[The effects of ERK1/2 pathway on the expression of calcium activated chloride channel in hypoxia in PASMCs rat model].

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.

[The regulation of MAPK signaling pathway on cell proliferation and apoptosis in hypoxic PASMCs of rats].

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.

[The effects and mechanisms of ligustrazine injection on pulmonary arterial hypertension in COPD patients].

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: ①After 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); ② 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.

[Effect of ERK1/2 on rat pulmonary artery smooth muscle cells Kv1.5 channel in the process of hypoxia].

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

[Ischemic postconditioning attenuates pneumocyte apoptosis after lung ischemia/reperfusion injury via inactivation of p38 MAPK].

OBJECTIVE: To investigate the role of p38 MAPK on ischemic postconditioning (IPO) attenuating pneumocyte apoptosis after lung ischemia/reperfusion injury (LIRI). METHODS: Forty adult male SD rats were randomly divided into 5 groups based upon the intervention (n = 8): control group (C), LIR group (I/R), LIR + IPO group (IPO), IPO + solution control group (D), IPO + SB203580 group (SB). Left lung tissue was isolated after the 2 hours of reperfusion, the ratio of wet lung weight to dry lung weight (W/D), and total lung water content (TLW) were measured. The histological structure of the left lung was observed under light and electron transmission microscopes, and scored by alveolar damage index of quantitative assessment (IQA). Apoptosis index (AI) of lung tissue was determined by terminal deoxynuleotidyl transferase mediated dUTP nick end and labeling (TUNEL) method. The mRNA expression and protein levels of and Bax were measured by RT-PCR and quantitative immunohistochemistry (IHC). RESULTS: Compared with C group, W/D, TLW, IQA, AI and the expression of Bax of I/R were significantly increased, the expression of Bcl-2 and Bcl-2/Bax were significantly decreased (P < 0.05, P < 0.01), and was obviously morphological abnormality in lung tissue. Compared with I/R group, all the indexes of IPO except for the expression of Bcl-2 and Bcl-2/ Bax were obviously reduced, the expression of Bcl-2 and Bcl-2/Bax were increased (P < 0.05, P < 0.01). All the indexes between D and IPO were little or not significant( P > 0.05). The expression of Bcl-2 and Bcl-2/Bax of SB were significantly increased and other indexes were reduced than those of IPO (P < 0.05, P < 0.01). CONCLUSION: IPO may attenuate pneumocyte apoptosis in LIRI by inactivation of p38 MAPK, up-regulating expression of Bcl-2/Bax ratio.

[Glybenclamide regulate ERK1/2 signal pathway during hypoxia hypercapnia pulmonary vasoconstriction in rats].

OBJECTIVE: To investigate the role and significance of ATP-sensitive K+ channels in the pathological process of hypoxia hypercapnia-induced pulmonary vasoconstriction (HHPV) and the relationship with ERK1/2 signal pathway in rats. METHODS: We made the third pulmonary artery rings of SD rats, used the model of pulmonary artery rings perfusion in vitro. Under acute hypoxia hypercapnia condition, and observed the effects of the three stages of HHPV incubated by glybenclamide(Gly) and the combined application of Gly and U0126. At the same time, the values of rings' tension changes were recorded via the method of hypoxia hypercapnia conditions reactivity. RESULTS: Under the normoxia condition, the values of the third pulmonary artery rings tension were relatively stable, but under the hypoxia hypercapnia condition, we observed a biphasic pulmonary artery contractile response compared with N group (P < 0.05, P < 0.01). When the third pulmonary artery rings incubated by Gly, it's phase II persistent vasoconstriction was enhanced compared with the H group (P < 0.05, P < 0.01), and the phase I vasoconstriction was also heightened. Moreover, under the hypoxia hypercapnia condition, U0126 could significantly relieve the phase II persistent vasoconstriction compared with HD group (P < 0.05, P < 0.01) induced by Gly, but the phase I acute vasoconstriction and the phase I vasodilation had no changes (P > 0.05). CONCLUSION: Gly may mediate HHPV via activating ERK1/2 signal transduction pathway.

[Expression of KATP in pulmonary artery smooth muscle cells under hypoxia-hypercapnia condition and the relationship with p38 MAPK pathway].

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.

[Effect of Xuebijing injection on TLR4--NF-kappaB--IL-1beta pathway of myocardial hypoxia/reoxygenation in rats].

OBJECTIVE: To investigate the role of Xuebijing injection(XBJI, traditional Chinese medicine), in inhibiting TLR4--NF-kappaB--IL-1beta pathway of myocardial hypoxia/reoxygenation in rats. METHODS: Thirty six male SD rats (280 +/- 30) g were randomly divided into six groups (n = 6): normal group (N group), balanced perfusion group (BP group), model group (M group), low dose XBJI group (XBJI(L) group), middle dose XBJI group (XBJI(M) group), high dose XBJI group (XBJI(H) group). By Langendorff isolated heart perfusion device to establish the model of myocardial hypoxia/reoxygenation in rats. ELISA was used to detect the concentration of interleukin-1beta (IL-1beta); Western blot was used to detect the expression of nuclear factor kappa B p65 (NF-kappaB p65) protein and toll like receptor 4 (TLR4) protein; and RT-PCR to determine the expression of NF-kappaB p65 mRNA and TLR4 mRNA;To observe microstructure changes of hypoxia/reoxygenation myocardial by light microscopy. RESULTS: Compared with M group, the IL-1beta concentration, NF-kappaB p65 and TLR4 protein,NF-kappaB p65 and TLR4 mRNA of XBJIL group, XBJI(M) group, XBJI(H) group expression decreased in varying degrees,and decreased most obviously all in XBJI(M) group (P < 0.05, P < 0.01); Myocardical structural damage was serious in M group, and improved after treatment XBJI, the most obvious was the XBJI(M). CONCLUSION: Different dose of XBJI can inhibit TLR4--NF-kappaB--IL-1beta signal transduction pathway and reduce several inflammatory reaction after myocardial hypoxia/reoxygenation injury, the 4 ml/100 ml of XBJI is the best.

[The effect of niflumic acid in hypoxic hypercapnia pulmonary vasoconstriction].

OBJECTIVE: To investigate the effect of chloride channel blocker--niflumic acid (NFA) on the pathological process of hypoxia hypercapnia-induced pulmonary vasoconstriction in rats. METHODS: We used the model of hypoxia hypercapnia-induced pulmonary vasoconstriction rats, and divided the second, third branch pulmonary artery rings randomly into four groups (n = 8): control group (N group), hypoxia hypercapnia group (H group), DMSO incubation group (HD group), niflumic acid group (NFA group). Under acute hypoxia hypercapnia conditions, we observed the effects of the three stages of hypoxia hypercapnia-induced pulmonary vasoconstriction (HHPV) incubated by NFA in the second, third brach pulmonary artery rings. At the same time, the values of rings' tension changings were recorded via the method of hypoxia hypercapnia conditions reactivity. And investigated the effect of NFA to HHPV. RESULTS: (1) Under the hypoxia hypercapnia condition, we observed a biphasic pulmonary artery contractile (the phase I rapid contraction and vasodilation; the phase II sustained contraction) response in both the second and the third branch pulmonary artery rings compared with the control group (P < 0.05 , P < 0.01); (2) The second and third pulmonary artery rings incubated by NFA which phase II persistent vasoconstriction were significantly attenuated compared with the H group (P < 0.05 , P < 0.01). CONCLUSION: The blocker of the chloride channels attenuates the second and third branch pulmonary artery rings constriction in rat, especially the phase II persistent vasoconstriction, so then have an antagonistic effect on HHPV.

[Calcium-activated chloride channels are involved in two-phase hypoxic pulmonary vasoconstriction in rat pulmonary arteries].

The aim of the present study was to investigate the roles of calcium-activated chloride channels (Cl(Ca)) in the two-phase hypoxic pulmonary vasoconstriction (HPV). The second pulmonary artery branches were dissected from male Sprague-Dawley rats, and the changes in vascular tone were measured by using routine blood vascular perfusion in vitro. The result showed that, under normoxic conditions, Cl(Ca) inhibitors (NFA and IAA-94) significantly relaxed second pulmonary artery contracted by norepinephrine (P < 0.01), but merely had effects on KCl-induced second pulmonary artery contractions. A biphasic contraction response was induced in second pulmonary artery ring pre-contracted with norepinephrine exposed to hypoxic conditions for at least one hour, but no biphasic contraction was observed in pulmonary rings pre-contracted with KCl. NFA and IAA-94 significantly attenuated phase II sustained hypoxic contraction (P < 0.01), and also attenuated phase I vasodilation, but had little effect on phase I contraction. These results suggest that Cl(Ca) is an important component forming phase II contraction in secondary pulmonary artery, but not involved in phase I contraction.