Oridonin ameliorates caspase-9-mediated brain neuronal apoptosis in mouse with ischemic stroke by inhibiting RIPK3-mediated mitophagy

Lei Li1, Jing-jing Song1, Meng-xue Zhang1, Hui-wen Zhang1, Hai-yan Zhu1, Wei Guo2, Cai-long Pan1, Xue Liu1, Lu Xu1,3, Zhi-yuan Zhang1,4
1 School of Basic Medical Sciences, Nanjing Medical University, Nanjing 211166, China
2 Department of Urology, The Affiliated Wuxi No.2 People’s Hospital of Nanjing Medical University, Wuxi 214002, China
3 Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing 211166, China
4 Department of Neurology, Sir Run Run Hospital, Nanjing Medical University, Nanjing 211166, China
Correspondence to: Lu Xu:, Zhi-yuan Zhang:,
DOI: 10.1038/s41401-022-00995-3
Received: 19 March 2022
Accepted: 2 September 2022
Advance online: 10 October 2022


Neuronal loss is a primary factor in determining the outcome of ischemic stroke. Oridonin (Ori), a natural diterpenoid compound extracted from the Chinese herb Rabdosia rubescens, has been shown to exert anti-inflammatory and neuroregulatory effects in various models of neurological diseases. In this study we investigated whether Ori exerted a protective effect against reperfusion injury- induced neuronal loss and the underlying mechanisms. Mice were subjected to transient middle cerebral artery occlusion (tMCAO), and were injected with Ori (5, 10, 20 mg/kg, i.p.) at the beginning of reperfusion. We showed that Ori treatment rescued neuronal loss in a dose-dependent manner by specifically inhibiting caspase-9-mediated neuronal apoptosis and exerted neuroprotective effects against reperfusion injury. Furthermore, we found that Ori treatment reversed neuronal mitochondrial damage and loss after reperfusion injury. In N2a cells and primary neurons, Ori (1, 3, 6 μM) exerted similar protective effects against oxygen-glucose deprivation and reoxygenation (OGD/R)-induced injury. We then conducted an RNA-sequencing assay of the ipsilateral brain tissue of tMCAO mice, and identified receptor-interacting protein kinase-3 (RIPK3) as the most significantly changed apoptosis-associated gene. In N2a cells after OGD/R and in the ipsilateral brain region, we found that RIPK3 mediated excessive neuronal mitophagy by activating AMPK mitophagy signaling, which was inhibited by Ori or 3-MA. Using in vitro and in vivo RIPK3 knockdown models, we demonstrated that the anti-apoptotic and neuroprotective effects of Ori were RIPK3-dependent. Collectively, our results show that Ori effectively inhibits RIPK3-induced excessive mitophagy and thereby rescues the neuronal loss in the early stage of ischemic stroke.
Keywords: ischemic stroke; oridonin; RIPK3; mitophagy; apoptosis; transient middle cerebral artery occlusion

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