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Glycyrrhizin protects against focal cerebral ischemia via inhibition of T cell activity and HMGB1-mediated mechanisms

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ABSTRACT

Background: Glycyrrhizin (Gly) protects against brain injury induced by stroke. We studied whether Gly achieves its protection by inhibiting T cell activity and high-mobility group box 1 (HMGB1) release in the ischemic brain.

Methods: Stroke was induced by transient middle cerebral artery occlusion in rats and mice. Gly was injected intraperitoneally before or after stroke. We measured infarction, neuroinflammatory cells, gene expressions of interferon-γ (IFNγ), IL-4, and IL-10 in CD4 T cells, HMGB1 release, and T cell proliferation in cultured splenocytes.

Results: Gly treatment reduced infarctions and neuroinflammation characterized by the infiltration of CD68-positive macrophages and myeloperoxidase-positive neutrophils, which corresponds to a reduction in the number of T cells and their subsets, CD4 and CD8 T cells, in the ischemic brain, as measured by flow cytometry. Unlike in wild-type animals, Gly did not offer protection in nude rats and severe combined immunodeficient (SCID) mice who had no T cells, while Gly reduced infarction in both nude rats and SCID mice whose T cells were reconstituted, suggesting that T cells should be the target of Gly. In addition, Gly administration inhibited T cell proliferation stimulated by ConA in in vitro assays and inhibited HMGB1 release from the ischemic brain. Furthermore, Gly attenuated gene expression of IFNγ, but not IL-4 and IL-10 in CD4 T cells. Lastly, HMGB1 promoted T cell proliferation stimulated by ConA, which was inhibited by the addition of Gly.

Conclusions: Gly blocks infarction by inhibiting IFNγ-mediated T cell activity, which is at least partly modulated by HMGB1 activity.

No MeSH data available.


Related in: MedlinePlus

The effect of Gly on rhHMGB1-mediated T cell proliferation in cultured splenocytes. a rhHMGB1 protein promoted splenocyte proliferation in vitro. Splenocytes harvested from naïve rats were cultured, and rhHMGB1 was added in a series of concentrations. The cell numbers were detected using Cell Counting Kit-8. The results suggest that rhHMGB1 dose-dependently increased splenocyte proliferation. *P < 0.05, ***P < 0.001 vs 0 ng/ml of rhHMGB1, respectively. @@@P < 0.001 between the two indicated groups. b The addition of Gly inhibited rhHMGB1-mediated T cell proliferation. Different concentrations of Gly were added to the cultured splenocytes with rhHMGB1 and ConA, and T cell proliferation ratios were measured. *P < 0.05, **P < 0.01, ***P < 0.001, respectively, between the two indicated groups. n = 5/group. OD optical density, CPM counts per minute
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Fig8: The effect of Gly on rhHMGB1-mediated T cell proliferation in cultured splenocytes. a rhHMGB1 protein promoted splenocyte proliferation in vitro. Splenocytes harvested from naïve rats were cultured, and rhHMGB1 was added in a series of concentrations. The cell numbers were detected using Cell Counting Kit-8. The results suggest that rhHMGB1 dose-dependently increased splenocyte proliferation. *P < 0.05, ***P < 0.001 vs 0 ng/ml of rhHMGB1, respectively. @@@P < 0.001 between the two indicated groups. b The addition of Gly inhibited rhHMGB1-mediated T cell proliferation. Different concentrations of Gly were added to the cultured splenocytes with rhHMGB1 and ConA, and T cell proliferation ratios were measured. *P < 0.05, **P < 0.01, ***P < 0.001, respectively, between the two indicated groups. n = 5/group. OD optical density, CPM counts per minute

Mentions: We then examined the hypothesis that HMGB1 promotes T cell activation. Indeed, when rhHMGB1 protein was added to a cell culture of splenocytes, it dose-dependently promoted T cell proliferation stimulated by ConA (Fig. 8a), suggesting that HMGB1 is a direct effector on T cell function. We then examined the effect of Gly on HMGB1-mediated T cell proliferation. T cell proliferation stimulated by ConA in the presence of HMGB1 was evaluated, and we showed that HMGB1 promoted T cell proliferation but that proliferation was inhibited by Gly in a dose-dependent pattern (Fig. 8b).Fig. 8


Glycyrrhizin protects against focal cerebral ischemia via inhibition of T cell activity and HMGB1-mediated mechanisms
The effect of Gly on rhHMGB1-mediated T cell proliferation in cultured splenocytes. a rhHMGB1 protein promoted splenocyte proliferation in vitro. Splenocytes harvested from naïve rats were cultured, and rhHMGB1 was added in a series of concentrations. The cell numbers were detected using Cell Counting Kit-8. The results suggest that rhHMGB1 dose-dependently increased splenocyte proliferation. *P < 0.05, ***P < 0.001 vs 0 ng/ml of rhHMGB1, respectively. @@@P < 0.001 between the two indicated groups. b The addition of Gly inhibited rhHMGB1-mediated T cell proliferation. Different concentrations of Gly were added to the cultured splenocytes with rhHMGB1 and ConA, and T cell proliferation ratios were measured. *P < 0.05, **P < 0.01, ***P < 0.001, respectively, between the two indicated groups. n = 5/group. OD optical density, CPM counts per minute
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016958&req=5

Fig8: The effect of Gly on rhHMGB1-mediated T cell proliferation in cultured splenocytes. a rhHMGB1 protein promoted splenocyte proliferation in vitro. Splenocytes harvested from naïve rats were cultured, and rhHMGB1 was added in a series of concentrations. The cell numbers were detected using Cell Counting Kit-8. The results suggest that rhHMGB1 dose-dependently increased splenocyte proliferation. *P < 0.05, ***P < 0.001 vs 0 ng/ml of rhHMGB1, respectively. @@@P < 0.001 between the two indicated groups. b The addition of Gly inhibited rhHMGB1-mediated T cell proliferation. Different concentrations of Gly were added to the cultured splenocytes with rhHMGB1 and ConA, and T cell proliferation ratios were measured. *P < 0.05, **P < 0.01, ***P < 0.001, respectively, between the two indicated groups. n = 5/group. OD optical density, CPM counts per minute
Mentions: We then examined the hypothesis that HMGB1 promotes T cell activation. Indeed, when rhHMGB1 protein was added to a cell culture of splenocytes, it dose-dependently promoted T cell proliferation stimulated by ConA (Fig. 8a), suggesting that HMGB1 is a direct effector on T cell function. We then examined the effect of Gly on HMGB1-mediated T cell proliferation. T cell proliferation stimulated by ConA in the presence of HMGB1 was evaluated, and we showed that HMGB1 promoted T cell proliferation but that proliferation was inhibited by Gly in a dose-dependent pattern (Fig. 8b).Fig. 8

View Article: PubMed Central - PubMed

ABSTRACT

Background: Glycyrrhizin (Gly) protects against brain injury induced by stroke. We studied whether Gly achieves its protection by inhibiting T cell activity and high-mobility group box 1 (HMGB1) release in the ischemic brain.

Methods: Stroke was induced by transient middle cerebral artery occlusion in rats and mice. Gly was injected intraperitoneally before or after stroke. We measured infarction, neuroinflammatory cells, gene expressions of interferon-&gamma; (IFN&gamma;), IL-4, and IL-10 in CD4 T cells, HMGB1 release, and T cell proliferation in cultured splenocytes.

Results: Gly treatment reduced infarctions and neuroinflammation characterized by the infiltration of CD68-positive macrophages and myeloperoxidase-positive neutrophils, which corresponds to a reduction in the number of T cells and their subsets, CD4 and CD8 T cells, in the ischemic brain, as measured by flow cytometry. Unlike in wild-type animals, Gly did not offer protection in nude rats and severe combined immunodeficient (SCID) mice who had no T cells, while Gly reduced infarction in both nude rats and SCID mice whose T cells were reconstituted, suggesting that T cells should be the target of Gly. In addition, Gly administration inhibited T cell proliferation stimulated by ConA in in vitro assays and inhibited HMGB1 release from the ischemic brain. Furthermore, Gly attenuated gene expression of IFN&gamma;, but not IL-4 and IL-10 in CD4 T cells. Lastly, HMGB1 promoted T cell proliferation stimulated by ConA, which was inhibited by the addition of Gly.

Conclusions: Gly blocks infarction by inhibiting IFN&gamma;-mediated T cell activity, which is at least partly modulated by HMGB1 activity.

No MeSH data available.


Related in: MedlinePlus