The Diagnostic Value of Gut Microbiota Analysis for Post-Stroke Sleep Disorders.

BACKGROUND: Gut microbiota have been associated with many psychiatric disorders. However, the changes in the composition of gut microbiota in patients with post-stroke sleep disorders (PSSDs) remain unclear. Here, we determined the gut microbial signature of PSSD patients. METHODS: Fecal samples of 205 patients with ischemic stroke were collected within 24 h of admission and were further analyzed using 16 s RNA gene sequencing followed by bioinformatic analysis. The diversity, community composition, and differential microbes of gut microbiota were assessed. The outcome of sleep disorders was determined by the Pittsburgh Sleep Quality Index (PSQI) at 3 months after admission. The diagnostic performance of microbial characteristics in predicting PSSDs was assessed by receiver operating characteristic (ROC) curves. RESULTS: Our results showed that the composition and structure of microbiota in patients with PSSDs were different from those without sleep disorders (PSNSDs). Moreover, the linear discriminant analysis effect size (LEfSe) showed significant differences in gut-associated bacteria, such as species of Streptococcus, Granulicatella, Dielma, Blautia, Paeniclostridium, and Sutterella. We further managed to identify the optimal microbiota signature and revealed that the predictive model with eight operational-taxonomic-unit-based biomarkers achieved a high accuracy in PSSD prediction (AUC = 0.768). Blautia and Streptococcus were considered to be the key microbiome signatures for patients with PSSD. CONCLUSIONS: These findings indicated that a specific gut microbial signature was an important predictor of PSSDs, which highlighted the potential of microbiota as a promising biomarker for detecting PSSD patients.

Fecal microbiota transplantation inhibited neuroinflammation of traumatic brain injury in mice via regulating the gut-brain axis.

Introduction: Recent studies have highlighted the vital role of gut microbiota in traumatic brain injury (TBI). Fecal microbiota transplantation (FMT) is an effective means of regulating the microbiota-gut-brain axis, while the beneficial effect and potential mechanisms of FMT against TBI remain unclear. Here, we elucidated the anti-neuroinflammatory effect and possible mechanism of FMT against TBI in mice via regulating the microbiota-gut-brain axis. Methods: The TBI mouse model was established by heavy object falling impact and then treated with FMT. The neurological deficits, neuropathological change, synaptic damage, microglia activation, and neuroinflammatory cytokine production were assessed, and the intestinal pathological change and gut microbiota composition were also evaluated. Moreover, the population of Treg cells in the spleen was measured. Results: Our results showed that FMT treatment significantly alleviated neurological deficits and neuropathological changes and improved synaptic damage by increasing the levels of the synaptic plasticity-related protein such as postsynaptic density protein 95 (PSD-95) and synapsin I in the TBI mice model. Moreover, FMT could inhibit the activation of microglia and reduce the production of the inflammatory cytokine TNF-α, alleviating the inflammatory response of TBI mice. Meanwhile, FMT treatment could attenuate intestinal histopathologic changes and gut microbiota dysbiosis and increase the Treg cell population in TBI mice. Conclusion: These findings elucidated that FMT treatment effectively suppressed the TBI-induced neuroinflammation via regulating the gut microbiota-gut-brain axis, and its mechanism was involved in the regulation of peripheral immune cells, which implied a novel strategy against TBI.

SP1 induced long non-coding RNA AGAP2-AS1 promotes cholangiocarcinoma proliferation via silencing of CDKN1A.

BACKGROUND: LncRNA can regulate gene at various levels such as apparent genetics, alternative splicing, and regulation of mRNA degradation. However, the molecular mechanism of LncRNA in cholangiocarcinoma is still unclear. This deserves further exploration. METHODS: We investigated the expression of AGAP2-AS1 in 32 CCA tissues and two CCA cell lines. We found a LncRNA AGAP2-AS1 which induced by SP1 has not been reported in CCA, and Knockdown and overexpression were used to investigate the biological role of AGAP2-AS1 in vitro. CHIP and RIP were performed to verify the putative targets of AGAP2-AS1. RESULTS: AGAP2-AS1 was significantly upregulated in CCA tumor tissues. SP1 induced AGAP2-AS1 plays an important role in tumorigenesis. AGAP2-AS1 knockdown significantly inhibited proliferation and caused apoptosis in CCA cells. In addition, we demonstrated that AGAP2-AS1 promotes the proliferation of CCA. CONCLUSIONS: We conclude that the long non-coding RNA AGAP2-AS1 plays a role in promoting the proliferation of cholangiocarcinoma.

Upregulated miR-96-5p inhibits cell proliferation by targeting HBEGF in T-cell acute lymphoblastic leukemia cell line.

INTRODUCTION: microRNAs (miRNAs) are critical for tumorigenesis and progression of T-cell acute lymphoblastic leukemia (T-ALL). MiR-96-5p has been shown to play important roles in the development of many cancers, but its roles in T-ALL have yet not been studied. MATERIALS AND METHODS: miR-96-5p expression was detected in T-leukemic cells from peripheral blood of 30 patients with T-ALL using real-time quantitative PCR (RT-qPCR). TargetScan database was utilized to identify the target genes for miR-96-5p, and their target relationship was verified by western blot, dual luciferase reporter assay and RT-qPCR. The effects of miR-96-5p on the viability and proliferation of T-leukemic cells (Jurkat cells) were respectively determined using MTT and BrdU incorporation assays. RESULTS: miR-96-5p presented low expression levels by qPCR in peripheral blood of T-ALL patients compared to healthy volunteers. Upregulated miR-96-5p by miR-96-5p mimic transfection markedly inhibited the viability and proliferation of Jurkat cells. Furthermore, miR-96-5p negatively regulated the expression of its target gene, HBEGF. The decreased viability and proliferation of Jurkat cells caused by miR-96-5p over-expression was suppressed after the introduction of HBEGF plasmid. CONCLUSIONS: The presented study showed that upregulation of miR-96-5p inhibited the viability and proliferation of Jurkat T-leukemic cells through suppressing HBEGF expression. Our study provides a novel sight for understanding the pathological mechanism of T-ALL and suggests that miR-96-5p may be a potential biomarker for the therapy and diagnosis of T-ALL.

MicroRNA-532 exerts oncogenic functions in t(4;14) multiple myeloma by targeting CAMK2N1.

Multiple myeloma (MM) is a plasma cell neoplasm which is characterized by widespread genetic heterogeneity. The MMs with t(4;14) translocation exhibit poor outcomes. However, the mechanism underlying has not been well dissected. Our study aimed to identify key microRNA involved in the oncogenesis of t(4;14) MM. We here performed an integrated analysis to screen important regulators in the pathogenesis of t(4;14) MM. We used real-time quantitative polymerase chain reaction and western blotting to evaluate the mRNA and protein expression of the indicated microRNA or protein. Cell proliferation assay, colony formation assay, and transwell assay were used to examine the cell growth and metastasis. More importantly, the tumor growth and metastasis were analyzed in nude mice injected with MM cells. The integrated analysis indicated that miR-532 functioned as a pivotal regulator in t(4;14) MM. miR-532 was upregulated in t(4;14) MMs and promotes cell growth and metastasis in vitro and in vivo. Notably, though combing bioinformatics analysis and functional assays, CAMK2N1 was revealed as a functional target of miR-532 in MM cells. CAMK2N1 plays an anti-proliferative and anti-migration role in MM cells, and miR-532 exerts its oncogenic role though inhibiting CAMK2N1 expression in MMs. miR-532 promotes cell proliferation and invasion in t(4;14) MMs by targeting CAMK2N1. Our study, thus, provides possible targets for t(4;14) MM therapy.

DUXAP8, a pseudogene derived lncRNA, promotes growth of pancreatic carcinoma cells by epigenetically silencing CDKN1A and KLF2.

BACKGROUND: Recent studies highlight pseudogene derived long non-coding RNAs (lncRNAs) as key regulators of cancer biology. However, few of them have been well characterized in pancreatic cancer. Here, we aimed to identify the association between pseudogene derived lncRNA DUXAP8 and growth of pancreatic cancer cells. METHODS: We screened for pseudogene derived lncRNAs associated with human pancreatic cancer by comparative analysis of three independent datasets from GEO. Quantitative real-time reverse transcription polymerase chain reaction was used to assess the relative expression of DUXAP8 in pancreatic cancer tissues and cells. Loss-of-function approaches were used to investigate the potential functional roles of DUXAP8 in pancreatic cancer cell proliferation and apoptosis in vitro and in vivo. RNA immunoprecipitation, chromosome immunoprecipitation assay and rescue experiments were performed to analyze the association of DUXAP8 with target proteins and genes in pancreatic cancer cells. RESULTS: Pancreatic cancer tissues had significantly higher DUXAP8 levels than paired adjacent normal tissues. High DUXAP8 expression was associated with a larger tumor size, advanced pathological stage and shorter overall survival of pancreatic cancer patients. Moreover, silencing DUXAP8 expression by siRNA or shRNA inhibited pancreatic cancer cell proliferation and promoted apoptosis in vitro and in vivo. Mechanistic analyses indicated that DUXAP8 regulates PC cell proliferation partly through downregulation of tumor suppressor CDKN1A and KLF2 expression. CONCLUSION: Our results suggest that tumor expression of pseudogene derived lncRNA DUXAP8 plays an important role in pancreatic cancer progression. DUXAP8 may serve as a candidate biomarker and represent a novel therapeutic target of pancreatic cancer.

Clostridium butyricum Attenuates Chronic Unpredictable Mild Stress-Induced Depressive-Like Behavior in Mice via the Gut-Brain Axis.

Abnormal gut microbiome has been associated with depression. The mechanism of probiotics against depression remains unclear. This study aimed to determine whether Clostridium butyricum (Cb) could attenuate chronic unpredictable mild stress-induced depressive-like behavior and its possible mechanisms. Male C57BL/6 mice were subjected to chronic unpredictable mild stress (CUMS) and were treated with Cb. Depressive-like behavior was evaluated by a series of behavioral tests. The levels of cerebral 5-hydroxytryptamine (5-HT), brain derived neurotrophic factor (BDNF), glucagon-like peptide-1 (GLP-1) receptor and intestinal were measured. Cb treatment significantly improved CUMS-induced depressive-like behavior in mice. Meanwhile, Cb treatment exhibited prominent effects, increasing 5-HT and GLP-1 and upregulating BDNF expression. Furthermore, Cb-treated mice showed increased secretion of GLP-1 and upregulated GLP-1R expression. Taken together, our results demonstrate an antidepressive effect of Cb in CUMS mice partially attributed to stimulation of intestinal GLP-1 secretion and activation of cerebral GLP-1R.

MiR-544 promotes immune escape through downregulation of NCR1/NKp46 via targeting RUNX3 in liver cancer.

OBJECTIVE: To study the potential role of miR-544 in the immune escape mechanism of hepatoma cells. METHODS: Natural killer (NK) cells were collected from healthy volunteers and patients with liver cancer. Interleukin (IL)-2 activated-NK-92 cells were transfected with miR-544 inhibitor/mimic or NC/pre-NC in HepG2 co-culture system. NK-92 cells were treated with control, IL-2, IL-2 + pre-NC, IL-2 + miR-544 mimic, IL-2 + miR-544 mimic + pcDNA and IL-2 + miR-544 mimic + pcDNA-runt-related transcription factor 3 (RUNX3) groups. Mice models of liver cancer were well established. Expression of miR-544, natural cytotoxicity receptor 1 (NCR1) and RUNX3 were evaluated by quantitative real-time PCR and western blotting. Flow cytometry and ELISA were used to determine NK cell cytotoxicity and the levels of INF-γ, respectively. RESULTS: MiR-544 was upregulated while NCR1 and RUNX3 was downregulated in NK cells of patients with liver cancer. The levels of IFN-γ and miR-544 expression were increased and decreased in IL-2 activated-NK cells, respectively. Inversely, miR-544 overexpression inhibited NK cell cytotoxicity by downregulating IFN-γ. However, miR-544 directly targeted RUNX3 and negatively regulated NCR1. Furthermore, miR-544 promoted immune escape of hepatoma cells in vivo and in vitro. CONCLUSION: miR-544 promoted the immune escape of liver cancer cells by downregulating NCR1 via targeting RUNX3.

Sodium butyrate exerts protective effect against Parkinson's disease in mice via stimulation of glucagon like peptide-1.

Sodium butyrate (NaB) has exhibited protective activity in neurological disorders. Here, we investigated the neuroprotective effect and potential mechanisms of NaB in a mouse model of Parkinson's disease (PD). A mouse was intraperitoneally treated with MPTP (30mg/kg) for 7 consecutive days to induce PD model and NaB (200mg/kg) was intragastrically treated for 3weeks. The behavioral tests were then conducted. Dopaminergic degeneration was evaluated by western blot and immunohistochemistry of tyrosine hydroxylase (TH) in the SN. Brain damage was assessed by histologic (Nissl staining for cell death), apoptosis-associated protein and tight junction (TJ) proteins studies. Meanwhile, the levels of colonic glucagon-like peptide-1 (GLP-1) and cerebral GLP-1 receptor (GLP-1R) expression were assessed. Our results showed that NaB improved neurobehavioral impairment including cognitive behavior and coordination performance. Moreover, NaB treatment prevented the MPTP-induced dopaminergic degeneration and decreased expression level of TH in the striatum. NaB treatment attenuated the PD-associated disruption of BBB by upregulation of Occludin and zonula occludens (ZO)-1. In addition, NaB resulted in increased level of Bcl-2 and decreased level of Bax. Particularly, NaB-treated mice with PD exhibited increased colonic GLP-1 level as well as upregulation of brain GLP-1R expression compared with PD group. Our findings suggest that NaB has potential as a novel therapeutic for treatment of PD, and its mechanism was associated with stimulating colonic GLP-1secretion.

Role of PDGF-D and PDGFR-ß in neuroinflammation in experimental ICH mice model.

OBJECTIVE: Inflammation plays a key role in the pathophysiological processes after intracerebral hemorrhage (ICH). Post-ICH macrophages infiltrate the brain and release pro-inflammatory factors (tumor necrosis factor-α), amplifying microglial activation and neutrophil infiltration. Platelet-derived growth factor receptor-ß (PDGFR-ß) is expressed on macrophages and it's activation induces the recruitment of macrophages. Platelet-derived growth factor-D (PDGF-D) is an agonist with a significantly higher affinity to the PDGFR-ß compared to another isoform of the receptor. In this study, we investigated the role of PDGF-D in the pro-inflammatory response after ICH in mice. METHODS: A blood injection model of ICH was used in eight-week old male CD1 mice (weight 30g). Some mice received an injection of plasmin or PDGF-D. Gleevec, a PDGFR inhibitor, was administered at 1, 3 or 6h post-ICH. Plasmin was administered with or without PDGF-D siRNAs mixture or scramble siRNA. A plasmin-antagonist, ε-Aminocaproic acid (EACA), was co-administrated with the blood. The effects of ICH and treatment on the brain injury and post-ICH inflammation were investigated. RESULTS: ICH resulted in the overexpression of PDGF-D, associated with the infiltration of macrophages. PDGFR-inhibition decreased ICH-induced brain injury, attenuating macrophage and neutrophil infiltration, reducing microglial activation and TNF-α production. Administration of recombinant PDGF-D induced TNF-α production, and PDGFR-inhibition attenuated it. A plasmin-antagonist suppressed PDGFR-ß activation and microglial activation. Plasmin increased PDGF-D expression, and PDGF-D inhibition reduced neutrophil infiltration. CONCLUSION: ICH-induced PDGF-D accumulation contributed to post-ICH inflammation via PDGFR activation and enhanced macrophage infiltration. The inhibition of PDGFR had an anti-inflammatory effect. Plasmin is a possible upstream effector of PDGF-D. The targeting of PDGF-D may provide a novel way to decrease brain injury after ICH.

Gambogenic acid alters chemosensitivity of breast cancer cells to Adriamycin.

BACKGROUND: Breast cancer remains a major health problem worldwide, and is becoming increasingly resistant to traditional drug treatments. For instance, Adriamycin (ADR) is beneficial for the treatment of breast cancer. However, its wide application often leads to drug resistance in clinic practice, which results in treatment failure. Gambogenic acid (GNA), a polyprenylated xanthone isolated from the traditional medicine gamboge, has been reported to effectively inhibit the survival and proliferation of cancer cells. Its effects on ADR resistance have not yet been reported in breast cancer. In this study, we examined the ability of GNA to modulate ADR resiatance and the molecular mechanisms underlying this process using a cell based in vitro system. METHODS: An MTT assay was used to evaluate the inhibitory effect of the drugs on the growth of MCF-7 and MCF-7/ADR cell lines. The effects of drugs on apoptosis were detected using Annexin-V APC/7-AAD double staining. The expression of apoptosis-related proteins and the proteins in the PTEN/PI3K/AKT pathway were evaluated by Western blot analysis. RESULTS: In the MCF-7/ADR cell lines, the IC50 (half maximal inhibitory concentration) of the group that received combined treatment with GNA and ADR was significantly lower than that in the ADR group, and this value decreased with an increasing concentration of GNA. In parallel, GNA treatment increased the chemosensitivity of breast cancer cells to ADR. The cell apoptosis and cell cycle anaysis indicated that the anti-proliferative effect of GNA is in virtue of increased G0/G1 arrest and potentiated apoptosis. When combined with GNA in MCF-7/ADR cell lines, the expression levels of the tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome ten) and the apoptosis-related proteins caspase-3 and capsese-9 were significantly increased, while the expression of phosphorylated AKT was decreased. CONCLUSIONS: Our study has indicated a potential role for GNA to increase the chemosensitivity of breast cancer cells to ADR. This modulatory role was mediated by suppression of the PTEN/PI3K/AKT pathway that led to apoptosis in MCF-7/ADR cells. This work suggests that GNA may be used as a regulatory agent for treating ADR resistance in breast cancer patients.

[The comparison of clinical manifestation of organophosphorus pesticide poisoning (OPP) between oral exposure and occupational exposure in field work].

OBJECTIVE: To investigate the differences of clinical manifestation and therapy of organophosphorus pesticide poisoning (OPP) between oral exposure and occupational exposure in field work. METHODS: From July 2007 to July 2010, 85 patients with acute severe OPP were treated in a hospital, which were divided into oral poisoning group (51 cases) and non-oral poisoning group (34 cases). The differences of clinical manifestations, curative effects and prognosis between two groups were compared. RESULTS: The rates of myoclonus and ataxia in cases with moderate poisoning of oral poisoning group were 86.4% and 90.9%, which were significantly higher than those (50.0% and 55.0%) of non-oral poisoning group (P<0.05 or P< 0.01). The rates of myoclonus, lung fluid and coma in cases with severe poisoning of oral poisoning group were 100.0%, 89.7% and 93.1%, respectively, which were significantly higher than those (71.4%, 64.3% and 50.0%) of non-oral poisoning group (P<0.05). The mean detoxification hours in cases with moderate poisoning and cases with severe poisoning of non-oral poisoning group were (35.0 +/- 6.2) and (45.0 +/- 11.1) hours which were significantly lower than those [(49.0 +/- 7.7) and (77.0 +/- 10.3) hours] in cases with moderate poisoning and cases with severe poisoning of oral poisoning group (P<0.05). In 24, 48 and 72 h after treatment, the cholinesterase (ChE) activities of non-oral poisoning group were higher than those of oral poisoning group (P< 0.05 or P<0.01). The used doses of pyraloxime methylchloride (PAM-Cl) or atropine and the used total dose of atropine in non-oral poisoning group were lower than those in oral poisoning group (P<0.05 or P<0.01). CONCLUSIONS: The clinical manifestation of non-oral poisoning group is different from the clinical manifestation of oral poisoning group due to the high morbidity of OPP occurred at field site in summer. The used doses of atropine and PAM-Cl are less and the ChE activity recovers quickly for non-oral poisoning group.

[Alterations of peripheral blood T cell subsets in patients with sepsis and the clinical implications].

OBJECTIVE: To observe the dynamic changes of CD3+, CD4+, and CD8+ T lymphocytes in the peripheral blood of patients with sepsis and discuss the clinical significance. METHODS: Sixty-four patients admitted in the Emergency Center and Emergency Intensive Care Unit of the Second Hospital of Wenzhou Medical University between August, 2007 and July, 2009 were enrolled in this study. CD3+, CD4+, and CD8+ T lymphocytes in the peripheral blood were detected by flow cytometry on days 1, 7 and 14 after admission, and the results were compared between the patients with improvement of the condition and those without improvement, with 20 healthy subjects as the control group. RESULTS: On day 1 after admission, CD3+ and CD4+ T lymphocytes and CD4+/CD8+ T cell ratio were obviously lower in the 2 groups of patients with sepsis than in the control group (P<0.05), but no significant difference was found in CD8+ T lymphocytes. The sepsis patients with clinical improvement showed significant higher CD3+ and CD4+ T lymphocyte percentages and CD4+/CD8+ T cell ratio than those without improvement on day 1. In the patients with clinical improvement, CD3+ and CD4+T lymphocytes and CD4+/CD8+ T cell ratio increased gradually with time and till day 14, they were comparable with the control levels; in the patients without improvement, CD3+ and CD4+ T lymphocytes and CD4+/CD8+ T cell ratio showed no obvious alterations in the course of observation. CONCLUSION: Immune imbalance occurs in patients with sepsis represented by lowered CD3+ and CD4+T lymphocyte percentages and CD4+/CD8+ T cell ratio in relation to the severity of the condition. CD3+ and CD4+ T lymphocytes and CD4+/CD8+ T cell ratio can be used as the indicators for assessing the severity of sepsis.