Piperine protects against cerebral ischemic injury by regulating the Caspase-1-mediated pyroptosis pathway

Abstract

Background: Ischemic stroke (IS) is a prevalent form of stroke and marked by high rates of morbidity, disability, and mortality. IS greatly threatens the physical health of people around the world. Oxidative stress triggered by IS can lead to inflammatory responses. Piperine (Pip) is a bioactive dietary phytochemical known for its pharmacological properties, including anti-inflammatory, anti-tumor, and antioxidant effects. Pip has attracted considerable interest among researchers. This study aims to investigate whether Pip attenuates cerebral ischemic injury by regulating the Caspase-1-mediated pyroptosis pathway.

Methods: In vivo and in vitro experimental models were employed. For the in vivo simulation of cerebral ischemia, the rat permanent middle cerebral artery occlusion (pMCAO) model was utilized. For the in vitro simulation, the BV-2 cells were subjected to oxygen-glucose deprivation (OGD). The recovery of neurological function in rats was assessed through multiple behavioral tests, including the Zea-Longa score, balance beam test, traverse beam test, forelimb grip pull test, postural reflex test, sensory test, and tail lifting test. Pathological changes in cerebral ischemic injury were observed using TTC staining, HE staining, and transmission electron microscopy. In in vivo and in vitro experiments, the potential protective mechanism of Pip in alleviating cerebral ischemic injury by regulating the Caspase-1-mediated pyroptosis pathway was investigated using Western blot and reverse transcription-polymerase chain reaction assays.

Results: In the in vivo experiments, compared with the Sham group, the Model group exhibited significant neurological damage, increased infarct volume, brain tissue edema, and elevated protein and mRNA expression levels of pyroptosis-associated factors. By contrast, the Pip group demonstrated notable improvements in behavioral function, brain tissue morphology, and the expression levels of pyroptosis-related factors compared with the Model group. In the in vitro experiments, the protein and mRNA expression of pyroptosis-associated factors in the OGD group were significantly upregulated compared with that in the Con group. However, the expression of these factors in the OGD+Pip group was markedly reduced compared with that in the OGD group. Furthermore, when cells were treated with the Caspase-1 inhibitor Ac-YVAD-cmk, the results revealed a significant decrease in the protein expression of Caspase-1 and its downstream factors, GSDMD-N and IL-1β, compared with that in the OGD group. Notably, the protein expression of GSDMD-N and IL-1β in the Pip+Ac-YVAD-cmk group was significantly higher than in the Pip group, which suggests that the inhibition of Caspase-1 attenuated the suppressive effect of Pip on GSDMD-N and IL-1β expression.

Conclusion: Pip exerts neuroprotective effects by modulating the Caspase-1-mediated pyroptosis pathway, which inhibits neuronal damage in the pMCAO model. These findings highlight the therapeutic potential of Pip in mitigating cerebral ischemic injury.

Keywords: Caspase-1; cerebral ischemic injury; natural product; neuroprotective; pyroptosis.

FIGURE 1

Effects of Pip on body weight, neurological function and motor performance in pMCAO rats. (A) Comparison of body weight among groups on day 14 post-model establishment and changes in body weight over time. (B) Comparison of Zea-Longa scores among groups on day 14 post-model establishment and changes in Zea-Longa scores over time. (C) Comparison of mNSS scores among groups on day 14 post-model establishment and changes in mNSS scores over time. (D) Comparison of Beam Waliking Test scores among groups on day 14 post-model establishment and changes in Beam Waliking Test scores over time. (E) Comparison of forelimb grip strength among groups on day 14 post-model establishment and changes in forelimb grip strength over time. (F) Comparison of posture reflex scores among groups on day 14 post-model establishment and changes in posture reflex scores over time. Compared to the Sham group, ^# P < 0.05, ^## P < 0.01; compared to the Model group, *P < 0.05, **P < 0.01 (n = 8 per group).

FIGURE 2

Effects of Pip on cerebral infarction volume and pathological damage in pMCAO rats. (A) Photographs of TTC-stained brain tissue sections. (B) Statistical analysis of cerebral infarction volume in each group. Compared to the Sham group, ^# P < 0.05, ^## P < 0.01; compared to the Model group, *P < 0.05, **P < 0.01 (n = 3 per group). (C) HE staining. Scale bar = 20 μm at ×400 magnification; scale bar = 50 μm at ×200 magnification; scale bar = 100 μm at ×100 magnification. The scale bars are indicated at the bottom left corner of the images (n = 3 per group).

FIGURE 3

Effects of Pip on neuronal cell damage in pMCAO rats. Ultrastructure of neurons in the ischemic penumbra. ①⑫⑮ represent the double-layered nuclear membrane structure; ②⑪ represent Golgi apparatus; ③⑬⑭ represent rough endoplasmic reticulum; ④⑩⑯ represent mitochondria; ⑤-⑨ respectively represent heterochromatin, swollen and structurally damaged mitochondria, fragmented nuclear membrane structures, and swollen Golgi apparatus. Scale bar = 5 μm at 5000× magnification; scale bar = 2 μm at ×10000 magnification; scale bar = 1 μm at ×30000 magnification. The scale bars are indicated at the bottom right corner of the images (n = 3 per group).

FIGURE 4

Effects of Pip on the protein and mRNA expression of pyroptosis-related factors in ischemic brain tissue. (A–C,G,H) Western blot bands of Caspase-1, cleaved Caspase-1, IL-1β, NLRP3 and GSDMD-N in each group. (D–F,I,J) Bar graphs showing the target protein/β-actin ratios in each group. (K–P) Bar graphs showing the mRNA expression levels of ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 in each group. Compared to the Sham group, ^# P < 0.05, ^## P < 0.01; compared to the Model group, *P < 0.05, **P < 0.01 (n = 4 per group).

FIGURE 5

Effects of Pip on the protein and mRNA expression of pyroptosis-related factors in BV-2 cells. (A–C,G,H) Western blot bands of Caspase-1, cleaved Caspase-1, IL-1β, NLRP3 and GSDMD-N in each group. (D–F,I,J) Bar graphs showing the target protein/β-actin ratios in each group. (K–P) Bar graphs showing the mRNA expression levels of ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 in each group. Compared to the Con group, ^# P < 0.05, ^## P < 0.01; compared to the OGD group, *P < 0.05, **P < 0.01 (n = 4 per group).

FIGURE 6

Effects of Ac-YVAD-cmk on Pip’s inhibition of pyroptosis. (A) Western blot bands and bar graphs of Caspase-1, GSDMD-N and IL-1β in the Pip + Ac-YVAD-cmk group, Pip group, Con group, Ac-YVAD-cmk group and OGD group. (B–D) Bar graphs showing the protein expression levels of Caspase-1, GSDMD-N and IL-1β in the Pip + Ac-YVAD-cmk group, Pip group, Con group, Ac-YVAD-cmk group and OGD group. Compared to the Con group, ^# P < 0.05, ^## P < 0.01; compared to the OGD group, *P < 0.05, **P < 0.01; compared to the Pip group, ^▲ P < 0.05, ^▲▲ P < 0.01 (n = 4 per group).