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Glycyrrhizin Prevents Hemorrhagic Transformation and Improves Neurological Outcome in Ischemic Stroke with Delayed Thrombolysis Through Targeting Peroxynitrite-Mediated HMGB1 Signaling

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Abstract

Peroxynitrite (ONOO−) and high mobility group box 1 protein (HMGB1) are important cytotoxic factors contributing to cerebral ischemia-reperfusion injury. However, the roles of ONOO− in mediating HMGB1 expression and its impacts on hemorrhagic transformation (HT) in ischemic brain injury with delayed t-PA treatment remain unclear. In the present study, we tested the hypothesis that ONOO− could directly mediate the activation and release of HMGB1 in ischemic brains with delayed t-PA treatment. With clinical studies, we found that plasma nitrotyrosine (NT, a surrogate marker of ONOO−) was positively correlated with HMGB1 level in acute ischemic stroke patients. Hemorrhagic transformation and t-PA-treated ischemic stroke patients had increased levels of nitrotyrosine and HMGB1 in plasma. In animal experiments, we found that FeTmPyP, a representative ONOO− decomposition catalyst (PDC), significantly reduced the expression of HMGB1 and its receptor TLR2, and inhibited MMP-9 activation, preserved collagen IV and tight junction claudin-5 in ischemic rat brains with delayed t-PA treatment. ONOO− donor SIN-1 directly induced expression of HMGB1 and its receptor TLR2 in naive rat brains in vivo and induced HMGB1 in brain microvascular endothelial b.End3 cells in vitro. Those results suggest that ONOO− could activate HMGB1/TLR2/MMP-9 signaling. We then addressed whether glycyrrhizin, a natural HMGB1 inhibitor, could inhibit ONOO− production and the antioxidant properties of glycyrrhizin contribute to the inhibition of HMGB1 and the neuroprotective effects on attenuating hemorrhagic transformation in ischemic stroke with delayed t-PA treatment. Glycyrrhizin treatment downregulated the expressions of NADPH oxidase p47 phox and p67 phox and iNOS, inhibited superoxide and ONOO− production, reduced the expression of HMGB1, TLR2, MMP-9, preserved type IV collagen and claudin-5 in ischemic brains. Furthermore, glycyrrhizin significantly decreased the mortality rate, attenuated hemorrhagic transformation, brain swelling, blood-brain barrier damage, neuronal apoptosis, and improved neurological outcomes in the ischemic stroke rat model with delayed t-PA treatment. In conclusion, peroxynitrite-mediated HMGB1/TLR2 signaling contributes to hemorrhagic transformation, and glycyrrhizin could be a potential adjuvant therapy to attenuate hemorrhagic transformation, possibly through inhibiting the ONOO−/HMGB1/TLR2 signaling cascades.

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Funding

This work is supported by Hong Kong General Research Fund (GRF No. 17102915, GRF No. 17118717), Research Grant Council, Hong Kong SAR and Health and Medical Research Fund, Hong Kong SAR (NO. 13142901), AoE/P-705/16 Areas of Excellence Scheme, RGC, Hong Kong SAR; SIRI/04/04/2015/06 Shenzhen Basic Research Plan Project. National Natural Science Foundation of China (No. 81671164).

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Authors and Affiliations

  1. School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, SAR, China

    Hansen Chen, Binghe Guan, Xi Chen & Jiangang Shen

  2. Institute of Research and Innovation (HKU-SIRI), The University of Hong Kong-Shenzhen, Shenzhen, China

    Hansen Chen & Jiangang Shen

  3. School of Medical Technology, Xuzhou Medical University, Xuzhou, 221002, China

    Bin Wang, Haiwei Pu & Suhua Qi

  4. Department of Chemistry, Morningside Laboratory for Chemical Biology, The University of Hong Kong, Hong Kong, SAR, China

    Xiaoyu Bai & Dan Yang

  5. Department of Core Facility, The People’s Hospital of Bao-an, Shenzhen, China

    Xi Chen

  6. Department of Neurology, Huizhou First Hospital, Huizhou, Guangdong, China

    Jihong Liu, Caiming Li & Jinhua Qiu

  7. Department of Pharmaceutical Sciences, College of Pharmacy, The University of New Mexico, Albuquerque, NM, 87131, USA

    Kejian Liu

  8. Institution of Clinical Pharmacology, Guangzhou University of Chinese Medicine, Guangzhou, China

    Qi Wang & Jiangang Shen

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  1. Hansen Chen
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Contributions

Hansen Chen performed in vivo and in vitro experiments, analyzed data, and wrote the manuscript; Binghe Guan, Bin Wang, and Haiwei Pu performed clinical investigations and analyzed the data; Caiming Li, Jihong Liu, and Jinhua Qiu recruited the ischemic stroke patients, made clinical diagnosis, collected blood samples, and performed clinical studies; Xiaoyu Bai and Xi Chen performed in vitro study and conducted data analysis; Dan Yang synthesized the probes for ONOO− detection and data analysis; Kejian Liu and Qi Wang contributed to the study design and discussion; Suhua Qi contributed to the study design in vivo study, clinical investigation and manuscript preparation; Jiangang Shen received the funding, guided and designed all experiments, interpreted the results, and prepared and revised the manuscripts.

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Chen, H., Guan, B., Wang, B. et al. Glycyrrhizin Prevents Hemorrhagic Transformation and Improves Neurological Outcome in Ischemic Stroke with Delayed Thrombolysis Through Targeting Peroxynitrite-Mediated HMGB1 Signaling. Transl. Stroke Res. 11, 967–982 (2020). https://doi.org/10.1007/s12975-019-00772-1

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