Zhen-Nan Ye

Sirtuin 1 activation protects against early brain injury after experimental subarachnoid hemorrhage in rats

Increasing evidence indicates that sirtuin 1 (SIRT1) is implicated in a wide range of cellular functions, such as oxidative stress, inflammation and apoptosis. The aim of this study was to investigate the change of SIRT1 in the brain after subarachnoid hemorrhage (SAH) and its role on SAH-induced early brain injury (EBI). In the first set of experiments, rats were randomly divided into sham group and SAH groups at 2, 6, 12, 24, 48 and 72 h. The expression of SIRT1 was evaluated by western blot analysis, immunohistochemistry and immunofluorescence. In another set of experiments, SIRT1-specific inhibitor (sirtinol) and activator (activator 3) were exploited to study the role of SIRT1 in SAH-induced EBI. It showed that the protein level of SIRT1 was markedly elevated at the early stage of SAH and peaked at 24 h after SAH. The expression of SIRT1 could be observed in neurons and microglia, and the enhanced SIRT1 was mainly located in neurons after SAH. Administration of sirtinol inhibited the expression and activation of SIRT1 pathways after SAH, while activator 3 enhanced the expression and activation of SIRT1 pathways after SAH. In addition, inhibition of SIRT1 could exacerbate forkhead transcription factors of the O class-, nuclear factor-kappa B- and p53-induced oxidative damage, neuroinflammation and neuronal apoptosis, leading to aggravated brain injury after SAH. In contrast, activator 3 treatment could reduce forkhead transcription factors of the O class-, nuclear factor-kappa B-, and p53-induced oxidative damage, neuroinflammation and neuronal apoptosis to protect against EBI. These results suggest that SIRT1 plays an important role in neuroprotection against EBI after SAH by deacetylation and subsequent inhibition of forkhead transcription factors of the O class-, nuclear factor-kappa B-, and p53-induced oxidative, inflammatory and apoptotic pathways. SIRT1 might be a new promising molecular target for SAH.

Resveratrol Attenuates Acute Inflammatory Injury in Experimental Subarachnoid Hemorrhage in Rats via Inhibition of TLR4 Pathway

Toll-like receptor 4 (TLR4) has been proven to play a critical role in neuroinflammation and to represent an important therapeutic target following subarachnoid hemorrhage (SAH). Resveratrol (RSV), a natural occurring polyphenolic compound, has a powerful anti-inflammatory property. However, the underlying molecular mechanisms of RSV in protecting against early brain injury (EBI) after SAH remain obscure. The purpose of this study was to investigate the effects of RSV on the TLR4-related inflammatory signaling pathway and EBI in rats after SAH. A prechiasmatic cistern SAH model was used in our experiment. The expressions of TLR4, high-mobility group box 1 (HMGB1), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) were evaluated by Western blot and immunohistochemistry. The expressions of Iba-1 and pro-inflammatory cytokines in brain cortex were determined by Western blot, immunofluorescence staining, or enzyme-linked immunosorbent assay. Neural apoptosis, brain edema, and neurological function were further evaluated to investigate the development of EBI. We found that post-SAH treatment with RSV could markedly inhibit the expressions of TLR4, HMGB1, MyD88, and NF-κB. Meanwhile, RSV significantly reduced microglia activation, as well as inflammatory cytokines leading to the amelioration of neural apoptosis, brain edema, and neurological behavior impairment at 24 h after SAH. However, RSV treatment failed to alleviate brain edema and neurological deficits at 72 h after SAH. These results indicated that RSV treatment could alleviate EBI after SAH, at least in part, via inhibition of TLR4-mediated inflammatory signaling pathway.

Upregulation of miR-183 expression and its clinical significance in human brain glioma

Glioma is the most common type of primary malignant tumor in the central nervous system (CNS) with a high incidence and a high mortality rate, as well as an extremely low 5-year survival rate. As a class of small non-coding RNAs, microRNAs (miRNAs) may be closely involved in carcinogenesis and might also be connected with glioma diagnosis and prognosis. In this study, we aimed at investigating the expression level of microRNA-183 (miR-183) in 105 cases of glioma tissues of four World Health Organization (WHO) grades and 10 cases of normal brain tissues and its potential predictive and prognostic values in glioma. We found that the expression levels of miR-183 were significantly higher in glioma tissues than that in normal brain tissues, and also higher in high-grade gliomas (WHO grade III and IV) compared with low-grade gliomas (WHO grade I and II). The miR-183 expression level was classified as low or high according to the median value. High expression of miR-183 was found to significantly correlate with larger tumor size, higher WHO grade, and worse Karnofsky performance score (KPS). Kaplan–Meier survival analysis showed that patients with high miR-183 expression had worse overall survival (OS) and progression-free survival (PFS) than patients with low miR-183 expression. Moreover, univariate and multivariate analyses indicated that miR-183 expression level was an independent prognostic parameter of a patient’s OS and PFS. In conclusion, our study indicated that miR-183 was upregulated in glioma, and that it may be used as a potential biomarker of poor prognosis in patients with glioma.

Novel β -carbolines against colorectal cancer cell growth via inhibition of Wnt/ β -catenin signaling

Wnt signaling pathway is aberrantly activated in a variety of cancers, especially in colorectal cancer (CRC), because of mutations in the genes encoding adenomatous polyposis coli (APC), β-catenin and Axin. Small-molecule antagonists of Wnt/β-catenin signaling are attractive candidates for developing effective therapeutics for CRC. In this study, we have identified a novel Wnt signaling inhibitor, isopropyl 9-ethyl-1- (naphthalen-1-yl)-9H-pyrido[3,4-b]indole-3- carboxylate (Z86). Z86 inhibited Wnt reporter activities and the expression of endogenous Wnt signaling target genes in mammalian cells and antagonized the second axis formation of Xenopus embryos induced by Wnt8. We showed that Z86 treatment inhibits GSK3β (Ser9) phosphorylation, leading to its overactivation and promoting the phosphorylation and degradation of β-catenin. In vitro, Z86 selectively inhibited the growth of CRC cells with constitutive Wnt signaling and caused obvious G1-phase arrest of the cell cycle. Notably, in a nude mouse model, Z86 inhibited dramatically the xenografted tumor growth of CRC. Daily intraperitoneal injection of Z86 at 5 mg/kg resulted in >70% reduction in the tumor weight of HCT116 cell origin that was associated with decreased GSK3β (Ser9) phosphorylation and increased β-catenin phosphorylation. Taken together, our findings provide a novel promising chemotype for CRC therapeutics development targeting the canonical Wnt signaling.

Inhibition of myeloid differentiation factor 88(MyD88) by ST2825 provides neuroprotection after experimental traumatic brain injury in mice.

Myeloid differentiation factor 88(MyD88) is an endogenous adaptor protein that plays an important role in coordinating intracellular inflammatory responses induced by agonists of the Toll-like receptor and interleukin-1 receptor families. MyD88 has been reported to be essential for neuronal death in animal models and may represent a therapeutic target for pharmacologic inhibition following traumatic brain injury (TBI). The purpose of the current study was to investigate the neuroprotective effect of MyD88 specific inhibitor ST2825 in an experimental mouse model of TBI. Intracerebroventricular (ICV) injection of high concentration (20μg/μL) ST2825 (15min post TBI) attenuated the development of TBI in mice, markedly improved neurological function and reduced brain edema. Decreased neural apoptosis and increased neuronal survival were also observed. Biochemically, the high concentration of ST2825 significantly reduced the levels of MyD88, further decreased TAK1, p-TAK1, nuclear p65 and increased IκB-α. Additionally, ST2825 significantly reduced the levels of Iba-1 and inflammatory factors TNF-α and IL-1β. These data provide an experimental rationale for evaluation of MyD88 as a drug target and highlight the potential therapeutic implications of ST2825 in TBI.

Roles of Pannexin-1 Channels in Inflammatory Response through the TLRs/NF-Kappa B Signaling Pathway Following Experimental Subarachnoid Hemorrhage in Rats

Background: Accumulating evidence suggests that neuroinflammation plays a critical role in early brain injury after subarachnoid hemorrhage (SAH). Pannexin-1 channels, as a member of gap junction proteins located on the plasma membrane, releases ATP, ions, second messengers, neurotransmitters, and molecules up to 1 kD into the extracellular space, when activated. Previous studies identified that the opening of Pannexin-1 channels is essential for cellular migration, apoptosis and especially inflammation, but its effects on inflammatory response in SAH model have not been explored yet. Methods: Adult male Sprague-Dawley rats were divided into six groups: sham group (n = 20), SAH group (n = 20), SAH + LV-Scramble-ShRNA group (n = 20), SAH + LV-ShRNA-Panx1 group (n = 20), SAH + LV-NC group (n = 20), and SAH + LV-Panx1-EGFP group (n = 20). The rat SAH model was induced by injection of 0.3 ml fresh arterial, non-heparinized blood into the prechiasmatic cistern in 20 s. In SAH + LV-ShRNA-Panx1 group and SAH + LV-Panx1-EGFP group, lentivirus was administered via intracerebroventricular injection (i.c.v.) at 72 h before the induction of SAH. The Quantitative real-time polymerase chain reaction, electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, immunofluorescence staining, and western blotting were performed to explore the potential interactive mechanism between Pannexin-1 channels and TLR2/TLR4/NF-κB-mediated signaling pathway. Cognitive and memory changes were investigated by the Morris water maze test. Results: Administration with LV-ShRNA-Panx1 markedly decreased the expression levels of TLR2/4/NF-κB pathway-related agents in the brain cortex and significantly ameliorated neurological cognitive and memory deficits in this SAH model. On the contrary, administration of LV-Panx1-EGFP elevated the expressions of TLR2/4/NF-κB pathway-related agents, which correlated with augmented neuronal apoptosis. Conclusion: Pannexin-1 channels may contribute to inflammatory response and neurobehavioral dysfunction through the TLR2/TLR4/NF-κB-mediated pathway signaling after SAH, suggesting a potential role of Pannexin-1 channels could be a potential therapeutic target for the treatment of SAH.

Two Natural ent-kauranoids as Novel Wnt Signaling Inhibitors

Constitutively active Wnt signaling frequently occurs in most colon cancers. Therefore, inhibitors of Wnt signaling pathway could provide rational therapeutic effects for colorectal malignancy. Within this paper, we identified two inhibitors of Wnt signaling pathway, rabdoternin B and maoecrystal I from a natural ent-kauranoid library by a dual-luciferase reporter gene assay. The two compounds inhibited Wnt signaling pathway in a concentration-dependent manner and exhibited selective cytotoxicity toward a number of colon carcinoma cell lines SW480, HCT116, and HT29, with only weak cytotoxicity towards the normal colonic epithelial cell line CCD-841-CoN. Rabdoternin B and maoecrystal I treatment induced G2/M phase arrest efficiently in SW480 cells as revealed by flow cytometry analysis. A further study found that maoecrystal I decreased the expression of Wnt signaling target genes, including c-myc, cyclin D1, survivin and Axin2 in colon cancer cells. Collectively our data suggests that rabdoternin B and maoecrystal I are novel inhibitors of canonical Wnt signaling pathway and may possess potentials for colon cancer therapy.

Bisleuconothine A, a bisindole alkaloid, inhibits colorectal cancer cell in vitro and in vivo targeting Wnt signaling

Wnt signaling pathway is aberrantly activated in a variety of cancers, especially in colorectal cancer and small molecule antagonists of Wnt/β-catenin signaling are attractive candidates for developing effective therapeutics. In the present study, we identified Bisleuconothine A, a bisindole alkaloid with an eburnane-aspidosperma type skeleton, as a novel and selective Wnt signaling inhibitor by using a cell-based luciferase assay system. Our study found that Bisleuconothine A down-regulated the endogenous Wnt target gene expression through promoting phosphorylation of β-catenin and the subsequent inhibition of its nuclear translocation in HCT116 and SW480 colorectal cancer cells. In vitro, Bisleuconothine A inhibited cell proliferation through induction of apoptosis by increasing the cleavage of caspases in HCT116 and SW480 colorectal cancer cells. Moreover, in vivo, Bisleuconothine A dramatically suppressed tumor growth in HCT116 Xenograft. And further analysis showed that Bisleuconothine A suppressed the Wnt target gene expression in HCT116 Xenograft, which was associated with up-regulation of β-catenin phosphorylation and subsequent Wnt signaling inhibition. Taken together, our study indicated that bisindole alkaloids could be included as a new chemotype of small-molecule Wnt signaling inhibitors, and have great potential to be further developed for anti-tumor agents.

Elevated cerebrospinal fluid levels of thrombospondin-1 correlate with adverse clinical outcome in patients with aneurysmal subarachnoid hemorrhage.

BACKGROUND Thrombospondin-1 (TSP-1) is a homotrimeric glycoprotein which modulates a wide range of biological functions. Elevated level of TSP-1 in plasma was reported to be correlated with intracerebral hemorrhage. Our study was designed to investigate the relationship between cerebrospinal fluid (CSF) TSP-1 levels and clinical outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH). METHODS CSF TSP-1 levels were measured in 31 aSAH patients on days 1-3, days 5-7 and days 8-10 after aSAH onset using enzyme-linked immunosorbent assay. Patients were under a close follow-up until death or completion of three months after aSAH. Binary logistic regression analyses were performed to determine independent risk factors for the clinical outcomes. RESULTS TSP-1 levels peaked on days 1-3 after aSAH, kept up high on days 5-7 and remained elevated until days 8-10 (p<0.05). Significant elevation of CSF TSP-1 levels were found in patients both with and without vasospasm. Modified Rankin Scale at 3months after aSAH showed a significant correlation with CSF TSP-1 levels on days 1-3 and days 5-7 (both p<0.01). Binary logistic regression analysis showed that higher TSP-1 level on days 1-3 (p<0.05) and on days 5-7 (p<0.05) was a predictive marker of cerebrovasospasm and poor outcome of patient with aSAH. CONCLUSIONS Upregulation of TSP-1 may involve in the pathological process of aSAH and might be a risk factor of future adverse prognosis of aSAH.

Sinomenine Provides Neuroprotection in Model of Traumatic Brain Injury via the Nrf2–ARE Pathway

The neuroprotective effect of sinomenine (SIN) has been demonstrated in several brain injury models. However, its role and molecular mechanism in traumatic brain injury (TBI) remain unknown. In this study, we investigated the neuroprotective effects of SIN in the weight-drop model of TBI in male ICR mice. Mice were randomly divided into the sham and TBI groups, SIN (10 mg/kg, 30 mg/kg and 50 mg/kg, administered intraperitoneally) or equal volume of vehicle was given at 30 min after TBI. Treatment with 30 mg/kg SIN significantly improved motor performance and alleviated cerebral edema. However, treatment with 10 mg/kg or 50 mg/kg SIN did not exhibit a better outcome. Therefore, we chose 30 mg/kg SIN for our subsequent experiments. SIN significantly increased the expression of Bcl-2 and decreased that of cleaved caspase-3, indicating that SIN is anti-apoptotic. This was confirmed by the observation that SIN-treated animals had fewer apoptotic neurons. Cortical malondialdehyde content, glutathione peroxidase (GPx) activity and superoxide dismutase (SOD) activity were restored in the group that received SIN. Furthermore, Western blot and immunofluorescence experiments showed that SIN enhanced the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus. SIN administration also significantly upregulated the expression of the downstream factors heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 at pre- and post-transcriptional levels. Together, these data demonstrate that SIN exerts a neuroprotective effect in a model of TBI, possibly by activating the Nrf2–antioxidant response element (ARE) pathway.