α‑lipoic acid inhibits cerulein/resistin‑induced expression of interleukin‑6 by activating peroxisome proliferator‑activated receptor‑γ in pancreatic acinar cells

Abstract

Cerulein‑induced pancreatitis resembles human acute pancreatitis in terms of pathological events, such as enzymatic activation and inflammatory cell infiltration in the pancreas. Cerulein is a cholecystokinin analog that increases levels of reactive oxygen species (ROS) and interleukin‑6 (IL‑6) expression level in pancreatic acinar cells. Serum levels of resistin, which is secreted from adipocytes, are reportedly higher in patients with acute pancreatitis than in healthy individuals. Previously, it was shown that the adipokine resistin can aggravate the cerulein‑induced increase in ROS levels and IL‑6 expression level in pancreatic acinar cells. Peroxisome proliferator‑activated receptor‑gamma (PPAR‑γ) is a key regulator of the transcription and expression of antioxidant enzymes, including heme oxygenase 1 (HO‑1) and catalase. α‑lipoic acid, a naturally occurring dithiol antioxidant, can prevent cerulein‑induced pancreatic damage in rats. In the present study, it was aimed to investigate whether α‑lipoic acid can attenuate the cerulein/resistin‑induced increase in IL‑6 expression and ROS levels via PPAR‑γ activation in pancreatic acinar AR42J cells. The anti‑inflammatory mechanism of α‑lipoic acid was determined using reverse transcription‑quantitative PCR, western blot analysis, enzyme‑linked immunosorbent assay, immunofluorescence staining and fluorometry. Treatment with cerulein and resistin increased ROS levels and IL‑6 expression level, which were inhibited by α‑lipoic acid in pancreatic acinar cells. α‑lipoic acid increased the nuclear translocation and expression level of PPAR‑γ and the expression levels of its target genes: HO‑1 and catalase. The PPAR‑γ antagonist GW9662 and HO‑1 inhibitor zinc protoporphyrin reversed the inhibitory effect of α‑lipoic acid on cerulein/resistin‑induced increase in ROS and IL‑6 levels. In conclusion, α‑lipoic acid inhibits the cerulein/resistin‑induced increase in ROS production and IL‑6 expression levels by activating PPAR‑γ and inducing the expression of HO‑1 and catalase in pancreatic acinar cells.

Keywords: acute pancreatitis; interleukin‑6; peroxisome proliferator‑activated receptor‑gamma; reactive oxygen species; α‑lipoic acid.

Figure 1.

Impact of α-lipoic acid on the expression of PPAR-γ, HO-1 and catalase in unstimulated AR42J cells. Cells were treated with 5 µM α-lipoic acid for the indicated time periods. Protein levels of PPAR-γ, catalase and HO-1 in whole-cell extracts were determined by western blot analysis. Actin was used as the loading control (left panel). The densitometry data represent the means ± standard error from three immunoblots and are shown as the relative density of the protein band normalized to actin level (right panel). *P<0.05. HO-1, heme oxygenase-1; PPAR-γ, peroxisome proliferator-activated receptor-γ.

Figure 2.

Impact of α-lipoic acid on expression of PPAR-γ and its target genes catalase and HO-1 and catalase activity in cerulein/resistin-stimulated AR42J cells. Cells were pretreated with the indicated concentrations of LA for 2 h and then stimulated with Cer and Res for 45 min. (A) Protein levels of PPAR-γ, catalase and HO-1 in whole-cell extracts were determined by western blot analysis. Actin was used as the loading control (left panel). Densitometry data are presented as the means ± SE from three immunoblots and are shown as the relative density of protein band normalized to actin level (right panel). (B) Catalase activity was determined using a catalase assay kit. Data are expressed as the mean ± SE (n=12 for each group). *P<0.05. PPAR-γ, peroxisome proliferator-activated receptor-γ; HO-1, heme oxygenase-1; LA, α-lipoic acid; Cer, cerulein; Res, resistin; SE, standard error.

Figure 3.

Impact of LA on nuclear translocation of PPAR-γ in cerulein/resistin-stimulated AR42J cells. Cells were pretreated with 5 µM LA for 2 h and then stimulated with Cer and Res for 45 min. (A) Immunofluorescence staining was performed to determine the levels of PPAR-γ in the cytosolic and nuclear extracts. PPAR-γ was visualized using fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (left panel) with DAPI counterstaining (middle panel) of the same field. Scale bars, 15 µm. For each coverslip, six fields were measured. Results were obtained from four independent measurements (n=4 for each group). The intensity ratio of green (PPAR-γ) to blue (DAPI) was assessed using ImageJ v.5.0 software. (B) Levels of PPAR-γ in cytosolic or nuclear extracts were determined by western blot analysis. Aldolase A is used as a marker of cytosolic marker, while lamin B1 was used as a nuclear marker (upper panel). The densitometry data represent the mean ± standard error from three immunoblots and are shown as the relative density of protein band normalized to aldolase A or lamin B1 levels (lower panel). *P<0.05. LA, α-lipoic acid; PPAR-γ, peroxisome proliferator-activated receptor-γ; Cer, cerulein; Res, resistin; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4.

Effect of LA on the levels of intracellular ROS and IL-6 expression in cerulein/resistin-stimulated AR42J cells. Cells were pretreated with LA for 2 h and stimulated with Cer and Res for (A) 45 min, (B) 4 h, or (C) 24 h. (A) Intracellular ROS levels were measured using dichlorofluorescein diacetate. ROS levels are expressed as the relative increase. The value for ROS levels in unstimulated cells was set at 100%. (B) The mRNA expression of IL-6 was assessed by reverse transcription-quantitative PCR analysis. (B) The protein level of IL-6 in the cultured media was determined by ELISA. Data are expressed as the mean ± standard error (n=12 for each group). *P<0.05. LA, α-lipoic acid; ROS, reactive oxygen species; IL-6, intereukin-6; Cer, cerulein; Res, resistin.

Figure 5.

Impact of PPAR-γ antagonist GW9662 on the levels of intracellular ROS, IL-6, HO-1, and catalase in cerulein/resistin-stimulated AR42J cells treated with α-lipoic acid. Cells were co-treated with 10 µM GW9662 and 5 µM LA for 2 h, followed by stimulation with Cer and Res for 45 min (A,C and D), or 24 h (B). (A) Intracellular ROS levels were measured using dichlorofluorescein diacetate. Data are expressed as the mean ± SE (n=12 for each group). (B) The protein level of IL-6 in the media was determined by ELISA. (C) Protein levels of HO-1 and catalase in whole-cell extracts were determined by western blot analysis. Actin was used as the loading control (left panel). Densitometry data represent mean ± SE from three immunoblots and are shown as the relative density of protein band normalized to actin level (right panel). (D) Catalase activity was determined using a catalase assay kit. Data are expressed as the mean ± SE (n=12 for each group). *P<0.05. PPAR-γ, peroxisome proliferator-activated receptor-γ; ROS, reactive oxygen species; IL-6, intereukin-6; HO-1, heme oxygenase-1; LA, α-lipoic acid; Cer, cerulein; Res, resistin; SE, standard error.

Figure 6.

Effect of the HO-1 inhibitor ZnPP on the levels of intracellular ROS and IL-6 in cerulein/resistin-stimulated AR42J cells treated with LA. Cells were co-treated with 1 µM ZnPP and 5 µM LA for 2 h, followed by stimulation with Cer and Res for (A) 45 min or (B) 24 h. (A) Intracellular ROS levels were measured using dichlorofluorescein diacetate. (B) The protein level of IL-6 in the media was determined by ELISA. Data are expressed as the mean ± standard error (n=12 for each group). *P<0.05. HO-1, heme oxygenase-1; ZnPP, zinc protoporphyrin; ROS, reactive oxygen species; IL-6, intereukin-6; LA, α-lipoic acid; Cer, cerulein; Res, resistin.