Melatonin ameliorates sleep-wake disturbances and autism-like behaviors in the Ctnnd2 knock out mouse model of autism spectrum disorders.

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder characterized by atypical patterns of social interaction and communication, as well as restrictive and repetitive behaviors. In addition, patients with ASD often presents with sleep disturbances. Delta (δ) catenin protein 2 (CTNND2) encodes δ-catenin protein, a neuron-specific catenin implicated in many complex neuropsychiatric diseases. Our previous study demonstrated that the deletion of Ctnnd2 in mice led to autism-like behaviors. However, to our knowledge, no study has investigated the effects of Ctnnd2 deletion on sleep in mice. In this study, we investigated whether the knockout (KO) of exon 2 of the Ctnnd2 gene could induce sleep-wake disorders in mice and identified the effects of oral melatonin (MT) supplementation on Ctnnd2 KO mice. Our results demonstrated that the Ctnnd2 KO mice exhibited ASD-like behaviors and sleep-wake disorders that were partially attenuated by MT supplementation. Overall, our current study is the first to identify that knockdown of Ctnnd2 gene could induce sleep-wake disorders in mice and suggests that treatment of sleep-wake disturbances by MT may benefit to autism-like behaviors causing by Ctnnd2 gene deletion.

Progranulin improves neural development via the PI3K/Akt/GSK-3ß pathway in the cerebellum of a VPA-induced rat model of ASD.

Autism spectrum disorder (ASD) is a neurodevelopmental disease featuring social interaction deficits and repetitive/stereotyped behaviours; the prevalence of this disorder has continuously increased. Progranulin (PGRN) is a neurotrophic factor that promotes neuronal survival and differentiation. However, there have not been sufficient studies investigating its effect in animal models of autism. This study investigated the effects of PGRN on autistic phenotypes in rats treated with valproic acid (VPA) and assessed the underlying molecular mechanisms. PGRN was significantly downregulated in the cerebellum at postnatal day 14 (PND14) and PND35 in VPA-exposed rats, which simultaneously showed defective social preference, increased repetitive behaviours, and uncoordinated movements. When human recombinant PGRN (r-PGRN) was injected into the cerebellum of newborn ASD model rats (PND10 and PND17), some of the behavioural defects were alleviated. r-PGRN supplementation also reduced cerebellar neuronal apoptosis and rescued synapse formation in ASD rats. Mechanistically, we confirmed that PGRN protects neurodevelopment via the PI3K/Akt/GSK-3ß pathway in the cerebellum of a rat ASD model. Moreover, we found that prosaposin (PSAP) promoted the internalisation and neurotrophic activity of PGRN. These results experimentally demonstrate the therapeutic effects of PGRN on a rat model of ASD for the first time and provide a novel therapeutic strategy for autism.

Protective Effects of Notoginsenoside R1 via Regulation of the PI3K-Akt-mTOR/JNK Pathway in Neonatal Cerebral Hypoxic-Ischemic Brain Injury.

Notoginsenoside R1 (NGR1) is a predominant phytoestrogen extracted from Panax notoginseng that has recently been reported to play important roles in the treatment of cardiac dysfunction, diabetic kidney disease, and acute liver failure. Studies have suggested that NGR1 may be a viable treatment of hypoxic-ischemic brain damage (HIBD) in neonates by reducing endoplasmic reticulum stress via estrogen receptors (ERs). However, whether NGR1 has other neuroprotective mechanisms or long-term neuroprotective effects is unclear. In this study, oxygen-glucose deprivation/reoxygenation (OGD/R) in primary cortical neurons and unilateral ligation of the common carotid artery (CCL) in 7-day-old postnatal Sprague Dawley (SD) rats followed by exposure to a hypoxic environment were used to mimic an HIBD episode. We assessed the efficacy of NGR1 by measuring neuronal damage with MTT assay and assessed brain injury by TTC staining and brain water content detection 24-48 h after OGD/HIE. Simultaneously, we measured the long-term neurophysiological effects using the beam walking test (5 weeks after HI) and Morris water maze test 5-6 weeks after HI. Expression of PI3K-Akt-mTOR/JNK (24 h after HI or OGD/R) proteins was detected by Western blotting after stimulation with HI, NGR1, LY294002 (PI3K inhibitor), 740Y-P (PI3K agonist), or ICI 182780(estrogen receptors inhibitor). The results indicated that NGR1 exerted neuroprotective effects by inhibiting neuronal apoptosis and promoting cell survival via the PI3K-Akt-mTOR/JNK signaling pathways by targeting ER in neonatal hypoxic-ischemic injury.

[Protective effect of notoginsenoside R1 on neuron injury induced by OGD/R through ATF6/Akt signaling pathway].

Notoginsenoside R1(NGR1),a critical compound in traditional herb Panax notoginseng, is a kind of estrogen receptor agonist.It is reported to exhibit anti-apoptotic,anti-oxidative and anti-inflammatory properties activity, so it is widely used for treatment of various diseases.In order to investigate the potential neuroprotective effect of NGR1 in hypoxic-ischemic brain damage(HIBD), primary cortical neurons were used in this study to establish oxygen-glucose deprivation/reoxygenation(OGD/R) injury models. They were treated with NGR1 and estrogen receptor inhibitor ICI-182780 respectively, then the neuronal survival, cell membrane integrity and apoptosis were assessed by MTT assay,lactate dehydrogenase test(LDH) and Hoechst 33342 stain respectively, while the protein expression levels of ATF6α,p-Akt,Akt,Bax and Cleaved Caspase-3 were measured by Western blotting. Results indicated that as compared with the blank control group,OGD/R could induce cell injury and apoptosis(P<0.05), reduce relative integrity of cell membrane(P<0.05), decrease protein expression of ATF6α,p-Akt(P<0.05), and increase protein expression of Bax and Cleaved Caspase-3(P<0.05) in the primary cortical cells. After NGR1 treatment, the expression levels of ATF6α,p-Akt were obviously increased, and the expression levels of Bax and Cleaved Caspase-3 and the apoptosis of neuron were decreased(P<0.05). However, these neuroprotective properties of NGR1 against ODG/R-induced cell damage could be blocked by ICI-182780. This finding indicated that NGR1 may protect the primary cortical neurons against OGD/R induced injury,and the mechanism may be associated with accelerating the activation of the ATF6/Akt signaling pathway via estrogen receptors.

[Study on molecular target promoting human neural stem cells of ginsenoside Rg1 by gene chip].

OBJECTIVE: To screen out main molecular target promoting human neural stem cells (NSCs) of ginsenoside Rg1 by using the gene chip technology. METHOD: First, MTT assay was adopted to screen out the optimal concentration of Rg1-promoted NSC proliferation (120 mg x L(-1)). Then, on the 7th day after the Rg1-promoted NSC proliferation, the expression of target genes was observed by the gene chip technology. The most important target gene and signal transduction pathways were screened out through the data calculations. RESULT: On the 7th day after the Rg1-promoted NSC proliferation, obtained 440 differential genes, 266 significantly upregulated genes and 174 significantly down-regulated genes. HES1 gene, CAMP (cyclic adenosine monophosphate)-PKA (protein kinase A) and PI3K (phosphatidylinositol 3 kinase)-AKT signal transduction pathways were closely related to the NSC proliferation. CONCLUSION: The differentially expressed genes screened out by gene chip may provide new clues for studies on molecular mechanism of ginsenoside Rg1-promoted NSCs proliferation.

[Study on mechanism of ginsenoside Rg1-induced human neural stem cells differentiation by genechip].

OBJECTIVE: The molecular targets of ginsenoside Rg1-induced neural stem cells (NSCs) differentiation were screened by genechip. METHOD: 7th day following ginsenoside Rg1 induced human neural stem cells to neurons the gene expression was observed by genechip. The purpose gene and signal transduction pathways were selected by the data calculations, and then confirmed by western blot and immunohistochemical method. RESULT: 7th day following Rg1-induced NSCs differentiation, there were about 675 different genes, 255 genes of which were up-regulated and 420 genes down-regulated obviously. Meanwhile the ERK1/2 (extracellular signal-regulated protein kinase) in MAPK (mitogen-activated protein kinase) pathway was related with the NSCs differentiation. The Western blot and immunohistochemistry detection confirmed that ERK 1/2 protein and its phosphorylation were significantly increased, which can be blocked by PD98059 (ERK1/2 inhibitor). In addition, differentiation rate of NSCs was also decreased obviously in ginsenoside Rg1-induced differentiated NSCs when ERK blocker PD98059 was used. CONCLUSION: ERK1/2 is an important molecular target in ginsenoside Rg1-induced NSC differentiation. The selected differentially expressed genes by genechip may provide new clues to study of ginsenoside Rg1-induced NSCs differentiation.

[Effect of ginsenoside Rg1 on functional expression of human neural stem cells: a patch clamp study].

OBJECTIVE: To observe the effects of ginsenoside Rg1 on the functional expression of human neural stem cells (hNSCs). METHOD: The membrane electrophysiological properties and sodium and potassium ion channels in the hNSCs induced by Rg1 were analyzed using the whole-cell patch-clamp. RESULT: On the 7th day, the neuron-like cells derived from ginsenoside Rg1 (20 mg x L(-1))-induced NSCs show: (1) The resting membrane potential: (-45.70 +/- 2.63) mV, the membrane capacitance: (26.89 +/- 1.91) pF, the membrane input impedance: (877.51 +/- 20.44) MH (P < 0.05 compared with the control group, respectively); (2) The detection rate of inward sodium current which is rapidly activated and inactivated in voltage-dependence was 50%, and its average peak value was (711.48 +/- 158.03) pA (P < 0.05 compared with the control group); (3) The outward potassium currents were composed of rapidly activated and inactivated transient outward potassium current and delayed rectifier outward potassium current, and its average peak value was (1 070.42 +/- 177.18) pA (P < 0.05 compared with the control group). CONCLUSION: Ginsenoside Rg1 can promote the functional expression and maturity of hNSCs.

Treatment with Z-ligustilide, a component of Angelica sinensis, reduces brain injury after a subarachnoid hemorrhage in rats.

Subarachnoid hemorrhage (SAH) is a devastating stroke subtype accounting for approximately 3 to 7% of cases each year. Despite its rarity among the various stroke types, SAH is still responsible for approximately 25% of all stroke fatalities. Although various preventative and therapeutic interventions have been explored for potential neuroprotection after SAH, a considerable percentage of patients still experience serious neurologic and/or cognitive impairments as a result of the primary hemorrhage and/or secondary brain damage that occurs. Z-ligustilide (LIG), the primary lipophilic component of the Chinese traditional medicine radix Angelica sinensis, has been shown to reduce ischemic brain injury via antiapoptotic pathways. Accordingly, in our study, we investigated the neuroprotective potential of LIG after experimental SAH in rats. Rats with SAH that was induced using the established double hemorrhage model were studied with and without LIG treatment. Mortality, neurobehavioral evaluation, brain water content, blood-brain barrier (BBB) permeability, and vasospasm assessment of the basilar artery were measured on days 3 and 7 after injury. Additional testing was done to evaluate for apoptosis using TdT-mediated dUTP-biotin nick end labeling staining as well as immunohistochemistry and Western blotting to identify key proapoptotic/survival proteins, i.e., p53, Bax, Bcl-2, and cleaved caspase-3. The results showed that LIG treatment reduced mortality, neurobehavioral deficits, brain edema, BBB permeability, and cerebral vasospasm. In addition, treatment reduced the number of apoptotic cells in the surrounding brain injury site, which accompanied a marked down-regulation of proapoptotic proteins, p53, and cleaved caspase-3. Our data suggest that LIG may be an effective therapeutic modality for SAH victims by altering apoptotic mechanisms.

Treatment with ginsenoside rb1, a component of panax ginseng, provides neuroprotection in rats subjected to subarachnoid hemorrhage-induced brain injury.

OBJECTIVE: recent trials have shown Ginsenoside Rb1 (GRb1), an active component of a well known Chinese medicine Panax Ginseng, plays a significant role in improving the complications seen after an ischemic brain event. In the present study, we investigated the use of GRb1 as a treatment modality to reduce brain edema, reduce arterial vasospasm, and improve neurobehavioral function after subarachnoid hemorrhage-induced brain injury (SAH) in rats. METHOD: male Sprague-Dawley rats weighing between 250 and 300 g were randomly assigned to three groups: (1) Sham group (n = 10), (2) Vehicle group (SAH + no treatment; n = 12); (3) Treatment group (SAH + GRb1 treatment at 20 mg/kg; n = 11). Subarachnoid hemorrhage was induced using the modified double hemorrhage model followed by treatment administration intravenously. Post-operative assessment included neurobehavioral testing using the spontaneous activity scoring system, brain water content, and histological examination of the basilar artery. RESULTS: post-operative findings indicated treatment with GRb1 had significantly reduced brain edema and improved neurobehavioral functioning. In addition, histological examination revealed a significant reduction in basilar artery vasospasm and lumen thickness with treatment. CONCLUSION: the results of the study suggest that GRb1 treatment reduces brain edema, improves neurobehavioral function, and blocks vasculature thickening and spasm after SAH in rats. Given the novelty of the study, further research will be needed to confirm the benefits of treatment and mechanisms behind neuroprotection.

Ginsenoside Rbeta1 reduces neurologic damage, is anti-apoptotic, and down-regulates p53 and BAX in subarachnoid hemorrhage.

Stroke is the second leading cause of death worldwide and the number one cause of adult disability in the United States and Europe. A subtype of stroke, subarachnoid hemorrhage (SAH), accounts for 7% of all strokes each year and claims one of the highest mortalities and morbidities. Many therapeutic interventions have been used to treat brain injury following SAH but none have reached the level of effectiveness needed to clinically reduce mortality. Ginsenoside Rb1 (GRb1), a major component of the Chinese traditional medicine Panax Ginseng, has been shown to reduce ischemic brain injury and myocardial injury via anti-apoptotic pathways. In the present study, we investigated the use of GRb1 on SAH induced brain injury in rats. Four groups were used: sham, vehicle (SAH), low dose treatment (SAH+ 5mg/kg GRb1), and high dose treatment (SAH+ 20mg/kg GRb1). Post assessment included wall thickness and mean cross-section area of basilar artery were measured for evaluating cerebral vasospasm, Evans blue extravasations to assess blood brain barrier (BBB) permeability, immunohistochemistry and Western Blot analysis looking for specific pro-apoptotic markers, and tunnel staining for cell death assessment. In addition, mortality, neurological function and brain edema were investigated. The results showed that high dose GRb1 treatment significantly enlarged mean cross-sectional area and decreased wall thickness of basilar artery, reduced neurological deficits, brain edema, BBB disruption, and TUNEL positive cell expression. Same time, we found that the proteins expression of P53, Bax and Caspase-3 were significantly reduced, whereas the expression of bcl-2 was up-regulated in Rb1 treatment. The results of this study suggest that GRb1 could relieve cerebral vasospasm and potentially provide neuroprotection in SAH victims. The underlying mechanisms may be partly related to inhibition of P53 and Bax dependent proapoptosis pathway. More studies will be needed to confirm these results and determine its potential as a long term agent.

[The effect of TSPG in vivo on transplantation of neural stem cells in treatment of Parkinson's disease mouse].

AIM: To observe the effect for the model of PD and the transplantation of NSCs after injection of TSPG into mouse in advance. METHODS: Firstly, we divided the mouse into 5 groups. For the group of 1-4, we established the model of PD with MPTP. For the group of 5, before the establishment of the model, we injected TSPG into mouse in advance for prevention. And then, we evaluated the effect by paralysis agitans score standard and praxiology marker. Secondly, we obtained the NSCs from the 7-12 week embryo cerebral cortex. Then we transplanted NSCs which pretreated by TSPG into the striatum of the 5 groups. After 60 days, we obtained the brain section, and detected the TH by ICC to analyse the differentiation status of NSCs. RESULTS: The prevention of TSPG could decrease the neural cells damage by MPTP, and could protect the nervous system. After we transplanted NSCs into the striatum of Parkinson' s disease mouse, we found that for the group of 5, the paralysis agitans, auto-activity and memory function had the most distinct amelioration. And the number of dopaminergic neurons increased most transparently in brain section, and the neurons contact was the most enriched with the adjacent nervous cells. CONCLUSION: TSPG can decrease the neural cells damage and can produce a marked effect in treatment of PD by transplanting NSCs invivo.

[Effect of TSPG on proliferation and differentiation of human embryonic neural stem cell into dopaminergic neuron].

OBJECTIVE: To investigate the effects of total saponins of panax ginseng (TSPG) on proliferation and differentiation of human embryonic neural stem cell (NSC) into dopaminergic neuron. METHOD: Isolation, cultivation and identification of human embryonic NSC from cerebral cortex of 7-12 week abortus. By using flow cytometry and MTT assay, the effects of various concentration of TSPG and TSPG cooperating with cytokines( EGF, bFGF) in NSC culture media for 3 days on proliferation of human embryonic NSC has studied. By employing immunocytochemistry assay of the expression of tyrosine hydroxylase (TH), the effect of different dilution of TSPG and TSPG cooperating with IL-1 on induced differentiation of human embryonic NSC into dopaminergic neuron has researched. RESULT: TSPG can significantly promote the proliferation of NSC. When TSPG cooperating with EGF and bFGF, the proliferation of NSC is much stronger than that of only using FGF and bFGF. TSPG also induces NSC to differentiate into dopaminergic neuron, especially when TSPG is cooperating with IL-1. CONCLUSION: TSPG can not only obviously accelerate the proliferation of NSC, but also significantly induce differentiation of NSC into dopaminergic neuron.

[Synergistic effects of total saponins of panax ginseng in combination with hematopoietic growth factor on proliferation and differentiation of CD34(+) cells ex vivo].

To investigate the effects of total saponins of panax ginseng (TSPG) in combination with hematopoietic growth factors (HGF) on proliferation and differentiation of CD34(+) cells ex vivo, the purified CD34(+) cells from cord blood and bone marrow were expanded by various concentrations of TSPG with combination of cytokines in liquid culture systems and the expanded cell number, CD34(+) cell number, CD33(+) cell ratio, the numbers of total CFC and hematopoietic progenitor cell number were detected. The results showed that TSPG (10 - 70 microg/ml) could raise the expanded cell number, CD34(+) cell number, and the numbers of total CFC, TSPG 50 microg/ml was identified as the most potent stimulating concentration, and increased total nucleated cells to (2470.5 +/- 79.96) x 10(3), CFC to (53.96 +/- 4.286) x 100% and CD34(+) cells to (21.86 +/- 3.094) x 100%; TSPG (10 - 50 microg/ml) could raise the colony formation rate of CFU-GM, TSPG (20 microg/ml) induced the best effect on granulocytopoietic differentiation committed of CD34(+) cells. It is concluded that the optimal concentration of TSPG can promote CD34(+) cells to proliferate and differentiate by cooperating with hematopoietic growth factors.

[Experimental study on expression of GM-CSF from human endothelial cells and monocytes induced by total saponins of panax ginseng].

OBJECTIVE: To study the effect of total saponins of Panax ginseng (TSPG) on the expression of granulocytemacrophage colony-stimulating factor (GM-CSF) from human endothelial cells and monocytes and the relationship between TSPG and human granulocytopoiesis and monocytopoiesis modulation. METHODS: Adopting the hematopoietic progenitor cells culture in vitro, hematopoietic growth factor biological assay, immunocytochemistry and nucleic acid in situ hybridization, the GM-CSF expression in the endothelial cells and monocytes were detected. RESULTS: The conditioned cultural media of endothelial and monocytes induced and prepared by TSPG, could significantly promote the proliferation and differentiation of human colony forming unit-granulocyte macrophage (CFU-GM), and enhance the protein and mRNA expression of GM-CSF in endothelial cells and monocytes. CONCLUSION: TSPG could possibly through direct or indirect route, promote hematopoietic, induce endothelial cells and monocytes in the microenvironment to synthetize and secrete GM-CSF, so as to further promote the proliferation and differentiation of human CFU-GM.

[Modulation of expression of human GM-CSF and GM-CSFRalpha by total saponins of Panax ginseng].

The purpose of the present study was to investigate the biological mechanism for modulating granulocytopoiesis by Panax ginseng. The techniques of culture of hematopoietic progenitor cells and hematopoietic stromal cells in vitro, biological assay of hematopoietic growth factor (HGF), immunocytochemistry, in situ hybridization of nucleic acid, immunoprecipitation and Western blot were used to explore the effect of total saponins of Panax ginseng (TSPG) on the expression of human granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte-macrophage colony stimulating factor receptor alpha (GM-CSFRalpha). The results indicated that (1) bone marrow stromal cell (BMSC), thymocyte (TC), splenocyte (SC), endothelial cells (EC), and monocyte (MO) conditioned media prepared with TSPG (50 microg/ml) could significantly enhance the proliferation of CFU-GM; (2) the expressions of GM-CSF in protein and mRNA level in BMSC, TC, SC, EC and MO induced by TSPG (50 microg/ml) were much higher than that of the control; (3) the expression of GM-CSFRalpha protein in hematopoietic cells induced by TSPG (50 microg/ml) was stronger than that of the control; (4) TSPG (50 microg/ml) could stimulate the transient tyrosine phosphorylation of GM-CSFR and Shc protein. We speculate that TSPG may directly and/or indirectly promote the stromal cells and lymphocytes to produce GM-CSF and other cytokine and induce bone marrow hematopoietic cells to express GM-CSF receptors (GM-CSFRalpha), leading to the regulation of the GM-CSFR-mediated signals transduction pathway and the proliferation of human CFU-GM.