Therapeutic potency of compound RMY-205 for pulmonary fibrosis induced by SARS-CoV-2 nucleocapsid protein.

Pulmonary fibrosis is a typical sequela of coronavirus disease 2019 (COVID-19), which is linked with a poor prognosis for COVID-19 patients. However, the underlying mechanism of pulmonary fibrosis induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Here, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 induced pulmonary fibrosis by activating pulmonary fibroblasts. N protein interacted with the transforming growth factor ß receptor I (TßRI), to disrupt the interaction of TßRI-FK506 Binding Protein12 (FKBP12), which led to activation of TßRI to phosphorylate Smad3 and boost expression of pro-fibrotic genes and secretion of cytokines to promote pulmonary fibrosis. Furthermore, we identified a compound, RMY-205, that bound to Smad3 to disrupt TßRI-induced Smad3 activation. The therapeutic potential of RMY-205 was strengthened in mouse models of N protein-induced pulmonary fibrosis. This study highlights a signaling pathway of pulmonary fibrosis induced by N protein and demonstrates a novel therapeutic strategy for treating pulmonary fibrosis by a compound targeting Smad3.

Thymosin ß4 reverses phenotypic polarization of glial cells and cognitive impairment via negative regulation of NF-κB signaling axis in APP/PS1 mice.

BACKGROUND: Thymosin ß4 (Tß4) is the most abundant member of the ß-thymosins and plays an important role in the control of actin polymerization in eukaryotic cells. While its effects in multiple organs and diseases are being widely investigated, the safety profile has been established in animals and humans, currently, little is known about its influence on Alzheimer's disease (AD) and the possible mechanisms. Thus, we aimed to evaluate the effects and mechanisms of Tß4 on glial polarization and cognitive performance in APP/PS1 transgenic mice. METHODS: Behavior tests were conducted to assess the learning and memory, anxiety and depression in APP/PS1 mice. Thioflavin S staining, Nissl staining, immunohistochemistry/immunofluorescence, ELISA, qRT-PCR, and immunoblotting were performed to explore Aß accumulation, phenotypic polarization of glial cells, neuronal loss and function, and TLR4/NF-κB axis in APP/PS1 mice. RESULTS: We demonstrated that Tß4 protein level elevated in all APP/PS1 mice. Over-expression of Tß4 alone alleviated AD-like phenotypes of APP/PS1 mice, showed less brain Aß accumulation and more Insulin-degrading enzyme (IDE), reversed phenotypic polarization of microglia and astrocyte to a healthy state, improved neuronal function and cognitive behavior performance, and accidentally displayed antidepressant-like effect. Besides, Tß4 could downregulate both TLR4/MyD88/NF-κB p65 and p52-dependent inflammatory pathways in the APP/PS1 mice. While combination drug of TLR4 antagonist TAK242 or NF-κB p65 inhibitor PDTC exerted no further effects. CONCLUSIONS: These results suggest that Tß4 may exert its function by regulating both classical and non-canonical NF-κB signaling and is restoring its function as a potential therapeutic target against AD.

The neurotoxic effect of isoflurane on age-defined neurons generated from tertiary dentate matrix in mice.

INTRODUCTION: Recent animal studies showed that isoflurane exposure may lead to the disturbance of hippocampal neurogenesis and later cognitive impairment. However, much less is known about the effect of isoflurane exposure on the neurons generated form tertiary dentate matrix, even though a great increase of granule cell population during the infantile period is principally derived from this area. METHODS: To label the new cells originated from the tertiary dentate matrix, the mice were injected with BrdU on postnatal day 6 (P6). Then, the mice were exposed to isoflurane for 4 hr at 1, 8, 21, and 42 days after BrdU injection, and the brains were collected 24 hr later. The loss of newly generated cells/neurons with different developmental stage was assessed by BrdU, BrdU + DCX, BrdU + NeuN, or BrdU + Prox-1 staining, respectively. RESULTS: We found that the isoflurane exposure significantly decreased the numbers of nascent cells (1 day old) and mature neurons (42 days old), but had no effect on the immature (8 days old) and early mature neurons (8 and 21 days old, respectively). CONCLUSION: The results suggested isoflurane exposure exerts the neurotoxic effects on the tertiary dentate matrix-originated cells with an age-defined pattern in mice, which partly explain the cognitive impairment resulting from isoflurane exposure to the young brain.

Status epilepticus induced Gadd45b is required for augmented dentate neurogenesis.

In animal models with temporal lobe epilepsy (TLE), the status epilepticus (SE) leads to a dramatic increase in number of newly born neuron in the subgranular zone (SGZ) of dentate gyrus. How the SE confers a modulation in the dentate neurogenesis is mostly unknown. Gadd45b is involved in epigenetic gene activation by DNA demethylation. This study was performed to present a novel mechanism underling SE-induced dentate neurogenesis. A transient induction (12 hrs to 3 days) of Gadd45b was observed in dentate gyrus of mice after pilocarpine-induced SE. Labeling the dividing cells with BrdU, we next found that the induction of Gadd45b was required to increase the rate of cell proliferation in the dentate gyrus at 7 and 14 days after SE. Afterward, the DNA methylation levels for candidate growth factor genes critical for the adult neurogenesis were assayed with Sequenom MassARRAY Analyzer. The results indicated that Gadd45b was necessary for SE-induced DNA demethylation of specific promoters and expression of corresponding genes in the dentate gyrus, including brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2). Using Timm staining, we further suggested that SE-induced Gadd45b might contribute to the subsequent mossy fiber sprouting (MFS) in the chronically epileptic hippocampus via epigenetic regulation of dentate neurogenesis at early stage after SE. Together, Gadd45b links pilocarpine-induced SE to epigenetic DNA modification of secreted factors in the dentate gyrus, leading to extrinsic modulation on the neurogenesis.

Blocking PPARγ interaction facilitates Nur77 interdiction of fatty acid uptake and suppresses breast cancer progression.

Nuclear receptor Nur77 participates in multiple metabolic regulations and plays paradoxical roles in tumorigeneses. Herein, we demonstrated that the knockout of Nur77 stimulated mammary tumor development in two mouse models, which would be reversed by a specific reexpression of Nur77 in mammary tissues. Mechanistically, Nur77 interacted and recruited corepressors, the SWI/SNF complex, to the promoters of CD36 and FABP4 to suppress their transcriptions, which hampered the fatty acid uptake, leading to the inhibition of cell proliferation. Peroxisome proliferator-activated receptor-γ (PPARγ) played an antagonistic role in this process through binding to Nur77 to facilitate ubiquitin ligase Trim13-mediated ubiquitination and degradation of Nur77. Cocrystallographic and functional analysis revealed that Csn-B, a Nur77-targeting compound, promoted the formation of Nur77 homodimer to prevent PPARγ binding by steric hindrance, thereby strengthening the Nur77's inhibitory role in breast cancer. Therefore, our study reveals a regulatory function of Nur77 in breast cancer via impeding fatty acid uptake.

Forced Physical Training Increases Neuronal Proliferation and Maturation with Their Integration into Normal Circuits in Pilocarpine Induced Status Epilepticus Mice.

Increased number of newly-born neurons produced at latent stage after status epilepticus (SE) contribute to aberrant rewiring of hippocampus and are hypothesized to promote epileptogenesis. Although physical training (PT) was reported to cause further increase in neurogenesis after SE, how PT affect their integration pattern is still elusive, whether they integrate into normal circuits or increase aberrant integrations is yet to be determined. To understand this basic mechanism by which PT effects SE and to elaborate the possible role of neuronal integrations in prognosis of SE, we evaluated the effect of 4 weeks of treadmill PT in adult male mice after pilocarpine-induced SE on behavioral and aberrant integrations' parameters. Changes in BDNF gene methylation and its protein level in hippocampus was also measured at latent stage (2-weeks) to explore underlying pathways involved in increasing neurogenesis. Our results demonstrated that although PT increased proliferation and maturation of neurons in dentate gyrus, they showed reduced aberrant integrations into hippocampal circuitry assessed through a decrease in the number of ectopic granular cells, hilar basal dendrites and mossy fiber sprouting as compared to non-exercised SE mice. While SE decreased the percentage methylation of specific CpGs of BDNF gene's promoter, PT did not yield any significant difference in methylation of BDNF CpGs as compared to non-exercised SE mice. In conclusion, PT increases hippocampal neurogenesis through increasing BDNF levels by some pathways other than demethylating BDNF CpGs and causes post SE newly-born neurons to integrate into normal circuits thus resulting in decreased spontaneous recurrent seizures and enhanced spatial memory.

Knockdown of TRIM32 Protects Hippocampal Neurons from Oxygen-Glucose Deprivation-Induced Injury.

Tripartite motif 32 (TRIM32) is a member of TRIM family that plays a potential role in neural regeneration. However, the biological function of TRIM32 in cerebral ischemia reperfusion injury has not been investigated. In the present study, we evaluated the expression level of TRIM32 in hippocampal neurons following oxygen-glucose deprivation/reperfusion (OGD/R). The results showed that TRIM32 expression was significantly elevated in hippocampal neurons subjected to OGD/R as compared to the neurons cultured in the normoxia condition. To further evaluate the role of TRIM32, hippocampal neurons were transfected with TRIM32 small interfering RNA (si-TRIM32) to knock down TRIM32. We found that knockdown of TRIM32 improved cell viability of OGD/R-stimulated hippocampal neurons. Generation of reactive oxygen species was decreased, while contents of superoxide dismutase and glutathione peroxidase were increased after si-TRIM32 transfection. Knockdown of TRIM32 suppressed cell apoptosis, as proved by the increased bcl-2 expression along with decreased bax expression and caspase-3 activity. We also found that TRIM32 knockdown enhanced OGD/R-induced activation of Nrf2 signaling pathway in hippocampal neurons. Furthermore, siRNA-Nrf2 was transfected to knock down Nrf2. SiRNA-Nrf2 transfection reversed the protective effects of TRIM32 knockdown on neurons. These data suggested that knockdown of TRIM32 protected hippocampal neurons from OGD/R-induced oxidative injury through activating Nrf2 signaling pathway.

MicroRNA-770 affects proliferation and cell cycle transition by directly targeting CDK8 in glioma.

BACKGROUND: MicroRNAs play crucial roles in tumorigenesis and tumor progression. miR-770 has been reported to be downregulated in several cancers and affects cancer cell proliferation, apoptosis, metastasis and drug resistance. However, the role and underlying molecular mechanism of miR-770 in human glioma remain unknown and need to be further elucidated. METHODS: The expression of miR-770 in glioma tissues and cell lines was measured by quantitative real-time PCR (qRT-PCR) to explore the association of miR-770 expression with clinicopathological characteristics. The expression of CDK8 was detected by qRT-PCR and Western blotting in glioma tissues. A target prediction program and a dual-luciferase reporter assay were used to confirm that CDK8 is a target gene of miR-770. MTT and cell counting assays were used to assess the effect of miR-770 on glioma cell proliferation. The cell cycle distribution and apoptosis were examined by flow cytometry. CDK8 siRNA and overexpression were used to further confirm the function of the target gene. RESULTS: We demonstrated that miR-770 expression was downregulated in human glioma tissues and cell lines. The overexpression of miR-770 inhibited glioma cell proliferation and cell cycle G1-S transition and induced apoptosis. The inhibition of miR-770 facilitated cell proliferation and G1-S transition and suppressed apoptosis. miR-770 expression was inversely correlated with CDK8 expression in glioma tissues. CDK8 was confirmed to be a direct target of miR-770 by using a luciferase reporter assay. The overexpression of miR-770 decreased CDK8 expression at both the mRNA and protein levels, and the suppression of miR-770 increased CDK8 expression. Importantly, CDK8 silencing recapitulated the cellular and molecular effects observed upon miR-770 overexpression, and CDK8 overexpression eliminated the effects of miR-770 overexpression on glioma cells. Moreover, both exogenous expression of miR-770 and silencing of CDK8 resulted in suppression of the Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our study demonstrates that miR-770 inhibits glioma cell proliferation and G1-S transition and induces apoptosis through suppression of the Wnt/ß-catenin signaling pathway by targeting CDK8. These findings suggest that miR-770 plays a significant role in glioma progression and serves as a potential therapeutic target for glioma.

Flightless-I Blocks p62-Mediated Recognition of LC3 to Impede Selective Autophagy and Promote Breast Cancer Progression.

p62 is a receptor that facilitates selective autophagy by interacting simultaneously with cargoes and LC3 protein on the autophagosome to maintain cellular homeostasis. However, the regulatory mechanism(s) behind this process and its association with breast cancer remain to be elucidated. Here, we report that Flightless-I (FliI), a novel p62-interacting protein, promotes breast cancer progression by impeding selective autophagy. FliI was highly expressed in clinical breast cancer samples, and heterozygous deletion of FliI retarded the development of mammary tumors in PyVT mice. FliI induced p62-recruited cargoes into Triton X-100 insoluble fractions (TI) to form aggregates, thereby blocking p62 recognition of LC3 and hindering p62-dependent selective autophagy. This function of Flil was reinforced by Akt-mediated phosphorylation at Ser436 and inhibited by phosphorylation of Ulk1 at Ser64. Obstruction of autophagic clearance of p62-recruited cargoes by FliI was associated with the accumulation of oxidative damage on proteins and DNA, which could contribute to the development of cancer. Heterozygous knockout of FliI facilitated selectively autophagic clearance of aggregates, abatement of ROS levels, and protein oxidative damage, ultimately retarding mammary cancer progression. In clinical breast cancer samples, Akt-mediated phosphorylation of FliI at Ser436 negatively correlated with long-term prognosis, while Ulk1-induced FliI phosphorylation at Ser64 positively correlated with clinical outcome. Together, this work demonstrates that FliI functions as a checkpoint protein for selective autophagy in the crosstalk between FliI and p62-recruited cargoes, and its phosphorylation may serve as a prognostic marker for breast cancer.Significance: Flightless-I functions as a checkpoint protein for selective autophagy by interacting with p62 to block its recognition of LC3, leading to tumorigenesis in breast cancer.Cancer Res; 78(17); 4853-64. ©2018 AACR.

Nuclear Receptor Nur77 Facilitates Melanoma Cell Survival under Metabolic Stress by Protecting Fatty Acid Oxidation.

Fatty acid oxidation (FAO) is crucial for cells to overcome metabolic stress by providing ATP and NADPH. However, the mechanism by which FAO is regulated in tumors remains elusive. Here we show that Nur77 is required for the metabolic adaptation of melanoma cells by protecting FAO. Glucose deprivation activates ERK2 to phosphorylate and induce Nur77 translocation to the mitochondria, where Nur77 binds to TPß, a rate-limiting enzyme in FAO. Although TPß activity is normally inhibited by oxidation under glucose deprivation, the Nur77-TPß association results in Nur77 self-sacrifice to protect TPß from oxidation. FAO is therefore able to maintain NADPH and ATP levels and prevent ROS increase and cell death. The Nur77-TPß interaction further promotes melanoma metastasis by facilitating circulating melanoma cell survival. This study demonstrates a novel regulatory function of Nur77 with linkage of the FAO-NADPH-ROS pathway during metabolic stress, suggesting Nur77 as a potential therapeutic target in melanoma.

Nur77 suppresses hepatocellular carcinoma via switching glucose metabolism toward gluconeogenesis through attenuating phosphoenolpyruvate carboxykinase sumoylation.

Gluconeogenesis, an essential metabolic process for hepatocytes, is downregulated in hepatocellular carcinoma (HCC). Here we show that the nuclear receptor Nur77 is a tumour suppressor for HCC that regulates gluconeogenesis. Low Nur77 expression in clinical HCC samples correlates with poor prognosis, and a Nur77 deficiency in mice promotes HCC development. Nur77 interacts with phosphoenolpyruvate carboxykinase (PEPCK1), the rate-limiting enzyme in gluconeogenesis, to increase gluconeogenesis and suppress glycolysis, resulting in ATP depletion and cell growth arrest. However, PEPCK1 becomes labile after sumoylation and is degraded via ubiquitination, which is augmented by the p300 acetylation of ubiquitin-conjugating enzyme 9 (Ubc9). Although Nur77 attenuates sumoylation and stabilizes PEPCK1 via impairing p300 activity and preventing the Ubc9-PEPCK1 interaction, Nur77 is silenced in HCC samples due to Snail-mediated DNA methylation of the Nur77 promoter. Our study reveals a unique mechanism to suppress HCC by switching from glycolysis to gluconeogenesis through Nur77 antagonism of PEPCK1 degradation.

MicroRNA-25 Negatively Regulates Cerebral Ischemia/Reperfusion Injury-Induced Cell Apoptosis Through Fas/FasL Pathway.

MicroRNA-25 (miR-25) has been reported to be a major miRNA marker in neural cells and is strongly expressed in ischemic brain tissues. However, the precise mechanism and effect of miR-25 in cerebral ischemia/reperfusion (I/R) injury needs further investigations. In the present study, the oxygen-glucose deprivation (OGD) model was constructed in human SH-SY5Y and IMR-32 cells to mimic I/R injury and to evaluate the role of miR-25 in regulating OGD/reperfusion (OGDR)-induced cell apoptosis. We found that miR-25 was downregulated in the OGDR model. Overexpression of miR-25 via miRNA-mimics transfection remarkably inhibited OGDR-induced cell apoptosis. Moreover, Fas was predicted as a target gene of miR-25 through bioinformatic analysis. The interaction between miR-25 and 3'-untranslated region (UTR) of Fas mRNA was confirmed by dual-luciferase reporter assay. Fas protein expression was downregulated by miR-25 overexpression in OGDR model. Subsequently, the small interfering RNA (siRNA)-mediated knockdown of Fas expression also inhibited cell apoptosis induced by OGDR model; in contrast, Fas overexpression abrogated the protective effects of miR-25 on OGDR-induced cells. Taken together, our results indicate that the upregulation of miR-25 inhibits cerebral I/R injury-induced apoptosis through downregulating Fas/FasL, which will provide a promising therapeutic target.

[Optimization of trypsin digestion intensity to obtain high-purity in vitro cultured astrocytes].

The aim of the present study was to observe the effect of trypsin digestion on the purity of in vitro cultured astrocytes and optimize the culture methods. The cerebral cortical tissue from newborn Sprague Dawley (SD) rats was isolated and digested with 0.25% trypsin for 20, 30, or 40 min. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each of 20 and 30 min groups was subdivided into two groups, the control group with normal digestion and two-time-digestion group, and the cells were passaged and purified. Through inverted phase contrast microscope and MTT assay, cell growth and proliferation were observed, respectively. Immunofluorescence for glial fibrillary acidic protein (GFAP) was used to observe the morphology of astrocytes and to assess their purity in different stages. Flow cytometric analysis was used to detect the apoptotic rates of purified astrocytes. The results showed that, the cells being digested for 20 min usually reached confluence at 9 d after seeding. When the digestion time was extended to 30 min, the cells grew faster and reached confluence at 7 d after seeding, meanwhile the morphology of astrocytes was normal, GFAP positive rate (70.2 ± 4.0)% being much higher than that of the 20 min group (P < 0.05). Compared with 20 min group, 40 min group showed higher GFAP positive rate, whereas the cell proliferation was slower, and cell injury was more obvious. After shaking at constant temperature, two times of trypsin digestion could decrease the number of contaminated cells after passage. The GFAP positive rates of two-time-digestion groups in passage 1 (P1) were higher than those of corresponding control groups, and the GFAP positive rate of 30 min + two-time-digestion group in P1 reached (98.1 ± 1.7)%, which was equivalent to that of the 20 min + control group in P3. However, the apoptotic rate showed no significant difference between these two groups. Based on above mentioned results, we conclude that 30 min + two-time of trypsin digestion effectively improves the purity of astrocytes and shortens the time of primary culture and purification, suggesting that it is a rapid and effective method to obtain astrocytes with high purity in vitro.

Low concentration of serum helps to maintain the characteristics of NSCs/NPCs on alkali-treated PHBHHx film in vitro.

OBJECTIVE: Efforts have been made by tissue engineers to create a permissive environment for neural regeneration, and to enhance the efficiency of neural stem cell (NSC) transplantation. However, to acquire sufficient number of seed cells on the material appears to be the main obstacle to constructing functional transplantable NSC-biomaterial complexes. A culture system has been optimized in the current study to maintain the specific characteristics of NSCs/neural progenitor cells (NPCs) on the material and achieve sustaining increased multipotent seed cells. METHODS: The PHBHHx film was selected as biomaterial and the surface was firstly modified with NaOH treatment. The NSCs/NPCs isolated from the cerebral cortex of rat embryos were cultured on the treated PHBHHx films in growth medium containing 1%, 5%, and 10% fetal bovine serum (FBS). Then the attachment, survival, proliferation, and differentiation of NSCs/NPCs were assessed. RESULTS: NaOH treatment significantly increased the hydrophilicity of PHBHHx and enhanced NSCs/NPCs attachment. On the treated PHBHHx film, NSCs/NPCs survived well and actively proliferated in the medium containing 1% FBS. After 7-14 days in culture, approximately two-thirds of cells remained as nestin and Sox2 positive NSCs/NPCs. However, in the medium containing 5% and 10% FBS, NSCs/NPCs proliferation was reduced and differentiation, particularly glial differentiation was significantly promoted. CONCLUSION: Growth medium containing low concentration of FBS is favorable for maintaining the characteristics, in terms of self-renewal and multiple differentiation, of NSCs/NPCs on NaOH-treated PHBHHx films. This could be a useful method to construct functional transplantable NSCs/NPCs-biomaterial complex.

Reduced expression of Phospholipase C beta in hippocampal interneuron during pilocarpine induced status epilepticus in mice.

We investigated localization of Phospholipase C beta (PLCß1 and PLCß4) in laminaes of dorsal hippocampus and different subtypes of hippocampal interneurons in normal Kunming mouse, and their progressive changes during pilocarpine induced status epilepticus (SE) by quantitative immunohistochemistry and real time PCR. PLCß1 was observed in the pyramidal layer of CA1-3 area, hilus of the dentate gyrus, whereas PLCß4 was mainly expressed in calcium binding protein positive interneurons, i.e. calbindin, calretinin, parvalbumin positive interneurons in the strata oriens, radiatum of the CA area and hilus of the dentate gyrus. During pilocarpine induced SE, a temporary down-regulation of PLCß4 in the interneurons of CA area at SE 30min, and a progressive reduction of PLCß1/PLCß4 in dentate hilar cells were demonstrated. These findings confirm and extend the regional specific distribution of PLCß1 and PLCß4 immunoreactivity in mouse hippocampus, and suggest that PLCß1 and PLCß4 may play an important role in maintenance of the status epilepticus.

Vasorelaxant and antihypertensive effects of ZCM298, a dihydropyridine derivative, are through inhibiting extracellular calcium influx.

BACKGROUND: ZCM298 is a novel 1,4-dihydropyridine derivative. The aim of the study was to investigate its vasodilation and hypotension, and the related mechanisms. METHODS: The isometric tension of artery ring segments was recorded using an in vitro myography system. The blood pressure of spontaneously hypertensive rats (SHRs) was measured in vivo using a non-invasive tail cuff blood pressure system. Changes in the intracellular calcium concentration ([Ca2+]i) in the mesenteric artery were surveyed using real-time confocal microscopy. Regional cerebral blood flow (rCBF) in the pia mater was monitored by laser-Doppler flowmetry (LDP). RESULTS: ZCM298 (10(-9)-10(-4) M) relaxed rat mesenteric artery obviously and concentration-dependently, which was not affected by the removal of the endothelium. ZCM298 shifted the concentration-contractile curves of mesenteric arteries in response to phenylephrine, U46619, KCl and CaCl2 towards the right in a non-parallel manner. The potency of ZCM298 on relaxing basilar artery was much higher than on mesenteric artery. ZCM298 did not depress the phenylephrine-induced vasoconstriction; however, it inhibited the contraction caused by the addition of CaCl2 in Ca2+-free solution. ZCM298 (10(-6) M) inhibited the increase of [Ca2+]i induced by KCl in the artery. ZCM298 improved the rCBF in the pia mater of rats at 0.03 and 0.06 mg/kg. ZCM298 depressed the systolic and diastolic blood pressure of SHRs in a dose-dependent manner. CONCLUSIONS: ZCM298 relaxes arteries probably through inhibiting extracellular calcium influx and decreases the blood pressure of SHRs. ZCM298 is more potent in the basilar artery than in the mesenteric artery and improves rCBF in the pia mater of rats.

Spatial relationship between NSCs/NPCs and microvessels in rat brain along prenatal and postnatal development.

Neurogenesis and angiogenesis are two parallel processes that occur in brain development and repair, and so share some molecular signals. In order to better understand the interaction between the genesis of neural cells and vessels during brain development, the density of microvessels and the number of nestin positive neural stem/neural progenitor cells (NSCs/NPCs) around microvasculature in various brain regions was quantified. Results showed that the density of microvessels remained at a relative low level during embryonic development and dramatically increased after postnatal day 3 (P3), especially in subventricular zone. The number of nestin positive NSCs/NPCs per microvessel in neurogenic brain regions continually increased with fetal brain development and then gradually dropped down during postnatal development. The highest density of NSCs/NPCs appeared at postnatal day 1 (P1) and dramatically decreased after P3. Similar pattern was observed in striatum. In the olfactory bulb, the cerebral cortex and cerebellum, the dramatic decrease of NSCs/NPCs density appeared after P7, especially in the cerebral cortex. Our results demonstrated that anatomically, the spatial relationship between NSCs/NPCs and microvessels changed during brain development. The alteration patterns in neurogenic brain regions differed from non-neurogenic brain regions.

The protective effects of tanshinone IIA on neurotoxicity induced by ß-amyloid protein through calpain and the p35/Cdk5 pathway in primary cortical neurons.

The characteristic pathological change of Alzheimer's disease (AD) include deposits of ß-amyloid protein (Aß) in brain, neurofibrillary tangles (NFTs), as well as a few neuronal loss. Evidence shows that Aß causes calcium influx and induces the cleavage of p35 into p25. Furthermore, the binding of p25 to cyclin-dependent kinase 5 (Cdk5) constitutively activates Cdk5. The p25/Cdk5 complex then hyperphosphorylates tau. Tanshinone IIA (tanIIA), a natural product extracted from Chinese herbal medicine Salvia miltiorrhiza BUNGE, has been reported to exert antioxidative activity. However, its neuroprotective activity remains unclear. The present study determined whether tanIIA protects neurons against Aß(25-35)-induced cytotoxicity and detected the association of this protective effect with calpain and the p35/Cdk5 pathway. The results showed that tanIIA protected neurons against the neurotoxicity of Aß(25-35), increased the viability of neurons, decreased expression of phosphorylated tau in neurons induced by Aß(25-35), improved the impairment of the cell ultrastructure (such as nuclear condensation and fragmentation, and neurofibril collapse). Further more, we found that tanIIA maintained the normal expression of p35 on peripheral membranes, and decreased p25 expression in the cytoplasm. TanIIA also inhibited the translocation of Cdk5 from the nucleus into the cytoplasm of primary neurons induced by Aß(25-35). These data suggested that tanIIA possessed neuroprotective action and the protection may involve in calpain and the p35/Cdk5 pathway.

Effect of histamine on regional cerebral blood flow of the parietal lobe in rats.

Histamine is a powerful modulator that regulates blood vessels and blood flow. The effect of histamine on the extracortical vessels has been well described, while much less is known about the effect of histamine on intracortical vessels. In this study, we investigated the effect of histamine on regional cerebral blood flow in rat parietal lobe with laser Doppler flowmetry. The pharmacological characteristics of distinct ways (intracerebroventricular injection, intraperitoneal injection, and cranial window infusion) in applying histamine to the brain were also obtained and compared. Histamine applied in three ways all produced a decrease of rCBF in parietal lobe in a concentration-dependent manner. Cranial window infusion was the most effective way and intraperitoneal injection of L-histidine was the most ineffective, although it is a simple and applied way. To determine which type of receptor takes part in the vessel contraction induced by histamine, H1 receptor antagonist, diphenhydramine, and H2 receptor antagonist, cimetidine, were applied, respectively, before histamine administration. When the injection of cimetidine was conducted in advance, histamine still resulted in a decrease of infusion amount; while the injection of diphenhydramine was conducted in advance, the infusion of blood amount wasn't changed. These findings indicated that histamine could result in a reduction of rCBF in the rat parietal lobe and this effect of histamine may attribute partly to its combination with H1 receptor.

Protective effects of [Gly14]-Humanin on beta-amyloid-induced PC12 cell death by preventing mitochondrial dysfunction.

Mitochondrial dysfunction is a hallmark of beta-amyloid (Abeta)-induced neuronal toxicity in Alzheimer's disease (AD), and is considered as an early event in AD pathology. Humanin (HN) and its derivative, [Gly14]-Humanin (HNG), are known for their ability to suppress neuronal death induced by AD-related insults in vitro and in vivo. In the present study, we investigated the neuroprotective effects of HNG on Abeta(25-35)-induced toxicity and its potential mechanisms in PC12 cells. Exposure of PC12 cells to 25 microM Abeta(25-35) caused significant viability loss and cell apoptosis. In addition, decreased mitochondrial membrane potential and increased cytochrome c releases from mitochondria were also observed after Abeta(25-35) exposure. All these effects induced by Abeta(25-35) were markedly reversed by HNG. Pretreatment with 100 nM HNG 6h prior to Abeta(25-35) exposure significantly elevated cell viability, reduced Abeta(25-35)-induced cell apoptosis, stabilized mitochondrial membrane potential, and blocked cytochrome c release from mitochondria. Furthermore, HNG also ameliorated the Abeta(25-35)-induced Bcl-2/Bax ratio reduction and decreased caspase-3 activity in PC12 cells. These results demonstrate that HNG could attenuate Abeta(25-35)-induced PC12 cell injury and apoptosis by preventing mitochondrial dysfunction. Furthermore, these data suggest that mitochondria are involved in the protective effect of HNG against Abeta(25-35).