Protective Effects Proanthocyanidin Nanoliposome Freeze-Dried Powder on Oxidative Injury in a p31-43 Induced Celiac Disease Cell Model

Abstract

Proanthocyanidins play a crucial role in celiac disease (CD) through their antioxidant and anti-inflammatory properties, effectively regulating oxidative stress and inflammatory responses. However, the poor stability and low bioavailability of proanthocyanidins significantly limit their therapeutic applications. The aim of this study was to investigate the effects of lyoprotectants on proanthocyanidin nanoliposome freeze-dried powder (PCNL-FD), characterize its structural properties, evaluate its in vitro antioxidant capacity and explore its protective effects in a celiac disease cell model. The addition of 4% trehalose as a lyoprotectant effectively maintained the encapsulation efficiency and redispersion stability of PCNL-FD. FTIR and TEM analyses confirmed the successful incorporation of proanthocyanidins into the phospholipid bilayer. In vitro antioxidant assays showed that PCNL-FD exhibited significantly higher DPPH, ABTS, and hydroxyl radical scavenging activities compared to free proanthocyanidins. In the p31-43 peptide-induced celiac disease Caco-2 cell model, PCNL-FD significantly reduced oxidative stress by decreasing ROS and MDA levels while increasing GSH levels and antioxidant enzyme activities (SOD and CAT). The formulation showed anti-inflammatory effects by inhibiting pro-inflammatory cytokines (TNF-α, IL-6) and promoting anti-inflammatory cytokines (IL-4, IL-10). In addition, PCNL-FD activated the Keap1/Nrf2 signaling pathway and upregulated the expression of NQO-1 and HO-1. This nanoliposomal delivery system effectively overcame the bioavailability and stability limitations of free proanthocyanidins, providing new insights into the management of oxidative stress in inflammatory diseases such as celiac disease.

Keywords: antioxidant; anti‐inflammatory; celiac disease; nanoliposomes; proanthocyanidins.

FIGURE 1

Effect of lyophilization protectant agent on the appearance of PC‐NL lyophilized powder.

FIGURE 2

Characterization of PCNL‐FD. (A, B) Transmission electron microscopy (TEM) image of PCNL‐FD. (C) FTIR spectrum of PCNL‐FD.

FIGURE 3

In Vitro antioxidant evaluation of PCNL‐FD with in vitro simulated digestion. (A) The effect of PC, PCNL‐FD, and VE on DPPH free radical scavenging rate. (B) The effect of PC, PCNL‐FD, and VE on ABTS radical scavenging rate. (C) The effect of PC, PCNL‐FD, and VE on OH radical scavenging rate. (D) In vitro simulated digestion of PCNL‐FD. Different letters indicate significant differences (p < 0.05).

FIGURE 4

Establishment of the in vitro celiac disease cell model and PCNL‐FD concentration screening. (A) The effect of gliadin peptide p31‐43 stimulation on the viability of Caco‐2 cells. (B) The impact of gliadin peptide p31‐43 on intracellular GSH levels in Caco‐2 cells. (C) The impact of gliadin peptide p31‐43 on intracellular MDA levels in Caco‐2 cells. (D) Quantitative analysis of ROS fluorescence expression in Caco‐2 cells induced by gliadin peptide p31‐43. (E) Fluorescence expression of ROS in Caco‐2 cells induced by gliadin peptide p31‐43. (F) The effect of BNLF on the viability changes of Caco‐2 cells. (G) The effect of PCNL‐FD on the viability changes of Caco‐2 cells. (H) The protective effect of PC, BNLF, and PCNL‐FD on the viability of model cells. ** indicates a statistically significant and highly significant difference compared to the CK group at *p < 0.05; **p < 0.001; ^## indicates a statistically significant and highly significant difference between two groups at ^# p < 0.05; ^## p < 0.001, the same as below.

FIGURE 5

The effect of PCNL‐FD on oxidative stress in the CD cell model. (A) The effect of PCNL‐FD treatment on intracellular GSH levels. (B) The effect of PCNL‐FD treatment on intracellular GSSG levels. (C) The effect of PCNL‐FD treatment on the intracellular GSH/GSSG ratio. (D) The effect of PCNL‐FD treatment on intracellular CAT activity. (E) The effect of PCNL‐FD treatment on intracellular SOD activity. (F) The effect of PCNL‐FD treatment on intracellular MDA levels. (G) Quantitative analysis of ROS fluorescence expression in cells. (H) Intracellular ROS fluorescence expression levels. (I) The level of TNF‐α in Caco‐2 cells. (J) The level of IL‐6 in Caco‐2 cells. (K) The level of IL‐4 in Caco‐2 cells. (L) The level of IL‐10 in Caco‐2 cells.

FIGURE 6

Effect of PCNL‐FD on the protein expression of Keap1/Nrf2 and NQO‐1/HO‐1 in CD cell model. (A) Intracellular Keap1, Nrf2, NQO‐1, and HO‐1 protein expression levels. (B) Relative protein expression levels of Keap1. (C) Relative protein expression levels of Nrf2. (D) Relative protein expression levels of NQO‐1. (E) Relative protein expression levels of HO‐1.