The present study received ethical approval from the Ethics Committee of Zhejiang Hospital. Blood samples were collected from 3 UC patients and 3 age-match healthy donors after written informed consent was obtained from all participants. Clinicopathological data of UC patients are listed in Table I. PBMCs were freshly isolated from blood samples by gradient centrifugation with lymphocyte cell separation media (Cedarlane Laboratories, Ontario, Canada) and grown in RPMI-1640 (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and penicillin/streptomycin. The PBMCs were maintained in a 37°C incubator with 5% CO₂.

PBMCs from UC patients were randomly treated with dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 10, 20 or 30 µg/ml of andrographolide (AG; Sigma-Aldrich; Merck KGaA). PBMCs from healthy donors were randomly divided into three groups: Control group, treated with DMSO; IL-23 group, treated with 50 ng/ml IL-23 (Sigma-Aldrich; Merck KGaA); and IL-23+AG group, treated with 20 µg/ml andrographolide for 2 h and then treated with 50 ng/ml IL-23. After 48 h of culture, the culture media were collected for enzyme-linked immunosorbent assay (ELISA), and PBMCs were harvested for flow cytometry and Western blot analysis.

The culture media were collected and the concentrations of IFNγ, IL-4, IL-23 and IL-17A were measured by using commercial ELISA kits (Bio-swap, Shanghai, China) according to the manufacturer's instructions. Optic densities were measured at 450 nm, and the concentrations of cytokines were calculated according to a standard curve.

The treated PBMCs were centrifuged at 1,000 rpm for 10 min. The pellet was resuspended in cultured media supplemented with PMA (100 ng/ml; Sigma-Aldrich; Merck KGaA)/ionomycin (1 µg/ml; Sigma-Aldrich; Merck KGaA) and monensin (1 µg/ml; Shanghai Aladdin Bio-Chem Technology Co., Shanghai, China) and plated onto 24-well plates (0.5×10⁵ cells/well). After incubation at 37°C for 4 h, the PBMCs were collected, resuspended in phosphate-buffered saline (PBS) and labeled with anti-CD4-FITC (BioLegend, Inc., San Diego, CA, USA) for 1 h at 4°C. Subsequently, the cells were fixed with 2% formaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Intracellular cytokine staining was then performed with anti-IFNγ-APC, anti-IL-4-PE or anti-IL-17A-PE (BioLegend, Inc.) for 1 h. The cells were detected by using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The proportions of CD4+IFNγ+ cells, CD4+IL-4+ cells and CD4+IL-17A+ cells (right upper quadrant) in CD4+ cells (right upper and lower quadrant) were calculated.

PBMCs was lysed in RIPA buffer and then centrifuged at 12,000 rpm for 20 min. The supernatant was collected, and the protein concentrations were quantified by using a BCA method. An equal amount of protein (30 µg) from each sample was loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto a nitrocellulose blotting membrane (EMD Millipore, Billerica, MA, USA). Following incubation with 5% skim milk at 4°C for 1 h, the membranes were incubated with anti-T-bet (cat. no. Ab91109, 1:500; Abcam, Cambridge, MA, USA), anti-GATA-3 (cat. no. Ab106625, 1:1,000; Abcam), anti-ROR-γt (cat.no. Ab78007, 1:1,500; Abcam) and anti-GAPDH (cat. no. 5174, 1:2,000; Cell Signaling Technology, Danvers, MA, USA) antibodies at 4°C overnight. Then, the membrane was washed three times with TBST buffer and incubated with horseradish peroxidase conjugated secondary antibody (Beyotime Institute of Biotechnology, Shanghai, China) for 1 h. Immunoreactive bands were detected using an ECL detection kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and semi-quantified by ImageJ software (http://rsb.info.nih.gov/ij/, National Institutes of Health, Bethesda, MD, USA).

Data are expressed as the means ± standard deviation (SD). Statistical analysis was performed with GraphPad Prism software (v6.0, San Diego, CA, USA). One-way analysis of variance with a Tukey's post hoc test was performed. P<0.05 was considered to indicate a statistically significant difference.

To examine the effect of andrographolide on the production of Th cell-specific cytokines, PBMCs were isolated from three UC patients and treated with 10, 20 or 30 µg/ml of andrographolide. The concentrations of cytokines in the culture media were determined by an ELISA assay. After 48 h of treatment, andrographolide decreased IFNγ, IL-23 and IL-17A but increased IL-4 in a dose-dependent manner (Fig. 1).

To analyze the effect of andrographolide on subtypes of Th cell populations, PBMCs treated with andrographolide were stained with IFNγ, IL-4 and IL-17A in CD4+ T cells, which are the respective signature cytokines of Th1, Th2 and Th17 cells. Andrographolide treatment resulted in a decreased percentage of Th1 and Th17 cells and an increased proportion of Th2 cells (Fig. 2).

The protein expression levels of the transcription factors, T-bet, GATA-3 and ROR-γt, of the T lymphocytes were measured, and the results showed that T-bet and ROR-γt expression was decreased (n = 3); however, GATA-3 expression was increased after andrographolide treatment (n=3, Fig. 3).

We next explored the effects of andrographolide pretreatment on IL-23-treated PBMCs from healthy donors. As shown in Fig. 4, IL-23 treatment significantly increased the concentrations of IFNγ, IL-23 and IL-17A but decreased the concentrations of IL-4. IL-23 exposure caused a notable increase in the percentages of IFNγ+CD4+ cells and IL-17+CD4+ but a decrease in the percentages of IL-4+CD4+ cells. Additionally, IL-23 treatment significantly increased the protein levels of T-bet and ROR-γt but reduced GATA-3 expression. Pretreatment with andrographolide significantly rescued the effects of IL-23 on PBMCs.