双歧杆菌对裸鼠腹腔巨噬细胞NO/cGMP信号系统的影响

双歧杆菌是哺乳动物肠道内的生理性主导菌。它能激活巨噬细胞和ＮＫ细胞等多种效应细胞。本文首次运用Ｇｒｉｅｓｓ试剂、激光共聚焦显微镜以及放免法检测了双歧杆菌刺激裸鼠腹腔巨噬细胞后产生的ＮＯ、ｉＮＯＳ和ｃＧＭＰ的含量 ,旨在探讨这些信号效应分子在双歧杆菌调节机体免疫反应中的作用。1　材料和方法1 1　菌种来源及其培养　双歧杆菌 (Ｂａｃｉｌｌｕｓｂｉｆｉｄｕｓ)系本所从健康婴儿粪便中分离培养所得 ,经ＡＰＩ及ＴＡＢ系统 (英国 )和多次生化反应鉴定为青春型。实验时将双歧杆菌接种至硫乙醇酸盐肉汤中 ,置厌氧培养箱 37℃培…     

双歧乐对双歧杆菌增殖的影响

本文介绍了唐山市生物化学制药厂生产的双歧乐活性的检验方法和结果.用含有双歧乐的培养基对双歧杆菌进行了定性、定量观察.其结果表明,唐山市生物化学制药厂生产的双歧乐对双歧杆菌有明显的促进作用;在双歧杆菌制剂的生产、实验过程中,如果添加一定量的双歧乐,不仅可以提高双歧杆菌收率,而且还可以缩短菌种发酵时间.     

氢溴酸樟柳碱对急性脑缺血再灌注损伤模型大鼠脑组织细胞凋亡及ERK1/2磷酸化水平的影响

目的:研究氢溴酸樟柳碱对急性脑缺血再灌注损伤模型大鼠脑组织细胞凋亡及细胞外信号调节蛋白激酶1/2(ERK1/2)磷酸化(p-ERK1/2)水平的影响。方法:将大鼠随机分为假手术组、模型组、阳性对照组(尼莫地平1.0 mg/kg)和氢溴酸樟柳碱高、中、低、极低剂量组(1.2、0.6、0.3、0.15 mg/kg),每组8只,采用线栓法建立大鼠急性脑缺血再灌注损伤模型。分别于脑缺血2 h和再灌注6 h时对各组大鼠尾iv给药1次,再灌注22 h后检测各组大鼠脑组织三磷酸腺苷(ATP)酶活性、Ca~(2+)含量、细胞凋亡情况、脑组织中p-ERK1/2蛋白表达和p-ERK1/2/总ERK1/2(t-ERK1/2)比例。结果:与假手术组比较,模型组大鼠脑组织ATP酶活性明显降低、Ca~(2+)含量明显增加、凋亡细胞密度明显增加,以上差异均有统计学意义(P<0.01)。与模型组比较,各给药组大鼠脑组织凋亡细胞密度均明显减小,阳性对照组和氢溴酸樟柳碱高、低剂量组大鼠脑组织Ca~(2+)含量均明显降低,氢溴酸樟柳碱高、低、极低剂量组大鼠脑组织中p-ERK1/2/t-ERK1/2比例均明显增加,以上差异均有统计学意义(P<0.05或P<0.01);其余差异均无统计学意义(P>0.05)。结论:氢溴酸樟柳碱能抑制急性脑缺血再灌注损伤模型大鼠脑组织细胞凋亡,其作用机制可能与激活ERK1/2信号通路和调节ATP酶活性,进而降低脑组织Ca~(2+)含量有关。     

ERK1/2在胶质瘤中的表达及意义

目的 探讨细胞外信号调节激酶1/2(ERK1/2)在人脑胶质瘤中的表达及其与胶质瘤的发生发展的关系.方法 收集具有明确病理分级的胶质瘤标本48例,WHO I～Ⅱ级15例,Ⅲ级21例,Ⅳ级12例;另取8例正常脑组织作正常对照.用免疫组织化学染色检测ERK1/2和磷酸化ERK1/2(p-ERK1/2)表达水平.结果 在胶质瘤组织中,ERK1/2和p-ERK1/2表达均增高,且与胶质瘤恶性程度呈正相关;在Ⅲ、Ⅳ级胶质瘤中,ERK1/2和p-ERK1/2表达水平显著强于I、Ⅱ级胶质瘤.结论 ERK1/2在胶质瘤组织中表达水平与胶质瘤的恶性程度有关.     

周期性压力对大鼠软骨细胞ERK1、ERK2的影响

目的 探讨周期性压力对大鼠软骨细胞细胞外信号调节激酶(ERK)1和ERK2的影响.方法 将软骨细胞置于压力为150 kPa,频率为0.1 Hz的周期性应力系统中.分为四组:A组不加压作为对照;B、C和D组分别加压0.5、1和2 h.用细胞免疫荧光法观察磷酸化ERK1和ERK2(pERK1和pERK2)在软骨细胞内的定位表达,同时用Western blot法检测ERK1和ERK2在软骨细胞内的磷酸化.结果 加压组的软骨细胞内pERK1和pERK2的荧光强度较A组明显增强;C组的荧光强度最大.pERK1和pERK2在B、C、D组内的表达较A组增高(P<0.01).结论 适当的压力刺激可导致软骨细胞内pERK1和pERK2表达增高,ERK1和ERK2可能在软骨细胞将压力信号转导为生物化学信号过程中起重要作用.     

白皮杉醇对糖尿病肾病大鼠ERK1/2通路及CTGF蛋白表达的影响

目的:探究白皮杉醇(PIC)对糖尿病肾病(DN)大鼠细胞外调节蛋白激酶(ERK1/2)通路及其调控的结缔组织生长因子(CTGF)蛋白表达的影响.方法:取健康雄性SD大鼠,采用高脂高糖喂养+腹腔注射链脲霉素建立大鼠DN模型,并将造模成功的50只SD大鼠随机分为模型组(DN组)、PIC低(50mg·kg-1)、中(100m...     

基于MAPK/ERK信号通路探讨当归多糖对脑缺血再灌注损伤大鼠的影响

目的 探讨当归多糖对脑缺血再灌注损伤大鼠丝裂原活化蛋白激酶(MAPK)/细胞外调节蛋白激酶(ERK)信号通路的影响.方法 选SPF级健康雄性SD大鼠65只,随机分为对照组12只和造模组53只.造模采用大脑中动脉阻塞法建立脑缺血再灌注损伤大鼠模型,对照组大鼠给予假手术.将造模成功的大鼠随机分为模型组、西药组及当归多糖低、...     

ERK1/2通路在哮喘大鼠的表达和作用

目的：探讨哮喘大鼠肺组织中磷酸化的ERK1/2（p-ERK1/2）的表达情况，以及其与肺组织炎性评分之间的相关性。方法清洁级雄性SD大鼠20只，随机分为正常对照组和哮喘组，每组10只。 HE染色，半定量评定大鼠肺组织炎症程度；用免疫组织化学染色法观察p-ERK1/2在哮喘大鼠肺组织的表达。结果哮喘组大鼠肺组织炎性评分及p-ERK1/2表达显著升高，差异有统计学意义（P＜0.01），p-ERK1/2主要表达于气道周围，两者呈显著正相关（r=0.779，P＜0.01）。结论哮喘大鼠的肺组织 p-ERK1/2表达水平显著增加，提示p-ERK1/2可能参与了哮喘的发病过程，且与肺部炎症有一定关系。     

辛伐他汀干预对急性心肌梗死大鼠ERK1／2信号通路及心室重塑的影响

目的：观察辛伐他汀对急性心肌梗死大鼠心功能、心室肌Ⅰ、Ⅲ型胶原含量变化、心脏细胞外信号调控蛋白激酶1/2（ERK1/2）和p-ERK1/2的影响。方法：20只建模后24h存活的雄性Wistar心肌梗死（AMI）大鼠,随机分为AMI（A）组,辛伐他汀（S）组[20mg/（kg.d）],每组10只,另设假手术（C）组（n=6）,手术方法同模型组,但不结扎冠状动脉。术后8周测定心功能和血流动力学参数。处死后测定左室重量指数（left ventricular weight index,LVWI）;Western blot法检测ERK1/2和p-ERK1/2的表达。结果：辛伐他汀组与AMI组比较心肌梗死面积差异无统计学意义（P〉0.05）;左心室截面直径、面积和左心室容积差异无统计学意义（P〉0.05）;LVW、LVW/BW和LVEDP显著降低（P〈0.01）,±dp/dt/LVSP绝对值升高（P〈0.01）。辛伐他汀组与AMI组相比左心室非梗死区Ⅰ型和Ⅲ型胶原含量和ERK1/2磷酸化水平显著降低（P〈0.01）,左心室非梗死区Ⅰ型、Ⅲ型胶原含量和心脏组织内ERK1/2磷酸化水平均呈显著正相关（r分别为0.916和0.983,P〈0.01）。结论：辛伐他汀能有效抑制AMI左心室非梗死区Ⅰ、Ⅲ型胶原的产生以及缓解大鼠AMI后心室重构、改善心功能。这种作用可能与抑制心脏内ERK1/2磷酸化水平有关。     

新型KATP开放剂埃他卡林对原代培养人肺动脉平滑肌细胞ERK1／2磷酸化的影响

目的：研究新型ATP敏感性钾通道（KATP）开放剂埃他卡林（I阴）对内皮素-1（ET-1）诱导的原代培养人肺动脉平滑肌细胞细胞外信号调节激酶1和2（ERK1／2）磷酸化的影响。方法：原代培养人肺动脉平滑肌细胞,用Western blot方法检测磷酸化细胞外信号调节激酶1和2（p-ERK1／2）。培养液中加入ET-1（10nmol／L）,孵育0、1、2、5、10、30、60min。培养液中加入ET-1（10nmol／L）前30min分别加入0．1、1．0和10．0umol／LIPT．孵育10min。培养液中加入ET-1（10nmol／L）和IPT（10umol／L）前30min加入格列本脲（CU）（10umol／L）,孵育10min。结果：在2min至30min之间,ET-1呈时间依赖性促进人肺动脉平滑肌细胞ERK1／2磷酸化,10min时最明显。IPI、呈浓度依赖性拮抗ET-1对ERK1／2磷酸化的影响。特异性KATP阻断剂GLI逆转IPT的作用。结论：IPT可能通过激活KATP通道,抑制ET-1诱导的原代培养人肺动脉平滑肌细胞ERK1／2磷酸化,可能可用于肺血管重构、肺动脉高压的治疗。     

ERK1/2及JNK通路在AcSDKP抑制PDGF诱导的大鼠心脏成纤维细胞增殖和胶原蛋白表达中的作用

目的探讨细胞外信号调节激酶1/2(ERK1/2)及C-Jun氨基末端激酶(JNK)通路在N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)抑制血小板源性生长因子(PDGF)诱导的大鼠心脏成纤维细胞增殖和胶原蛋白表达中的作用。方法将新生Wistar大鼠心脏成纤维细胞分为对照组(A组)、10ng/ml PDGF刺激组(B组)、10-9 mol/L AcSDKP+10ng/ml PDGF干预组(C组)、25μmol/L PD98059+10ng/ml PDGF干预组(D组)和10nmol/L SP600125+10ng/ml PDGF干预组(E组)。MTT法检测心脏成纤维细胞代谢活性的变化,Western blot法检测大鼠心脏成纤维细胞中ERK1/2、JNK及磷酸化-ERK1/2、磷酸化-JNK蛋白表达和Ⅰ型、Ⅲ型胶原蛋白的表达。结果与A组相比,B组心脏成纤维细胞代谢活性、Ⅰ型及Ⅲ型胶原蛋白表达、磷酸化-ERK1/2及磷酸化-JNK蛋白表达均增强(P<0.05);而C、D、E组能明显逆转B组上述各观察指标的增加过程(P<0.05)。结论 AcSDKP能够通过阻断PDGF介导的ERK1/2及JNK通路的激活,进而抑制大鼠心脏成纤维细胞增殖和胶原蛋白表达。     

灵芝多糖对小鼠腹腔巨噬细胞蛋白激酶C活性的影响

目的　观察灵芝多糖在体外对小鼠腹腔巨噬细胞(MΦ)蛋白激酶C(PKC)活性是否有影响。方法　采用反相离子对高效液相色谱法测定PKC活力。结果　灵芝多糖GLB7( 40mg·L-1)能引起小鼠腹腔MΦ中PKC活性明显升高 ,30min达峰值 ,2h恢复到基础水平 ;GLB7还可引起MΦ中PKC发生质膜转位 ,并拮抗Staurosporine对MΦ中PKC的抑制作用。结论　灵芝多糖的免疫增强作用与其增强PKC活性有关     

ERK信号蛋白在吗啡预处理减轻大鼠全心缺血/再灌注损伤中的作用

目的探讨细胞外信号调节激酶(extracellular signaling-regulated kinase,ERK)在吗啡预处理减轻大鼠全心缺血/再灌注损伤中的作用。方法健康成年♂SD大鼠,采用随机数字表法分为6组(n=10):对照组(control,CON)、缺血/再灌注组(ischemia/reperfusion,I/R)、缺血预处理组(ischemia preconditioning,IPC)、吗啡1μmol·L^-1预处理组(morphine preconditioning,MPC)、MPC+ERK抑制剂PD98059组(MPD)、ERK抑制剂PD98059对照组(PD)。利用Langendorff灌流系统对各组大鼠离体心脏在进行相应处理后,化学比色法检测基线、再灌注5、10 min时冠脉流出液乳酸脱氢酶(lactate dehydrogenase,LDH)活性;同时利用充水球囊置入左心室持续监测左心室发展压(left ventricular developed pressure,LVDP)。2,3,5-氯化三苯基四氮唑(triphenyltetrazolium chloride,TTC)染色测量梗死区(infarct size,IS)、缺血危险区(area at risk,AAR)体积及IS/AAR比值;Western blot检测心肌组织ERK磷酸化水平。结果与I/R组比较,MPC组IS和IS/AAR减小,再灌注5、10 min时LDH活性降低,再灌注结束时LVDP升高,心肌组织磷酸化ERK(p-ERK)相对表达量增加;ERK抑制剂PD98059阻断MPC的保护作用,增加心肌梗死面积,升高再灌注5、10 min时LDH活性,降低再灌注末LVDP,此外,PD98059抑制MPC诱导的ERK磷酸化。结论吗啡预处理可能通过诱导ERK磷酸化水平升高而减轻大鼠全心缺血/再灌注损伤。     

Ginsenoside Rd prevents and rescues rat intestinal epithelial cells from irradiation-induced apoptosis

Panax ginseng has been shown to have a protective effect for irradiated animals or cells. Ginsenosides are the most active components isolated from ginseng, and ginsenoside Rd has been identified as one of the effective compounds responsible for the pharmaceutical actions of ginseng. In the present study, we studied the molecular mechanisms for the radio-protective action of ginsenoside Rd in rat intestinal epithelial IEC-6 cells. Cells were irradiated with gamma-ray, and apoptosis was examined using Hoechst staining and Western blot analysis. Treatment with ginsenoside Rd before gamma-irradiation inhibited irradiation-induced apoptosis in IEC-6 cells. Administration of Rd after irradiation also inhibited apoptosis in these cells. Irradiation of IEC-6 cells resulted in inactivation of Akt phosphorylation that was abrogated by Rd. On the other hand, irradiation activated phosphorylation of ERK1/2 but did not affect that of p38 MAPK. Inhibition of Akt phosphorylation prevented the reduction of apoptosis by Rd following irradiation. Pretreatment with an inhibitor of the MEK pathway further decreased the number of apoptotic cells. Rd decreased the ratios of Bax/Bcl-2 and Bax/Bcl-xL, the levels of cytochrome c, and the cleaved form of caspase-3 in irradiated IEC-6 cells. Our results suggest that ginsenoside Rd protects and rescues rat intestinal epithelial cells from irradiation-induced apoptosis through a pathway requiring activation of PI3K/Akt, inactivation of MEK, and also inhibition of a mitochondria/caspase pathway.     

Tanshinone IIA suppresses lung injury and apoptosis, and modulates protein kinase B and extracellular signal-regulated protein kinase pathways in rats challenged with seawater exposure

1. Tanshinone IIA (TIIA) is one of the main active components of the Chinese herb, Danshen. In the present study, we investigated the role of apoptosis in seawater exposure-induced acute lung injury (ALI), and explored the effects of TIIA on lung injury, apoptosis, and protein kinase B (Akt) and extracellular signal-regulated protein kinase (ERK) pathways in seawater-challenged rats. The rats were randomly divided into four groups: (i) naive group, no drug was given; (ii) TIIA control group, TIIA (50 mg/kg) was given intraperitoneally; (iii) seawater (SW) group, seawater (4 mL/kg) was given; and (iv) TIIA/SW group, TIIA (50 mg/kg) was injected intraperitoneally 10 min after seawater instillation. 2. The results showed that TIIA treatment significantly improved seawater exposure-induced lung histopathological changes, alleviated the decrease in PaO(2) , and reduced lung oedema, vascular leakage and cell infiltration. As shown by terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) assay, seawater exposure induced apoptosis in lung tissue cells. Furthermore, seawater exposure also changed apoptosis-related factors Bcl-2 and caspase-3, and caused a reduction in the activation of Akt and ERK1/2 pathways. Furthermore, TIIA treatment decreased the number of apoptotic cells, reversed changes in Bcl-2 and caspase-3, and upregulated the activation of Akt and ERK1/2 in seawater-challenged rats. 3. In conclusion, the data suggest that apoptosis might play an important role in seawater exposure-induced lung injury and that TIIA could significantly attenuate the severity of ALI and apoptosis in seawater-challenged rats, which is possibly through modulation of Akt and ERK1/2 pathways.     

Calcium and the replication of human vascular smooth muscle cells: studies on the activation and translocation of extracellular signal regulated kinase (ERK) and cyclin D1 expression

Since the precise role of sarcoplasmic reticular Ca2+ in mediating vascular smooth muscle cells (VSMC) proliferation is unknown, the effect of pre-incubation with thapsigargin on extracellular signal regulated kinase (ERK) activation, the translocation of activated of ERK 1/2 to the nucleus, cyclin D1 expression, the onset of S phase and cytosolic Ca2+ levels were studied. Human saphenous vein VSMCs (hVSMC) were incubated with 10 nM thapsigargin for 24 h followed by stimulation with fetal calf serum and the activation of ERK1/2 and cyclin D1 assessed by western blotting, the intracellular distribution of ERK1/2 using indirect immunofluorescence, the onset of S-phase with the incorporation of bromodeoxyuridine and sarcoplasmic reticular Ca2+ status using FURA-2. Thapsigargin had a marginal effect on ERK1/2 activation only at 5 min and 10 min after stimulation with fetal calf serum. In contrast, the rapid translocation of ERK1/2 to the nucleus was completely blocked by thapsigargin. S phase was delayed by 8 h by thapsigargin which co-incided with the recovery of cytosolic Ca2+ levels and cyclin D1 expression. It is concluded that the inhibitory effect of thapsigargin (depletion of Ca2+ pools) on hVSMC replication is mediated through the inhibition of translocation of activated ERK1/2 to the nucleus and not to the phosphorylation of ERK, per se, which in turn prevents cyclin D1 expression and thus progression of the cell cycle.