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. 2021 Aug 9;18(1):174.
doi: 10.1186/s12974-021-02216-w.

Celastrol exerts a neuroprotective effect by directly binding to HMGB1 protein in cerebral ischemia-reperfusion

Dan-Dan Liu #  1 , Piao Luo #  1 , Liwei Gu  1 , Qian Zhang  1 , Peng Gao  1 , Yongping Zhu  1 , Xiao Chen  2 , Qiuyan Guo  1 , Junzhe Zhang  1 , Nan Ma  3   4 , Jigang Wang  5   6   7   8   9   10
Affiliations

Celastrol exerts a neuroprotective effect by directly binding to HMGB1 protein in cerebral ischemia-reperfusion

Dan-Dan Liu et al. J Neuroinflammation. .

Abstract

Background: Celastrol (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.

Methods: We 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.

Results: We 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.

Conclusion: Cel exhibited neuroprotective and anti-inflammatory effects by targeting HSP70 and NF-κB p65 and directly binding to HMGB1 in cerebral I/R injury.

Keywords: Celastrol; Cerebral ischemia–reperfusion; Chemical proteomics; High mobility group protein 1; Target identification.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1
Fig. 1
A Chemical structure and specific synthesis route of cel-p. B IC50 values of cel and cel-p against living primary neurons. C, D Cel and cel-p showed similar neuroprotective effects in an OGD model of primary neurons in vitro. The optimal dose of cel and cel-p was 0.1–0.8 μM, and the optimal administration time was continuous administration for 48 h immediately after OGD. Cel-p closely mimicked the original compound in biological activity. Error bars represent SEM. ###p < 0.001 vs. control group, *p < 0.05, **p < 0.01, ***p < 0.001 vs. model group based on a one-way analysis of variance (n = 6)
Fig. 2
Fig. 2
Cel significantly reduced the volume of cerebral infarction, improved the behavior indexes, and decreased Nissl staining injury of MCAO rats. A Representative brain slices stained by TTC. B Quantitative evaluation of infarct volume. C Zea-Longa score results indicated that cel and eda injection improved the behavior indexes of MCAO rats at 24 h, 48 h, and 72 h. D, E Compared with the sham group, extensive neuronal cell loss, nuclear shrinkage, and dark staining (at the red arrowhead) were visualized in the cortex of the model group. Administration of cel or eda markedly reduced these pathological changes. Error bars represent SEM. ###p < 0.001 vs. sham group, *p < 0.05, **p < 0.01, ***p < 0.001 vs. model group based on a one-way analysis of variance was used (n = 8, Nissl staining n = 3). Scale bar = 200 μm
Fig. 3
Fig. 3
Labeling profiles of cel-p in living primary neurons. A Concentration-dependent (0–1.6 μM) labeling profiles of cel-p in normal and OGD injury primary neurons. B Competitive labeling profiles of cel-p (0.8 μM) in normal and OGD injury primary neurons in the presence of cel (2 × , 4 × , 6 × , 8 ×). C Cel-p mainly localized in the cell cytoplasm within 2 h and gradually entered the cell nucleus after 2 h. FL, in-gel fluorescence scanning; CBB, coomassie brilliant blue gel
Fig. 4
Fig. 4
A Quantitative mass spectrometry-based profiling of cel-p (4 μM) in the presence of excess cel (8 × , 32 μM). HMGB1 protein had a high degree of credibility. B–D Target validation of HMGB1 by pull-down, WB, and IF assays in living primary neurons under normal or OGD conditions verified that 8 × cel could completely compete the binding of cel-p to HMGB1 protein. E, F CETSA results demonstrated that cel directly bound to HMGB1 protein to decrease protein degradation with increasing temperature. G High concentrations of cel or cel-p did not affect the expression of HMGB1 in primary neurons lysate or living neuronal cells within 5 h. H–K Cel had no effect on HMGB1 expression in normal primary neurons within 48 h at 0.1–0.8 μM. L–N HMGB1 levels were decreasing with time in the M and M + cel groups. Error bars represent SEM. #p < 0.05 vs. control group based on a one-way analysis of variance was used (n = 6). Scale bar = 25 μm
Fig. 5
Fig. 5
A–J Changes of HMGB1, HSP70, and NF-κB p65 levels affected by cel under the OGD model were observed at 48 h by WB and IF in vitro. H–N Changes in HMGB1, HSP70, and NF-κB p65 levels were observed in the rat cerebral cortex by WB and rat cerebral cortex slices by IF. Error bars represent SEM. ###p < 0.001 vs. control group (in vitro) or sham group (in vivo), *p < 0.05, **p < 0.01, ***p < 0.001 vs. model group based on a one-way analysis of variance was used (n = 4). Scale bar = 25 μm
Fig. 6
Fig. 6
A Secreted HMGB1 in the OGD model group supernatant medium evolved over time and reached a peak at 24 h. B Cel did not affect the level of secreted HMGB1 in supernatant medium 48 h after OGD injury. The disulfide form (not the fully reduced) HMGB1 isoform occupied the vast majority of HMGB1 isoforms in the OGD supernatant (C) and neuron cells (D), and cel did not affect the redox states of HMGB1. E Structure and function of HMGB1, a 25-kDa protein consisting of 214 amino acids
Fig. 7
Fig. 7
A, B Cel exhibited strong combining ability with HMGB1 protein in a concentration-dependent manner. C, D Cel almost completely competed for the binding of IAA-yne with HMGB1, and DTT reduced HMGB1. E, F The binding ability of cel to the HMGB1 protein was stronger than that of GA and Met. G Cel could bind to other sites of HMGB1 in addition to Cys 23, 45 and 106
Fig. 8
Fig. 8
A Cel significantly inhibited the secretion of TNF-α induced by HMGB1 in RAW 264.7 cells. B Cel obviously decreased the secretion of TNF-α induced by the B box in RAW 264.7 cells. C Cel remarkably blocked the binding of TLR4 and RAGE receptors to the B box. ###p < 0.001 vs. control group, **p < 0.01, ***p < 0.001 vs. HMGB1 or B box group based on a one-way analysis of variance (n = 3)
Fig. 9
Fig. 9
A OGD model of primary neurons and MCAO model of adult rats were established to demonstrate that cel had a neuroprotective effect and its modified product (cel-p) was with retained the biological activity. B HMGB1 was verified as an important target directly bound by cel based on biorthogonal reaction, TMT labeling, LC–MS/MS, and CETSA. Cel had an anti-inflammatory effect by targeting HSP70 and NF-κB p65 and did not affect the expression levels of HMGB1. Cel blocked the cytokine activity of HMGB1 and B box by directly binding them to disrupt their binding with inflammatory receptors
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