Exploring The Effects And Mechanisms Of Capsaicin On Gastric Cancer Metastasis

Background and Purposes:Diet dominates human existence and evolution.The changes of eating habits and dietary structure directly affect human evolution and health.As an indispensable condiment in Chinese daily diet,huge amounts of chili peppers are produced and then consumed every year.Therefore,it is even more significant to study influences of chili peppers on the occurrence and development of diseases.As many epidemiological studies shown,long-term and large amounts of chili peppers intake can promote the progression of gastric cancer.Patients with gastric cancer often exhibit poor prognosis and higher fatality rate compared with other digestive system tumors.Gastric cancer shows unobvious early clinical symptoms,while the tumor process will advance rapidly and endanger patients’ lives once metastasis occurs.Thus,it also enlightens us to find out those potential factors affecting gastric cancer metastasis in daily diet.In this thesis,we explored the effect of capsaicin on gastric cancer metastasis and possible molecular mechanism and biological basis through bioinformatics analysis combined with a series of in vivo and in vitro experiments.Methods and results:(1)Firstly,the promotion effect of excessive intake of capsaicin on the metastasis of gastric cancer was determined by two in vivo mouse models:in situ tumor model and tail vein metastasis model.Subsequently,a serious of in vitro experiments such as wound-healing,transwell and 3D invasion were also used to confirm the effect of capsaicin on gastric cancer metastasis.(2)Secondly,bioinformatics analysis as well as the analysis of clinical gastric cancer samples both showed that the expression level of TRPV1 was negatively correlated with the survival time of gastric cancer patients.Then,the expression of TRPV1 in several types of gastric cancer cells were confirmed by Western-blot and qPCR.Treatment with capsaicin could increase the expression of TRPV1 protein of gastric cancer cells,and promote TRPV1-mediated intracellular Ca2+flow of MKN-45 cells in calcium imaging experiment.It is indicated that TRPV1 may play a key role in the promoting effects of capsaicin on gastric cancer metastasis.(3)Thirdly,further experiments(wound-healing,transwell and 3D invasion experiment)verified that pre-incubation with TRPV1 inhibitors could reverse the promotion effect of capsaicin on gastric cancer metastasis in vitro.Then,CRISPR-Cas9 technology was applied to construct TRPV1 knock out cell line using MKN-45 cells.Similarly,in vivo and in vitro experiments both proved that the pro-gastric cancer metastasis effect of capsaicin was partially inhibited after TRPV1 knocked out.Furthermore,the correlation between TRPV1 and the motility of gastric cancer cells was found through the application of the Linked Omics database.The function of TRPV1 is closely related to cell motility by living cell imaging experiment and immunofluorescence.On this basis,through immunofluorescence,it was found that the regulation of TRPV1 on the motility of gastric cancer cells might be related to its effect on the perinuclear actin aggregation.(4)Finally,16S rDNA technology was used to sequence intestinal flora in the feces of mice fed 100mg/kg capsaicin diet for a month.The results showed that 100mg/kg capsaicin diet for a month affected the composition and abundance of intestinal microorganisms,and increased the abundance of TPH1-related bacteria,a key enzyme in the process of 5-HT synthesis.The abundance of Clostridium,Lactaobacilus and Peptostreptococcus can promote the combination of 5-HT and HTR3 A receptor on gastric cancer cells and accelerate the process of gastric cancer metastasis.Conclusion:Combined with bioinformatics analysis,clinical samples and multiple experiments in vivo and in vitro,this study confirmed from multiple perspectives that excessive intake of capsaicin is negatively correlated with the occurrence and development of gastric cancer,and that it is also correlated with the expression and function of TRPV1.In addition,Peripheral serotonin also plays an important role in the metastasis of gastric cancer.Further investigation of the mechanism showed that capsaicin could affect the progression of gastric cancer by regulating the motor ability of gastric cancer cells.This study plays a helpful role in avoiding excessive consumption of pepper and achieving the effect of more systematic and accurate treatment of malignant tumors.