Zinc Ameliorates Nogo-A Receptor and Osteocalcin Gene Expression in Memory-Sensitive Rat Hippocampus Impaired by Intracerebroventricular Injection of Streptozotocin.

Metabolic dysfunction is a critical step in the etiopathogenesis of Alzheimer's disease. In this progressive neurological disorder, impaired zinc homeostasis has a key role that needs to be clarified. The aim of this study was to investigate the effect of zinc deficiency and administration on hippocampal Nogo-A receptor and osteocalcin gene expression in rats injected with intracerebroventricular streptozotocin (icv-STZ). Forty male Wistar rats were divided into 5 groups in equal numbers: Sham 1 group received icv artificial cerebrospinal fluid (aCSF); Sham 2 group received icv a CSF and i.p. saline; STZ group received 3 mg/kg icv STZ; STZ-Zn-deficient group received 3 mg/kg icv STZ and fed a zinc-deprived diet; STZ-Zn-supplemented group received 3 mg/kg icv STZ and i.p. zinc sulfate (5 mg/kg/day). Hippocampus tissue samples were taken following the cervical dislocation of the animals under general anesthesia. Nogo-A receptor and osteocalcin gene expression levels were determined by real-time-PCR method. Zinc supplementation attenuated the increase in hippocampal Nogo-A receptor gene expression, which was significantly increased in zinc deficiency. Again, zinc supplementation upregulated the intrinsic protective mechanisms of the brain by activating osteocalcin-expressing cells in the brain. The results of the study show that zinc has critical effects on Nogo-A receptor gene expression and hippocampal osteocalcin gene expression levels in the memory-sensitive rat hippocampus that is impaired by icv-STZ injection. These results are the first to examine the effect of zinc deficiency and supplementation on hippocampal Nogo-A receptor and osteocalcin gene expression in icv-STZ injection in rats.

Xuesaitong exerts long-term neuroprotection for stroke recovery by inhibiting the ROCKII pathway, in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuesaitong (XST) is a traditional Chinese medicine injection with neuroprotective properties and has been extensively used to treat stroke for many years. The main component of XST is Panax notoginseng saponins (PNS), which is the main extract of the Chinese herbal medicine Panax notoginseng. AIM OF THE STUDY: In this study, we investigated whether XST provided long-term neuroprotection by inhibiting neurite outgrowth inhibitor-A (Nogo-A) and the ROCKII pathway in experimental rats after middle cerebral artery occlusion (MCAO) and in SH-SY5Y cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R). MATERIALS AND METHODS: Rats with permanent MCAO were administered XST, Y27632, XST plus Y27632, and nimodipine for 14 and 28 days. Successful MCAO onset was confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Neurological deficit score (NDS) was used to assess neurological impairment. Hematoxylin-eosin (HE) staining and immunohistochemical (IHC) analysis of synaptophysin (SYN) and postsynaptic density protein-95 (PSD-95) were performed to evaluate cerebral ischemic injury and the neuroprotective capability of XST. Nogo-A levels and the ROCKII pathway were detected by IHC analysis, western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR) to explore the protective mechanism of XST. OGD/R model was established in SH-SY5Y cells. Cell counting kit 8 (CCK8) was applied to detect the optimum OGD time and XST concentration. The expression levels Nogo-A and ROCKII pathway were determined using western blotting. RESULTS: Our results showed that XST reduced neurological dysfunction and pathological damage, promoted weight gain and synaptic regeneration, reduced Nogo-A mRNA and protein levels, and inhibited the ROCKII pathway in MCAO rats. CCK8 assay displayed that the optimal OGD time and optimal XST concentration were 7 h and 20 µg/mL respectively in SH-SY5Y cells. XST could evidently inhibit OGD/R-induced Nogo-A protein expression and ROCKII pathway activation in SH-SY5Y cells. CONCLUSIONS: The present study suggested that XST exerted long-term neuroprotective effects that assisted in stroke recovery, possibly through inhibition of the ROCKII pathway.

Blockade of Nogo-A/Nogo-66 receptor 1 (NgR1) Inhibits Autophagic Activation and Prevents Secondary Neuronal Damage in the Thalamus after Focal Cerebral Infarction in Hypertensive Rats.

Focal cerebral infarction leads to autophagic activation, which contributes to secondary neuronal damage in the ipsilateral thalamus. Although Nogo-A deactivation enhances neuronal plasticity, its role in autophagic activation in the thalamus after ischemic stroke remains unclear. This study aimed to investigate the potential roles of Nogo-A/Nogo-66 receptor 1 (NgR1) in autophagic activation in the ipsilateral thalamus after cerebral infarction. Focal neocortical infarction was established using the middle cerebral artery occlusion (MCAO) method. Secondary damage in the ipsilateral thalamus was assessed by Nissl staining and immunostaining. The expression of Nogo-A, NgR1, Rho-A and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) as well as autophagic flux were evaluated by immunofluorescence and immunoblotting. The roles of Nogo-A-NgR1 signaling in autophagic activation were determined by intraventricular delivery of an NgR1 antagonist peptide, NEP1-40, at 24 h after MCAO. The results showed that Nogo-A and NgR1 overexpression temporally coincided with marked increases in the levels of Beclin1, LC3-II and sequestosome 1 (SQSTM1)/p62 in the ipsilateral thalamus at seven and fourteen days after MCAO. In contrast, NEP1-40 treatment significantly reduced the expression of Rho-A and ROCK1 which was accompanied by marked reductions of LC3-II conversion as well as the levels of Beclin1 and SQSTM1/p62. Furthermore, NEP1-40 treatment significantly reduced neuronal loss and gliosis in the ipsilateral thalamus, and accelerated somatosensory recovery at the observed time-points after MCAO. These results suggest that blockade of Nogo-A-NgR1 signaling inhibits autophagic activation, attenuates secondary neuronal damage in the ipsilateral thalamus, and promotes functional recovery after focal cerebral cortical infarction.

Houshiheisan and its components promote axon regeneration after ischemic brain injury.

Houshiheisan, a classic prescription in traditional Chinese medicine, contains Flos Chrysanthemi, Radix Saposhnikoviae, Ramulus Cinnamomi, Rhizoma Chuanxiong, Radix et Rhizoma Asari, Radix Platycodonis, Rhizoma Atractylodis macrocephalae, Poria, Rhizoma Zingiberis, Radix Angelicae sinensis, Radix et Rhizoma Ginseng, Radix Scutellariae and Concha Ostreae. According to traditional Chinese medicine theory, Flos Chrysanthemi, Radix Saposhnikoviae, Ramulus Cinnamomi, Rhizoma Chuanxiong, Radix et Rhizoma Asari and Radix Platycodonis are wind-dispelling drugs; Rhizoma Atractylodis macrocephalae, Poria, Rhizoma Zingiberis, Radix Angelicae sinensis and Radix et Rhizoma Ginseng are deficiency-nourishing drugs. A large number of randomized controlled trials have shown that Houshiheisan is effective in treating stroke, but its mechanism of action is unknown. Axonal remodeling is an important mechanism in neural protection and regeneration. Therefore, this study explored the effect and mechanism of action of Houshiheisan on the repair of axons after cerebral ischemia. Rat models of focal cerebral ischemia were established by ligating the right middle cerebral artery. At 6 hours after model establishment, rats were intragastrically administered 10.5 g/kg Houshiheisan or 7.7 g/kg wind-dispelling drug or 2.59 g/kg deficiency-nourishing drug. These medicines were intragastrically administered as above every 24 hours for 7 consecutive days. Houshiheisan, and its wind-dispelling and deficiency-nourishing components reduced the neurological deficit score and ameliorated axon and neuron lesions after cerebral ischemia. Furthermore, Houshiheisan, and its wind-dispelling and deficiency-nourishing components decreased the expression of proteins that inhibit axonal remodeling: amyloid precursor protein, neurite outgrowth inhibitor protein A (Nogo-A), Rho family small GTPase A (RhoA) and Rho-associated kinase 2 (Rock2), and increased the expression of growth associated protein-43, microtubule-associated protein-2, netrin-1, Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42). The effect of Houshiheisan was stronger than wind-dispelling drugs or deficiency-nourishing drugs alone. In conclusion, Houshiheisan, and wind-dispelling and deficiency-nourishing drugs promote the repair of axons and nerve regeneration after cerebral ischemia through Nogo-A/RhoA/Rock2 and Netrin-1/Rac1/Cdc42 signaling pathways. These effects are strongest with Houshiheisan.

Improved functional outcome after chronic stroke with delayed anti-Nogo-A therapy: A clinically relevant intention-to-treat analysis.

Many preclinical treatment strategies for stroke have failed when tested in human trials. Although the reasons for these translation failures are multifactorial, one potential concern is the statistical analysis of the preclinical data. One way to rigorously evaluate new therapies is to use an intention-to-treat analysis in preclinical studies. Therefore, in this study, we set out to evaluate the treatment efficacy of a potential clinically relevant therapeutic agent for stroke, i.e., anti-Nogo-A immunotherapy, using an intention-to-treat analysis. Adult rats were trained on the skilled forelimb reaching task and subsequently underwent an ischemic stroke. Nine weeks later, the rats either received intracerebroventricular anti-Nogo-A antibody, control antibody, or no treatment. Skilled reaching performance was assessed by a non-linear model using both an intention-to-treat and per-protocol analysis. Following testing, dendritic complexity was evaluated in the contralesional and perilesional sensorimotor cortex. Both intention-to-treat and per-protocol analysis showed that anti-Nogo-A immunotherapy resulted in statistically significant improved recovery on the skilled forelimb reaching task, although treatment effect was less (though statistically significant) in the intention-to-treat group. Improved functional performance was not shown to be associated with dendritic changes. In conclusion, this study provides evidence for the importance of using intention-to-treat paradigms in testing preclinical therapeutic strategies.

Electroacupuncture promotes axonal regeneration in rats with focal cerebral ischemia through the downregulation of Nogo-A/NgR/RhoA/ROCK signaling.

The purpose of the present study was to evaluate the effect of electroacupuncture (EA) on the axonal regeneration environment following cerebral ischemia injury and to investigate whether it was associated with Nogo-A/Nogo receptor (NgR)/RhoA/Rho-associated protein kinase (ROCK) signaling. Using a rat model of focal cerebral ischemia, the effects of EA at the Quchi (LI11) and Zusanli (ST36) acupoints on axonal growth inhibitory protein and axonal growth factors were assessed and the underlying molecular mechanisms were investigated. It was found that EA at the Quchi and Zusanli acupoints significantly improved neurological deficit scores following ischemia (P<0.05), and reduced the cerebral infarct volume. Moreover, it was demonstrated that crucial signaling molecules in the Nogo-A signaling pathway were regulated by EA. These results suggest that EA provides a less inhibitory environment for axonal regeneration following cerebral ischemia through inhibition of Nogo-A/NgR/RhoA/ROCK signaling.

BDNF/PI3K/Akt and Nogo-A/RhoA/ROCK signaling pathways contribute to neurorestorative effect of Houshiheisan against cerebral ischemia injury in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Houshiheisan (HSHS), a classic traditional medicine prescription, has notable effects on patients with stroke AIM OF THE STUDY: To investigate the neurorestorative effects of HSHS on ischemic stroke and explore its mode of action. MATERIALS AND METHODS: Focal cerebral ischemia models were induced by permanent middle cerebral artery occlusion (pMCAO). Male Sprague-Dawley (SD) rats were randomly divided into 5 experimental groups: sham vehicle, ischemia vehicle, pMCAO+HSHS at 5.1, 10.2g/kg, and pMCAO+Ginaton 0.028g/kg. HSHS or Ginaton was administrated 6h after pMCAO onset. Neurological function was assessed and then rats were sacrificed 7 days after MCAO. Cerebral ischemic injury was evaluated by hematoxylin and eosin (HE) staining and Neuronal nuclear antigen (NeuN) immunofluorescence analysis. The levels of BDNF were detected by enzyme linked immunosorbent assay (ELISA), and the expression levels of PI3K/Akt and Nogo-A/RhoA/ROCK2 signaling pathway were detected by western blot and quantitative real-time PCR (qRT-PCR). RESULTS: Compared with those results of pMCAO group, HSHS 5.1 and HSHS 10.2 groups markedly improved neurological function, alleviated pathological damage, promoted the neuronal survival, increased the expression of BDNF, PI3K, Akt, in protein and mRNA, decreased the expression of Nogo-A, NgR, RhoA and ROCK2 in protein and mRNA 7 days after pMCAO. CONCLUSIONS: The findings demonstrate that HSHS had significant therapeutic effects on ischemic stroke and it perhaps worked through the activation of BDNF/PI3K/Akt and down-regulation of Nogo-A/RhoA/ROCK signaling pathways.

Neuroprotective effects of electroacupuncture on early- and late-stage spinal cord injury.

Previous studies have shown that the neurite growth inhibitor Nogo-A can cause secondary neural damage by activating RhoA. In the present study, we hypothesized that electroacupuncture promotes neurological functional recovery after spinal cord injury by inhibiting RhoA expression. We established a rat model of acute spinal cord injury using a modification of Allen's method. The rats were given electroacupuncture treatment at Dazhui (Du14), Mingmen (Du4), Sanyinjiao (SP6), Huantiao (GB30), Zusanli (ST36) and Kunlun (BL60) acupoints with a sparse-dense wave at a frequency of 4 Hz for 30 minutes, once a day, for a total of 7 days. Seven days after injury, the Basso, Beattie and Bresnahan (BBB) locomotor scale and inclined plane test scores were significantly increased, the number of apoptotic cells in the spinal cord tissue was significantly reduced, and RhoA and Nogo-A mRNA and protein expression levels were decreased in rats given electroacupuncture compared with rats not given electroacupuncture. Four weeks after injury, pathological tissue damage in the spinal cord at the site of injury was alleviated, the numbers of glial fibrillary acidic protein- and neurofilament 200-positive fibers were increased, the latencies of somatosensory-evoked and motor-evoked potentials were shortened, and their amplitudes were increased in rats given electroacupuncture. These findings suggest that electroacupuncture treatment reduces neuronal apoptosis and decreases RhoA and Nogo-A mRNA and protein expression at the site of spinal cord injury, thereby promoting tissue repair and neurological functional recovery.

Polyphenols from green tea prevent antineuritogenic action of Nogo-A via 67-kDa laminin receptor and hydrogen peroxide.

Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5 µM) or more effectively through a steady-state generation (1-2 µM), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.>

Effect of Chinese herb Suifukang on expression of NoGo-A protein in the brain of MACO rat / 中国药学杂志

OBJECTIVE: To study the effect of Chinese herb Suifukang on the expression of Nogo protein-A in the brain of MCAO rats and explore the action mechanism of its promoting neurogenesis. METHODS: SD rats were selected and randomized into six groups: normal group, control group, treatment group of methylprednisolone, treatment group of Chinese herb Suifukang in large-does, middle-does and small-does. The models of MCAO rats were successfully established by the method of Koizumi. After the surgery, the rats of drug group had been taken orally Suifukang by 10 g · kg-1 crude drug one time daily, and the rest groups had been taken orally equivalence saline once a day. The brain of rats were investigated and the expression of NoGo-A was detected by immunohistology method and RT-PCR method. RESULTS: The expression of NoGo-A in the injured brain was apparently inhibited by Suifukang (P < 0.01). CONCLUSION: Suifukang can inhibit the expression of NoGo-A. So this function may be one of the mechanisms that Suifukang can promote the functional recovery after brain injury. Copyright 2013 by the Chinese Pharmaceutical Association.