A pilot study: Gut microbiota, metabolism and inflammation in hypertensive intracerebral haemorrhage.

AIMS: In recent years, the incidence rate of hypertensive intracerebral haemorrhage (HICH) has been increasing, accompanied by high mortality and morbidity, which has brought a heavy burden to the social economy. However, the pathogenesis of HICH is still unclear. This study intends to explore the mechanism of gut microbiota metabolism and inflammation in the process of HICH to provide a theoretical basis for the diagnosis and treatment of HICH. METHODS AND RESULTS: HE staining showed that the brain tissues of model group had obvious oedema injury, which indicated that the HICH model was successfully constructed. ELISA analysis showed that IL-1ß and TNF-α levels in blood and brain tissues were significantly increased, and IL-10 level was significantly decreased in blood. IHC analysis showed that microglia and macrophages were activated in the model group. 16S rRNA sequence showed that the diversity of gut microbiota in HICH patients decreased. Also, the microbiota belonging to Firmicutes, Proteobacteria and Verrucomicrobia changed significantly. LC-MS/MS analysis showed that the metabolic phenotype of HICH patients changed. Also, the 3,7-dimethyluric acid- and 7-methylxanthine-related metabolic pathways of caffeine metabolism pathways were downregulated in patients with HICH. Bacteroides was negatively correlated with the IL-1ß and TNF-α levels. Blautia was negatively correlated with the IL-1ß and TNF-α levels, and positively correlated with the IL-10 level. Akkermansia was negatively correlated with the 3,7-dimethyluric acid and 7-methylxanthine. CONCLUSION: Our study suggested that HICH was accompanied by the increased inflammation marker levels in peripheral blood and brain, decreased gut microbiota diversity, altered gut metabolic phenotype and downregulation of caffeine metabolism pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reported that HICH accompanied by the increased inflammation, decreased gut microbiota diversity and altered gut metabolic phenotype. Due to the number of patients, this work was a pilot study.

4,5-Dimethoxycanthin-6-one is a novel LSD1 inhibitor that inhibits proliferation of glioblastoma cells and induces apoptosis and pyroptosis.

BACKGROUND: Glioblastoma is one of the most common fatal intracranial malignancies. Lysine-specific demethylase 1 (LSD1) reportedly has therapeutic effects on a variety of tumors. This study explored the therapeutic effect of LSD1 inhibition on glioblastoma cell lines and the possible underlying mechanisms. METHODS: The MTT assay was utilized to screen for the sensitivity of U87, U251 and T98G cells to 4, 5-dimethoxycarrageenin-6-one. qRT-PCR and western blot were used to measure the proliferation, apoptosis, and pyroptosis signaling pathway expression to observe the effect of LSD1 inhibition on U251 and T98G cells. Flow cytometry, immunofluorescence, immunohistochemistry, wound scratch, clone formation, and TUNEL assay were used to analyze the effects of 4, 5-dimethoxycanthin-6-one on glioblastoma cells. The effect of 4, 5-dimethoxycanthin-6-one was examined in vivo in BALB/c nude mice injected with U251 cells. HE staining was used to detect the histopathology of the tumor. RESULTS: LSD1 specifically catalyzes the demethylation of monomethylated and demethylated histone H3 lysine at position 4 (h3k4me1, h3k4me2, h3k4me3) and lysine at position 9 (h3k9me1). This regulated the transcriptional activity of proliferation, apoptosis, and pyroptosis signaling pathway genes. In vitro, the proliferation of glioblastoma cells was decreased in the 4, 5-dimethoxycanthin-6-one group. The expression of Caspase1 in glioblastoma cells treated with 4, 5-dimethoxycanthin-6-one increased, and the number of apoptotic cells increased. The tumor volume of mice injected with 4, 5-dimethoxycanthin-6-one decreased significantly. CONCLUSION: 4, 5-Dimethoxycanthin-6-one could act as a novel inhibitor of LSD1 to regulate glioblastoma, which could inhibit the proliferation of U251 and T98G cells and induce their apoptosis and pyroptosis. It is a potential drug for the treatment of glioblastoma.

LncRNA SNHG12 inhibits miR-199a to upregulate SIRT1 to attenuate cerebral ischemia/reperfusion injury through activating AMPK signaling pathway.

Cerebral ischemia caused severe disability, and associated with a series of neurological events. Long non-coding RNA SNHG12 was found to be upregulated in mouse brain microvascular endothelial cells by cerebral ischemia. Moreover, it was reported that SNHG12 could directly interact with miR-199a and sirtuin 1 (SIRT1) as a direct target of miR-199a in other diseases. However, the function and mechanism of SNHG12 in cerebral ischemia and reperfusion (I/R) injury of neuronal cells remains unclear. The present study was thus designed to explore the potential effect of SNHG12 and to investigate the underlying mechanism in I/R neuronal cells. we found that SNHG12 was upregulated in primary neuronal cells and N2a cells and peaked at 12 h and 24 h after OGD/R treatment, respectively. Meanwhile, MTT assay showed that knockdown SNHG12 inhibited cell proliferation under OGD/R condition. And flow cytometry analyses revealed more apoptosis rate was caused by SNHG12 knockdown. Mechanistically, SNHG12 interacted with miR-199a and decreased the expression of miR-199a. Overexpression miR-199a largely inhibited the cell proliferation and induced the cell apoptosis. Meanwhile, SNHG12 was proven to target miR-199a and then activated SIRT1 expression, which finally led to activation of AMPK signaling pathway. In summary, we demonstrate SNHG12 targets miR-199a to upregulate SIRT1 expression, which attenuates cerebral ischemia/reperfusion injury through AMPK pathway activation. Our findings provide molecular mechanism by which SNHG12 attenuates cerebral I/R injury and facilitate development of therapeautical strategies for treating ischemia-induced stroke.

Sodium hydrosulfide attenuates cerebral ischemia/reperfusion injury by suppressing overactivated autophagy in rats.

Ischemic stroke is a leading cause of death and disability worldwide, and autophagy may be involved in the pathological process of cerebral ischemia/reperfusion injury. Hydrogen sulfide (H2S) is an endogenous gasotransmitter with protective effects against multiple diseases. Here, we tested the effect of H2S on cerebral ischemia/reperfusion injury in rats. Sodium hydrosulfide (NaHS), an H2S donor, improved neurological function and reduced the size of the infarcts induced by transient middle cerebral artery occlusion (MCAO) followed by reperfusion in rats. NaHS treatment reduced the lactate dehydrogenase (LDH) activity in the serum (a marker of cellular membrane integrity) and the expression of cleaved caspase-3 (a marker for apoptosis) in the brains of MCAO rats. We also found that autophagy was overactivated in the brains of MCAO rats, as indicated by an increased ratio of LC3 II to I, decreased expression of p62, and transmission electron microscope detection. NaHS treatment significantly inhibited the autophagic activity in the brains of MCAO rats. Furthermore, PC12 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic MCAO in vitro. We found that NaHS treatment reduced cellular injury and suppressed overactivated autophagy induced by OGD/R in PC12 cells. An autophagy stimulator (rapamycin) eliminated the protective effect of NaHS against LDH release and caspase-3 activity induced by OGD/R in PC12 cells. An autophagy inhibitor (3-methyladenine, 3-MA) also reduced the cellular injury induced by OGD/R in PC12 cells. In conclusion, the results indicate that overactivated autophagy accelerates cellular injury after MCAO in rats and that exogenous H2S attenuates cerebral ischemia/reperfusion injury via suppressing overactivated autophagy in rats.

Neuroprotective effects of lentivirus-mediated cystathionine-beta-synthase overexpression against 6-OHDA-induced parkinson's disease rats.

Parkinson's disease (PD) is age-related neurodegenerative disorder by a progressive loss of dopaminergic(DA) neurons in the substantia nigra (SN) and striatum, which is at least partly associated with α-synuclein protein accumulation in these neurons. Hydrogen sulfide (H2S) plays an important role in the nervous system. Studies have shown that H2S has a protective effect on PD. However, as a kind of gas molecules, H2S is lively, volatile, and not conducive to scientific research and clinical application. Cystathionine-beta-synthase(CBS) is the main enzymes of synthesis of H2S in the brain. In order to examine the neuroprotective effects of CBS on PD, we detected the effects of CBS overexpression on 6-Hydroxydopamine (6-OHDA)-lesioned PD rats using lentivirus-mediated gene transfection techniques. In the injured SN of 6-OHDA-induced PD rats, the CBS expression and the endogenous H2S level markedly decreased, while administration of lentivirus-mediated CBS overexpression increased the CBS expression and the endogenous H2S production.CBS overexpression dramatically reversed apomorphine-induced rotation of the 6-OHDA model rats, decreased the number of TUNEL-positive neurons and the loss of the nigral DA neurons，specifically inhibited 6-OHDA-induced oxidase stress injury, and down-regulated the expression of α-synuclein(α-SYN) in the injured SN. NaHS (an H2S donor) had similar effects to CBS overexpression, while Amino-oxyacetate(AOAA, a CBS inhibitor) had opposite effects on PD rats. In summary, we demonstrated that CBS overexpression was able to provide neuroprotective on PD rats and improving the expression of CBS may be a potential therapeutic method for PD.

MiR-223/PAX6 Axis Regulates Glioblastoma Stem Cell Proliferation and the Chemo Resistance to TMZ via Regulating PI3K/Akt Pathway.

Chemotherapy is a standard strategy for glioma, while chemoresistance remains a major therapeutic challenge in current clinical practice. Our present study was aimed to determine whether inhibition of the miR-223/paired box 6 (PAX6) pathway could increase the sensitivity of glioma to Temozolomide. An elevated level of miR-223 was observed in glioma tissues. Exogenous miR-223 promoted cell survival when exposed to Temozolomide (TMZ), while miR-223 inhibition could reverse this process. The RNA and protein levels of PAX6 were significantly decreased by exogenous miR-223, and the 3'-untranslated region of PAX6 was shown to be a target of miR-223. Besides, it has also been reported that PI3K/Akt signaling pathway is pivotal to regulate glioma growth and proliferation. In the present study, we revealed that miR-223/PAX6 axis regulated the growth, invasion, and chemo resistance of glioblastoma stem cells to TMZ via regulating PI3K/Akt signaling pathway, which present a novel potential therapy for intervention of glioblastoma. Taken together, our findings shed new light on the miR-223/PAX6 pathway in glioma and this pathway might modulate the sensitivity of glioma to TMZ via regulating PI3K/Akt signaling pathway. J. Cell. Biochem. 118: 3452-3461, 2017. © 2017 Wiley Periodicals, Inc.

[Construction of a lentiviral vector carrying short?hairpin RNA targeting PAX6 and its effect on proliferation of glioma U251 cells in vitro].

OBJECTIVE: To construct a lentiviral vector for delivering short hairpin RNA (shRNA) targeting PAX6 and investigate its effect on the proliferation of glioma U251 cells in vitro. METHODS: Two small interfering RNA sequences targeting PAX6 gene were designed based on the reported sequence of PAX6 and annealed to form a double?stranded chain, which was inserted into a lentiviral vector to construct the recombinant lentiviral vector shRNA?PAX6. The recombinant vector was infected into U251 cells, and the expression of PAX6 mRNA and protein in the cells was detected by real?time PCR and Western blotting, respectively. The changes in the proliferation of U251 cells after the infection was assessed using MTT assay. RESULTS: Double enzyme digestion of the lentiviral vector pLKD?CMV?G&NR?U6?shRNA yielded an 8208?bp fragment, and colony PCR and sequencing analysis confirmed successful construction of the lentiviral vector shRNA?PAX6. Infection of the cells with shRNA?PAX6 caused a significant reduction of the expressions of PAX6 mRNA and protein (P<0.05) and resulted in obviously increased proliferation of U251 cells (P<0.05). CONCLUSION: We successfully constructed the recombinant vector shRNA?PAX6 for silencing PAX6 gene. PAX6 gene silencing results in increased proliferation of U251 cells in vitro.

microRNA-223 promotes the growth and invasion of glioblastoma cells by targeting tumor suppressor PAX6.

Glioblastoma is the most common primary central nervous system malignancy and its unique invasiveness hinders effective treatment. Its high invasiveness may be controlled partly by microRNAs (miRNAs, miRs) and their target genes. In the present study, we found that increased miR-223 expression and reduced PAX6 expression coexisted in glioblastoma as detected by quantitative PCR or tissue microarrays. We confirmed that miR-223 directly targets PAX6 through binding to its 3'-UTR using dual luciferase reporter assay. In U251 and U373 glioblastoma cells, overexpression of miR-223 decreased PAX6 mRNA and protein expression; however, inhibition of miR-223 increased PAX6 mRNA and protein expression. Moreover, overexpression of miR-223 led to effects similar to those of PAX6 knockdown: increased cell viability, increased percentage of cells in the G1 phase and increased cell invasiveness parallel with increased MMP2, MMP9 and VEGFA expression. In addition, inhibition of miR-223 resulted in effects similar to those of PAX6 overexpression: decreased cell viability, decreased percentage of cells in the G1 phase and decreased cell invasiveness parallel with reduced MMP2, MMP9 and VEGFA expression. The data presented here suggest that miR-223 promotes the growth and invasion of U251 and U373 glioblastoma cells by targeting PAX6, which serves as a tumor suppressor in glioblastoma exerting the functions of inhibition of cell cycle transition, and the expression of MMP2, MMP9 and VEGFA. In conclusion, the present study supports miR-223 and PAX6 as novel therapeutic targets for glioblastoma.

[Effects of hypoxic preconditioning on hypoxia tolerance of astrocytes in vitro].

AIM: To explore the mechanisms of hypoxic preconditioning on protecting cultured astrocytes from hypoxia injury. METHODS: Cultured astrocytes were divided randomly into several groups: control(C), hypoxia(H) and hypoxic preconditioning (HP). Cells MTT metabolic activity, qualitation of apoptosis and modality to explore the protection effects of hypoxic preconditioning. Immunocytochemistry of Bcl-2 and Bax to explore the mechanisms of hypoxic preconditioning on protecting astrocytes from hypoxia. RESULTS: Compared with H group there was marked increase of MTT metabolic activity in HP48 and HP72 groups. Immunocytochemistry of Bcl-2 and Bax showed that compared with H group, expression of Bcl-2 was increased in HP group, while expression of Bax was decreased in HP group. CONCLUSION: Hypoxic preconditioning can protect astrocytes from hypoxia. One possible mechanism maybe concerned with inhibition of Bax and maintain of Bcl-2 to depress apoptosis procedure.

[Correlation between MHC class I-related chain A gene *008 allele and human cytomegalovirus infection].

OBJECTIVE: To investigate the correlation between MHC class I-related chain A (MICA) gene *008 allele and human cytomegalovirus (HCMV) infection. METHODS: MICA*008 allele was detected in 86 patients with chronic granulocytic leukemia and 81 unrelated normal individuals by way of sequence-specific primers (PCR-SSP). Anti-HCMV IgM was also detected in the sera of these subjects with enzyme linked immunosorbent assay (ELISA). RESULTS: MICA*008 allele frequency was lower in patients with chronic granulocytic leukemia than in the control group (22.2% vs 34.3%, Chi(2)=4.98, P<0.05). The infection rate of HCMV was significantly higher in those individuals with genotype of MICA*008 (-) than in those with MICA*008 (+), and moderate correlation was suggested between MICA*008 and HCMV infection (C=0.5829, 0.6142). CONCLUSION: Individuals with MICA*008 positivity is not liable to HCMV infection, but those with MICA*008 (-) can be vulnerable to HCMV infection, suggesting an inverse correlation between MICA*008 allele with HCMV.

[MICA*008/A5.1 allele and HCMV infection in kidney transplanted donees of Hunan Han nationality].

OBJECTIVE: To investigate the relationship between MICA*008/A5.1 allele and human cytomegalovirus (HCMV) infection in kidney transplanted donees of Hunan Han nationality. METHODS: The MICA*008/A5.1 allele based on 91 kidney transplanted donees and 81 unrelated normal individuals of Han nationality in Hunan Province were analyzed by PCR/SSP assay. At the same time, anti-HCMV antibody IgM was detected in the serum by ELISA method. RESULTS: The positive rate of MICA*008/A5.1 allele was significantly higher in the control group (56.79%) than that in the kidney transplanted donee group (34.07%) (P <0.05). The infection rate of HCMV in those individuals whose genotype was MICA*008/A5.1 (-) was significantly higher than that in the MICA*008/A5.1(+). CONCLUSION: The individual whose genotype is MICA*008/A5.1 (+) is not liable to HCMV infection, but the individual whose genotype is MICA*008/A5.1 (-) is liable to HCMV infection.