Caffeic acid phenethyl ester improves peritoneal dialysis-associated peritoneal fibrosis by alleviating oxidative stress injury through activating nuclear factor erythroid-2-related factor 2/heme oxygenase-1 pathway / 中华肾脏病杂志

ABSTRACT

Objective:To investigate whether caffeic acid phenethyl ester (CAPE) would improve peritoneal dialysis (PD)-associated peritoneal fibrosis by alleviating oxidative stress through activating nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway.Methods:Thirty-two male Sprague-Dawley rats were randomly divided into four groups by the random number table control (CON) group (0.9% normal saline 20 ml/d intraperitoneal injection), CAPE group (0.9% normal saline 20 ml/d+CAPE 10 mg·kg -1·d -1 intraperitoneal injection), PD group [4.25% glucose peritoneal dialysis fluid (PDF) 20 ml/d intraperitoneal injection with lipopolysaccharide 0.6 mg/kg intraperitoneal injection at day 1, 3, 5 and 7], and PD+CAPE group (CAPE 10 mg·kg -1·d -1 intraperitoneal injection in addition to PD group), with 8 rats per group. On day 28, rats were euthanized after peritoneal equilibration test, and then the parietal peritoneum and omentum were collected for follow-up tests. To further investigate the mechanism, primary peritoneal mesothelial cells (PMCs) of rats were isolated and cultured. The PMCs were stimulated with 2.5% glucose PDF and added with 5 μmol/L CAPE intervention. The Nrf2 inhibitor (ML385) was used to identify whether CAPE protected PMCs from PDF by activating the Nrf2/HO-1 pathway. Histopathological staining was used to detect structural changes of the peritoneum, and immunohistochemical analysis was performed on cleaved caspase-3, Bax, α-smooth muscle actin (α-SMA), fibronectin (FN), and typeⅠ collagen (Col-Ⅰ) protein. Western blotting was used to detect the protein expression of α-SMA, FN, transforming growth factor-β1 (TGF-β1), HO-1 and nuclear Nrf2 (N-Nrf2). The apoptosis detection kit was used to detect apoptosis and flow cytometry was used to detect reactive oxygen species (ROS) in PMCs. The malondialdehyde (MDA) and superoxide dismutase (SOD) activity detection kit were used to detect MDA content and SOD activity. Cell immunofluorescence was used to analyze the protein expression of Nrf2 in PMCs. Results:Compared with the CON group, the PD group had thicker peritoneum, and the expression levels of cleaved caspase-3, Bax, α-SMA, FN, Col-Ⅰand MDA in peritoneum were significantly higher, while HO-1, N-Nrf2 protein expression and SOD activity were lower (all P<0.05). Compared with the PD group, the parietal peritoneum morphology of CAPE+PD group was improved, accompanied by reduced cleaved caspase-3, Bax, α-SMA, FN, Col-Ⅰ protein expression, and MDA content, while N-Nrf2, HO-1 protein expression, and SOD activity were higher (all P<0.05). Compared with the CON group, the PD group had significantly lower ultrafiltration volume and higher peritoneal permeability (both P<0.05). After CAPE intervention, the peritoneal transport function of the rats was significantly improved ( P<0.05). In cultured PMCs, PDF inhibited nuclear translocation of Nrf2 and protein expression of HO-1, and upregulated intracellular ROS level. In addition, PDF increased cell apoptosis and the protein expression levels of α-SMA, TGF-β1 and FN (all P<0.05). CAPE activated nuclear translocation of Nrf2, increased HO-1 protein expression, downregulated intracellular ROS level, and partially reversed PDF-induced cell apoptosis and epithelial- mesenchymal transition (all P<0.05). The protective effects of CAPE on PMCs were partially abolished by ML385 (all P<0.05). Conclusions:CAPE can reduce PD-induced PMCs apoptosis and epithelial-mesenchymal transition by attenuating oxidative stress, and significantly improve peritoneal fibrosis and ultrafiltration function. The beneficial effects of CAPE on peritoneum are related to activation of Nrf2/HO-1 pathway.