Abstract
Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome featuring ectopic lipid accumulation in hepatocytes. NAFLD has been a severe threat to humans with a global prevalence of over 25% yet no approved drugs for the treatment to date. Previous studies showed that procyanidin B2 (PCB2), an active ingredient from herbal cinnamon, has an excellent hepatoprotective effect; however, the mechanism remains inconclusive. The present study aimed to investigate the protective effect and underlying mechanism of PCB2 on PA-induced cellular injury in human hepatoma HepG2 cells. Our results showed that PA-induced oxidative stress, calcium disequilibrium, and subsequent endoplasmic reticulum stress (ERS) mediated cellular injury, with elevated protein levels of GRP78, GRP94, CHOP, and hyperphosphorylation of PERK and IRE1α as well as the increased ratio of Bax/Bcl-2, which was restored by PCB2 in a concentration-dependent manner, proving the excellent antiapoptosis effect. In addition, 4-phenylbutyric acid (4-PBA), the ER stress inhibitor, increased cell viability and decreased protein levels of GRP78 and CHOP, which is similar to PCB2, and thapsigargin (TG), the ER stress agonist, exhibited conversely meanwhile partly counteracted the hepatic protection of PCB2. What is more, upregulated protein expression of p-IKKα/β, p-NF-κB p65, NLRP3, cleaved caspase 1, and mature IL-1β occurred in HepG2 cells in response to PA stress while rescued with the PCB2 intervention. In conclusion, our study demonstrated that PA induces ERS in HepG2 cells and subsequently activates downstream NLRP3 inflammasome-mediated cellular injury, while PCB2 inhibits NLRP3/caspase 1/IL-1β pathway, inflammation, and apoptosis with the presence of ERS, thereby promoting cell survival, which may provide pharmacological evidence for clinical approaches on NAFLD.
Highlights
Nonalcoholic fatty liver disease (NAFLD) is a chronic pathological syndrome caused by excessive lipid accumulation in hepatocytes and steatosis [1, 2], including a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH), and may progress to cirrhosis and hepatocellular carcinoma [1, 3]
For 8h-treated groups, there was no significance v.s. control. e cell viability of 16h-treated groups was all above 80%, and that of 24h-treated groups decreased further to 60%–70% importantly, cell viability of 125 μM palmitic acid (PA) was 66.48 ± 4.9% (P < 0.001 vs control, Figure 1(b)). e data in Figure 1(c) showed that intervention of procyanidin B2 (PCB2) from 2.5 to 100 μM led to decrease in cell viability in a concentration-dependent manner, and when it is below 20 μM of PCB2, there was no significance vs control
We used Cell Counting Kit-8 (CCK-8), a modified method of MTT assay, to assess the protective effect of PCB2 against PA. e data showed that PA (125 μM) caused significant cell damage, whereas PCB2 (2.5, 5, and 10 μM) reversed the damage in a concentration-dependent manner, with cell viability rising to 102.28 ± 15.0% in the high concentration group (P < 0.01 vs model, Figure 1(d))
Summary
Nonalcoholic fatty liver disease (NAFLD) is a chronic pathological syndrome caused by excessive lipid accumulation in hepatocytes and steatosis [1, 2], including a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH), and may progress to cirrhosis and hepatocellular carcinoma [1, 3]. Elevated circulating free fatty acids (FFAs) derived from adipose contribute to hepatic lipid accumulation [7,8,9,10]. Lipotoxicity of FFA and its metabolites leads to mitochondrial dysfunction, oxidative stress, endoplasmic reticulum stress, and apoptosis [1, 11, 12], thereby contributing to the development of NASH. Previous studies have shown that human hepatoma HepG2 cells exposed to PA are susceptible to oxidative stress and apoptosis [15]; Ca2+ disequilibrium, ERS, and subsequent apoptosis occurred in OUMS-29 (human hepatocyte line) and Huh (human hepatocellular carcinomaderived cell line) cells in response to PA stimulation [16]; PA-induced apoptosis in mouse hepatocyte AML-12 cells
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