Baicalein alleviates fibrosis and inflammation in systemic sclerosis by regulating B-cell abnormalities

Abstract

Background: Systemic sclerosis (SSc; also known as "scleroderma") is an autoimmune disorder characterized by extensive fibrosis, vascular changes, and immunologic dysregulation. Baicalein (phenolic flavonoid derived from Scutellaria baicalensis Georgi) has been used to treat the pathological processes of various fibrotic and inflammatory diseases. In this study, we investigated the effect of baicalein on the major pathologic characteristics of SSc: fibrosis, B-cell abnormalities, and inflammation.

Methods: The effect of baicalein on collagen accumulation and expression of fibrogenic markers in human dermal fibroblasts were analyzed. SSc mice were produced by injecting bleomycin and treated with baicalein (25, 50, or 100 mg/kg). The antifibrotic features of baicalein and its mechanisms were investigated by histologic examination, hydroxyproline assay, enzyme-linked immunosorbent assay, western blotting and flow cytometry.

Results: Baicalein (5-120 μM) significantly inhibited the accumulation of the extracellular matrix and fibroblast activation in transforming growth factor (TGF)-β1- and platelet derived growth factor (PDGF)-induced human dermal fibroblasts, as evidenced by abrogated deposition of total collagen, decreased secretion of soluble collagen, reduced collagen contraction capability and downregulation of various fibrogenesis molecules. In a bleomycin-induced model of dermal fibrosis in mice, baicalein (25-100 mg/kg) restored dermal architecture, ameliorated inflammatory infiltrates, and attenuated dermal thickness and collagen accumulation in a dose-dependent manner. According to flow cytometry, baicalein reduced the proportion of B cells (B220⁺ lymphocytes) and increased the proportion of memory B cells (B220⁺CD27⁺ lymphocytes) in the spleens of bleomycin-induced mice. Baicalein treatment potently attenuated serum levels of cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-17A, tumor necrosis factor-α), chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1 beta) and autoantibodies (anti-scleroderma 70 (Scl-70), anti-polymyositis-scleroderma (PM-Scl), anti-centromeres, anti-double stranded DNA (dsDNA). In addition, baicalein treatment can significantly inhibit the activation of TGF-β1 signaling in dermal fibroblasts and bleomycin-induce mice of SSc, evidenced by reducing the expression of TGF-β1 and IL-11, as well as inhibiting both small mother against decapentaplegic homolog 3 (SMAD3) and extracellular signal-related kinase (ERK) activation.

Conclusions: These findings suggest that baicalein has therapeutic potential against SSc, exerting modulating B-cell abnormalities, anti-inflammatory effects, and antifibrosis.

Keywords: Autoimmunity; B cell; Baicalein; Fibrosis; Inflammation; Scleroderma.

Fig. 1

Baicalein inhibits TGF-β1- (A) or PDGF (B)-induced fibrosis in CCC-ESF-1 cells. Cells were seeded in collagen I-coated plates and treated/not treated with TGFβ1 (5 ng/mL) or PDGF (40 ng/mL) in the presence or absence of baicalein (5–120 μM) for 48 h. Total collagen deposition was visualized by PSR staining and quantified by spectrophotometry. Soluble collagen in the supernatant was detected by the Sircol Soluble Collagen Assay. Cytotoxicity was assessed using the lactate dehydrogenase release (LDH) assay. Representative images were shown, scale bar = 200 μm. Quantitative data were the mean ± SD from one representative experiment out of ≥ 3 experiments with similar results, n ≥ 4 wells per group. ^#P < 0.05, ^##P < 0.01 vs. no TGF-β1 or PDGF group; *P < 0.05, **P < 0.01 vs. TGF-β1- or PDGF-only group

Fig. 2

Baicalein suppresses the expression of fibrogenesis markers and TGF-β1 signaling pathway in dermal fibroblasts. Cells were treated with TGFβ1 (5 ng/mL) or PDGF (40 ng/mL) in the presence or absence of baicalein (5–120 μM) for 48 h, and collected for the collagen contractibility of dermal fibroblasts by collagen gel contraction assay (A), RT-qPCR (B, C), ELISAs (D) and western blotting (E). Quantitative data were the mean ± SD of 3–5 replicates. Each experiment was repeated thrice. ^#P < 0.05, ^##P < 0.01 vs. no TGF-β1 or PDGF group; *P < 0.05, **P < 0.01 vs. TGF-β1- or PDGF-only group

Fig. 3

Baicalein attenuates fibrosis in mice suffering from bleomycin-induced SSc. Bleomycin (1 mg/mL; 100 μL/mice) was injected subcutaneously into the back of mice once daily for 4 weeks. Mice underwent oral gavage with vehicle control (0.5% carboxymethylcellulose sodium), D-penicillamine (125 mg/kg), or baicalein (BA, 25–100 mg/kg) once daily for 4 weeks. Representative images of the H&E and Masson’s-trichrome staining of the skin of mice in each group were shown. In H&E, scale bar = 100 μm (upper) and scale bar = 50 μm (lower). In Masson’s-trichrome, scale bar = 100 μm (A). Dermal thickness (n = 9), hydroxyproline content (n = 9) and inflammation score (n = 6–7) were quantified (B). C protein from lesional skins was analyzed by western blotting for COL1A1, α-SMA, TGF-β1/SMAD3 activation and ERK activation in randomly selected mice of each group. Levels of expression were measured by densitometry and expressed as normalized density (n = 3–4). Representative immunoblots for 3 mice of each group were shown. Quantitative data were the mean ± SEM. ^#P < 0.05, ^##P < 0.01 vs. NaCl-treated mice. * P < 0.05, ** P < 0.01 vs. bleomycin (BLM)-treated mice

Fig. 4

Baicalein reduces serum levels of cytokines and chemokines in mice with bleomycin-induced SSc. Bleomycin (1 mg/mL; 100 μL/mice) was injected subcutaneously into the back of mice once daily for 4 weeks. Mice were oral gavage with vehicle control (0.5% carboxymethylcellulose sodium) or baicalein (BA, 25–100 mg/kg) once daily for 4 weeks. Serum was collected for cytokine/chemokine analysis using MILLIPLEX™ MAP. Quantitative data were the mean ± SEM from 7–10 mice in each group. ^#P < 0.05, ^##P < 0.01 vs. NaCl-treated mice. * P < 0.05, ** P < 0.01 vs. bleomycin (BLM)-treated mice

Fig. 5

Baicalein inhibits bleomycin-induced autoantibody overproduction in serum. Quantitative data were the mean ± SEM from 7–10 mice in each group. ^#P < 0.05, ^##P < 0.01 vs. NaCl-treated mice. * P < 0.05, ** P < 0.01 vs. bleomycin (BLM)-treated mice

Fig. 6

Baicalein inhibits abnormalities of B-cell distribution in a bleomycin-induced SSc model in mice. Mice were treated for 4 weeks. Spleen cells were collected to calculate the proportion of B cells (A) and CD27⁺ memory B cells (B). Quantitative data were the mean ± SEM from 9 mice in each group. ^#P < 0.05, ^##P < 0.01 vs. NaCl-treated mice. * P < 0.05, ** P < 0.01 vs. bleomycin (BLM)-treated mice