Berberine affects activity of HSV-1 virus infected HEp-2 cells and its molecular mechanism / 中国药理学通报

Aim To discuss the effect of berberine ( BE) on the activity of HSV-1 virus infected HEp-2 cells and its related molecular mechanisms.Methods Hie infected cell model was constructed and divided into control group, infection group, low concentration group ( 5 (xmol • L 1 -BE) , medium concentration group ( 10 (xmol • L '-BE) and high concentration group ( 15 (xmol • L '-BE) ) , and then incubated for 24 hours.qRT-PCR was used to determine HSV-1 infection-related genes ( gD, ICP-4, ICP-8, ICP-27 ) and mRNA expression levels of LncBNA NRAV, miR- 299-3p, RAB5C.CCK-8 method and flow cytometry were applied to detect cell viability and apoptotic rate.The expression levels of PI3K/AKT signaling pathway and JNK/p38 MAPK signaling pathway related protein were analysed by WB.Results It was found that BE j j reduced the mRNA expression of gD, ICP-4, ICP-8, anrl ICP-27, improved cell viability, and inhibited eell apoptosis.BE promoted the expression of miR-299-3p by inhibiting LncRNA NRAV and RAB5C.BE inhibited the protein expression levels of PBK/AKT signaling pathway and JNK/p38 MAPK signaling pathway proteins PI3K, AKT, JNK, and P38.Conclusions The mechanism that BE enhances the activity of HEp-2 cells after HSV-1 infection and suppresses its apoptosis may be related to LncRNA NRAV and RAB5C targeting competitive binding to miH-299-3p, inhibiting the activation of PBK/AKT signaling pathway and JNK/ p38 MAPK signaling pathway.

Expression and Significance of Rab5A in Colorectal Cancer / 胃肠病学

Background:Colorectal cancer is one of the commonly seen malignant tumors in digestive system. Most patients with colorectal cancer were diagnosed in advanced stage and the prognosis is poor. Early diagnosis is crucial to improve the overall survival of patient. Currently,molecular markers for diagnosis of colorectal cancer has attracted more and more attention of the investigators. Aims:To investigate the expression and significance of Rab5A in colorectal cancer. Methods:Colorectal cancerous tissue and the paired para-cancer noncancerous tissue of 135 patients who underwent resection for colorectal cancer were collected. Expression of Rab5A mRNA in cancerous tissue,para-cancer noncancerous tissue,colon cancer cell lines HCT116,HT-29,LoVo and SW480 and normal colonic epithelial cell line NCM460 was determined by real-time PCR. Protein expression of Rab5A in cancerous tissue and para-cancer noncancerous tissue of 4 patients was detected by Western blotting. Protein expression of Rab5A in cancerous tissue of 110 patients was detected by immunohistochemistry. Correlations between expression of Rab5A and clinicopathological characteristics and prognosis of colorectal cancer were analyzed. Results:Expression of Rab5A mRNA in cancerous tissue was significantly higher than that in para-cancer noncancerous tissue(P=0. 003). Expression of Rab5A mRNA in colon cancer cell lines HCT116, HT-29,LoVo and SW480 was significantly higher than that in normal colonic epithelial cell line NCM460(P﹤0. 05). Protein expression of Rab5A in cancerous tissue was significantly higher than that in para-cancer noncancerous tissue. In 110 cancerous tissues,51(46. 4%)had low expression of Rab5A and 59(53. 6%)had high expression of Rab5A. Protein expression of Rab5A was significantly correlated with tumor size,serum carcinoembryonic antigen(CEA)level and TNM stage(P =0. 008;P =0. 002;P =0. 010). 3-year survival rate of patients with high Rab5A expression was significantly lower than that of patients with low Rab5A expression(52. 1% vs. 77. 5%,P ﹤0. 05). Rab5A protein expression,serum CEA level and TNM stage were correlated with the prognosis of patients with colorectal cancer( P =0.009;P =0. 006;P =0. 017)and were independent factors for prognosis(P =0. 026;P =0. 032;P=0. 014). Conclusions:Expression of Rab5A correlates with the malignant behavior of colorectal cancer and its high expression indicates a poor prognosis. Rab5A might be used as a predictor for prognosis of colorectal cancer.

Rab5a promotes expression of pro-inflammatory cytokines and type Ⅰ IFN in CpG induced macrophages / 中国免疫学杂志

Objective:Using the macrophage cell lines RAW264.7 stably expressing Rab5a and its dominant negative mutant Rab5aN133I as models to analyze the effect and the mechanism of Rab 5a,Rab5aN133I on CpG-induced production of pro-inflammatory cytokines and type Ⅰ IFN.Methods: The eukaryotic expression vectors of Rab5a and Rab5aN133I were transfected into RAW264.7 cells by liposome,and screened with G418.The G418-resistant colonies were obtained and amplified.The transformed cell lines were i-dentified by RT-PCR,Real time-PCR and Western blot.The production of cytokines were measured after transformed cell lines of Rab5a and Rab5aN133I was stimulation with CpG for 8 h.Results: Rab5a expression in transfected cells was significantly higher than the control cell group (P<0.05).Overexpression of Rab5a significantly promoted the production of TNF -α,IL1-β(P<0.01) and IFN-β( P<0.05) in CpG stimulated RAW264.7.The production of cytokines was restored in Rab 5aN133I transfected cell line.Conclusion:Rab 5a promotes CpG-induced pro-inflammatory cytokines and typeⅠIFN in macrophages,it may be act as a positive regulator in TLR9 signaling pathway.

Rab5a promotes LPS-induced cytokine expression in macrophages / 中国免疫学杂志

Objective:To establish cell lines stably expressing Rab5a and its the inactive mutant Rab5aN133I,analyze the effect of Rab5a on the expression of cytokines in LPS-stimulated RAW264.7 cells .Methods:RAW264.7 cells were transfected with Rab5a and its inactive mutant vector Rab5a N133I separately,and then screened by G418.Rab5a stable expressing cell lines were identified by Real time-PCR.The growth of the stable cell lines was analyzed by MTT assay.After the stable cell lines were stimulated by LPS for different time periods,the expression of iNOS,TNF-αand IL-6 was detected.Results:Rab5a and Rab5aN133I transfection resulted in elevated Rab5a mRNA expression compared with the control cells ( P<0.05 ).Rab5a overexpression enhanced the proliferation of RAW264.7 cells.However,the proliferation of Rab5aN133I cells was significantly slower than the control cells ( P<0.05).Overexpression of Rab5a promoted LPS-induced production of iNOS,TNF-αand IL-6 in RAW264.7 cells (P<0.01). Conversely,overexpression of Rab5aN133I abolished the stimulating effects of Rab5a.Conclusion: Rab5a promoted LPS-induced expression of iNOS,TNF-αand IL-6 in RAW264.7 macrophages in a GTP-binding ability-dependent manner.

Coordinate Regulation of Neurite Outgrowth by LRRK2 and Its Interactor, Rab5

Neurite outgrowth and its maintenance are essential aspects of neuronal cells for their connectivity and communication with other neurons. Recent studies showed that over-expression of either leucine-rich repeat kinase 2 (LRRK2), whose mutations are associated with familial Parkinson's disease (PD), or Rab5b, an early endosomal marker protein, induces reduction in neurite length. Based on our recent findings that LRRK2 co-localizes and interacts with Rab5, we tested the hypothesis that LRRK2 and Rab5 may functionally interplay while controlling neurite outgrowth. Firstly, we confirmed previous reports that over-expression of either the LRRK2 PD-specific G2019S mutant or the Rab5 constitutively active Q79L mutant, but not of dominant negative N133I mutant, significantly reduces neurite outgrowth. Secondly, when over-expression of either LRRK2 wild type (WT) or G2019S was accompanied with over-expression of one of the Rab5 variants (WT, Q79L and N133I), or with down-regulation of Rab5, the reduction extent of its neurite length was similar to that of cells over-expressing LRRK2 alone, regardless of Rab5's status. Finally, we observed similar patterns of neurite length regulation in embryonic rat hippocampal neuron cultures. Taken together, our results suggest that LRRK2 and Rab5 functionally coordinate their regulation of neurite outgrowth and that LRRK2 is a more critical factor than Rab5.

Regulation of Notch1 activity by Rab5 in pancreatic cancer BxPC3 cells / 基础医学与临床

Objective To elucidates the regulation of Notch1 activity by Rab5 which controls endosome fusion.Methods Rab5 expression of BxPC3 cells were inhibited by siRNA interference,the changes of Notch ICD protein were measured by Western blot.We use Wortmannin and LY294002 which inhibit the acticity of Phosphatidylinositol(PI) 3-kinases to investigate Notch ICD protein level.Results After inhibiting Rab5 expression or the activity of Phosphatidylinositol(PI) 3-kinases,Notch ICD protein level and the growth of BxPC3 cells decreased.Conclusion In BxPC3 cells,Rab5 and Phosphatidylinositol(PI) 3-kinases are important to regulate Notch1 activity and the activation of Notch is dependent on endocytosis.

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.