Glycolytic Inhibitor 3-Brpa Inhibits Proliferation and Induces Apoptosis of Mouse Splenic Lymphocytes in Mixed Lymphocytes Culture
Authors: Zhou Ruiqing , He Huiqing , Guo Ziwen , Qiu Dafa , Li Weihua , Lin Shuhua , Xu Xiaojun , Liu Qifa 
Published: October 2019

Abstract
Background: T-cell activation plays a critical role in the pathogenesis of acute graft-versus-host disease (GVHD). Quiescent T cells utilize oxidative phosphorylation to generate ATP, whereas activated T cells utilize glycolysis, so use glycolysis inhibitor may be a metabolically regulator needed to control T cells induced GVHD. The mixed lymphocytes culture (MLC) was used as a model to evaluate the effect of treatment for GVHD in vitro. Glucolysis inhibitor 3-Bromopyruvic acid (3-BrPA), a glucolysis inhibitor, can effectively induce multidrug resistance leukemia cell lines apoptosis and enhanced chemotherapy-induced cytotoxity to leukemia cells.
Objective : This study aimed to study the effects of glycolytic inhibitor 3-Bromopyruvate (3-BrPA) on the proliferation, the apoptosis, the T lymphocyte subsets and the contents of cytokine IL-4 and IFN-γ in mouse spleen cells harvested from mixed lymphocyte culture.
Methods An one-way mixed lymphocyte culture system characterized by labeled responder cells with BALB/c mouse spleen cells (H-2kd) and stimulator cells with C57BL/6 mouse spleen cells (H-2kb) was established. With treatment of 3-BrPA at different concentrations (0-200 μmol/L), the CCK-8 method was applied for lymphoproliferation activity, flow cytometry for cell surface markers of CD3, CD4 and CD8, and ELISA method for the levels of cytokine IL-4 and IFN-γ in the supernatant.
Results: The CCK-8 test revealed that 3-BrPA in middle or high concentrations (over IC 30, 20 μmol/L) displayed a dose-dependent inhibitory effect on T-cell proliferation of MLC system. The IC50 were 48.6、41.2 and 41.9 μmol/L after 24 h, 36 h and 48 h of culture, respectively. FCM test discovered that the inhibitory effect mainly occurred in the CD4+ cells. After 48 h of culture, the apoptosis rate of 0, 10, 20, 50 and 100 μmol/L group were 4.86±0.88%, 5.2±1.13%, 12.63±2.97%, 18.55±4.06% and 22.47±3.61%, respectively. With treatment of 20 or 50μmol/L 3-BrPA, the levels of IFN-γ decreased obviously to 243.37±15.64 ng/L and 164.25±20.14 ng/L, compared with the control group (277.61±18.46 ng/L). The levels of IL-4 increased mildly to 33.18±5.69 ng/L and 31.06±6.06 ng/L, compared with the control group (28.64±3.97ng/L). Thus, the IFN-γ/IL-4 ratio decreased significantly.
Conclusions :The results indicated that 3-BrPA could inhibit T cells proliferation, induce apoptosis and contribute to the Th2 cytokine environment in murine mixed lymphocyte culture system.

Disclosures
Liu: National Natural Science Foundation of China (81270647, 81300445, 81200388): Research Funding; National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; National Public Health Grand Research Foundation (201202017): Research Funding; Natural Science Foundation of Guangdong Province (S2012010009299): Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; the Technology Plan of Guangdong Province of China (2012B031800403): Research Funding; the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding.