【作者】 滑永志；

【导师】 田耀洲；

【作者基本信息】 南京中医药大学 ， 中西医结合临床（专业学位）， 2021， 博士

【摘要】 慢性萎缩性胃炎(Chronic Atrophic Gastritis,CAG)是以固有腺体萎缩为特征的慢性炎症性疾病,早在1980年,WHO即将本病纳入癌前病变的范畴,近年来随着研究深入及普及,CAG越发受到消化学界以及患者的重视。数据显示,我国CAG发病率为7.5-13.8%,其中,约2.55-7.46%的患者可能转化为胃癌。CAG的发病一般在中年以后,与年龄的增长成正比,是中老年人的常见疾病,而随着患病时间延长及年龄增长,肠上皮化生、上皮内瘤变,癌变等风险逐渐提高,有数据显示,CAG患者10年以上每年的癌变率为0.5-1%。这与胃癌的发病年龄亦相符合。在临床实践中我们观察到,青年人中CAG发病比例逐年升高,且发病特点及病因病机方面与老年人CAG有显著区别,但是在青年人CAG中,很少有合并肠化、上皮内瘤变等情况。但是,因为其患病年龄较低,加之青年人生活节奏较快,工作压力大,生活作息不规律等因素,远期癌变风险变大,故就针对消化道肿瘤早诊早治方面,青年人CAG有更高的临床诊治价值。但目前尚没有专门针对青年人萎缩性胃炎的相关研究。为此,本项目拟在前期工作基础上,进一步归纳总结青年人萎缩性胃炎中医证型特点,评价和优化已有研究基础而形成的治疗方案,总结提高,以进一步发挥中医药对CAG疗效优势,促进临床规范用药,提高中医药治疗CAG的临床水平。临床实践中,我们发现CAG伴有肠上皮化生、上皮内瘤变的治疗非常棘手,一旦没有及时发现和治疗,通常在3-5年即进展为进展期胃癌,而目前尚没有有效的方法阻止这一病情进展。针对这一部分患者,田耀洲教授在临床实践中,不断探索并结合络病学说总结认为,癌前状态以脾胃气虚为根本病机,瘀阻血络为关键环节立法,气虚则运化乏源,内蕴不消,而腑病以通为用,久病多瘀,病机发展,入里伤络,故可将病机概括为胃络虚滞,运用益气通络法治疗胃癌前状态取得了较好的疗效。故拟方石丹颗粒,基于多年疗效总结,石丹颗粒可显著改善患者临床症状,亦可改善胃粘膜萎缩、肠化腺体形态异常[11]。课题组前期已完成部分动物实验,初步观察了“石丹颗粒”对CAG模型大鼠血清增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、胃粘膜再生基因 I(regenerating gene I,Reg I)及胃粘膜病理的影响,发现“石丹颗粒”对控制或逆转萎缩确有疗效,但其具体作用机制并不明确,仍需从多层次、多方面进行深入探讨。基于以上,本项目研究纳入了两项实验,一项是针对青年人CAG的临床研究,旨在通过研究,探析青年人萎缩性胃炎的中医证治规律。一项是针对石丹颗粒逆转癌前状态的基于核因子 E2 相关因子 2(Nuclear factor-elytrorid derived factor 2-related factor 2,NRF2)及下游重要的抗氧化酶血红素加氧酶1(heme oxygenase 1,HO-1)和醌氧化还原酶1(quinone oxidoreductase 1,NQO-1)探索石丹颗粒治疗癌前状态的机制研究。第一部分理论研究从中西医方面分别对CAG的病因病机、诊治的进展进行总结,现代医学目前多从幽门螺旋杆菌(Helicobacterpylori,Hp)感染,免疫功能变化,胃黏膜屏障受损、遗传和饮食生活等因素认识CAG的产生,胃镜及病理学的检查更是强有力的诊断证明,而治疗方面尚未发现可从根本上逆转萎缩的方法,仍以缓解症状为主要治疗方案,如根除Hp,强固黏膜,减少反流,补充维生素等,因此,中医药研究在国内外成为新的热点,在单味中药、复方汤剂、中成药等均有所涉及,而其繁杂的治疗方法和方药,使CAG的中医药诊疗方案和治疗原则难以规范和统一。目前国际公认的肠型胃癌发病模式为Correa模式,即慢性浅表性胃炎一萎缩性胃炎一肠上皮化生一上皮内瘤变一胃癌。这提示CAG作为胃癌前状态,具有一定癌变可能,与“炎一癌转化”理论相符。“炎一癌转化”提示炎症性疾病通过癌前状态进展致肿瘤的过程,而疾病的转化并非一蹴而就,大多呈现为动态的变化过程,范围广泛,并非局限于某一疾病。归纳总结可知“炎-癌转化”为脾胃由气渐渐累及阴津,再致阳气的变化,其治疗方法为活血解毒,兼顾扶正。其中,在正虚的内因基本上,各种病理因素可作为实邪,为CAG的变化进展提供条件,最终形成胃癌,病性总属正虚邪实。因此,在临床治疗中应辨证论治,在兼顾正气的基础上祛除邪实,如活血化瘀、解毒通络等,以改善甚则逆转CAG进程,近年来“癌毒学说”和“浊毒学说”等理论的提出对于认识此过程以及指导临床诊疗有着重要作用。第二部分青年人萎缩性胃炎的中医证治规律探析目的:通过对青年人(20-44岁)慢性萎缩性胃炎(CAG)病因病机、辨证分型、治疗方案等的研究,探索青年人CAG中医证治规律和疾病特点;客观评价中药对青年人CAG临床疗效,进一步发挥中医诊疗优势,规范临床用药,提高中医药治疗青年人CAG的临床水平,为后续CAG发病机理和疗效机制的研究打下基础。方法:收集2016年04月～2018年06月江苏省中西医结合医院消化科就诊的200例青年(20-44岁)CAG患者,经胃镜检查且病理学提示为CAG,并根据胃黏膜分级评估萎缩程度,同时,经中医辨证分析,符合纳排标准后,依据中医证型分别予以左金丸合温胆汤加味方(脾胃湿热)、柴胡疏肝散加减(肝胃不和)、香砂六君子汤加味(脾胃虚弱)、仁术健胃方(气虚热瘀)等治疗,6个月后根据患者治疗前后症状、胃镜病理、不良反应等分析处理数据,以探索青年人CAG的证治规律和疾病特点,进一步评价中医药辨证治疗对青年人CAG的临床疗效。结果:200例慢性CAG患者中中医最常见证型为脾胃湿热证,约占总比的35%,以男性较为多见,其后为占比30.5%的肝胃不和证,以女性多见,脾胃虚弱证以及气虚瘀热证分别为25.5%和9%。经治后,青年人CAG中医证候积分均较前有所下降,临床总有效率高达92.59%。此外,因治疗后胃镜复查者较前减少,数据显示胃镜及组织病理学总有效率分别为77.7%、69.2%。除部分患者出现腹泻外,无其他特殊不适。结论:青年人CAG证型分析提示,男性患者最常见脾胃湿热证型,女性则以肝胃不和证为首要,并确立了以清热化湿、疏肝和胃为本,以益气健脾、滋阴养胃为之常法的青年人CAG中医治则理论。中医药在青年人CAG中疗效确切,症状学的改善作用显著,经胃镜及病理学证实,部分病例萎缩得到了控制,显示了辨证方案的治疗优势。第二部分 实验研究(基于NRF2/ARE/HO-1信号通路探讨石丹颗粒治疗胃癌前状态的作用机制)目的:观察石丹颗粒基于NRF2/ARE/HO-1信号通路对癌前状态大鼠的疗效,并探究石丹颗粒治疗癌前状态的作用机制。方法:Wistar大鼠适应性饲养后,随机分为10只空白组、10只模型组、9只阳性药组(维酶素0.3 g/kg),石丹颗粒高剂量(5.6 g/ml)、中剂量(2.8 g/ml)、低剂量(1.4 g/ml)各9只。大鼠造模方法为饮用0.1%氨水溶液、喂食0.03%雷尼替丁的饲料,并予150 μg/ml 1-甲基-3硝基-1-亚硝基胍(MNNG)溶液,5 ml/kg,每日一次灌胃。第28周末大鼠胃粘膜萎缩、固有腺体减少、上皮内瘤变出现,说明造模成功。模型组大鼠予灌胃生理盐水(2ml/只),各给药组按计划每天一次灌胃相应药物,直至40周后解剖收集胃组织,通过western blot法检测细胞浆和细胞核中NRF2蛋白,采用qRT-PCR法检测胃黏膜NRF2、谷氨酰半胱氨酸连接酶调节亚基(GCLM)、谷氨酰半胱氨酸连接酶催化亚基(GCLC)、HO-1和NQO-1的表达,NRF2-ARE结合力则可运用EMSA法检测,荧光素酶活性检测抗氧化反应原件(ARE)活性。结果:与空白组相比癌前状态模型组大鼠反应迟钝,毛发粗糙无光,尾巴淡白色,部分大鼠稀便,食欲不振,饮水减少。在细胞核中,模型组的NRF2蛋白含量较空白组明显增加(P<0.05),石丹颗粒各组较模型组增加显著(P<0.05,P<0.01);在细胞浆中,空白组和模型组的的NRF2蛋白基本没有差别,较前者而言,石丹颗粒高、中剂量组蛋白表达水平明显提高。此外,模型组NRF2蛋白表达水平较空白组增加明显(P<0.05),其中石丹颗粒高剂量组可极显著提高NRF2、NQO-1和HO-1蛋白含量(P<0.001),中剂量组则明显提高下游蛋白NQO-1、HO-1含量(P<0.05)。同时,模型组NRF2mRNA的表达较空白组显著提高(P<0.05);而较癌前状态模型组,石丹颗粒高、中剂量组中NRF2、HO-1 mRNA的表达显著增加(P<0.05,P<0.01),高、中、低三组显著增加NQO-1 mRNA表达(P<0.05,P<0.01),石丹颗粒对GCLM nmRNA、GCLC mRNA表达则没有影响。模型组ARE荧光素酶活性较空白组明显增强(P<0.05),NRF2-ARE结合力则几乎没有差别。与模型组比较,石丹颗粒高、中、低剂量呈剂量依赖性地激活ARE荧光素酶活性。但能明显增强NRF2与ARE的结合力,其中石丹颗粒高剂量作用最强结论:石丹颗粒可明显改善癌前状态大鼠的的一般状态和胃黏膜病理形态学变化,其作用可能与激活NRF2/ARE/HO-1信号通路有关,增加NRF2核内积累,上调下游的HO-1、NQO-1含量,且不影响谷胱甘肽(GSH)生物合成,GCLC mRNA和GCLM mRNA含量并未改变,从而改善胃粘膜萎缩、肠化腺体形态异常,减轻细胞损害。更多还原

【Abstract】 Chronic atrophic gastritis(Chronic Atrophic Gastritis,CAG)is a chronic inflammatory disease characterized by atrophy of the inherent glands.As early as 1980,the WHO included this disease in the category of precancerous lesions.In recent years,with the deepening and popularization of research,CAG has received more and more attention from the gastroenterology community and patients.Data show that the incidence of CAG in my country is 7.5-13.8%,of which about 2.55-7.46%of patients may be transformed into gastric cancer.The onset of CAG generally occurs after middle age and is directly proportional to the increase of age.It is a common disease of middle-aged and elderly people.With the prolonged disease time and age,the risk of intestinal metaplasia,intraepithelial neoplasia,and canceration gradually increases.According to statistics,the annual cancer rate of CAG patients for more than 10 years is 0.5-1%.This is also consistent with the age of onset of gastric cancer.In clinical practice,we have observed that the incidence of CAG among young people is increasing year by year,and the characteristics and etiology and pathogenesis of CAG are significantly different from those of the elderly.Tumors and other conditions.However,because of the younger age of the disease,the faster pace of life of young people,heavy work pressure,and more bad habits,the risk of long-term cancer becomes greater.Therefore,the early diagnosis and treatment of gastrointestinal tumors is aimed at young people.CAG has higher clinical diagnosis and treatment value.However,there is no relevant research specifically aimed at young people with atrophic gastritis.For this reason,this project intends to further summarize the characteristics of TCM syndromes of young people with atrophic gastritis based on the previous work,evaluate and optimize the treatment plan formed by the existing research foundation,summarize and improve,so as to further exert the advantages of TCM on CAG.,Promote the clinical standard use of drugs,and improve the clinical level of Chinese medicine in the treatment of CAG.In clinical practice,we found that the treatment of CAG accompanied by intestinal metaplasia and intraepithelial neoplasia is very difficult.Once it is not detected and treated in time,it usually progresses to advanced gastric cancer within 3-5 years.There is no effective method yet.Stop the progression of this condition.Aiming at this part of patients,Professor Tian Yaozhou has continuously explored and combined with the theory of collateral disease in clinical practice and concluded that the precancerous state is based on the basic pathogenesis of spleen and stomach qi deficiency and blood stasis blocking blood collaterals as a key link to legislate.No problem,but the organs are used for general purpose,chronic disease with multiple blood stasis,pathogenesis develops,enters the collaterals,so the pathogenesis can be summarized as gastric collateral deficiency and stagnation,and the method of replenishing qi and dredging collaterals has been used to treat the precancerous state of the stomach.Efficacy.Therefore,based on the summary of curative effects for many years,Shidan granules can be used to improve the clinical symptoms of patients,as well as gastric mucosal atrophy and abnormal intestinal gland morphology[11].The research group has completed some animal experiments in the early stage,and initially observed the effects of "Shidan Granule" on the serum proliferating cell nuclear antigen(PCNA),gastric mucosal regeneration gene I(regenerating gene I,Reg I)and gastric mucosa in CAG model rats.The influence of pathology has been found that"Shidan Granules" are effective in controlling or reversing atrophy,but the specific mechanism of action is not clear,and it still needs to be discussed in depth from multiple levels and aspects.Based on the above,this project research included two experiments,one is a clinical study on CAG of young people,aiming to analyze the law of TCM syndrome and treatment of young people’s atrophic gastritis through research.One is for Shidan granules to reverse the precancerous state based on nuclear factor E2-related factor 2(NRF2)and the downstream important antioxidant enzyme heme oxygenase 1(heme oxygenase).1,HO-1)and quinone oxidoreductase 1(quinone oxidoreductase 1,NQO-1)explore the mechanism of Shidan granules in the treatment of precancerous states.Part Ⅰ Theoretical ResearchFrom the aspects of Chinese and Western medicine,the etiology,pathogenesis,diagnosis and treatment of CAG are summarized.Modern medicine is currently mostly based on Helicobacter pylori(Hp)infection,changes in immune function,impaired gastric mucosal barrier,genetics,and dietary life.The production of CAG,gastroscopy and pathological examinations are even more powerful diagnostic proofs.However,treatment has not yet found a method that can fundamentally reverse atrophy.Relieving symptoms is still the main treatment plan,such as eradicating Hp,strengthening mucosa,and reducing reaction.Therefore,Chinese medicine research has become a new hot spot at home and abroad,involving single Chinese medicine,compound decoction,Chinese patent medicine,etc.,and its complicated treatment methods and prescriptions make CAG Chinese medicine Diagnosis and treatment plans and treatment principles are difficult to standardize and unify.At present,the internationally recognized pattern of intestinal gastric cancer is the Correa pattern,that is,chronic superficial gastritis—atrophic gastritis—intestinal metaplasia—intraepithelial neoplasia—gastric cancer.This suggests that CAG,as a precancerous state of the stomach,has a certain possibility of cancerous transformation,which is consistent with the theory of "inflammation-carcinoma transformation"."Inflammation-cancer transformation" suggests that inflammatory diseases progress to tumors through precancerous states,while disease transformation does not happen overnight.Most of them appear as a dynamic process of change,with a wide range and not limited to a certain disease.The summary shows that "inflammation-cancer transformation" means that the spleen and stomach gradually involve the yin and fluid from the qi,and then cause the change of the yang.Among them,in the internal cause of the deficiency of righteousness,various pathological factors can be used as the actual evil,which provides conditions for the change and progression of CAG,and eventually forms gastric cancer.The disease is always the righteous deficiency and evil.Therefore,clinical treatment should be based on syndrome differentiation and treatment.On the basis of taking into account the righteousness,the evils should be eliminated,such as promoting blood circulation and removing blood stasis,detoxification and dredging collaterals,so as to improve or reverse the process of CAG.In recent years,"cancer toxin theory" and "turbid toxin theory" The proposing of other theories plays an important role in understanding this process and guiding clinical diagnosis and treatment.Part Ⅱ Exploration and Analysis of TCM Syndromes of Atrophic Gastritis in Young PeopleObjective:Through the research on the etiology,pathogenesis,syndrome differentiation,treatment plan,etc.of chronic atrophic gastritis(CAG)in young people(20-44 years old),to explore the law of CAG syndrome treatment and disease characteristics of young people;objectively evaluate the effects of traditional Chinese medicine on young people The clinical efficacy of human CAG will further utilize the advantages of TCM diagnosis and treatment,standardize clinical medication,and improve the clinical level of TCM treatment of CAG in young people,laying a foundation for subsequent research on the pathogenesis and efficacy mechanism of CAG.Methods:Collected 200 young CAG patients(20-44 years old)who attended the Gastroenterology Department of Jiangsu Provincial Hospital of Integrated Traditional Chinese and Western Medicine from April 2016 to June 2018.They were examined by gastroscopy with pathological findings indicating CAG,and evaluated according to the gastric mucosal classification.At the same time,according to the syndrome differentiation analysis of TCM,after meeting the standard of intake and discharge,Zuojin Pill and Wendan Decoction Modified Recipe(spleen and stomach damp-heat),Chaihu Shugan powder(disharmony of liver and stomach),Xiangsha Six months later,it is treated with modified Liujunzi Decoction(weakness of the spleen and stomach)and Renshu Jianwei Recipe(qi deficiency and heat stasis).After 6 months,the data will be analyzed and processed according to the patient’s symptoms,gastroscopy pathology,adverse reactions,etc.,in order to explore the treatment of CAG in young people Regularity and disease characteristics,and further evaluate the clinical efficacy of TCM syndrome differentiation treatment on CAG in young people.Results:The most common TCM syndrome in 200 patients with chronic CAG was spleen and stomach damp-heat syndrome,which accounted for about 35%of the total,and was more common in men,followed by liver-stomach discordance,which accounted for 30.5%,and was more common in women.,Spleen and stomach weakness syndrome and Qi deficiency and stasis heat syndrome were 25.5%and 9%,respectively.After the treatment,the CAG TCM syndrome scores of young people decreased compared with before,and the total clinical effective rate was as high as 92.59%.In addition,because the number of gastroscopy reviewers after treatment decreased compared with before,the data showed that the total effective rates of gastroscopy and histopathology were 77.7%and 69.2%,respectively.Except for diarrhea in some patients,there was no other special discomfort.Conclusion:The analysis of CAG syndrome types of young people suggests that male patients are the most common type of spleen and stomach damp-heat syndrome,while female patients take liver-stomach discordant syndrome as the first.,Young people’s CAG,which is the common method of nourishing yin and nourishing stomach.Traditional Chinese medicine has definite curative effect in CAG of young people,and its symptomatic improvement effect is remarkable.It is confirmed by gastroscopy and pathology that the atrophy of gastric mucosal glands in some cases has been controlled,showing the therapeutic advantages of the syndrome differentiation program.Part Ⅲ Experimental Study(Based on NRF2/ARE/HO-1 signal pathway to explore the mechanism of Shidan granule in the treatment of gastric precancerous state)Objective:To observe the therapeutic effect of Shidan granules on precancerous rats based on the NRF2/ARE/HO-1 signaling pathway,and to explore the mechanism of Shidan granules in the treatment of precancerous conditions.Methods:After adaptive feeding,Wistar rats were randomly divided into 10 blank group,10 model group,9 positive drug group(Vitamin 0.3 g/kg),high dose of Shidan granules(5.6 g/ml),The middle dose(2.8 g/ml)and the low dose(1.4 g/ml)were 9 mice each.The method of modeling rats is to drink 0.1%ammonia solution,feed 0.03%ranitidine feed,and give 150 μg/ml 1-methyl-3nitro-1-nitrosoguanidine(MNNG)solution,5 ml/kg,once a day by gavage.At the end of the 28th week,the rat gastric mucosa was atrophy,proper glands decreased,and intraepithelial neoplasia appeared,indicating that the model was successful.Rats in the model group were given normal saline(2 ml/mouse).Each administration group was given the corresponding drugs once a day as planned,until 40 weeks later,the gastric tissue was dissected and collected,and the NRF2 protein in the cytoplasm and nucleus was detected by western blot.,Using qRT-PCR to detect gastric mucosal NRF2,glutamylcysteine ligase regulatory subunit(GCLM),glutamylcysteine ligase catalytic subunit(GCLC),HO-1 and NQO-1 The expression of NRF2-ARE can be detected by the EMSA method,and the luciferase activity can be detected by the antioxidative reaction element(ARE)activity.Results:Compared with the blank group,the rats in the precancerous state model group were slow to respond,with rough and dull hair,pale white tails,loose stools in some rats,loss of appetite,and reduced drinking water.In the nucleus,the NRF2 protein content of the model group was significantly higher than that of the blank group(P<0.05),and the Shidan granule groups were significantly higher than the model group(P<0.05,P<0.01);in the cytoplasm,the blank group and the model group There was basically no difference in the NRF2 protein between the two groups.Compared with the former,the expression levels of histones at high and medium doses of Shidan granules were significantly increased.In addition,the expression level of NRF2 protein in the model group was significantly higher than that in the blank group(P<0.05).The high-dose Shidan granule group could significantly increase the protein content of NRF2,NQO-1 and HO-1(P<0.001).The middle-dose group It significantly increased the content of downstream proteins NQO-1 and HO-1(P<0.05).At the same time,the expression of NRF2 mRNA in the model group was significantly higher than that of the blank group(P<0.05);and compared with the precancerous state model group,the expression of NRF2 and HO-1 mRNA in the Shidan granule high and medium dose groups were significantly increased(P<0.05,P<0.01),the three groups of high,medium and low significantly increased the expression of NQO-1 mRNA(P<0.05,P<0.01).Shidan granules had no effect on the expression of GCLM mRNA and GCLC mRNA.The ARE luciferase activity of the model group was significantly higher than that of the blank group(P<0.05),and there was almost no difference in the binding capacity of NRF2-ARE.Compared with the model group,high,medium and low doses of Shidan granules activated ARE luciferase activity in a dose-dependent manner.But it can significantly enhance the binding force of NRF2 and ARE,among which Shidan granules have the strongest effect at high dosesConclusion:Shidan granules can significantly improve the general state of precancerous rats and the pathological changes of gastric mucosa.Its effect may be related to the activation of the NRF2/ARE/HO-1 signaling pathway,increasing the accumulation of NRF2 in the nucleus,and upregulating downstream The content of HO-1 and NQO-1 does not affect the biosynthesis of glutathione(GSH),and the content of GCLC mRNA and GCLM mRNA remains unchanged,thereby improving gastric mucosal atrophy,abnormal intestinal gland morphology,and reducing cell damage.更多还原