Study On The Damage Of Colon Barrier Function By Lung Adenocarcinoma And The Role And Mechanism Of TMAO In Promoting The Progression Of Lung Adenocarcinoma

Objective: The aim of this study was to clarify the changes of colon permeability and microenvironment in lung cancer mice,explore the influence and mechanism of TMAO on the development of lung adenocarcinoma,and further understand the lung-to-intestinal axis interaction in the occurrence and development of lung cancer,providing a new experimental basis for the clinical systemic treatment of lung cancer.Methods: 6-week-old C57BL/6J male mice were injected through the tail vein with LLC tumor cells,and samples were collected at 14 and 30 days after inoculation.Histological changes of the colon were analyzed by HE and Alcian blue staining,and relative expression of MUC2,AQP2-5 ZO-1 and inflammatory factor were analyzed by immunofluorescence assay and Quantitative real-time PCR.Colonic permeability was quantified through detecting the level of FD4 in the serum.Stool samples were collected,and microbiomes were analyzed by Miseq sequencing.We used the colon enteroids cultured in conditioned medium from Lewis to investigate the effects of lung adenocarcinoma on growth,permeability and inflammation of colon.A549 conditioned medium and Caco2 cells were used to simulate the environment of lung cancer in vitro.The growth of Caco2 cells treated with A549 conditioned medium was observed dynamically and stereoscopically by matrigel suspension.The transmembrane resistance of Caco2 cells co-cultured with different concentrations of A549 conditioned medium was measured by EVOM3,and its permeability was evaluated.The mouse lung cancer model was established by tail vein injection and subcutaneous injection of LLC cells,and every 4 mice were randomly divided into a group.Tumor bearing mice were divided into TMAO group and control group according to their drinking water.And according to mice types and drinking water,subcutaneous tumor bearing mice were divided into control group,TMAO group,DT+DTR group and DT+DTR+TMAO group.By observing the growth of lung tumor in vein group and the volume and weight of subcutaneously transplanted tumor in subcutaneous tumor-bearing group,the effects of TMAO and clearance of macrophages on tumor growth of lung adenocarcinoma and its potential mechanism were analyzed.PCR amplification was used to determine the expression of Sry in LLC cells and the liver of male and female mice,and to identify the gender origin of LLC cells.The effects of Brd U labeling on the proliferation and migration of LLC cells were evaluated by CCK8 test and scratch test respectively.The proportion and fluorescence intensity of Brd U positive LLC cells in each experimental group were counted after immunofluorescence staining.LLC cells and Brd U-treated LLC cells were subcutaneously injected into the upper and lower back of the same mouse.The effect of Brd U labeling on the proliferation of LLC cells in vivo was observed.Brd U-labeled LLC cell suspension was slowly injected into the tail vein of mice,and the samples were taken at 1 day,2 days and3 days respectively to explore the persistence of Brd U labeling of LLC cells in vivo.The cell fragment suspension prepared by was slowly injected into the tail vein of mice in the cell fragment inoculation group,and the samples were taken at 1 day,2 days and 3days after injection to study the non-specific uptake of Brd U in mice.Results: Here we found that mice with advanced lung adenocarcinoma had shorter colon,lighter cecum and thicker submucosa.Compared with control mice,the quantity and volume of goblet cells,the relative expression of MUC2,AQP2-5,TJP1 in tumor-bearing mice were reduced significantly,which were reversed at the late stage,causing the corresponding permeability change.In addition,Miseq sequencing from the lung adenocarcinoma group showed that the ratio of Firmicutes/Bacteroidota were descending,and the abundance of flora associated with inflammation such as Clostridia,Deferribacteres and Proteobacteria were increased.Furthermore,lung cancer upregulated the m RNA levels expression of IL1β,i NOS,TNFα,downregulated the relative expression of antiinflammatory cytokine(IL4,IL10,IL13),whereas it reversed these variations in the advanced stages.In addition,we confirmed the promoting effect of lung tumor metabolites on colon growth at the cellular and organoid level,and the changes of permeability and inflammation were also verified at the cellular and organoid level,respectively.In the in vitro cell model,subcutaneous tumor-bearing model and intravenous tumor-bearing model,we observed that TMAO,a metabolite of intestinal flora,could promote the growth of lung adenocarcinoma.In the lung adenocarcinoma mouse model established by DTR transgenic mice,it was proved that the lung tumor load of mice was higher after macrophage clearance.LLC cells were derived from male C57 mice.10 μ M Brd U treatment for 24 hours could effectively label LLC cells and the inhibitory effect on their activity was relatively insignificant.The effective labeling effect could be maintained for 3 days in vitro and 24-48 hours in vivo.Conclusion: Lewis lung adenocarcinoma changes the structure of colon and the composition of intestinal flora,aggravates colonic inflammation and destroys colonic permeability,which may be achieved by exocrine bodies.Changes in intestinal microenvironment may have an adverse effect on lung tumors through the "lungintestinal axis".Intestinal metabolite TMAO can directly promote the proliferation of tumor cells,and may also promote tumor effect by acting on macrophages.The specific mechanism remains to be further studied.LLC cells were derived from male C57 mice,which could be identified by detecting Y chromosome after transplantation into female mice.Brd U could successfully label LLC cells in vitro,but attention should be paid to labeling concentration and time,and this method was effective for early tracer after transplantation in vivo.