Neferine Ameliorates Severe Acute Pancreatitis-Associated Intestinal Injury by Promoting NRF2-mediated Ferroptosis

Abstract

Severe acute pancreatitis (SAP) is a life-threatening abdominal condition often complicated by intestinal barrier dysfunction, which further exacerbates disease progression. Neferine has demonstrated potent anti-inflammatory and antioxidant properties; however, its role in ameliorating SAP and associated intestinal barrier damage remains unclear. In this study, we found that neferine administration significantly alleviates SAP severity by reducing pancreatic and ileal pathological damage, oxidative stress, inflammatory cell infiltration, and intestinal flora translocation. Additionally, neferine enhances the expression of tight junction proteins, increases short-chain fatty acid levels, and improves intestinal dysbiosis, thereby contributing to intestinal homeostasis restoration. Mechanistically, neferine upregulates Nrf2 expression and promotes its nuclear translocation by competitively binding to the Cys-288 site on Keap1. This activation enhances the Nrf2/FPN and Nrf2/xCT/GPX4 axes, thereby preventing ferroptosis and ultimately protecting against pancreatic and intestinal injury in SAP mice. Furthermore, the protective effects of neferine were largely reversed by the Nrf2 inhibitor ML385 and the ferroptosis inducer erastin. This study demonstrates that neferine effectively alleviates SAP by inhibiting ferroptosis and restoring intestinal homeostasis, providing insights into new treatment options for SAP.

Keywords: Acute pancreatitis; Ferroptosis; Gut microbiota; Intestinal injury; Iron export; Neferine.

Figure 1

Neferine mitigates the severity of SAP. (A) Experimental schedule for SAP establishment. (B) H&E staining of pancreatic tissue and corresponding histologic scores. (C) Serum levels of amylase and lipase. (D) Pancreatic edema assessed by the wet-to-dry ratio. (E) Serum levels of IL-1β, IL-6, TNF-α, and IL-10. (F, G) Immunohistochemical staining of pancreatic IL-1β, IL-6, and TNF-α (F), along with average optical density calculations (G). (H) Pancreatic mRNA levels of il-1β, il-6, and tnf-α. (I) Immunofluorescence staining of pancreatic TUNEL and quantification of TUNEL⁺ cells. (J) Immunohistochemical staining of pancreatic MPO and quantification of MPO⁺ cells. (K) Immunofluorescence staining of pancreatic Ly-6G and quantification of Ly6G⁺ neutrophils. (L) Immunofluorescence staining of F4/80 and INOS in the pancreas, with quantification of F4/80⁺ iNOS⁺ macrophages. AOD, average optical density; NE, Neferine; Data were expressed as mean ± SD; n = 6 in each group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Neferine attenuates ileal damage in SAP. (A) Representative H&E staining of the ileum and corresponding histopathology scores. (B) Immunofluorescence staining of ileal MUC2 and its quantification. (C) Immunofluorescence staining of ileal lysozyme and its quantification. (D) Immunofluorescence staining of ileal Ly-6G and quantification of Ly6G⁺ neutrophils. (E) FITC-dextran distribution detected by small-animal imaging, and serum FITC-dextran levels measured. (F) Serum DAO levels measured by ELISA. (G, H) Detection of total bacterial hybridization signals using the EUB338 probe in the pancreas (G) and ileum (H), with quantification of EUB338-positive bacteria in the epithelium per field. (I) Western blot analysis of ZO-1, Occludin, and Claudin-1 protein levels in the ileum. (J) Distributional differences in gut microbiota profiles assessed using PCoA. (K) Community richness estimated by Chao1 and Observed_features. (L) Differentially abundant taxa between the SAP and GAA/SAP groups identified by linear discriminant analysis effect size (LEfSe) analysis. (M) Fecal levels of acetic acid, propionic acid, and butyric acid. NE, Neferine; Data are presented as mean ± SD; n = 6 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Neferine restores iron metabolism by regulating FPN-mediated iron export. (A) Volcano plot: gray, red, and blue dots represent genes with no significant difference, significantly upregulated genes, and significantly downregulated genes, respectively (fold change ≥ 1.5 and FDR < 0.05). (B) Heatmap of differentially expressed genes: each row represents a gene, and each column represents a sample, with red indicating upregulation and blue indicating downregulation. (C) Gene set enrichment analysis (GSEA) of the ferroptosis signaling pathway. (D) Serum levels of GSH, SOD, T-AOC, MDA, and LPO. (E) Western blot analysis of ACSL4, xCT, and GPX4 protein levels in the pancreas and ileum. (F) Serum, pancreatic, and ileal Fe²⁺ levels. (G) Relative quantification of Tfr1, Fth1, Ftl, Fpn, and Ncoa4 mRNA levels in the pancreas. (H) Western blot of FPN protein levels in the pancreas and ileum. NE, Neferine; Data were expressed as mean ± SD; n = 6 in each group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Upregulation of ferroptosis reversed the protective effects of neferine on pancreatic and ileal injury. (A) Experimental schedule for SAP establishment. (B) Serum levels of amylase and lipase. (C) Pancreatic edema assessed by the wet-to-dry ratio. (D) H&E staining and corresponding histologic scores; Immunofluorescence staining of TUNEL and quantification of TUNEL⁺ cells; Positive hybridization signals of total bacteria detected by the EUB338 probe, with quantification of EUB338-positive bacteria in the per field. (E) Serum levels of IL-1β, IL-6, and TNF-α. (F) Serum levels of GSH, SOD, T-AOC, MDA, LPO, and Fe²⁺. (G) Pancreatic levels of SOD, GSH, and MDA. (H) Western blot of ACSL4, xCT, and FPN protein levels in the pancreas and ileum. NE, Neferine; Data were expressed as mean ± SD; n = 6 in each group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Neferine protects against SAP-induced ferroptosis in an Nrf2-dependent manner. (A) Western blot analysis of total Nrf2, Keap1, HO-1, NQO-1, and nuclear Nrf2 protein levels in the pancreas and ileum. (B) Experimental schedule for SAP establishment. (C) Serum levels of amylase and lipase. (D) Pancreatic edema assessed by the wet-to-dry ratio. (E) H&E staining and corresponding histologic scores; Immunohistochemical staining of IL-1β, IL-6, and TNF-α, along with average optical density calculations; Immunohistochemical staining of pancreatic MPO; Immunofluorescence staining of MUC2, and TUNEL; Positive hybridization signals of total bacteria detected by the EUB338 probe, with quantification of EUB338-positive bacteria in the per field. (F) Serum DAO levels measured by ELISA. (G) Serum FITC-dextran levels measured by ELISA. (H) Western blot of ZO-1, occludin, and claudin-1 protein levels in the ileum. (I) Fecal levels of acetic acid, propionic acid, and butyric acid. AOD, average optical density; NE, Neferine; ML, ML385. Data were expressed as mean ± SD; n = 6 in each group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Inhibition of Nrf2 reverses the inhibitory effects of neferine on ferroptosis. (A) Serum levels of GSH, SOD, T-AOC, MDA, LPO, and Fe²⁺. (B) Pancreatic and ileal levels of MDA, SOD, and GSH. (C) Western blot analysis and corresponding relative gray values of Nrf2, Keap1, HO-1, NQO-1, and nuclear Nrf2 protein levels in the pancreas and ileum. (D) Western blot analysis and relative gray values of ACSL4, xCT, GPX4, and FPN protein levels in the pancreas and ileum. NE, Neferine; ML, ML385. Data are presented as mean ± SD, with n = 6 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Neferine inhibits ferroptosis by upregulating the Nrf2 pathway in vitro. (A) Western blot analysis was performed to detect the expression of Nrf2, Keap1, FPN, ACSL4, IL-1β, IL-6, ZO-1, and Occludin in IEC-6 cells subjected to different neferine treatments. (B) Immunofluorescence staining was conducted to visualize Nrf2, FPN, ZO-1, and Occludin in IEC-6 cells; The neferine concentration used is 4 μM. (C) Western blot analysis was performed to measure the expression of Nrf2, Keap1, FPN, ACSL4, IL-1β, IL-6, ZO-1, and Occludin in IEC-6 cells treated with si-Nrf2 and si-FPN. (D) Amino acid binding sites of neferine within the crystal structure of the Keap1 complex. (E) Co-immunoprecipitation analysis illustrating the interaction between Keap1 and Nrf2 in wild-type and Keap1-mutant IEC-6 cells. (F) Western blot analysis was performed to measure the expression of nuclear Nrf2, FPN, ACSL4, IL-1β, IL-6, ZO-1, and Occludin in wild-type and Keap1-mutant IEC-6 cells. NE, Neferine; Data were expressed as mean ± SD; n = 6 in each group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

A proposed model illustrates the mechanism by which neferine protects against SAP and its associated intestinal injury. In SAP, iron metabolism becomes dysregulated, and ROS production increases. Neferine administration not only upregulates Nrf2 but also promotes the dissociation of Keap1-Nrf2 and enhances Nrf2 nuclear translocation by competitively binding to the Cys-288 site of Keap1. This activation strengthens the Nrf2/FPN and Nrf2/xCT/GPX4 axes, thereby preventing ferroptosis and ultimately protecting against pancreatic and intestinal injury in SAP mice. Additionally, neferine helps restore intestinal homeostasis by increasing the abundance of beneficial Lactobacillus, reducing harmful Escherichia-Shigella, and inhibiting intestinal flora migration. Ultimately, neferine administration disrupts the positive feedback loop in pancreatitis, where pancreatic inflammation triggers systemic inflammation, leading to intestinal barrier damage, subsequent gut microbiota translocation, and further exacerbation of pancreatic injury.