Pimecrolimus protects neuron-like SH-SY5Y cells against anti-inflammatory and anti-oxidant effects of both microglial secretome and hydrogen peroxide.

Calcineurin inhibitors have been found to exhibit a preventive role against neuroinflammation, which represents a crucial underlying mechanism in neurodegenerative diseases (ND). Additionally, they possess suppressive effects on the activation of apoptotic pathways, which constitute another mechanism underlying such diseases. Given that pimecrolimus, a calcineurin inhibitor, impedes the synthesis of pro-inflammatory cytokines, such as interleukin (IL)-2, IL-4, and IL-10, and influences apoptotic processes, it is noteworthy to test its potential neuroprotective properties. Thus, the objective of this investigation was to assess the potential protective effects of pimecrolimus against the degenerative consequences of both microglial secretomes and hydrogen peroxide (H2O2), an oxidant agent. The survival rates of HMC3 microglia cells, neuron-like differentiated SH-SY5Y (d-SH-SY5Y) cells, and their co-culture were determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) method. Furthermore, the levels of pro-inflammatory cytokines IL-1ß and IL-6, and anti-inflammatory cytokine IL-10 were measured using ELISA kits, besides total antioxidant and oxidant capacities in conditioned media of cells. Additionally, the effect of pimecrolimus on neurite length in these cell groups was evaluated through morphological observations. This study revealed, for the first time, that pimecrolimus exerts preventive effects on neurodegenerative processes by virtue of its anti-inflammatory and -antioxidant activities. It holds promise as a potential treatment option for ND.

Adalimumab combined with erythropoietin improves recovery from spinal cord injury by suppressing microglial M1 polarization-mediated neural inflammation and apoptosis.

OBJECTIVE: Adalimumab (ADM), a humanized antibody against tumour necrosis factor (TNF), is widely applied in treating inflammatory and autoimmune diseases, but its usage in spinal cord injury (SCI) is rarely reported. Hence, this study aimed to explore the effect of ADM with or without erythropoietin (EPO) on microglial polarization, neuroinflammation, neural apoptosis, and functional recovery in SCI. METHODS: Primary microglia were stimulated with lipopolysaccharide (LPS) and then treated with ADM, EPO, or ADM combined with EPO. Then, primary neurons were incubated in the microglial culture medium. SCI rats were established and then treated with ADM, EPO or ADM combined with EPO. RESULTS: ADM suppressed LPS-induced microglial M1 polarization, as reflected by downregulated iNOS and CD86 expression, and neuroinflammation, as reflected by decreased TNF-α, IL-1ß, and IL-6 expression, in a dose-dependent manner. Moreover, ADM inhibited microglia-induced neural apoptosis, as reflected by TUNEL assay results and the expression of apoptotic markers (C-Caspase3 and Bcl2), in a dose-dependent manner. EPO monotherapy displayed an effect similar to that of ADM monotherapy. Furthermore, ADM combined with EPO therapy exhibited greater effects than either monotherapy in terms of inhibiting microglial M1 polarization, neuroinflammation, and neural apoptosis. In vivo experiments confirmed the findings of the in vitro experiments and showed that ADM combined with EPO improved SCI functional recovery and neural injury compared with monotherapy. CONCLUSION: ADM combined with EPO improves recovery from SCI by suppressing microglial M1 polarization-mediated neural inflammation and apoptosis.

TREM2 activation alleviates neural damage via Akt/CREB/BDNF signalling after traumatic brain injury in mice.

BACKGROUND: Neuroinflammation is one of the most important processes in secondary injury after traumatic brain injury (TBI). Triggering receptor expressed on myeloid cells 2 (TREM2) has been proven to exert neuroprotective effects in neurodegenerative diseases and stroke by modulating neuroinflammation, and promoting phagocytosis and cell survival. However, the role of TREM2 in TBI has not yet been elucidated. In this study, we are the first to use COG1410, an agonist of TREM2, to assess the effects of TREM2 activation in a murine TBI model. METHODS: Adult male wild-type (WT) C57BL/6 mice and adult male TREM2 KO mice were subjected to different treatments. TBI was established by the controlled cortical impact (CCI) method. COG1410 was delivered 1 h after CCI via tail vein injection. Western blot analysis, immunofluorescence, laser speckle contrast imaging (LSCI), neurological behaviour tests, brain electrophysiological monitoring, Evans blue assays, magnetic resonance imaging (MRI), and brain water content measurement were performed in this study. RESULTS: The expression of endogenous TREM2 peaked at 3 d after CCI, and it was mainly expressed on microglia and neurons. We found that COG1410 improved neurological functions within 3 d, as well as neurological functions and brain electrophysiological activity at 2 weeks after CCI. COG1410 exerted neuroprotective effects by inhibiting neutrophil infiltration and microglial activation, and suppressing neuroinflammation after CCI. In addition, COG1410 treatment alleviated blood brain barrier (BBB) disruption and brain oedema; furthermore, COG1410 promoted cerebral blood flow (CBF) recovery at traumatic injury sites after CCI. In addition, COG1410 suppressed neural apoptosis at 3 d after CCI. TREM2 activation upregulated p-Akt, p-CREB, BDNF, and Bcl-2 and suppressed TNF-α, IL-1ß, Bax, and cleaved caspase-3 at 3 d after CCI. Moreover, TREM2 knockout abolished the effects of COG1410 on vascular phenotypes and microglial states. Finally, the neuroprotective effects of COG1410 were suppressed by TREM2 depletion. CONCLUSIONS: Altogether, we are the first to demonstrate that TREM2 activation by COG1410 alleviated neural damage through activation of Akt/CREB/BDNF signalling axis in microglia after CCI. Finally, COG1410 treatment improved neurological behaviour and brain electrophysiological activity after CCI.

Resveratrol inhibits neural apoptosis and regulates RAX/P-PKR expression in retina of diabetic rats.

PURPOSE: This study was to investigate the effect of resveratrol (RSV) administration on diabetes-induced neural apoptosis and on RNA-dependent-protein-kinase (PKR)-associated protein X (RAX), PKR and phosphorylated PKR (P-PKR) expression and distribution in retina of diabetic rats. METHODS: Retina was obtained from normal and diabetic Sprague-Dawley rats with or without RSV (5 and 10 mg/kg/d) treatment at 30-, 32-, 34- and 36-weeks. Apoptosis of retinal neural cells and distribution of RAX/P-PKR was assessed by TUNEL and immunofluorescence methods. Expression of RAX, PKR and P-PKR was evaluated by qRT-PCR and western-blotting methods. RESULTS: Our study showed that the TUNEL-positive cells were mainly localized in ganglion cells layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL) of the diabetic rat's retina at 30-, 32-, 34- and 36-weeks. RSV administration effectively suppressed the neural apoptosis in GCL, INL and ONL. Almost no TUNEL-positive cells were observed in retina of normal control and RSV-treated normal control rats. Our study also showed that the expression level of RAX, P-PKR in diabetic rats retina at 30-, 32-, 34-, and 36-weeks was elevated. With supplementation of 5 and 10 mg/kg/d RSV, the expression level of RAX and P-PKR was decreased (P < 0.05). The expression level of RAX and P-PKR in the retina of normal control rats was not altered by RSV. The expression level of PKR was not altered by streptozotocin injection and RSV treatment. CONCLUSIONS: Our results suggested that RSV attenuates retinal neural apoptosis in diabetic rats retina may be via regulation RAX/P-PKR expression.

Encephalitis is mediated by ROP18 of Toxoplasma gondii, a severe pathogen in AIDS patients.

The neurotropic parasite Toxoplasma gondii is a globally distributed parasitic protozoan among mammalian hosts, including humans. During the course of infection, the CNS is the most commonly damaged organ among invaded tissues. The polymorphic rhoptry protein 18 (ROP18) is a key serine (Ser)/threonine (Thr) kinase that phosphorylates host proteins to modulate acute virulence. However, the basis of neurotropism and the specific substrates through which ROP18 exerts neuropathogenesis remain unknown. Using mass spectrometry, we performed proteomic analysis of proteins that selectively bind to active ROP18 and identified RTN1-C, an endoplasmic reticulum (ER) protein that is preferentially expressed in the CNS. We demonstrated that ROP18 is associated with the N-terminal portion of RTN1-C and specifically phosphorylates RTN1-C at Ser7/134 and Thr4/8/118. ROP18 phosphorylation of RTN1-C triggers ER stress-mediated apoptosis in neural cells. Remarkably, ROP18 phosphorylation of RTN1-C enhances glucose-regulated protein 78 (GRP78) acetylation by attenuating the activity of histone deacetylase (HDAC), and this event is associated with an increase of neural apoptosis. These results clearly demonstrate that both RTN1-C and HDACs are involved in T. gondii ROP18-mediated pathogenesis of encephalitis during Toxoplasma infection.

PI3K/AKT/JNK/p38 signalling pathway-mediated neural apoptosis in the prefrontal cortex of mice is involved in the antidepressant-like effect of pioglitazone.

Numerous studies have reported that inflammation is involved in the pathophysiology of depression. Pioglitazone, a PPAR-γ agonist, has potential anti-inflammatory and antidepressive effects. However, the underlying molecular mechanisms of the antidepressant-like effect of pioglitazone on an inflammation-related mouse model of depression remain to be fully elucidated. Herein, we aimed to explore the effects of pioglitazone on depressive-like behaviours of mice exposed to lipopolysaccharides (LPS), and elucidate the underlying mechanisms. We assessed behaviour changes of mice pretreated with pioglitazone exposed to LPS. Additionally, neural apoptosis, and the expression of apoptosis-related (cleaved caspase-3, Bax, Bcl-2, cyt c) and signalling proteins (AKT, JNK, p38) were assessed in the prefrontal cortex (PFC) of these mice. Furthermore, we assessed the influence of anisomycin, a JNK/p38 agonist, and LY294002, a PI3K/AKT inhibitor, on the antidepressant-like effect of pioglitazone in mice. We show that pioglitazone pretreatment (20 mg/kg, intragastrically) attenuated LPS-induced (10 ng/µL per site) depressive-like behaviours. GW9662, a PPAR-γ antagonist, significantly blocked the antidepressant-like effect of pioglitazone. Furthermore, at the molecular level, pioglitazone significantly reversed, via PPAR-γ-dependent increase in neural apoptosis in the PFC of mice, accompanied by upregulation of the PI3K/AKT pathway and down-regulation of the JNK/p38 pathway. Moreover, both anisomycin and LY294002 abrogated the antidepressant-like effect of pioglitazone.; In conclusion, our results showed that PI3K/AKT/JNK/p38 signalling pathway-mediated neural apoptosis in the PFC of mice may be involved in the antidepressant-like effect of pioglitazone. This provides novel insights into and therapeutic targets for inflammation-related depression.

Involvement of the delayed rectifier outward potassium channel Kv2.1 in methamphetamine-induced neuronal apoptosis via the p38 mitogen-activated protein kinase signaling pathway.

Methamphetamine (Meth) is an illicit psychostimulant with high abuse potential and severe neurotoxicity. Recent studies have shown that dysfunctions in learning and memory induced by Meth may partially reveal the mechanisms of neuronal channelopathies. Kv2.1, the primary delayed rectifying potassium channel in neurons, is responsible for mediating apoptotic current surge. However, whether Kv2.1 is involved in Meth-mediated neural injury remains unknown. In the present study, the treatment of primary cultured hippocampal neurons with Meth indicated that Meth induced a time- and dose-dependent augmentation of Kv2.1 protein expression, accompanied by elevated cleaved-caspase 3 and declined bcl-2/bax ratio. The blockage of Kv2.1 with the inhibitor GxTx-1E or the knockdown of the channel noticeably abrogated the pro-apoptotic effects mediated by Meth, demonstrating the specific roles of Kv2.1 in Meth-mediated neural damage. Additionally, the p38 mitogen-activated protein kinase (MAPK) signaling was demonstrated to be involved in Meth-mediated Kv2.1 upregulation and in the subsequent pro-apoptotic effects, as treatment with a p38 MAPK inhibitor significantly attenuated Meth-mediated Kv2.1 upregulation and cell apoptosis. Of note, PRE-084, a sigma-1 receptor agonist, obviously attenuated Meth-induced upregulation of Kv2.1 expression, neural apoptosis and p38 MAPK activation. Taken together, these results reveal a novel mechanism involved in Meth-induced neural death with implications for therapeutic interventions for Meth users.

Roles of microRNA-1 in hypoxia-induced apoptotic insults to neuronal cells.

Hypoxia is a common occurrence in brain tumors and traumatic brain injury. microRNA (miR)-1 participates in the regulation of brain development and neuronal function. Interestingly, miR-1 can mediate ischemia-induced injury to cardiomyocytes. This study was designed to evaluate the roles of miR-1 in hypoxia-induced insults to neurons and the possible mechanisms. Exposure of neuro-2a cells to oxygen/glucose deprivation (OGD) or cobalt chloride decreased cell viability and induced cell apoptosis in time-dependent manners. In parallel, OGD caused augmentation of cellular Bax and cytochrome c levels, a reduction in the mitochondrial membrane potential (MMP), activation of caspase-3, and fragmentation of DNA. miR-1 was induced in neuro-2a cells by OGD. Knocking down miR-1 expression using specific antisense inhibitors significantly alleviated OGD-induced neuronal death. Administration of OGD to neuro-2a cells induced heat-shock protein (HSP)-70 messenger (m)RNA and protein expressions. A bioinformatic search revealed that miR-1-specific binding elements exist in the 3'-untranslated region of HSP-70 mRNA. Overexpression of miR-1 simultaneously attenuated OGD-induced HSP-70 mRNA and protein expressions. In comparison, knocking down miR-1 expression synergistically enhanced OGD-induced HSP-70 mRNA. As to the mechanism, reducing miR-1 expression lowered OGD-induced alterations in the MMP, caspase-3 activation, DNA fragmentation, and cell apoptosis. Taken together, this study shows that miR-1 can target HSP-70 expression and consequently mediate hypoxia-induced apoptotic insults to neuro-2a cells via an intrinsic Bax-mitochondrion-caspase protease pathway.

Long Non-Coding RNA MALAT1 Protects Against Spinal Cord Injury via Suppressing microRNA-125b-5p Mediated Microglial M1 Polarization, Neuroinflammation, and Neural Apoptosis.

Our previous studies have discovered that long non-coding RNA (lncRNA) MALAT1 and its target microRNA-125b-5p (miR-125b-5p) are implicated in neurological diseases via regulating neuroinflammation and neuronal injury. This study aimed to further explore the relationship between lncRNA MALAT1 and miR-125b-5p, as well as their effect on microglial activation, neuroinflammation, and neural apoptosis in spinal cord injury (SCI). Primary microglia from Sprague Dawley rats were stimulated with lipopolysaccharide (LPS). Then, microglia were transfected with lncRNA MALAT1 overexpression or knock-down adenovirus-associated virus with or without miR-125b-5p mimic. The culture medium of microglia was incubated with primary neurons. SCI rats were established for in vivo validation. LncRNA MALAT1 expression was reduced by LPS treatment in a dose-dependent manner. LncRNA MALAT1 overexpression suppressed the microglial M1 polarization (decreased iNOS but increased ARG1), neuroinflammation (declined PTGS2, TNF-α, IL-1ß, and IL-6), and microglia-induced neural apoptosis (lower TUNEL positive cells and C-caspase3 but higher BCL2) under LPS treatment; its knock-down displayed the opposite trend. Moreover, lncRNA MALAT1 directly bound to and negatively regulated miR-125b-5p. MiR-125b-5p mimic promoted microglial M1 polarization, neuroinflammation, and microglia-induced neural apoptosis following LPS treatment; also, it could attenuate the effect of lncRNA MALAT1. Further in vivo study displayed that lncRNA MALAT1 overexpression elevated the Basso-Beattie-Bresnahan motor function score and improved neural injury. Also, in vivo validation indicated a similar effect of lncRNA MALAT1 on microglial polarization and neuroinflammation as in vitro. LncRNA MALAT1 improves SCI recovery via miR-125b-5p mediated microglial M1 polarization, neuroinflammation, and neural apoptosis.

Cerebral Cortex Apoptosis in Early Aged Hypertension: Effects of Epigallocatechin-3-Gallate.

This study aimed to investigate cerebral cortex apoptosis on the early aged hypertension and the effects of green tea flavonoid epigallocatechin-3-gallate (EGCG). Twenty-four rats were divided into three groups: a control Wistar-Kyoto group (WKY, n = 8), a spontaneously early aged hypertensive group (SHR, n = 8), and an early aged hypertension with EGCG treatment group (SHR-EGCG, n = 8; daily oral EGCG 200 mg/kg-94%, 12 weeks). At 48 weeks old, blood pressures (BPs) were evaluated and cerebral cortexes were isolated for TUNEL assay and Western blotting. Systolic, diastolic, and mean blood pressure levels in the SHR-EGCG were reduced compared to the SHR. The percentage of neural cell deaths, the levels of cytosolic Endonuclease G, cytosolic AIF (Caspase-independent apoptotic pathway), Fas, Fas Ligand, FADD, Caspase-8 (Fas-mediated apoptotic pathway), t-Bid, Bax/Bcl-2, Bak/Bcl-xL, cytosolic Cytochrome C, Apaf-1, Caspase-9 (Mitochondrial-mediated apoptotic pathway), and Caspase-3 (Fas-mediated and Mitochondria-mediated apoptotic pathways) were increased in the SHR relative to WKY and reduced in SHR-EGCG relative to SHR. In contrast, the levels of Bcl-2, Bcl-xL, p-Bad, 14-3-3, Bcl-2/Bax, Bcl-xL/Bak, and p-Bad/Bad (Bcl-2 family-related pro-survival pathway), as well as Sirt1, p-PI3K/PI3K and p-AKT/AKT (Sirt1/PI3K/AKT-related pro-survival pathway), were reduced in SHR relative WKY and enhanced in SHR-EGCG relative to SHR. In conclusion, green tea flavonoid epigallocatechin-3-gallate (EGCG) might prevent neural apoptotic pathways and activate neural survival pathways, providing therapeutic effects on early aged hypertension-induced neural apoptosis.

Therapeutic modulation of the phosphatidylinositol 3-kinases (PI3K) pathway in cerebral ischemic injury.

The cerebral ischemic reperfusion injury may leads to morbidity and mortality in patients. phosphatidylinositol 3-kinase (PI3K) signaling pathway has been believed to work in association with its downstream targets, other receptors, and pathways that may offer antioxidant, anti-inflammatory, anti-apoptotic effects, neuroprotective role in neuronal excitotoxicity. This review elaborates the mechanistic interventions of the PI3K pathway in cerebral ischemic injury in context to nuclear factor erythroid 2-related factor 2 (Nrf2) regulation, Hypoxia-inducible factor 1 signaling (HIF-1), growth factors, Endothelial NOS (eNOS) proinflammatory cytokines, Erythropoietin (EPO), Phosphatase and tensin homologous protein of chromosome 10 gene (PTEN) signaling, NF-κB/Notch signaling, c-Jun N-terminal kinase (JNK) and Glycogen synthase kinase-3ß (GSK-3ß) signaling pathway. Evidences showing the activation of PI3K inhibits apoptotic pathway, which results in its neuroprotective effect in ischemic injury. Despite discussing the therapeutic role of the PI3K pathway in treating cerebral ischemic injury, the review also enlighten the selective modulation of PI3K pathway with activators and inhibitors which may provide promising results in clinical and preclinical settings.

The protective role of microRNA-133b in restricting hippocampal neurons apoptosis and inflammatory injury in rats with depression by suppressing CTGF.

OBJECTIVE: Increasing evidence has proved the functions of microRNAs (miRNAs) in human diseases, our research was designed to explore the effects of miR-133b on the progression of depression with the involvement of connective tissue growth factor (CTGF). METHODS: Depression rat models were established by chronic unpredictable mild stress, then the ethology of rats in each group was observed, and the morphological changes as well as the apoptosis of hippocampal neurons was measured. Subsequently, the expression of miR-133b, CTGF, glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF), Bax, Bcl-2, interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α) and neurotransmitters was determined. The target relation between miR-133b and CTGF was assessed. RESULTS: We have found in this study that miR-133b was poorly expressed, and CTGF was highly expressed in hippocampal tissues of depression rats. Additionally, elevated miR-133b and inhibited CTGF could restrain apoptosis of hippocampal neurons, repress inflammatory reaction, and increase the expression of GFAP, BDNF and neurotransmitters in hippocampal tissues of depression rats, resulting in a protective impact on neural injury in depression rats. CONCLUSION: This study demonstrates that elevated miR-133b could suppress the expression of CTGF to protect the hippocampal neurons from apoptosis and inflammatory injury in depression rats.

Quietness Circ 0000962 promoted nerve cell inflammation through PIK3CA/Akt/NF-κB signaling by miR-302b-3p in spinal cord injury.

BACKGROUND: In this study, we analyzed whether the neuroprotection of Circ 0000962 promoted neural inflammation in spinal cord injury (SCI) and its possible mechanism. METHODS: Inflammation factors (TNF-α, IL-1ß, IL-6 and IL-18) were measured using ELIS kit, and NF-κB, PI3K and phosphorylation-(p)-Akt protein expression were analyzed by Western blot analysis. RESULTS: Circ 0000962 expression was decreased in SCI model rat and vitro model. Over-expression of Circ 0000962 decreased inflammation in vitro model of SCI via activation of PI3K/Akt and suppression of NF-κB by down-regulation of miR-302b-3p. Down-regulation of Circ 0000962 promotion inflammation, suppressed NF-κB protein expression, and induced PI3K and p-Akt protein expression in vitro model of SCI by up-regulation of miR-302b-3p. MiR-302b-3p reduced the effect of Circ 0000962 on inflammation in vitro model. CONCLUSIONS: This study showed that Circ 0000962 promoted nerve cell inflammation through Akt/ NF-κB signaling by PI3K in SCI.

CDK5: Key Regulator of Apoptosis and Cell Survival.

The atypical cyclin-dependent kinase 5 (CDK5) is considered as a neuron-specific kinase that plays important roles in many cellular functions including cell motility and survival. The activation of CDK5 is dependent on interaction with its activator p35, p39, or p25. These activators share a CDK5-binding domain and form a tertiary structure similar to that of cyclins. Upon activation, CDK5/p35 complexes localize primarily in the plasma membrane, cytosol, and perinuclear region. Although other CDKs are activated by cyclins, binding of cyclin D and E showed no effect on CDK5 activation. However, it has been shown that CDK5 can be activated by cyclin I, which results in anti-apoptotic functions due to the increased expression of Bcl-2 family proteins. Treatment with the CDK5 inhibitor roscovitine sensitizes cells to heat-induced apoptosis and its phosphorylation, which results in prevention of the apoptotic protein functions. Here, we highlight the regulatory mechanisms of CDK5 and its roles in cellular processes such as gene regulation, cell survival, and apoptosis.

miR-29b affects neurocyte apoptosis by targeting MCL-1 during cerebral ischemia/reperfusion injury.

The present study aimed to determine whether an miRNA (miR)-29b inhibitor protected against cerebral ischemia/reperfusion (I/R) injury in vitro and to investigate the underlying mechanisms. As a model for induced cerebral IR injury, N2a cells were exposed to an oxygen-glucose deprivation/reoxygenation (OGD/R) environment. Using this model, it was demonstrated that miR-29b was significantly upregulated compared with cells in a normal environment. The interactions between miR-29b and myeloid cell leukemia sequence (MCL)-1 were then investigated using dual-luciferase assays, revealing a strong regulation of MCL-1 through the 3'untranslated region. Using the OGD/R model, the present study additionally examined the effects of miR-29b and miR-29b inhibitor on cell viability and apoptosis using Cell Counting kit 8 and flow cytometry assays, respectively. miR-29b transfection led to increased N2a cell apoptosis and reduced cell viability under an OGD/R environment. However, this effect was reversed by the miR-29b inhibitor. Finally, the effects of miR-29b on the expression of several Wnt-associating proteins were examined. It was observed that B cell lymphoma-2 was inhibited by miR-29b, as was MCL-1, whereas caspase-3 expression was promoted. The miR-29b inhibitor demonstrated the opposite effect. Overall, miR-29b promoted neurocyte apoptosis by targeting MCL-1 during cerebral I/R injury. The results of the present study suggest a potential novel therapeutic target for the treatment of ischemic stroke.

[ROLE OF EXTRACELLULAR SIGNAL-RELATED PROTEIN KINASE 1/2 PATHWAY IN GINSENOSIDE Rg1 MEDIATED ANTI-APOPTOTIC EFFECT ON NEURON AFTER HYPOXIA ISCHEMIA BRAIN DAMAGE IN NEONATAL RATS].

OBJECTIVE: To investigate the anti-apoptotic effect of ginsenoside Rg1 in neonatal rats with hypoxia ischemia brain damage (HIBD), and to explore the possible signaling pathway involved in anti-apoptosis. METHODS: Forty-eight 10-day-old Sprague Dawley (SD) rats (weighing 17-21 g, male or female) were randomly allocated into 4 groups (12 rats in each group): sham-operation group (sham group), HIBD group (HI group), HIBD+ginsenoside Rg1 group (HI+Rg1 group), and HIBD+ginsenoside Rg1+U0126 group (HI+Rg1+U0126 group). SD rats in HI group, HI+Rg1 group, and HI+Rg1+U0126 group underwent ligation of the right common carotid artery (CCA) and hypoxic ventilation (8%O2+92%N2) for 2.5 hours to prepare the HIBD model, and rats in sham group underwent only separation of the right CCA. SD rats in HI+Rg1+U0126 group received intraventricular injection of 5 µL phosphate buffer saline (PBS) containing U0126 (25 µg/kg) at 1 hour before HIBD, and rats in the other three groups received intraventricular injection of PBS at the same time. The rats in HI+Rg1 group and HI+Rg1+U0126 group received intraperitoneal injection of 0.1 mL normal saline (NS) containing Rg1 (40 mg/kg) at immediate after HIBD, while rats in HI group and sham group received intraperitoneal injection of 0.1 mL NS at immediate after HIBD. At 4 and 24 hours after HIBD, the right hemisphere and hippocampus were collected to detect the protein expression and distribution of extracellular signal-related protein kinase 1/2 (Erk1/2), phospho-Erk1/2 (p-Erk1/2), hypoxia inducible factor 1α (HIF-1α), and cleaved Caspase-3 (CC3) by Western blot and immunohistochemistry staining. TUNEL staining was used to evaluate neural apoptosis in situ. RESULTS: Western blot results showed that there were expressions of Erk1/2, p-ERK1/2, HIF-1α, and CC3 proteins at 4 and 24 hours after HIBD in each group. The expressions of HIF-1α and CC3 protein at 4 and 24 hours, and expression of p-Erk1/2 protein at 4 hours were significantly increased in HI group when compared with sham group (P<0.05). When compared with HI group, the expressions of p-Erk1/2 and HIF-1α protein in HI+Rg1 group were significantly increased (P<0.05), while the expression of CC3 protein was significantly decreased at 4 and 24 hours (P<0.05). When compared with HI+Rg1 group, the expressions of p-Erk1/2 and HIF-1α proteins in HI+Rg1+U0126 group were significantly decreased (P<0.05), while expression of CC3 protein was significantly increased at 4 and 24 hours (P<0.05). There was no significant difference in Erk1/2 protein expression between groups at different time points (P>0.05). Immunohistochemistry staining displayed that HIF-1α and CC3 proteins mainly distributed in the nucleus and cytoplasma, while Erk1/2 and p-Erk1/2 proteins mainly distributed in the cytoplasma. The expression levels of protein by immunohistochemistry results were similar to the results by Western blot. TUNEL staining showed that the apoptotic neurons were characterized by yellow or brown particle in the nucleus. The apoptotic index (AI) of neurons at 4 and 24 hours was significantly increased in HI group when compared with sham group (P<0.05), and the AI of neurons was significantly decreased in HI+Rg1 group when compared with HI group and HI+Rg1+U0126 group at 24 hours (P<0.05). CONCLUSIONS: Rg1 could enhance HIBD induced HIF-1α expression and inhibit activation of Caspase-3 by Erk1/2 signaling pathway, and play an anti-apoptotic role in neonatal rats with HIBD.

Expression of p75 neurotrophin receptor in human neuroblastoma cells under condition of oxygen-glucose deprivation and its significance / 第二军医大学学报

Objective: To explore the expression and effect of p75 neurotrophin receptor (p75NTR) in human neuroblastoma cell line (SH-SY5Y cells) under the condition of oxygen-glucose deprivation (OGD). Methods: The OGD model of SH-SY5Y cells was established by glucose-free and serum-free culturing using tri-gas incubator, and then was assigned to 3 groups, including serum-free regular culturing group (control group), OGD group and OGD + LM11A-31 (a competitive blocker of p75NTR) group. After 12 h of culturing, the cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release activity was determined by LDH activity assay kit, cell apoptosis proportion was detected by flow cytometry, and p75NTR protein expression was detected by Western blotting. Results: The OGD model of SH-SY5Y cells was successfully established. Twelve hours after cell culture, the cell viability of the control, OGD and OGD + LM11A-31 groups was (94.80 ± 4.06)%, (50.34 ± 5.55)% and (64.68 ± 4.59)%, the LDH release activities were (46.93 ± 5.49) U/L, (353.09 ± 30.67) U/L and (282.20 ± 25.60) U/L, the proportions of apoptosis cells were (1.82 ± 0.45)%, (14.98 ± 2.59)% and (7.36 ± 1.98)%, and the relative expression levels of p75NTR were 0.06 ± 0.01, 0.41 ± 0.02 and 0.19 ± 0.03, respectively, and the differences were all significant (F=67.94, 142.10, 36.28 and 221.20, all P<0.05). Post hoc analysis showed that the cell viability of the OGD group was significantly lower than that of the control group, and the LDH release activity, the proportion of apoptosis cells and the relative protein expression level of p75NTR of the OGD group were significantly higher (Bonferroni test, all P' < 0.05). After treatment with LM11A-31, the cell viability of the OGD + LM11A-31 group was significantly higher than that of the OGD group, and the LDH release activity, the proportion of apoptosis cells and the relative protein expression level of p75NTR of the OGD LM11A-31 group were significantly lower (Bonferroni test, all P' < 0.05). Conclusion: The expression of p75NTR is increased in human neuroblastoma cell line SH-SY5Y cells under OGD condition, which may promotes neuronal injury and apoptosis.

Sevoflurane induced amnesia inhibits hippocampal Arc expression partially through 5-hydroxytryptamine-7 receptors in the bilateral basolateral amygdala in rats.

This study aimed to investigate whether the regulation of 5-hydroxytryptamine-7 (5-HT7) receptors in the bilateral basolateral amygdala (BLA) could alter the amnesic effects of sevoflurane and change the hippocampal expression of Arc and neural apoptosis. Male Sprague-Dawley rats were randomized into ten groups. First, the animals received bilateral injection of SB269970 (20, 50, or 100 pmol/0.2 µl) or saline (0.2 µl) or AS-19 (2, 10, or 50 pmol/0.2 µl), followed by inhalation of 2% sevoflurane or air for 2h. Then, fear conditioning training was carried out, and the percentage of freezing was detected 24h later. Furthermore, hippocampal Arc protein level and neural apoptosis were measured. Pre-training inhalation of sevoflurane reduced the extent of freezing, and hippocampal Arc expression. The largest dose of SB269970 (100 pmol) could block sevoflurane-induced amnesia and reverse the inhibitive effect of sevoflurane on Arc expression, while the maximal dose of AS-19 could exacerbate the amnesic effect, and further inhibit Arc expression. Furthermore, pre-training inhalation of 2% sevoflurane for 6h could not induce neural apoptosis in the hippocampus. The amnesic effect of sevoflurane might partly attribute to its impairment of memory formation in the hippocampus via activation of 5-HT7 receptors in the BLA.

Tauroursodeoxycholic acid protects retinal neural cells from cell death induced by prolonged exposure to elevated glucose.

Diabetic retinopathy is one of the most frequent causes of blindness in adults in the Western countries. Although diabetic retinopathy is considered a vascular disease, several reports demonstrate that retinal neurons are also affected, leading to vision loss. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has proven to be neuroprotective in several models of neurodegenerative diseases, including models of retinal degeneration. Since hyperglycemia is considered to play a central role in retinal cell dysfunction and degeneration, underlying the progression of diabetic retinopathy, the purpose of this study was to investigate the neuroprotective effects of TUDCA in rat retinal neurons exposed to elevated glucose concentration. We found that TUDCA markedly decreased cell death in cultured retinal neural cells induced by exposure to elevated glucose concentration. In addition, TUDCA partially prevented the release of apoptosis-inducing factor (AIF) from the mitochondria, as well as the subsequent accumulation of AIF in the nucleus. Biomarkers of oxidative stress, such as protein carbonyl groups and reactive oxygen species production, were markedly decreased after TUDCA treatment as compared to cells exposed to elevated glucose concentration alone. In conclusion, TUDCA protected retinal neural cell cultures from cell death induced by elevated glucose concentration, decreasing mito-nuclear translocation of AIF. The antioxidant properties of TUDCA might explain its cytoprotection. These findings may have relevance in the treatment of diabetic retinopathy patients.

The multifunctional and multi-system influence of Ghrelin in the treatment of diabetic and spinal cord injury induced Neuropathy.

Ghrelin (Gh), a small peptide, which was originally discovered as a gastrointestinal (GI) tropic hormone, has shown to have a presence and function within multiple tissue systems. Recently, Gh has shown to exhibit anti-inflammatory and regenerative abilities in response to both chemical and mechanical stressors within neural tissues. By continuing to elucidate the potential applications of Gh on pathological neural states, the viability of this peptide hormone for therapeutic uses can be explored for future clinical application.