Celastrol exerts a neuroprotective effect by directly binding to HMGB1 protein in cerebral ischemia\u2013reperfusion

Dan-Dan Liu,Piao Luo,Yongping Zhu,Xiao Chen,Jigang Wang,Peng Gao,Nan Ma,Liwei Gu,Qiuyan Guo,Junzhe Zhang,Qian Zhang

doi:10.1186/s12974-021-02216-w

Abstract

BackgroundCelastrol (cel) was one of the earliest isolated and identified chemical constituents of Tripterygium wilfordii Hook. f. Based on a cel probe (cel-p) that maintained the bioactivity of the parent compound, the targets of cel in cerebral ischemia–reperfusion (I/R) injury were comprehensively analyzed by a quantitative chemical proteomics method.MethodsWe constructed an oxygen–glucose deprivation (OGD) model in primary rat cortical neurons and a middle cerebral artery occlusion (MCAO) model in adult rats to detect the direct binding targets of cel in cerebral I/R. By combining various experimental methods, including tandem mass tag (TMT) labeling, mass spectrometry, and cellular thermal shift assay (CETSA), we revealed the targets to which cel directly bound to exert neuroprotective effects.ResultsWe found that cel inhibited the proinflammatory activity of high mobility group protein 1 (HMGB1) by directly binding to it and then blocking the binding of HMGB1 to its inflammatory receptors in the microenvironment of ischemia and hypoxia. In addition, cel rescued neurons from OGD injury in vitro and decreased cerebral infarction in vivo by targeting HSP70 and NF-κB p65.ConclusionCel exhibited neuroprotective and anti-inflammatory effects by targeting HSP70 and NF-κB p65 and directly binding to HMGB1 in cerebral I/R injury.

Highlights

Celastrol was one of the earliest isolated and identified chemical constituents of Tripterygium wilfordii Hook. f
For the cell viability assay, primary neurons were incubated in 96-well plates for 7 days and cultured in an experienced oxygen–glucose deprivation (OGD) model to evaluate the neuroprotective effect of cel and cel-p
Discussions Our studies demonstrated the following: (1) cel exhibited a neuroprotective effect against cerebral I/R injury in vitro and in vivo; (2) cel directly bound to high mobility group box 1 (HMGB1) protein; (3) cel did not affect the expression of HMGB1, it increased HSP70 and decreased NF-κB p65 expression to exert a neuroprotective effect in vitro and in vivo; (4) cel bound to Cys 106 of the disulfide bond HMGB1 isoform or Cys 23, 45, 106 of the fully reduced HMGB1 isoform; (5) cel scavenged the overproduced tumor necrosis factor (TNF)-α induced by the disulfide bond HMGB1 isoform

Summary

Introduction

Celastrol (cel) was one of the earliest isolated and identified chemical constituents of Tripterygium wilfordii Hook. f. Celastrol (cel) was one of the earliest isolated and identified chemical constituents of Tripterygium wilfordii Hook. Of the many bioactive constituents isolated from Tripterygium wilfordii, celastrol (cel) has attracted close attention for more than 70 years because. Few have focused on determining whether cel has a neuroprotective effect against cerebral ischemia–reperfusion (I/R) injury or identifying its specific binding protein targets. Neuroinflammatory processes have been implicated in the pathophysiology of multiple stages of cerebral I/R injury, and targeting neuroinflammation has always been an attractive treatment strategy for stroke [9]. The nonhistone DNA binding protein HMGB1 is primarily located in the cell nucleus and has different biological functions according to the cellular location, binding receptors and redox states. The three cysteines (Cys) located at positions 23, 45 (A box), and 106 (B box) mainly determine the redox states and physiological functions of HMGB1. The disulfide bond HMGB1 isoform is a biomarker of inflammation, which indicates that blocking the extracellular disulfide bond HMGB1 isoform may be a potential direction for the treatment of inflammation and immune-related diseases, including stroke

Methods

Results

Conclusion