Fig 1: PTUPB attenuates PA-induced hepatocyte senescence in vitro. AML12 cells were treated with a series of concentrations of PA (0, 20, 200, and 2000 μM) for 48 h, and then the changes in cell morphology and number were photographed under a microscope ((A), scale bars = 100 μm, n = 3). The fluorescence intensity of Ki67 and PCNA was detected by immunofluorescence ((A), scale bars = 100 μm, n = 3). AML12 cells were treated with PTUPB (1 μM) for 1 h before treatment with PA (200 µM). Forty-eight hours after the PA administration, the protein expressions of p53, p21, γH2AX, COX-2, and sEH in AML12 cells were measured by Western blot and quantitatively analyzed ((B–G), n = 3). Representative images of Oil Red O staining in AML-12 hepatocytes treated with PA with/without PTUPB for 48 h ((H), bar = 50 µm). Senescence was confirmed by SA-β-gal staining ((I), bar = 50 µm). The protein expressions of γH2AX, p53, and p16 in AML12 cells were measured by Western blot and quantitatively analyzed after PA stimulation with/without PTUPB for 48 h ((J–M), n = 3). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. Comparisons between the two-group were made with an unpaired t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.
Fig 2: Dysregulation of CYPs/COX-2 metabolism of ARA occurs in the lungs and macrophages under the LPS challenge. Cyp2j9 was the most abundant CYP isoform expressed in the lungs, whereas Cyp2c29 and Cyp2c44 mRNA were undetectable (A, n = 6). Cyp2j9, Cyp2j6, and Cyp2j5 mRNAs were robustly decreased at 12 h after LPS administration (5 mg/kg, i.t.) in the lungs (B, n = 6). Western blotting and RT-qPCR results showing increased sEH and COX-2 proteins, and Cox-2 mRNA at 12 h after LPS administration. (C-F, n = 4-8). Cyp2j6 was the most abundant CYP isoform expressed in primary murine macrophages after treatment with LPS (100 ng/mL), whereas Cyp2c29 and Cyp2c44 mRNA were undetectable (G, n = 3). Cyp2j6 and Cyp2j9 mRNA were remarkably decreased at 6 h after LPS administration (H, n = 3). Expression of sEH and COX-2 protein and Cox-2 mRNA were detected by Western blotting and RT-qPCR (I-L, n = 3). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.
Fig 3: Decreased EETs content is associated with the senescence of AECs. Heatmap showing the levels of arachidonic acid metabolites in MLE12 cells treated with BLM compared to normal saline. The content of metabolites was calculated in log2 (A, n = 3). Absolute quantification of 14,15-EET in MLE12 cells (B, n = 3). Cyps mRNA expression in MLE12 cells was detected by real-time PCR (C, n = 3). MLE12 cells were treated with BLM (0.1 U/mL) for 2 h. Cells were then washed with PBS and subsequently incubated for 22 h. The mRNA expression of Cyp2j6 and Cyp2j9 in MLE12 cells was detected by real-time PCR (D, n = 3). MLE12 cells were treated with different concentrations of BLM (0, 0.01, 0.033, and 0.1 U/mL) for 2 h. Cells were then washed with PBS and subsequently incubated for 46 h. The protein expression of sEH, p16, and ?H2AX in MLE12 cells was detected by Western blot (E-F, n = 3). The linear associations between sEH and p16 or ?H2AX protein expression were analyzed with Pearson correlations analysis (G–H). MLE12 cells were treated with different concentrations of doxorubicin (0, 100, 200, and 400 nM) or H2O2 (0, 100, 250, and 500 µM) for 2 h. Cells were then washed with PBS and subsequently incubated for 46 h. The protein expression of sEH and p53 in MLE12 cells was detected by Western blot (I–K, n = 3). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey's test was used as a post hoc test for pairwise comparisons. Comparisons between the two-group were made with an unpaired t-test. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 4: sEH levels in selected tissues from sham, anhedonia-susceptible, and anhedonia-resilient mice. A Levels of the sEH protein in selected tissues. ACC [H2 = 1.865, P = 0.394]; mPFC [F (2, 21) = 8.615, P < 0.01]; NAc [H2 = 0.620, P = 0.733]; Striatum [H2 = 0.695, P = 0.706]; Hippo [F (2, 21) = 6.145, P < 0.01]; Cerebellum [F (2, 21) = 2.75, P = 0.087]; Spinal cord [F (2, 21) = 3.457, P = 0.05]; Heart [H2 = 2.945, P = 0.229]; Liver [F (2, 21) = 6.214, P < 0.01]; Kidney [F (2, 21) = 4.945, P < 0.05]; Muscle [F (2, 21) = 1.554, P = 0.235]; Gut [H2 = 7.338, P < 0.05]; Vessels [F (2, 21) = 0.4566, P = 0.64]. *P < 0.05; **P < 0.01; ***P < 0.001. B Correlations between sEH levels and MWT scores in the mPFC (R2 = 0.4302, P < 0.01); C correlations between sEH levels and MWT scores in the hippocampus (R2 = 0.547, P < 0.01); D correlations between sEH levels and MWT scores in the spinal cord (R2 = 0.3576, P < 0.05); E correlations between sEH levels and MWT scores in the liver (R2 = 0.2829, P < 0.05); F correlations between sEH levels and MWT scores in the kidney (R2 = 0.8007, P < 0.001); G correlations between sEH levels and MWT scores in the gut (R2 = 0.476, P < 0.01); H correlations between MWT and SPT scores (R2 = 0.7731, P < 0.001). ACC anterior cingulate cortex, mPFC medial prefrontal cortex, MWT mechanical withdrawal threshold, NAc nucleus accumbens, NS not significant, SNI spared nerve injury, SPT sucrose preference test
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