【作者】 刘晓玲；

【导师】 王汝俊；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2011， 博士

【摘要】 一研究背景现代研究认为,胃粘膜防御体系是一个多级复杂体系,主要可以分为三级。在这三级防御体系中,每一级体系都涉及到了各种细胞因子,它们在胃粘膜防御体系中发挥了重要作用,并且这些因子互相影响,相互作用。三叶因子1(TFF1)是三叶家族成员之一,拥有高度保守的三叶肽结构,相当稳定,耐酸、耐热分解。有研究发现,在空肠中过度表达人类TFF1的转基因小鼠,胃肠道中诱发溃疡的发生率偏低,提示TFF1在胃肠道的修复和防护中发挥了重要作用。粘蛋白是一组高度糖基化的不同糖蛋白的总称,在胃粘膜的防御和修复过程中扮演着重要的角色。正常状态下,TFF1通过与MUC5AC粘蛋白结合或与其相应受体或转运蛋白结合发挥生理功能。促进细胞迁移是胃粘膜修复重建过程的重要步骤,TFF1在促细胞移行过程中发挥了重要作用,其启动、促迁移或诱导作用主要依赖于有功能的Ras和ERK1/2的激活,并与EGFR酪氨酸激酶信号途径紧密相关。EGFR是一种具有酪氨酸蛋白激酶活性的细胞表面受体,与配体结合后,可自发磷酸化配体,活化各种信号路径的下游蛋白,包括有丝分裂原激活的蛋白激酶(MAPK)等。Ras-Raf-MEK-ERK通路是MAPK级联信号通路之一,是介导细胞外信号从细胞表面传导至细胞内部的重要信号传递途径,即细胞受到刺激后,需通过一系列级联反应,引起特定蛋白的表达或活性改变,才能产生特定的生物学反应,诱导细胞的移行、增殖或分化。可以看出,TFF1-MUC5AC-EGFR及MEK/ERK目关通路与胃粘膜屏障保护、修复功能关系密切。补中益气汤是金元一代名医李东垣根据“内伤脾胃,百病由生”的学术思想而创制的代表方剂,主要由黄芪、人参、白术、甘草、升麻、柴胡、当归、陈皮等药味组成,全方功在补中益气,升阳举陷,是临床用于脾虚证治疗的经典方。脾虚证是中医临床常见的证候之一,在胃肠道粘膜疾病如消化性溃疡、慢性胃炎等中脾虚证患者占有一定比重,在各种亚健康人群当中更为多见。脾虚是以消化系统形态与功能异常为特征的多系统多功能异常的综合,胃肠粘膜的病变与其相应的功能障碍可能是脾虚证的病理基础之一。已有研究发现,脾虚大鼠胃粘膜组织确有损伤,这为“脾胃内伤,百病由生”提供了现代医学的组织形态学依据。脾为后天之本,为气血生化之源,“四季脾旺不受邪”,这说明脾胃与人体防御功能密切相关。脾失健运,不能运化水谷精微,必然会导致机体防御抵抗机能的下降,导致各种疾证的出现,这提示机体处于脾虚状态时,其胃肠粘膜本身防御功能是下降的,因而易出现各种病症。目前对补中益气汤的研究多见于临床疗效的观察,基础研究较薄弱,并多以药效学研究为主。补中益气汤对脾虚大鼠胃粘膜易损伤性有复健作用,但其机制缺乏深入探讨。因此,观察补中益气汤对胃粘膜的保护作用,并从TFF1-MUC5AC-EGRFR及MEK/ERK相关通路等角度探讨其可能机制,不仅有助于脾虚证本质的现代阐释,而且有望揭示补脾方剂药理作用的分子机制。二方法与结果1.补中益气汤对脾虚大鼠胃粘膜TFF1mRNA与蛋白表达的影响TFF1是由胃粘膜上皮细胞分泌的一种小分子多肽,为三叶因子家庭成员之一,在胃粘膜的屏障保护及损伤修复中发挥重要作用。目的：本实验观察脾虚大鼠胃粘膜TFF1mRNA与蛋白表达变化及补中益气汤对其的影响,并探讨相关作用机制。方法：SD大鼠称重随机分组,分为空白对照组、脾虚损伤组、补中益气汤组、非脾虚损伤组。脾虚损伤组、补中益气汤组大鼠给予100%大黄水煎剂灌胃造成脾虚,一天2次,连续10天,其余两组灌服等体积蒸馏水。第11天开始,补中益气汤组给予补中益气汤灌胃治疗7天,其余三组灌服等体积蒸馏水。第18天(术前禁食24h),除空白对照组外的另外三组灌服消炎痛溶液造成胃粘膜损伤。7h后处死大鼠取出胃,取出胃,冰上刮取胃粘膜,采用逆转录-聚合酶链式反应(RT-PCR法)检测TFF1mRNA,用双抗体两步夹心酶联免疫吸附法(ELISA)检测TFF1蛋白表达的变化。结果：与非脾虚损伤组比较,脾虚损伤组大鼠胃粘膜TFF1mRNA与蛋白表达明显降低(P<0.05,P<0.01)；与脾虚损伤组比较,补中益气汤组大鼠胃粘膜TFF1mRNA、蛋白明显上调(P<0.05)。结论：脾虚损伤组大鼠胃粘膜TFF1mRNA与蛋白表达显著下降,这提示脾虚大鼠胃粘膜防御能力减弱,经补中益气汤治疗后两者表达明显增加,提示该方通过提高TFF1的表达来增强胃粘膜防御能力,促进受损胃粘膜细胞的重建和修复,这可能是补中益气汤降低胃粘膜易损性的机理之一。2.补中益气汤对脾虚大鼠胃粘膜MUC5ACmRNA与蛋白表达的影响胃粘液保护层是胃粘膜的重要防御体系,其主要成分为高分子量的粘蛋白。粘蛋白是一组高度糖基化的不同糖蛋白的总称,广泛存在于消化道,是维持粘液层厚度及胶体形态的主要成分,在胃粘膜的防御和修复过程中扮演着重要的角色。分泌型MUC5AC是胃肠道凝胶主要成分之一,生理状态下与TFF1配对结合,促使胃肠粘膜更好发挥防御保护作用。目的：本实验在前期研究基础上,进一步观察脾虚大鼠胃粘膜MUC5ACmRNA与蛋白表达的变化及补中益气汤对其的影响,探讨相关作用机制。动物分组、造模、治疗、消炎痛处理、取材、检测方法同实验一。结果：与非脾虚损伤组比较,脾虚损伤组大鼠胃粘膜MUC5ACmRNA与蛋白表达有一定下调,但无统计学意义；与脾虚损伤组比较,补中益气汤组大鼠胃粘膜MUC5AC蛋白表达显著升高(P<0.05)。结论：脾虚损伤组大鼠胃粘膜MUC5AC蛋白表达下调,提示脾虚时胃粘液凝胶层防御能力下降,补中益气汤可提高MUC5AC蛋白的表达,促进胃粘液凝胶的形成,同时与TFF1发挥协同增效作用,这可能是补中益气汤保护胃粘膜机制之一。3.补中益气汤对脾虚大鼠胃粘膜EGFR蛋白表达的影响EGFR是一种具有酪氨酸蛋白激酶活性的细胞表面受体,其被配体激活后启动胞内信号传导,经过细胞质中衔接蛋白、酶的级联反应,调节转录因子,激活基因的转录,指导细胞迁移、黏附、增殖、分化或凋亡。目的：本实验在前期实验基础上进一步观察脾虚大鼠胃粘膜EGFR蛋白表达变化及补中益气汤对其的影响,并探讨相关机制。动物分组、造模、治疗及消炎痛处理同实验一。消炎痛灌胃7h后处死大鼠,取出胃,沿胃大弯剪开,用0.9%NaCL冲洗干净胃粘膜,选取胃粘膜病变相同部位剪取小块,放入10%中性福尔马林缓冲溶液中固定,采用免疫组化法(immunohistochemistry, IHC)检测EGFR蛋白表达的变化。结果：与非脾虚损伤组比较,脾虚损伤组大鼠胃粘膜EGFR蛋白表达明显下调,(P<0.01)；与脾虚损伤组比较,补中益气汤组大鼠胃粘膜EGFR蛋白表达明显升高(P<0.05)。结论：补中益气汤可提高EGFR蛋白表达,促进受损粘膜的重建修复,并可能激活下游信号传导通路,这可能是该方对脾虚大鼠胃粘膜易损性复健作用的机制之一。4.补中益气汤对脾虚大鼠胃粘膜MEK、ERKmRNA与蛋白表达的影响丝裂原激活的蛋白激酶(mitogen-activated protein kinase, MAPK)介导的细胞信号传导系统是一条重要信号转导通路,广泛存在于真核细胞内。细胞外信号调节激酶(MAPK/ERK)是MAPK家族成员中最早得到证实的转导途径。各种生长因子或其他刺激因素等细胞外信号能促使ERK通路的激活,引起特定蛋白的表达或活性改变,最终影响细胞代谢功能。目的：本实验在前期研究基础之上,进一步观察脾虚大鼠MEK、ERKmRNA与蛋白表达变化及补中益气汤对其表达的影响,深入探讨相关机制。动物分组、造模、治疗、消炎痛处理、取材同前实验一。采用荧光定量PCR(FQ-PCR法,SYBR Green I)检测MEK、ERKmRNA,用双抗体两步夹心酶联免疫吸附法(ELISA)检测MEK、ERK蛋白表达的变化。结果：与非脾虚损伤组比较,脾虚损伤组大鼠胃粘膜MEKmRNA与蛋白表达明显下降(P<0.01)；与脾虚损伤组比较,补中益气汤组大鼠胃粘膜MEKmRNA与蛋白表达明显上调(P<0.05),胃粘膜ERKmRNA与蛋白表达显著升高(P<0.05,P<0.01))。结论：补中益气汤可通过上调TFF1、EGFR的表达来激活MEK/ERK信号传导途径,从而促进细胞迁移、增殖或分化,这可能是该方胃粘膜保护及促进胃粘膜修复重建的深层次作用机制之一。三结论TFFl对胃肠粘膜有重要的保护作用,并可促进受损胃粘膜的重建修复,其机制涉及到TFF1与其对应粘蛋白MUC5AC的相互作用,促进胃粘液凝胶层的形成,并通过提高EGFR的表达进一步激活下级MEK/ERK信号传导通路,从而调节细胞代谢,促进细胞的移行、增殖、分化等。从TFF1-MUC5AC-EGFR及MEK/ERK信号传导途径等角度深入研究脾虚状态下胃粘膜防御功能及补中益气汤相关作用机制,对阐释脾虚证的病理机制及健脾方药的治疗机理有重要的理论意义。本实验研究结果表明：①补中益气汤能提高脾虚大鼠胃粘膜TFF1mRNA与蛋白的表达；②补中益气汤可提高脾虚大鼠胃粘膜MUC5AC蛋白的表达；③补中益气汤可提高脾虚大鼠胃粘膜EGFR蛋白表达；④补中益气汤可上调脾虚大鼠胃粘膜MEKmRNA与蛋白及ERKmRNA与蛋白的表达水平。综合研究结果分析,脾虚大鼠经消炎痛攻击后胃粘膜TFF1mRNA与蛋白、MUC5AC蛋白、EGFR蛋白、MEKmRNA与蛋白、ERKmRNA与蛋白表达显著下降,提示脾虚时机体胃粘膜防御功能下降,并处于高应激状态,对各种攻击因素反应敏感。经过补中益气汤治疗后,它们的表达有显著的上调,提示该方可通过提高TFF1、MUC5AC的表达,加强二者间的相互作用,增强胃粘膜的屏障保护作用。同时可增加EGFR表达,协同TFF1,激活下游MEK/ERK信号转导通路,刺激胃粘膜上皮细胞移行、增殖、分化,加速受损胃粘膜细胞的重建和修复,这可能是补中益气汤保护胃粘膜,降低脾虚胃粘膜易损性的作用靶标,这在一定程度上也体现了中医药多靶点,多层次的作用特点。更多还原

【Abstract】 Modern studies suggest that gastric mucosal defense system is a multi-level complex system that can be divided into three levels. In this three-tier defense system, every level system is related to various cytokines, which play an important role in the gastric mucosal defense and influence on each other.Trefoil factor 1 (TFF1) is a member of tefoil factor family with highly conserved structure, which is quite stable and can endure acid and decomposition caused by heat. Studies have found that the rate of incidence of ulcer is lower in the jejunum in transgenic mice with human TFFl overexpressing, which indicates that TFF1 plays an important role in protection and repairment in the gastrointestinal tract. Mucin is a general name of a group of different highly glycosylated glycoprotein which is of great value in the gastric mucosa defense and repair process.In normal conditions, TFF1 has important physiological functions with he combination of MUC5AC mucin or the corresponding receptors or transporters. Promoting cell migration is an important step in the gastric mucosa repairation and reconstitution. The launch, and inducing the migration of TFF1 mainly depend on the activation of Ras and ERK1/2, which is closely related to the signaling pathway of EGFR tyrosine kinase. EGFR is a kind of cell surface receptors with tyrosine kinase activity. The ligand can be spontaneously activiated after bond with EGFR, which can activate the downstream of proteins siganl pathways, including mitogen-activated protein kinase (MAPK). Ras/Raf/MEK/ERK pathway is one of the MAPK signaling cascade, which is an important pathway that mediates the extracellular signal transmission from the cell surface to the inside. It can cause the expression of specific proteins or activity changes through a cascade reaction, resulting in specific biological response, including the induction of cell migration, proliferation or differentiation. It can be seen that TFF1-MUC5AC-EGFR and MEK/ERK pathway is related to gastric mucosalbarrier.LiDongYuan, a famous doctor in JingYuan Dynasty, created buzhongyiqi decoction, which is a representative prescription, according to his academic thought "all diseases are caused by internal damage of the spleen and stomach". For invigorating spleen-stomach, replenishing qi, elevating yang and raising the drooping, this decoction contains primarily these herbs: Huangqi, Renshen, Baizhu, Danggui, Chenpi, Chaihu, Shengma, Gancao. It is a classical prescription for the Syndrome of Spleen Deficiency. The Syndrome of Spleen Deficiency is one of commonly clinical diseases, such as peptic ulcer, chronic gastritis, etc. It is more common in sub-healthy groups. Spleen Deficiency is a disease which is characterized by abnormal morphology and dysfunction. Gastrointestinal mucosal lesions and its corresponding dysfunction may be one of the pathological basis of Spleen Deficiency. Many studies have found that gastric mucosal tissue does have damage, which provides a morphological proof for the thought of "all diseases are caused by internal damage of the spleen and stomach".Spleen is the source of qi and blood, "No spleen deficiency, no disease", which indicats that the spleen and stomach are closely related to human defense. Spleen can’t translate, or can not ship cereal essence, which will inevitably lead to a decline in host defense against function and the emergence of various diseases. That is to say, Spleen Deficiency decreases the defense capabilities of gastrointestinal mucosa. So it is easier for the emergence of various diseases.Now most of the researches of Buzhongyiqitang are about clinical observation. The basic study is rather weak, which is more focused on pharmacodynamics. The protection of Buzhongyiqitang on injuried gastric mucosal is certain and precise, but the relevant mechanisms are lack of depth. Therefore, we observed the TFF1-MUC5AC-EGRFR and ERK/EK pathway in gastric mucosa in rats with Spleen Deficiency, and discussed the effect of Buzhongyiqitang. Our work was to explain the nature of Spleen Deficiency Syndrome, and was expected to reveal the molecular pharmacological mechanism of this prescription. 1. Effect of Buzhongyiqitang on the expression of gastric mucosa TFF1mRNA and protein in rats with the Syndrome of Spleen DeficiencyAs a member of the trefoil factor family, TFF1 is secreted by the gastric mucous cells, which is one kind of molecule peptides, and plays an important role in the gastric mucosal barrier protection and repair. The expression of gastric mucosa TFF1mRNA and protein in the Spleen Deficient rats and the effect of Buzhongyiqitang were observed, then the related mechanisms were discussed. Methods:The rats were randomly divided into the spleen deficiency with injury group, the Buzhongyiqitang group, the blank control group, and the non-spleen deficiency with injury group according to their weight. The spleen deficiency with injury group and the Buzhongyiqitang group were administrated by 100% DaHuang solution, twice a day, for 10 days, to copy the model of spleen deficiency. The other two groups were administrated by distilled water. Then the Buzhongyiqitang group was treated by Buzhongyiqitang for 7 days, twice a day, and the other three groups were administrated by distilled water. In addition to the control group, the other three groups were fed with indomethacin solution to cause mucosa injruy after treatment.7 hours later, all the rats were killed, gastric mucosa was scraped on the ice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to assay TFF1mRNA, ELISA was used to detect the expression of TFF1 protein. Results:Compared with the non-spleen deficiency with injury group, the expression of gastric mucosa TFF1mRNA and protein in the spleen deficiency with injury group were decreased significantly (P<0.05, P<0.01); Compared with the spleen deficiency with injury group, the expression of gastric mucosa TFF1mRNA and protein in the Buzhongyiqitang group were increased significantly (P<0.05). Conclusion: The expression of gastric mucosa TFF1mRNA and protein in the spleen deficiency with injury group were decreased significantly, suggesting the decreasing mucosal defense capabilities in rats with spleen deficiency. Buzhongyiqitang could increase their expression greatly, which suggested the prescription raise the expression of TFF1 to enhance gastric mucosal barrier and promote the reconstruction of damaged gastric mucosal, which may be one of the mechanisms of reducing gastric mucosal’s vulnerability of Buzhongyiqitang.2.Effect of Buzhongyiqitang on the expression of gastric mucosa MUC5ACmRNA and protein in rats with the Syndrome of Spleen DeficiencyGastric mucus belongs to important gastric mucosa protective system, the main component is mucin with high molecular weight. Widely in the digestive tract, mucin is to maintain the mucus layer thickness and colloidal, which plays an important role in the gastric mucosa defense and repair process. MUC5AC mucin is a major component of the gastrointestinal tract colloidal, play a more important role in gastric mucosa protection with the combination of TFFl in physiological conditions. In this experiment, the expression of gastric mucosa MUC5ACmRNA and protein in the Spleen Deficient and the effect of Buzhongyiqitang were observed, then the related mechanisms were discussed. The rats grouping, the model copying,, the drug treatment and indomethacin treatment and reciping essay were the same as the Experiment One. Reverse transcription-polymerase chain reaction (RT-PCR) was used to assay MUC5ACmRNA, ELISA was used to detect the expression of MUC5AC protein. Results: Compared with the non-spleen deficiency with injury group, the expression of gastric mucosa MUC5AC mRNA and protein in the spleen deficiency with injury group were decreased in some degree (P>0.05); Compared with the spleen deficiency with injury group, the expression of gastric mucosa MUC5AC protein in the Buzhongyiqitang group was increased significantly (P<0.05). Conclusion: The expression of gastric mucosa MUC5AC protein in the spleen deficiency with injury group was decreased significantly, suggesting the decreasing mucosal defense capabilities in rats with spleen deficiency. Buzhongyiqitang could increase the expression of MUC5AC protein greatly, to promote the formation of gastric mucosal gellayer. MUC5AC was bound to TFF1, which may be one of gastric mucosa’s protective mechanisms of Buzhongyiqitang.3. Effect of Buzhongyiqitang on the expression of gastric mucosa EGFR protein in rats with the Syndrome of Spleen DeficiencyEGFR is one kind of cell surface receptors with tyrosine kinase activity. It starts signal transduction inside the cells to adust transcription factors and direct cell migration, proliferation or differentiation through the cascade of adapter proteins and enzymes after its ligand is activated. The expression of gastric mucosa EGFR protein in the Spleen Deficient rats and the effect of Buzhongyiqitang were observed, then the related mechanisms were discussed.The rats grouping,the model copying,, the drug treatment and indomethacin treatment were the same as the Experiment One. All the rats were killed after the administration of indomethacin for 7 hours, and the same site of gastric mucosa was collected and put into 10% neutral formalin solution to be fixed. IHC was adopt to detect the protein expression of EGFR. Results: Compared with the non-spleen deficiency with injury group, the expression of gastric mucosa EGFR protein in the spleen deficiency with injury group was decreased significantly (P<0.01); Compared with the spleen deficiency with injury group, the expression of gastric mucosa EGFR protein in the Buzhongyiqitang group was increased significantly (P<0.05). Conclusion: Buzhongyiqitang could increase the expression of EGFR protein greatly,to promote the respairation of gastric mucosa, which maybe activate the downstream of proteins siganl pathways, which may be one of gastric mucosa’s protective mechanisms of Buzhongyiqitang.4.Effect of Buzhongyiqitang on the expression of gastric mucosa MER/ERKmRNA and protein in rats with the Syndrome of Spleen DeficiencyMAPK is an important pathway that mediats cellular signal transmission, which widely exists in eukaryotic cells. MAPK/ERK is a kind of transcription channel which is proved first. Various kinds of growth factors or other stimulation can induce the activation of ERK pathway and the expression of specified protein or the change of its activity to influence cell metabolism functions. The expression of gastric mucosa MEK, ERK mRNA and protein in the Spleen Deficient rats and the effect of Buzhongyiqitang were observed,then the related mechanisms were discussed. The rats grouping,the model copying,, the drug treatment and indomethacin treatment and reciping essay were the same as the Experiment One. FQ-PCR was used to examine MEKmRNA、ERKmRNA and ELISA was adopt to detect the expression of ME、ERK protein. Results: Compared with the non-spleen deficiency with injury group, the expression of gastric mucosa MEKmRNA and protein in the spleen deficiency with injury group were decreased significantly (P<0.01); Compared with the spleen deficiency with injury group, the expression of gastric mucosa MEKmRNA and protein in the Buzhongyiqitang group were increased significantly (P<0.05), the expression of gastric mucosa ERK mRNA and protein in the Buzhongyiqitang group were increased significantly (P<0.05, P<0.01). Conclusion:Buzhongyiqitang could increase the expression of TFF1、EGFR to activate the MEK/ERK pathway and promote cell migration, proliferation or differentiation, which may be one of the deeper protective mechanisms of gastric mucosa of Buzhongyiqitang.5. ConclusionTFF1 plays an important protective role in the gastrointestinal tract and promotes the re-establishment and reparation in gastric mucosa.The involved mechanisms included that TFF1 was combinated with MUC5AC and influenced on each other to make mucosal fluid strengthen the resistance to noxious substance, which enhanced the expression of EGFR to activite the MEK/ERK signal pathway, and adust cell metabolism, induce its migration, proliferation or differentiation. We studied the correlated targets of Buzhongyiqitang deeply from the TFF1-MUC5AC-EGFR and MEK/ERK signal pathway, which was of great value in study and application.Our empirical study showed:①Buzhongyiqitang could increase the expression of TFF1mRNA and protein of gastric mucosa in the Spleen Deficient rats;②Buzhongyiqitang could raise the expression of MUC5AC protein of gastric mucosa in the Spleen Deficient rats;③Buzhongyiqitang could increase the expression of EGFR protein of gastric mucosa in the Spleen Deficient rats;④Buzhongyiqitang could increase the expression of MEK/ERKmRNA and protein of gastric mucosa in the Spleen Deficient rats.In a word, compared with the non-spleen with injury group, the expression of TFF1mRNA、MEKmRNA. ERKmRNA and their protein, MUC5AC、EGFR protein of gastric mucosa were decreased in the rats of spleen deficient with injury group, which indicated gastric mucosal defense function subsided when the body was deficient,and the gastric mucosa was in high stringent state and was more sensitive to all kinds of attack factors. Their expression was increased through Buzhongyiqitang’s administration, which indicated this prescription could increase the expression of TFF1, MUC5AC and reinforce their mutual effect to strengthen the barrier defense protection of mucosal fluid. And this recipe could raise the expression of EGFR, and activite the MEK/ERK signal pathway, and stimulate the migration, proliferation or differentiation of Ep cells in gastric mucosa to quicken the reconstitution and recovery in injuried Ep cells, which may be the corresponding target of protecting gastric mucosa and decreasing its vulnerability of Buzhongyiqitang. In some degree, our study reflected the feature of TCM-multitarget and multistrata.更多还原