Roles of STIM2 and TRPC3 in the CaR mediated Ca2+ entry and NO generation in human umbilical vein endothelial cells / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the roles of stromal interaction molecule 2 (STIM2) and transient receptor potential canonical 3 (TRPC3) in extracellular Ca(2+)-sensing receptor (CaR)-induced extracellular Ca2+ influx and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVEC).</p><p><b>METHODS</b>(1) The interaction of STIM2 and TRPC3 was determined using the immunofluorescence technique. (2) The expressions of STIM2 and TRPC3 genes were silenced in HUVEC by transfection constructed STIM2 and TRPC3 RNA interference plasmids. The interference efficiency of STIM2, TRPC3 protein and mRNA levels were determined by Western blot and real time RT-PCR, respectively. (3) The second to fifth passage of HUVEC were divided into: STIM2-002 short hairpin RNA (STIM2-002 shRNA ) + spermine + Ca2+ group and TRPC3-004 short hairpin RNA (TRPC3-004 shRNA ) + spermine + Ca2+ group; control group (spermine + Ca2+ group) and vehicle+ spermine + Ca2+ group. The four groups of cells were incubated with CaR agonist spermine, the intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, and the production of NO was determined by DAF-FM (NO fluorescent probe) of each group in HUVEC.</p><p><b>RESULTS</b>(1) Immunofluorescence technique results showed that STIM2 and TRPC3 proteinswere present in the cytoplasm of HUVEC. (2) The results of transfection constructed STIM2 and TRPC3 RNA interference plasmids demonstrated that shRNA targeted to the STIM2 and TRPC3 genes decreased STIM2 and TRPC3 mRNA levels by 88.2% and 74.0%, respectively (P < 0.05), simultaneously, the STIM2 and TRPC3 protein levels were decreased by 79.9% and 71.8%, respectively (P < 0.05). (3) Compared with spermine + Ca2+ group, the [Ca2+]i and the net NO fluorescence intensity of spermine + Ca(2+) + ShSTIM2-002 group, spermine + Ca(2+) + ShTRPC3-004 group and spermine + Ca2+ Vehicle group were not changed (P > 0.05).</p><p><b>CONCLUSION</b>STIM2 and TRPC3 do not participate in CaR-mediated Ca2+ influx and NO production individually.</p>

Roles of gap junctions in proliferation mediated by Hcy in the spontaneous hypertensive rat vascular smooth muscle cells / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate whether homocysteine (Hcy) participates the proliferation of the spontaneously hypertensive rat(SHR) vascular smooth muscle cell (VSMCs) and the molecular mechanism.</p><p><b>METHODS</b>The rat's arota were removed. The primary SHR VSMCs were isolated and cultured in vitro, then the SHR VSMCs were divided into four groups: (1) control group, (2) Hcy group, (3) 18alpha-glycyrrhetinic acid (GA) group, (4) Hcy + 18alpha-GA group. We detected proliferation of the SHR VSMCs by MTT and flow cytometry. The expression and co-localization of the connexin (Cx) 43 and Cx40 proteins in the SHR VSMCs were deteced by immunofluorescence. The expression of the Cx43 and Cx40 proteins in SHR VSMCs were detected by Western blot. The molecular dye transfer method (scrape dye transfer method) was applied to detect the gap junction function in the SHR VSMCs.</p><p><b>RESULTS</b>(1) The Cx43 and Cx40 proteins expression in the SHR VSMCs were positive, confocal microscopy supported the co-localization of Cx43 and Cx40 in the cytoplasm. (2) The S value deteced by cell cycle and A value detected by MTT in the Hcy group were increased obviously compared with those in the control group (P < 0.05), decreased in 18alpha-GA group (P < 0.05). Compared with the Hcy group, the S and A value in the Hcy + 18alpha-GA group were significantly decreased, respectively (P < 0.05). (3) The expression of Cx43 and Cx40 proteins in Hcy group were increased compared with the control group (P < 0.05), decreased in 18alpha-GA group (P < 0.05). Compared with the Hcy group, the expression of Cx43 and Cx40 proteins in the Hcy + 18alpha-GA group were significantly decreased, respectively (P < 0.05). (4) The function of gap junction detected by scrape dye transfer method in the Hcy group were enhanced compared with the control group (P < 0.05), weakened in the 18alpha-GA group (P < 0.05). Compared with the Hcy group,the function of gap junction in the Hcy + 18alpha-GA group was significantly weakened (P < 0.05).</p><p><b>CONCLUSION</b>Hcy can enhance the function of gap junctional to stimulate the proliferation of SHR VSMCs through the expression of Cx43 and Cx40 proteins promoted.</p>

Interaction between TGF-beta1/Smad pathway and ERK pathway in vascular smooth muscle cells / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate if the interaction between TGF-beta1/Smad pathway and ERK pathway in vascular smooth muscle cells exists.</p><p><b>METHODS</b>The rat arota was removed. The primary VSMC were isolated and cultured in vitro, then the VSMC were divided into four groups: (1) control group, (2) (TGF-beta1 group, (3) ERK blocking agent group, (4) TGF-beta1 + ERK blocking agent group. The expression of Smad2/3, ERK1/2 proteins, the content of phosphorylated ERK1/2 and Smad2/3 proteins were detected by Western blot, and the expression of Smad2/3 mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR) .</p><p><b>RESULTS</b>(1) In contrast to control group, the content of phosphorylated Smad2/3 and phosphorylated ERK1/2 proteins in TGF-beta1 group was increased (P < 0.05), that in ERK blocking agent group was decreased (P < 0.05). There was no difference between control group and TGF-beta1 + ERK blocking agent group. Compared with TGF-beta1 group, the contents of phosphorylated Smad2/3 and phosphorylated ERK1/2 proteins in TGF-beta1 + ERK blocking agent group was decreased (P < 0.05). There was no difference in the expression of Smad2/3 and ERK1/2 proteins among different groups. (2) There were no differences in expression of Smad2 and Smad3 mRNA among different groups.</p><p><b>CONCLUSION</b>(1) TGF-beta1 can induce Smad2/3 proteins to be phosphorylated dependent on the activated ERK pathway. (2) ERK pathway does not effect the expression of Smad2/3 at the level of protein and mRNA.</p>

Change of JNK and c-Jun in lung injury associated with paraquat poisoning of rats / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the change of JNK and c-Jun in lung injury associated with paraquat poisoning of rats.</p><p><b>METHODS</b>46 Rats were randomly divided into four groups: PQ group (n = 12), control group (n = 10), PQ + ZnPP group (n = 12) and PQ + Hm group (n = 12). The rats were injected with 2% PQ (25 mg/kg, ip) in PQ group. ZnPP and Hemin (10 mg/kg, 10 mg/ml) were injected through inguinal vein before intraperitoneal administration of 2% paraquat in PQ + ZnPP group and PQ + Hm group respectively. The rats were injected NS (1 ml/kg, ip) in control group. HE dyeing of lung tissue and MDA content of plasma were used for estimating the injury of lung tissue. The content of CO in the lung tissue was determined. The expression of HO-1 mRNA of the lung tissue was detected by the reverse transcription-polymerase chain reaction. The phosphorylation of JNK and c-Jun was evaluated by Western blot analysis.</p><p><b>RESULTS</b>The degree of lung injury in PQ group and PQ + ZnPP group was higher than that in control group and PQ + Hm group. But in PQ + Hm group the degree of lung injury was lower. The content of MDA in PQ group and PQ + ZnPP group was higher than that in control group and PQ + Hm group (P < 0.01). The content of MDA in PQ + Hm group was higher than that in control group (P < 0.05). The content of CO in lung tissue in PQ group, PQ + ZnPP group and PQ + Hm group was and (1.08 +/- 0.15 mg/L) respectively, and higher than that in control group (P < 0.01). The content of CO in lung tissue in PQ + Hm group was significantly higher than that in PQ + ZnPP group (P < 0.01). The expression of HO-1 and the phosphorylation of JNK (55.24 +/- 9.34, 38.15 +/- 10.71, 128.55 +/- 19.43) and c-Jun (23.16 +/- 4.85, 15.49 +/- 3.13, 44.89 +/- 10.37) were increased remarkably in PQ group, PQ + ZnPP group and PQ + Hm group. Those in PQ + Hm group were higher significantly than PQ group and PQ + ZnPP group (P < 0.01). Those in PQ + ZnPP group were lower than PQ group (P < 0.05).</p><p><b>CONCLUSION</b>The increase of CO of lung tissue in rats at the lung injury associated with paraquat poisoning reduces the acute lung injury of rats. The level of JNK and c-Jun phosphorylation increases obviously, especially after Hemin is utilized.</p>

Effects of high dose ambroxol on lung injury induced by paraquat in rats / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To evaluate the protective effect of high dose ambroxol, a mucoactive drug, on acute lung injury caused by paraquat in rats.</p><p><b>METHODS</b>One hundred and thirty-six healthy male Sprague-Dawley rats were randomly divided into three groups: control group (n = 24) injected with normal saline intraperitoneally, PQ group (n = 56) [(2% paraquat (25 mg/kg) injected into peritoneal cavity on the first day)] and AT group (n = 56) ambroxol 35 mg/kg was injected into peritoneum daily after paraquat intoxication once daily for 7 consecutive days. The arterial gas was determined and the extent of lung injury was assessed by measuring the ratio of wet to dry weight (W/D) and protein content in BALF, the WBC count, the percentage of PMN, the content of malondialdehyde (MDA) and the levels of superoxide dismutase (SOD) in the blood and BALF respectively. Left lung tissue was observed through both light microscope and electron microscope (TEM).</p><p><b>RESULTS</b>The white cell count and the content of protein in the blood and the BALF of PQ group were significantly higher than those of the control group (P < 0.05 or P < 0.01). On the 7th day, the content of MDA 9 [(8.12 +/- 1.12) nmol/ml] in the serum of PQ group was significantly higher than the control group and the GSH-Px activity [(1256.8 +/- 133.2) U/ml] was significantly lower than the control group (P < 0.01). The white cell count and the content of protein in the blood and the BALF of AT group were significantly lower than the PQ group (P < 0.05 or P < 0.01). On the 7th day, the content of MDA in the serum of the AT group [(4.86 +/- 0.75) nmol/ml] was significantly lower than the PQ group and the GSH-Px activity [(1509.5 +/- 183.0) U/ml] and the SOD activity [(3903.2 +/- 374.7) U/ml] were significantly higher than the PQ group (P < 0.01). Under optical and electronic microscopes, the injury of lung tissue was reduced after large dose of ambroxol was administered.</p><p><b>CONCLUSION</b>Treatment with ambroxol (35 mg/kg) could influence the status of oxidative stress in lung and alleviate lung injury induced by paraquat. Ambroxol has obviously therapeutic effect on paraquat poisoning.</p>

Effect of baicalin on expression of heme oxygenase-1 in lung injury of rats associated with paraquat poisoning / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effect of baicalin (Bai) on lung injury, the level of TNF-alpha in cultured liquid of pulmonary interstitial macrophage and the expression of heme oxygenase-1 (HO-1) in lung injury associated with paraquat poisoning.</p><p><b>METHODS</b>Rats were randomizedly divided into four groups: control group, PQ group, Bai group (Bai, 300 mg.kg(-1).d(-1)) and simple Bai group (Bai, 300 mg. kg(-1).d(-1)) (n = 10 in each group). The 2% PQ was injected (25 mg/kg) in PQ group. Bai was injected in the rats (300 mg.kg(-1).d(-1) x 3 d) through caudal vein after paraquat poisoning in Bai group. In simple Bai group, Bai was injected in the healthy rats (300 mg.kg(-1).d(-1) x 3 d). The samples were obtained three days after intraperitoneal administration of 2% paraquat (25 mg/kg). The injury of lung was estimated with HE dyeing and electron microscope. Pulmonary interstitial macrophage (PIM) were obtained, and then cultured for 24 hours. The content of TNF-alpha was evaluated. The expression of HO-1 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression of HO-1 protein was evaluated by Western blot analysis.</p><p><b>RESULTS</b>The lung tissue was normal in control group and simple Bai group. The degree of lung injury in PQ group was higher than that in control group by HE dyeing and electron microscope observation. The level of TNF-alpha expression in cultured PIM in Bai group [(484.2 +/- 39.5) microg/L] was lower than that in PQ group [(790.2 +/- 35.0) microg/L], but higher than that in the control group [(121.6 +/- 19.2) microg/L] (P < 0.05). The expression of HO-1 mRNA and protein [(59.8 +/- 5.40) and (122.0 +/- 31.98)] in Bai group were higher than those in PQ group [(45.9 +/- 5.82) and (77.92 +/- 10.23)] (P < 0.05).</p><p><b>CONCLUSION</b>The lung injury associated with paraquat poisoning was alleviated by baicalin, which was possibly related to the decrease of level of TNF-alpha in cultured PIM and the increase of the expression of HO-1 mRNA and protein.</p>

Influence of ambroxol on paraquat-induced lung tissue injury and change of pulmonary surfactant-associated protein A in the experimental rats / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the influence of ambroxol on paraquat poisoning induced acute lung tissue injury and the change of pulmonary surfactant associated protein A in the experimental rats.</p><p><b>METHODS</b>One hundred and twenty healthy adult male Sprague-Dawley rats were randomizedly assigned into normal saline (NS) group (n = 24), paraquat poisoning induced lung tissue injury model (PQ) group (n = 48) and ambroxol treatment (AT) group (n = 48). The indexes were observed among the three groups comprising the mortality rate, the change of arterial blood PaCO(2) and PaO(2), the ratio of wet to dry lung tissue (W/D), the change of the lung tissue under light and electric microscope respectively, and the expression of pulmonary surfactant associated protein A.</p><p><b>RESULTS</b>The mortality rate of rats in the PQ group was 50.0% on the seventh day while the mortality rate in the AT group was 25.0%. The level of arterial blood PaCO(2) in the PQ group (6.94 +/- 0.8) kPa was significantly higher than that in the AT group (6.12 +/- 0.5) kPa and the NS group (4.6 +/- 0.4) kPa. The level of arterial blood PaO(2) in the PQ group (6.98 +/- 1.1) kPa was significantly lower than that in the AT group (8.25 +/- 0.7) kPa and the NS group (12.7 +/- 0.8) kPa. There were significant differences among the groups (P < 0.05). The degree of lung tissue injury was severe in PQ group and relieved in AT group. The expression of pulmonary surfactant associated protein A was significantly decreased in PQ group 13.22% +/- 2.21% on the seventh day, compared with that in the AT group (21.82% +/- 3.67%) (P < 0.05). The expression of pulmonary surfactant associated protein A in AT group was significantly higher in the AT group (18.97% +/- 0.91%) than that in the PQ group on the seventh day (P < 0.05).</p><p><b>CONCLUSION</b>Ambroxol plays a role in facilitating synthesis and secretion of pulmonary surfactant protein A and relieves the lung tissue injury induced by paraquat poisoning.</p>

Effect of arginine vasopressin on membrane potential of dorsal root ganglion neurons in rats / 生理学报

The effect of arginine vasopressin (AVP) on membrane potential of neurons from dorsal root ganglion (DRG) was examined in the rat by means of intracellular recording technique. The results showed that (1) AVP induced hyperpolarization in the membrane of most DRG neurons. (2) The membrane conductance of the DRG neurons increased by 19.32% following application of AVP (p<0.05). (3) Perfusion with balance sodium solution (BSS) containing Cd(2+) (blocker of Ca(2+) channel) instead of Na+ failed to affect the AVP-induced membrane hyperpolarization of the DRG neurons (p> 0.05). After perfusion with BSS containing tetraethylammonium (TEA), however, the extent of AVP-induced hyperpolarization was reduced (p<0.05). (4) The AVP-induced hyperpolarization of the neurons was blocked by the antagonist of AVP V(1) receptors. The results demonstrate that AVP induces hyperpolarization of most DRG neurons, which might be caused by K(+) outflow mediated by AVP V(1) receptors in the membrane of the neurons.

Effects of taurine on NOS activity in myocardium and plasma of hemorrhagic shock resuscitation in rabbits / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effect of taurine on nitric oxide synthase (NOS) activity and nitric oxide products (NO2 /NO3 ) content in myocardium and plasma during shock resuscitation.</p><p><b>METHODS</b>Twenty-four rabbits were divided randomly into 3 groups (n=8): control group, shock group, taurine group. The model of hemorrhagic shock resuscitation was used. The activities of nitric oxide synthase (NOS), lactate dehydrogenase (LDH) and the contents of nitric oxide products (NO2- /NO3-) in plasma were observed before shock and shock 1.5 hours, after resuscitation 1 hour, 2 hours and 3 hours. The activities of NOS and the contents of NO2-/NO3- in myocardium homogenate were measured after resuscitation 3 hours. Meanwhile, pathologic samples treated routinely.</p><p><b>RESULTS</b>(1) During resuscitation, the activities of NOS, LDH and the contents of NO2- /NO3- in plasma of shock group were significantly higher than that of before shock and shock 1.5 hours (P < 0.01). (2) After resuscitation 3 hours, the activity of NOS and the contents of NO2- / NO3 in myocardium of shock group were significantly higher than that of control group (P < 0.01). The cardiac myocyte appeared edema, fatty degeneration. (3) All the changes of above mentioned could be attenuated by intravenous injection taurine (40 mg/kg) (P < 0.01).</p><p><b>CONCLUSION</b>These results suggest that the NOS activation and NO release may mediated myocardium injury induced by shock resuscitation, taurine can ameliorate the myocardium injury, which may be related to decreasing the generation of NO.</p>

Relationship between lung injury induced by hemorrhagic shock/reperfusion and nitric oxide and beneficial effect of taurine / 中国应用生理学杂志

<p><b>AIM</b>To approach the relationship between lung injury induced by shock/reperfusion and nitric oxide as well as the beneficial effect of taurine.</p><p><b>METHODS</b>Twenty four rabbits were divided randomly into 3 groups (n = 8): control group, shock group, taurine group. The model of lung injury induced by shock/reperfusion was used. The activities of nitric oxide synthase (NOS), superoxide dismutase (SOD), the contents of malondialdehyde (MDA), nitric oxide products (NO2-/NO3-) in plasma and lung homogenate, lung wet/dry weight, lung water content, lung permeability index, and protein content in the pulmonary alveolar lavage fluid were measured. Meanwhile, pathologic samples treated routinely.</p><p><b>RESULTS</b>(1) At 3 hours after reperfusion, the activities of SOD in plasma and lung homogenate decreased markedly, but the other indexes above mentioned were increased significantly compared with the control group (P < 0.01). (2) A close correlation was shown between MDA content and NO2-/NO3- content in plasma and lung. Furthermore, the content of NO2-/NQ3- in lung homogenate showed strong positive correlation with the lung injury parameters. (3) Taurine (40 mg x kg(-1) i.v.) could attenuate all the changes above mentioned at the same time points of reperfusion.</p><p><b>CONCLUSION</b>NO may play an important role in lung injury induced by shock/reperfusion. Taurine can ameliorate the lung injury, mechanism of which may be related to decreasing the generation of NO and anti-lipoperoxidation.</p>