Berberine safeguards sepsis-triggered acute gastric damage and inhibits pyroptosis in gastric epithelial cells via suppressing the ubiquitination and degradation of Nrf2

Abstract

Berberine (BBR), a widely recognized traditional Chinese medicine, has attracted considerable attention for its promising anti-inflammatory effects. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2) effectively safeguards against organ damage stemming from sepsis-induced oxidative stress and inflammatory responses. This study examined the potential of BBR in alleviating sepsis-induced acute gastric injury, with a particular focus on elucidating whether its mechanism of action involves the activation of the Nrf2 signaling pathway. Following intraperitoneal injection of BBR, mice were subjected to the cecal ligation and puncture (CLP) method to induce sepsis. In vitro experiments involved pre-treating the normal gastric epithelial cells (GES-1) with BBR, followed by treatment with lipopolysaccharide (LPS). Functional assays were then performed to assess cell proliferation and apoptosis. To validate the role of Nrf2 in pyroptosis and inflammation, siRNA targeting Nrf2 (si-Nrf2) was transfected into LPS-treated GES-1 cells. Additionally, mice were administered the Nrf2 inhibitor ML385 to confirm the protective effects of BBR in vivo. BBR displayed a dose-dependent effect in mitigating gastric tissue damage, suppressing the release of inflammatory cytokines, and reducing the expression of NLRP3, ASC, and GSDMD-N. In vitro, BBR fostered GES-1 cell proliferation, hindered apoptosis, and suppressed the levels of TNF-α, IL-18, IL-1β, NLRP3, ASC, and GSDMD-N. Further analysis revealed that knocking down Nrf2 reversed BBR's inhibitory effect on pyroptosis in LPS-treated GES-1 cells. Through binding to Keap1, BBR efficiently prevented the ubiquitination and degradation of Nrf2, ultimately promoting its nuclear translocation. In vivo experiments confirmed that ML385 reversed the protective effect of BBR on pyroptosis and inflammation. Our research reveals that BBR interacts with Keap1 to activate the Keap1/Nrf2 signaling pathway in gastric epithelial cells, thereby suppressing pyroptosis and inflammation in sepsis-induced acute gastric injury.

Keywords: Nrf2; acute gastric injury; berberine; inflammation; pyroptosis; sepsis.

FIGURE 1

BBR ameliorates sepsis‐related mouse acute gastric injury. Mice were injected intraperitoneally with BBR at low (20 mg/kg), medium (50 mg/kg), and high (100 mg/kg) doses before CLP surgery. N = 6. (A) The schematic diagram of experimental process progress. (B) H&E staining was performed to examine histopathological damage in gastric tissues (200×). Scale bar: 100 μm. (C) ELISA detection of TNF‐α, IL‐18, and IL‐1β in gastric tissue. (D) Survival rate of mice in each group. Log‐rank test was employed for the comparison among groups. N = 13. **p < 0.01 versus Sham group; ^# p < 0.05, ^## p < 0.01 versus CLP group.

FIGURE 2

BBR inhibits NLRP3‐mediated pyroptosis in sepsis‐related mouse acute gastric injury. (A) Western blotting detection of NLRP3, ASC, and GSDMD‐N in gastric tissues. (B) Immunohistochemical staining of GSDMD‐N protein in gastric tissues (200×). Scale bar: 100 μm. N = 6.

FIGURE 3

BBR suppresses pyroptosis and inflammation in LPS‐induced gastric epithelial cells. GES‐1 cells were pre‐treated with BBR (2, 5, and 10 μM) for 6 h followed by LPS (1 μg/mL) treatment for 24 h. (A) CCK‐8, (B) Edu, and (C) flow cytometry assays were performed to measure cell proliferation and apoptosis. Scale bar: 100 μm. (D) ELISA was used to measure TNF‐α, IL‐18, and IL‐1β levels in cell supernatants. (E) Western blotting assay for the protein levels of NLRP3, ASC, and GSDMD‐N. (F) The fluorescence intensity of caspase‐1 was measured. N = 3. Scale bar: 100 μm. **p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.01 versus LPS group.

FIGURE 4

BBR represses pyroptosis in LPS‐induced gastric epithelial cells via activation of Keap1/Nrf2 signaling pathway. GES‐1 cells were pre‐treated with BBR (5 μM) for 6 h followed by LPS (1 μg/mL) treatment for 24 h. (A) Western blotting detection of Keap1, Nrf2, HO‐1, and NOQ‐1. (B–D) The si‐Nrf2 or si‐Con was transfected into GES‐1 cells, following BBR and LPS stimulation. CCK‐8, Edu, and flow cytometry assays were employed to detect cell proliferation and apoptosis. Scale bar: 100 μm. (E) ELISA detection of TNF‐α, IL‐18, and IL‐1β in cell supernatants. (F) Western blotting for NLRP3, ASC, and GSDMD‐N. (G) The fluorescence intensity of caspase‐1 was measured. N = 3. Scale bar: 100 μm. **p < 0.01 versus control group; ^## p < 0.01 versus LPS group; ^& p < 0.05, ^&& p < 0.01 versus LPS + 5 μM BBR group.

FIGURE 5

BBR inhibits the ubiquitination of Nrf2 through binding to Keap1. GES‐1 cells were pre‐treated with BBR (5 μM) for 6 h followed by LPS (1 μg/mL) treatment for 24 h. (A) qRT‐PCR analysis for mRNA expressions of Keap1, Nrf2, HO‐1, and NOQ‐1. (B) Autodock analysis for the binding of BBR and Keap1. (C) Co‐IP assay was conducted to confirm the interaction of BBR and Keap1. (D) CHX (10 μM) was added to cells and incubated for a specified period. Nrf2 protein level was measured. (E) MG132 (10 μM) was used to incubate with cells. Nrf2 protein level was measured. (F) The ubiquitination level of Nrf2 was validated. (G) The nuclear and cytoplasm of GES‐1 cells was separated. Nrf2 protein level in nuclear and cytoplasm was measured. N = 3. *p < 0.05, **p < 0.01 versus LPS group.

FIGURE 6

Nrf2 inhibitor reverses the effect of BBR on pyroptosis and inflammation in sepsis‐related mouse acute gastric injury. (A) H&E staining on gastric tissue sections (200×). Scale bar: 100 μm. (B) ELISA detection of TNF‐α, IL‐18, and IL‐1β in gastric tissues. (C) Western blotting detection of NLRP3, ASC, and GSDMD‐N. (D) Immunohistochemical staining of GSDMD‐N protein in gastric tissues (200×). Scale bar: 100 μm. (E) Western blotting detection of Nrf2, HO‐1, and NOQ‐1. N = 6. **p < 0.01 versus Sham group; ^## p < 0.01 versus CLP group; ^& p < 0.05 versus CLP + 50 mg/kg BBR group.

FIGURE 7

The protective effect and mechanism of berberine for sepsis‐related gastric injury. Berberine binds to Keap1, inhibiting the ubiquitination of Nrf2, thereby activating the Keap1/Nrf2 signaling pathway and subsequently reducing sepsis‐induced gastric injury.