Ginkgo biloba extract attenuates the disruption of pro-and anti-inflammatory T-cell balance in peripheral blood of arsenicosis patients

Abstract

Endemic arsenicosis is a public health problem that affects thousands of people worldwide. However, the biological mechanism involved is not well characterized, and there is no specific treatment. Exposure to arsenic may be associated with immune-related problems. In the present work, we performed an investigation to determine whether the Th17/Treg balance was abnormal in peripheral blood mononuclear cells (PBMCs) of patients with arsenicosis caused by burning coal. Furthermore, we investigated the effect of Ginkgo biloba extract (GBE) on the Th17/Treg imbalance in patients with arsenicosis. In this trial, 81 arsenicosis patients and 37 controls were enrolled. The numbers of Th17 and Treg cells, as well as related transcription factors and serum cytokines, were determined at the beginning and end of the study. Patients with arsenicosis exhibited higher levels of Th17 cells, Th17-related cytokines (IL-17A and IL-6), and the transcription factor RORγt. There were lower levels of Treg cells, a Treg-related cytokine (IL-10), and the transcription factor Foxp3 as compared with controls. There was a positive correlation between the levels of Th17 cells and IL-17A and the levels of arsenic in hair. Arsenicosis patients were randomly assigned to a GBE treatment group or a placebo group. After 3 months of follow-up, 74 patients completed the study (39 cases in the GBE group and 35 in the placebo group). Administration of GBE to patient upregulated the numbers of Treg cells and the level of IL-10 and downregulated the numbers of Th17 cells and the levels of cytokines associated with Th17 cells. The mRNA levels of Foxp3 and RORγt were increased and decreased, respectively. These results indicated that exposure to arsenic is associated with immune-related problems. The present investigation describes a previously unknown mechanism showing that an imbalance of pro- and anti-inflammatory T cells is involved in the pathogenesis of arsenicosis and that a GBE exerts effects on arsenicosis through regulation of the pro- and anti-inflammatory T cell balance.

Keywords: Arsenicosis; Ginkgo biloba extract; Regulatory T cells.; T helper 17 cells.

Figure 1

Study-flow diagram.37 normal controls and 81 arsenicosis patients were investigated for the effects of arsenicosis on the balance of Th17/Treg cell in PBMCs and the levels of IL-17A, IL-6, and IL-10 levels in serum. The 81 arsenicosis patients were divided into two randomly allocated groups, with 39 in the placebo group and 42 in the GBE group. After 3 months, 35 (loss of 4 from 39) subjects in the placebo group and 39 (loss of 3 from 42) subjects in the GBE group were examined for the effects of GBE on the Th17/Treg cell balance of PBMCs and on the levels of IL-17A, IL-6, and IL-10 in serum.

Figure 2

Arsenicosis disrupts the balance of Th17/Treg cell in PBMCs.PBMCs: Peripheral blood mononuclear cells; Th17: T helper 17 cells; Treg: Regulatory T cell; RORγt: Retinoic acid-related orphan nuclear receptor γt; Foxp3: Forkhead/winged helix transcription factor 3. PBMCs were obtained from normal subjects (n=37) and from arsenicosis patients (n=81). (A) Representative flow cytometry (FCM) pictures of CD4⁺IL-17A⁺ Th17 cells and CD4⁺CD25⁺FoxP3⁺Treg cells in PBMCs of subjects. (B) The percentages of Th17 and Treg cells in PBMCs were measured by FCM. The mRNA levels of RORγt (C) and Foxp3 (D) were determined by qRT-PCR. Results are given as means ±SD. * P < 0.05, arsenicosis patients versus controls.

Figure 3

Arsenicosis causes increases of IL-17A and IL-6 levels and decreases of IL-10 levels in serum.IL: interleukin. Serum were obtained from normal subjects (n=37) and arsenicosis patients (n=81). The levels of IL-17A (A), IL-6 (B), and IL-10 (C) in serum were measured using ELISA. Results are given as means ±SD. * P < 0.05, arsenicosis patients versus controls.

Figure 4

GBE reduces arsenicosis-induced disruption of the Th17/Treg cell balance in PBMCs.After arsenicosis patients were administered placebo (group, n=35) or GBE (group, n=39) for 3 months, PBMCs were obtained as described in Methods. (A) Representative FCM pictures of CD4⁺IL-17A⁺ Th17 cells and CD4⁺CD25⁺FoxP3⁺Treg cells in the two groups. The percentage of Th17 cells (B) and Treg cells (C) in PBMCs were measured by FCM. The mRNA levels of RORγt (D) and Foxp3 (E) were measured by qRT-PCR. Results are given as means ±SD. * P <0.05 versus before treatment.

Figure 5

GBE prevents increases of IL-17A and IL-6 levels and decreases of IL-10 levels in serum of arsenicosis patients. After arsenicosis patients were administered placebo (group, n=35) or GBE (group, n=39) for 3 months, sera were obtained as described in Methods. The levels of IL-17A (A), IL-6 (B), and IL-10 (C) in serum were measured using ELISA. Results are given as means ±SD; * P <0.05 versus before treatment.

Figure 6

A schematic diagram of how GEB attenuates the arsenicosis-induced imbalance of Th17/Treg cells.Arsenicosis causes increases of the transcription factor RORγt, which elevates the levels of Th17 cells and Th17-related cytokines (IL-17A and IL-6). However, arsenicosis induces decreases of the transcription factor Foxp3, which decreases the levels of Treg cells and a Treg-related cytokine (IL-10). GEB attenuates the arsenicosis-induced imbalance of Th17/Treg cells, which indicates that the imbalance of Th17/Treg cells may be involved in the pathogenesis of arsenicosis and that GEB may be involved in the treatment of arsenicosis through influencing the Th17/Treg balance.