Materials

Thymoquinone (purity > 98%) purchased from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and BU-224 purchased from Tocris Bioscience (Fisher Scientific, Leicestershire, UK) were dissolved in normal saline. Additionally, the present study also purchased NISCH cDNA ORF clone (Origene, Rockville, MD, USA), anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA), and anti-NISCH antibody (Abcam, Cambridge, MA, USA).

Cell cultures

Human NCI-H716 cells (BCRC No. CCL-251) and CHO-K1 cells (BCRC No. CCL-61) were obtained from the Culture Collection and Research Center (BCRC) of the Food Industry Institute (Hsin-Chiu City, Taiwan). Cells subcultured every 3 to 4 days in medium containing 10% fetal bovine serum (FBS) were applied between passages 18 and 25 in the present study.

Transfection of NISCH in CHO-K1 cells

Nischarin is generally used as a functional imidazoline receptor because it is a mouse homolog of the human imidazoline receptor antisera-selective (IRAS) protein [15]. According to a previous report [16], an expression vector containing human NISCH cDNA (NISCH) (Myc-DDK-tagged human nischarin, NM_007184; Origene Technologies, Inc., Rockville, MD, USA) was stably transfected into CHO-K1 cells using the TurboFect transfection reagent (Thermo Fisher Scientific, Rockford, IL, USA). After 24 h of transfection, expression in cells was confirmed using Western blotting analysis (described below) and the cells were used to incubate with TQ for further experiments.

Western blotting analysis

The cells were lysed to extract the protein and thirty micrograms of the cell lysates were separated using 10% sodium dodecylsulfate polyacrylamide gel electrophoresis. Then, they were transferred to a polyvinyl difluoride membrane. After blocking with 10% skim milk for 1 h, the blots were developed with a primary antibody specific for NISCH. The secondary antibodies were then incubated with membranes for an additional 3 h. The mixed complexes were determined using an ECL kit (Thermo Fisher Scientific). Using a marker to verify specificity, immunoblots indicating NISCH (37 kDa) and β-actin (43 kDa) were obtained.

Estimation of intracellular calcium level

Intracellular calcium levels have been widely determined using the fluorescent probe fura-2 [14]. According to our previous report [17], NISCH-CHO-K1 cells were maintained in physiological saline solution (PSS). Fura-2 (5 mM) was added to 1 ml of the cell suspension (1 × 10⁶ cells) to incubate at 37°C for 30 min in the dark. After addition of a testing substance of vehicle, the fluorescence was continuously recorded using a fluorescence spectrofluorometer (Hitachi F-2000). Values of [Ca²⁺]_i were detected and the background in unloaded cells was subtracted from all measurements.

Silencing of imidazoline receptors in GLP-1 secreting cells

Following a previous report [18], a validated small interfering RNA (siRNA) targeting human nischarin (NISCH) was purchased from a commercial source (GE Healthcare Dharmacon, Inc. Lafayette, CO, USA). Pooled non-targeting siRNA was used as a negative control to distinguish the silencing from nonspecific effects. Lipofectamine 2000 (Thermo Fisher Scientific, Pittsburgh, PA, USA) was applied to transfect the siRNA into NCI-H716 cells. After 24 h of transfection, Western blotting analysis was then used to evaluate the success of this silencing before assays.

Evaluation of GLP-1 secretion in vitro

NCI-H716 cells were seeded in 24-well plates (5 × 10⁵ cells per well) to incubate in assay medium (DMEM 1 g/l glucose supplemented with 1 mmol/l pyruvate, 1% PenStrep, 0.1 mmol/l, DPP-IV inhibitor) containing desired concentration of TQ at 37°C for 1 h. Supernatants were collected and centrifuged (1,500 g, 5 min) to remove floating cells and debris for GLP-1 measurement. The concentrations of GLP-1 in the supernatants were then measured using an ELISA kit (Millipore, Burlington, MS, USA).

Animals

Male Sprague-Dawley (SD) rats, weighing 250 to 280 g, were supplied from the National Laboratory Animal Center (Taipei, Taiwan). Before the experiments, all protocols were approved by the Institutional Animal Ethics Committee (105051901) in Chi-Mei Medical Center. All experiments conformed to the Guide for the Care and Use of Laboratory Animals, as well as the guidelines of the Animal Welfare Act. Animals under sodium pentobarbital (35 mg/kg, i.p.) anesthesia were used in the present study to reduce their suffering.

Induction of diabetic rats

To induce the model of type 1-like diabetes, overnight fasted rats were used to receive an intravenous injection of streptozotocin (STZ) at 65 mg/kg. Success of the model was identified once they showed hyperglycemia, the plasma glucose level not less than 300 mg/dl measured from an automatic analyzer, in addition to polyuria and other diabetic features at 1 week later. The plasma insulin levels determined by a rat insulin ELISA kit (Mercodia, Uppsala, Sweden) were also extremely lowered in these rats. Then, experiments were started 2 weeks after the preparation of the diabetic model.

Determination of plasma GLP-1 levels in vivo

The plasma GLP-1 levels were estimated after the treatment with TQ at the desired dose. The treated fasting rats were received 2.5 g/kg of glucose by gavage as described previously [19]. Under anesthesia with sodium pentobarbital (35 mg/kg, i.p.), at 30 min following glucose load, blood samples (about 300 ml) collected from the femoral artery were kept in Eppendorf tubes containing EDTA and DPP-IV inhibitor (10 μl; Millipore). Blood samples were centrifuged at 1500 g for 10 min and the GLP-1 levels in plasma were then estimated using an ELISA kit (Millipore).

Changes of plasma glucose in diabetic rats that received TQ treatment

Diabetic rats received sitagliptin (10 mg/kg) daily through oral gavage to inhibit DPP-4 or the same volume of vehicle for 2 weeks. Then, TQ at the indicated dose was administered by intraperitoneal injection (i.p.) in overnight-fasted diabetic rats with or without pretreatment with sitagliptin. Additionally, varying doses of exendin 9-39 (Ex9-39; Sigma-Aldrich) were pretreated 30 min before the injection of TQ in diabetic rats with or without pretreatment with sitagliptin. Under anesthesia, blood samples were collected at 1 h after TQ administration. Then, plasma glucose levels were determined as above.

Statistical analysis

Data shown are the mean ± SEM from the sample number (n) in each group. Statistical analysis regarding the one-way analysis of variance (ANOVA), followed by Tukey’s post hoc comparison, was performed with the software SPSS 21. A p-value of 0.05 or less was defined as significant.

Direct effect of TQ on imidazoline receptors in cells transfecting NISCH

The exogenous NISCH gene transfected into CHO-K1 cells following our previous method [17] was confirmed using Western blot and it is indicated in the upper section of Figure 1. The imidazoline receptor (I-R) expressed in NISCH-CHO-K1 cells is proven to be functional [17].

Figure 1

Direct effect of thymoquinone (TQ) on imidazoline receptors (I-Rs) in vitro. Western blotting analysis confirmed the success of I-R gene transfection in CHO-K1 cells. Dose-dependent elevations in cellular calcium concentrations induced by TQ in I-R-transfected CHO-K1 cells (NISCH-CHO-K1 Cells) were observed compared with cells transfected with empty vector (CHO-K1 Cells). Values (means ± SEM) were obtained from 8 independent experiments

Then, the possible effects of TQ on I-Rs were investigated. In NISCH-CHO-K1 cells incubated with TQ, the intracellular calcium concentration was markedly elevated in a dose-dependent manner (Figure 1). However, similar changes induced by TQ treatment were not obtained in the original CHO-K1 cells that were not transfected with an exogenous I-R gene (Figure 1). Therefore, a direct effect of TQ on I-Rs could be determined.

TQ enhances GLP-1 secretion via imidazoline receptors in intestinal cells

Intestinal NCI-H716 cells have been widely used to evaluate GLP-1 secretion [20]. TQ enhanced GLP-1 secretion in NCI-H716 cells, as shown in Figure 2, and this effect of TQ was inhibited after silencing of the I-R gene, which dominated in NCI-H716 cells. Activation of I-Rs by TQ therefore enhanced GLP-1 secretion. Interestingly, this effect of TQ was also blocked by BU-224 pre-treatment in a dose-dependent manner (Figure 2).

Figure 2

Thymoquinone (TQ) treatment stimulated GLP-1 secretion in NCI-H716 cells. A – Western blots showing successful I-R silencing in NCI-H716 cells, as indicated in the upper panel. TQ treatment markedly induced GLP-1 secretion in NCI-H716 cells, as shown in scrambled RNA-treated controls. However, the effects of TQ were abolished by siRNA specific for I-Rs in NCI-H716 cells. B – TQ-induced GLP-1 secretion in NCI-H716 cells was blocked by BU-224 treatment. Each value (mean ± SEM) was obtained from 8 independent experiments

*P < 0.05 compared with the vehicle-treated group (1^st column). #P < 0.05 compared with the scramble-treated, TQ-stimulated group.

Elevation of plasma GLP-1 levels by TQ in diabetic rats

In the type 1-like diabetic rats, TQ treatment induced an increase in the plasma GLP-1 levels in a dose-dependent manner (Figure 3). Moreover, BU-224 pre-treatment also dose-dependently reversed the TQ-induced changes in these diabetic rats (Figure 3). However, similar treatment with BU-224 only at the most effective dose (1 mg/kg) did not influence the GLP-1 level in these diabetic rats compared with the vehicle-treated group (7.83 ±0.34 pg/ml vs. 7.24 ±0.65 pg/ml, p > 0.05, n = 8).

Figure 3

Effects of thymoquinone (TQ) on plasma GLP-1 levels in type 1-like diabetic rats. A – The plasma GLP-1 levels were reduced in diabetic rats (diabetes) as compared with the normal rats (first column) and administration of TQ at the indicated dose increased the plasma GLP-1 levels more markedly than in the vehicle-treated group (2nd column). B – BU-224 pretreatment inhibited the TQ-induced increase of plasma GLP-1 levels in diabetic rats. Diabetic rats were pretreated with BU-224 at the indicated dose for 30 min. Values are expressed as the mean ± SEM of 8 samples

*P < 0.05 compared with the vehicle-treated diabetic group. #P < 0.05 compared with the basal diabetic group without TQ treatment.

Functional identification of the effects of TQ on GLP-1 associated action in vivo

In type 1-like diabetic rats, TQ treatment may reduce the hyperglycemia in a dose-dependent manner (Figure 4). The plasma insulin levels in these diabetic rats (46.24 ±14.35 pmol/l, n = 8) were significantly different (p < 0.01) from those in normal rats (185.65 ±25.20 pmol/l, n = 8). TQ treatment at the most effective dose did not influence the plasma insulin levels in these diabetic rats (42.67 ±18.06 pmol/l, n = 8). Therefore, mediation of endogenous insulin in the effect of TQ seems negligible in the type-1 diabetic model. Additionally, hyperglycemia was reduced in these diabetic rats by sitagliptin treatment due to inhibition of DPP-4. The decrease in hyperglycemia by TQ was markedly potentiated by sitagliptin in these diabetic rats (Figure 4). Moreover, Ex9-39 treatment reversed the TQ-induced changes in these diabetic rats whether they received sitagliptin pre-treatment or not (Figure 4). However, Ex9-39 alone at the most effective dose did not influence the plasma glucose level in the diabetic rats compared with that in the vehicle-treated group (387.18 ±16.13 mg/dl vs. 394.53 ±17.68 mg/dl, n = 8, p > 0.05). Also, the same treatment with Ex9-39 did not modify the plasma glucose in diabetic rats treated with sitagliptin compared that in vehicle-treated rats receiving sitagliptin pre-treatment (342.53 ±16.44 mg/dl vs. 336.14 ±14.5 mg/dl, p > 0.05, n = 8).

Figure 4

Effects of thymoquinone (TQ) treatment on plasma glucose levels in type 1-like diabetic rats. A – The plasma glucose levels were measured 1 h after administration of TQ at the indicated dose in diabetic rats that received vehicle treatment or sitagliptin (10 mg/kg per day orally) for 14 days. B – Exendin 9-39 treatment inhibits the TQ-induced reduction in hyperglycemia in diabetic rats that received vehicle or sitagliptin for 14 days. Animals were pretreated with exendin 9-39 at the indicated dose for 30 min. The values are expressed as the mean ± SEM of 8 samples

*P < 0.05 compared with the vehicle-treated diabetic group (each white column at same condition). #P < 0.05 compared with the vehicle-treated diabetic rats (first white column). †P < 0.05 compared with the diabetic rats that received sitagliptin treatment (first black column).