CCK-8对大鼠肺间质巨噬细胞cAMP-PKA信号通路的激活作用

目的探讨八肽胆囊收缩素(CCK-8)对大鼠肺间质巨噬细胞(PIMs)cAMP-PKA信号通路的激活作用。方法分离纯化大鼠PIMs,采用放射免疫分析法测定细胞内cAMP含量,放射激酶法测定PKA活性,受体拮抗剂的IC50值由对数-几率单位法求得。结果正常对照组大鼠静息状态下PIMscAMP含量和PKA活性分别为(2.04±0.13)nmol·g-1和(118.3±11.2)nmol·min-1·g-1。低浓度CCK-8[(10-12～10-10)mol·L-1]对细胞内cAMP含量和PKA活性没有影响(与正常对照组比较:P>0.05);高浓度CCK-8[(10-9～10-5)mol.L-1]可明显提高细胞内cAMP含量和PKA活性(与正常对照组比较:P<0.05)。10mg·L-1脂多糖(LPS)刺激大鼠PIMs,可明提高细胞内cAMP含量和PKA活性,分别为(5.15±0.12)nmol·g-1和(188.6±13.5)nmol·min-1·g-1。不同浓度的CCK-8与LPS共同孵育PIMs,细胞内cAMP含量和PKA活性的变化趋势与CCK-8作用于静息状态下大鼠PIMs的变化趋势完全相同。CCK受体拮抗剂丙谷胺、CR-1409、CR-2945可呈剂量依赖性地抑制CCK导致的cAMP含量的升高,它们的IC50值分别为(0.5×10-6、4.1×10-6、7.2×10-4)mol·L-1。丙谷胺、CR-1409、CR-2945也可明显减弱CCK-8所导致的PKA活性的升高;其中,丙谷胺的抑制作用最强,CR-1409次之,CR-2945的抑制作用最小。结论CCK-8可剂量依赖性地激活静息状态和LPS诱导的大鼠PIMscAMP-PKA信号转导途径,这可能是CCK抗炎作用的分子机制之一。CCK激活cAMP-PKA通路是通过CCK受体来实现的,且CCK-AR的作用比CCK-BR的作用更为明显。     

cAMP/PKA信号通路在肾上腺肿瘤中的作用

目的：探讨cAMP/PKA信号通路在肾上腺肿瘤中的作用。方法将人肾上腺肿瘤H295R细胞随机分为对照组、实验组A及实验组B,对照组不进行处理,实验组A加入cAMP衍生物db-cAMP,实验组B加入db-cAMP及cAMP/PKA信号通路抑制剂H-89,分别检测细胞PKA活性;应用Western Blot方法检测cAMP/PKA信号通路下游CREB蛋白的磷酸化水平;CCK8实验检测细胞72 h的增殖水平;糖皮质激素分泌实验检测3组H295R的激素分泌能力。结果实验组A的PKA活性水平、CREB蛋白的磷酸化水平、细胞增殖水平、糖皮质激素分泌水平均较对照组和实验组B显著升高(P<0．05);而实验组B与对照组比较差异无统计学意义(P<0．05)。结论 cAMP/PKA信号通路的异常活化能够促进肾上腺肿瘤的形成与功能的发挥。     

桂枝、荆芥挥发油对大鼠急性肺损伤模型核因子KB信号通路影响的比较

目的 探讨桂枝、荆芥挥发油对核因子KB/IKB信号通路的影响.方法 用大肠肝菌内毒素(LPS)制作大鼠急性肺损伤模型,通过桂枝挥发油和荆芥挥发油治疗后,采用ELISA法检测肺组织细胞中核蛋白NF-KB P65的含量和肺组织溶浆中磷酸化IKB-a、TNF-a和IL-β的含量.结果 正常大鼠肺组织中,NF-κB P65、磷酸化IKB-α、TNF-α和IL-1β有微量表达,LPS经尾静脉注射6 h后,各指标表达均显著增高,与空白对照组比较有显著差异(P<0.01),桂枝、荆芥挥发油组NF-KB P65、磷酸化IKB-α、TNF-α和IL-1β的含量均较模型组显著降低(P<0.01).结论 桂枝、荆芥挥发油对急性肺损伤时高度活化的核因子KB/IKB信号通路有显著的抑制或拮抗作用,提示核因子KB/IKB信号通路可能是桂枝、荆芥挥发油抗炎作用的主要机制之一.     

丹参酮ⅡA对缺血再灌注损伤的乳鼠心肌细胞Notch信号通路的影响

目的 研究丹参酮ⅡA预处理对缺血再灌注后乳鼠心肌细胞丙二醛(MDA)水平、细胞凋亡及细胞内Notch1、Hes1及HIF-1α mRNA表达的影响,从Notch信号通路活化水平阐明丹参酮ⅡA预处理防治缺血再灌注损伤(ischemia/ reperfusion,I/R)的分子机制。方法 培养乳鼠原代心肌细胞,建立心肌细胞I/R模型。并分为对照组、I/R模型组(先用正常培养液孵育1 h,然后模拟缺血2 h,正常DMEM再灌注4 h)、DAPT处理组(先用含DAPT的DMEM中孵育1 h,然后模拟缺血2 h,正常DMEM再灌注4 h)和丹参酮ⅡA处理组(先用含丹参酮IIA的DMEM中孵育1 h,然后模拟缺血2 h,正常DMEM再灌注4 h)。应用硫代巴比妥酸法测定MDA水平,用磷脂酰丝氨酸外翻分析法检测细胞凋亡,并用荧光定量PCR技术检测反映Notch信号通路活化水平的Notch信号通路受体Notch1 mRNA、下游信号分子Hes1 mRNA和HIF-1α mRNA表达。结果 与对照组比较,乳鼠I/R模型组心肌细胞MDA水平升高(P<0.01),细胞早期凋亡率增加,Notch1、Hes1、HIF-1α mRNA表达水平增加(P<0.05,P<0.01,P<0.01)。与I/R模型组比较,DAPT处理组和丹参酮ⅡA处理组心肌细胞MDA水平降低(P<0.01),心肌细胞早期凋亡率降低,Notch1、Hes1、HIF-1α mRNA表达水平下降(P均<0.01)。结论 丹参酮ⅡA在乳鼠心肌细胞I/R损伤中起到心肌保护作用,其可能的分子机制为抑制Notch信号通路的活化及调节细胞凋亡。     

桂枝、荆芥挥发油对大鼠急性肺损伤模型核因子κB信号通路影响的比较

目的探讨桂枝、荆芥挥发油对核因子κB/IκB信号通路的影响。方法用大肠肝菌内毒素(LPS)制作大鼠急性肺损伤模型,通过桂枝挥发油和荆芥挥发油治疗后,采用ELISA法检测肺组织细胞中核蛋白NF-κB P65的含量和肺组织溶浆中磷酸化IκB-α、TNF-α和IL-1β的含量。结果正常大鼠肺组织中,NF-κB P65、磷酸化IκB-α、TNF-α和IL-1β有微量表达,LPS经尾静脉注射6 h后,各指标表达均显著增高,与空白对照组比较有显著差异(P<0.01),桂枝、荆芥挥发油组NF-κB P65、磷酸化IκB-α、TNF-α和IL-1β的含量均较模型组显著降低(P<0.01)。结论桂枝、荆芥挥发油对急性肺损伤时高度活化的核因子κB/IκB信号通路有显著的抑制或拮抗作用,提示核因子κB/IκB信号通路可能是桂枝、荆芥挥发油抗炎作用的主要机制之一。     

基于Ca2+-CaMKⅡ信号通路的济川煎治疗阳虚便秘的作用及分子机制

目的 基于Ca²⁺-CaMKⅡ通路观察济川煎对阳虚便秘大鼠的治疗作用及分子机制。方法 采用白醋+活性炭冰水联合复方地芬诺酯片复制阳虚便秘大鼠模型。将模型成功大鼠随机分为5组,即模型组、莫沙必利组(1.88 mg·kg^-1)、济川煎高(8.26 g·kg^-1)、中(4.31 g·kg^-1)、低(2.16 g·kg^-1)剂量组,灌胃给药每日1次,连续7 d。末次给药,记录大鼠状态并评分,进行排便功能测定;分离模型大鼠结肠平滑肌细胞并鉴定,FCM检测结肠平滑肌细胞内Ca²⁺浓度变化;采用ELISA法检测大鼠血清中cAMP、cGMP含量及其比值;采用RT-PCR及Western blot法检测大鼠结肠平滑肌细胞CaM、CaMKⅡ亚型的表达情况。结果 济川煎可明显缓解模型大鼠腹胀,摄食、饮水量、排便减少及体质量降低的症状(P<0.05),评分明显升高(P<0.01);能明显缩短首粒排便时间(P<0.01),增加排便粒数(P<0.05);济川煎...     

胞外pH值通过BMP-2信号通路影响高磷诱导的血管平滑肌细胞钙化

目的 探讨骨形态发生蛋白2（bone morphogenetic protein 2,BMP-2）信号通路在不同pH值环境影响高磷诱导的大鼠血管平滑肌细胞（vascular smooth muscle cells, VSMCs）钙化中的作用。方法 选取5～8周龄健康雄性SD大鼠,体外分离培养大鼠胸主动脉VSMCs,采用免疫细胞化学方法鉴定。将VSMCs用随机抽样法分为正常对照组(pH7.4)、pH7.4+高磷组、pH7.1+高磷组及pH7.7+高磷组。采用茜素红染色及邻甲酚酞络合酮比色法检测细胞钙化情况,酶联免疫吸附法检测细胞碱性磷酸酶(ALP)活性,用反转录聚合酶链反应（RT-PCR）法检测BMP-2、Smad1及 Runt 相关转录因子2（Runx2） mRNA的表达。结果 与正常对照组相比,pH7.4+高磷组大鼠VSMCs的钙含量增加（P<0.05）、茜素红染色钙化结节增多,Runx2 mRNA表达及ALP活性均增高（P<0.05）;与pH7.4+高磷组相比,pH7.1+高磷组大鼠VSMCs的钙含量减低（P<0.05）、茜素红染色钙化结节减少,Runx2 mRNA表达及ALP活性均降低（P<0.05）,BMP-2、Smad1mRNA表达均降低（P<0.05）,pH7.7+高磷组大鼠VSMCs的钙含量增加（P<0.05）、茜素红染色钙化结节增多,Runx2 mRNA表达及ALP活性均增高（P<0.05）,BMP-2、Smad1mRNA表达均增高（P<0.05）。结论 胞外酸性环境（pH7.1）抑制高磷诱导的大鼠VSMCs钙化,胞外碱性环境（pH7.7）促进高磷诱导的大鼠VSMCs钙化,其机制可能是通过BMP-2信号通路介导了VSMCs的表型转化。     

cAMP/PKA/CREB信号通路调控组织器官细胞纤维化及中医药干预作用的研究进展

环磷酸腺苷（cAMP）通过活化cAMP依赖的蛋白激酶A（PKA）,使cAMP反元件结合蛋白（CREB）磷酸化,从而调节基因转录,广泛参与神经系统的学习记忆过程,而近年来研究显示cAMP/PKA/CREB信号通路参与组织器官细胞的纤维化过程。机体损伤后在其组织器官细胞修复过程中,细胞外基质异常增生谓之纤维化,纤维化可使肝、肺、肾及心等脏器组织功能下降。中医药在治疗纤维化等慢性复杂疾病过程中有独特的优势,而调节cAMP/PKA/CREB信号通路是其防治组织器官细胞纤维化的机制之一。     

TAL对慢支模型大鼠肺泡巨噬细胞凋亡的影响及部分机制研究

目的探讨枇杷叶三萜酸(TAL)对慢支模型(CB)大鼠肺泡巨噬细胞(AM)活性及凋亡的影响及其可能的作用机制。方法应用BCG联用LPS的方法复制大鼠CB模型,采用体内外给药的方法,观察TAL及MAPK3条通路对CB大鼠AM凋亡及活性的影响。MTT法检测细胞活性,电镜观察AM凋亡,流式细胞技术检测AM凋亡率及AM中凋亡相关基因Bcl-2、Bax的表达。Western blot法检测AM中ERK蛋白磷酸化水平。结果①与正常对照组比较,模型大鼠支气管肺泡灌洗液中AM数增加(P<0.01),而TAL灌胃给药可抑制模型大鼠异常增多的AM数量(P<0.01)。②慢支模型组AM活性较正常组相比明显增强(P<0.01),TAL可抑制CB模型组大鼠AM的活性(P<0.05)。③与正常组比较,模型大鼠AM凋亡率明显降低(P<0.01);TAL给药组及ERK通路抑制剂PD98059组AM凋亡率较模型组比较升高(P<0.05,P<0.01)。而JNK通路抑制剂Curcumin组AM凋亡率降低(P<0.05)。④模型大鼠AMERK磷酸化水平明显升高(P<0.01);TAL(5mg.L-1)体外给药可抑制慢支大鼠AM内ERKMAPK通路的磷酸化(P<0.05)。⑤模型大鼠AMBax表达明显降低而Bcl-2表达升高(P<0.01),TAL给药组可降低慢支大鼠AMBcl-2表达,升高Bax表达。结论慢支模型大鼠AM活性增强、凋亡减少,TAL可诱导AM凋亡,抑制AM活性,使AM内Bcl-2/Bax的表达趋于平衡。ERK、JNKMAPK信号传导通路参与慢支大鼠AM凋亡的调节,JNKMAPK信号通路促进AM凋亡而ERKMAPK信号通路抑制AM凋亡。TAL的促AM凋亡作用可能与其抑制慢支大鼠AMERKMAPK信号通路的磷酸化活化、调节Bcl-2/Bax的表达有关。     

腺苷酸环化酶及蛋白激酶A在β受体激动增加eNOS活性信号通路中的作用

目的阐明β受体激动增加内皮型一氧化氮合酶(eNOS)活性信号通路中腺苷酸环化酶(AC)及蛋白激酶A(PKA)的作用。方法体外培养人脐静脉内皮细胞;运用同位素两步色谱法([3H]-L-精氨酸转化法)检测eNOS活性。本研究设置空白对照组、AC组和PKA组,每组因素下再分ISO及BSS两个干预因素组,观察AC特异性阻断剂SQ-2256(5×10-5mol/L)及PKA特异性阻断剂H-89(1×10-7mol/L)分别与人脐静脉内皮细胞孵育10min,再与异丙肾上腺素(ISO)孵育30min后eNOS活性变化。结果ISO明显增加eNOS活性(30.7±3.9)%,P<0.01;分别阻断AC、PKA,ISO诱导的eNOS活性增加均受到抑制(12.1±3.53)%、(4.73±2.19)%,P<0.01。结论ISO激活eNOS活性的信号通路上,AC和PKA均有参与。     

Effect of cholecystokinin octapeptide on diacylglycerol-PKC signaling pathway in rat pulmonary interstitial macrophages stimulated by lipopolysaccharide

AIM: To investigate the effect of cholecystokinin octapeptide (CCK-8) on the diacylglycerol-protein kinase C (DAG-PKC) signaling pathway in rat pulmonary interstitial macrophages (PIM) stimulated by lipopolysaccaride (LPS). METHODS: The PIM from rat lung tissues were isolated using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. DAG content and PKC activity were measured by radioenzymatic assay. The translocation of PKCzeta was determined by semi-quantitative immunoblot analysis. RESULTS: CCK-8, at high concentrations (1 x 10(-6) - 1 x 10(-5) mol/L), decreased DAG content and inhibited PKC activity and PKCzeta translocation compared with that in rat resting PIM of a control group (P< 0.01). LPS increased DAG content, and promoted PKC activity and PKCzeta translocation (P< 0.01). CCK-8 decreased LPS-induced DAG content and inhibited LPS-induced PKC activity and PKCzeta translocation significantly at 1 x 10(-8) - 1 x 10(-5) mol/L (P< 0.01). This inhibitory effect of CCK-8 could be abrogated partly by proglumide (non-selective CCK receptor antagonist), CR-1409 (selective CCK-A receptor antagonist), and CR-2945 (selective CCK-B receptor antagonist) in a concentration-dependent manner (P< 0.01). CONCLUSION: CCK-8 was a negative modulator of the DAG-PKC signaling pathway in rat resting PIM, which is very important for maintaining body homeostasis. It significantly inhibited LPS-induced DAG content, PKC activity and PKCzeta translocation in a concentration-dependent manner. The CCK receptor, especially the CCK-A receptor, might play a major role in this process.     

基于NF-κB信号通路筛选产抗炎活性次级代谢产物的红树林真菌

摘要：目的 以福建漳江口红树林沉积物来源真菌为对象,筛选产抗炎活性次级代谢产物的红树林真菌。方法 将pNFκB-luc 与pRL-SV40-C报告基因质粒共转染Raw264.7细胞,脂多糖(LPS)刺激该细胞建立体外炎症模型,构建双荧光素酶报告基因筛选体 系,对福建漳江口红树林沉积物来源真菌的发酵粗提物进行抗炎活性筛选;CCK-8法检测粗提物对Raw264.7细胞活力影响;光学显 微镜观察粗提物对LPS刺激的Raw264.7细胞分化作用。结果 从102株红树林沉积物来源真菌中获得5株阳性菌株,其发酵粗提物可 明显抑制LPS诱导的Raw264.7细胞中NF-κB信号通路活化,菌株编号分别为H788、H1458、H515、XT20-8和T2-3'。结论 5株红树 林沉积物来源真菌产生的次级代谢产物可通过抑制NF-κB信号通路发挥抗炎作用,其活性成分值得开展进一步化学研究。     

己酮可可碱通过Hedgehog信号通路对日本血吸虫病肝纤维化形成的抑制作用

目的：探讨己酮可可碱（PTX）通过Hedgehog信号通路抑制日本血吸虫病肝纤维化的作用机制。方法：PMA诱导单核细胞THP-1分化成巨噬细胞,用可溶性日本血吸虫虫卵抗原（SEA）刺激活化THP-1源性巨噬细胞,留取培养上清用于检测其活化状况。CCK-8检测培养上清及PTX对肝星状细胞LX-2的活性影响。设空白对照组、上清刺激组（加入活化的巨噬细胞培养上清）、PTX干预组（加入活化的巨噬细胞培养上清和PTX）。收集以上3组LX-2培养上清及细胞,上清用于ELISA检测TGF-β蛋白表达,细胞用于提取总RNA后RT-PCR检测TGF-β,shh,gli-1的基因表达。结果：SEA可活化THP-1源性巨噬细胞,其活化后培养上清可活化LX-2细胞。上清刺激组TGF-β,shh,gli-1的基因表达空白对照组明显上调,而PTX干预组相对于上刺激组则明显降低。结论：Hedgehog信号通路可能在血吸虫肝纤维化形成过程中起重要作用,己酮可可碱可通过该通路对血吸虫肝纤维化的形成起抑制作用。     

石蒜碱通过TLR4/NF-κB途径抑制LPS诱导的原代小胶质细胞炎症反应

目的 观察石蒜碱（LYC）对脂多糖（LPS）诱导的原代小胶质细胞炎症反应和表型变化的影响,并探讨其作用机制。方法 将培养的原代小胶质细胞分为对照组、LYC组（0.1、0.5、1和5 μmol/L LYC）、LPS组（0.1 mg/L LPS）和LPS（0.1 mg/L LPS）+LYC组（5 μmol/L LYC）。倒置显微镜观察细胞形态变化;流式细胞术检测原代小胶质细胞的纯度及LPS诱导原代小胶质细胞向两种表型转化的情况;CCK-8法检测细胞活性;实时荧光定量PCR（qPCR）检测白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）以及不同表型小胶质细胞表面标志物mRNA表达;Griess法检测一氧化氮（NO）含量;免疫印迹法检测细胞TLR4、p-NF-κB p65蛋白表达情况。结果 原代小胶质细胞纯度达95%以上。对照组及各浓度LYC组细胞活性差异无统计学意义。后续实验以5 μmol/L的LYC为药物浓度。与LPS组相比,LPS+LYC组的阿米巴样小胶质细胞数量明显减少,IL-1β、IL-6、TNF-α mRNA表达水平以及NO含量降低,CD86 mRNA表达水平和表型比例降低,CD206 mRNA表达水平和表型比例升高,TLR4和p-NF-κB p65蛋白表达水平降低（P<0.01）。结论 LYC通过TLR4/NF-κB信号通路抑制LPS诱导的原代小胶质细胞炎症反应,并促进其向“抑炎型”极化。     

Involvement of cAMP/cAMP-dependent protein kinase signaling pathway in regulation of Na+,K+-ATPase upon activation of opioid receptors by morphine

The depolarization of neurons induced by impairment of Na+,K+-ATPase activity after long-term opiate treatment has been shown to involve the development of opioid dependence. However, the mechanisms underlying changes in Na+,K+-ATPase activity after opioid treatment are unclear. The best-established molecular adaptation to long-term opioid exposure is up-regulation of the cAMP/cAMP-dependent protein kinase (PKA) signaling pathway; this study, therefore, was undertaken to investigate the role of up-regulation of cAMP/PKA signaling pathway in alteration of the mouse hippocampal Na+,K+-ATPase activity. The results demonstrated that short-term morphine treatment dose dependently stimulated Na+,K+-ATPase activity. This action could be significantly suppressed by adenylyl cyclase activator 7beta-acetoxy-8,13-epoxy-1alpha,6beta,9alpha-trihydroxylabd-14-en-11-one (forskolin), or the cAMP analog dibutyryl-cAMP. Contrary to short-term morphine treatment, long-term treatment significantly inhibited Na+,K+-ATPase activity. Moreover, an additional decrease in Na+,K+-ATPase activity was observed by naloxone precipitation. The effects of both short- and long-term morphine treatment on Na+,K+-ATPase activity were naltrexone-reversible. The regulation of Na+,K+-ATPase activity by morphine was inversely correlated with intracellular cAMP accumulation. N-[2-(4-Bromocinnamylamino)ethyl]-5-isoquinoline (H89), a specific PKA inhibitor, mimicked the stimulatory effect of short-term morphine but antagonized the inhibitory effect of long-term morphine treatment on Na+,K+-ATPase activity. However, okadaic acid, a protein phosphatase inhibitor, suppressed short-term morphine stimulation but potentiated long-term morphine inhibition of Na+,K+-ATPase activity. The regulation of Na+,K+-ATPase activity by morphine treatment seemed to associate with the alteration in phosphorylation level but not to be relevant to the change in abundance of Na+,K+-ATPase. These findings strongly demonstrate that cAMP/PKA signaling pathway involves regulation of Na+,K+-ATPase activity after activation of opioid receptors.     

Involvement of spinal PKA/CREB signaling pathway in the development of bone cancer pain

BackgroundIt has been shown that spinal PKA/CREB signaling pathway is involved in neuropathic and inflammatory pain, but its effects on bone cancer pain have not previously been investigated. The aim of this study was to examine the potential role of the spinal PKA/CREB signaling pathway in the development of bone cancer pain.MethodsAbone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia.Western blot analysis examined the expression of PKAca (PKA catalytic subunit) and phospho-CREB (p-CREB) protein levels. The authors further investigated effects of intrathecal treatment with H-89 (a PKA inhibitor, 8 nmol) or forskolin (a PKA agonist, 10 nmol) on nociceptive behavior and the expression of PKAca and p-CREB.ResultsOn days 6, 9, and 15 after inoculation, the expression of PKAca and p-CREB protein levels were higher in the bone cancer pain rats compared to the sham rats. On day 9, intrathecal administration of H-89 significantly attenuated bone cancer-induced mechanical allodynia as well as upregulation of PKAca and p-CREB protein levels. These effects were completely abolished by intrathecal pretreatment with the PKA agonist forskolin.ConclusionThe results suggest that the spinal PKA/CREB signaling pathway may participate in the development of bone cancer pain. The findings of this study may provide an evidence for developing novel analgesics to block bone cancer pain.     

Pertussis toxin inhibits cholecystokinin- and epidermal growth factor-induced mitogen-activated protein kinase activation by disinhibition of the cAMP signaling pathway and inhibition of c-Raf-1

Pertussis toxin (PTx), which inactivates G(i/o) type G proteins, is widely used to investigate the involvement of G(i/o) proteins in signal transduction. Activation of extracellular-regulated kinases 1 and 2 (ERK1/2) by G protein-coupled receptors has been described to occur either through a PTx-insensitive pathway involving activation of phospholipase C and protein kinase C (PKC), or through a PTx-sensitive pathway involving G(i)betagamma-mediated activation of Src. Cholecystokinin (CCK) activates ERK1/2 by a PKC-dependent, and thus presumably PTx-insensitive, pathway. However, CCK has recently been shown to induce activation of G(i) proteins in addition to G(q/11). In the present study, PTx partially inhibited CCK-induced ERK1/2 activation in pancreatic AR42J cells, although activation of phospholipase C was not reduced. PTx also inhibited ERK1/2 activation in response to the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) as well as activation of c-Raf-1 by EGF and CCK. In contrast, PTx, CCK, and EGF had only minor effects on A-Raf and B-Raf activity. Forskolin, a direct activator of adenylyl cyclase, inhibited CCK- and EGF-induced activation of c-Raf-1 and ERK1/2 in a manner similar to that of PTx. In PTx-treated cells, the cAMP content was increased and forskolin did not further inhibit CCK- and EGF-induced activation of c-Raf-1 or ERK1/2. In conclusion, the present study shows that PTx-sensitivity of receptor-induced ERK1/2 activation could be a consequence of disinhibition of the adenylyl cyclase signaling pathway, which in turn causes inhibition of c-Raf-1 activation rather than indicating involvement of a PTx-sensitive G protein in this signaling pathway.     

中药干预高脂血症相关信号通路的研究进展

高脂血症是因多种原因导致脂质代谢紊乱的一种病理状态,可引起动脉粥样硬化等多种疾病的发生,严重影响患者的生活质量。目前化学药治疗存在不良反应大、耐药等缺点。而中药用于治疗高脂血症的历史悠久,临床经验丰富,且以不良反应少、用药依从性好的优点成为近几年研究的热点。通过查阅分析国内外有关中药干预高脂血症信号通路的文献,归纳总结出6条中药干预高脂血症相关信号通路,分别为过氧化物酶体增殖物激活受体（PPAR）信号通路、腺苷酸激活蛋白激酶（AMPK）信号通路、环磷酸腺苷（cAMP）信号通路、脂肪细胞因子（Adipocytokine）信号通路、法尼酯衍生物X受体（FXR）-小异二聚体伴侣（SHP）信号通路及磷脂酰肌醇-3-激酶（PI3K）-丝氨酸/蛋白激酶B（Akt）信号通路,以期为治疗高脂血症的药物研发提供参考。     

芍药苷对胶原性关节炎大鼠滑膜细胞G蛋白偶联信号的调节作用

目的研究芍药苷(paeoniflorin,Pae)对胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠滑膜细胞G蛋白偶联信号转导的调节作用。方法用鸡Ⅱ型胶原诱导胶原性关节炎。在免疫后d16~23,胶原性关节炎大鼠灌胃给予Pae(25、50、100mg·kg-1)。通过足爪肿胀和关节炎指数评价关节炎。采用Western blot技术检测滑膜细胞中抑制性G蛋白(Gi)表达。用放免法检测滑膜细胞中cAMP水平,用激酶发光分析法测定滑膜细胞中蛋白激酶A(PKA)的活性。结果胶原性关节炎大鼠出现明显的继发性炎症反应。滑膜细胞中Gi蛋白表达增加,而cAMP水平和PKA活性明显降低。Pae抑制胶原性关节炎大鼠的炎症应答。在100mg·kg-1,Pae抑制Gi的表达。在50mg·kg-1和100mg·kg-1剂量,Pae提高cAMP水平和PKA活性。体外研究发现,Pae(2·5、12·5、62·5mg·L-1)降低滑膜细胞中Gi的表达,而提高降低的cAMP水平和PKA活性。结论胶原性关节炎大鼠滑膜细胞中G蛋白偶联的信号转导与滑膜炎病理机制密切相关;Pae对胶原性关节炎大鼠的抗炎作用是通过调节G蛋白偶联的信号转导实现的。