| IntroductionGastric cancer is a malignant tumor of digestive tract,which seriously endangers people’s health and life.The incidence rate of gastric cancer ranks the fourth in the incidence rate of malignant tumors worldwide,ranks the second in China,and the first in Yangzhou,Jiangsu.The majority of gastric cancer patients in our country are in the late stage,the treatment effect is poor,and the prognosis is poor.However,the general condition of patients with advanced gastric cancer is poor,coupled with the toxic and side effects of chemotherapy drugs on digestive tract,bone marrow function,immune system,liver,kidney,etc.,the chemotherapy tolerance of patients is poor How to optimize the use of chemotherapy is an important work for tumor clinicians The combination of Taditional Chinese Medicine and chemotherapy drugs can reduce toxicity,improve life quality and prolong survival time.According to the poor transport and chemical function of patients with gastric cancer,the function of transporting,excreting and eliminating toxic pathogens is not smooth.Yunhuaduxie recipe is a prescription for treating gastric cancer independently developed according to the characteristics of gastric cancer for many years(composed of Astragalus membranaceus,ginseng,fried Atractylodes macrocephala,fried semen lachryma,Hedyotis,Curcuma,Solanum nigrum,dandelion,Poria cocos,roasted licorice).On the basis of constructing tumor data bank of Traditional Chinese Medicine and transplanting tumor model of human gastric cancer tissue,the anti-gastric cancer effect of Yunhuaduxie recipe was systematically studied from the clinical,cellular and animal levels,and the possible anti gastric cancer mechanism of Yunhuaduxie recipe was preliminarily studied from the regulation of Claudin-4/VEGF-C/GSK3 β signal network.This subj ect is divided into four parts:Part Ⅰ:Theoretical framework of treating gastric cancer from toxin and pathogen1.1、The origin and development of the theory of toxin and pathogen in the treatment of tumorFrom the point of view that the treatment of toxin and pathogen is one of the core issues in cancer treatment,this paper systematically combs the origin and development of the theory of toxin and pathogen:the Qin and Han Dynasties were the embryonic stage of the theory of toxin and pathogen,and the Jin,Sui,Tang and song Dynasties roughly constructed the framework of the theory of toxin and pathogen,namely the classification,connotation,etiology and pathogenesis characteristics of toxin and pathogen.In the Ming and Qing Dynasties,the concept of "cancer toxin"has been put forward by many doctors since Ming and Qing Dynasties.The treatment of malignant tumor should be based on the general treatment principle of strengthening the body and eliminating pathogenic factors,and pay special attention to the important pathological factor of toxin and pathogen.According to the syndrome elements,syndrome characteristics and pathogenesis law of toxin in the occurrence and development of malignant tumor,this paper expounds strengthening the body and removing toxin,clearing away heat and detoxification,regulating Qi and expelling toxin,removing blood stasis and toxin,resolving phlegm and toxin,dispelling wind and toxin,discharging turbid and detoxifying,calming pathogenic factors and eliminating insects at the same time,combined with the results of modern scientific research,gradually improve the framework of the theoretical system of treating malignant tumor from toxin and pathogen.1.2、Treatment of gastric cancer based on the theory of "spleen dominates transport and transformationBased on the continuous development of classical Chinese Medicine theory and medical science,there is a close relationship between gastric cancer and the theory of"spleen dominating transportation".In order to improve the curative effect of diagnosis and treatment of gastric cancer and improve the prognosis of gastric cancer.Part Ⅱ:Clinical study of Yunhuaduxie Recipe on recurrence and metastasis of advanced gastric cancerObjective:To investigate the clinical effect of Yunhuaduxie Recipe on advanced gastric cancer patients,and to observe the effect of Yunhuaduxie Recipe on serum metabolites and metabolic pathway in patients with gastric cancer.Methods:Based on the Chinese Medicine tumor web information database,the structured design of TCM tumor patient information was carried out,and the basic information of patients,clinical information of Traditional Chinese Medicine and other related information were integrated to realize the online query and statistics function.98 patients with advanced gastric cancer admitted to Yangzhou Hospital of Traditional Chinese Medicine from February 2016 to March 2019 were selected as the research objects.Meanwhile,non randomized clinical research methods were used The eligible cases were included in the Traditional Chinese Medicine group and chemotherapy group,with 49 cases in each group.The control group was treated with oxaliplatin and tegafur,while the Traditional Chinese Medicine group was given Yunhuaduxie recipe on the basis of the control group.16 serum samples were derivatized and analyzed by GC-TOFMS detectorResults:After 4 courses of TCM and chemotherapy,19 cases of PR and 17 cases of SD in the chemotherapy group,the clinical effective rate was 38.77%,and the disease control rate was 73.47%;in the Traditional Chinese Medicine group,there were 24 cases of PR and 21 cases of SD,and the clinical effective rate was 48.98%,The disease control rate was 91.84%,the difference between the two groups was statistically significant(P<0.05);after treatment,KPS and ECOG scores of the Chinese Medicine group were significantly increased,the expression levels of CEA,CA199 and CA724 were significantly decreased in the same group(P<0.05);the incidence of liver and kidney function injury,gastrointestinal reaction,bone marrow suppression in the chemotherapy group was lower than that in the control group,the difference was statistically significant(P<0.05);the one-year survival rate of chemotherapy group was significantly lower than that of Traditional Chinese Medicine group,and the difference was statistically significant between the two groups(TCM intervention,age,weight,TNM stage,Karnofsky’s score,surgical method and other factors were related to the progression free survival of advanced gastric cancer,and TCM treatment,operation mode and weight were independent related factors affecting the survival period of advanced gastric cancer.Yunhuaduxie recipe intervenes the treatment of gastric cancer,mainly involving galactose metabolic pathway,branched chain amino acid metabolic pathway,and intervening the metabolism of L-isoleucine,L-serine,L-leucine,D-galactose,sucrose,3-methyl-2-oxo-valeric acid.Conclusion:Yunhuaduxie recipe can prolong the survival time,improve the quality of life,and reduce the side effects of oxaliplatin and tegafur chemotherapy.It is an independent protective factor for the prognosis of advanced gastric cancer.The influence of Yunhuaduxie Recipe on serum metabonomics of patients with gastric cancer mainly involves galactose metabolic pathway and branched chain amino acid metabolic pathway.Part Ⅲ:Effect of Yunhuaduxie Recipe on the growth of gastric cancer and the regulation of Claudin-4 on epithelial mesenchymal transition signal pathwayObjective:To investigate the effect of Yunhuaduxie Recipe on the growth and expression of related proteins and mRNA in gastric cancer cells and human tumor xenografts.Methods:MTT assay was used to detect the effect on the proliferation of human gastric cancer cells.The migration of gastric cancer cells was detected by scratch test.The model of patient-derived xenograft model(PDTX)was established.The expression of GSK3 β,VEGF-C and Claudin-4 were detected by Western blot,and GSK3 β,VEGF-C and claudin-4 mRNA were detected by qPCR.Results:The cells were inhibited to different degrees and had certain concentration dependence after treatment with Yunhuaduxie Recipe 1,2,4,8,16,32 mg/ml for 24 and 48 hours.Compared with the blank group,the scratch distance of the 3 and 6 mg/mL groups decreased significantly after 24 hours after the intervention of AGS,BGC-803 and SGC-7901 cells(P<0.05).Compared with the negative control group,the average tumor volume of the high dose group of Yunhuaduxie Recipe was significantly different,and there was significant difference in statistical analysis;the average tumor volume of the low dose group and the middle dose group of Yunhuaduxie Recipe was lower than that of the negative control group,but there was no significant difference in statistical analysis.Compared with the negative control group,the average tumor weight of the high dose group of Yunhuaduxie Recipe was significantly different,and there was significant difference in statistical analysis.The expression level of GSK3 β and mRNA in AGS,BGC-803,SGC-7901 cells and tumor tissues increased after the treatment of Yunhuaduxie Recipe and the expression level of VEGF-C,claudin-4 protein and mRNA decreasedConclusion:Yunhuaduxie recipe can inhibit the growth,invasion and metastasis of gastric cancer by inhibiting the expression level of epithelial mesenchymal transformation related protein and promoting the expression level of tumor suppressor gene related proteinPart Ⅳ:Chemical Constituents of Yunhuaduxie recipe based on HPLC-MSObjective:to analyze the chemical constituents of Yunhua duxie prescription and explore the chemical constituents of its efficacyMethods:the decoction was prepared according to the proportion of Chinese herbal medicine in Yunhua duxie prescription,and the chemical components in the decoction were identified by HPLC-MSResults:a total of 112 chemical active components were detected in the water decoction of Yunhua duxie Decoction under positive and negative ion mode,among which 59 active components with a response intensity greater than 50000 were identified in the order of liquiritin,apigenin,apigenin,formononetin,glycyrrhizic acid,7-hydroxy-4 ’-methoxyisoflavone,4’,7-Dihydroxyflavone,2 ’,4,4,-trihydroxychalcone,liquiritigenin,isoliquiritigenin,liquiritigenin,calycosin,(3R)-2’,3 ’-dihydroxy-7,4 ’-dimethoxyisoflavone,wogonin,sakurain,genkwanin,formononetin,ginsenoside,rutin,chlorogenic acid,ginsenoside RO,p-coumaric acid,ginsenoside Rg7,ginsenoside RF,Ginsenoside Rgl,perloside F2,perloside F4,quercetin-7-o-β-D-glucoside,Isoquercitrin,scopoletin,scopolactone,Ginsenoside Rg3,ginseng Saponin F2,ginsenoside Rg2,andrographolide,α-santalol,luteolin,linarin,curcumin,glycyrrhizin,caffeic acid,trigonoside,ginsenoside R2,ginsenoside F3,bamboo saponin L8,hemiglycyrrhizin B,naringin,ginsenoside Rbl,atractylolide,ginsenoside Rd,ginsenoside Re,Gypenoside XVII,palmitic acid,hexadecanoic acid,poria Lingxinic acid g,24 hydroxyglycyrrhetinic acid methyl ester,ginsenoside RH4,ginsenoside rk3,glycyrrhetinic acid.The content of Ginsenoside Rg3 in the sample was 3.67ppm.Conclusion:Yunhuaduxie recipe contains 31 kinds of Ginsenoside Rg3 and more than 30 kinds of flavonoids.PartV:Effects of Ginsenoside Rg3,one of the main active components of Yunhuaduxie recipe,on the growth of gastric cancer and the regulation of claudin-4/VEGF-C/GSK3 β pathwayObjective:Ginseng is one of the main components of Yunhua duxie prescription.Ginsenoside Rg3 is one of the main active components of ginseng.It was confirmed that Ginsenoside Rg3 was one of the chemical constituents of Yunhuaduxie recipe by ultra performance liquid chromatography time of flight high resolution mass spectrometry.To further explore the anti-tumor effect and possible mechanism of the active component of Yunhua duxie recipe.Methods:MTT assay was used to detect the proliferation of human gastric cancer cells(AGS,BGC-803,SGC-7901).Scratch test was used to detect cell migration.Western blot and qPCR were used to detect the expression of claudin-4/VEGF-C/GSK3 β pathway protein and mRNA.Results:Ginsenoside Rg3 1,2,4,8,16,32 mg/L could inhibit the growth of gastric cancer cells in different degrees for 24 and 48 h.After administration of Ginsenoside Rg3 for 24 h,the growth of SGC-7901,AGS and BGC-803 cells was significantly inhibited,and the 24-hour IC50 values were 12.48,31.53 and 13.05 mg/L,respectively.The scratch distance of BGC-803,AGS and SGC-7901 cells was significantly reduced after treated with 8 mg/L Ginsenoside Rg3 for 24 h.Compared with the negative control group,the average tumor weight of the high-dose Ginsenoside Rg3 group(20mg/kg)was significantly different from that of the negative control group.Compared with the control group,AGS,BGC-803 and SGC-7901 cells were treated with Ginsenoside Rg3 4 and 8 mg/L for 24 h.The expression levels of GSK3 β protein and mRNA in AGS,BGC803,SGC7901 cells and tumor tissues were increased with the increase of drug concentration,VEGF-C and claudin-4 were increased the expression of protein and mRNA decreased.Conclusion:Ginsenoside Rg3,one of the effective components of Yunhuaduxie recipe,has anti-gastric cancer effect and has certain regulatory effect on Claudin-4/VEGF-C/GSK3 β signaling pathway.In particular,Ginsenoside Rg3 has anti-tumor effect on human gastric cancer animal model,which further confirms the reliability of Ginsenoside Rg3 on human gastric cancer inhibition.PartⅥ:Anti-metastatic Efficacy of Traditional Chinese Medicine(TCM)Ginsenoside Conjugated to a VEFGR-3 Antibody on Human Gastric Cancer in an Orthotopic Mouse ModelObjective::The aim of the present study was to investigate the efficacy of a traditional Chinese medicine(TCM),VEGFR-3 antibody-conjugated ginsenoside Rg3 nanoemulsion(VRIN),targeting lymphangiogenesis,on the inhibition of tumor growth and metastasis in an orthotopic mouse model of human gastric cancer.Methods:An orthotopic nude-mouse model of gastric cancer was established with the red fluorescent protein(RFP)-expressing human gastric cancer cell line NUGC-4-RFP.The tumorbearing mice were treated with vehicle(0.2 ml normal saline every other day,iv),5-FU(20 mg/kg once a week,i.p.)and VRIN(1 mg/kg every other day,i.v.).Real-time fluorescence imaging was performed to assess tumor inhibition in each group.Metastasis was evaluated by open fluorescence imaging at autopsy.The expression of lymphangiogenesis-related factors VEGF-C,VEDF-D and VEGFR-3 in the tumors were analyzedby immunohistochemistry and real-time RCP.Results:VRIN and 5-FU significantly inhibited primary tumor growth as compared to vehicle control(p<0.05).However,significant inhibition of lymph-node metastasis was only found in the VRIN-treated group(p<0.05).The expression of VEGF-C,VEGF-D and VEGFR-3 in the tumor was suppressed by VRIN treatment(p<0.05).Expression of VEGF-D and VEGFR-3 in the 5-FUtreated group was not significantly increased(p>0.05).No obvious toxicity was found in VRIN-and 5-FU-treated groups.Conclusion:Lymphangiogenesis-targeted ginsenoside Rg3 immune-nanoemulsion inhibited tumor growth and reduced lymphatic metastasis by suppressing expression of VEGF-C,VEGF-D and VEGFR-3 in an orthotopic mouse model of human gastric cancer.Our study demonstrates the potential of TCM as an effective targeted treatment for metastatic gastric cancer... |
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