Trimethylamine N-Oxide (TMAO) Inducing Endothelial Injury: UPLC-MS/MS-Based Quantification and the Activation of Cathepsin B-Mediated NLRP3 Inflammasome

Figure 1. Effects of TMAO on endothelial cell junction in vivo and in vitro. (A) Location of ZO-2 (green) in the endothelial cells of thoracic aorta was estimated by immunofluorescence microscopy, the nucleus was labeled with DAPI (blue) and endothelial marker CD31 was labeled with red. (B) Mice serum TMAO concentration was determined by UPLC-MS/MS. (C) Cell viability was determined by CCK8. (D) Western blot analysis of ZO-2 and OCLN in HUVECs treated with TMAO for 24 h in different concentrations. (E,F) Bar charts show the quantification of the indicated proteins. *, Compared with the control group, * p < 0.05, ** p < 0.01. n = 3.

Figure 2. Effects of TMAO on endothelial function in vivo and in vitro. (A) Location of eNOS (green) in the endothelial cells of thoracic aorta was estimated by immunofluorescence microscopy, the nucleus was labeled with DAPI (blue) and Endothelial marker CD31 was labeled with red. (B) Western blot analysis of p-eNOS/eNOS in HUVECs treated with TMAO for 24 h in different concentrations. (C,D) Bar charts show the quantification of the indicated proteins. *, Compared with the control group, * p < 0.05, ** p < 0.01. n = 3.

Figure 3. Effects of TMAO on the expression and activity of Cathepsin B in vivo and in vitro. (A) Location of Cathepsin B (green) in the endothelial cells of thoracic aorta was estimated by immunofluorescence microscopy, the nucleus was labeled with DAPI (blue) and Endothelial marker CD31 was labeled with red. (B) Cells were treated with TMAO for 24 h with different concentrations, the expression of Cathepsin B was analyzed by western blot. (C) Bar charts show the quantification of the indicated proteins. (D) Cells were treated with TMAO at the indicated concentrations, the Cathepsin B activity in the HUVECs was detected by Cathepsin B activity assay. *, Compared with the control group, * p < 0.05, ** p < 0.01. n = 3.

Figure 4. TMAO causes endothelial cell disjunction via Cathepsin B in vivo and in vitro. (A) Location of ZO−2 (green) in the endothelial cells of thoracic aorta was estimated by immunofluorescence microscopy, the nucleus was labeled with DAPI (blue), and endothelial marker CD31 was labeled with red. (B) Cells were pretreated with CA-074Me and then exposed to TMAO (600 μmol/L) for 24 h. Expression of Cathepsin B, ZO-2 and OCLN was detected via Western blot. (C–E)) Bar charts show the quantification of the indicated proteins. *, Compared with the control group, ** p < 0.01. #, Compared with the TMAO group, # p < 0.05. ## p < 0.01. n = 3.

Figure 5. TMAO disrupts endothelial function through Cathepsin B in vivo and in vitro. (A) Location of eNOS (green) in the endothelial cells of thoracic aorta was estimated by immunofluorescence microscopy, the nucleus was labeled with DAPI (blue), and Endothelial marker CD31 was labeled with red. (B) Cells were pretreated with CA−074Me and then exposed to TMAO (600 μmol/L) for 24 h. The levels of p-eNOS/eNOS was detected via Western blot. (C,D) Bar charts show the quantification of the indicated proteins. *, Compared with the control group, * p < 0.05. #, Compared with the TMAO group, # p < 0.05. n = 3.

Figure 6. TMAO promotes NLRP3 inflammasome activation in vivo and in vitro. (A,B) Location of NLRP3 and Cleaved Caspase-1 in endothelial cell of TMAO drinking mice was estimated by immunofluorescence microscopy, the nucleus was labeled with DAPI (blue) and endothelial marker CD31 was labeled with red. (C) Cells were treated with TMAO at different concentrations for 24 h, and the expression of NLRP3, Cleaved Caspase-1 and Cleaved IL-1β detected via Western blot. (D–F) Bar charts show the quantification of the indicated proteins. (G) supernatant IL-1B levels were detected by Elisa assay. *, Compared with the control group, * p < 0.05, ** p < 0.01. n = 3.

Figure 7. Effects of NLRP3 inhibition by MCC950 on TMAO−induced endothelial injury. (A) Cells were pretreated with MCC950 (10 μmol/L) for 2 h, and then exposed to TMAO (600 μmol/L) for a further 24 h. Expression of NLRP3, Cleaved Caspase−1 and Cleaved IL−1β were detected via Western blot. (B–D) Bar charts show the quantification of the indicated proteins. (E) Cells were pretreated with MCC950 and then exposed to TMAO (600 μmol/L) for 24 h. Expression of ZO−2, OCLN and p−eNOS/eNOS were detected via Western blot. (F–I) Bar charts show the quantification of the indicated proteins. *, Compared with the control group, * p < 0.05. #, Compared with the TMAO group, # p < 0.05. n = 3.

Figure 8. Effects of Cathepsin B inhibition by CA−074Me on TMAO−induced activation of NLRP3 inflammasome in vivo and in vitro. (A,B) Location of NLRP3 and Cleaved Caspase−1 (green) in the endothelial cells of thoracic aorta was estimated by immunofluorescence microscopy, the nucleus was labeled with DAPI (blue), and Endothelial marker CD31 was labeled with red. (C) Cells were pretreated with CA−074Me (10 μmol/L) for 2 h, and then exposed to TMAO (600 μmol/L) for a further 24 h, expression of NLRP3, Cleaved Caspase−1 and Cleaved IL−1β was detected via Western blot. (D–F) Bar charts show the quantification of the indicated proteins. *, Compared with the control group, * p < 0.05. ** p < 0.01. #, Compared with the TMAO group, ## p < 0.01. n = 3.