哮喘气道重塑中钙调神经磷酸酶与蛋白激酶活性的相互调节

目的:研究钙调神经磷酸酶(CaN)信号途径与蛋白激酶C(PKC)、丝裂素活化蛋白激酶(MAPK)和蛋白激酶A(PKA)的相互调节在哮喘气道重塑发病中的作用.方法:建立卵蛋白诱导的哮喘豚鼠模型,实验分为:哮喘组、CaN抑制剂环孢霉素A(CsA)组和对照组,采用磷酸化和去磷酸化方法测定肺组织CaN活性、MAPK活性、PKC活性、PKA活性.在离体培养的大鼠气道平滑肌细胞(ASMC)中,以尾加压素Ⅱ(UⅡ)为刺激因素,测定CaN,PKC和MAPK活性以及它们之间的相互调节.结果:(1)哮喘组CaN活性、MAPK活性和PKC活性分别较对照组高19%(P<0.01)、28%(P<0.01)和35%(P<0.05),PKA活性较对照组低53%(P<0.01).CsA组CaN活性、MAPK活性和PKC活性分别较哮喘组低52%(P<0.01)、18%(P<0.05)和52%(P<0.01),PKA活性较哮喘组高2.65倍(P<0.01).(2)UⅡ 10-7 mol/L孵育20 min引起ASMC PKC和MAPK活性分别较对照组增加44%和24%(P<0.01),并呈时间依赖性引起ASMC中CaN活性增加,24 h为对照组的1.67倍(P<0.01).(3)与单用UⅡ 10-7 mol/L组比,CaN抑制剂CsA 10-6 mol/L使CaN活性降低了45%(P<0.01),MAPK抑制剂PD98059 50 μmol/L对CaN活性无明显影响(P>0.05),PKC抑制剂H7 50 μmol/L使CaN活性下降21%(P<0.05).(4)CsA 10-6 mol/L 作用使UⅡ 10-7 mol/L刺激的ASMC PKC活性下降了14%(P<0.05),对MAPK活性无明显影响(P>0.05).结论:在哮喘气道重塑的发生过程中,CaN与MAPK、PKC和PKA之间存在相互调节.     

Nuclear factor- κB in signal conduction of protein kinase C in T lymphocytes from an asthmatic guinea pig model

Objective To explore the role of nuclear factor- κB (NF- κB) in the signal conduction of protein kinase C (PKC) regulated proliferation, apoptosis and expression of Th2 cytokines - interleukin- 4 (IL- 4) and interleukin- 5 (IL- 5) of T lymphocytes in the bronchial alveolus lavage fluid (BALF).Methods T lymphocytes were isolated and purified from BALF of asthmatic guinea pigs in normal and asthmatic groups, and were stimulated with PKC agitator phorbol 12- myristate 13- acetate (PMA) and NF- κB inhibitor pyrrolidine dithiocarbamate (PDTC), respectively.The expressions of NF- κB, IL- 4 and IL- 5 mRNA and protein, the proliferation and apoptosis of T lymphocytes were observed by immunohistochemistry, in situ hybridization, ELISA, MTT and TUNEL, respectively. Results The activation of NF- κB, proliferation response, and expression of IL- 4 and IL- 5 mRNA and protein in T lymphocytes stimulated by PMA were significantly higher than those of their blank control (P&lt;0.01), while those indexes of T lymphocytes stimulated by PMA and PDTC simultaneously were significantly lower than those stimulated by PMA alone (P&lt;0.01).The apoptotic index of T lymphocytes stimulated with PMA were significantly lower than that of their blank control (P&lt;0.01), and the apoptotic index of asthmatic guinea pig T lymphocytes stimulated with PMA and PDTC simultaneously were significantly higher than that stimulated by PMA alone (P&lt;0.01).The significant positive correlations were found between the activation of NF- κB and the proliferation (r=0.64, P&lt;0.001), and the expression of IL- 4 and IL- 5 mRNA and protein of T lymphocytes, respectively (r=0.55-0.68, P&lt;0.001).There was also significant negative correlation between the activation of NF- κB and apoptosis of T lymphocytes (r=0.62, P&lt;0.001). Conclusions NF- κB may participate in the signal conduction of PKC regulated proliferation, apoptosis and expression of IL- 4 and IL- 5 of T lymphocytes in asthma.The activation of NF- κB in PKC signal conduction pathway of T lymphocytes may play an important role in the pathogenesis of asthma.     

Angiotensin Ⅱ induced upregulation of Gαq/11, phospholipase Cβ3 and extracellular signal-regulated kinase 1/2 via angiotensin Ⅱtype 1 receptor

Background The role of the Gαq/11-mediated signal transduction pathway in angiotensin Ⅱ (Ang Ⅱ) induced cardiac hypertrophy remains unclear. This study was to investigate the role of the Gαq/11 signal transduction pathway in the development of cardiac hypertrophy in 2K1C hypertensive rats and in cultured neonatal rat ventricular myocytes (NRVMs) and to elucidate the effects of the pathway on Ang Ⅱ induced cardiac hypertrophy.Methods Renal hypertension was induced in 2K1C hypertensive rats by placing a silver clip around the left renal artery. At 8 weeks after operation, the systolic blood pressure, the ratio of left ventricular weight to body weight (LV/BW), and the concentration of AngⅡ in the heart were measured. The protein levels of Gαq/11 and extracellular signal-regulated kinase 1/2 (ERK1/2) were assayed by Western blot analysis, and the activity of phospholipase C (PLC) in the myocardium was detected using ［3H］-PIP2 as a substrate. Changes in ［3H］-leucine incorporation and in the protein levels of the signal molecules Gαq/11, PLCβ3, and ERK1/2 were measured after NRVMs were stimulated with 10-7mol/L AngⅡ.Results The protein levels of Gαq/11 and ERK1/2 in the hearts of 2K1C rats increased by 35.8% and 31.9%, respectively, compared with the sham group. The PLC activity in the 2K1C group was also significantly increased (P&lt;0.05). The levels of Gαq/11, PLCβ3, and ERK1/2 increased significantly after NRVMs were stimulated by AngⅡ. The upregulation of Gαq/11, PLCβ3 and ERK1/2 in NRVMs occurred prior to ［3H］-leucine incorporation increases, and could be inhibited with losartan. Conclusion AngⅡ can initiate cardiac hypertrophy and upregulate signal molecules in the Gαq/11-mediated signal transduction pathway, such as Gαq/11, PLCβ3 and ERK1/2, at both tissue and cellular levels.     

Effect of advanced glycosylation end products on activity of protein kinase C in human peripheral blood mononuclear cells

Objectives To investigate the effect of advanced glycosylation end products (AGEs) on the a ctivity of protein kinase C (PKC) in human peripheral blood mononuclear cells (P BMC) and to observe whether aminoguanidine (AG) can influence the effect of AGEs .Methods After PBMC were isolated from human peripheral blood and incubated with differen t concentrations of AGEs-BSA for various periods, total PKC activity in PBMC wa s determined by measuring the incorporation of (32)P from ［γ-(32) P］ ATP into a special substrate using Promega PKC assay kit.Results AGEs-BSA increased the total PKC activity in PBMC from 83.43±6.57 pmol/min/ mg protein to 116.8±13.82 pmol/min/mg protein with a peak at 15 min. AGEs -BSA also increased the total PKC activity in a concentration-dependent manner fro m 83.1±6.4 pmol/min/mg protein (control) to 119.1±13.3 pmol/min/mg prote in (control vs AGEs-BSA 400 mg/L, P&lt;0.01).Furthermore, AGEs-BSA induc ed an elevation of PKC activity in a glycosylating time-related manner, from 80 .9±8.2 (control) to 118.3±11.5 pmol/min/mg protein (glycosylation for 12 wk, P&lt;0.01).The total PKC activity stimulated by AGEs-BSA pretreated wi th AG (100, 200 mg/L) was markedly lower than that of AGEs-BSA group not pretr eated with AG (P&lt;0.05, P&lt;0.01).Conclusions AGEs-BSA increased the total PKC activity in PBMC in a concentration and incuba tion time dependent manner. The ability of AGEs-BSA to stimulate PKC activity was markedly decreased by pretreatment of AGEs-BSA with AG.     

Changes in delayed rectifier K+ channel function and its regulation by protein kinase C pathway in bronchial myocytes from asthmatic rats

Objective To investigate changes in the delayed rectifier K+ channel (Kv) function and the regulation of Kv by the protein kinase C (PKC) pathway in bronchial myocytes from asthmatic rats. Methods The Kv currents and membrane potentials in bronchial myocytes from asthmatic rats and from controls were observed, using whole cell voltage- and current-patch clamp techniques.Results Bronchial myocytes from asthmatic rats showed a significant reduction in Kv-current density (51.6±9.4 pA/pF, n=14, P&lt;0.01) in comparison with those from control rats (72.4±12.3 pA/pF, n=14) at +50 mV. The current-voltage relationship curve exhibited a significant downward shift. Bronchial myocytes from asthmatic rats had no significantly different capacitances (P&gt;0.05), but had more positive membrane potential ( P&lt;0.01) compared with those from controls. 1 μmol/L phorbol 12-myristate 13-acetate, a PKC activator, caused an obvious reduction in Kv-current density (P&lt;0.01) and a significant downward shift in the current-voltage relationship curve, an effect which was partly abolished by 1 μmol/L Ro31-8220 (a PKC inhibitor); 1 μmol/L phorbol 12-myristate 13-acetate caused more positive membrane potential (Em), from -36.8±5.7 mV to -30.4±7.3 mV, in rat bronchial myocytes (P&lt;0.05). This effect was partly abolished by 1 μmol/L Ro31-8220. Conclusions Bronchial myocytes from asthmatic rats have inhibited Kv function, more positive membrane potential, and higher excitability, all of which can also be induced by PKC activation. These characteristics may contribute to the development of airway hyperreactivity in asthma.     

Comparative study of the effect of basic fibroblast growth factor and vascular endothelial growth factor on limb ischemia/reperfusion injury of rats

Objective To investigate the effect of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on limb ischemia/reperfusion injury of rats and the mechanism. Methods The hind limb ischemia/reperfusion injury of male SD rats was induced by tourniquet for 2 hours and then reperfusing for 12 hours with administration of different agents. Animals were divided into control, bFGF-10 and bFGF-50, VEGF-10 and VEGF-50 group by infusing physiological saline, 10 and 50 μg/kg bFGE, 10 and 50 μg/kg VEGF, respectively. Blo od was collected to determine malonyldialdehyde (MDA), and the ischemic-reperfused gastrocnemius muscle and the contralateral control one were harvested together for measurement of tissue viability, water content, myeloperoxidose (MPO) activity, ATP and MDA concentration.Results Compared with control group, tissue viability of ischemia/reperfusion limb in bFGF-10 and bFGF-50 group increased by 16.0% (P&lt;0.05) and 32.8% (P&lt;0.01), ATP content increased by 14.8% and 35.6% (P&lt;0.01), and plasma MDA level decreased by 45.2% and 56.2% (P&lt;0.01). 10 μg/kg bFGF had no significant effect on tissue water content, MPO activity, MDA concentration of ischemia/reperfusion limb, while 50 μg/kg of bFGF lowered these values by 15.7%, 32.5% and 13.6% (P&lt;0.05) and 14.7% (P&lt;0.01), MPO activity augmented by 44.9% and 96.1% (P&lt;0.01), ATP content decreased by 13.1% (P&lt;0.05) and 33.3% (P&lt;0.01). Plasma and tissue MDA concentrations in VEGF-10 group had no significant changes (P&gt;0.05), while in VEGF-50 group, these values were elevated by 46.4% and 38.6% (P&lt;0.01) . Conclusion bFGF attenuated, while VEGF exacerbated ischemia/reperfusion injury of rat limb significantly, the mechanism of which was probably related to preventing or enhancing lipid peroxide, and increasing or decreasing energy store.     

Anti-endotoxic shock effects of cyproheptadine in rats

Objective To investigate the antagonistic effect and mechnism of the effect of cyprohe ptadine (Cyp) on endotoxic shock in rats. Methods Endotoxic shock was produced in rats by iv injection of lipopolysaccharides (LPS) (5 mg/kg). Tumor necrosis fator (TNFα) mRNA expression was assessed by Northern blot. Plasma TNFα content was measured by radioimmunoassay. P lasma superoxide dismutase (SOD) activity and malondialdehyde (MDA) content wer e measured. The intracellular free calcium concentration (［Ca&lt;sup&gt;2+&lt;/sup&gt;&lt;sub&gt;i&lt;/sub&gt;) in single endothelial cells was determined by laser scanning confocal microscop y (LSCM).Results Cyp 5 mg/kg injected immediately after iv LPS raised the mean arterial blood p ressure (MABP) of shocked rats and improved their 24 h survival rate. Meanwhil e, Cyp markedly decreased TNFα mRNA levels in rat liver (18±10 vs LPS+sal ine 38±10, P&lt;0.01) as well as plasma TNFα content ［(7.8±2.4) μg/L vs LPS+saline (21.5±3.2) μg/L, P&lt;0.01)］. It enhanced plasma SOD act ivity ［(1037.2±112.8) NU/L vs LPS+saline (615.4±92.6) NU/L, P&lt;0.01］ , reduced the MDA content ［(5.2±1.1) μmol/L vs LPS+saline (9.8±1.5) μmol/L, P&lt;0.01］, and inhibited TNFα- induced Ca(2+)(i) eleva tion.Conclusion Cyp exerts an anti- endotoxic shock effect by inhibiting TNFα gene expression , enhancing SOD activity, reducing lipid peroxidation, and preventing ［Ca［2+］］i overload.     

Effects of ribozyme targeting platelet-derived growth factor receptor β subunit gene on the proliferation and apoptosis of hepatic stellate cells in vitro

Background Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor β subunit (PDGFR-β) is the predominant signal transduction pathyway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-β mRNA in HSC and the effect on biological characteristics of HSC.Methods Expression vector of anti-PDGFR-β ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-β, α-smooth muscle actin, and typeⅠand type Ⅲ collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.Results The expression of PDGFR-β at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49%-57% (P&lt;0.05-0.01). The proliferation and α-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased (P&lt;0.05-0.01), and the type Ⅰ and type Ⅲ collagen synthesis were also reduced (P&lt;0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC (P&lt;0.01), and typical apoptotic cells could be found under transmission electron microscopy.Conclusions The anti-PDGFR-β ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-β expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-β expression.     

The role of cyclooxygenase-2/prostanoid pathway in visceral

Background Cyclooxygenase (COX) is the rate-limiting enzyme in the production of prostanoids from arachidonic acid. COX-2 is the inducible enzyme in the COX family, together with the prostanoids forms the COX-2/prostanoid pathway. Research showed that the COX-2/prostanoid pathway is activated in hepatic diseases and liver stress reaction, such as fibrogenesis, portal hypertension, carcinogenesis, and ischemic/reperfusion injury. But there was no report on visceral pain induced liver stress. This study was to investigate the role of the COX-2/prostanoid pathway in liver stress response in rat acute colitis visceral pain liver stress model. Methods Fifty-three male SD rats were randomly divided into Naive, Model, NS398 treatment, and Morphine treatment groups. The rat acute colitis visceral pain liver stress model was established under anesthesia by the colonic administration of 0.5 ml of 6% acetic acid using a urethral catheter. NS398 and morphine were administrated 30 minutes prior to model establishment in NS398 and Morphine treatment groups respectively. Spontaneous activities and pain behavior were counted and the extent of colonic inflammation was assessed histologically. Liver tissue levels of Glutathione-S-Transferase (GST) activity, COX-2 mRNA, prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and 6-Ketone-prostaglandin F1α (6-K-PGF1α) contents were assessed.Results Thirty minutes after the colonic administration of acetic acid, a significant decrease in spontaneous activities and an increase in pain behaviors were observed in Model group (P&lt;0.01 and P&lt;0.05 respectively), accompanied by colonic inflammation. Liver GST activity levels significantly dropped (P&lt;0.05). Liver COX-2 mRNA expression significantly increased, accompanied by an increase in liver concentrations of PGE2 and TXB2, but no obvious change in 6-K-PGF1α concentrations. NS398 and morphine both ameliorated post-stress liver GST activity (P&lt;0.05 and P&lt;0.01 respectively), decreased stress-induced COX-2 expression, decreased PGE2 and TXB2 production, but increased liver 6-K-PGF1α levels. Morphine attenuation in colonic tissue inflammation was apparent at 24 hours (P&lt;0.05).Conclusions Acute colitis visceral pain liver stress can induce liver injury. Liver injury might have occurred through the activation of the COX-2/prostanoid pathway and increased production of PGE2 and TXB2. Effective analgesia might offer protective effect during visceral pain stress.     

Inhibitory effect of PD98059 on proliferation of EJ cells induced by acidic fibroblast growth factor

Objective To investigate the inhibitory effect of PD98059 （ERK kinase inhibitor） on proliferation and deduce the intracellular signal transduction pathway of EJ cells induced by acidic fibro-blast growth factor （aFGF）.Methods EJ cells were treated with variable doses of aFGF and the activity of ERK was measured;the EJ cells were then treated with different doses of PD98059,     

钙调神经磷酸酶信号通路在AngⅡ刺激的大鼠VSMCs增殖中的作用

目的:探讨钙调神经磷酸酶（calcineurin,CaN）依赖的信号通路在血管紧张素Ⅱ（Ang Ⅱ）刺激的大鼠血管平滑肌细胞（VSMCs）增殖中的作用。方法:采用组织贴块法,体外原代培养大鼠胸主动脉平滑肌细胞;Ang Ⅱ刺激培养的大鼠 VSMCs增殖;CaN特异性抑制剂环孢素 A（CsA）阻断Ang Ⅱ刺激的大鼠VSMCs中CaN依赖的信号转导通路;观察各因素对CaN活性、细胞增殖活度、增殖细胞核抗原（PCNA）表达水平的影响。结果:在一定范围内,Ang Ⅱ（10-8～10-6mol/L）以浓度依赖性方式增加VSMCs的CaN活性,同时细胞增殖活度（AMTT值）增高,与对照组相比,差异有统计学意义（P<0.01或P<0.001）。CsA（1 μmol/L）抑制CaN活性后,明显降低Ang Ⅱ（10-7mol/L）刺激的VSMCs增殖活度及核内PCNA的表达水平（OD值）,与Ang Ⅱ组比较,差异显著（P<0.001）。结论:CaN依赖的信号通路在Ang Ⅱ刺激的血管平滑肌细胞增殖中发挥重要作用。     

三磷酸肌醇剌激心肌成纤维细胞的增殖

目的　探讨钙调神经磷酸酶 (CaN)依赖的信号通路在三磷酸肌醇 (IP3)剌激的乳鼠心肌成纤维细胞 (FBs)增殖中的作用。方法　以培养的FBs为模型 ,用IP3剌激FBs内Ca2 +释放 ,环孢素A(CsA)阻断CaN ,维拉帕米 (Ver)阻断FBs钙通道 ,检测FBs的CaN、丝裂素活化蛋白激酶 (MAPK)、蛋白激酶C(PKC)活性 ,用3H 亮氨酸及3H 胸腺嘧啶掺入量作为反应FBs增殖的指标。结果　IP3剌激组FBs蛋白核酸合成速率明显增高 ,与对照组相比差异显著 (P <0 .0 1 ) ;CsA及Ver能明显抑制IP3介导的FBs蛋白核酸合成速率增高 ,与IP3剌激组相比差异显著 (P <0 .0 1 )。同时发现IP3剌激组CaN、PKC活性与对照FBs相比差异显著 (P <0 .0 5,P <0 .0 1 )。CsA和Ver抑制IP3介导的FBs的CaN活性增高 ,Ver抑制IP3介导的FBs的PKC活性增高。结论　CaN在IP3剌激的FBs增殖中起重要作用 ,但CaN信号通路不是IP3剌激FBs增殖的唯一信号通路 ,其它信号通路也可能参与了IP3剌激的FBs增殖。     

UROTENSIN II RECEPTOR IN THE RAT AIRWAY SMOOTH MUSCLE AND ITS EFFECT ON THE RAT AIRWAY SMOOTH MUSCLE CELLS PROLIFERATION

INTRODUCTIONUrotensinII（U－II）isavasoactive“somatostatin－like”cyclicpeptidewhichwasoriginallyisolatedfromfishspinalcordsandwhichhasrecentlybeenclonedfromman（１,２）．Ames,etal．recently（in１９９９）identifiedanorphanhumanG－protein－coupledreceptorthatishomol－ogoustoratGPR１４andexpressedpredominantlyincar－diovasculartissue,whichfunctionsasaU－IIreceptor．HumanU－IIisfoundwithinbothvascularandcardiactissue,includingcoronaryatheroma．ThepotencyofvasoconstrictionofU－IIi…     

bFGF对NIH3T3细胞TPK,PKC,ERK活性的影响

目的:观察碱性成纤维细胞生长因子(bFGF)对NIH3T3细胞酪氨酸蛋白激酶(TPK)、蛋白激酶C(PKC)及胞外调节蛋白激酶(ERK)活性的影响,探讨其信号转导机制.方法:分别加入bFGF及PKC抑制剂H-7、MEK1抑制剂PD98059孵育细胞,利用[γ-32P]ATP掺入外源性底物的方法测定活性.结果:bFGF可使细胞膜TPK、细胞质PKC及ERK活性明显升高.H-7 bFGF、PD98059 bFGF组与只加bFGF组比较TPK活性保持不变,而PKC及ERK活性均下降.结论:TPK、PKC、ERK介导bFGF的信号转导.ERK与PKC是TPK受体下游的两个分支,PKC与ERK可以互相激活.     

钙调神经磷酸酶对血管平滑肌细胞增殖中蛋白激酶G表达的影响

目的:探讨钙调神经磷酸酶(CaN)对血管平滑肌细胞(VSMCs)增殖中蛋白激酶G(PKG Iα)表达的影响.方法:对大鼠VSMCs培养并行细胞鉴定,分为对照组、低浓度环孢菌素A(CsA,0.5 mg/L)干预组、高浓度CsA(5 mg/L)干预组以及苯肾上腺素(PE)刺激组(5 mg/L CsA 10 μmol/L PE).其中CsA为CaN特异性抑制剂,PE为已知的CaN激活剂,能够刺激细胞增殖.应用RT-PCR、免疫细胞化学及Western blot法定性及定量测定VSMCs增殖中PKG Iα mRNA以及蛋白的表达水平.结果:0.5 mg/L CsA组PKG Iα mRNA以及蛋白的表达水平与对照组相比差异无统计学意义,而5 mg/L CsA组PKG Iα mRNA及蛋白的表达明显高于对照组,5 mg/L CsA 10 μmol/L PE组中PKG Iα mRNA的表达比5 mg/L CsA组减少了32.2%,蛋白的表达减少了36.7%,但仍明显高于对照组.结论:CaN能够调节培养VSMCs中PKG Iα的表达,增殖细胞用CaN特异性抑制剂CsA灭活CaN后,PKG Iα的表达明显增加,给予PE再次激活CaN后,PKG Iα的表达明显减少.     

The role of calcineurin in the lung fibroblasts proliferation and collagen synthesis induced by basic fibroblast growth factor

Objective To investigate the role of calcineurin (CaN) in the lung fibroblast proliferation and collagen synthesis induced by basic fibroblast growth factor (bFGF).Methods We used Western blot and immunohistochemical methods for investigating the content and distribution of calcineurin in the lung tissue. Calcineurin activity in different tissues was measured using 32P-labelled substrate. In the primary culture of lung fibroblasts, 3H-thymidine (3H-TdR) and 3H-proline incorporation methods were used to study the effect of cyclosporin A (CsA), an inhibitor of calcineurin, on the lung fibroblast DMA and collagen synthesis stimulated by bFGF.Results We found that calcineurin was expressed in lung tissue and has phosphatase activity (7.1±2.0 pmol Pi/mg pr/min). CsA (10~(-8)-10~(-6) mol/L) inhibited lung fibroblast 3H-TdR incorporation induced by bFGF in a dose-dependent manner, with the inhibitory rates by 20% , 46% and 66% (P< 0.01). CsA (10~(-7) -10~(-6) mol/L) inhibited 3H-proline incorporation in lung     

Increased activity of protein kinase C in alveolar macrophages in idiop athic pulmonary fibrosis

目的　探讨特发性肺纤维化 (IPF)患者肺泡巨噬细胞蛋白激酶C(PKC)活性的变化。方法　应用放射活性测定法检测 9例健康对照者和 15例临床诊断为IPF的患者经纤维支气管镜检查获得的支气管肺泡灌洗液 (BALF)中肺泡巨噬细胞的PKC活性。结果　IPF患者BALF肺泡巨噬细胞总PKC活性 (57 86± 8 6 0pmol·min 1·mg 1vs 4 6 0 2± 11 73pmol·min 1·mg 1)、胞浆 (89 83± 2 1 94pmol·min 1·mg 1vs 6 5 73± 2 7 91pmol·min 1·mg 1)和胞膜PKC活性 (39 6 7± 8 98pmol·min 1·mg 1vs 32 77± 4 96pmol·min 1·mg 1)均明显高于对照组 (P <0 0 1、P <0 0 5和P <0 0 5) ,AM的总PKC活性与BALF中细胞总数呈正相关 (r=0 8135,P <0 0 1) ,胞膜PKC活性也与之呈正相关 (r=0 5917,P <0 0 5)。结论　PKC作为细胞活化的信号传导通路与肺间质病的发生发展密切相关     

p38丝裂原活化蛋白激酶在β淀粉样肽+缺氧缺糖所致SHSY5Y细胞损伤中的作用

目的探讨p38丝裂原活化蛋白激酶(p38MAPK)活性在β淀粉样肽(Aβ)+缺氧缺糖(OGD)所致SHSY5Y细胞损伤中的作用。方法体外培养SHSY5Y神经母细胞瘤株,以OGD、伴或不伴Aβ(5μmol/L)处理,以MTT检测SHSY5Y细胞活性,Westernblot方法检测p38MAPK活性,fluo-3/AM的荧光强度检测SHSY5Y细胞内钙离子的浓度。结果与对照组相比,OGD、Aβ、OGD+Aβ组SHSY5Y细胞活性显著下降,分别为对照组的(63±4)%、(68±7)%、(56±1)%,(P<0.05或P<0.01);p38MAPK活性在OGD、Aβ、OGD+Aβ组显著升高,分别为对照组的(121±3)%、(132±4)%、(165±5)%,(P<0.05或P<0.01);fluo-3/AM的荧光强度在OGD、Aβ、OGD+Aβ组显著升高,分别为照组的(169±4)%、(176±4)%、(220±7)%,(P<0.05或P<0.01)。结论与OGD、Aβ比较,OGD+Aβ对SHSY5Y细胞具有更显著的细胞毒性作用。OGD+Aβ通过激活p38MAPK,进而促进细胞内钙超载,最终导致SHSY5Y细胞死亡。     

神经细胞牵张损伤后CsA对CaN活性和APP表达的影响

目的 研究神经细胞牵张损伤后环孢霉素A (cyclosporin A, CsA)对钙调神经磷酸酶(calcineurin, CaN)活性和淀粉样前体蛋白(amyloid precursor protein, APP)表达的影响.方法 在Bioflex培养皿原代培养大鼠大脑皮层神经元.细胞分为正常对照组、损伤组和CsA组.多功能小型生物撞击机25 kPa的压力牵张细胞.损伤后损伤组不予以处理,而CsA组则给以10 nM CsA. 各组伤后1、6、24h,3d标本的CaN活性和APP蛋白表达,分别采用酶底物显色和Western blot方法进行检测.结果 牵张损伤后1h神经元的CaN活性已较正常对照组显著增加(P＜0.05),伤后6h更为明显(P＜0.01),并持续到伤后3d(P＜0.01);而伤后10nM CsA干预能在各时相点非常显著地降低CaN的活性(P＜0.01).APP伤后1h有增加趋势,但在伤后6h差异才具有统计学意义(P＜0.05),伤后24h、3d这种增加则更加明显(P＜0.01).伤后10nM CsA干预能在6、24h,3d显著地降低APP的表达增加(P＜0.05),其APP的表达仍显著高于正常对照组(P＜0.05).结论 CsA能降低神经细胞牵张损伤后CaN活性的增加并减少APP表达的增加,可能起到神经保护作用.