The Study Of Gut Microbiota And Enteric Neurotransmitters Influence Colorectal Cancer Liver Metastasis By Inducing Polarization Of Kupffer Cells In Liver

Part one Enteric neurotransmitter affects colorectal cancer liver metastasis in view of gut microbiota Objective:1.To investigate the effect of gut microbiota regulates colorectal cancer（CRC）liver metastasis and hepatic Kupffer cells（KCs）;2.To explore whether gut microbiota influence CRC liver metastasis and hepatic KC levels are related to the change of enteric neurotransmitter noradrenaline（NE）or vasoactive intestinal peptide（VIP）.Methods:Vivo experiment:40 SPF male BALB/c mice were randomly divided into Control group（received sterile drinking water）;Vanc group（received 0.25mg/m L vancomycin solution）;Coli group（received 2mg/m L colistin solution）and ASC group（received a mix of 1mg/m L ampicillin,5mg/m L streptomycin and 1mg/m L colistin）.After 2 weeks of feeding,the antibacterial efficacy of antibiotics was evaluated;a model of CRC liver metastasis was made via splenic inoculation of colon cancer 26（CT26）cells.Continue to feed antibiotics until to the end of experiment（19 days）.The liver weight,volume was measured and the hepatic metastatic nodules were counted to observe the effect of gut microbiome on CRC liver metastasis.Gene sequencing of 16S r DNA was performed using mouse fecal pellets collected after two weeks of antibiotic administration.KCs and cytokines in liver were examined by immunohistochemistry.The changes of enteric neurotransmitters in different antibiotic groups were observed by enzyme-linked immunosorbent assay（ELISA）and immunofluorescence.Results:1.Gut microbiota alteration had an effect on the CRC liver metastasis.Among the groups that treated with different antibiotics,the lowest number of liver metastases were ASC group,followed by Coli group;the greatest number of liver metastases was Vanc group.Colistin treatment could inhibit CRC liver metastasis,however,vancomycin treatment promoted CRC liver metastasis（P=0.041,P=0.028）.Analysis of 16S r DNA sequencing was revealed that rich and diversity of bacteria in Coli group were superior to Vanc group;at species level,the predominant bacteria,Parabacteroides.goldsteinii,Bacteroides.vulgatus,Bacteroides.uniformis and Bacteroides.thetaiotaomicron,etc.were performed in Coli group,on the contrary,Parabacteroides.distasonis and Proteus.mirabilis were enriched in Vanc group.2.The change of serum neurotransmitters was selected in different antibiotic groups,and norepinephrine（NE）or vasoactive intestinal peptide（VIP）level found that was coincidence with the results of liver metastasis.The immunofluorescence results showed that NE or VIP was expressed in the enteric nervous system（ENS）,NE contents raised obviously in Vanc group,while VIP contents increased remarkably in Coli and ASC group.Pearson correlation analysis performed serum NE level was negative correlation with Proteus.mirabilis（r=0.591,P=0.043）.3.KCs was distributed in the hepatic sinusoids and central veins or portal ducts of the hepatic lobules,the contents of KCs were lower in Vanc group,while were higher in Coli group and ASC group（P=0.027,P=0.038,P=0.001）.Consistence with the results of liver metastasis nodule in different antibiotics,Proteus.mirabilis was negatively related to KCs（P=0.028,r=-0.632）,while Bacteroides.vulgatus was positively correlated with KCs（P=0.011,r=0.705）.IFN-g,IL-2 were distributed in hepatocyte cytoplasm,the contents of IFN-gwere less in Vanc group than that in Coli group and ASC group（P=0.011,P=0.001）.Conclusions:1.Gut microbiota alteration could be one of factor to influence the CRC liver metastasis.Proteus.mirabilis,Parabacteroides.distasonis were increased,Parabacteroides.Goldsteinii,Bacteroides.vulgatus,Bacteroides.thetaiotaomicron,Bacteroides.uniformis were decreased in the gut,which might be important pathogenic bacteria causing CRC liver metastasis.2.The changes of gut microbiota might affect the secretion of VIP or NE.P.mirabilis was positive with NE levels.3.The gut microbiota alteration might be related to the content of KCs and the level of INF-g.Increasing of Proteus.mirabilis could inhibit hepatic KCs expression,while amount of Bacteroides.vulgatus promoted KCs levels.Part two Proteus.mirabilis and Bacteroides.vulgatus influence colorectal cancer liver metastasis and its hepatic kupffer cellsObjective:To confirm Proteus.mirabilis（P.mirabilis）and Bacteroides.vulgatus（B.vulgatus）affect CRC liver metastasis and its hepatic KCs.Methods:1.Vivo experiment:56 SPF male BALB/c mice were administrated with broad-spectrum antibiotics through sterile drinking water containing 0.2g/L ampicillin,neomycin,and metronidazole and 0.1g/L vancomycin daily for 2weeks before the experiment.Then mice were divided into seven groups based on intragastric gavage twice weekly with 2×10⁸CFU/0.2m L P.mirabilis or heat-killed P.mirabilis,2×10⁸CFU/0.2m L B.vulgatus or heat-killed B.vulgatus,1×10⁸CFU/0.1m L P.mirabilis and 1×10⁸CFU/0.1m L B.vulgatus or1×10⁸CFU/0.1m L heat-killed P.mirabilis and 1×10⁸CFU/0.1m L heat-killed B.vulgatus,and 0.2m L stroke-physiological saline solution（control group）.After 1 week of P.mirabilis and B.vulgatus administration,a CRC liver metastasis model was established and we continued to administer P.mirabilis or B.vulgatus via gavage until the end of experiment.The liver weight,volume was measured and the hepatic metastatic nodules were counted to observe the effect of P.mirabilis and B.vulgatus on CRC liver metastasis.Hepatic KCs was examined immunohistochemistry.2.Vitro experiment:P.mirabilis and B.vulgatus was separately treated at concentrations of 1×10⁰,1×10³,1×10⁴,1×10⁵,1×10⁶,and 1×10⁷CFU/m L and cultured for 12h,24h,48h and 72h.Cell Counting Kit-8（CCK8）was used to count the cell numbers that were viable.Then,KCs treated with different concentrations of P.mirabilis or B.vulgatus at 12h,24h,48h and 72h,CT26cells were cultured in lower and upper of transwell chamber respectively,transwell analysis was applied to perform the migration of CT26 cells after24h con-culture.Results:1.Whether P.mirabilis and B.vulgatus affected CRC liver metastasis,in vivo,the numbers of liver metastases in P.mirabilis group were more than the Control group（P=0.046）.Similarly,the liver weight and volume were larger in P.mirabilis group（P=0.006,P=0.003）;In contrast,the numbers of liver metastases in B.vulgatus were less than the Control group（P=0.001）,the liver weight and volume were smaller in B.vulgatus group（P=0.031,P=0.039）.In accordance with our liver metastasis nodules data,the expression of KCs was significantly lower in liver tissues of P.mirabilis group,while,the expression of hepatic KCs obviously higher in B.vulgatus group as compared with the control group（P=0.037,P=0.047）.2.In vitro,P.mirabilis markedly inhibited KC proliferation relative to that in the control group at 12h,24h,48h and 72h（P<0.001,P<0.001,P=0.021,P=0.039）.The half maximal inhibitory doses(Log IC₅₀)of P.mirabilis at 12h,24h,48h,and 72h were 4.013,4.085,2.988,and 2.481CFU/m L respectively.Next,an increased number of CT26 cells was detected for P.mirabilis pre-treated with KCs at 12h,24h,48h,and 72h compared to that in the group pre-treated with PBS（P<0.001,P<0.001,P<0.001,P<0.001）.The Log IC₅₀of P.mirabilis-induced KC-promoted CT26 cell migration at different times was 4.700,3.886,3.986 and 4.105 CFU/m L.In vitro,compared with that in the control group,KC proliferation increased remarkably with increasing doses of B.vulgatus in dose-and time-dependent manners（P<0.001,P<0.001,P<0.033,P<0.039）.The Log IC₅₀of B.vulgatus inducing KCs proliferation at 12h,24h,48h and 72h was 6.384,6.708,2.863,and 3.113 CFU/m L,respectively.Moreover,B.vulgatus treatment ameliorated CT26 cell migration in dose-and time-dependent manners at 12h,24h,48h and 72h,with KC proliferation（P<0.001,P<0.001,P<0.001,P<0.001）.The Log IC₅₀of B.vulgatus-induced KC effects on CT26cell migration at 12h,24h,48h and 72h was 3.990,3.978,3.378,and 3.055CFU/m L,respectively.Conclusions:1.A increased P.mirabilis populations and decreased B.vulgatus populations could play key roles in CRC liver metastasis,which ultimately might be related to the exhausted number of KCs.2.P.mirabilis appeared to exhaust the phagocytotic capacity of KCs and promoted CT26 cell migration;B.vulgatus potentially had an essential role in preventing CT26 cell migration in response to KC proliferation.Part three Enteric neurotransmitter norepinephrine and vasoactive intestinal peptide influences colorectal cancer liver metastasis and its hepatic kupffer cellsObjective:To confirm the enteric neurotransmitter norepinephrine,vasoactive intestinal peptide affects CRC liver metastasis and hepatic KCs.Methods:1.Vivo experiment:30 SPF male BALB/c mice were divided into Control group,low dose of VIP group（2.5nmol）,high dose of VIP group（5nmol）,low dose of NE group（0.28nmol）and high dose of NE group（2.8nmol）.At the 3th day of CRC liver metastasis models were established with CT26 cells intrasplenic transplant,mice were injected 0.1 m L of NE or VIP every other day for a total of 8 times.The liver weight,volume was measured and the hepatic metastatic nodules were counted to observe the effect of NE and VIP on CRC liver metastasis.Hepatic KCs was examined immunohistochemistry.2.Vitro experiment:VIP and NE were treated with respectively the following concentration of KCs and divided into Control group（PBS treatment）,VIP group with concentration gradient(10^-6mol/L 10^-7mol/L,10^-8mol/L,10^-9mol/L,10^-10mol/L of VIP treatment)and NE group with concentration gradient(10^-5mol/L,10^-6mol/L,10^-7mol/L,10^-8mol/L,10^-9mol/L of NE treatment).After pre-treatment for 24h,the expressions of the subtype-KCs,CD86（M1）and CD206（M2）,were examined by flow cytometry.The secretion of cytokine from KCs was detected by ELISA.The expression of PI3K,Akt and p-Akt in KCs were reduced by NE with concentration gradient for 24h were tested by western blotting.3.Bioinformatics analysis:The gene associated with CRC liver metastasis was screened from GEO database with GSE 81980 dataset and GSE 18105 dataset,then was performed by KEGG enrichment analysis.Results:1.Among the effect of different doses of VIP or NE on CRC liver metastasis,the number of liver metastases in low and high doses of VIP were lower than Control group（P=0.025,P=0.011）,while the number of livers metastases in low and high doses of NE were more than Control group（P=0.019,P=0.032）.2.KCs treated with a concentration gradient of NE for 24h,the result performed that 10^-9,10^-8,10^-7,10^-6mol/L of VIP with concentration-dependent manner could promote CD86 expression in KCs and suppress the expression of CD206 in KCs（P<0.001,P<0.001）;Each concentration of VIP had an inhibitory effect on the secretion of TGF-b（P=0.015）.However,each concentration of NE not only inhibited CD86 expression in KCs,but also promote CD206 expression in KCs（P<0.001,P<0.001）,among which,10^-9,10^-8,10^-7mol/L of NE-induced KCs exhibited a significantly increased expression of CD206;Each concentration of NE augmented the secretion of VEGF in the KCs and inhibited the secretion of IL-6 in the KCs comparison to Control group（P=0.035,P=0.001）.3.The adrenergic receptor（AR）in KCs treated with 10^-9,10^-5mol/L of NE for 24h was tested by western blotting.The result found that NE induced the increase ofβ₂-AR in KCs.4.The gene between CRC with liver metastasis and CRC without liver metastasis samples was compared from the datasets GSE81980 and GSE18105,and the differential genes were analyzed for function and pathway enrichment analysis,the result exhibited that the occurrence of CRC liver metastasis was related to PI3K/Akt signal pathway.Compared with Control group,10^-9,10^-7,10^-5mol/L of NE induced KCs had higher PI3K expression and p-Akt expression（P=0.002,P=0.018）.Conclusions:1.Enteric neurotransmitter VIP and NE might influence the CRC liver metastasis.VIP inhibited CRC liver metastasis,while NE promoted CRC liver metastasis.2.VIP and NE could affect the polarization and secretion of KCs.VIP enhanced the polarization of KCs to M1-subtype,while NE induced the polarization of KCs to M2-subtype.3.NE combined withb₂-AR on KCs via PI3K/Akt signal pathway activating,which might result into M2-subtype KCs polarization.Part four Effect and mechanism of norepinephrine promotes colon cancer cells migration by remodeling M2-subtype Kupffer cells polarizationObjective:To explore the mechanism of enteric neurotransmitter NE influences CT26 cells migration.Methods:1.Vitro experiment:KCs were pre-treated with NE for 24h were co-cultured with CT26 cells for 24h,and transwell chamber was applied to comprehend the migration of CT26 cells.The expression of chemokines in KCs were reduced with a concentration gradient of NE was examined by RT-q PCR.2.Bioinformatics analysis:The chemokines related to macrophages in CRC were screen by TISIDB website.The influence and prognosis of high expression of CXCR4 on CRC patients were analyzed by Progno Scan website.3.Clinical research:the levels of serum NE,VIP,VEGF,TGF-b,IL-6and CXCL12 were detected by ELISA between 20 CRC patients with liver metastasis and 20 CRC patients without liver metastasis.Results:1.Among the migration of CT26 cells induced by different concentration of NE,the migration of CT26 was strongly improved by KCs treated with 10^-9,10^-8,10^-7,10^-6mol/L of NE（P<0.001）.2.Eight chemokines screened from TISIDB website were related to macrophage,10^-9mol/L of NE-induced KCs（M2-subtype）was up-regulation of the CXCL12 gene（P=0.006）,while down-regulation of the CXCL9,CCL2,CCL20,CXCL8,CXCL16,CX3CL1 and CCL17 gene（P<0.05）,were detected by RT-q PCR.3.The levels of serum NE,CXCL12,VEGF,TGF-bin CRC with liver metastasis were higher than that in CRC without liver metastasis（P=0.020,P=0.009,P=0.011,P=0.031）,while,the level of serum IL-6 in former was lower than that in latter（P=0.034）.The correlation of CXCR 4 expression and prognosis of CRC in GSE17537,GSE14333,GSE17536 and GSE12945datasets was analyzed by Progno Scan website,result showed that compared with lower expression CXCR4 in CRC patients,the OS,DFS,DSS of the higher expression CXCR4 in CRC patients were decreased（P=0.018,P=0.018,P=0.029）.Conclusions:1.M2-subtype KCs induced by NE might drive the migration of CT26cells via up-regulating CXCL12 expression.2.The levels of serum NE,CXCL12,VEGF,TGF-bin CRC with liver metastasis were higher than that in CRC without liver metastasis.Therefore,the mechanism of gut microbiota on CRC liver metastasis is complex,partly through changes of hepatic Kupffer cell contents are regulated by increased P.mirabilis and decreased B.vulgatus resulting in CRC liver metastasis;partly through secretion of noradrenaline may be induced by gut microbiota,which regulates Kupffer cell to M2-subtype Kupffer cell polarization viab₂-AR/PI3K/Akt signal pathway and cytokines secretion,further metastasizes to liver,may be associated with higher CXCL12expression in hepatic Kupffer cell;partly through secretion of vasoactive intestinal peptide is induced by gut microbiota,which controls CRC liver metastasis via promoting Kupffer cell to M1-subtype Kupffer cell polarization.