Baicalein and baicalin (Fig. 1A) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). For the in vitro study, the drugs were used at a concentration of 100 µM baicalein and 200 µM baicalin. The compounds were added 1 h prior to cell activation. For the in vivo study, baicalein and baicalin were both used at a dose of 20 mg/kg.

All animal procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of the Animal Biosafety Level 3 Laboratory (of Wuhan University (Wuhan, China). The protocol was approved by the Committee on the Ethics of Animal Experiments of Wuhan University (permit no. SCXK 2008-0004).

A total of 50 C57BL/6J male mice (6–8 weeks of age; weight, 25 g) were obtained from the Animal Biosafety Level 3 Laboratory of Wuhan University. The mice were housed under standard laboratory conditions (a 12-h light/dark cycle with an average room temperature of 23°C and a relative humidity of 55%, with food and water available ad libitum). Mice received a subcutaneous injection of 0.3 ml collagen (Sigma-Aldrich; Merck KGaA) at the base of the tail. The vehicle group was treated with PBS. Affected joints were counted daily for 35 days, mice were sacrificed at the end of the treatment with baicalein/baicalin and the synovial fluids (SF) were processed for ELISA analysis.

Colitis was induced in mice with 2.5% w/v DSS (MP Biomedicals, LLC, Santa Ana, CA, USA) provided ad libitum for 9 days. DSS induces mucosal damage in the colon, resulting in an influx of commensal flora to the submucosal layers; this induces an immune response that closely parallels human colitis. Control groups received tap water only, DSS group received 2.5% DSS, DSS + baicalein group received 2.5% DSS and baicalein treatment, and DSS + baicalin group was treated with 2.5% DSS and baicalin.

Cells The cells are colon epithelial cells isolated from DSS mice and the lymphocytes isolated from the synovial fluid of CIA mice. For isolation of colon epithelial cells, the entire colon was dissected out and cut longitudinally. The feces were removed and the lumen was cleaned with PBS. The colon was subsequently cut into 1-cm-long pieces, transferred into a 50 ml conical flask containing PBS and stirred vigorously at 37°C for 30 min. The tissue was subsequently strained through a tea strainer and the debris was washed in PBS to isolate any residual epithelial cells. The collected PBS solution was centrifuged at 1,000 × g for 5 min at room temperature. The cells were then suspended in 1 ml PBS, added to a Percoll gradient (40 and 25% Percoll solutions, GE Healthcare, Chicago, IL, USA), and subjected to density gradient centrifugation for 25–30 min at 1,000 × g at room temperature. Subsequently, the cells at the interface (colon epithelial cells) were collected, washed twice with PBS and used for isolation of RNA. CD4⁺ T cells were sorted by fluorescence-activated cell sorting from the SF of CIA mice. FACS for CD4⁺ T cells was performed using the BD FACS Aria flow cytometer (BD Biosciences, San Jose, CA, USA) by gating on CD4⁺ cells after Fc block. Cells were washed once with staining buffer [Pharmingen Stain Buffer containing bovine serum albumin (BSA) proteins; BD Biosciences; cat. no. 554657]. The supernatant was discarded and cells were resuspend in the staining buffer at a density up to 50×10⁶ cells/ml to maximize the efficiency of staining. For lymphocytes expressing high levels of Fc receptor (FcR), a monoclonal antibody that binds to FcgR (Purified Rat Anti-Mouse CD16/CD32; Mouse BD Fc Block™; cat. no. 553142; BD Biosciences) was used to bind the receptor on ice for 10–15 min. Subsequently, CD4 mAb (FITC Rat Anti-Mouse CD4; cat. no. 553046; BD Biosciences) was used to detect the desired cell population by incubation for 20–30 min on ice in the dark, followed by two washes with Pharmingen Stain Buffer containing BSA proteins. Finally, the cell sorting analysis was performed at 4°C. The data were analyzed with the FlowJo software version 6.3.2 (Tree Star, Inc., Ashland, OR, USA). Cells were treated with baicalein/baicalin in vitro and the culture period was 8 h. Total RNA was extracted from the cells using the RNeasy mini kit (Qiagen China Co., Ltd., Shanghai, China), followed by cDNA synthesis using the Superscript III first strand synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 25°C for 10 min, 50°C for 30 min and 85°C for 5 min. qPCR was performed on a Bio-Rad amplifier using the Bio-Rad real time PCR mix, including SYBR Green dye (both Bio-Rad Laboratories, Inc., Hercules, CA, USA). The following thermocycling conditions were used for the PCR: 50°C for 2 min, 10 min at 95°C; 40 cycles of 95°C for 15 sec and 60°C for 1 min. Data were analyzed using the Cq value normalized to the endogenous reference gene GAPDH (14). The following primers were used for the PCR: Ki-67 (forward: 5′-ACCGTGGAGTAGTTTATCTGGG-3′; reverse: 5′-TGTTTCCAGTCCGCTTACTTCT-3′); STAT3 (forward: 5′-CAATACCATTGACCTGCCGAT-3′; reverse: 5′-GAGCGACTCAAACTGCCCT-3′); STAT4 (5′-forward: TGGCAACAATTCTGCTTCAAAAC-3′; reverse: 5′-GAGGTCCCTGGATAGGCATGT-3′); STAT6 (5′-forward: CTCTGTGGGGCCTAATTTCCA-3′; reverse: 5′-CATCTGAACCGACCAGGAACT-3′); GAPDH (5′-forward: AGGTCGGTGTGAACGGATTTG-3′; forward: 5′-TGTAGACCATGTAGTTGAGGTCA-3′).

Cytokine production was measured using commercial kit for interleukin (IL)-2 (cat. no. DY402), interferon (IFN)-γ (cat. no. DY485), tumor necrosis factor (TNF)-α (cat. no. DY410; all R&D Systems, Inc., Minneapolis, MN, USA), IL-6 (cat. no. 550950) and IL-17 (cat. no. 555067; both BD Pharmingen; BD Biosciences), according to the manufacturers' protocols.

Cells were incubated with MTT reagent (5 µg/ml final concentration) at 37°C for 4 h. Formazan was solubilized by adding 100 µl DMSO into each well. The extent of formazan production was determined by an ELISA reader at a wavelength of 550 nm, while 630 nm served as the reference wavelength. The results were calculated according to the manufacturer's instructions (Vybrant™ MTT Cell Proliferation Assay kit; Thermo Fisher Scientific, Inc.).

The colon was washed with cold PBS, measured and weighed. It was Swiss-rolled and sections were stained with hematoxylin and eosin as described previously (15). A histopathological score was generated in a blinded fashion using a widely used grading tool (16) examining crypts, epithelia, goblet cells, cellular infiltration and edema. Scores of 0–1 reflect normal morphology, and 2–4, 5–7 and 8–10 represent mild, moderate, and severe colitis, respectively.

The data were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) to evaluate the differences between the groups. The data are reported as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference. One-way analysis of variance with the Student-Newman-Keuls post hoc test was used to calculate the differences between the multiple comparison groups. The Mann-Whitney U test was used to analyze the differences between two groups.

Baicalein and baicalin administration exhibited protective activity in two animal models in vivo. Firstly, the effect on the mouse CIA model was investigated. Mice developed stable arthritis symptoms on day 10 and the severity of the disease in the vehicle-treated group worsened continuously with time. The baicalein-treated group had a lower arthritis score and fewer affected joints compared with the vehicle group during the entire course of treatment, while baicalin exerted no significant effect on the CIA model (Fig. 1B and C). This result was confirmed by measuring cytokine production in the SF. Baicalein was able to suppress Th1-type cytokine production, including IFN-γ, TNF-α and IL-2, in addition to the Th17-type cytokine IL-17, suggesting that it may inhibit T cell function in vivo (Fig. 1D).

Furthermore, the role of baicalein in T cell proliferation was investigated. Treatment with 100 nM baicalein in vitro significantly inhibited the proliferation of CD4⁺ T cells sorted from the SF of CIA mice upon anti-CD3 Ab activation, demonstrated via the MTT assay, and supported by the Ki67 expression analysis using qPCR (Fig. 2A and B). To elucidate the primary mechanism underlying the role of baicalein in proliferation, JAK/STAT pathway gene expression was assessed and the cells were treated with baicalein in vitro. As presented in Fig. 2C, baicalein significantly reduced the expression of STAT3 and STAT4, although it did not exert the same effect on STAT6 expression in infiltrated SF T cells (Fig. 2C), suggesting that baicalein regulated joint inflammatory signaling by suppressing STAT3/STAT4 expression in the JAK/STAT pathway.

The efficacy of baicalein and baicalin was investigated in another type of autoimmune disease. A DSS-induced colitis model in mice was produced via treatment with 3% DSS. The body weight of the baicalin-treated group was increased compared with the vehicle-treated group at the peak of the disease, which was consistent with the colon length and histology scores, while baicalein exerted no effect on the DSS model (Fig. 3A-C). Similarly, the serum IL-2, IFN-γ and IL-17 levels were reduced by baicalin (Fig. 3D).

To determine the effect of baicalin on epithelial proliferation, colon epithelial cells (CECs) were isolated and treated with baicalin in vitro. Baicalin was able to promote the proliferation of CECs as measured by CCK8 staining, suggesting that it was able to repair the epithelial barrier and promote mucosal wound healing in the disease state (Fig. 4A). This was consistent with the proliferation marker Ki67 analysis using qPCR (Fig. 4B). In order to elucidate the possible mechanism underlying the effect of baicalin on CEC proliferation, the JAK-STAT signaling pathway was selected for analysis and the mRNA expression of STAT subtype genes was assessed by qPCR. It was demonstrated that baicalin may reduce the expression of the STAT4 gene locus, while having no significant effect on STAT3/STAT6 (Fig. 4C), suggesting that baicalin mediated CEC proliferation through the JAK-STAT signaling pathway.