Baicalein selectively induces apoptosis in activated lymphocytes and ameliorates concanavalin a-induced hepatitis in mice

Abstract

Background: Insufficient 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 findings: The 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/significance: The 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.

Figure 1. BE preferentially induces apoptosis in mitogen-activated splennocytes.

Murine splenocytes were treated with indicated concentrations of BE for 24 h in the absence or presence of 5 µg/ml of Con A, and the percentages of apoptosis were detected using Annexin V/PI staining. (A) is a representative of three independent assays. (B) represents mean ± SEM of three independent experiments. *P<0.05 versus untreated controls.

Figure 2. BE selectively induces apoptosis in activated lymphocytes.

(A) Different types of immune cells as indicated were incubated with 10 µM of BE for 24 h in the absence or presence of activator, and the percentages of apoptosis were detected using Annexin V/PI staining. The increase in the percentages of apoptosis in each type of cells upon BE exposure was calculated. Following activators were used: 5 µg/ml of Con A for splenocytes and CD3⁺ splenocytes; 500 ng/ml of LPS for CD19⁺ splenocytes, BM-derived DCs, peritoneal macrophages, and RAW264.7 cells; 25 ng/mL of PMA and 1 µM of ionomycin for Jurkat T cells. Data are mean ± SEM of three independent experiments. *P<0.05 versus BE only control. (B) CD3⁺ splenocytes, CD19⁺ splenocytes, and Jurkat T cells were treated with indicated concentrations of BE for 24 h in the absence or presence of respective activator, and the percentages of apoptosis were detected using Annexin V/PI staining. Data are mean ± SEM of three independent experiments. *P<0.05 versus untreated controls.

Figure 3. BE selectively induces apoptosis of Con A-activated CD3⁺ T cells through mitochondrial pathway.

CD3⁺ T cells were isolated from murine splenocytes using Miltenyi MACS Purification and incubated with indicated concentrations of BE for 24 h in the absence or presence of 5 µg/ml of Con A. (A–E) the percentages of Annexin V⁺, Fas⁺, and FasL⁺ cells were analyzed using PE-anti-Fas mAb/annexin V-FITC or PE-anti-FasL mAb/annexin V-FITC staining. A is a representative of three independent assays with PE-anti-Fas mAb/annexin V-FITC staining. B is a representative of three independent assays with PE-anti-FasL mAb/annexin V-FITC staining. C–E represents mean ± SEM of three independent experiments. (F, G) Loss of ΔΨm in T cells was analyzed using JC-1 staining. F is a representative of three independent assays, and G represents mean ± SEM of three independent experiments. (H) The release of cytochrome c (Cyto-c) from mitochondria in T cells after BE treatment in the presence of Con A was examined by Western blotting. (I) Protein levels of Bcl-2 and Bax in T cells after BE treatment in the presence of Con A were examined by Western blotting. The results shown in H and I are representative of three experiments. (J) The activities of caspase-3, 8, 9 in T cells after BE treatment in the presence of Con A was measured using colorimetric assay. Each column represents the mean ± SEM of 3 experimental values. *P<0.05 versus untreated controls.

Figure 4. BE selectively induces apoptosis of LPS-activated CD19⁺ B cells through mitochondrial pathway.

CD19⁺ B cells were isolated from murine splenocytes using Miltenyi MACS Purification and incubated with indicated concentrations of BE for 24 h in the absence or presence of 10 µg/ml of LPS. (A, B) The percentages of cells with low ΔΨm were analyzed by flow cytometry using JC-1 staining. (C, D) The percentages of active caspase-3+ cells were analyzed by flow cytometry using FITC-anti-active caspase-3 mAb staining. A and C demonstrate representative experiment of three assays. B and D show mean ± SEM of three independent experiments. (E) The release of cytochrome c (Cyto-c) from mitochondria in B cells after BE treatment in the presence of LPS was examined by Western blotting. (F) Protein levels of Bcl-2 and Bax in B cells after BE treatment in the presence of LPS were examined by Western blotting. The results shown in E and F are representative of three experiments. (G) The activities of caspase-3, 8, 9 in B cells after BE treatment in the presence of LPS was measured using colorimetric assay. Each column represents the mean ± SEM of 3 experimental values. *P<0.05 versus untreated controls.

Figure 5. BE protected mice against Con A-induced hepatitis and increased the incidences of apoptosis in liver-infiltrating MNCs and splenocytes, as well as in CD3⁺ and CD19⁺ splenocytes.

Murine hepatitis was induced by an intravenous injection of Con A at a dose of 15 mg/kg. BE (100 mg/kg) was administrated intraperitoneally immediately after Con A injection. Blood samples were collected at 0, 8, and 24 h after Con A injection. Livers and spleens were collected 24 h after Con A injection. (A) Serum ALT levels at indicated time points after Con A injection. (B) Serum levels of IFN-γ and TNF-α at indicated time points after Con A injection. (C) Photomicrographs of representative H&E stained liver sections (×200). (D) Cell numbers of MNCs, CD3⁺ T cell and CD19⁺ B cells in livers at 24 h after Con A injection. (E) Percentages of apoptosis in liver MNCs detected by flow cytometry using FITC-annexin V/PI staining. (F and G) Flow cytometry analysis of apoptosis in splenic T and B cells using PE-anti-CD3/staining and PE-anti-CD19/FITC-annexin V staining, respectively. F shows representative results of 10 mice in each experimental group. Data in A, B D, E, and G are mean ± SEM of 10 mice/group. *P<0.05 versus Con A only control, ^# P<0.05 versus Non-treatment control.

Figure 6. The lymphocytes exposed to BE in vivo underwent increased apoptosis upon subsequent culture in vitro.

Murine hepatitis was induced by an intravenous injection of Con A at a dose of 15 mg/kg. BE (100 mg/kg) was administrated intraperitoneally immediately after Con A injection. The liver MNCs and splenocytes were prepared at 8 h after Con A injection. After 2 hours of in-vitro culture, the adherent macrophages were removed and the nonadherent cells were further cultured in vitro for 12 and 24 h. (A) The percentages of apoptosis in the in vitro cultured nonadherent cells at each time point were determined by flow cytometry using FITC-annexin V/PI staining. (B) After in vitro culture for 24 h, the absolute numbers of apoptotic cells in liver MNCs and splenocytes, as well as in CD3⁺ T cell and CD19⁺ B cell subsets, were determined by cell counting and flow cytometry using PE-anti-CD3/FITC-annexin V staining and PE-anti-CD19/FITC-annexin V staining, respectively. Data are mean ± SEM of 6–8 mice/group. *P<0.05 versus Con A only control, ^# P<0.05 versus Non-treatment control.