| BackgroundMalignant ascites is a common complication in patients with advanced colon cancer and always a difficult problem for clinical doctors.At present,western medicine mainly adopts the methods of abdominal puncture and drainage,systemic chemotherapy or combined whole-body and intraperitoneal infusion chemotherapy,but most of the patients with malignant ascites are in the late stage of tumor and have poor physique after multi-course radiotherapy and chemotherapy.Sensitivity and tolerance of re-chemotherapy are reduced,and most of the malignant ascites is bloody,which can not drain repeatedly,so that the symptoms persist and seriously affect the quality of life.Therefore,it is necessary to explore a more mild and effective treatment.Intraperitoneal perfusion with traditional Chinese medicine can avoid gastrointestinal discomfort caused by oral decoction with small side effects,it has been widely used in the clinical treatment of malignant ascites in recent years.Our team has committed to the study of intracavitary infusion of Huachansu injection in the treatment of malignant pleural and ascites for many years,and found that Huachansu injection has a certain effect on malignant pleural and ascites.Zhuang et al found that the effective rate of intracavitary infusion of Huachansu injection in the treatment of malignant serous effusion was 66.42%.Zhou et al found that the effective rate of intracavitary infusion of Huachansu injection was 42.57%for malignant pleural effusion,for malignant ascites was 50%.Yuan found that Huachansu injection had an effective rate of 75.4%for malignant ascites derived from digestive system tumors,and the curative effect was better,but there is no specific stratification discussion for patients with different types of cancer.The formation of malignant ascites is closely related to angiogenesis.Our previous basic research found that Huachansu injection could reduce the number of red blood cells in malignant ascites and make the ascites color lighter.It is speculated that Huachansu injection may interfere the formation of malignant ascites by inhibiting tumor angiogenesis.Vasculogenic mimicry(VM)is a new model of tumor angiogenesis proposed in recent years,which may promote the formation of malignant ascites together with traditional endothelial cell angiogenesis.Most of the previous studies focused on the effect of Huachansu injection on tumor angiogenesis involving endothelial cells,while few studies explored the effect of Huachansu injection on the formation of VM.ObjectivesClinical research:To determine the efficacy of intraperitoneal infusion of Huachansu injection in the treatment of malignant ascites of colon cancer and analysis the characteristics of the benefit population,in order to provide more individual clinical guidance for intraperitoneal infusion of Huachansu injection in the treatment of malignant ascites of colon cancer.Experimental research:Taking VM as a new breakthrough point and explore the mechanism of Huachansu injection on VM formation of HCT116 cells in vivo and in vitro,so as to explain the mechanism of intraperitoneal infusion of Huachansu injection on malignant ascites of colon cancer from the point of view of tumor angiogenesis.more comprehensively.MethodsClinical research:The clinical data of 135 patients with malignant ascites of colon cancer treated by intraperitoneal infusion of Huachansu injection in the Department of Oncology,Oriental Hospital of Beijing University of traditional Chinese Medicine from January 1,2010 to December 31,2020 were collected.The curative effect was evaluated from the control rate of ascites volume,the improvement rate of ascites quality,the improvement of KPS score and the survival time of the patients.The safety was evaluated at the same time.Further compare the effects of different factors(such as the characteristics of primary tumor,metastasis,overall and local TCM syndrome differentiation,combined with systemic treatment,etc.)on the curative effect,select the effective cases,and summarize the characteristics of the benefit population.Experimental research:(1)The model of bloody ascites of colon cancer in nude mice was established by in situ inoculation of HCT116 cells into abdominal cavity and spleen,and the general signs,body weight,abdominal girth,ascites volume,number of red blood cells in ascites and the weight of abdominal metastatic tumor before and after modeling and intervention of Huachansu injection were observed.(2)The hypoxia model of HCT116 cells was established by CoCl2 chemical induction in vitro,and the effects of hypoxic microenvironment and Huachansu injection on the proliferation,migration and invasion of HCT116 cells were detected by CCK-8 test,cell scratch test and Transwell test.(3)The three-dimensional culture of Matrigel stromal glia was used to establish the VM model of HCT116 cells in vitro.PAS-CD31 histochemistry and immunohistochemistry were used to show the formation of VM HCT116 cells in vivo.The number of VM formation was counted under microscope to observe the effects of hypoxia microenvironment and Huachansu injection on VM formation of HCT116 cells in vivo and in vitro.(4)RT-qPCR and Western-blot experiments were used to detect the effects of hypoxia microenvironment and Huachansu injection on the expression of HIF-1α,VEGF,MMP2,MMP9,VE-cadherin mRNA and proteins related to VM formation in HCT116 cells in vivo and in vitro.ResultsClinical research:(1)A total of 135 patients were enrolled.Evaluation of the curative effect of ascites:the abdominal girth after perfusion was significantly lower than before(P<0.01).2 cases complete remission,25 cases partial remission,56 cases stable,total 83 cases effective,52 cases ineffective,the total effective rate was 61.5%.(2)Evaluation of curative effect of ascites quality:after perfusion,the number of red blood cells in ascites,tumor markers in ascites and the level of LDH in ascites decreased significantly as compared with those before perfusion(P<0.01),and the number of red blood cells in ascites decreased by more than 25%compared with that before treatment in 94 cases,with a total effective rate of 74.0%.The tumor markers decreased by more than 25%compared with those before treatment in 70 cases,with a total effective rate of 55.1%,the levels of CEA,CA199 and CA724 decreased significantly,and the level of ferritin had no significant difference compared with that before perfusion(P>0.05).50 cases of ascites lactate dehydrogenase decreased by more than 25%compared with before treatment,with a total effective rate of 39.4%.(3)Evaluation of curative effect of KPS score:The KPS score after perfusion was significantly higher than that before treatment(P<0.01),41 cases were higher than before treatment,82 cases were stable than before treatment,and 12 cases were lower than that before treatment.(4)Evaluation of curative effect of survival time:As of the last follow-up,2 of the 135 patients were still alive,the survival time of was 1～31 months,the average survival time was 5.66±4.59 months,and the median survival time was 4.00 months.The 1-year survival rate was 9.6%,the 2-year survival rate was 3.7%,and no one with more than 3 years survival was found.(5)Safety evaluation:Adverse reactions occurred in 27 cases(20.0%).The main adverse reactions were abdominal pain(10 cases),fever(11 cases),nausea and vomiting(3 cases)and diarrhea(3 cases).No drug-induced myelosuppression,abnormal liver and renal function and ECG changes were found,and no serious complications such as abdominal infection,intestinal obstruction and gastrointestinal bleeding were found.(6)Analysis of the characteristics of benefit population with excellent short-term efficacy:for male,drinking history,left colon,blood NLR≤2.81,diagnosis of malignant ascites,bloody ascites,systemic syndrome differentiation containing stasis and poison syndrome,systemic syndrome differentiation other than liver-kidney yin deficiency syndrome,local syndrome differentiation as damp-heat toxin syndrome,combined with systemic treatment,the effect of ascites volume control is better.The location of colon cancer,the nature of ascites,combined with systemic treatment were independent prognostic factors affecting the control rate of ascites volume.For drinking history,no liver metastasis,abdominal lymph node metastasis,no increase of bilirubin,bloody ascites,local syndrome differentiation as damp-heat toxin syndrome,combined with systemic therapy,the effect of ascites quality is better.Liver metastasis,elevated bilirubin,ascites property and local syndrome differentiation were independent prognostic factors affecting ascites color improvement rate.(7)Analysis of the characteristics of people with excellent long-term curative effect:the patients with family history,left colon,no liver metastasis,no brain metastasis,less than 2 metastatic sites,no increase of LDH in blood before perfusion,diagnosis of malignant ascites at first diagnosis,systemic differentiation of non-liver and kidney yin deficiency syndrome,no adverse reactions and ascites volume control,the survival time of ascites was longer,but compared with the results of external studies,the survival time of ascites was longer.Experimental research:(1)HCT116 cells were inoculated into abdominal cavity and spleen in situ could establish a stable BALB/C model of colon cancer bloody ascites in nude mice.Three-dimensional culture with Matrigel stromal glia could establish a VM model of HCT116 cells in vitro.(2)Intraperitoneal injection of Huachansu injection can inhibit the formation of bloody ascites,reduce the number of red blood cells in ascites and inhibit the formation of abdominal metastases of colon cancer.(3)Hypoxia microenvironment can promote the migration and invasion of HCT116 cells and enhance the ability of VM formation in vitro.(4)Huachansu injection could reverse the adverse effects of hypoxia on HCT116 cells and inhibit the proliferation,migration,invasion and VM formation in vivo and in vitro.(5)Hypoxia microenvironment could up-regulate the expression of HIF-1α,VEGF,MMP2,MMP9,VE-cadherin mRNA and protein in HCT116 cells.Huachansu injection could inhibit the expression of HIF-1α,VEGF,MMP2,VE-cadherin mRNA and protein,but had no significant effect on MMP9 mRNA and protein.ConclusionsClinical research:(1)Intraperitoneal infusion of Huachansu injection could effectively inhibit the production of malignant ascites of colon cancer,delay the progression of the disease,reduce the number of red blood cells in ascites,increase KPS score,improve the quality of life of patients,and showed good safety.(2)The patients with left colon cancer,no liver metastasis,no increase of bilirubin,bloody ascites,local syndrome differentiation with dampness-heat toxin syndrome,combined with systemic TCM treatment achieved better effect in the treatment of malignant ascites of colon cancer.Through the preliminary screening of population characteristics,the clinical effect could be improved.Experimental research:The mechanism of inhibitory effect of Huachansu injection on bloody ascites of colon cancer may be related to its reversal of tumor hypoxia microenvironment and down-regulation of HIF-1α,VEGF,MMP2,VE-cadherin mRNA and protein expression to inhibit the formation of VM in colon cancer cells in vivo and in vitro,as well as the proliferation,migration and abdominal invasion of colon cancer cells. |
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