【作者】 邱鹏程；

【导师】 王四旺；

【作者基本信息】 第四军医大学 ， 药理学， 2013， 博士

【摘要】 目的：研究元胡止痛片治疗溃疡型胃痛的物效机制。方法：从细胞和整体动物水平，采用MTT、组织病理学、比色法检测、酶联免疫吸附测定、计算机辅助药物设计、RT-PCR以及多巴胺受体阻断剂等多种方法深入探讨元胡止痛片（母方）和延胡索乙素、欧前胡素及其延胡索乙素+欧前胡素（成分组方）治疗溃疡型胃痛的物效机制。结果：①母方及其成分组方可促进胃黏膜细胞增殖。组织病理学证实母方和成分组方能够有效促进乙酸致雄性SD大鼠胃黏膜损伤愈合。在体外，与模型组比较，3μg/ml延胡索乙素可促进GES-l细胞增殖14.7%（P<0.05）；2.4×10^-1μg/ml欧前胡素促进GES-l细胞增殖12.4%（P<0.05）；100μg/ml母方促进GES-l细胞增殖52.1%（P<0.05）。与8%乙醇损伤1h的GES-1模型组比较，3μg/ml延胡索乙素促进GES-l增殖26.5%（P<0.05），2.4μg/ml欧前胡素促进GES-l增殖26.9%（P<0.05），100μg/ml母方促进GES-l增殖27.1%（P<0.05）。在体内，对于乙酸致雄性SD大鼠胃溃疡的作用，与模型组相比，PCNA在以下各组中分别增加，50mg/kg延胡索乙素+40mg/kg欧前胡素组（组方I；151.2%；P<0.05），50mg/kg延胡索乙素+20mg/kg欧前胡素组（组方II组；144.6%；P<0.05），母方组（141.9%；P<0.05）。与50mg/kg延胡索乙素和40mg/kg欧前胡素组比较，组方I组分别上调447.8%（P<0.05）和298.7%（P<0.05）。②母方及其成分组方上调多种防御因素保护胃黏膜。对于乙酸致雄性SD大鼠胃溃疡，与模型组相比，SP在以下几组显著性上升，组方I（58.6%；P<0.01），组方II（40.4%；P<0.01），母方（41.8%；P<0.01）。与模型组相比，bFGF在以下处理组中增加，组方I（146.6%；P<0.01），组方II（59.0%；P<0.05）和母方（182.8%；P<0.01）。与延胡索乙素或者欧前胡素单用相比或者与组方II相比，组方I均能显著提高bFGF含量（P<0.05）。这与以上组织病理学和抑制胃酸的结果一致。母方和成分组方能够减轻无水乙醇致雄性SD大鼠胃黏膜损伤。这与其清除自由基、维护ATPase、提高NOS以及EGF的作用机制相关。与模型组相比，母方提高总SOD含量125%（P<0.001），组方I提高127.6%（P<0.001），组方II提高97.0%（P<0.001）。延胡索乙素或欧前胡素分别单独使用，不能达到母方和成分组方的效果。并且与50mg/kg延胡索乙素，20mg/kg欧前胡素相比，组方I提高总SOD60.6%（P<0.05）和70.3%（P<0.05）。与模型组相比，母方降低MDA46.7%（P<0.05），组方I降低MDA到51.6%（P<0.05）；组方II，延胡索乙素或欧前胡素分别单独使用，均不能显著性降低MDA。与模型组相比，母方提高Na+K+-ATPase43.2%（P<0.05），组方I提高52.1%（P<0.05）。与50mg/kg延胡索乙素相比，组方I提高Na+K+-ATPase38.1%（P<0.05）。组方II，延胡索乙素或欧前胡素分别单独使用，均不能显著性提高Na+K+-ATPase。与模型组相比，母方提高Ca²⁺Mg²⁺-ATPase47.8%（P<0.05），组方I提高58.0%（P<0.001）。与50mg/kg延胡索乙素相比，组方I提高Ca²⁺Mg²⁺-ATPase37.5%（P<0.05）。组方II，延胡索乙素或欧前胡素分别单独使用，均不能显著性提高Ca²⁺Mg²⁺ATPase。与模型组相比，TNOS在以下各组显著性提高：母方（105.5%, P<0.05），组方I（149.2%, P<0.05）和组方II（94.5%，P<0.05）。延胡索乙素或欧前胡素分别单独使用，均不能显著性上调TNOS。与模型组相比，iNOS在以下用药组显著性提高：母方（115.6%, P<0.01），组方I（120.9%, P<0.01）和组方II（63.4%，P<0.05）。延胡索乙素或欧前胡素分别单独使用，均不能显著性上调iNOS。与模型组相比，EGF在以下用药组显著性提高：母方（13.7%, P<0.05），组方I（19.8%, P<0.01）。组方II，延胡索乙素或欧前胡素分别单独使用，均不能显著上调EGF。与模型组相比，TNF-α在以下用药组显著性提高：母方（39.6%, P<0.001），组方I（44.9%, P<0.01），组方II（85.3%, P<0.001），50mg/kg延胡索乙素组（30.6%,P<0.05），20mg/kg欧前胡素组（36.3%, P<0.05）。与组方I相比，组方II提高TNF-α含量27.9%（P<0.05）。③成分组方促进胃溃疡粘膜愈合与其上调乙酰胆碱酯酶活力、下调乙酰胆碱、降低H⁺-K⁺ATPase和抑制胃酸有关。对于乙酸致雄性SD大鼠胃溃疡，与模型组相比，组方I升高pH27.0%（P<0.05），降低H⁺-K⁺ATPase活力47.9%（P<0.01），上调乙酰胆碱酯酶活力90.4%（P<0.01），下调乙酰胆碱37.2%（P<0.05）。其他给药处理组不能达到组方I的这些作用。④成分组方抑制胃黏膜损伤作用可被多潘立酮阻断。与无水乙醇致大鼠胃粘膜损伤模型组相比，组方I+0.2ml/只多潘立酮组的总SOD、MDA、Na+K+-ATPase、Ca²⁺Mg²⁺-ATPase均无显著性差异；但不干扰组方I对TNOS、iNOS、TNF-α的作用。0.2ml/只多潘立酮未干扰母方的以上作用机制(总SOD、MDA、Na+K+-ATPase、Ca²⁺Mg²⁺-ATPase、TNOS、iNOS、TNF-α)。0.2ml/只多潘立酮未干扰成分组方和母方对EGF的上调作用。与模型组相比，PGE2在以下用药组显著性下降：0.2ml/只多潘立酮组（45.4%，P<0.001），母方（28.4%，P<0.05），组方I+0.2ml/只多潘立酮（53.2%，P<0.001）。结论：①发现成分组方可显著促进乙酸所致大鼠胃溃疡损伤的愈合，减轻乙醇诱发的应激性伤害。该效果与元胡止痛片相一致；而且单独使用延胡索乙素或欧前胡素不能达到成分组方的效果。②首次揭示延胡索乙素和欧前胡素极有可能是元胡止痛片治疗溃疡型胃痛的物效成分。其理由，一是源自元胡止痛片的延胡索中延胡索乙素和白芷中欧前胡素，合并组成“成分组方”时具有协同起效的作用，并非化学物质药理作用的简单叠加；二是组织病理学、胃黏膜完整性，抑制胃酸，清除自由基，提高ATPase，和其他一些有益于胃黏膜的防御因素检测结果，均证实该“成分组方”发挥了元胡止痛片治疗溃疡型胃痛相一致的功效。③元胡止痛片治疗胃痛的作用机制可能与其主成分即延胡索乙素和欧前胡素促进胃溃疡粘膜愈合与其上调乙酰胆碱酯酶活力、下调乙酰胆碱、降低H⁺-K⁺ATPase和抑制胃酸多因素有关。④成分组方的有效特性为元胡止痛片的二次开发或新药创制提供新的思路和重要的实验依据。⑤首次采用外周D2受体拮抗剂观察到成分组方失去功效，而母方依然有效。可见成分组方的药物靶点是外周D2受体，母方中尚存作用于其它靶点发挥治疗溃疡型胃痛的有效成分。综上，元胡止痛片中存在已知的化学成分至少十余种，故不能排除更多成分组成的“成分组方”产生更好的功效，或还有更复杂的机制尚需进一步研究。更多还原

【Abstract】 Purpose：Study of active ingredients and mechanisms for the yuanhu analgesia tablet astraditional Chinese medicine treatment of gastric ulcer.Methods:In vitro and in vivo, combination use of MTT, pathological examination, colorimetricmethod, enzyme-linked immunosorbent assay, computer-aided drug design, RT-PCR anddopamine D2receptor antagonist domperidone for the study of active ingredients andmechanisms for the yuanhu analgesia tablet as traditional Chinese medicine treatment ofgastric ulcer.Results:①Yuanhu analgesia tablet （YAT） and ingredients component （IC） promoted theproliferation of gastric mucosa cell. pathological examination demonstrated that YAT and IC-I promoted proliferation of gastric mucosa cell in rats with gastric ulcer induced byacetic acid. in vitro, compared to the normal control group,3μg/ml tetrahydropalmatine（THP） promoted GES-1proliferation14.7%（P<0.05）.2.4×10^-1μg/ml imperatorin （IMP）promoted GES-1proliferation12.4%（P<0.05）.100μg/ml YAT promoted GES-1proliferation52.1%（P<0.05）. Compared to the control group damaged by8%ethanol for1h,3μg/ml THP promoted GES-1proliferation26.5%（P<0.05）.2.4μg/ml promoted GES-1proliferation26.9%（P<0.05）.100μg/ml YAT promoted GES-1proliferation27.1%（P<0.05）. In vivo, compared to the control group （gastric ulcer in rats induced by aceticacid）, PCNA were up regulated in following groups:50mg/kg THP+40mg/kg IMP（T50+I40）（151.2%, P<0.05）,50mg/kg THP+20mg/kg IMP （T50+I20）（144.6%,P<0.05）,YAT（141.9%, P<0.05）. Compared to50mg/kg THP （T50） or40mg/kg IMP （I40）, T50+I40up regulated PCNA447.8%（P<0.05） or298.7%（P<0.05）, respectively.②YAT and IC up regulated manydefense factors to protect gastric mucosa.Compared to the gastric ulcer induced by acetic acid control group in rats, SP were upregulated in the following groups: T50+I40（58.6%, P<0.01）, T50+I20（40.4%,P<0.01）,YAT （41.8%, P<0.01）. bFGF were up regulated in the following groups: T50+I40（146.6%, P<0.01）, T50+I20（59.0%, P<0.05） and YAT （182.8%, P<0.01）.Compared toT50or I40or I20or T50+I20, T50+I40significantly （P<0.05） up regulated bFGF,respectively. This was in line with the results of pathological examination and inhibitinggastric acid. YAT and IC protected the gastric mucosa from the damage caused by absoluteethyl alcohol in rats. Compared to the control group, TSOD were up regulated in thefollowing groups: YAT （125%, P<0.001）, T50+I40（127.6%, P<0.001）, T50+I20（97.0%,P<0.001）. Individually using THP or IMP did not achieve the same effect, respectively.And compared to T50or I20, T50+I40up regulated TSOD60.6%（P<0.05） or70.3%（P<0.05）. Compared to the control group, MDA were down regulated in the followinggroups: YAT （46.7%, P<0.05）, T50+I40（51.6%, P<0.05）. The other treatments did notdown regulated MDA significantly. Compared to the control group, Na+K+-ATPaseactivities were up regulated in the following groups: YAT （43.2%, P<0.05）, T50+I40（52.1%, P<0.05）. The other treatments did not up regulated Na+K+-ATPase activity significantly. And compared to T50, T50+I40up regulated Na+K+-ATPase activity38.1%（P<0.05）. Compared to the control group, Ca²⁺Mg²⁺-ATPase activities were up regulatedin the following groups: YAT （47.8%, P<0.05）, T50+I40（58.0%, P<0.0001）. Comparedto T50, T50+I40up regulated Ca²⁺Mg²⁺-ATPase activity37.5%（P<0.05）. The othertreatments did not up regulated Ca²⁺Mg²⁺-ATPase activity significantly. Compared to thecontrol group, TNOS was up regulated in the following groups: YAT （105.5%, P<0.05）,T50+I40（149.2%, P<0.05）, T50+I20（94.5%, P<0.05）. Individually using THP or IMP didnot achieve the same effect,respectively. Compared to the control group, iNOS was upregulated in the following groups: YAT （115.6%, P<0.01）, T50+I40（120.9%, P<0.01）,T50+I20（63.4%, P<0.05）. Individually using THP or IMP did not achieve the same effect,respectively. Compared to the control group, EGF was up regulated in the followinggroups: YAT （13.7%, P<0.05）, T50+I40（19.8%, P<0.01）. The other treatments did not upregulated EGF significantly. Compared to the control group, TNF-α was up regulated inthe following groups: YAT （39.6%, P<0.001）, T50+I40（44.9%, P<0.01）, T50+I20（85.3%,P<0.001）, T50（30.6%, P<0.05）, I20（36.3%, P<0.05）. Compared to T50+I40, T50+I20upregulated TNF-α27.9%（P<0.05）.③IC promoted the healing of gastric ulcer was due to up regulating AHCE, downregulating ACH, inhibiting H⁺-K⁺ATPase activity and antiacid. In vivo, compared to thecontrol group （gastric ulcer in rats induced by acetic acid）, T50+I40increased pH27.0%（P<0.05）, decreased H⁺-K⁺ATPase activity47.9%（P<0.01）, up regulated ACHE90.4%（P<0.01） and down regulated ACH37.2%（P<0.05）. The other treatments did not achievethe same effect as T50+I40did.④The protection of IC to gastric mucosa was blocked by domperidone. Compared tothe control group （induced by absolute ethyl alcohol in rats）,0.2ml/ratdomperidone+T50+I40did not change TSOD, MDA, Na+K+-ATPase activity andCa²⁺Mg²⁺-ATPase activity. However0.2ml/rat domperidone did not block the effect ofT50+I40to TNOS, iNOS, EGF and TNF-α.0.2ml/rat domineering did not block theeffect of YAT to these mechanisms. Compared to control group, PGE2was downregulated in the following groups:0.2ml/rat domineering （45.4%, P<0.001）, YAT （28.4%, P<0.05）,0.2ml/rat domineering+T50+I40（53.2%, P<0.001）.Conclusions:①we found that IC promoted the healing of gastric ulcer induced by acetic acid inrats and protected gastric mucosa from the damage caused by absolute ethyl alcohol. Thiswas in line with YAT. However individually using THP or IMP did not achieve the sameeffect as IC did.②First time discovery that THP and IMP are probably active ingredients of YAT astraditional Chinese medicine treatment of gastric ulcer. There are two reasons:1. THP isfrom rhizoma corydalis and IMP is from radix angelicae. Combination of THP and IMP asIC got a mutual reinforcement of compounds.2. IC achieved the same effect as YAT didto gastric ulcer according to the assays （pathological examination, gastric mucosa cellproliferation, antiacid, scavenging free radical, up regulating ATPase, many defencefactors for gastric mucosa, and dopamine D2receptor antagonist domperidone）.③Mechanisms of YATas traditional Chinese medicine treatment of gastric ulcerprobably via THP and IMP were promoting the healing of gastric ulcer due to upregulating ACHE, down regulating ACH, decreasing H⁺-K⁺ATPase and antiacid.④The pharmacologicalmechanisms and actions of IC provided a new thinking andexperiment data for new drug discovery based on YAT.⑤It was first observed that dopamine D2receptor blocked effect and mechanisms ofIC as treatment of gastric ulcer but did not block YAT. Thus dopamine D2receptor was thetarget of IC. There were many compounds in YAT playing more performance via multipletargets. Above all, there are at least dozens of compounds in YAT. Therefore maybe morecompounds involved in IC achieve better effect, and the pharmacological mechanisms andactions respected to further research..更多还原