Capsaicin attenuates sepsis‑associated encephalopathy by inhibiting neuroinflammation and apoptosis whilst activating mitophagy through the BNIP3/NIX pathway

Abstract

Sepsis‑induced abnormalities in brain function or sepsis‑associated encephalopathy (SAE) can manifest as cognitive dysfunction and other neuropsychiatric symptoms; however, the underlying mechanisms remain unclear. The aim of the present study was to elucidate the possible effects and mechanism of capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, on the pathological features of SAE. A model of SAE in C57BL/6 mice was generated using cecal ligation and puncture (CLP). Capsaicin (1 mg/kg) was injected subcutaneously before surgery. Cognitive function in mice was evaluated using the novel object recognition test (NORT) and Morris water maze (MWM). Immunofluorescence staining, ELISA, western blotting and transmission electron microscopy were performed to detect the degree of microglial activation (ionized calcium‑binding adapter molecule 1), proinflammatory cytokine levels (TNF‑α), autophagy and apoptosis‑related protein expression, and autophagosomes. Autophagic flux was monitored using the LC3‑GFP‑mCherry fluorescent reporter. Compared with that in the sham group mice, the expression levels of TRPV1 were significantly reduced in the hippocampal tissue of mice with sepsis. Mice with sepsis also exhibited cognitive dysfunction. Notably, a single administration of capsaicin reduced the mortality rate, but did not improve cognitive function in mice with sepsis. Furthermore, repeated administration of capsaicin was revealed to enhance the recognition index of novel objects among mice with sepsis, to reduce the latency to locate the platform and to augment the duration of mouse platform quadrant movements, according to the NORT and MWM tasks. Increased microglial activation, release of proinflammatory cytokines and expression levels of apoptosis‑related proteins were all observed in mice with CLP‑induced sepsis, as was brain tissue destruction in the hippocampal regions. By contrast, capsaicin treatment ameliorated CLP‑induced microglial activation, inflammation, neuronal apoptosis (cleaved caspase 3 expression increased) and brain tissue destruction. Furthermore, application of capsaicin increased the expression levels of LC3, reduced the expression of p62 and elevated autophagic flux compared with those in the CLP group. Finally, treatment with capsaicin effectively enhanced the levels of Bcl‑2‑interacting protein 3 (BNIP3) and BNIP3‑like (NIX) expression. These findings suggested that capsaicin may be considered a potential drug for the treatment of SAE, and BNIP3/NIX‑mediated mitophagy may be involved in this process.

Keywords: capsaicin; mitophagy; neuroin-flammation; sepsis‑associated encephalopathy.

Figure 1.

Effects of subcutaneous CAP administration on mouse survival rate and cognitive function after CLP surgery. (A) Western blot analysis and semi-quantitative analysis of TRPV1 protein expression levels in hippocampal tissues after CLP surgery (n=6 mice/group; unpaired Student's t-test; t₍₁₀₎=3.221; P<0.01). (B) Survival rate of sham (n=10), CLP + Veh (n=15) and CLP + 1 mg/kg CAP (n=15) mice were monitored for 7 days. Log-rank Mantel-Cox test, P=0.035. (C) Recognition index of the novel objective in the novel object recognition test [Sham (n=8), CLP (n=6) and CLP + CAP (n=7) mice; one-way ANOVA with Bonferroni post hoc test; F_(2,18) =3.670; P=0.046]. (D) Time spent in the target quadrant in the Morris water maze test phase [Sham (n=8), CLP (n=10) and CLP + CAP (n=12) mice]. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. sham. CAP, capsaicin; CLP, cecal ligation and puncture; TRPV1, transient receptor potential vanilloid 1; Veh, vehicle; CAP-1*1, capsaicin 1 mg/kg * 1 application.

Figure 2.

CAP-induced protection in mice with sepsis. (A) Recognition index of the novel object in the novel object recognition test [Sham (n=10), CLP (n=12) and CLP + CAP (n=11) mice; n=10-12 mice/group; one-way ANOVA with Bonferroni post hoc test; F_(2,30)=12.45; P=0.0001]. (B) Escape latency in the MWM during the training phase [Sham (n=10), CLP (n=15) and CLP + CAP (n=14) mice]. (C) Time spent in the target quadrant in the MWM during the test phase (one-way ANOVA with Bonferroni post hoc test; F_(2,35)=8.925; P=0.0007). (D) Representative swimming traces in the MWM during the learning and test phase. (E) Average swimming speed in the MWM during the test phase. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001. CAP, capsaicin; CLP, cecal ligation and puncture; MWM, Morris water maze; Veh, vehicle; CAP-1*3, capsaicin 1 mg/kg * 3 application.

Figure 3.

CAP inhibits CLP-induced microglia activation and level of pro-inflammatory factors. (A) Immunofluorescence images of Iba1 (green) fluorescent signals. Scale bars, 10 µm. (B) Quantification of microglia cell numbers (n=6 mice/group) (one-way ANOVA with Bonferroni post hoc test; F_(2,15)=15.72; P=0.0002). (C) Quantification of the microglial cell area (n=6 mice/group) (one-way ANOVA with Bonferroni post hoc test; F_(2,24)=10.48; P=0.0005). (D) Levels of TNF-α in the hippocampus (n=6 mice/group) (one-way ANOVA with Bonferroni post hoc test; F_(2,15)=10.73; P=0.0013). (E) Relative density of NLRC4 expression. (n=8 mice/group) (one-way ANOVA with Bonferroni post hoc test; F_(2,21)=10.37; P=0.0007). (F) NLRC4 expression was detected by western blotting. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001. CAP, capsaicin; CLP, cecal ligation and puncture; Iba1, ionized calcium-binding adapter molecule 1; NLRC4, nucleotide-binding oligomerization domain, leucine rich repeat and CARD domain-containing 4; Veh, vehicle.

Figure 4.

CAP inhibits CLP-induced neuronal damage in mice with sepsis. (A) Representative image of hematoxylin and eosin staining showing the morphological features of neurons in the hippocampal CA1 region. Scale bars, 50 µm. (B) Qualitative analysis of neuronal damage of the hippocampus in septic mice, n=6 mice/group. ***P<0.001, Kruskal-Wallis test with Dunn's post-hoc analysis. (C) Pro-caspase 3 and cleaved caspase 3 expression was detected by western blotting. (D) Relative density of pro-caspase 3 and cleaved caspase 3 expression (n=4 mice/group) (one-way ANOVA with Bonferroni post hoc test; pro-caspase 3: F_(2,9)=16; P=0.0010; cleaved caspase 3: F_(2,9)=9.571; P=0.0059). Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. CAP, capsaicin; CLP, cecal ligation and puncture; Veh, vehicle.

Figure 5.

Autophagy level in mice with CLP-induced sepsis. (A) LC3 and p62 protein expression levels in the hippocampus were detected by western blotting. Relative density of (B) LC3 and (C) p62 expression (n=8 mice/group) (one-way ANOVA with Bonferroni post hoc test; LC3: F_(2,21)=20; P<0.0001; p62: F_(2,21)=12.76; P=0.0002). (D) Mitophagy was observed under a transmission electron microscope. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001. CAP, capsaicin; CLP, cecal ligation and puncture; Veh, vehicle.

Figure 6.

CAP enhances autophagy in LPS-treated BV2 cells. (A) BV2 cells were transfected with LC3-GFP-mCherry. The fluorescence images were captured by confocal microscopy. (B) Quantification of autophagosomes (yellow) and autolysosomes (red) from BV2 cells (one-way ANOVA with Bonferroni post hoc test; F_(5,90)=44.77; P<0.0001). Data are presented as the mean ± standard deviation. **P<0.01 and ****P<0.0001. CAP, capsaicin; Ctrl, control; LPS, lipopolysaccharide.

Figure 7.

Capsaicin promotes mitophagy via the BNIP3/NIX pathway. (A) PINK1, Parkin, BNIP3 and NIX proteins were detected by western blotting. Relative density of (B) PINK1, (C) Parkin, (D) BNIP3 and (E) NIX expression; n=4 mice/group. (one-way ANOVA with Bonferroni post hoc test; BNIP3: F_(2,9)=22.79; P=0.0003; NIX: F_(2,9)=22; P=0.0003). Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. BNIP3, Bcl-2-interacting protein 3; CAP, capsaicin; CLP, cecal ligation and puncture; NIX, BNIP3-like; PINK1, PTEN-induced kinase 1; Veh, vehicle.