Celastrol alleviates esophageal stricture in rats by inhibiting NLR family pyrin domain containing 3 activation

Figure 8 The effect of celastrol on the thioredoxin reductase 1/reactive oxygen species axis. A: Representative fluorescence image and of intracellular reactive oxygen species (ROS) production detected by DCFH-DA. Scale bar = 500 μm; B: Quantitative analysis of ROS fluorescence intensity (presented as mean ± SE). Statistical analysis was performed using Dunnett’s multiple comparison test, with the “lipopolysaccharide (LPS)” group as the control, ^aP < 0.001; ^bP < 0.001; C: Flow cytometry of ROS levels in cells. The mean value of the BL1-A channel represents the ROS production level (presented as mean ± SE). Statistical analysis was performed using Dunnett’s multiple comparison test, with the “LPS” group as the control, ^cP < 0.001; ^dP < 0.001; D: Western blot analysis of nuclear factor erythroid 2-related 2 (Nrf2) in the nucleus and cytoplasm indicates nuclear translocation (presented as mean ± SE). Statistical analysis was performed using Dunnett’s multiple comparison test, with the “LPS” group as the control, ^eP = 0.005; ^fP = 0.03; ^fP = 0.03; E and F: mRNA, protein expression levels, and activity of thioredoxin reductase 1 (TXNRD1) (presented as mean ± SE). Statistical analysis was performed using Dunnett’s multiple comparison test, with the “LPS” group as the control, ^hP = 0.01; ⁱP = 0.03; ^jP = 0.02; ^kP < 0.001; G: Flow cytometry of ROS levels in cells transfected with shNC or TXNRD1 shRNA. The mean value of the BL2-A channel represents the ROS production level (presented as mean ± SE). Statistical analysis was performed using the Student’s t test, ^LP = 0.002 vs shNC-LPS; ^mP < 0.001 vs shTXNRD1-LPS; H: Western blot analysis of NLR family pyrin domain containing 3 protein expression level in cells with shNC or shRNA of TXNRD1 (presented as mean ± SE). Statistical analysis was performed using Dunnett’s multiple comparison test, with the “shTXNRD1-LPS” or “shNC-LPS” as the control. ⁿP = 0.04 vs shTXNRD1-LPS; ^oP = 0.007 vs shNC-LPS; ^pP = 0.04 vs shNC-LPS. NLRP3: NLR family pyrin domain containing 3; CEL: Celastrol; PRED: Prednisolone; LPS: Lipopolysaccharide; TXNRD1: Thioredoxin reductase 1.