Phthalide derivative CD21 ameliorates ischemic brain injury in a mouse model of global cerebral ischemia: involvement of inhibition of NLRP3.

The activation of NLRP3 inflammasome is closely related to ischemic brain injury and inhibition of NLRP3 inflammasome activation may be a new therapeutic strategy for ischemic stroke. Our previous studies showed that ligustilide (LIG) had a dose-dependent neuroprotective effect on various models of cerebral ischemia and dementia in vivo and in vitro. CD21, a kind of phthalide derivative, was modified from LIG. In this study, we established a global cerebral ischemia-reperfusion model in mice by bilateral common carotid artery ligation (2VO), and explored the neuroprotective effect of CD21 and its anti-inflammatory mechanism on cerebral ischemia mice. CD21 significantly improved weight loss, neurobehavioral deficits and neurons loss in hippocampal CA1 and caudate putamen (CPu) subregions, which were induced by 2VO in mice. CD21 significantly inhibited the overactivation of astrocyte and microglia, and decreased the mRNA level of IL-6, TNF-α and IL-1ß. Moreover, CD21 significantly inhibited the activation of TLR4/NF-κB signaling pathway mediated by HMGB1 and NLRP3/ASC/Caspase-1 signaling pathway mediated by Cathepsin B, thus inhibiting the activation of NLRP3 inflammasome. Our results demonstrated that CD21 may exert a neuroprotection by inhibiting NLRP3 inflammasome activation after cerebral ischemia. These findings provide a new strategy for the treatment of ischemic stroke.

The neuroprotective effects and probable mechanisms of Ligustilide and its degradative products on intracerebral hemorrhage in mice.

BACKGROUND: Intracerebral hemorrhage (ICH) is a common neurological emergency with higher mortality and disability rate than cerebral ischemia. Although diverse therapeutic interventions have been explored for potential neuroprotection from ICH, no effective drugs until now are available for improvement of survival rate or the life quality of survivors after ICH. Just like cerebral ischemia, inflammatory mechanism is highly thought to play a vital role in hemorrhagic brain injury. Ligustilide (LIG) has potent anti-inflammatory effects, which were shown to be closely related to its neuroprotective effects against ischemic brain injury. Senkyunolide H (SH) and senkyunolide I (SI) are natural degradation products of LIG, which contain the mother nucleus structure of LIG as that of phthalide. However, no reports have been retrieved about the neuroprotective effects of the three phthalide compounds on ICH, especially from the perspectives of inflammatory pathways. Accordingly, this study investigated the neuroprotective potentials and mechanisms of LIG, SH and SI on experimental ICH in mice. METHODS: ICH was induced in adult male CD-1 mice by intracerebral injection of autologous blood. LIG, SH and SI, respectively, was administrated after ICH induction. Neurological deficits, brain edema, injury volume, the number of surviving/dying neurons and inflammatory gene expression were evaluated at 3 days after ICH. RESULTS: Neurological deficits, brain edema, neuronal injury, microglia and astrocytes activation as well as peripheral immune cells infiltration were all significantly improved by LIG and SH, yet SI not. Moreover, the expression of TLR4, p-NF-kB p65, TNF-α and IL-6, was significantly downregulated by LIG and SH treatment. So was Prx1 expression and release. CONCLUSIONS: LIG and SH provide the potent neuroprotective effects against hemorrhagic stroke by inhibiting Prx1/TLR4/NF-kB signaling and the subsequent immune and neuroinflammation lesions.

Klotho upregulation contributes to the neuroprotection of ligustilide against cerebral ischemic injury in mice.

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.

Protective Effect of Klotho against Ischemic Brain Injury Is Associated with Inhibition of RIG-I/NF-κB Signaling.

Aging is the greatest independent risk factor for the occurrence of stroke and poor outcomes, at least partially through progressive increases in oxidative stress and inflammation with advanced age. Klotho is an antiaging gene, the expression of which declines with age. Klotho may protect against neuronal oxidative damage that is induced by glutamate. The present study investigated the effects of Klotho overexpression and knockdown by an intracerebroventricular injection of a lentiviral vector that encoded murine Klotho (LV-KL) or rat Klotho short-hairpin RNA (LV-KL shRNA) on cerebral ischemia injury and the underlying anti-neuroinflammatory mechanism. The overexpression of Klotho induced by LV-KL significantly improved neurobehavioral deficits and increased the number of live neurons in the hippocampal CA1 and caudate putamen subregions 72 h after cerebral hypoperfusion that was induced by transient bilateral common carotid artery occlusion (2VO) in mice. The overexpression of Klotho significantly decreased the immunoreactivity of glial fibrillary acidic protein and ionized calcium binding adaptor molecule-1, the expression of retinoic-acid-inducible gene-I, the nuclear translocation of nuclear factor-κB, and the production of proinflammatory cytokines (tumor necrosis factor α and interleukin-6) in 2VO mice. The knockdown of Klotho mediated by LV-KL shRNA in the brain exacerbated neurological dysfunction and cerebral infarct after 22 h of reperfusion following 2 h middle cerebral artery occlusion in rats. These findings suggest that Klotho itself or enhancers of Klotho may compensate for its aging-related decline, thus providing a promising therapeutic approach for acute ischemic stroke during advanced age.