Study On Anti-Colon Cancer Effect Of Aconitum Coreanum Polysaccharide Based On Epithelial Mesenchymal Transition

Objective:Aconitum aconitum is a traditional Chinese medicine commonly used in the treatment of stroke,epilepsy,wind phlegm headache,vertigo and convulsion.Based on the isolation,structure analysis and pharmacological study of Aconitum coreanum homogeneous polysaccharides(ACP),we found that ACP has potential clinical application prospects.In this project,from the perspective of new drug development and utilization,ACPC was eluded by DEAE cellulose column with 0.2 mol / L Na Cl solution to explore the therapeutic effect and mechanism of ACP02 on inhibiting liver metastasis of colon cancer based on epithelial mesenchymal transition,and to provide reference value for the extraction,purification and clinical application of Aconitum aconitum.Methods:1.ACPC was extracted from Aconitum aconitum by distilled water extraction ethanol precipitation.The crude polysaccharide was further purified by DEAE cellulose chromatographic column to obtain ACP02.Phenol sulfuric acid test total polysaccharide content as an indicator.The sample loading volume,elution time and elution flow rate were considered as a single factor to study,and the response surface method was used to optimize the purification process.Finally,the content of single molecular weight ACP in ACP02 was determined by HPLC as the quality verification.2.The EMT model was established by inducing colon cancer cell(HT-29)with TGF-β1(10 ng / m L)for 48 hours.The effect of ACP02 on cell viability,metastasis and invasion of HT-29 were observed by toxicity test,invasion test and scratch test;The changes of EMT related marker protein and Smad pathway protein were detected by Western blot to explore the inhibitory effect of ACP02 on HT-29 cells in the process of EMT Its mechanism of action.3.By injecting fluorescently transfected HT-29 cells into the spleen,a nude mouse colon cancer liver metastasis model was established.Group and untreated group every two days 200 μL saline by intraperitoneal injection;Positive controls received 200 μL oxaliplatin(2.5 mg / m L)every 3 days intraperitoneally;Intraperitoneal injection every two days 200 μL ACP02(100 mg / kg,200 mg / m L)polysaccharide group.After 21 days,all mice were killed,dissect liver tumor tissues of all mice and collect dead serum,and the number of tumor nodules transferred from spleen to liver was observed.Western blot detects EMT associated protein,HE staining of liver tissue pathology,ELISA kits nude mouse serum TNF-α content detection.Results:1.The total polysaccharides consistence of ACPC detected by phenol sulfuric acid method was 47.83 %(RSD = 2.05 %);before the optimization of the best process of purification,the total polysaccharides consistence of ACP02 was 65.63 %(RSD =1.05 %),after the optimization of the best purification process,the total polysaccharide consistence of ACP02 was 90.05 %.The results indicated that the total polysaccharide concentration of ACP02 was greatly improved by the best extraction process;the best extraction process of ACP02 was obtained by single factor experiment and BBD response surface analysis: the sample loading was 5 g,the elution time was 70 min,and the speed of elution flow was 7 m L/min.The examination indicated that the maximum absorption peak of ACP02 was ACP,and the consistence of ACP was 94.67%(RSD =1.02 %).2.CCK-8 kit showed that ACP02 was between 0-400 μg/m L in the model of EMT induced by TGF-β1,ACP02 could significantly inhibit the invasion and metastasis of HT-29 cells by cell invasion test and scratch test;Western blot analysis showed that ACP02 could significantly inhibit the EMT process of HT-29 cells,the mechanism may be through down regulating the phosphorylation of Smad2 and Smad3.3.ACP02 could significantly inhibit the metastasis of tumor cells from spleen to liver in nude mice,and the body weight of nude mice remained unchanged during administration.The number and volume of tumor tissue in each group were observed.It was found that ACP02 could significantly reduce the number and volume of tumor tissue in nude mice.According to statistics,the tumor inhibition rates of low-dose ACP02 group,high-dose ACP02 group and positive drug oxaliplatin group were67.91%,62.60% and 75.90%.After the intervention of ACP02,the fluorescence intensity of tumor cells in spleen and liver in the drug group was obviously below to the model group(P < 0.05),and indicated a dose-dependent;Western blotting was used to investigate the changes of EMT related marker proteins in tumor tissues of nude mice.ACP02 could obviously inhibit the procedure of EMT;The results of HE staining showed that the cell morphology changed significantly after drug intervention,the cell arrangement was tight and regular,the nuclear heteromorphism decreased obviously,and the cell growth activity increased significantly;The results showed that ACP02 could obviously promote the release of TNF-α in serum of nude mice(P < 0.05).Conclusion:ACP02 was the main effective part of Aconitum aconitum polysaccharide.The experimental results showed that the best extraction process of ACP02 was stable and feasible,with high potency ratio.The proportion of homogeneous polysaccharide ACP was higher(94.67%,RSD = 1.02)%.This process can be widely used in the industrial production of anti-tumor drugs of Aconitum aconitum polysaccharide;In vitro experiments show that ACP02 may inhibit the process of EMT and inhibit the growth of tumor cells through Smad pathway;In vivo experiments show that ACP02 can significantly inhibit the liver metastasis trend of colon cancer based on EMT,and its mechanism may be in the tumor microenvironment It works by enhancing the body’s immunity.This study laid a foundation for the clinical research of Aconitum aconitum in the treatment of liver metastasis of colon cancer.