| Background and Objects:Atmospheric particulate matter is one of the most complex and harmful pollutants in the environment.At present,particulate matter pollution in the atmosphere has become a global health problem,and about 2.9 million people die from atmospheric particulate matter(PM)pollution every year.Studies have shown that inhalable particles can cause diseases in the respiratory system,cardiovascular system,nervous system,reproductive system,immune system,and even induce cancer and gene mutations.However,the source,composition,and harm to human health of PM of different particle size are also different.PM in the atmosphere is quickly cleared from the lungs into the intestines by the mucociliary transport of the respiratory tract.Meanwhile,PM can directly enter the intestines by polluting food and water sources,thereby causing intestinal diseases.Therefore,the digestive tract is one of the potential target organs of PM expousre.From the perspective of protecting health,studying the damaging effects of atmospheric particulates on the human intestine and its molecular mechanism will help to fully understand the toxic effects of PM and explore potential diseases prevention strategy.This study first explored the damage effect of PM long-term exposure on murine colonic epithelia,and used the mouse colon tumor model driven by chronic colitis as a positive Control to study the molecular mechanism of PM long-term exposure involved in the formation of colon tumors.Combined with RNA-seq high-throughput sequencing analysis,it was found that the differentially expressed molecules and phosphatidylinositol-3 kinase(PI3K)/AKT pathway of human colonic mucosal epithelial cells exposed to long-term PM were activated.The upstream driving factors of PI3K/AKT pathway,fibroblast growth factor receptor 4(FGFR4),was knocked down to explore the mechanism of PM exposure in the inflammation and tumor formation of colorectal cancer.We further further evaluated the role of dietary supplements in mouse colorectal injury.Our results suggested the role of PM exposure played in promoting the tumorigenesis of colorectal cancer,and potential nutritional supplements,providing a new preventive intervention for colorectal cancer.Methods:1.Long-term exposure of atmospheric particulates induced mouse colonic epithelium damage1.1 To observe the damage effects of long-term PM exposure on mouse colon,the mice were exposed to PM continuously for 12 months,and the colorectal was examined at the end of each month.The macroscopic view of the colonic tissues has been recorded,then the intact colon tissue was immersed in 4%paraformaldehyde(PFA)solution in a“Swiss-roll”style,and the colon tissue section was stained with H&E to observe the pathological changes.Alician blue and Periodic Acid-Schiff(PAS)staining were used to detect the expression of acid mucin(sulfated and carboxylated)and neutral mucin in goblet cells of mouse colonic epithelium.1.2 Enzymelinked immunosorbent assay(ELISA)experiment to detect the levels of pro-inflammatory cytokines in mouse serum;combined with Quantitative realtime polymerase chain reaction(q RT-PCR)analysis and immunohistochemistry(IHC)staining to discover and verify the expression of key molecules in the colorectal inflammation pathway.2.Long-term exposure to atmospheric particulates promotes the occurrence and development of colorectal cancer2.1 To explore the effect of long-term PM exposure on the tumor proliferation ability of colonic epithelia,100?g/m L PM was used to continuously treat normal human colonic mucosal epithelial cells(NCM460)for 48-72 hours per generation for 30 generations.The Transwell experiments were used to evaluate the migration and invasion abilities of NCM460 cells after long-term PM exposure,and the colony formation experiment was used to analyze the changes in the colony forming ability of NCM460 cells exposed to PM for a long time.A nude mouse subcutaneous tumorigenesis model was constructed.The Control group NCM460 cells and long-term PM-treated NCM460 cells were injected subcutaneously into nude mice to detect the tumorigenesis ability of the cells in vivo;the luciferase biochemical method was used to evaluate the metastatic potential of flanking tumors in vivo.To construct a mouse lung metastasis model,mice were injected with the Control group NCM460 cells and long-term PM-treated NCM460 cells through the tail vein,and the mouse lung tissue was collected to observe the mouse lung metastasis.2.2 We constructed a chronic colitis-drived colorectal cancer model.A total of 5 groups have been devided as follow:clean air(filtered room air,FRA)/Vehicle group,FRA/azoxymethane(AOM)group,PM/Vehicle group,PM/AOM group,and AOM/Dextran Sulfate Sodium Salt(DSS)positive Control group.After intraperitoneal injection of AOM or Vehicle,the mice were exposed to PM or FRA for 12 consecutive months.During this period,the weight of the mice was recorded,and the diet and stool conditions of the mice were observed;the mouse colonic tissues were dissected and collected for histopathological examination.Ki67immunohistochemical staining was used to count cell proliferation and explore the effect of PM exposure on the occurrence of colorectal cancer.3.The molecular mechanism of atmospheric particulate matter exposure on the malignant transformation of colonic epithelial cells3.1 In order to explore the differential gene expression profile induced by long-term PM exposure in NCM460 cells,the total RNA of Control and PM-exposed NCM460 cells was extracted for RNA-seq analysis.The cut-off was set as fold change(FC)>1.5 and P-value<0.05.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis found that 22 differentially expressed genes were significantly enriched in the PI3K/AKT pathway.Combined with the TCGA database to retrieve the expression of related differentially expressed genes in colorectal cancer tissues and normal tissues;q RT-PCR experiments were used to verify the differential expressed gene levels in Control and PM-exposed NCM460 cells.Western Blot(WB)was further used to verify the protein expression levels of PI3K/AKT pathway genes.Use DAVID Bioinformatics Resources 6.8 for protein interaction analysis and immunoprecipitation experiments to explore the potential interaction relationship between FGFR4 and PDGFB.Using the mouse colonic tumor model constructed in Chapter 1.3,IHC was employed to detect the expression levels of FGFR4 and p-AKT in the colon tissue of the AOM+/PM-and AOM+/PM+groups of mice to explore the activation of PI3K/AKT pathway in PM exposure and its role in the development of colorectal cancer.3.2 Lentiviral infection was further used to establish stable FGFR4 knockout NCM460 cells.Wild-type and stable FGFR4 knockout NCM460 cells were treated with PM for 30 generations.The Transwell method was used to detect the migration and invasion ability of the two NCM460cell grouops.A nude mouse subcutaneous tumor model and a mouse lung metastasis model were constructed,to evaluate the role of FGFR4 on the formation of subcutaneous and lung metastases in mice.4.Curcumin supplements prevented against atmospheric particulate matter exposure-induced colon inflammation4.1 The dietary supplements,including Curcumin,DHA,1?25(OH2)D3,were used to treat NCM460 cells exposed to PM for a long time,and the expression levels of FGFR4,PDGFB,CASP9 and SGK1 in the cells were detected by q RT-PCR experiment.4.2 Wild-type C57BL/6 mice were fed with diet containing 0.2%(W/W)Curcumin,and continuously exposed to PM for a total of 12 months.At the end of 3rd,6th,9th,and 12th months,mouse colon tissues were collected.H&E,PAS,and alcian blue staining were used to detect the effect of Curcumin treatment on the colon injury of mice exposed to PM.q RT-PCR experiment and IHC staining were used to detect the expression levels of p65 and FGFR4 in the colon epithelium of mice in the Vehicle/PM group and Curcumin/PM treated group at both messenger RNA(m RNA)and protein levels.Results:1.Long-term exposure of atmospheric particulates induced mouse colonic epithelium damage1.1 After exposure to PM for 1 month,sporadic epithelial damage appeared in the murine colorectal tissues;after 3 months of exposure to PM,more than half of the mice showed sporadic colorectal damage,such as epithelial lesions and inflammatory infiltrations.After 12months of PM exposure,epithelial damage and inflammatory cell aggregation were observed in the colon and rectum of all PM-exposed mice.Alician Blue and PAS staining showed a decrease in acid mucin and neutral mucin in the colonic mucus.The results of epithelial damage score and inflammatory cell infiltration score indicate that PM exposure significantly increases colorectal damage.1.2 Compared with the mice exposed to filtered room air(FRA),the levels of pro-inflammatory cytokine IL-1?in the serum of mice exposed to PM for 2 months or 10 months were significantly increased(P<0.05).The results of q RT-PCR analysis showed that,compared with the Control group,the key molecules(Stat3 and p65)in the colorectal inflammation pathway were significantly up-regulated(P<0.05)after the mice were exposed to PM for 3months and 12 months.However,the results of IHC staining indicated that in mouse colonic epithelium,only p65 expression increased at the protein level(P<0.05).2.Long-term exposure to atmospheric particulates promotes the occurrence and development of colorectal cancer2.1 Construct a malignant transformed human colonic epithelial cell(NCM460)model following long-term PM exposure.Compared with the Control group,long-term PM exposure can significantly enhance the migration and invasion abilities of NCM460 cells(P<0.01);the colony formation experiment results show that the colony-forming ability of NCM460 cells exposed to PM for a long time increases significantly(P<0.001).The results of in vivo tumor formation experiments showed that the mice in the NCM460 cell group exposed to PM under subcutaneous injection developed subcutaneous tumors,while the Control group cells injected nude mice did not find any obvious tumor formation.The results of luciferase assay showed that,compared with the Control group,the luciferase activity in the liver and lung of the mice after PM exposure was significantly increased(P<0.01),indicating that the load of liver and lung metastasis increased.In the mouse lung metastasis model,macroscopic and H&E staining results of lung tissue sections confirmed tumor formation in the lungs of mice injected with PM-exposed NCM460 cells through the tail vein.The above results indicate that long-term PM exposure can improve the tumorigenesis capability and metastasis ability of NCM460 cells.2.2 The results of chronic colitis-driven tumor progression model showed that 3 months after the mice were injected with AOM,AOM/DSS-induced colon tumor formation was observed.Compared with the Vehicle/FRA group,AOM/FRA group and the Vehicle/PM group,the tumor growth rate of the mice in the AOM/PM treatment group was accelerated,and the tumor formed at the 6th months after treatment.Compared with the Vehicle/FRA group,the length of the colon tissue of the mice treated with Vehicle/PM,AOM/PM,and AOM/DSS was significantly shorter(P<0.05);compared with the Vehicle/PM group and the Vehicle/FRA group,the AOM/The overall survival rate of the PM group mice decreased and the number of tumors increased(P<0.05);the histopathological results of the tumors in the AOM and AOM/PM treatment groups were similar.However,an increase in the number of Ki67+cells was observed in tumor tissues induced by AOM/PM(P<0.05).The above results suggest that PM exposure can accelerate the occurrence of colon tumors induced by AOM.3.The molecular mechanism of atmospheric particulate matter exposure on the malignant transformation of colonic epithelial cells3.1 RNA-seq results showed that there was a total of 496 differentially expressed m RNAs between Control and PM exposure group,of which 237 were up-regulated and 259 were down-regulated.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis results showed that 22 differentially expressed genes were enriched in PI3K/AKT pathway(P=0.011).In addition,the data retrieved from the TCGA database showed that the expression levels of FGFR4 and platelet-derived growth factor B(PDGFB)in colorectal cancer tissues were significantly higher than those in normal tissues(P<0.001),and Cysteincontaining aspartate specific protease 9(CASP9)and Serum/Glucocorticoid Regulated Kinase 1(SGK1)expression levels in colorectal cancer tissues were significantly lower than normal tissues(P<0.001).The q RT-PCR assay verified the expression levels of these 4 genes in the Control or PM-exposed NCM460 cells,which was consistent with the RNA-seq results.WB results showed that,compared with the Control group,the expression levels of FGFR4,PDGFB and p-AKT in PM-exposed NCM460 cells were significantly increased(P<0.05),while the expression level of SGK1 was significantly reduced(P<0.05),indicating PI3K/AKT The signal pathway is activated.Protein interaction analysis and Co-immunoprecipitation(Co-IP)assay suggested that there was a potential interaction between FGFR4 and PDGFB,and the former played a major role in the interaction.Further in vivo research results showed that compared with adjacent normal tissues,the expression levels of FGFR4 and p-AKT were significantly higher in AOM/PM-induced mouse colorectal cancer tissues(P<0.01).It is suggested that the activation of PI3K/AKT pathway plays a key role in the occurrence of CRC tumors induced by PM exposure.3.2 Construction of FGFR4 stable knockdown NCM460 cells and followed long-term PM exposure,wild-type NCM460 cells were used as a Control.The results of Transwell experiment found that compared with wild-type NCM460 cells,the migration,invasion abilities and the tumor formation ability of subcutaneous and lung tumors of NCM460 cells stably knocked down FGFR4 were significantly inhibited(P<0.05).The above results indicate that FGFR4 is an upstream driving factor that triggers PM-induced tumor formation in vivo and in vitro.4.Curcumin supplements prevented against atmospheric particulate matter exposure-induced colon inflammation4.1 Using the long-term PM exposed NCM460 cell model,we tested the anti-inflammation capacities of long-chain fatty acids(DHA)rich in omega-3,vitamin D(1?25(OH2)D3)and turmeric,respectively.The results showed that,compared with the PM exposure group,20×10-6M Curcumin could effectively restore the gene expression levels of FGFR4,PDGFB,CASP9and SGK1 in NCM460 cells to the Control levels.While DHA and 1?25(OH2)D3 partially restored gene expression levels.Therefore,Curcumin was chosen for subsequent in vivo studies.4.2 The histopathological results of a long-term PM exposure mouse model showed that oral Curcumin supplementation can alleviate the colon inflammation induced by PM exposure in mice.The expression levels of p65 and FGFR4 m RNA and protein in the colon mucosa of the PM/Curcumin treatment group were reduced,compared with the Vehicle/PM treatment group.The above research results confirm that oral Curcumin can improve colorectal inflammation induced by PM exposure.Conclusions:1.PM exposure promoted tumorigenesis of colorectal cancer induced by the chemical carcinogen AOM.2.PI3K/AKT pathway activation plays a key role in the process of PM accelerating the formation of colorectal cancer.FGFR4 is an up-stream driver of PI3K/AKT pathway.3.Dietary supplementation with Curcumin can protect mice againist inflammatory damage in the colon caused by PM exposure. |
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