糖宁孜亚比土斯片基于高糖人结直肠腺癌细胞模型对小克里斯滕森菌-TαMCA-FXR/TGR5轴的调控作用

Regulatory effect of TZT on Christensenella minuta-TαMCA-FXR/TGR5 axis based on a high-glucose human colorectal adenocarcinoma cell model

目的探讨糖宁孜亚比土斯片(TZT)基于高糖人结直肠腺癌细胞模型对小克里斯滕森菌科-牛磺-α鼠胆酸钠盐(TαMCA)-法尼醇X受体(FXR)/G蛋白偶联受体5轴的调控作用。方法配制菌株液体培养基、高糖培养基、TZT溶液、TαMCA溶液,培养菌株,制备灭活小克里斯滕森菌及其发酵液,常规培养人结直肠腺癌细胞(Caco-2细胞)。取部分细胞随机分为对照组、灭活菌体组、106 CFU/mL活菌组、10^(7)CFU/mL活菌组、10^(8)CFU/mL活菌组、10^(9)CFU/mL活菌组,对照组用无菌Caco-2专用培养基培养,灭活菌体组用灭活小克里斯滕森菌菌体悬液干预,106 CFU/mL活菌组、10^(7)CFU/mL活菌组、10^(8)CFU/mL活菌组、10^(9)CFU/mL活菌组分别在含有完全分化的Caco-2细胞培养板孔中加入2 mL 10^(9)CFU、10^(8)CFU、10^(7)CFU、10^(6)CFU的小克里斯滕森菌活菌干预。取部分细胞随机分为对照组、发酵培养液组,对照组用无菌Caco-2专用培养基培养,发酵培养液组用小克里斯滕森菌发酵液干预。取部分细胞随机分为对照组、高糖组及TZT低、中、中高、高剂量组,除对照组外其他各组加入8 g/L高糖培养基干预24 h,TZT低、中、中高、高剂量组分别加入10、25、50、100μg/mL的TZT含药培养基干预24 h。取部分细胞随机分为对照组、25μmol/L TαMCA组、50μmol/L TαMCA组,后两组换入25、50μmol/L的含TαMCA培养基干预24 h。实时荧光定量PCR法检测FXR、TGR5、IL-8、IL-10 mRNA,Western blotting法检测FXR、TGR5蛋白。结果与对照组比较,10^(6)CFU/mL活菌组、10^(7)CFU/mL活菌组、10^(8)CFU/mL活菌组、10^(9)CFU/mL活菌组TGR5 mRNA表达高(P均<0.05),FXR、IL-8、IL-10 mRNA表达差异无统计学意义(P均>0.05)。与对照组比较,菌发酵液组FXR mRNA表达高(P均<0.05),TGR5 mRNA表达差异无统计学意义(P均>0.05)。与对照组比较,高糖组FXR mRNA表达高(P<0.05),TGR5 mRNA表达低(P<0.05),FXR、TGR5蛋白表达差异无统计学意义(P均>0.05)。与高糖组比较,各TZT组FXR mRNA表达低(P均<0.05),TGR5 mRNA表达高(P均<0.05),FXR、TGR5蛋白表达差异无统计学意义(P均>0.05)。与对照组比较,25μmol/L TαMCA组FXR mRNA、蛋白表达低(P均<0.05),TGR5 mRNA、蛋白表达高(P均<0.05);50μmol/L TαMCA组FXR蛋白表达低(P<0.05)。结论小克里斯滕森菌具有一定的抗炎效果,TZT可能通过促进小克里斯滕森菌的生长,产生代谢产物影响胆汁酸代谢,促进TαMCA肠道内累积,进一步抑制肠FXR表达,促进TGR5表达。

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,10^(6)CFU/mL live bacteria group,107 CFU/mL live bacteria group,10^(8)CFU/mL live bacteria group and 10^(9)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,10^(7)CFU/mL live bacteria group,10^(8)CFU/mL live bac⁃teria group and 10^(9)CFU/mL live bacteria group were intervened by adding 2 mL of 10^(9)CFU,108 CFU,10^(7)CFU and 10^(6)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 100μg/mL TZT drug-containing medium for 24 h.Some cells were randomly divided into the con⁃trol group,25μmol/L TαMCA group and 50μmol/L TαMCA group,respectively.The latter two groups were intervened by adding 25 and 50μmol/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 10^(6)CFU/mL live bacteria group,10^(7)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 25μmol/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 50μmol/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.