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Background: Upregulation of mitochondrial E3 ubiquitin ligase 1 (Mul1) contributes to brain injury in ischemic stroke due to disturbance of mitochondrial dynamics, and bioinformatics analysis predicts that Mul1 is a potential target of Dipsacoside B. Objective: The aim of the study was to explore whether Dipsacoside B can exert a beneficial effect on brain injury in the ischemic stroke rat via targeting Mul1. Methods: The SD rat brains or PC12 cells were subjected to 2 h-ischemia or 8 h-hypoxia plus 24 h-reperfusion or 24 h-reoxygenation to establish the ischemic stroke rat model in vivo or in vitro, which were treated with Dipsacoside B at different dosages. The brain or PC12 cell injury, relevant protein levels and mitochondrial functions were measured by methods of biochemistry, flow cytometry or Western blot. Results: The neurological dysfunction and brain injury (such as infarction and apoptosis) observed in the ischemic stroke rats were accompanied by increases in Mul1 and dynamin-related protein 1 (Drp1) levels along with decreases in mitofusin 2 (Mfn2) level and ATP production. These effects were attenuated by Dipsacoside B. Consistently, cell injury (necroptosis and apoptosis) occurred in the PC12 cells exposed to hypoxia concomitant with the upregulation of Mul1 and Drp1 along with downregulation of Mfn2 and mitochondrial functions (such as increases in reactive oxygen species production and mitochondrial fission and decreases in mitochondrial membrane potential and ATP production).These phenomena were reversed in the presence of Dipsacoside B. Conclusion: Dipsacoside B can protect the rat brain against ischemic injury via inhibition of Mul1 due to the improvement of mitochondrial function.

Necroptosis has been demonstrated to contribute to brain injury in ischemic stroke, whereas A20 can exert anti-necroptosis effect via deubiquitinating receptor-interacting protein kinase (RIPK3) at k63 and it can be cleaved by MALT1. This study aims to explore whether MALT1 is upregulated in the brain during ischemic stroke and promotes brain cell necroptosis through enhancing the degradation of A20. Ischemic stroke model was established in Sprague Dawley rats by occlusion of the middle cerebral artery (MCA) for 2 h, followed by 24 h reperfusion, which showed brain injury (increase in neurological deficit score and infarct volume) concomitant with an upregulation of MALT1, a decrease in A20 level, and increases in necroptosis-associated protein levels [RIPK3, mixed lineage kinase domain-like protein (MLKL) and p-MLKL] and k63-ubiquitination of RIPK3 in brain tissues. Administration of MALT1 inhibitor (Ml-2) at 8 or 15 mg/kg (i.p.) at 1 h after ischemia significantly improved neurological function and reduced infarct volume together with a downregulation of MALT1, an increase in A20 level and decreases in necroptosis-associated protein levels and k63-ubiquitination of RIPK3. Similarly, knockdown of MALT1 could also reduce oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in the cultured HT22 cells coincident with an increase in A20 level and decreases in necroptosis-associated protein levels and k63-ubiquitination of RIPK3. Based on these observations, we conclude that MALT1 promotes necroptosis in stroke rat brain via enhancing the degradation of A20, which leads to a decrease in the capability of A20 to deubiquitinate RIPK3 at k63 and a subsequent compromise in counteraction against the brain cell necroptosis.

Purpose Pellino3, an ubiquitin E3 ligase, prevents the formation of the death-induced signaling complex in response to TNF-α via targeting receptor-interacting protein kinase 1 (RIPK1), and bioinformatics analysis predicts the interaction between Pellino3 and caspofungin, a common antifungal drug used in clinic. This study aims to explore the effect of caspofungin on brain injury in ischemic stroke and the underlying mechanisms. Methods The SD rat brains (or nerve cells) were subjected to 2h-ischemia (or 8h-hypoxia) plus 24h-reperfusion (or 24h-reoxygenation) to establish the I/R (or H/R) injury model. The cerebral injury was assessed by the methods of triphenyltetrazolium chloride (TTC) staining and Hematoxylin & eosin (H&E) staining. The correlations among caspofungin, Pellino3 and necroptosis in I/R-treated brain or H/R-treated nerve cells were evaluated by biochemistry, molecular and gene overexpression assays. Results The I/R-treated brain showed the injuries (increase in neurological deficit score and infarct volume), downregulation of Pellino3, decreased ubiquitination of RIPK1 and up-regulation of necroptosis-associated proteins [RIPK1, RIPK3, mixed lineage kinase domain-like protein (MLKL), p-RIPK1, p-RIPK3 and p-MLKL]. Caspofungin treatment improved neurological function, reduced infarct volume, up-regulated Pellino3, increased RIPK1 ubiquitination and down-regulated levels of RIPK1, p-RIPK1, p-RIPK3 and p-MLKL. In PC12 cells, H/R treatment caused cellular injury (LDH release and necroptosis), downregulation of Pellino3, decreased ubiquitination of RIPK1 and up-regulation of necroptosis-associated proteins, these phenomena were reversed by overexpression of Pellino3. Conclusion Pellino3 has an important role in counteracting necroptosis via ubiquitination of RIPK1 and caspofungin can suppress the brain cell necroptosis in ischemic stroke via upregulation of Pellino3.