Interventional Effect Of Intraperitoneal Injection Of Huachansu Injection On VEGF Expression In Bloody Ascites Of Colon Cancer

BackgroundMalignant Ascites(MA)is caused by various malignant tumors,which is caused by cancer progression or cancer metastasis.Malignant ascites can occur in 10%-15%of gastrointestinal tumors,and the prognosis of malignant ascites is poor,especially the malignant ascites derived from gastrointestinal tract.The survival time is only 12-20 weeks.The occurrence of ascites in the cancer patients not only brings great pain to the patients,but also delays the positive treatment opportunities,even causes the deficiency of nutrition and the deterioration of the condition,which makes the survival period obviously shortened.Therefore,how to effectively reduce malignant ascites and reduce the related clinical symptoms and improve the quality of life is particularly important.At present,the treatment methods of modern medicine include diuresis,abdominal puncture and drainage,intraperitoneal chemotherapy or combined hyperthermia,immunotherapy and so on.Because of its side effects and good curative effect,Chinese medicine is showing more and more advantages in cancer treatment.Cinobucine(CINO),as the main extract of dried toad skin,has been widely applied in the treatment of malignant ascites,and has a good clinical curative effect,especially for the local syndrome differentiation belonging to the damp and hot toxin syndrome one.CINO can not only effectively alleviate the occurrence and development of malignant ascites,but also make the ascites clear and reduce the number of red cells in the ascites as well.The mechanisam is possibly related to the inhibition of CINO on neovascularization.VEGF and its receptors play an important role in the formation and treatment of malignant ascites.Studies have shown that CINO can reduce the expression of VEGF and VEGFR-2 in tumor and inhibit the formation of neovascularization.Therefore,the study of the relationship between CINO and VEGF,VEGFR-2 in the malignant ascites of colon cancer is very important for making clear of the treatment mechanism of CINO on malignant bloody ascetics and improving curative effects of malignant ascites in colon cancer.Based on the above,we speculate that CINO can inhibit the expression of VEGF in malignant ascites,block the combination of VEGF and VEGFR-2,slow down the formation of neovascularization of tumor,and thus reduce the production of malignant ascites.ObjectiveTo understand the effect of CINO on the signal pathways of VEGF,VEGF-mRNA and VEGFR-2.To clarify the regulatory role of CINO on the expression of VEGF in the cell signaling pathway of colon cancer peritoneum metastasisTo explain the specific target and pathway of CINO in the treatment of colon cancer ascites.Research MethodsThis study mainly includes two parts--animal and cell experiments.using the injection of toad injection.With the intervention of CINO in animal and cell model respectively,the expression of VEGF and its receptor from the protein and gene level will be revealed,and the possible mechanism of CINO to the malignant hemorrhagic ascites of colon cancer will be explored.The animal experiment(effects of CINO on the expression of VEGF in hemorrhagic colon cancer ascites of tumor bearing mice)mainly includes the following four part:1.Establishment of the animal model:various cell types,inoculation routes and injection doses were attempted on mice,and finally the model of bloody ascites in the tumor bearing mice was set up.2.Determination of the CINO dosage:the acute toxicity test of the intraperitoneal perfusion of CINO was designed and carried out.By judging the pathological changes of the organs in the abdominal cavity of the mice after different doses,the safe dosage of the intraperitoneal perfusion of the injection was determined.3.Calculation of ascites volume:with the intervention of CINO,the Intraperitoneal ascites volume in each group was collected and calculated.4.Expression of VEGF,VEGF-mRNA and VEGFR-2 in the tumor bearing mice:ELISA was used to detect the VEGF expression level in the bloody ascites of colon cancer,while WB for the content of VEGF and VEGFR2 in mesenteric metastases of mice.qPCR was applied to manifest VEGF-mRNA and VEGFR2-mRNA in the mesenteric metastases of mice.The cell experiment(the metabolism of human umbilical vein endothelial cells by CINO)is comprised of the three parts below:1.Increment of HUVEC under the intervention of CINO:the apoptosis of HUVEC cells was detected with MTT assay,and the morphological changes of cells were observed by fluorescence microscope.2.Expressions of VEGF,VEGFR-2 and VEGF-mRNA in HUVEC under the intervention of CINO:ELISA was used to detect the content of VEGF in the supernatant of HUVEC cells and the content of VEGFR-2 on the surface of the cell membrane,while qPCR to determine VEGF-mRNA and VEGFR2-mRNA expressions in HUVEC cells.Results1.the establishment of animal models of ascites due to colon cancer mainly included two kinds--inoculation of tumor cells into the spleen and intraperitoneal inoculation of tumor cells.The first method,inoculation of tumor cells into the spleen,was better in the collection and measurement of ascites.The latter method of intraperitoneal inoculation was suitable for the study of peritoneal and intestinal metastases,which were closely related to the ascites of colon cancer.2.It was safe when the dose of intraperitoneal perfusion of CINO did not exceed 1ml.It did not cause acute necrosis of liver,spleen and kidney in mice,and did not affect the weight and diet of mice as well.3.The concentration of CINO inhibited the ascites content in abdominal cavity of tumor bearing mice in a concentration dependent manner.4.CINO failed to decrease the expression of VEGF in bloody ascites of nude mice for no difference appeared in each group(P>0.05).The expression of VEGF and VEGFR-2 in the mesenteric metastatic tumor of BALB/c mice decreased in varying degrees under the action of different concentrations of CINO,and the expression levels from high to low were low concentration group,medium group and high group,and the change was statistically significant(P<0.05).Therefore,the expression of VEGF and VEGFR-2 in mice mesenteric metastases was inhibited in a concentration dependent manner.5.The concentration of VEGF-mRNA and VEGFR2-mRNA in peritoneal metastasis of mice was decreased in a concentration dependent manner.The change was statistically significant(P<0.05).Among them,high dose of CINO had the strongest inhibitory effect on the two kinds of mRNA,followed by low dose and the lowest dose.6.CINO had obvious inhibitory effect on the growth of HUVEC cells.The inhibition rate of CINO on HUVEC was between 10-80%.The 50%inhibitory concentration(IC50)was lmg/ml,and the bestactuation duration of CINO was within 24h.In 24h,the intervention of HUVEC was dependent on time concentration.7.The expressions of VEGF and VEGFR-2 in HUVEC cells were inhibited in a concentration dependent manner,and the difference was statistically significant(P<0.05).The inhibitory effect on VEGFR-2 was higher than that of VEGF at the same dose(P<0.001).8.CINO downregulated the expressions of VEGF-mRNA and VEGFR2-mRNA in HUVEC cells,whose difference was statistically significant(P<0.05),and the inhibitory effect was concentration dependent.ConclusionThe tumor ascites mice model and human umbilical vein endothelial cell model were successfully established.In both animal and cell experiments,the downregulations of VEGF and its receptor were showed under the intervention of CINO from the gene and protein levels.Meanwhile the inhibitory effect on VEGFR-2 was higher than that of VEGF at the same dose--the inhibition of VEGFR-2 was particularly obvious.The results indicated that the treatment of the bloody ascites of colon cancer could be inclined to the downregulation of VEGF and its receptors.This study provides an experimental basis for the promotion of the peritoneal perfusion of CINO in the clinical treatment of the bloody ascites of colon cancer,andsets up a new way for the study of the antitumor mechanisms of CINO.we will continue to further verify and probe into the molecular mechanism of the effect of cinobufotalin injection against tumor angiogenesis.Validation of effects of CINO on the anti-tumor neovascularization and its molecular mechanism will be future researched.