Flavonoids from Citrus paradise cv. Changshan-huyou exerts protective effect on ischemia-induced cerebral injury in mice via inhibiting RhoA-ROCK2 signaling pathway. / èƒ¡æŸšé»„é ®é€šè¿‡æŠ‘åˆ¶RhoA-ROCK2信号通路发挥脑缺血保护作用.

OBJECTIVES: To investigate the protective effect and mechanism of total flavonoids from Citrus paradise cv. Changshan-huyou extracts (TFC) on oxygen-glucose deprivation (OGD) of primary neurons and chronic ischemia-induced cerebral injury in mice. METHODS: Primary hippocampal neurons of 18-day fetal rats were isolated and cultured for 1 week, then treated with 0.25, 0.50 and 1.00 mg/mL TFC. After oxygen-glucose deprivation for 1 h, cells were reperfused for 6 and 24 h, respectively. The cytoskeleton was observed by phalloidin staining. In animal study, 6-week ICR male mice were randomly divided into sham operation group, model group, low-dose (10 mg/kg), medium-dose (25 mg/kg) and high-dose (50 mg/kg) TFC treatment groups, with 20 mice in each group. After 3 weeks, chronic cerebral ischemia was induced by unilateral common carotid artery ligation in all groups except sham operation group. Mice were treated with different concentrations of TFC in the three TFC treatment groups for 4 weeks. Open field test, novel object recognition test and Morris water maze test were used to evaluate anxiety, learning and memory of these mice. Nissl, HE and Golgi stainings were used to detect neuronal degeneration and dendritic spine changes in the cortex and the hippocampus. The expression levels of Rho-associated kinase (ROCK) 2, LIM kinase (LIMK) 1, cofilin and its phosphorylation, as well as the expression of globular actin (G-actin) and filamentous actin (F-actin) protein in hippocampus of mice were detected by Western blotting. RESULTS: Neurons subjected to OGD showed that neurites displayed shortening and breakage; while treatment with TFC reversed OGD-induced neurite injury, especially in the 0.50 mg/mL TFC group. Compared with the sham operation group, the mice in the model group showed a significant decline in anxiety and cognitive ability (P<0.01), whereas treatment with TFC significantly reversed anxiety and cognitive deficits (P<0.05). Improvement in the medium-dose TFC group was the most obvious. Histopathological analysis indicated that the number of Nissl bodies and dendritic spines in hippocampus and cortex were decreased in the model group (all P<0.01). However, after treatment with medium dose of TFC, the number of Nissl bodies and dendritic spines (all P<0.05) was significantly recovered. Compared with the sham operation group, the phosphorylation level of ROCK2 in the brain tissue of the model group was significantly increased (P<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly decreased (P<0.05), and the relative content ratio of G-actin/F-actin was significantly increased (P<0.05). After administration of TFC, the phosphorylation level of ROCK2 in brain tissue of each group was significantly decreased (P<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly up-regulated (P<0.05) and the relative content ratio of G-actin/F-actin was significantly decreased (P<0.05). CONCLUSIONS: TFC protects from ischemia-induced cytoskeletal damage, reduces neuronal dendritic spine injury and protects mice against chronic cerebral ischemia through RhoA-ROCK2 signaling pathway, indicating that TFC might be a potential candidate for treatment of chronic ischemic cerebral injury.

Roseburia intestinalis-derived flagellin is a negative regulator of intestinal inflammation.

Our previous study showed that the Roseburia intestinalis (R. intestinalis), one of the dominant intestinal bacterial microbiota, was significantly decreased in Crohn's disease patients and protected colon epithelial cells from inflammatory damage. However, the roles of lncRNAs in R. intestinalis flagellin-mediated anti-inflammation remain unclear. In this study, we investigate global lncRNA expression profiles using microarray analysis of ulcerative colitis samples from DSS/Flagellin-challenged mice and identified a Flagellin-induced upregulated lncRNA (HIF1A-AS2). Flagellin induced HIF1A-AS2 expression in a dose- and time-dependent manner via p38-stat1 activation. Selective pharmacological inhibitors of Stat1 and p38, and genetic knockdown of these genes abolished Flagellin-induced HIF1A-AS2 expression. In addition, luciferase reporter assay showed that Flagellin activated HIF1A-AS2 promotor via increasing stat1 phosphorylation. Silencing of HIF1A-AS2 abolished Flagellin-mediated anti-inflammatory effects, evaluating by upregulation of cytokines expression, including TNF-α, IL-1ß, IL-6 and IL-12, but not TNFß. In addition, knockdown of HIF1A-AS2 significantly increased p65 and Jnk phosphorylation, and sufficiently abolished Flagellin-mediated anti-inflammatory affects in vivo. Our study provides new insights into the mechanisms that lncRNAs regulate flagellin-mediated alleviation of colonic inflammation. It is indicated that HIF1A-AS2 may be a modulator of intestinal inflammation and represent a novel target for future therapeutics.

Roseburia intestinalis inhibits interleukin­17 excretion and promotes regulatory T cells differentiation in colitis.

Roseburia intestinalis (R. intestinalis) is one of the dominant intestinal bacterial microbiota and is decreased in patients with inflammatory bowel disease (IBD). It helps protect colonic mucosa against the development of inflammation and subsequent IBD, however its underlying mechanisms are unclear. The aim of the present study was to evaluate the anti­inflammatory properties of R. intestinalis in vitro and in an animal model of IBD. The effects of R. intestinalis on disease activity index (DAI) scores, intestinal pathology, the expression of interleukin (IL)­17 and the frequency of CD4+CD25+Foxp3+ regulatory T cells (Treg) were evaluated in vivo in a model of 2,4,6­trinitrobenzenesulfonic acid solution (TNBS)­induced colitis. Compared with the control group, TNBS­treated mice had significantly higher secretion of IL­17, higher DAI scores, a lower ratio of Treg, reduced colon lengths and higher histological scores for colon inflammation. The administration of R. intestinalis significantly downregulated the expression of IL­17, increased the ratio of Treg and ameliorated the high DAI scores and the pathological signs of inflammation in the colon compared with mice treated with TNBS alone. Gene expression profiling was also used to detect the expression of IL­17 in human IBD and healthy control specimens. To extend these findings to an in vitro model of inflammation the human colon epithelial cell line NCM460 was stimulated with lipopolysaccharide (LPS) to induce inflammation and co­cultured with R. intestinalis and changes in IL­17 expression were evaluated. R. intestinalis inhibited the LPS­induced secretion of IL­17 by NCM460 cells. In conclusion, these results demonstrate that R. intestinalis inhibits IL­17 secretion and promotes Treg differentiation in colitis, suggesting that R. intestinalis could be of potential use in the treatment of IBD.