Regulatory effect of TZT on Christensenella minuta-TαMCA-FXR/TGR5 axis based on a high-glucose human colorectal adenocarcinoma cell model
Objective To explore the regulatory effect of Tangning Ziyabitus tablets(TZT)on the Christensenella minuta-taurine-α-muricholate(TαMCA)-farnesoid X receptor(FXR)/G protein-coupled receptor 5 axis based on a high-glu-cose human colorectal adenocarcinoma cell model.Methods The liquid medium for strains,high-glucose medium,TZT solution and TαMCA solution were prepared.The strains were cultured.Inactivated Christensenella minuta and its fermenta-tion broth were prepared.Human colorectal adenocarcinoma cells(Caco-2 cells)were routinely cultured.Some cells were randomly divided into the control group,the inactivated bacteria group,106 CFU/mL live bacteria group,107 CFU/mL live bacteria group,108 CFU/mL live bacteria group and 109 CFU/mL live bacteria group,respectively.The control group was cultured with sterile Caco-2 special medium.The inactivated bacteria group was intervened with inactivated Christensenella minuta bacterial suspension.The 106 CFU/mL live bacteria group,107 CFU/mL live bacteria group,108 CFU/mL live bac-teria group and 109 CFU/mL live bacteria group were intervened by adding 2 mL of 109 CFU,108 CFU,107 CFU and 106 CFU of live Christensenella minuta bacteria to the wells of the culture plate containing completely differentiated Caco-2 cells,respectively.Some cells were randomly divided into the control group and fermentation broth group.The control group was cultured with sterile Caco-2 special medium,and the fermentation broth group was intervened with the fermenta-tion broth of Christensenella minuta.Some cells were randomly divided into the control group,high-glucose group and low-,medium-,medium-to-high-and high-dose TZT groups.Except the control group,the other groups were intervened by add-ing 8 g/L high-glucose medium for 24 h.The low-,medium-,medium-to-high-and high-dose TZT groups were intervened by adding 10,25,50 and 100 μg/mL TZT drug-containing medium for 24 h.Some cells were randomly divided into the con-trol group,25 μmol/L TαMCA group and 50 μmol/L TαMCA group,respectively.The latter two groups were intervened by adding 25 and 50 μmol/L TαMCA-containing medium for 24 h.The expression levels of FXR,TGR5,IL-8 and IL-10 mRNA were detected by real-time fluorescent quantitative PCR,and the expression levels of FXR and TGR5 proteins were detected by Western blotting.Results Compared with the control group,the 106 CFU/mL live bacteria group,107 CFU/mL live bacteria group,108 CFU/mL live bacteria group and 109 CFU/mL live bacteria group had higher levels of TGR5 mRNA expression(all P<0.05),while there were no statistically significant differences in the FXR,IL-8 or IL-10 mRNA expression levels(all P>0.05).Compared with the control group,the bacterial fermentation broth group had higher expres-sion of FXR mRNA(all P<0.05),and there was no statistically significant difference in the expression of TGR5 mRNA(P>0.05).Compared with the control group,the high-glucose group had higher expression of FXR mRNA(P<0.05),and lower expression of TGR5 mRNA(P<0.05);there were no statistically significant differences in the expression levels of FXR or TGR5 proteins(all P>0.05).Compared with the high-glucose group,each TZT group had lower expression of FXR mRNA(all P<0.05),and higher expression of TGR5 mRNA(all P<0.05);there were no statistically significant differences in the expression levels of FXR or TGR5 proteins(all P>0.05).Compared with the control group,in the 25 μmol/L TαMCA group,the expression levels of FXR mRNA and protein were lower(both P<0.05),and the expression levels of TGR5 mRNA and protein were higher(both P<0.05);in the 50 μmol/L TαMCA group,the expression of FXR protein was lower(P<0.05).Conclusions Christensenella minuta has a certain anti-inflammatory effect.TZT may promote the growth of Christensenella minuta and produce metabolites that affect bile acid metabolism,promote the accumulation of TαMCA in the intestine,further inhibit the expression of intestinal FXR and promote the expression of TGR5.