Quercetin Alleviated Inflammasome-Mediated Pyroptosis and Modulated the mTOR/P70S6/P6/eIF4E/4EBP1 Pathway in Ischemic Stroke

Abstract

Stroke ranks as the world's second most prevalent cause of mortality, and it represents a major public health concern with profound economic and social implications. In the present study, we elucidated the neuroprotective role of quercetin on NLRP3-associated pyroptosis, Nrf2-coupled anti-inflammatory, and mTOR-dependent downstream pathways. Male Sprague Dawley rats were subjected to 72 h of transient middle cerebral artery ischemia, followed by the administration of 10 mg/kg of quercetin. Our findings demonstrated that MCAO induced elevated ROS which were coupled to inflammasome-mediated pyroptosis and altered mTOR-related signaling proteins. We performed ELISA, immunohistochemistry, and Western blotting to unveil the underlying role of the Nrf2/HO-1 and PDK/AKT/mTOR pathways in the ischemic cortex and striatum. Our results showed that quercetin post-treatment activated the Nrf2/HO-1 cascade, reversed pyroptosis, and modulated the autophagy-related pathway PDK/AKT/mTOR/P70S6/P6/eIF4E/4EBP1. Further, quercetin enhances the sequestering effect of 14-3-3 and reversed the decrease in interaction between p-Bad and 14-3-3 and p-FKHR and 14-3-3. Our findings showed that quercetin exerts its protective benefits and rescues neuronal damage by several mechanisms, and it might be a viable neuroprotective drug for ischemic stroke therapy.

Keywords: NLRP3; Nrf2/HO-1; ischemic stroke; mTOR pathway; quercetin.

Figure 1

Quercetin attenuated neuronal cell death: (A) Images illustrating the immunofluorescence staining of FJB in the cortex (n = 5/group) pictured at magnification 10×, scale bar = 100 μm. (B) Representative TUNEL histochemistry pictures demonstrate apoptotic cells in the striatum; scale bar = 100 μm. The administration of MCAO resulted in notable neuronal apoptosis, whereas the application of quercetin exhibited a mitigating effect on apoptotic injury. The data reported are related to sham (n = 5/group). (C) Panels of Nissl staining to evaluate neuronal viability, scale bar = 50 μm. The symbol * is relative to the sham group while the symbol #—to the MCAO group.

Figure 2

Quercetin attenuated 8-oxo guanine in the MCAO model. 8-oxo guanine was used as a ROS marker. Representative 8-oxo guanine histochemistry pictures demonstrate ROS in the cortex and the striatum (n = 5) pictured at magnification 10×, scale bar = 100 μm. Rhodamine was employed for detecting 8-oxo guanine, whereas blue coloration (DAPI) was indicative of nuclear staining. The statistical analysis involved the presentation of data as the means ± SEM. The symbol * is relative to the sham group while the symbol #—to the MCAO group.

Figure 3

Quercetin modulates the mTOR pathway. (A) Western blot analysis of p-PDK, p-AKT, p-mTOR, p-P70S6, p-P6, p-eIF4E, and p-4EBP1 in the cortex and the striatum. The Western blot bands were quantified by ImageJ and the densitometric analysis was quantified relative to β-actin. (B) Representative p-P70S6 histochemistry pictures in the cortex and the striatum (n = 5) pictured at magnification 40×, scale bar = 30 μm. FITC was employed for detecting p-P70S6, which showed cytoplasmic localization, whereas blue coloration (DAPI) was indicative of nuclear staining. The statistical analysis involved the presentation of data as the means ± SEM. The symbol * is relative to the sham group while the symbol #—to the MCAO group.

Figure 3

Quercetin modulates the mTOR pathway. (A) Western blot analysis of p-PDK, p-AKT, p-mTOR, p-P70S6, p-P6, p-eIF4E, and p-4EBP1 in the cortex and the striatum. The Western blot bands were quantified by ImageJ and the densitometric analysis was quantified relative to β-actin. (B) Representative p-P70S6 histochemistry pictures in the cortex and the striatum (n = 5) pictured at magnification 40×, scale bar = 30 μm. FITC was employed for detecting p-P70S6, which showed cytoplasmic localization, whereas blue coloration (DAPI) was indicative of nuclear staining. The statistical analysis involved the presentation of data as the means ± SEM. The symbol * is relative to the sham group while the symbol #—to the MCAO group.

Figure 4

Quercetin dimerizes p-Bad and p-FKHR with 14-3-3. (A) Western blot analysis of p-FKHR and 14-3-3 in the cortex and the striatum. The Western blot bands were quantified by ImageJ and the densitometric analysis was quantified relative to beta-actin. The symbol * is relative to the sham group while the symbol #—to the MCAO group. (B) Representative p-FKHR and 14-3-3 co-localization histochemistry pictures in the cortex and the striatum (n = 5) pictured at magnification 40×, scale bar = 50 μm. FITC and TRITC were employed for detecting p-FKHR and 14-3-3, respectively, and both showed cytoplasmic localization, whereas blue coloration (DAPI) was indicative of nuclear staining. (C) Representative p-BAD and 14-3-3 co-localization histochemistry pictures in the cortex and the striatum (n = 5), pictured at magnification 40×, scale bar = 50 μm. FITC and TRITC were employed for detecting 14-3-3 and p-BAD, respectively, and both showed cytoplasmic localization, whereas blue coloration (DAPI) was indicative of nuclear staining.

Figure 5

Quercetin augments the Nrf2/HO-1 expression. (A) Western blot analysis of Nrf2 and HO-1 in the cortex and the striatum. The Western blot bands were quantified by ImageJ and the densitometric analysis was quantified relative to beta-actin. (B) Representative TRX histochemistry pictures in the striatum (n = 5) pictured at magnification 10×, scale bar = 100 μm. TRITC was employed for detecting TRX and showing cytoplasmic localization, whereas blue coloration (DAPI) was indicative of nuclear staining. (C) Western blot analysis of p-NF-κb and GFAP in the cortex. The Western blot bands were quantified by ImageJ and the densitometric analysis was quantified relative to beta-actin. The statistical analysis involved the presentation of data as the means ± SEM and the utilization of two-way ANOVA with post-hoc Tukey’s test; * p < 0.05 is compared to the sham while # p < 0.05 is compared to MCAO.

Figure 6

Quercetin administration reversed pyroptosis by the NLRP3 pathway. (A) Pictures of NLRP3 (scale bar = 80 um) and iNOS (scale bar = 50 um) in the striatum. MCAO induced the expression of these markers while quercetin treatment attenuated the expression level. Both NLRP3 and iNOS represent cytoplasmic localization. TRITC was employed for detecting iNOS and NLRP3, whereas blue coloration (DAPI) was indicative of nuclear staining. The statistical analysis involved the presentation of data as the means ± SEM and the utilization of one-way ANOVA with post-hoc Tukey’s test; * p < 0.05 is compared to the sham while # p < 0.05 is compared to MCAO. (B) IL-1β expression in the cortex, magnification 40×, scale bar = 30 μm. FITC was employed for detecting IL-1, whereas blue coloration (DAPI) was indicative of nuclear staining. The statistical analysis involved the presentation of data as the means ± SEM and the utilization of two-way ANOVA with post-hoc Tukey’s test; * p < 0.05 is compared to the sham while # p < 0.05 is compared to MCAO. (C) ELISA analysis of NLRP3 in the cortex. The symbol * is relative to the sham group while the symbol #—to the MCAO group. (D) Serum LDH evaluation for demonstrating leakage in the cell membrane. The symbol * is relative to the sham group while the symbol #—to the MCAO group.

Figure 6

Quercetin administration reversed pyroptosis by the NLRP3 pathway. (A) Pictures of NLRP3 (scale bar = 80 um) and iNOS (scale bar = 50 um) in the striatum. MCAO induced the expression of these markers while quercetin treatment attenuated the expression level. Both NLRP3 and iNOS represent cytoplasmic localization. TRITC was employed for detecting iNOS and NLRP3, whereas blue coloration (DAPI) was indicative of nuclear staining. The statistical analysis involved the presentation of data as the means ± SEM and the utilization of one-way ANOVA with post-hoc Tukey’s test; * p < 0.05 is compared to the sham while # p < 0.05 is compared to MCAO. (B) IL-1β expression in the cortex, magnification 40×, scale bar = 30 μm. FITC was employed for detecting IL-1, whereas blue coloration (DAPI) was indicative of nuclear staining. The statistical analysis involved the presentation of data as the means ± SEM and the utilization of two-way ANOVA with post-hoc Tukey’s test; * p < 0.05 is compared to the sham while # p < 0.05 is compared to MCAO. (C) ELISA analysis of NLRP3 in the cortex. The symbol * is relative to the sham group while the symbol #—to the MCAO group. (D) Serum LDH evaluation for demonstrating leakage in the cell membrane. The symbol * is relative to the sham group while the symbol #—to the MCAO group.