A novel small-molecular CCR5 antagonist promotes neural repair after stroke.

Chemokine receptor 5 (CCR5) is one of the main co-receptors of HIV-1, and has been found to be a potential therapeutic target for stroke. Maraviroc is a classic CCR5 antagonist, which is undergoing clinical trials against stroke. As maraviroc shows poor blood-brain barrier (BBB) permeability, it is of interest to find novel CCR5 antagonists suitable for neurological medication. In this study we characterized the therapeutic potential of a novel CCR5 antagonist A14 in treating ischemic stroke mice. A14 was discovered in screening millions compounds in the Chemdiv library based on the molecular docking diagram of CCR5 and maraviroc. We found that A14 dose-dependently inhibited the CCR5 activity with an IC50 value of 4.29 µM. Pharmacodynamic studies showed that A14 treatment exerted protective effects against neuronal ischemic injury both in vitro and vivo. In a SH-SY5Y cell line overexpressing CCR5, A14 (0.1, 1 µM) significantly alleviated OGD/R-induced cell injury. We found that the expression of CCR5 and its ligand CKLF1 was significantly upregulated during both acute and recovery period in focal cortical stroke mice; oral administration of A14 (20 mg·kg-1·d-1, for 1 week) produced sustained protective effect against motor impairment. A14 treatment had earlier onset time, lower onset dosage and much better BBB permeability compared to maraviroc. MRI analysis also showed that A14 treatment significantly reduced the infarction volume after 1 week of treatment. We further revealed that A14 treatment blocked the protein-protein interaction between CCR5 and CKLF1, increasing the activity of CREB signaling pathway in neurons, thereby improving axonal sprouting and synaptic density after stroke. In addition, A14 treatment remarkably inhibited the reactive proliferation of glial cells after stroke and reduced the infiltration of peripheral immune cells. These results demonstrate that A14 is a promising novel CCR5 antagonist for promoting neuronal repair after ischemic stroke. A14 blocked the protein-protein interaction between CKLF1 and CCR5 after stroke by binding with CCR5 stably, improved the infarct area and promoted motor recovery through reversing the CREB/pCREB signaling which was inhibited by activated CCR5 Gαi pathway, and benefited to the dendritic spines and axons sprouting.

Regulatory T cells in ischemic stroke.

Recent evidence shows that when ischemic stroke (IS) occurs, the BBB would be destructed, thereby promoting the immune cells to migrate into the brain, suggesting that the immune responses can play a vital role in the pathology of IS. As an essential subpopulation of immunosuppressive T cells, regulatory T (Treg) cells are involved in maintaining immune homeostasis and suppressing immune responses in the pathophysiological conditions of IS. During the past decades, the regulatory role of Treg cells has attracted the interest of numerous researchers. However, whether they are beneficial or detrimental to the outcomes of IS remains controversial. Moreover, Treg cells exert distinctive effects in the different stages of IS. Therefore, it is urgent to elucidate how Treg cells modulate the immune responses induced by IS. In this review, we describe how Treg cells fluctuate and play a role in the regulation of immune responses after IS in both experimental animals and humans, and summarize their biological functions and mechanisms in both CNS and periphery. We also discuss how Treg cells participate in poststroke inflammation and immunodepression and the potential of Treg cells as a novel therapeutic approach.

Neuronal chemokine-like-factor 1 (CKLF1) up-regulation promotes M1 polarization of microglia in rat brain after stroke.

The phenotypic transformation of microglia in the ischemic penumbra determines the outcomes of ischemic stroke. Our previous study has shown that chemokine-like-factor 1 (CKLF1) promotes M1-type polarization of microglia. In this study, we investigated the cellular source and transcriptional regulation of CKLF1, as well as the biological function of CKLF1 in ischemic penumbra of rat brain. We showed that CKLF1 was significantly up-regulated in cultured rat cortical neurons subjected to oxygen-glucose deprivation/reoxygenation (ODG/R) injury, but not in cultured rat microglia, astrocytes and oligodendrocytes. In a rat model of middle cerebral artery occlusion, we found that CKLF1 was up-regulated and co-localized with neurons in ischemic penumbra. Furthermore, the up-regulated CKLF1 was accompanied by the enhanced nuclear accumulation of NF-κB. The transcriptional activity of CKLF1 was improved by overexpression of NF-κB in HEK293T cells, whereas application of NF-κB inhibitor Bay 11-7082 (1 µM) abolished it, caused by OGD/R. By using chromatin-immunoprecipitation (ChIP) assay we demonstrated that NF-κB directly bound to the promoter of CKLF1 (at a binding site located at -249 bp to -239 bp of CKLF1 promoter region), and regulated the transcription of human CKLF1. Moreover, neuronal conditional medium collected after OGD/R injury or CKLF1-C27 (a peptide obtained from secreted CKLF1) induced the M1-type polarization of microglia, whereas the CKLF1-neutralizing antibody (αCKLF1) or NF-κB inhibitor Bay 11-7082 abolished the M1-type polarization of microglia. Specific knockout of neuronal CKLF1 in ischemic penumbra attenuated neuronal impairments and M1-type polarization of microglia caused by ischemic/reperfusion injury, evidenced by inhibited levels of M1 marker CD16/32 and increased expression of M2 marker CD206. Application of CKLF1-C27 (200 nM) promoted the phosphorylation of p38 and JNK in microglia, whereas specific depletion of neuronal CKLF1 in ischemic penumbra abolished ischemic/reperfusion-induced p38 and JNK phosphorylation. In summary, CKLF1 up-regulation in neurons regulated by NF-κB is one of the crucial mechanisms to promote M1-type polarization of microglia in ischemic penumbra.

Korean red ginseng alleviate depressive disorder by improving astrocyte gap junction function.

ETHNOPHARMACOLOGICAL RELEVANCE: Korean red ginseng (KRG), a processed product of Panax ginseng C. A. Mey, show significant anti-depressive effect in clinic. However, its mechanism is still unclear. AIM OF THE STUDY: Gap junction intercellular communication (GJIC) dysfunction is a potential pathogenesis of depression. Therefore, this study's objective is to investigate whether the antidepressant effect of KRG is related to GJIC. MATERIALS AND METHODS: Rat were restraint 8 h every day for 28 consecutive days to prepare depression models, and meanwhile, rats were intragastrically administrated with normal saline, KRG solutions (25, 50 or 100 mg/kg) or fluoxetine (10 mg/kg) 1 h before stress. The behavioral performance was determined by forced swimming test, sucrose preference test and open field test. GJIC was determined by the Lucifer yellow (LY) diffusion distance in prelimb cortex (PLC). In addition, the level of Cx43, one of executors of GJIC, was tested by Western blot. To find out the protective effect of KRG against GJIC dysfunction directly, rats were intracranially injected with carbenoxolone (CBX, blocker of GJIC), and meanwhile normal saline, KRG (100 mg/kg) or fluoxetine (10 mg/kg) was administered daily. The behavioral performance of these rats was detected, and the LY localization injection PLC area was used to detect the gap junction function. RESULTS: Chronic resistant stress (CRS) induced depressive symptoms, as manifested by prolonged immobility time in forced swimming test and decreased sucrose consumption ratio. Administration of KRG alleviated these depressive symptoms significantly. GJIC determination showed that KRG improved the LY diffusion and increased Cx43 level in prefrontal cortex (PFC) significantly, indicated that GJIC dysfunction was alleviated by the treatment of KRG. However, the astrocytes number was also increased by the treatment of KRG, which maybe alleviate depression-like symptoms by increasing the number of astrocytes rather than improving GJIC. Injection of CBX produced depressive symptoms and GJIC dysfunction, as manifested by decreased sucrose consumption ratio and prolonged immobility time in forced swimming test, but no astrocytes number changes, KRG also reversed depressive symptoms and GJIC dysfunction, suggested that the improvement of depressive-like symptoms was improved by GJIC. CONCLUSIONS: KRG alleviate depressive disorder by improving astrocytic gap junction function.

Low corticosterone levels attenuate late life depression and enhance glutamatergic neurotransmission in female rats.

Sustained elevation of corticosterone (CORT) is one of the common causes of aging and major depression disorder. However, the role of elevated CORT in late life depression (LLD) has not been elucidated. In this study, 18-month-old female rats were subjected to bilateral adrenalectomy or sham surgery. Their CORT levels in plasma were adjusted by CORT replacement and the rats were divided into high-level CORT (H-CORT), low-level CORT (L-CORT), and Sham group. We showed that L-CORT rats displayed attenuated depressive symptoms and memory defects in behavioral tests as compared with Sham or H-CORT rats. Furthermore, we showed that glutamatergic transmission was enhanced in L-CORT rats, evidenced by enhanced population spike amplitude (PSA) recorded from the dentate gyrus of hippocampus in vivo and increased glutamate release from hippocampal synaptosomes caused by high frequency stimulation or CORT exposure. Intracerebroventricular injection of an enzymatic glutamate scavenger system, glutamic-pyruvic transmine (GPT, 1 µM), significantly increased the PSA in Sham rats, suggesting that extracelluar accumulation of glutamate might be the culprit of impaired glutamatergic transmission, which was dependent on the uptake by Glt-1 in astrocytes. We revealed that hippocampal Glt-1 expression level in the L-CORT rats was much higher than in Sham and H-CORT rats. In a gradient neuron-astrocyte coculture, we found that the expression of Glt-1 was decreased with the increase of neural percentage, suggesting that impairment of Glt-1 might result from the high level of CORT contributed neural damage. In sham rats, administration of DHK that inhibited Glt-1 activity induced significant LLD symptoms, whereas administration of RIL that promoted glutamate uptake significantly attenuated LLD. All of these results suggest that glutamatergic transmission impairment is one of important pathogenesis in LLD induced by high level of CORT, which provide promising clues for the treatment of LLD.

The role of chemokines and chemokine receptors in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease that is characterized by leukocyte infiltration and subsequent axonal damage, demyelinating inflammation, and formation of sclerosing plaques in brain tissue. The results of various studies in patients indicate that autoimmunity and inflammation make an important impact on the pathogenesis of MS. Chemokines are key mediators of inflammation development and cell migration, mediating various immune cell responses, including chemotaxis and immune activation, and are important in immunity and inflammation, therefore we focus on chemokines and their receptors in multiple sclerosis. In this article, we summarize the study of the role of prominent chemokines and their receptors in MS patients and MS animal modelsand discuss their potential significance in inflammatory injury and repair of MS. We have also summarized the progress in the treatment of multiple sclerosis antagonists in recent years with chemokine receptors as targets.

Rg1 improves LPS-induced Parkinsonian symptoms in mice via inhibition of NF-κB signaling and modulation of M1/M2 polarization.

Ginsenoside Rg1 is one of the most active ingredients in ginseng, which has been reported to protect dopaminergic neurons and improve behavioral defects in MPTP model, 6-OHDA model and rotenone model. However, it is unclear whether Rg1 exerted neuroprotection in LPS-induced sub-acute PD model. In this study, we investigated the neuroprotective effect of Rg1 in the sub-acute PD mouse model and explored the related mechanisms. Rg1 (10, 20, 40 mg·kg-1·d-1) was orally administered to mice for 18 days. A sub-acute PD model was established in the mice through LPS microinjection into the substantia nigra (SN) from D8 to D13. We found that Rg1 administration dose-dependently inhibited LPS-induced damage of dopaminergic neurons and activation of glial cells in the substantia nigra pars compacta (SNpc). The neuroprotective effects of Rg1 were associated with the reduction of pro-inflammatory cytokines and the improvement of anti-inflammatory cytokines and neurotrophin in the midbrain. Rg1 shifted the polarization of microglia towards the M2 phenotype from M1, evidenced by decreased M1 markers (inducible NO synthase, CD16, etc.) and increased M2 markers (arginase 1 (Arg1), CD206, etc) in the midbrain. Furthermore, Rg1 administration markedly inhibited nuclear translocation of NF-κB in midbrain microglia. In conclusion, Rg1 protects PD mice induced by continuous LPS injection by inhibiting the nuclear entry of NF-κB and regulating the polarization balance of microglia, shedding new light on a disease-modifying therapy of PD.

Caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis through modulating GSK3ß/ß-catenin pathway.

In this study, we investigate the anti-cancer activity of caudatin in carcinomic human alveolar basal epithelial cell line A549 and anti-angiogenic activity in human umbilical vein endothelial cells (HUVECs). We show that caudatin impairs the cell viability and induces G(0) /G(1) phase arrest in A549 cells with a dose dependent manner. A549 cells, not HUVECs, dealing with caudatin exhibited typical characteristics of apoptosis, which were accompanied by activation of caspase-3, caspase-9 and Poly(ADP-Ribose) Polymerase (PARP). In addition, caudatin treatment resulted in a decrease of ß-catenin and increase of phosphorylation of ß-catenin, and inhibited phosphorylation levels of GSK3ß (Ser 9) in A549 cells. Conditional medium of A549 cells-induced or growth factors-induced tube formation of HUVECs was markedly inhibited by caudatin treatment, which was associated with the inhibiting VEGF secretion from A549 cells by caudatin. Our findings suggest that caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis by targeting GSK3ß/ß-catenin pathway and suppressing VEGF production.