[Effect of moxibustion on Nogo/neurotrophin receptor signaling pathway in rats with cerebral ischemia/reperfusion injury].

OBJECTIVE: To observe the effect of moxibustion treatment on the expression of Nogo-A, Nogo receptor (NgR), neurotrophin receptor p75 (p75NTR) and leucine rich repeat and Ig domain containing 1 (Lingo-1) in brain tissue of rats with cerebral ischemia/reperfusion injury (CI/RI), so as to analyze its mechanism underlying improvement of CI/RI. METHODS: Male SD rats were randomly divided into sham operation group (16 rats), model group (17 rats), NEP1-40 (extracellular peptide residues 1-40, a blocker targeting NgR) group (model+blocker, 17 rats) and moxibustion group (model+moxibustion, 17 rats). The CI/RI model was established by occlusion of the left middle cerebral artery (MCAO). Moxibustion was applied to "Baihui"(GV20), right "Quchi"(LI11) and "Zusanli"(ST36) for 20 min, once a day for 14 days, with 2 days' rest after the top 7 days' intervention. For rats of the NEP1-40 group, 30 µL PBS containing 18 µg NEP 1-40 was injected into the epidural inferior vena (L5-S1) via a polyvinyl chloride conduit. The neurological deficit state in each group was evaluated by Longa's 5-point scale and Feeney's 7-point scale of beam walking test (BWT). The cerebral infarct volume was assessed by 2,3,5-triphenyltetrazole chloride staining. The brain tissue between the central anterior and posterior sulcus was taken for observing the expression of NgR and Lingo-1 by fluorescence double-label method, and for determining the expression levels of Nogo-A, NgR, p75NTR and Lingo-1 mRNAs and proteins by real-time quantitative PCR and Western blot, respectively. RESULTS: After modeling, the Longa's score, infarct volu-me percent, expression levels of Nogo-A, NgR, Lingo-1 and p75NTR mRNAs and proteins were significantly increased (P<0.01) and BWT score was obviously decreased (P<0.01) in the model group relevant to the sham operation group. In comparison with the model group, the increase of Longa's score, infarct volume percentage, expression levels of Nogo-A, NgR, Lingo-1 and p75NTR mRNAs and proteins and decrease of BWT score in NEP1-40 and moxibustion groups were reversed (P<0.01) except Nogo-A protein in the NEP1-40 group. The effect of moxibustion was significantly superior to that of blocker NEP1-40 in redu-cing the infarct volume percentage, and down-regulating the expression of Nogo-A mRNA and protein, p75NTR mRNA and protein, NgR and Lingo-1 proteins (P<0.01, P<0.05). CONCLUSION: Moxibustion, similar to blocker NEP1-40 of NgR, can improve neurological dysfunction in CI/RI rats, which may be related to its functions in reducing cerebral infarction and down-regulating the activity of Nogo/neurotrophin receptor signaling pathway.

[Effects of electroacupuncture on myelin-related protein in corpus callosum of rats with focal cerebral ischemia].

OBJECTIVE: To observe the effect of electroacupuncture(EA)on the expression of myelin basic protein (MBP), axon growth inhibitor Nogo-A and Nogo receptor (NgR) in corpus callosum of rats with focal cerebral ischemia, so as to explore the mechanism of EA underlying improving ischemic white matter injury. METHODS: Fourty male SD rats were randomly divided into normal, sham operation, model and EA groups, with 10 rats in each group. The focal cerebral ischemia rat model was established by occlusion of the middle cerebral artery (MCAO). EA was applied to "Baihui"(GV20) and "Zusanli"(ST36) on the left side for 30 min, once daily for 14 days. Neurological function score and the adhensive removal test were used to evaluate neurological deficit severity; Hematoxylin-esion staining was used to observe the pathological changes in myelin of corpus callosum and luxol fast blue（LFB） staining was used to observe the myelin of corpus callosum. Immunohistochemical staining and Western blot were used to detect the expressions of MBP、Nogo-A and NgR in the ischemic corpus callosum. RESULTS: After MCAO, the neurological function score was significantly increased (P<0.05), the time required for contact with tape and tape removal was longer (P<0.001), the intensity of LFB staining and the expression of MBP decreased, while the veside area and the expression of Nogo-A and its receptor NgR increased (P<0.01, P<0.05) in the model group relevant to the normal and sham operation groups. The fiber arrangement of the corpus callosum on the ischemic side was disordered and a large amount of myelin sheath was lost in the model group. Following the treatment, the neurological deficit score of EA group was gradually decreased and significantly decreased on the 3rd, 7th and 14th day (P<0.05), and the time to remove the adhesive tape was shortened at the 7th and 14th day (P<0.001). The shape of the corpus callosum in the EA group was close to normal, and the myelin structure was relatively complete. The intensity of LFB staining and the expression of MBP was increased (P<0.05, P<0.01) while the expression of Nogo-A and its receptor NgR were decreased in the EA group relevant to the model group (P<0.01). CONCLUSION: EA can play a protective role in myelin of the corpus callosum after cerebral ischemia, which may be related to down-regulating the expressions of Nogo-A and NgR.

Effect of Naoluoxintong on the NogoA/RhoA/ROCK pathway by down-regulating DNA methylation in MCAO rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Naoluoxintong (NLXT) is a traditional Chinese Medicine (TCM) prescription that is clinically used in the treatment of ischemic stroke (IS). However, its therapeutic mechanism remains unclear. AIM OF THE STUDY: To obtain the mechanism of NLXT by observing the protective effects of NLXT on the NogoA/RhoA/Rock pathway in a rat model of IS by regulating DNA methylation. MATERIALS AND METHODS: Rats were divided into five groups using a random number table: normal group, model group, NLXT group, blocker group I (NLXT + SGI-1027) and blocker group II (NLXT + Y27632). The right middle cerebral artery occlusion-reperfusion (MCAO/R) rat model was made, and the regional cerebral blood flow (rCBF) of each group was detected using laser Doppler. The methylation levels of CpG sites of neurite outgrowth inhibitor protein-A (Nogo-A), Nogo receptor (NgR), ras homolog gene family member A (RhoA) and rho-associated coiled-coil protein kinase 2 (ROCK2) genes in rat brain tissue were detected using the bisulfite method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect NogoA, RhoA, NgR1, NgR2 and ROCK2 mRNA expression in rat brain tissue. NogoA, RhoA, NgR1, NgR2 and ROCK2 proteins were detected using immunoblotting in rat brain tissue. RESULTS: After the modeling of middle cerebral artery occlusion (MCAO), neurological deficit test was made to ensure the success of the modeling. At each time point after surgery, the rCBF of the other groups decreased compared with the normal group (P < 0.01 or P < 0.05). Meanwhile, the rCBF increased in blocker group I as well as blocker group II after 3 days (P < 0.05). There were differences in the DNA methylation sites of NogoA, RhoA, NgR and ROCK2 genes between the model group and the NLXT group (P < 0.05). Compared with the normal group, NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression in the model group increased observably (P < 0.01). In comparison with the model group, NogoA and NgR1 gene expression in the blocker group II was prominently observed on the 1st day. NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression remarkably reduced (P < 0.01) on the 3rd and 7th days. Compared with the normal group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the model group increased observably (P < 0.01). In comparison with the model group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the other groups declined prominently (P < 0.01). CONCLUSION: NLXT can reduce the DNA methylation level of NogoA pathway after IS, thus inhibit the expression of NogoA/RhoA/ROCK pathway from producing anti-cerebral ischemia pharmacological effect.

Spirulina platensis reduces the schizophrenic-like symptoms in rat model by restoring altered APO-E and RTN-4 protein expression in prefrontal cortex.

AIMS: Schizophrenia (SZ) is recognized as a neuropsychiatric disorder in humans with accelerated mortality and profound morbidity followed with impairments in social as well as vocational functioning. Though various antipsychotics are being considered as approved treatment therapy for the psychotic symptoms of SZ but they also exert adverse effects and also lack efficacy in treating full spectrum of the disorder. Spirulina platensis (blue-green algae), a nutritional supplement, constitutes a variety of multi-nutrients and possesses a large number of neuroprotective activities. Therefore, present experimental work was designed to evaluate the neuroprotective effects of spirulina in ameliorating the psychosis-like symptoms in dizocilpine-induced rat model of SZ. MATERIALS AND METHODS: The spirulina was tested as preventive and therapeutic regimen at the dose of 180 mg/kg. After pre- and post-treatment with spirulina, rats were subjected to behavioral assessments followed by biochemical and neurochemical estimations. Biomarkers including APO-E, RTN-4, TNF-α, and IL-6 were also estimated using ELISA. KEY FINDINGS: Present results showed that administration of spirulina not only improved behavioral deficits induced by dizocilpine but it also regulates neurotransmission, oligodendrocyte dysfunction and APO-E over expression. Moreover, it also restores the immune response dysfunction by reducing inflammatory cytokines. SIGNIFICANCE: Thus, from present findings it may be suggested that spirulina aids in ameliorating the psychosis-like symptoms induced by dizocilpine in animal model possibly via regulation of neurotransmission and other biomarkers that are extensively used to uncover the etiopathology of SZ. Hence, blue-green algae can be used as an effective therapy for preventive or therapeutic measures in SZ.

Anti-apoptotic efficacy of Qingnao Yizhi formula in hypoxia/reoxygenation primary cortical neurons through the promotion of synaptic plasticity by modulating the NogoA-Nogo receptor/Rho-Rho kinase signaling pathway.

OBJECTIVE: To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula (，QNYZ) in cultured cerebral cortical neuronal cells (CNCs) and the regulation of the NogoA-Nogo receptor (NgR)/Rho-Rho kinase (ROCK) signaling pathway. METHODS: Primary cultured CNCs were randomly divided into the following groups: normal control group (N-C), hypoxia-reoxygenation group (H/R), high-dose QNYZ group (Q-H), low-dose QNYZ group (Q-L) butylphthalide (NBP) group, and Y-27632 (a selective ROCK transduction pathway inhibiter) group. Except those in the N-C group, CNCs were placed in hypoxic conditions for 24 h and then in reoxygenation conditions for 24 h. Cell media was changed every 48 h, and various assays were performed on the 7th day. Cell viability was evaluated by measuring mitochondrial dehydrogenase activity, using a CCK-8 assay, in triplicate. Synapsin (SYN) protein concentrations were evaluated by enzyme-linked immunosorbent assay. NogoA and RhoA protein expression were evaluated through Western blotting. The gene expression of NogoA, NgR, RhoA, and ROCK was evaluated by reverse transcription-polymerase chain reaction. Cell apoptosis was measured using a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. RESULTS: Compared with the N-C group, the cell viability of the H/R group decreased significantly (P < 0.05). The cell viability values for the Q-H and Q-L groups increased compared with that for the H/R group, and the difference was significant for the Q-H group (P < 0.05). The NogoA and RhoA protein levels and the NogoA, NgR, RhoA, and ROCK mRNA expression levels increased in the H/R group, compared with the N-C group, and decreased significantly in the Q-H and Q-L groups (P < 0.05) and in the Y-27632 group (P < 0.05) compared with the H/R group. The SYN levels in the Q-H, Q-L, and NBP groups significantly increased compared with that in the H/R group (P < 0.05). Compared with the H/R group, the numbers of apoptotic cells in the Q-H, Q-L, and NBP groups significantly decreased (P < 0.05). CONCLUSION: The presented study demonstrated that QNYZ exerted anti-apoptotic effects on H/R-induced CNCs, possibly through the modulation of the NogoA-NgR/Rho-ROCK signaling pathway and the promotion of synaptic plasticity in H/R CNCs.

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.

Effects of Bu Shen Yi sui capsule on NogoA/NgR and its signaling pathways RhoA/ROCK in mice with experimental autoimmune encephalomyelitis.

BACKGROUND: Axon growth inhibitory factors NogoA/Nogo receptor (NgR) and its signaling pathways RhoA/Rho kinase (ROCK) play a critical role in the repair of nerve damage in multiple sclerosis (MS). Bu Shen Yi Sui Capsule (BSYSC) is an effective Chinese formula utilized to treat MS in clinical setting and noted for its potent neuroprotective effects. In this study, we focus on the effects of BSYSC on promoting nerve repair and the underlying mechanisms in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. METHODS: The EAE mouse model was induced by injecting subcutaneously with myelin oligodendrocyte glycoprotein (MOG) 35-55 supplemented with pertussis toxin. BSYSC was orally administrated at dose of 3.0 g/kg once a day for 40 days. The levels of protein gene product (PGP) 9.5, p-Tau, growth associated protein (GAP) -43, KI67 and Nestin in the brain or spinal cord on 20 and 40 day post-induction (dpi) were detected via immunofluorescence and Western blot analysis. Furthermore, NogoA/NgR and RhoA/ROCK signaling molecules were studied by qRT-PCR and Western blot analysis. RESULTS: Twenty or 40 days of treatment with BSYSC increased markedly PGP9.5 and GAP-43 levels, reduced p-Tau in the brain or spinal cord of mice with EAE. In addition, BSYSC elevated significantly the expression of KI67 and Nestin in the spinal cord 40 dpi. Further study showed that the activation of NogoA/NgR and RhoA/ROCK were suppressed by the presence of BSYSC. CONCLUSIONS: BSYSC could attenuate axonal injury and promote repair of axonal damage in EAE mice in part through the down-regulation of NogoA/NgR and RhoA/ROCK signaling pathways.

Nogo receptor complex expression dynamics in the inflammatory foci of central nervous system experimental autoimmune demyelination.

BACKGROUND: Nogo-A and its putative receptor NgR are considered to be among the inhibitors of axonal regeneration in the CNS. However, few studies so far have addressed the issue of local NgR complex multilateral localization within inflammation in an MS mouse model of autoimmune demyelination. METHODS: Chronic experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. Analyses were performed on acute (days 18-22) and chronic (day 50) time points and compared to controls. The temporal and spatial expression of the Nogo receptor complex (NgR and coreceptors) was studied at the spinal cord using epifluorescent and confocal microscopy or real-time PCR. Data are expressed as cells/mm2, as mean % ± SEM, or as arbitrary units of integrated density. RESULTS: Animals developed a moderate to severe EAE without mortality, followed by a progressive, chronic clinical course. NgR complex spatial expression varied during the main time points of EAE. NgR with coreceptors LINGO-1 and TROY was increased in the spinal cord in the acute phase whereas LINGO-1 and p75 signal seemed to be dominant in the chronic phase, respectively. NgR was detected on gray matter NeuN+ neurons of the spinal cord, within the white matter inflammatory foci (14.2 ± 4.3 % NgR+ inflammatory cells), and found to be colocalized with GAP-43+ axonal growth cones while no ß-TubIII+, SMI-32+, or APP+ axons were found as NgR+. Among the NgR+ inflammatory cells, 75.6 ± 9.0 % were microglial/macrophages (lectin+), 49.6 ± 14.2 % expressed CD68 (phagocytic ED1+ cells), and no cells were Mac-3+. Of these macrophages/monocytes, only Arginase-1+/NgR+ but not iNOS+/NgR+ were present in lesions both in acute and chronic phases. CONCLUSIONS: Our data describe in detail the expression of the Nogo receptor complex within the autoimmune inflammatory foci and suggest a possible immune action for NgR apart from the established inhibitory one on axonal growth. Its expression by inflammatory macrophages/monocytes could signify a possible role of these cells on axonal guidance and clearance of the lesioned area during inflammatory demyelination.