Baicalein Selectively Induces Apoptosis in Activated Lymphocytes and Ameliorates Concanavalin A-Induced Hepatitis in Mice

Yan Zhang,Dan Wang,Lei Shan,Weidong Zhang,Yaping Hua,Zhenlin Hu,Huawu Zeng,Runhui Liu,Helene F Rosenberg

doi:10.1371/journal.pone.0069592

Yan Zhang, Dan Wang + Show 7 more

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https://doi.org/10.1371/journal.pone.0069592

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Abstract

BackgroundInsufficient apoptosis in activated lymphocytes contributes to the development of autoimmune hepatitis (AIH). Baicalein (BE), a flavonoid originally isolated from the root of Scutellaria baicalensis Georgi, possesses anti-inflammatory properties. However, whether BE can selectively induce apoptosis in activated lymphocytes and exert therapeutic effect on AIH has not been studied.Methodology/Principal FindingsThe pro-apoptotic properties of BE were evaluated in vitro on different types of immune cells, and in vivo effects of BE were examined in a murine model of Concanavalin A (Con A)-induced hepatitis. In vitro treatment with BE resulted in a higher increase in the level of apoptosis in Con A-stimulated murine splenocytes, Con A-stimulated CD3+ splenocytes, lipopolysaccharide (LPS)-stimulated CD19+ splenocytes, and phorbol 12-myristate 13-acetate/ionomycin-stimulated Jurkat T cells, compared with that in unstimulated naïve ones. Murine bone marrow-derived dentritic cells, peritoneal macrophages, and RAW264.7 cells, either stimulated with LPS or unstimulated, were all insensitive to the BE-induced apoptosis. BE treatment also led to a loss of mitochondrial membrane potential, an increase of cytochrome c release from mitochondria to the cytosol, a decrease in the ratio of Bcl-2/Bax, and activation of caspase-9,-3 in Con A-stimulated CD3+ splenocytes and LPS-stimulated CD19+ splenocytes, while showing no impact on Fas/FasL expressions and caspase-8 activation. In vivo administration of BE alleviated Con A-induced liver injury, suppressed serum level of TNF-α and IFN-γ, and reduced liver infiltration of mononuclear cells (MNCs). Furthermore, BE treatment increased the incidences of apoptosis in liver-infiltrating MNCs and splenocytes, as well as in CD3+ and CD19+ splenocytes. When liver MNCs and splenocytes from BE-treated mice were cultured in vitro for 24 h, they exhibited marked increase in apoptosis compared to vehicle-treated control.Conclusions/SignificanceThe present study demonstrates the ability of BE to promote apoptosis in activated lymphocytes through mitochondrial pathway and its potential use in the treatment of AIH.

Highlights

Autoimmune hepatitis (AIH) is a disease characterized by progressive liver inflammation of unknown etiology that may advance to fibrosis and cirrhosis [1]
For induction of cell activation, splenocytes and T lymphocytes were stimulated with 5 mg/ml of Concanavalin A (Con A); B lymphocytes, Bone marrow (BM)-derived Dendritic cells (DCs), peritoneal macrophages, and RAW264.7 cells were stimulated with 500 ng/ml of LPS; Jurkat T cells were stimulated with 25 ng/mL of phorbol 12-myristate 13-acetate (PMA) plus 1 mM of ionomycin
The results showed that during the in vitro culture period, the frequency of apoptotic cells raised more readily in the macrophage-depleted live mononuclear cells (MNCs) and splenocytes obtained from mice treated with Con A and BE than in the cells from mice treated with Con A only, so that by 24 h of in vitro culture, the increase in the frequency of apoptotic cells in Con A+BE group became more apparent compared with Con A only control (Fig. 6A)

Summary

Introduction

Autoimmune hepatitis (AIH) is a disease characterized by progressive liver inflammation of unknown etiology that may advance to fibrosis and cirrhosis [1]. The pathogenic mechanisms of AIH still remain unclear. The liver inflammation in AIH encompasses both cell-mediated cytotoxicity by infiltrating lymphocytes and the production of autoantibodies. Abnormality in immune regulation is thought to be implicated in the pathogenesis of this disease [2]. The only viable treatments of AIH are immunosuppressant application and liver transplantation. Long term applications of currently available immunosuppressive drugs carries serious risks [3]. It is significantly important to develop new specific drugs. Insufficient apoptosis in activated lymphocytes contributes to the development of autoimmune hepatitis (AIH). Whether BE can selectively induce apoptosis in activated lymphocytes and exert therapeutic effect on AIH has not been studied

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