| Background:Gastric cancer(GC)ranks the fifth in the incidence of malignant tumors worldwide,and the third among cancer-related deaths worldwide,and China is a country with high incidence and mortality of GC.Because the early symptoms of GC are not obvious,and it can not be treated by surgery on the late stage,as well as chemotherapy and drug resistance of targeted drugs and other factors,the therapeutic effect of GC is not good.In recent years,it has attracted widespread attention to seek treatment methods and drugs for reducing toxicity and increasing efficacy from the perspective of traditional Chinese medicine.Professor Liu Shenlin,a national famous Chinese medicine practitioner,of Nanjing University of Traditional Chinese Medicine,summarized the prescription of Yiqi Jianpi Huaji Fang(YJHF)in many years of clinical work,which is an empirical prescription for the treatment of GC,has a prominent curative effect,and has the effects of synergy and attenuation in combination with chemotherapeutic drugs.The main function of this prescription is to tonify and relieve stasis;The prescription combines the tonifying and catharsis,deficiency and excess,diseases differentiation and syndrome differentiation,which embodies the therapeutic principle of "the combination of disease and syndrome".In order to further evaluate the effects of synergy and attenuation of this prescription on chemotherapy,and explore some of its mechanism of action,and provide theoretical and experimental basis for clinical application,this thesis conducts relevant research in three aspects:theoretical literature,clinical effect evaluation and in vitro and in vivo experiments.Part Ⅰ:Theoretical ResearchFirstly,from the perspective of modern medicine,the epidemiology,risk factors,pathogenesis and stages,diagnosis status,individual prevention and treatment status of GC were summarized and analyzed,the document research and summary shows that timely surgical treatment of early GC can effectively remove the lesion and control the progression and spread.Traditional chemotherapies and new targeted drugs are effective,but according to the research,there is no significant improvement in the survival time of active post-operative adjuvant radiotherapy and multi drug combined chemotherapy.The single target and drug resistance of targeted drugs seriously affect the efficacy of GC medical treatment.The toxicity and side effects and immunosuppression caused by chemotherapy also restrict the normal course of treatment.Therefore,searching for new therapies and drugs that can improve the effect of chemotherapy and alleviate toxic and side effects has become one of the key research goals.Recent research findings show that TGF-β NF-κB Wnt、Notch and other signaling pathways are closely related to cell apoptosis,in particular,the NF-κB signaling pathway can control many aspects of the initiation and progression of gastric cancer after being activated.The application or development of effective inhibitors of NF-κB can selectively regulate the activity of NF-κB,bringing new improvement to the diagnostic level of GC,new breakthroughs to targeted therapy and new prospects for the treatment of gastric cancer.TCM literature shows that the pathogenesis of GC is generally believed to be caused by the stagnation of Qi,phlegm,blood stasis and toxin in the stomach and Fu-organs on the basis of spleen deficiency;the main pathogenesis is the deficiency of spleen and stomach and the pathogenic toxins existing in body;therefore,the therapeutic method is to replenish the spleen and invigorate Qi on the basis of dialectics,and to soothe the liver and regulate Qi,promote blood circulation and remove blood stasis,remove phlegm and disperse stagnation,clear away heat and toxic materials,etc.Numerous studies have shown that TCM has comprehensive advantages in improving clinical symptoms and quality of life of GC patients,alleviating toxic and side effects of chemotherapy,improving the effect of chemotherapy drugs,regulating immunity,inhibiting GC growth and metastasis,etc.Most scholars believe that therapy of Chinese medicine,integrated Chinese and Western Medicine Therapy,or combination of Chinese and Western medicine may be one of the important ways to treat GC in the future.Part Ⅱ:Clinical ResearchObjective:To observe the clinical effect on GC of Yiqi Jianpi Huaji Fang(YJHF)combined with chemotherapy,evaluate its synergistic and attenuating effects and immune regulation,and provide an objective basis for clinical application.Methods:①According to the GCP criteria,100 GC patients(volunteers)with chemotherapy in the middle and late period were divided into 5 centers and assigned according to the blind coding method,using a multi-center,double-blind,randomized,single-simulator parallel control experiment design method.②Divided the patients into Treatment Group(YJHF+DC chemotherapy regimen)and Control Group(YJHF simulator+ DC chemotherapy regimen).Five time windows(Day0%15、29、43、57)were used to observe the clinical effect(including recent objective curative effect,quality of life,physical condition and TCM syndrome scores)of two cycles of chemotherapy,toxic and side effects(hematology and non-hematology)and immune indexes(T lymphocyte subsets and NK cells in peripheral blood)during the two chemotherapy cycles.Results:①A total of 93 cases were collected,and 85 patients were observed effectively(with 15 expulsion cases,and the shedding rate was<20%).②After unblinding,wherein 44 patients were in the Treatment Group(with 3 expulsion cases)and 41 patients were in the Control Group(with 5 expulsion cases).There was no difference in age,sex,family history,operation history,chemotherapy history and staging between the two groups(P>0.05),which were comparable.③Clinical benefit rate:the Treatment Group and Control Group were 90.91%and 75.61%respectively(P<0.05).④TCM syndrome efficacy:the Treatment Group were 93.18%and 70.73%respectively(P<0.05).⑤TCM syndrome scores:the score of Day57 Treatment Group was significantly improved,which was better than that of the Control Group(P<0.05),particularly in the remedial effects of food less anorexia,fatigued body and lack of strength,belching,acid regurgitation and emaciation.⑥Physical condition and quality of life:From Day43,the Treatment Group was significantly better than the Control Group(P<0.05).⑦The side effects of chemotherapy(elevated transaminase,constipation,vomiting,anorexia,alopecia,abdominal pain)in the Treatment Group were significantly less than those in the Control Group(P<0.05).⑧Comparing the effect before and after treatment,the T cell subset Treatment Group had the trend of immunoregulation,which was more obvious than that of the Control Group,but there was no statistical significance;the level of NK cells in the Treatment Group was significantly higher than that before the treatment and that of Control Group(P<0.05).Conclusion:YJHF,Traditional Chinese Herb Compound,could improve the short-term objective curative effect,improve the quality of life,physical condition,the TCM symptoms and signs scores,and reduce the side effects of GC chemotherapy.YJHF played a role in immune regulation and could improve the efficiency of GC treatment by stabilizing the immune function of the body.This study showed that YJHF combined with chemotherapy had the effects of synergy and attenuation.Part Ⅲ:Experiment ResearchExperiment Ⅰ:In vitro cell testObjective:To explore the effect of Yiqi Jianpi Huaji Fang(YJHF)on the proliferation and apoptosis of human’s gastric cancer BGC-823 cells and its possible mechanism.Methods:after 24 hours of the application of 0,30,60,90,120 and 150 ug/mL YJHF to BGC-823 cells.We used MTT assay to detect cell proliferation,used flow cytometry(FCM)to measure the apoptosis rate of cell,and used Real-time Q-PCR and Western blotting to detect the levels of caspase 3,Bcl-2 and NF-κB signaling pathway-related genes and proteins in cells.Results:①By observation of cell morphology,after the application of YJHF to BGC-823 cells,cell apoptosis such as cell shrinkage and irregular changed in cell morphology appear.②MTT assay indicated that after 24 hours of the intervention of YJHF on BGC-823 cells,it had obvious concentration-dependent inhibitory effect on proliferation.The concentration of 120 and 150μg/mL had statistical significance(P<0.05),IC50=100.80μg/mL.③FCM indicated that YJHF could promote the apoptosis of BGC-823 cells,the apoptosis rate in concentration of 90ug/mL(P<0.05),120ug/mL and 150u/mL(P<0.01)increased significantly,with obvious concentration-dependent.④ Western blot assay showed that YJHF induced apoptosis of BGC-823 cells,compared with Control Group,the expression of Caspase 3 in YJHF 120 ug/mL and 150 ug/mL(P<0.01)decreased significantly.The expression of Bcl-2 in YJHF 150ug/mL(P<0.01)was significantly decreased.⑤ NF-κB signaling pathway-related protein expression results show that,the expression of NF-κB protein decreased at YJHF 150 ug/mL,the expression of IκBα,p-IKBα and IKKa protein decreased at the concentration of 120ug/mL、150ug/mL,and the expression of p-IKBa protein decreased at the concentration of 90ug/mL,significant difference compared with the Control Group(P<0.05/0.01).Q-PCR showed that the mRNA expression of NF-κB in YJHF 120 ug/mL and 150 ug/mL decreased significantly,and the mRNA expression of IKBa at the concentration of 60ug/mL and 150ug/mL decreased,significant difference compared with the Control Group(P<0.05/0.01).C.onclusion:When YJHF concentration was more than 90 ug/mL,it could inhibit the proliferation and promote apoptosis of human’s gastric cancer BGC-823 cells.YJHF blocked the process of NF-κB and inhibited the proliferation of gastric cancer BGC-823 cells by reducing the expression of NF-κB,triggering downstream exogenous Caspase 3 cleavage and endogenous Bcl-2 expression,leading to cell apoptosis.This study showed that YJHF could promote the apoptosis of BGC-823 by inhibiting the expression of NF-κB signaling pathway.Experiment Ⅱ:in vivo animal testObjective:To explore the possible mechanism of Yiqi Jianpi Huaji Fang(YJHF)and combined with chemotherapy drug cisplatin on promoting apoptosis of GC cells,interfering with proliferation,development and other function.Methods:A human GC nude mouse tumor model was established,which was divided into 5 groups:Model group(M group),Cisplatin group(C group),Cisplatin and YJHF(high dose 62.31 g·kg-1,medium dose 41.53 g·kg-1,low dose 20.77g·kg-1)group.In addition to group M,cisplatin was administered intraperitoneally at 2 mg-kg-1 twice per week,C group was administered with normal saline,and Chinese medicine group was treated with different concentrations of YJHF for a total of 31 days.The changes of body weight,survival time and tumor size of nude mice were observed before and after intervention.HE staining,Tunel method,Western blot and Q-PCR were used to detect the apoptosis,distribution and expression of NF-κB-related protein and mRNA in transplanted tumor tissues.Results:①The weight of tumor-bearing nude mice:YJHF combined with cisplatin can reduce the effect of cisplatin on weight loss in nude mice bearing human GC tumors,there is no significant difference between groups(P>0.05).②Tumor changes:On Day7,compared with Group M,the tumor of C+YJHF medium,high groups were different(P<0.05),on Day 13,compared with Group C,there was a significant difference(P<0.05).Tumor inhibition rate:Group C was similar with C+YJHF low group.The rate of moderate and high group was significantly increased and up to over 50%.③Intratumoral cell changes:The number of tumor cells decreased with the concentration of traditional Chinese medicine,loose arranged,the expression of apoptotic cells such as slice necrosis could be seen.It could also observe apoptosis in tumor tissue through Tunel method.The apoptotic index of C+YJHF moderate and high group was obviously higher than that of Group M and Group C(P<0.05).④YJHF concentration-dependent decreased the expression of Caspase 3 and Bcl-2 proteins in tumor tissues,and the YJHF high group was significantly lower than that in the C group(P<0.05/0,01);YJHF increased the expression of Bax,which was higher than that in the C group.The YJHF medium group was significantly higher than the M group(P<0.05).⑤YJHF concentration-dependent decreased the expression of IKK-β NF-κB、p-IKB-α proteins in tumor tissues,The YJHF high group was lower than the M and C groups(P<0.01),and the YJHF medium group was lower than the M group(P<0.05).YJHF concentration-dependent decreased the expression of p-NF-κB,IKB-κ,and IKK-α protein,and the YJHF high group was lower than the M and C groups,and was significantly different from the M group(P<0.05).The expression of p-IKB-αprotein in YJHF high group was lower than that in YJHF low group(P<0.05).Q-PCR indicated that compared with Group M,the mRNA expression of NF-κB in C+YJHF medium group(P<0.05)and C+YJHF high group(P<0.01)decreased significantly.Compared with Group C,the IKKa and IKBa mRNA expression of C+YJHF low,medium and high group decreased significantly(P<0.05),lower than that of Group M.Conclusion:YJHF could inhibit GC tissue growth in combination with cisplatin chemotherapy,and suggested that YJHF might decrease the activation of NF-κB,block the anti-apoptotic pathway of NF-κB,increase the apoptosis of GC cells,and improve the chemotherapeutic effect.This research confirms that:①YJHF could improve the quality of life and physical condition of patients with GC chemotherapy,improve the immune function of patients,and reduce the side effects of GC chemotherapy to a certain extent;YJHF combined with chemotherapy had the effects of synergy and attenuation and attenuated the effects of short-term effects.②Experimental studies had shown that YJHF could inhibit the proliferation of human gastric cancer BGC-823 cells and promote cell apoptosis;and down-regulate the expression of NF-κB.YJHF combined with chemotherapeutic drugs promoted the regulation of host NF-κB signaling pathway and increased the apoptosis of gastric cancer cells.We believed that this might be one of the molecular mechanisms of YJHF’s efficacy,synergistic and attenuating effects.This study provided a scientific basis for explaining the therapeutic effect of YJHF on clinical treatment of GC. |
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