ligustilide and Brain-Ischemia

Ischemic stroke is a major global cause of death and permanent disability. Studies have suggested that mitochondria play a critical role in maintaining cellular energy homeostasis and inevitably involved in neuronal damage during cerebral ischemic. Ligustilide is the main active ingredient of Angelica sinensis and Ligusticum chuanxiongs with neuroprotective activity.. These study sought to exlopre the role of LIG in improving mitochondrial function and the relationship between LIG induced mitochondrial fission and mitophagy in ischemic stroke.. Cerebral I/R injury was established by the model of Oxygen-glucose deprivation/reperfusion (OGD/R) in HT22 cells and middle cerebral artery occlusion (MCAO) in rats. Mitochondrial functions of were detected by flow cytometry and immunofluorescence, and mitochondrial fission were detected by western blots. Furthermore, we studied the role of AMPK pathway in the neuroprotective effect of LIG.. Our study indicate that LIG attenuated the injury of ischemic stroke by improving mitochondrial function and highlight the critical role of LIG in the regulation of LIG-induced mitochondrial fission and mitophagy via an AMPK-dependent manner. These findings indicate that LIG protects nerve damage against ischemic stroke by inducing Drp1-mediated mitochondrial fission via activation of AMPK signaling pathway in vivo and in vitro.

Topics: 4-Butyrolactone; AMP-Activated Protein Kinases; Animals; Apoptosis; Brain Ischemia; Dynamins; Ischemic Stroke; Mitochondrial Dynamics; Rats

Mitophagy plays a critical role in cerebral ischemia/reperfusion by timely removal of dysfunctional mitochondria. In mammals, PINK1/Parkin is the most classic pathway mediating mitophagy. And the activation of PINK1/Parkin mediated mitophagy exerts neuroprotective effects during cerebral ischemia reperfusion injury (CIRI). Ligustilide (LIG) is a natural compound extracted from ligusticum chuanxiong hort and angelica sinensis (Oliv.) diels that exerts neuroprotective activity after cerebral ischemia reperfusion injury (CIRI). However, it still remains unclear whether LIG could attenuates cerebral ischemia reperfusion injury (CIRI) through regulating mitophagy mediated by PINK1/Parkin.. To explore the underlying mechanism of LIG on PINK1/Parkin mediated mitophagy in the hippocampus induced by ischemia reperfusion.. The results show that LIG improved mitochondrial functions by mitophagy enhancement in vivo and vitro to alleviate CIRI. Whereas, mitophagy enhanced by LIG under CIRI is abolished by PINK1 deficiency and midivi-1, a mitochondrial division inhibitor which has been reported to have the function of mitophagy, which could further aggravate the ischemia-induced brain damage, mitochondrial dysfunction and neuronal injury.. LIG could ameliorate the neuronal injury against ischemia stroke by promoting mitophagy via PINK1/Parkin. Targeting PINK1/Parkin mediated mitophagy with LIG treatment might be a promising therapeutic strategy for ischemia stroke.

Topics: 4-Butyrolactone; Animals; Brain Ischemia; Hippocampus; Infarction, Middle Cerebral Artery; Ischemic Stroke; Mammals; Mitophagy; Protein Kinases; Rats; Reperfusion; Reperfusion Injury; Ubiquitin-Protein Ligases

Following stroke, angiogenic strategy has been proposed to alleviate ischemia-induced injury by promoting angiogenesis and improving cerebrovascular function in the ischemic regions. Ligustilide (LGSL) is known to have neuroprotection against ischemic stroke-induced injury. But the effect of LGSL on promoting angiogenesis after ischemic stroke is unknown. The purpose of this study was to determine the effects of LGSL on neuroprotection and angiogenesis after stroke.. BrdU cell proliferation assay, transwell, wound healing, and tube-formation experiments were performed to evaluate the effects of LGSL on mouse microvascular endothelial cells (bEnd.3). Male adult C57/BL6 mice were subjected to focal transient cerebral ischemia followed by intragastric LGSL administration. Immunostaining was performed to assess angiogenesis. VEGF level was assayed by ELISA. eNOS expression was performed by Western blot.. LGSL significantly improved neurological function. LGSL promoted angiogenesis. LGSL promoted focal angiogenesis and attenuated ischemia-induced brain injury, suggesting that LGSL is a potential agent that improves angiogenesis and promotes recovery from ischemic stroke.

Topics: 4-Butyrolactone; Angiogenesis Inducing Agents; Animals; Behavior, Animal; Brain Ischemia; Cell Line; Male; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Neuroprotective Agents

Microglia can be activated to become the classic phenotype (M1) or alternative phenotype (M2), which play an important role in regulating neuroinflammatory response and tissue repair after ischemic stroke. CD21, a novel phthalide derivative, is a potential neuroprotectant against ischemic brain injury. The present study further investigated the effects of CD21 on post-ischemic microglial polarization and the underlying mechanisms. Transient middle cerebral artery occlusion (tMCAO) was used as a mouse model of ischemic stroke, while BV2 cells stimulated with conditioned medium collected from oxygen-glucose deprivation-treated HT22 cells were used in in vitro ischemic studies. The current results showed that CD21 dose-dependently and significantly improved neurological outcomes in tMCAO mice. Biochemical analyses revealed that CD21 decreased the expression of M1 phenotype markers (CD86, interleukin-1β and inducible nitric oxide synthase) and increased the expression of M2 phenotype markers (CD206, interleukin-10 and YM1/2) in both ischemic brain tissues and BV2 cells. Meanwhile, CD21 decreased the production of proinflammatory cytokines (interleukin-1β, interleukin-6 and tumor necrosis factor-α), promoted the release of the antiinflammatory cytokine (interleukin-10), and enhanced the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) in ischemic brain tissue and BV2 cells. Furthermore, the AMPK inhibitor (compound C) reversed these effects of CD21 in BV2 cells. These findings indicate that CD21 alleviates post-ischemic neuroinflammation through induction of microglial M2 polarization that is at least in part medicated by AMPK activation, suggesting that CD21 may be a promising candidate for protecting against ischemic brain injury.

Topics: 4-Butyrolactone; AMP-Activated Protein Kinases; Animals; Behavior, Animal; Benzofurans; Brain Ischemia; Cell Line; Cell Polarity; Cytokines; Dose-Response Relationship, Drug; Encephalitis; Enzyme Activation; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Microglia; Neuroprotective Agents; Phenotype; Psychomotor Performance

Klotho, an aging-suppressor gene, encodes a protein that potentially acts as a neuroprotective factor. Our previous studies showed that ligustilide minimizes the cognitive dysfunction and brain damage induced by cerebral ischemia; however, the underlying mechanisms remain unclear. This study aims to investigate whether klotho is involved in the protective effects of ligustilide against cerebral ischemic injury in mice. Cerebral ischemia was induced by bilateral common carotid arterial occlusion. Neurobehavioral tests as well as Nissl and Fluoro-Jade B staining were used to evaluate the protective effects of ligustilide in cerebral ischemia, and Western blotting and ELISA approaches were used to investigate the underlying mechanisms. Administration of ligustilide prevented the development of neurological deficits and reduced neuronal loss in the hippocampal CA1 region and the caudate putamen after cerebral ischemia. The protective effects were associated with inhibition of the RIG-I/NF-κB p65 and Akt/FoxO1 pathways and with prevention of inflammation and oxidative stress in the brain. Further, downregulation of klotho could attenuate the neuroprotection of ligustilide against cerebral ischemic injury. Ligustilide exerted neuroprotective effects in mice after cerebral ischemia by regulating anti-inflammatory and anti-oxidant signaling pathways. Furthermore, klotho upregulation contributes to the neuroprotection of LIG against cerebral ischemic injury. These results indicated that ligustilide may be a promising therapeutic agent for the treatment of cerebral ischemia.

Topics: 4-Butyrolactone; Animals; Brain Ischemia; Glucuronidase; HEK293 Cells; Humans; Klotho Proteins; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Oxidative Stress; Signal Transduction; Up-Regulation

Z-Ligustilide (Z-LIG) is a major component in Rhizoma Chuanxiong, which has been traditionally used as a health food supplement for the prevention of cerebrovascular disease in China. This study investigates the ability of intranasal Z-LIG pretreatment to enhance protection against neuronal damage in rats with middle cerebral artery occlusion (MCAO) and the role of cellular stress response mechanisms Nrf2 and HSP70. Z-LIG significantly mitigated infarct volume, neurological dysfunction, blood-brain barrier disruption, and brain edema (p < 0.01). Moreover, Z-LIG prevented the loss of collagen IV, occludin, and ZO-1 (p < 0.05) and decreased MMP-2 and -9 levels (p < 0.01). Meanwhile, Z-LIG up-regulated NQO1 and HSP70. Notably, blockage of Nrf2-driven transcription or down-regulation of HSP70 remarkably attenuated the preventive effect of Z-LIG (p < 0.05). Together, intranasal delivery of Z-LIG enhanced protection against ischemic injury via Nrf2 and HSP70 signaling pathways and has prophylactic potential in the population at high risk of stroke.

Topics: 4-Butyrolactone; Administration, Intranasal; Animals; Blood-Brain Barrier; Brain Ischemia; Drugs, Chinese Herbal; HSP70 Heat-Shock Proteins; Humans; Male; Neuroprotective Agents; NF-E2-Related Factor 2; Rats, Sprague-Dawley; Rhizome; Signal Transduction

The mechanisms involved in the antioxidant and anti-apoptotic properties of (Z)-ligustilide (LIG) are not fully elucidated. Based on the accumulated data, we hypothesized that LIG might be able to reduce ischemia/reperfusion-induced increase in brain iron by regulating expression of iron transport proteins. We therefore investigated the effects of LIG on iron uptake protein transferrin receptor 1, iron exporter protein ferroportin 1, iron storage protein ferritin light chain and also hypoxia inducible factor-1 alpha (HIF-1 alpha) in oxygen-glucose deprivation/reoxygenation (OGD/R)-treated SH-SY5Y cells, using Western blot analysis. We demonstrated that LIG completely reversed the OGD/R-induced reduction of ferroportin 1, increased ferritin light chain content, and also suppressed the OGD-induced increase in HIF-1 alpha in SH-SY5Y cells. These findings imply that LIG might reduce the OGD/R-induced increase in brain iron by promoting cell iron release and iron corporation into ferritin, and also by inhibiting the HIF-1 alpha-induced increase in transferrin-bound iron uptake and iron accumulation in the brain, consequently attenuating iron-mediated free radical formation, oxidative stress and apoptosis.

Topics: 4-Butyrolactone; Antigens, CD; Apoferritins; Brain Ischemia; Cation Transport Proteins; Cell Line, Tumor; Cell Survival; Down-Regulation; Gene Expression Regulation; Glucose; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Neuroprotective Agents; Oxygen; Receptors, Transferrin

Blocking TLR4/peroxiredoxin (Prx6) signaling is proposed to be a novel therapeutic strategy for ischemic stroke because extracellular Prx6 released from ischemic cells may act as an endogenous ligand for TLR4 and initiate destructive immune responses in ischemic brain. Our previous studies showed that ligustilide (LIG) exerted antineuroinflammatory and neuroprotective effects against ischemic insult, but the underlying mechanisms remain unclear. This study investigated whether the TLR4/Prx6 pathway is involved in the protective effect of LIG against postischemic neuroinflammation and brain injury induced by transient middle cerebral artery occlusion (MCAO) in rats. Intraperitoneal LIG administration (20 and 40 mg/kg/day) at reperfusion onset after MCAO resulted in a reduction of brain infarct size and improved neurological outcome over 72 h. LIG-induced neuroprotection was accompanied by improvement of neuropathological alterations, including neuron loss, astrocyte and microglia/macrophage activation, neutrophil and T-lymphocyte invasion, and regulation of inflammatory mediators expression. Moreover, LIG significantly inhibited the expression and extracellular release of Prx6 and activation of TLR4 signaling, reflected by decreased TLR4 expression, extracellular signal-regulated kinase 1/2 phosphorylation, and transcriptional activity of NF-κB and signal transducer and activator of transcription 3 in the ischemic brain. Our results demonstrate that LIG may provide an early and direct neuroprotection by inhibiting TLR4/Prx6 signaling and subsequent immunity and neuroinflammation after cerebral ischemia. These findings support the translational potential of blocking TLR4/Prx6 signaling for the treatment of ischemic stroke.

Topics: 4-Butyrolactone; Animals; Anti-Inflammatory Agents, Non-Steroidal; Astrocytes; Brain Ischemia; Cell Movement; Gene Expression Regulation; Inflammation; Injections, Intraperitoneal; Macrophage Activation; Male; Microglia; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; NF-kappa B; Oxidative Stress; Peroxiredoxin VI; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; STAT3 Transcription Factor; T-Lymphocytes; Toll-Like Receptor 4

Z-ligustilide (LIG), the main lipophilic component of Radix Angelica sinensis, has been shown to protect against brain ischemic damage in rodents by oral and intra-peritoneal treatments. The present study aimed to confirm the therapeutic effect of LIG administered intravenously on 2h middle cerebral artery occlusion (MCAO) and 22 h reperfusion injury in rats since oral administration has low bioavailability, slow absorption and distribution. Moreover, whether LIG activated the NF-E2-related factor 2/ heme oxygenase-1 (Nrf2/HO-1) pathway was also investigated in vivo and in vitro to further elucidate the precise protective mechanisms. In vivo, rats treated intravenously with LIG immediately after the surgery was finished had less neurological dysfunction and smaller infarct volume than that of the vehicle-treated rats. Additionally, LIG promoted Nrf2 nuclear translocation, and further remarkably increased Nrf2 and HO-1 protein expression. In vitro, LIG induced Nrf2 nuclear translocation and up-regulated HO-1 expression in a time-dependent and concentration-dependent manner. Furthermore, LIG treatment reduced cell death induced by OGD, however, the protective action was abolished while Nrf2/HO-1 expression was knockdown by RNA interference. These results noted that intravenous post-treatment with LIG exhibits noticeable neuroprotective properties against brain damage by ischemia-reperfusion and the ability of LIG to activate Nrf2/HO-1 pathway may be partly responsible for it.

Topics: 4-Butyrolactone; Animals; Blotting, Western; Brain Ischemia; Cell Line; Disease Models, Animal; Fluorescent Antibody Technique; Heme Oxygenase-1; Humans; Male; Neuroprotective Agents; NF-E2-Related Factor 2; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Ligustilide (LIG), a main lipophilic component of Danggui (Chinese Angelica root, Radix Angelica sinensis) which is a popular used herb to treat menstrual disorders in traditional chinese medicine, has been reported to possess some neuroprotective effects on permanent focal ischemia and transient forebrain ischemia.. Based on previous work, we intended to investigate the protective effects of LIG on parietal cortex and hippocampus of rats in chronic cerebral hypoperfusion model.. Chronic cerebral hypoperfusion was induced by permanent, bilateral common carotid artery's occlusion (2VO). The rats were treated with LIG (80mg/kg, by oral) from the eighth day after surgery for seven consecutive days. Their spatial learning and memory abilities were assessed using the Morris water maze. After six days for maze test, rats were sacrificed. Coronal sections in cortex and hippocampus were stained with cresyl violet or labeled with NeuN (Neuronal Nuclei), MAP-2 (Microtubule-Associated Protein-2), Caspase-3 and GFAP (Glial Fibrillary Acidic Protein) antibodies.. LIG treatment for seven days decreased escape latency and swimming distance of 2VO rats from the third day in maze tests, and increased percent time in the target quadrant. LIG prevented neuronal loss, dendrites damage and neuronal apoptosis in both parietal cortex and hippocampus of 2VO rats; and it also inhibited astrocytic activation and proliferation stimulated by hypoperfusion.. These results demonstrate that LIG show obvious neuroprotective potential for treating chronic cerebral hypoperfusion injury, which may be attributed to its anti-apoptosis of neuron and anti-proliferation of astrocyte both in cortex and in hippocampus of 2VO rats. We suggest that LIG can be developed as an effective drugs for the prevention of vascular dementia (VD).

Topics: 4-Butyrolactone; Animals; Brain Ischemia; Cognition; Hippocampus; Male; Maze Learning; Memory; Neuroprotective Agents; Parietal Lobe; Phytotherapy; Rats; Rats, Sprague-Dawley

Previous studies have demonstrated that Z-Ligustilide (LIG), a characterized phthalide constituent present in numerous medical Umbelliferae plants, has significant neuroprotective effects in transient forebrain ischemia and permanent cerebral focal ischemia. The present study further investigated the effect of LIG on chronic cerebral hypoperfusion. Male Wistar rats were subjected to permanent ligation of both common carotid arteries (2VO). On Days 8-12 postsurgery, rat cognition was assessed in the Morris water maze. Rats with significantly impaired acquisition of spatial information were randomly allocated to three groups and orally administered LIG (10 or 40 mg/kg/day) or volume-matched vehicle on Days 13-40 post-2VO surgery. The sham-operated group served as controls. After long-term treatment with LIG, the impaired animals' behavioral, biochemical, and histopathological features were examined. Compared to the sham-operated group, significant cognitive impairment was observed in the vehicle-treated group 40 days after 2VO. Shortened mean escape latency was detected in the Morris water maze in rats treated with LIG (p<0.01 vs. vehicle-treated group) during the same trial days. Chronic 2VO-induced pathological changes included neuronal loss and an increase of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes in the hippocampus. These effects were prevented with LIG treatment (p<0.01 vs. vehicle-treated group). LIG also significantly reduced malondialdehyde levels and increased superoxide dismutase activity in ischemic brain tissue (p<0.05 and p<0.01 vs. vehicle-treated group). In addition, LIG significantly increased choline acetyltransferase activity and inhibited acetylcholinesterase activity in ischemic brain tissues (p<0.05 and p<0.01 vs. vehicle-treated group). The present data demonstrate that LIG significantly prevented chronically hypoperfused cognitive deficits and brain damage at least partly through an antioxidant effect and improved cholinergic activity. The present findings suggest that LIG may have therapeutic potential in treating vascular dementia and cerebrovascular insufficiency.

Topics: 4-Butyrolactone; Acetylcholinesterase; Angelica sinensis; Animals; Antioxidants; Brain Chemistry; Brain Ischemia; Cerebrovascular Circulation; Choline O-Acetyltransferase; Cognition Disorders; Male; Maze Learning; Parasympathetic Nervous System; Prosencephalon; Rats; Rats, Wistar; Reversal Learning

The present study investigated the effect of Z-Ligustilide (LIG), a characterized 3-n-alkyphthalide derivative existed in many medical Umbelliferae plants, on permanent focal ischemic brain injury in rats. Focal cerebral ischemia was induced by the occlusion of middle cerebral artery (MCA) for 24 h. LIG (20, or 80 mg/kg), orally administered at 2 h after ischemia, reduced the cerebral infarct volumes by 48.29% and 84.87% respectively compared to control group as visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining (p<0.01). Treatment with LIG could dose-dependently reduce brain swelling by 68.62% and 82.08% (p<0.01), and significantly improve behavioral deficits (p<0.01). In addition, LIG at the above used doses had no significant effect on rat body temperature. These data, along with previous findings in our lab demonstrating the neuroprotective effects of LIG in transient cerebral ischemia, suggest that LIG may be a potential neuroprotective agent for the treatment of ischemic stroke in future.

Topics: 4-Butyrolactone; Animals; Body Temperature; Brain Edema; Brain Ischemia; Infarction, Middle Cerebral Artery; Male; Neurologic Examination; Neuroprotective Agents; Rats; Rats, Sprague-Dawley