Catalpol ameliorates depressive-like behaviors in CUMS mice via oxidative stress-mediated NLRP3 inflammasome and neuroinflammation

Abstract

The purpose of the present study was to investigate whether catalpol exhibited neuroprotective effects in chronic unpredictable mild stress (CUMS) mice through oxidative stress-mediated nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin-domain-containing 3 (NLRP3) inflammasome and neuroinflammation. Deficits in behavioral tests, including open field test (OFT), forced swim test (FST), and elevated plus-maze test (EPM), were ameliorated following catalpol administration. To study the potential mechanism, western blots, quantitative real-time PCR (qRT-PCR) analysis and immunofluorescence imaging were performed on the hippocampus samples. We found that the defects of behavioral tests induced by CUMS could be reversed by the absence of NLRP3 and NLRP3 inflammasome might be involved in the antidepressant effects of catalpol on CUMS mice. Similar to the NLRP3 inflammasome, the expression of interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and inducible nitride oxide synthase (iNOS) were increased after CUMS. The current study demonstrated that catalpol possessed anti-inflammatory effect on CUMS mice and inhibited microglial polarization to the M1 phenotype. In addition, the activity of mitochondrial oxidative stress might be involved in the NLRP3 activation, which was proved by the downregulation of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and cleaved IL-1β, after the administration of mitochondrion-targeted antioxidant peptide SS31. Taken together, we provided evidence that catalpol exhibited antidepressive effects on CUMS mice possibly via the oxidative stress-mediated regulation of NLRP3 and neuroinflammation.

Fig. 1. Catalpol conferred antidepressive effects on behavioral tests.

a The experimental paradigm and the experimental group. b The time spent immobile in the FST (n = 8/group). c Rearing numbers in the OFT (n = 8/group). d Total distance traveled in the OFT (n = 8/group). e Open-arm time proportion in the EPM test (n = 8/group). All data are expressed as the mean ± SD. **p < 0.01, ***p < 0.001, compared with control group; ^#p < 0.05, ^###p < 0.001, compared with vehicle group.

Fig. 2. The NLRP3 inflammasome might be involved in antidepressive effect of catalpol.

a–c Detection of NLRP3 inflammasome in the hippocampus of CUMS mice. Western blot analysis of (a) NLRP3 (n = 6–8/group), (b) cleaved caspase-1 (n = 6–8/group), and (c) Il-1β (n = 6–8/group). All data are expressed as the mean ± SD. *p < 0.05, compared with control group; ^##p < 0.01, compared with vehicle group. d–h In order to detect whether or not NLRP3 inflammasome plays a key role in CUMS-induced depression, NLRP3 KO mice were used in this experiment. d The experimental paradigm and the experimental group. e The time spent immobile in the FST (n = 10/group). f Rearing numbers in the OFT (n = 10/group). g Total distance traveled in the OFT (n = 10/group). h Open-arm time proportion in the EPM test (n = 10/group). All data are expressed as the mean ± SD. **p < 0.01, ***p < 0.001, compared with wild-type group.

Fig. 3. Catalpol dampened neuroinflammation and mitochondrial oxidative stress in the hippocampus of CUMS mice.

a–e The expression of cytokines were assessed by qRT-PCR, the level of IL-1β, TNF-α, iNOS, IL-6 was downregulated significantly by catalpol, and the level of CD206 was also detected. (n = 5–8/group). f To detect the mitochondrial oxidative stress level, the average fluorescence intensity of DCF was analyzed (n = 5–8/group). g Immunofluorescence staining of hippocampal sections. Iba-1 (green), DAPI (blue); scale bar, 100 μm. h Qualification of Iba-1 immunofluorescence density (n = 5–8/group). All data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control group; ^#p < 0.05, ^##p < 0.01, compared with vehicle group.

Fig. 4. Mitochondrial oxidative stress might be involved in the effects of catalpol on CUMS-induced depressive-like behavior and NLRP3 activation.

a The experimental paradigm and the experimental group. b The time spent immobile in the FST (n = 10/group). c Rearing numbers in the OFT (n = 10/group). d Total distance traveled in the OFT (n = 10/group). e Open-arm time proportion in the EPM test (n = 10/group). f–i Detection of NLRP3 inflammasome in the hippocampus of CUMS mice. Western blot analysis of (f) NLRP3 (n = 6–8/group), (g) cleaved caspase-1 (n = 6–8/group), (h) ASC (n = 6–8/group), and (i) cleaved IL-1β (n = 6–8/group). All data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control group; ^##p < 0.01, ^###p < 0.001, compared with vehicle group.

Fig. 5. Mitochondrial oxidative stress might be involved in the downregulation of proinflammatory cytokines by catalpol.

The expression of cytokines in hippocampus were assessed by qRT-PCR, the level of (a) IL-1β (n = 5–8/group), (b) IL-6 (n = 5–8/group), and (c) iNOS (n = 5–8/group) was downregulated significantly by catalpol. d, e There are no significant differences in the expression of (d) IL-10 (n = 5–8/group) and (e) CD206 (n = 5–8/group) between groups. All data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control group; ^#p < 0.05, ^###p < 0.001, compared with vehicle group.