHPV). METHODS: In the present study, our specific objective was to investigate the role of ginsenoside Rg1, a major component of PNS, in this process and the possible underlying mechanism. The second order pulmonary rings isolated from the Sprague-Dawley rats were treated with different dosage of ginsenoside Rg1 at 8, 40, or 100 mg/L respectively, both before and during the conditions of hypoxia and hypercapnia. Contractile force changes of the rings were detected. Furthermore, SB203580, the selective inhibitor for p38 activation was applied to the rings. Pulmonary arterial smooth muscle cells (PASMCs) were cultured under hypoxic and hypercapnic conditions, and ginsenoside Rg1 was administered to detect the changes induced by p38. RESULTS: Under the hypoxic and hypercapnic conditions, we observed a biphasic pulmonary artery contractile response to the second pulmonary artery rings. It is hypothesized that the observed attenuation of vasoconstriction and the production of vasodilation could have been induced by ginsenoside Rg1. This effect was significantly reinforced by SB203580 (P<0.05 or P<0.01). The expression of p38 in the PASMCs under hypoxic and hypercapnic conditions was significantly activated (P<0.05 or P<0.01) and the observed activation was attenuated by ginsenoside Rg1 (P<0.05 or P<0.01). CONCLUSIONS: Our findings strongly support the significant role of ginsenoside Rg1 in the inhibition of hypoxia and hypercapnia-induced vasoconstriction by the p38 pathway.
RESUMEN
The aim of the present study was to investigate the effect of notoginsenoside Rb1 (Rb1) on the ERK and p38 MAPK pathways in primary cultured pulmonary arterial smooth muscle cells (PASMCs) exposed to hypoxia and hypercapnia, in order to elucidate the mechanism underlying the effect of Rb1 on hypoxia and hypercapnia-induced pulmonary vasoconstriction (HHPV). PASMCs were isolated from Sprague-Dawley rats. The cells were divided into five groups: Normal (N), hypoxia and hypercapnia (H), RbL, RbM and RbH groups. N group cells were cultured under 5% CO2 and 21% O2. H, RbL, RbM and RbH groups were cultured under 6% CO2 and 1% O2. Prior to the hypoxia and hypercapnia exposure, RbL, RbM and RbH groups were treated with 8, 40 and 100 mg/ml Rb1 for 30 min, respectively. Phosphorylated extracellular signal-regulated kinase (P-ERK) and P-p38 protein, and ERK1/2 and p38 mRNA expression levels were detected using western blot and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses, respectively. The correlations between P-ERK protein and ERK1/2 mRNA, and between P-p38 protein and p38 mRNA were evaluated. Results of western blot and RT-PCR showed hypoxia and hypercapnia increased P-ERK and P-p38 protein, and ERK1/2 mRNA, respectively (P<0.05). Rb1 suppressed the increased P-ERK and P-p38 protein, and ERK1/2 and p38 mRNA by hypoxia and hypercapnia (P<0.05). P-ERK protein was positively correlated with ERK1 (r=0.5, P<0.01) and ERK2 mRNA (r=0.977, P<0.01). P-p38 protein was positively correlated with p38 mRNA (r=0.884, P<0.01). Thus, the present results indicate that Rb1 may ameliorate HHPV by suppressing ERK and p38 pathways. The study provides an experimental basis for investigating the clinical use of Rb1 in the management of HHPV-related disorders.
RESUMEN
OBJECTIVE: To observe the effects of ligustrazine hydrochloride injection(LHI) on pulmonary arterial hypertension in chronic obstructive pulmonary disease(COPD) patients and to investigate its possible mechanisms. METHODS: Twenty-two cases of patients with COPD were randomly divided into conventional treatmentgroup (group C) and ligustrazine treatment group(group L), 11 persons were randomly selected from healthy subjects without lung disease served as normal control group(group N). Group C was given bed rest, low flow oxygen inhalation, bronchial diastolic agent, glucocorticoid and antibiotics and other conventional treatment, and group L was added with ligustrazine hydrochloride injection on the above mentioned basis treatment, group N was given no treatment. After 2 weeks, lung function, blood gas analysis and pulmonary arterial pressure were compared among the three groups, and the content of H2S in plasma was tested with sensitive sulfur electrode method. RESULTS: â After two weeks treatment, in group L and group C pulmonary function, blood gas analysis, pulmonary artery pressure were obviously improved, and group L was better than group C (P<0.05); â¡ In group L the content of H2S was increased (P<0.01), group C had no significant difference (P>0.05), and there was a significant difference between the two groups (P<0.01). CONCLUSIONS: Combination with LHI can effectively improve lung function. LHI mayrelieve hypoxic hypercapnia pulmonary hypertension induced by COPD through raising the content of H2S.
Asunto(s)
Hipertensión Pulmonar/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Pirazinas/uso terapéutico , Humanos , Hipercapnia/tratamiento farmacológicoRESUMEN
The effect of puerarin (Pur) on expressions of Fas/FasL mRNAs in pulmonary ischemia and reperfusion injury (PIRI) in rabbit was investigated. The sole side lung ischemia and reperfusion model was used. Rabbits were randomly divided into three groups, a sham operated group (sham, n = 10), PIR group (IR, n = 30) and PIR + Pur group (Pur, n = 30). Changes of several parameters including apoptotic index (AI), wet to dry ratio of lung tissue weight (W/D) and index of quantitative assessment of histologic lung injury (IQA) were measured after 60, 180 and 300 minutes of reperfusion. Meanwhile, the location and expression of Fas/FasL mRNA were investigated. Lung tissue was prepared for light microscopic and electron microscopic observation after 60, 180 and 300 minutes of reperfusion. Compared with group IR, Fas/FasL mRNAs were slightly expressed in intima and extima of small pulmonary artery, alveoli, and bronchiole epithelia in group Pur. The values of AI, W/D and IQA were significantly lower than those in group IR after 60, 180, and 300 minutes of reperfusion in lung tissue (P <0.01 or P <0.05). Meanwhile, the abnormal changes in lung tissue morphology were markedly less in group Pur. Puerarin notably protects lung from PIRI by inhibiting Fas/FasL mRNA expression and decreasing lung cell apoptosis in rabbits.
Asunto(s)
Proteína Ligando Fas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Isoflavonas/uso terapéutico , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Daño por Reperfusión/complicaciones , Animales , Proteína Ligando Fas/genética , Femenino , Lesión Pulmonar/tratamiento farmacológico , Lesión Pulmonar/metabolismo , Masculino , ARN Mensajero/genética , Conejos , Receptores del Factor de Necrosis Tumoral/genética , Vasodilatadores/uso terapéuticoRESUMEN
OBJECTIVE: To explore the effect of ERK1/2 MAPK pathway on the expression of Kv1.5 channel, a voltage-gated potassium ion channel, in rat pulmonary artery smooth muscle cells (PASMCs) and its mechanisms during the process of hypoxia. METHODS: The PASMCs derived from SD rats were cultivated primarily. The third to sixth generation of PASMCs were divided into 5 groups randomly: (1) Normal group (N); (2) Hypoxic group (H); (3) Demethy sulfoxide(DMSO) group (HD); (4) U0126 group (HU): 10 micromol/L U0126; (5) Anisomycin group (HA): 10 micromol/L anisomycin. There were three dishes of cells in each group. The cells in normal group were cultured in normoxic incubator (5% CO2, 37 degrees C), the cells in other groups were added to 0.05% DMSO in the hypoxic incubator (5% CO2, 2% O2, 37 degrees C), all cells were cultured for 60 h. RT-PCR and Western blot were used to detected the espressions of Kv1.5 mRNA and protein in PASMCs. RESULTS: Compared with N group, the expressions of Kv1.5 mRNA and protein in H, HD and HA groups were reduced significantly (P < 0.05); Compared with H group and HD groups, Kv1.5 mRNA and protein expressions in HU group were increased sharply (P < 0.05). Compared with the HU group, Kv1.5 mRNA and protein expressions in HA groups were significantly lower (P < 0.05). CONCLUSION: Low oxygen reduced Kv1.5 mRNA and protein expressions, U0126 could resistant the Kv1.5 channel lower expression caused by hypoxia. Anisomycin had no significant effect on Kv1.5 channel expression under hypoxia, but the expression of Kv1.5 was still significantly lower than the normal oxygen group. These data suggest that hypoxia may cause hypoxic pulmonary hypertension by interfering ERK1/2 signaling pathway to inhibit Kv1.5
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Canal de Potasio Kv1.5/metabolismo , Sistema de Señalización de MAP Quinasas , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Animales , Hipoxia de la Célula , Hipertensión Pulmonar , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Oxígeno , Arteria Pulmonar/citología , ARN Mensajero , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To investigate the effect of panax notoginseng saponins (PNS) injection on pulmonary artery pressure and the expression of p38MAPK in lung tissue of rats subjected to chronic hypoxia. METHODS: Thirty adult male Sprague Dawley rats were randomly divided into three groups (ten in each group): rats in control group were exposed to normoxic condition and the rats in hypoxia group and PNS group were subjected to 4-week hypoxia, and PNS injection (50 mg · kg(-1) · d(-1)) was administrated intraperitoneally at 30 min in the PNS group daily before the rats were kept in the hypoxic chamber, while rats in the other two groups received equal dose of normal saline instead. After chronic hypoxia, mean pulmonary artery pressure (mPAP) and mean carotid artery pressure (mCAP) were measured. The heart and lung tissues were harvested, and right ventricle (RV) and left ventricle plus ventricular septum (LV+S) were weighed to calculate the ratio of RV/(LV+S). The expression of p38MAPK mRNA was determined by reverse transcription-polymerase chain reaction, the quantity of phosphorylated p38MAPK (p-p38MAPK) in rat lung tissues and pulmonary arterioles was determined by Western blot and immunohistochemistry. RESULTS: Compared with the control group, mPAP and the ratio of RV/(LV+S) in the hypoxia group were increased, the expression of p-p38MAPK in pulmonary arterioles and p38MAPK mRNA in the lung were higher (P<0.05). The changes of these parameters in the hypoxia group were significantly attenuated by PNS treatment (P<0.05). CONCLUSION: PNS injection was shown to prevent hypoxic pulmonary hypertension at least partly by regulating p38MAPK pathway.
Asunto(s)
Hipertensión Pulmonar/enzimología , Hipoxia/enzimología , Pulmón/enzimología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Panax notoginseng/química , Saponinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Arteriolas/efectos de los fármacos , Arteriolas/metabolismo , Presión Sanguínea/efectos de los fármacos , Western Blotting , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/fisiopatología , Modelos Animales de Enfermedad , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/fisiopatología , Hemodinámica/efectos de los fármacos , Hipertensión Pulmonar/complicaciones , Hipertensión Pulmonar/fisiopatología , Hipoxia/complicaciones , Hipoxia/fisiopatología , Inyecciones , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/fisiopatología , Masculino , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiopatología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Saponinas/administración & dosificación , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Pulmonary arterial hypertension (PAH) is a disease of the small pulmonary arteries characterized by increased vascular resistance. Pulmonary vasoconstriction has been proven to play a pivotal role in PAH. We have previously hypothesized that Panax notoginseng saponins (PNS) might attenuate hypoxia-hypercapnia-induced pulmonary vasoconstriction. The specific objective of the present study was to investigate the role of notoginsenoside R1, a main ingredient of PNS, in this process and the possible underlying mechanism. The third order pulmonary rings from the Sprague-Dawley rats were treated with different concentrations of notoginsenoside R1 (8, 40, and 100 mg/L, respectively) both before and during the conditions of hypercapnia and hypoxia. Contractile force changes in the rings were detected and the optimal concentration (8 mg/L) was selected. Furthermore, an ERK inhibitor, U0126, was applied to the rings. In addition, pulmonary arterial smooth muscle cells (PASMCs) were cultured under hypoxic and hypercapnic conditions, and notoginsenoside R1 was administered to detect the changes induced by ERK1/2. The results revealed biphasic vasoconstriction in rings under hypoxic and hypercapnic conditions. It is hypothesized that the observed attenuation of vasoconstriction and the production of vasodilation could have been induced by notoginsenoside R1. This effect was found to be significantly reinforced by U0126 (p < 0.05 or p < 0.01). ERK expression in the PASMCs under hypoxic and hypercapnic conditions was significantly activated (p < 0.05 or p < 0.01) and the observed activation was attenuated by notoginsenoside R1 (p < 0.05 or p < 0.01). Our findings strongly support the significant role of notoginsenoside R1 in the inhibition of hypoxia-hypercapnia-induced vasoconstriction by the ERK pathway.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ginsenósidos/farmacología , Hipercapnia/fisiopatología , Hipoxia/fisiopatología , Sistema de Señalización de MAP Quinasas/fisiología , Músculo Liso Vascular/efectos de los fármacos , Arteria Pulmonar/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Animales , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Técnicas In Vitro , Masculino , Panax notoginseng , Ratas Sprague-DawleyRESUMEN
The aim of the present study is to investigate the expressions of ATP-sensitive K(+) channels (KATP) in pulmonary artery smooth muscle cells (PASMCs) and the relationship with p38 MAPK signal pathway in rats. Male SD rat PASMCs were cultured in vitro, and a model of hypoxia and hypercapnia was reconstructed. PASMCs were divided to normal (N), hypoxia-hypercapnia (H), hypoxia-hypercapnia+DMSO incubation (HD), hypoxia-hypercapnia+SB203580 (inhibitor of p38 MAPK pathway) incubation (HS) and hypoxia-hypercapnia+Anisomycin (agonist of p38 MAPK pathway) incubation (HA) groups. Western blot was used to detect the protein expression of SUR2B and Kir6.1; semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of SUR2B and Kir6.1. The results demonstrated that: (1) Compared with N, H, HD and HS groups, the expressions of Kir6.1 mRNA and protein in PASMCs of HA group were decreased significantly (P < 0.01), but there were no differences among N, H, HD and HS groups (P > 0.05); (2) Compared with N group, the expressions of SUR2B mRNA and protein in H, HD, HS and HA groups were increased significantly (P < 0.05), but there were no differences among H, HD, HS and HA groups (P > 0.05). The results imply that: (1) Hypoxia-hypercapnia, SB203580 didn't change the expressions of Kir6.1 mRNA and protein in PASMCs, but Anisomycin decreased the expressions of Kir6.1 mRNA and protein, so Kir6.1 may be regulated by the other subfamily of MAPK pathway; (2) Hypoxia-hypercapnia raised SUR2B mRNA and protein expressions in PASMCs, but SB203580 and Anisomycin did not affect the changes, so the increasing of SUR2B mRNA and protein induced by hypoxia-hypercapnia may be not depend on p38 MAPK pathway.