【作者】 王慧；

【导师】 孙志良；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2012， 博士

【摘要】 胃肠功能失调或胃肠道器质性病变可引起胃肠痉挛。当胃肠痉挛所致的疼痛为功能性时,应使用调节肠管运动的药物,临床上常见的有抗胆碱药和钙离子通道阻断药,如阿托品或硝苯地平等。传统的抗胆碱药由于其不良反应较大在临床应用中受到一定的限制,钙通道阻断剂硝苯地平长期使用也可引起负性肌力和负性传导作用,因此开发具有安全、高效、低残留的抗胆碱类或钙通道阻断性的药物迫在眉睫。血根碱(sanguinarine, SA),为苯菲啶异喹啉类生物碱,主要存在于蓝堇科、罂粟科和芸香科的植物中。近年来,血根碱被应用于治疗胃肠道疾病,包括腹痛,腹泻或肠道炎症。研究表明,血根碱添加到仔猪饲料中,可有效防治仔猪腹泻,提高仔猪饲料转化率和日增重。但关于血根碱抑制胃肠运动、缓解胃肠痉挛的作用机理研究甚少,因此本文采用细胞生物学,分子生物学和免疫荧光等技术系统研究SA对大鼠肠平滑肌细胞(Intestinal smooth muscle cells, ISMC)收缩的抑制作用及信号传导通路的影响。主要研究内容和结果为：1.离体肠实验,取大鼠的十二指肠组织,应用生物机能分析系统记录血根碱对正常及工具药(乙酰胆碱、组胺、CaCl2)作用下肠平滑肌收缩的频率和振幅；并应用阻断剂(阿托品、异丙嗪及硝苯地平)作用后,血根碱对肠平滑肌的作用。结果表明,血根碱浓度依赖性的抑制了肠平滑肌活动的频率和振幅,对频率的半数抑制浓度(the concentrations to inhibit contractions by50%),IC50=28.31μM,对振幅的IC50=11.37μM；对由乙酰胆碱、组胺、CaCl2引起的肠平滑肌收缩的频率和振幅有明显的抑制作用,其频率的IC50分别为：25.14μM、28.33μM、27.90μM；其对振幅的IC50分别为：13.77μM、14.72μM、3.35μM。血根碱可增强阿托品对M受体、异丙嗪对H1受体及硝苯地平对Ica-L的阻断作用。因此,血根碱对肠平滑肌活动的抑制作用可能与M受体、H1受体及Ica-L有关。2.应用酶消化法分离并培养大鼠原代ISMC,经免疫组化鉴定后进行细胞收缩反应试验,利用倒置相差显微镜测定SA及对照药阿托品对大鼠肠平滑肌细胞长度的影响,并应用乙酰胆碱、组胺和KCl诱导ISMC(?)缩后,血根碱对细胞长度的影响。结果表明,血根碱对大鼠肠平滑肌细胞的自发性收缩有直接抑制作用,且对由乙酰胆碱、组胺和KCl诱导的ISMC收缩具有明显的抑制作用。同时,血根碱与阿托品具有协同作用,两者同时应用可显著抑制肠平滑肌细胞的收缩。3. ISMC与不同浓度SA作用24h后,应用实时荧光定量PCR(real-time reverse transcription PCR, RT-PCR)技术,测定SA对大鼠肠平滑肌细胞M2和M3受体基因表达的影响。结果表明：M2R和M3R在ISMC中均有特异性表达,且M2R表达量较高。通过RT-PCR分析,SA可显著下调大鼠ISMC M2R和M3R的基因表达水平。4.经钙离子荧光探针Fura-2/AM标记ISMC后,再应用SA及工具药KC1和卡巴胆碱(carbachol, CCh)作用细胞,通过荧光分光光度计测定SA对白发性及KCl或CCh诱导细胞内钙离子浓度升高后的影响。结果显示：SA可浓度依赖性抑制静息状态下ISMC内钙离子的浓度(P<0.05),在有钙环境下,SA可显著抑制由KC1或CCh诱导的ISMC内钙离子浓度的升高,但在无钙细胞外液中,SA对KCl或CCh作用后的细胞内钙离子浓度无影响。表明,SA对细胞外钙离子内流有明显的抑制作用,对内钙释放无影响。5.采用RT-PCR和蛋白免疫印迹杂交(Western-bolt, WB)方法检测SA对大鼠ISMC中PKC亚型及CPI-17基因和蛋白表达的影响。PKC(δ、ε、η)及CPI-17在大鼠ISMC中均可高效表达。CCh(10μM,10min)可显著上调ISMC PKCs和PKCη mRNA表达,CCh(100μM,60min)可明显诱导ISMC PKC (δ、η)和CPI-17基因表达水平(P<0.05)。μM和3μM的SA作用细胞30min可显著下调ISMC PKC(δ、ε、η)及CPI-17基因表达水平；SA(1μM,30min)对CCh(100μM,60min)诱导的ISMC PKC(δ、η)和CPI-17的基因表达具有显著下调作用。1μM的SA作用ISMC30min可显著下调ISMC PKCδ的蛋白表达水平,作用24h时,明显下调CPI-17的蛋白表达水平。同时,SA可显著下调CCh诱导的ISMC PKCδ和CPI-17的蛋白表达水平。因此,SA对ISMC PKC亚型和CPI-17的基因和蛋白表达均具有明显的抑制作用。综上所述,本课题以调控胃肠平滑肌兴奋-收缩偶联的PKC-CPI-17信号传导通路的研究为中心,应用荧光标记技术、RT-PCR和Western blot技术,阐明了SA对大鼠肠平滑肌细胞的抑制作用机制。其研究结果证明,SA对大鼠ISMC收缩的抑制作用与调控平滑肌细胞收缩的M受体、细胞内钙离子浓度及PKC-CPI-17信号传导通路的改变有关。更多还原

【Abstract】 Gastrointestinal spasms and cramps can result from gastrointestinal dysfunction and organic lesion. Atropine, a nonselective muscarinic receptors blocker, and the Ca2+channel blocker nifedipine can be used in the treatment of gastrointestinal diseases. As is true for all antimuscarinic agents, the clinical uses are limited due to severe side-effects. However, nifedipine has a potent negative dromotropic effect with dose increase after long-term administration. It’s an important to develop the safe, efficient and no residue of anticholinergic or calcium channel antagonistic of natural medicine. Sanguinarine (SA) is a benzophenanthridine alkaloid which is derived mainly from the roots of Sauguinaria Canadensis L., Chelidonium majus L. and the seeds of Argemone mexicana L., and has been revealed for the treatment of gastrointestinal (GI) diseases by immediate the motility of the gastrointestinal (GI), including abdominal pain, diarrhea, and intestinal inflammation for recent of years. In our previous study, supplement of SA into the animal feed could improve the feed conversion and growth rate, and decrease the piglet diarrhea (Cai et al.,2010). However, the underlying mechanisms of action of SA on GI motility are unclear. Therefore, inhibitory effect of SA on smooth muscle cells contractions and the signal pathway in isolated rat small intestine were demonstrated by using the methods of cytobiology, molecular biology, and immunofluorescence.1. Duodenum tissue was isolated from rat for in vitro intestine movement experiment and biologic function analysis system was used to record the frequency and amplitude of the spontaneous intestinal muscle contractions induced by ACh/Atropine, HA/Proazamine and CaCl2/Nifedipine. SA dose dependently inhibited the frequency and amplitude of spontaneous contractions with a50%inhibition (IC50). The frequency and amplitude were28.31and11.37μM, respectively. In addition, SA inhibited the contractions of intestinal smooth muscle which were induced by acetylcholine (ACh), histamine (HA) and calcium chloride (CaCl2). The IC50of Ach’s frequency and amplitude were25.14and13.77μM, respectively. The IC50of HA’s frequency and amplitude were28.33and14.72μM, respectively. The IC50of CaCl2’s frequency and amplitude were27.90and3.35μM, respectively. Also, SA significantly strengthened the relaxant effect caused by atropine, promethazine. and nifedipine. Therefore, the inhibitory effects of SA might be involved in Muscarinic acetylcholine receptors. H1receptor and an L-type Ca2+channel that may mediate the relaxation in intestinal muscle. 2. The smooth muscle cells were isolated from rat intestines using collagenase digestion, and then cells were treated with SA or agonists (ACh, HA, or KCl) after immunohistochemistry identification. Cell lengths were measured using a phase-contrast microscope. The result showed that SA dose-dependently inhibited spontaneous cell contractions and induced by ACh, HA, or KC1. Meanwhile, the inhibitory effect of atropine in cells was enhanced by SA.3. ISMC were incubated with different concentrations of SA for24h. The mRNA expression of M2and M3receptors were measured using Real-Time PCR analysis. The mRNA of M2and M3receptors in ISMC was expressed by RT-PCR, especially M2receptor. RT-PCR analysis showed that mRNA expression of M2and M3receptors were significantly inhibited in smooth muscle cells isolated from rat intestines exposed to SA.4. ISMC were respectively treated with SA or agonists (CCh or KCl), and then intracellular Ca2+concentration was measured using a Ca2+-sensitive fluorescent indicator fura-2/AM by spectrofluorophotometer. The result showed that SA dose-dependently inhibited the concentration of intracellular [Ca2+]i. Meanwhile, SA inhibited KCl or CCh mediated increase in resting [Ca2+]i in1.8mM Ca2+-Hepes buffer, but SA did not inhibited CCh or KCl induced raise [Ca2+]i under Ca2+-free conditions. These results demonstrated that SA was mainly inhibited extracellular Ca2+influx through the Ca2+channel, also known as receptor-operative Ca2+channel.5. We examined the effect of SA on the mRNA and protein expression of PKC isoforms and CPI-17using RT-PCR and Western blot analysis in ISMC. PKC isoforms (δ,ε,η) and CPI-17were detected in rat ISMC. CCh(10μM,10min) rapidly caused an increase in PKCs and PKCη mRNA, and at the concentration of100μM for60min significantly increased mRNA levels of PKCS, PKCη and CPI17. RT-PCR analysis showed that treatment of ISMC for30min with SA (1or3μM) significantly decreased mRNA expression of PKCδ, PKCε, PKCη and CPI17. SA (1μM) markedly inhibited CCh-mediated increase in PKCδ, PKCη and CPI17mRNA. Treatment of ISMC with SA for a short period (30min) caused a decrease in PKCδ protein expression; however, the amount of CPI-17was significantly inhibited for24h by Western bolts analysis. SA markedly inhibited CCh-mediated increase in PKCδ and CPI17protein. This result demonstrated that the inhibitory effect of SA by down regulates PKC and CPI-17mRNA and protein expression in rat ISMC.In conclusion, the GI smooth muscle excitation-contraction coupling is mediated by G protein regulation of PKC-CPI-17. The underlying mechanisms of the inhibitory effect of SA on intestinal smooth muscle contracture were determined by fluorescence labeling, real-time RT-PCR and Western bolt analysis. The results demonstrated that the inhibitory effect of SA is associated with alteration of M receptors, intracellular Ca2+concentration and PKC-CPI-17signal transduction that regulate smooth muscle contraction.更多还原