Acupuncture alleviates hemorrhagic transformation after delayed rt-PA treatment for acute ischemic stroke by regulating the mitophagy-NLRP3 inflammasome pathway

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Acupuncture alleviates hemorrhagic transformation after delayed rt-PA treatment for acute ischemic stroke by regulating the mitophagy-NLRP3 inflammasome pathway

Tao Jiang^1,2Qianqian Liu^1,2Huanhuan Liu^1,2Zheng Huang^1,2Mengning Yang^1,2Peiyan Huang^1,2Yiting Shen^1,3Yangyang Song^1,2Wentao Xu^1,2Xinchang Zhang^1,2*Guangxia Ni^1,2*

• ¹College of Acupuncture-Moxibustion and Tuina, Nanjing University of Chinese Medicine, Nanjing, China
• ²Key Laboratory of Acupuncture and Medicine Research of Ministry of Education, Nanjing University of Chinese Medicine, Nanjing, China
• ³College of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, China

Background: The clinical application of recombinant tissue plasminogen activator (rt-PA) is significantly constrained by hemorrhagic transformation (HT), a common and severe complication following thrombolysis for ischemic stroke. Notably, the mitochondrial injury-mediated NLRP3 inflammasome plays a crucial role in HT after delayed rt-PA thrombolysis in acute ischemic stroke. Although acupuncture has demonstrated antioxidant and anti-inflammatory effects in acute cerebral infarction, its impact on delayed rt-PA thrombolysis, especially concerning mitophagy and the NLRP3 inflammasome, remains unclear. This study investigates how acupuncture protects against HT resulting from mitochondrial damage and NLRP3 inflammasome activation after delayed rt-PA thrombolysis in acute cerebral stroke.

Methods: We selected an embolic stroke model in rats and assessed brain injury after delayed rt-PA in acute ischemic stroke using neurological deficit score, volume of brain infarct, the permeability assay of the blood–brain barrier (BBB), and HT. Then, the levels of proteins and mRNA involved in mitophagy and the NLRP3 inflammasome pathway were measured by western blot and real-time PCR. The levels of interleukin-18 (IL-18) and interleukin-1β (IL-1β) were assessed using enzyme-linked immunosorbent assay (ELISA). Morphological changes in the BBB and mitochondria of neurons were observed via transmission electron microscopy.

Results: Acupuncture significantly improved neurological deficit scores, volume of cerebral infarction, BBB destruction, and HT in an embolic stroke model rat. Furthermore, acupuncture induced mitophagy and substantially downregulated the activity of the NLRP3 inflammasome. Additionally, the use of mitochondrial inhibitors significantly reversed the suppressive impact of acupuncture on the NLRP3 inflammasome.

Conclusion: Acupuncture can promote mitophagy and suppress NLRP3 inflammasome activation to decrease HT after delayed rt-PA therapy for acute ischemic stroke.

1 Introduction

Stroke is a leading cause of disability and death in China, with ischemic stroke being the most common type, representing approximately 86.8% of all cases (1). Currently, the best treatment for ischemic stroke is intravenous recombinant tissue fibrinogen activator (rt-PA). Nevertheless, this approach is constrained by a short time window (within 4.5 h) (2). Delayed rt-PA thrombolysis may lead to several severe complications, including hemorrhagic transformation (HT), cerebral edema, and reperfusion injury, with HT being the most frequently observed complication (3, 4).

The inflammasome is strongly implicated in the development of ischemic stroke (5). The Nod-like receptor protein 3 (NLRP3) inflammasome is a complex multiprotein structure that includes the NLRP3 receptor, apoptosis-associated speck-like protein containing the caspase-recruiting domain (ASC), and the precursor form of Caspase-1 (Pro-caspase-1) (6). The activated NLRP3 complex eventually triggers the release of interleukin-18 (IL-18) and interleukin-1β (IL-1β). These cytokines trigger downstream signaling pathways and initiate inflammatory responses. This process disrupts tight junction (TJ) proteins, thereby enhancing the permeability of the blood–brain barrier (BBB) and increasing the incidence of HT (7, 8).

Mitophagy can selectively eliminate defective or impaired mitochondria and is a critical mechanism for maintaining the quality of mitochondria (9). Extensive research has demonstrated that the Pink1 (PTEN-inducible putative kinase 1)/Parkin (E3 ubiquitin ligase) pathway constitutes the canonical mitophagy mechanism (10, 11). Upon mitochondrial injury, Pink1 is activated and subsequently recruits Parkin to the outer membrane (12). Activated Parkin is identified by P62 (an autophagy junction protein) and facilitates the transport of mitochondria to autophagic vesicles through its interaction with the protein LC3, which induces the generation of mitochondrial autophagic vesicles that ultimately merge with lysosomes to eliminate damaged mitochondria (9). Meanwhile, some reports have suggested that mitophagy mediated by Pink1/Parkin is an essential mechanism for suppressing the activity of the NLRP3 inflammasome (13, 14). Moreover, mitophagy may mitigate brain damage in ischemic stroke rats via its inhibitory effect on NLRP3 inflammasome activation (15).

Accumulating evidence indicates that acupuncture may substantially decrease neurological impairment score and cerebral infarction volume in ischemic stroke rats while also inhibiting NLRP3 and Caspase-1 expression, thereby providing a neuroprotective effect (16). Meanwhile, acupuncture can protect neurons from damage through enhancing mitophagy through the Pink1/Parkin-dependent pathway (17).

Although acupuncture has been demonstrated to exert neuroprotective effects in cerebral ischemia, its effects on mitophagy and interactions with NLRP3 inflammasomes remain unknown. This study examined the potential of acupuncture to mitigate HT following delayed rt-PA thrombolysis in acute ischemic stroke rats by modulating the mitophagy-NLRP3 inflammasome pathway.

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