Calcium-sensing receptor mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells through PI3K pathways

AIM:To explore the signal transduction pathways of calcium-sensing receptor(CaSR) that mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells(PASMCs). METHODS:The expression of cyclin D1 and phosphorylated protein kinase B(p-Akt) was analyzed by Western blotting. Cell proliferation was tested using a BrdU incorporation assay, and cell cycle analysis was carried out using a flow cytometric assay. RESULTS:Hypoxia significantly increased the expression of cyclin D1 and p-Akt, the BrdU incorporation and the cell proliferation index. GdCl₃, an agonist of CaSR, amplified the effect of hypoxia. LY294002,a PI3K inhibitor, decreased the up-regulation of cyclin D1 expression and the BrdU incorporation, and also inhibited the increase in the cell proliferation index induced by hypoxia and GdCl₃ in PASMCs. CONCLUSION: The CaSR mediates hypoxia-induced proliferation of rat PASMCs through PI3K pathways.     

Role of α1 adrenoceptor in proliferation of hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the role of α₁ and β₂ adrenoceptors(α₁AR and β₂AR) in the proliferation of hypoxic pulmonary artery smooth muscle cells (PASMCs).METHODS: PASMCs were isolated by an explant method from neonatal bovine pulmonary arteries. The cultured PASMCs were exposed to 6.6% O₂ for 6 h, 12 h and 24 h. The method of -TdR incorporation was used to measure the proliferation of PASMCs. _i was assayed with Fura-2/AM. The mRNA expression of α₁AR, β₂AR, c-fos and c-myc was determined by Northern blotting. The effects of activation of α₁AR and β₂AR, and inhibition of α₁AR on the above indexes were observed by treating PASMCs with different AR agonists and antagonists under hypoxic condition.RESULTS: Significant increase in TdR incorporation in hypoxic PASMCs with α₁AR activation was observed, and marked decrease in that was induced by α₁AR inhibition. However, no significant change was found after β₂AR activation. _i , the mRNA expression of c-fos, c-myc, α₁AR and β₂AR in PASMCs were increased after hypoxia.CONCLUSION: Hypoxia induces the increase in _i and mRNA expression of c-fos and c-myc, leading to the proliferation of PASMCs. The hypoxic proliferation of PASMCs is intervened by α₁AR, but not β₂AR. The remodeling of pulmonary arteriole and pulmonary hypertension may be involved in the processes of pulmonary arteriole constriction and proliferation induced by hypoxia through up-regulation of α₁AR.     

Expression of bFGF mRNA in pulmonary artery of rat with chronic hypoxia and its effect on tension of rat pulmonary artery in vitro

AIM: To explore the role of basic-fibroblast growth factor (bFGF) in the development of pulmonary hypertension induced by hypoxia. METHODS: 1) The pulmonary arteries of SD rats with hypoxia for one and two weeks were isolated, from which the total RNA were extracted by acid guanidinium thiocyanate-phenol-chlorform .Then the levels of mRNA were measured by RT-PCR. 2) About 3mm-long arterial rings cut from SD rat pulmonary arterial stem were suspended between stainless steelhooks in chamber with warmed (37℃) Kreb's solution. Different concentrations of bFGF were added in a cumulative fashion into the chamber where the rings were suspended. The cumulative concentration response curve was obtained. RESULTS: 1)The levels of bFGF mRNA in pulmonary artery of rats with hypoxia were increased significantly compared with those that without hypoxia (2578±384 counts·min^-1 (control) vs 5303±756 (hypoxia) for 1 week and 4054±547 (hypoxia) for 2 weeks, P all ＜0.05). 2) bFGF at concentrations ranged from 5.56×10^-10~2.78×10^-7mol/L caused dose-dependent contraction of vessel rings of rat pulmonary artery (r=0.695,P＜0.05), with EC₅₀ being 2.62×10^-7mol/L. CONCLUSION: bFGF may play an important role in the hypoxic pulmonary hypertension.     

Mechanism of dimerization and activation of CB1 and CB2 receptor

Cannabinoid receptor 1 (CB₁) and cannabinoid receptor 2 (CB₂) are important members of G protein-coupled receptors (GPCRs). Numerous studies have shown that CB₁ receptor can form heterodimers with dopamine receptors (D₂), μ-opioid receptor (μOR), orexin-1 receptor, adenosine receptor (A_2A) or β2 adrenergic receptors, and then forming an essential functional entity. This review summarizes the research progress on heterodimers of cannabinoid CB₁ or CB₂, the function of heterodimers as well as the downstream signalings. The different pharmacological properties of the receptor heterodimer lead to bringing a change in receptor pharmacology, which will have a profound impact on drug development.     

Effects of A2a adenosine receptor on hypoxic pulmonary hypertension in rats treated with salidroside

AIM: To study the protective effect of A_2a adenosine receptor (A_2aAR) on hypoxic pulmonary hypertension in the rats treated with salidroside. METHODS: Sprague-Dawley rats were randomly divided into 6 groups: normal control group, hypoxia group, hypoxia+salidroside (low dose) group, hypoxia+salidroside (median dose) group, hypoxia+salidroside (high dose) group, and hypoxia+CGS-21680 (a selective agonist of A_2aAR) group. Pulmonary hypertension in the rats was produced for 4 weeks. Mean pulmonary artery pressure (mPAP), mean carotid arterial pressure (mCAP) and the weight ratio of right ventricle/(left ventricle+septum)［RV/(LV+S)］ were measured. The expression of A_2aAR in the pulmonary arterioles was determined by immunohistochemistry and in situ hybridization. The mRNA expression of A2aAR in the lung tissues was detected by real-time RT-PCR. The protein level of A_2aAR in the lung tissues was analyzed by Western blotting. RESULTS: The mPAP in hypoxia group was significantly higher than that in normal control group. The mPAP in hypoxia+salidroside (high dose) group and CGS-21680 group was significantly lower than that in hypoxia group. RV/(LV+S) in hypoxia group were significantly higher than that in normal control group. RV/(LV+S) in hypoxia+salidroside (median dose) group, hypoxia+salidroside (high dose) group and CGS-21680 group were lower than that in hypoxia group. The ratio of vessel wall area/vessel total area (WA/TA) in hypoxia group was significantly higher than that in normal control group. WA/TA in hypoxia+salidroside (low dose) group, hypoxia+salidroside (median dose) group, hypoxia+salidroside (high dose) group and CGS21680 group were obviously lower than that in hypoxia group. The expression of A_2aAR was significantly higher in hypoxia group than that in normal control group. The expression of A_2aAR in hypoxia+salidroside (high dose) group and CGS-21680 group was obviously higher than that in hypoxia group. CONCLUSION: The A_2aAR attenuates pulmonary vessel remodeling and pulmonary hypertension induced by hypoxia. Salidroside protects the pulmonary vessel from remodeling and inhibits the development of hypoxia-induced pulmonary hypertension by up-regulation of A_2aAR expression.     

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

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

Ca2+-activated Cl- channels and pulmonary hypertension induced by high pulmonary blood flow

Pulmonary hypertension induced by high pulmonary blood flow involves a variety of complex mechanisms, including endothelial damage, pulmonary artery smooth muscle relaxation-contraction disorder and vascular remodeling. Besides, the factor of ion channels in pulmonary artery smooth muscle cells is also highly correlated to vasoconstriction. In recent years, many studies have shown that activation of Ca²⁺-activated Cl^- channels is responsible for the membrane depolarization of pulmonary artery smooth muscle cells, and plays an important role in the regulation of vascular tone and vasoconstriction. This article reviews the biophysical and pharmacological characteristics of Ca²⁺-activated Cl^- channels as well as the influence of Ca²⁺-activated Cl^- channels in high pulmonary blood flow-induced pulmonary hypertension.     

Role of potassium channels in the regulation of intracellular free Ca2+ concentration of pulmonary artery smooth muscle cells in rats

AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration ( [Ca²⁺]_i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition, [Ca²⁺]_i was (156.91±8.60) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (294.01±16.81) nmol/L. 2. In normoxic condition, the voltage-dependent K⁺-channel antagonist 4-aminopyridine (4AP), but not the Ca²⁺-activated K⁺-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K⁺-channel antagonist glibenclamide (Glib) increased [Ca²⁺]_i. 3. In hypoxic condition, 4AP and TEA caused the rise in [Ca²⁺]_i , but Glib had no effect on [Ca²⁺]_i. 4. MTT assay showed that 4AP increased the value of absorbing light degree (A value) in normoxic and hypoxic condition (0.582±0.062,0.873±0.043,respectively, P＜0.01), TEA increased A value only in hypoxic condition, and Glib had no effect on the proliferation of PASMCs. CONCLUSIONS: K_V plays an important role in the regulation of [Ca²⁺]_i and proliferation of PASMCs. K_Ca serves as distinct responsive roles in the regulation of proliferation of PASMCs in hypoxic condition. K_ATP has no effect on [Ca²⁺]_i and proliferation of PASMCs in normoxic and hypoxic conditions.     

Role of K+ channels in hypoxic pulmonary vasoconstriction in normal and chronic hypoxic rats

AIM:To investigate the role of K⁺ channels in the decreased hypoxic pulmonary vasoconstriction(HPV) in chronic hypoxic rats. METHODS:Blockers of three kinds of K⁺ channels, 4-AP(voltage dependent K⁺ channel blocker), TEA(Ca²⁺ activated K⁺ channel blocker), GLIB(ATP sensitive K⁺ channel blocker) were used in isolated perfused rat lungs to detect the role of K⁺ channels in HPV. RESULTS:In normal rats, 4-AP and TEA, but not GLIB, both elicited a significant increase in pulmonary artery baseline pressure, and also potentiated the acute hypoxic pulmonary vasoconstriction. In chronic hypoxic rats, the HPV is significantly decreased, while 4-AP, TEA, GLIB all elicited a significant but smaller increase in pulmonary artery baseline pressure. Additionally, all these three blockers potentiated the HPV stronger in chronic hypoxic rats than in control rats. CONCLUSION:The opening of Kv, K_Ca, K_ATP might modulate the hypoxic pulmonary vasoconstriction in isolated rat lungs, and the increase in this modulation by potassium channel in chronic hypoxic rats might play a role in its decrease in HPV.     

Role of K+ channels in attenuation of pulmonary vasoreactivity following chronic hypobaric hypoxia in rats

AIM and METHODS: To determine the role of different K⁺-channels in attenuation of vasoreactivity of intrapulminary artery rings induced by chronic hypoxia. RESULTS: ①Acute hypoxia-induced pulmonary vasoconstriction (HPV) could be significantly attenuated by chronic hypobaric hypoxia for 15 days and for 30 days. ②HPV could be significantly potentiated by ATP-sensitive K⁺-channel (K_ATP) blocker or Ca²⁺-activated K⁺-channel (K_Ca) blocker, and the potentiated scope in chronic hypoxic group was much higher than that observed in control group. ③Delayed rectifier K⁺-channel (K_DR) blocker showed no effect on HPV in both control group and chronic hypoxic group. CONCLUSION: Both K_ATP and K_Ca play an important modulating role in HPV and its potentiation may be a critical mechanism for the attenuated vasoreactivity to acute hypoxia following chronic hypobaric hypoxia.     

Role of intracellular free Ca2+ concentration in the regulation of calcium-activated chloride channels in rat pulmonary artery smooth muscle cells

AIM: To investigate the role of intracellular free Ca²⁺ concentration (［Ca²⁺］_i) in the regulation of calcium-activated chloride (Cl_Ca) channels in pulmonary artery smooth muscle cells (PASMCs) of rats under normoxic, acute and chronic hypoxic conditions. METHODS: Acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe ［Ca²⁺］_i of rat PASMCs in normal and chronic hypoxic condition. The influences of Cl_Ca channels on PASMCs proliferation were assessed by MTT assay. RESULTS: (1) The Cl_Ca channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) produced inhibitory effects on acute hypoxia-evoked contractions in pulmonary artery. (2) Under chronic hypoxic condition, ［Ca²⁺］_i was increased. In normoxic condition, ［Ca²⁺］_i was (123.63±18.98) nmol/L, and in hypoxic condition, ［Ca²⁺］_i was (281.75±16.48)nmol/L (P<0.01). (3) In normoxic condition, ［Ca²⁺］_i had no significant change and no effect on Cl_Ca channels was observed (P>0.05). (4) Chronic hypoxic increased ［Ca²⁺］_i which opened Cl_Ca channels. The NFA and IAA-94 blocked them and decreased ［Ca²⁺］_i from (281.75±16.48)nmol/L to (117.66±15.36)nmol/L (P<0.01). (5) MTT assay showed that in chronic hypoxic condition NFA and IAA-94 decreased the value of absorbing light degree (A value) from 0.459±0.058 to 0.224±0.025 (P<0.01). CONCLUSION: Hypoxia increased ［Ca²⁺］_i which opened Cl_Ca channels and had a positive-feedback to ［Ca²⁺］_i. This may play an important role in hypoxic pulmonary hypertension. In chronic hypoxic condition, Cl_Ca channel may play a role in the regulation of PASMCs proliferation.     

Effect of Zhengtian pills on P2X3 receptor expression in trigeminal gan-glion of migraine rats

AIM: To explore the effect of Zhengtian pills on P2X₃ receptor expression in trigeminal ganglion (TG) of migraine rat. METHODS: Sprague-Dawley (SD) rats were randomly divided into control group, migraine group, Zhengtian pills (ZTP) group and A-317491 group. After given corresponding drugs for 7 d, migraine rat model was established by subcutaneously injection of nitroglycerin (10 mg/kg), while the control rats were injected with saline. The beha-vioral manifestations of the rats were observed. The expression of P2X₃ receptor in rat TG was detected by the methods of immunofluorescence, Western blot and rea-time PCR. RESULTS: About 5 min after subcutaneousl injection, the behavioral manifestations such as scratching head and climbing cage were observed. The behavioral manifestations were observed within the first 30 min in control group, but the erythroid ears did not appear. After 2 h of molding, the behavioral manifestations disappeared in ZTP group and A-317491 group, while those in migraine group lasted for 3 h. Immunofluorescence results showed that the expression of P2X₃ receptor in TG was positive in each group. The expression level in migraine group was significantly higher than that in other groups. The P2X₃ receptor protein and mRNA levels in the TG of migraine group were higher than those in control group (P<0.01), while those in ZTP group were lower than those in migraine group (P<0.01). No difference of the P2X₃ receptor expression between ZTP group and A-317491 group was observed. CONCLUSION: Zhengtian pills may effectively alleviate migraine by inhibiting the expression of P2X₃ receptor in TG.     

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.     

Microinjection of AT1 and AT2 receptor antagonists into PVN affects renal sympathetic nerve activity in chronic heart failure rats

AIM: To examine the renal sympathoexcitation affected by microinjection of angiotensin Ⅱ type 1 (AT₁) receptor antagonist L-158809 and angiotensin Ⅱ type 2 (AT₂) receptor antagonist PD123319 into paraventricular nucleus (PVN) in heart failure rats.METHODS: Left anterior descending coronary artery ligation was used to induce rat heart failure (HF) . Four weeks after operation, the left ventricular end-diastolic pressure (LVEDP), the ratios of heart weight/body weight and lung weight/body weight, and the ratio of infarct area of the left ventricle were observed. Under anesthesia, SD rats were fixed into the brain stereo controller to locate PVN for microinjection and the artificial cerebrospinal fluid (ACSF) was used for control. The left kidney was exposed by retroperitoneal approach and the renal sympathetic nerve was separated under surgical microscope. The heart rate, blood pressure and the activity of renal sympathetic nerve discharge (RSNA) were recorded by POWERLAB 8/30 system. RESULTS: Microinjection of AT₁ receptor antagonist into PVN induced a decrease in RSNA in both HF rats and sham rats. The RSNA responses to L-158809 in the HF rats were significantly greater (P<0.05) than those in the sham rats. However, microinjection of AT₂ receptor antagonist and ACSF into PVN induced no change of RSNA in both HF and sham rats. CONCLUSION: There are some differences of sympathetic nerve outputs between using AT₁ receptor antagonist and AT₂ receptor antagonist on PVN, indicating the up-regulation of AT₁ receptors in PVN during HF. The central renin-angiotensin-aldosterone system(RAAS) may be affected by AT₁ receptor, not by AT₂ receptor.     

Neonatal rat cardiomyocyte hypertrophy is induced by tumor necrosis factor-α through PI3K-IP3R-Ca2+ pathways

AIM: To investigate the role of PI3K-IP₃R-Ca²⁺ pathways in cardiomyocyte hypertrophy induced by tumor necrosis factor-α (TNF-α). METHODS: Myocardial cells of neonatal rats were cultured in vitro. The hypertrophic model was induced by TNF-α. The protein content was assayed with Lowry's method. The volumes of the cardiomyocytes were detected by computer photograph analysis system. The protein synthesis was determined by the method of[³H]-leucine incorporation.[Ca²⁺]_i transient was measured by Till image system with cell-loading Fura-2/AM. RESULTS: LY294002, a PI3K inhibitor, significantly suppressed the amplitude elevation of the spontaneous[Ca²⁺]_i transients induced by TNF-α in cultured ventricular myocytes from neonatal rats. The effect was similar to that of LY294002+2-APB (P>0.05), but lower than that in LY294002+ryanodine group (P<0.05). LY294002 significantly reduced the enhancements of protein content,[³H]-leucine incorporation and cell size induced by TNF-α. The effect was similar to that in 2-APB+LY294002 group, but higher than that in 2-APB group and lower than that in ryanodine+LY294002 group. CONCLUSION: TNF-α induces cardiac hypertrophy through PI3K-IP₃R-Ca²⁺ pathways.     

Effects of P2X4 receptor in spinal microglia on rrTNF-induced pathological pain

AIM: To investigate the effects of P2X₄ receptor on peri-sciatic administration of recombinant rat TNF-α (rrTNF)-induced mechanical allodynia. METHODS: Male Sprague-Dawley rats (180~200 g) were used in the experiments. The levels of P2X₄ receptor on day 3, day 7 and day 14 after peri-sciatic administration of rrTNF were examined by Western blot, and the location of P2X₄ receptor in the spinal dorsal horn was observed by double immunofluorescence staining. The changes of 50% paw-withdrawal thresholds of the rat were detected by behavioral test, and the level of TNF-α in the spinal dorsal horn was also examined by Western blot when TNP-ATP was intrathecally injected before the administration of rrTNF. RESULTS: Compared with control group, the expression of P2X₄ receptor in the spinal dorsal horn on the ipsilateral side significantly increased on day 3, day 7 and day 14 (P<0.01) after rrTNF (100 ng/L) administration. P2X₄ receptor was co-localized only with microglia, but not with neurons or astrocytes. Intrathecal injection of TNP-ATP before rrTNF administration prevented mechanical allodynia induced by rrTNF and inhibited the upregulation of TNF-α in the spinal dorsal horn. CONCLUSION: P2X₄ receptors in microglia may be involved in rrTNF-induced mechanical allodynia by the upregulation of TNF-α in the spinal dorsal horn.     

SO2 derivatives stimulate inflammation of airway epithelial cells through NLRP3 inflammasome

AIM:To investigate the effect of sulfur dioxide (SO₂) derivatives (sodium sulfite and sodium bisulfate) on NLRP3 inflammasome in airway epithelial cells. METHODS:SO₂ derivatives at different concentrations were applied to bronchial epithelial 16HBE cells for 12 h. The production of reactive oxygen species (ROS) was detected by flow cytometry. The protein levels of NLRP3 and caspase-1 p20 were analyzed by Western blot. The level of interleukin-1β(IL-1β) in the cell culture supernatant was measured by ELISA. The cell viability was measued by MTT assay, and the concentration of SO₂ derivatives used in the following experiments was 2 mmol/L. When the NLRP3 gene in 16HBE cells was silenced by RNA interference technique or N-acetyl cysteine (NAC) was used to pretreat 16HBE cells, the intracellular ROS was detected by flow cytometry, and the protein levels of NLRP3 and caspase-1 p20 and the secretion of IL-1β were determined by Western blot and ELISA, respectively. RESULTS:Compared with the control group, the level of intracellular ROS, the protein levels of NLRP3 and caspase-1 p20, and the secretion of IL-1β in cell supernatant were increased significantly in 2 mmol/L and 4 mmol/L SO₂ derivative groups (P<0.05). Compared with the 2 mmol/L group, the protein levels of NLRP3 and caspase-1 p20 were significantly inhibited in NLRP3 siRNA group (P<0.05). The concentration of IL-1β in the cell culture supernatant was significantly decreased (P<0.05). No significant difference of ROS level was observed. Significantly decreased protein levels of NLRP3 and caspase-1 p20, and the concentration of IL-1β in NAC group were found (P<0.05). CONCLUSION:SO₂ derivatives directly promote the production of IL-1β through NLRP3 inflammasome in bronchial epithelial cells.     

H2S inhibits neuron apoptosis induced by anoxia-reoxygenation through cAMP-mediated PI3-K/Akt/P70S6K kinase cell-survival signaling pathways

AIM: To investigate the effect of hydrogen sulfide on neuron apoptosis through PI₃-K/Akt/P70S6K cell-survival signal transduction pathways after neuron anoxia-reoxygenation.METHODS: Newborn (24-48 h) Wistar rats were decapitated.The hippocampus tissue was dissected and cells were suspended.Cells were plated at 1.0×10⁸ cells/L on poly-dlysine-treated 96-well (100 μL/well) plates and 6-well (2 mL/well) plates.Cells were used after 7 days.For anoxia-reoxygenation (oxygen glucose deprivation,OGD) experiments,cells were washed three times in a glucose-free balanced salt solution (BSS).They were then placed in deoxygenated glucose-free medium and cultured under 95% N₂,5% CO₂ in an anaerobic chamber equilibrated to 37 ℃ and 100% humidity for 45 min.OGD was terminated by replacement of stored medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 95% air,5% CO₂.In experimental group,cells were respectively carried out OGD,OGD+150 μmol/L NaHS,OGD+150 μmol/L NaHS+10 μmol/L triciribin,OGD+150 μmol/L NaHS+10 nmol/L rapamycin and OGD+150 μmol/L NaHS+10 μmol/L triciribin+10 nmol/L rapamycin.Control cells were cultured normally.24 h later,neuron viability and apoptosis were measured.The level of cAMP and protein expression of PI₃-K,Akt and P70S6K were detected.RESULTS: NaHS enhanced concentration of cAMP and expression of PI₃-K,Akt and P70S6K.Meanwhile,increased neuron viability and decreased neuron apoptosis (P<0.01 vs group C or group I/R) were observed.Triciribin inhibited Akt and P70S6K,as well as increased neuron apoptosis and decreased neuron viability (P<0.05,P<0.01 vs group NaHS).Rapamycin inhibited P70S6K,as well as increased neuron apoptosis and decreased neuron viability (P<0.05,P<0.01 vs group NaHS).CONCLUSION: H₂S inhibits hippocampus neuron apoptosis and protects neuron from anoxia-reoxygenation injury through cAMP-mediated PI₃-K/Akt/P70S6K kinase cell-survival signaling pathways.     

Sphingosine-1-phosphate receptor 2 attenuates influenza A virus-induced viral pneumonia through PI3K/AKT/eNOS pathway

AIM: To investigate the effects of sphingosine-1-phosphate receptor 2 (S1PR2) on influenza A virus-induced viral pneumonia.METHODS: The animal model of influenza A virus pneumonia was established by infecting wild-type C57BL/6 mice and S1pr2^-/- mice with influenza virus subtype FM1 mouse lung adaptable strain through nose drops. The pathological changes of the lung tissues of wild-type mice (model group), JTE-013 (S1PR2 effective antagonist)-challenged mice and S1pr2^-/- mice were observed, and the protein concentration, total cell number, and interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) levels were determined in the bronchoalveolar lavage fluid (BALF) at 4 d and 6 d after virus infection. The phosphorylation levels of AKT and eNOS in the lung tissues were determined by Western blot. RESULTS: Compared with the wild-type mice of control group, the influenza A virus pneumonia in JTE treatment group and S1pr2^-/- mice were more serious, and the protein concentration, total cell number and inflammatory cytokines in the BALF were remarkably increased. Moreover, the phosphorylation levels of AKT and eNOS, the downstream targets of PI3K, were significantly increased (P<0.01). CONCLUSION: S1PR2 mediates PI3K/AKT/eNOS signaling transduction pathway to regulate NO generation, and inhibit vascular permeability and inflammatory cytokine release, thus attenuating the viral pneumonia induced by influenza A virus.     

Effects of several harmful factors on expression of glycine receptor α1 subunit in neonatal rat myocardial cells

AIM: To study the expression of glycine receptor α₁ subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells. METHODS: Neonatal rat myocardial cells were cultured in vitro. The expression of glycine receptor α₁ subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell viability was measured by CCK-8 assay, and the expression of glycine receptor α₁ subunit was determined by Western blotting. RESULTS: The expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells was positively detectable by Western blotting. Compared with control group, no significant difference of the cell viability (P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed. The expression of glycine receptor α₁ subunit was increased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION: Glycine receptor α₁ subunit exists in the neonatal rat myocardial cells. A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptor α₁ subunit, but HG downregulates the expression of glycine receptor α₁ subunit in cultured neonatal rat myocardial cells.