Objective: To investigate the role of calcium/calmodulin-dependent protein kinase Ⅱ delta (CaMKⅡδ) in platelet activation mediated by thrombin and thrombin receptor activator peptide (TRAP). Methods: The CaMKⅡδ-specific inhibitor Hesperadin was used to assess its inhibitory effects on platelet activation using multiple detection methods. These included: analyzing the inhibitory effect of different concentrations of Hesperadin on thrombin- and TRAP-induced platelet aggregation using a platelet aggregometer; measuring CD62P expression levels and calcium mobilization via flow cytometry; and determining dense granule release activity using a microplate reader. Results: Hesperadin at concentrations of 40 μM and 80 μM significantly inhibited thrombin- and TRAP-induced platelet aggregation (P<0.05). Within the concentration range of 20–80 μM, it significantly inhibited α-granule release and TRAP-induced calcium ion mobilization (P<0.05); at 80 μM, it markedly suppressed thrombin-induced platelet calcium ion mobilization and significantly inhibited dense granule release (P<0.05). Conclusion: The experimental results indicate that CaMKⅡδ in platelets is activated and plays a regulatory role in the process of platelet activation induced by thrombin and TRAP.

The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.

Objective To evaluate the relationship between annexin 1 (ANXA1) and the endogenous protective mechanism during intestinal epithelial cell injury induced by endotoxiu.Methods The intestinal epithelial cells at the logarithmic growth phase were seeded in culture palates and randomly divided into 4 groups (n =36 each) using a random number table:control group (group C),cell injury group (group I),ANXA1 overexpression group (group OE),and ANXA1 silencing group (group S).Lentivirus with ANXA1 overexpression and silencing was transfected into intestinal epithelial cells to construct a stable cell line.In I,OE and S groups,endotoxin was added with the final concentration of 100 μg/ml,and the cells were then incubated for 24 h to establish the cell injury model.The culture medium was changed,and the cells were then incubated for 24 h in group C.The cell apoptosis was detected by flow cytometry,the cell permeability was determined by Transwell assay,and the cell viability was evaluated by methyl thiazolyl tetrazolium assay.The apoptosis rate was calculated.Results Compared with group C,the apoptosis rate was significantly increased,and the cell permeability and viability were significantly decreased in I,OE and S groups (P＜0.05).Compared with group Ⅰ,the apoptosis rate was significantly decreased,the cell permeability and viability were significantly increased in group OE,and the apoptosis rate was significantly increased,and the cell permeability and viability were significantly decreased in group S (P＜0.05).Conclusion ANXA1 is involved in the endogenous protective mechanism during intestinal epithelial cell injury induced by endotoxin.

Objective To explore the expression of heat shock protein (HSP) 90α in outside of different metastatic hepatocellular carcinoma (HCC) cell lines and its role in the cells migration and invasion.Methods The expression of HSP90α was detected by Western blot analysis in conditioned media of MHCC97L and MHCC97H with low and high metastatic HCC cell lines.A small molecule cell-impermeant HSP90 inhibitor DMAG-N-oxide was used to inhibit extracellular HSP90α.Changes of the cells migratory and invasive capability were assessed by in vitro motility and invasion assay.The endogenous matrix metalloproteinase 2 (MMP-2) was demonstrated by Zymography.The expression of extracellular co-chaperone HSP70 and MMP-2 were tested by Western blots and the association between HSP90α,HSP70 and MMP-2 was analyzed by immunoprecipitation.The effects of HSP70 knockdown by siRNA,with or without MMP-2 inhibitor Batimastat,on the level of active MMP-2 and cell migration and invasion were also evaluated.Results HSP90α can express both inside and outside of different metastatic HCC cell lines,and the level of expression was consistent with metastasis potentials.After MHCC97-H cells were treated with a special HSP90α inhibitor DMAG-N-oxide for 24 h,the average migratory cell numbers (28.11 ±3.56) had a significantly reduction,compared with those without treatment group (80.12±4.16) and empty control group (82.24±4.12),respectively (P ＜ 0.01).In vitro invasion assay showed the average invaded cell numbers in treatment group (36.54±4.12) were more fewer than without treatment group (95.12±3.48) and empty control group (101.1 1±3.36),respectively (P =0.017),and accompanying with decreasing of the extracellular MMP-2 activity.HSP70 and MMP-2 could express outside of MHCC97-H cells and interact with HSP90α.Small molecular interfere RNA (siRNA) dramatically inhibited HSP70 expression and reduced the interaction HSP90α with MMP-2 and MMP-2 activity outside MHCC97-H cells,and also suppressed MHCC97-H cells migration and invasion.In addition,combining MMP-2 inhibitor had additive inhibition effects.Conclusion Extracellular HSP90α and HSP70 form chaperone complex to assist in MMP-2 activation and increases HCC cells migration and invasion,which maybe a novel therapeutic target against metastatic HCC.

Objective To evaluate the relationship between extracellular signal-regulated kinase (ERK)and ketamine-induced apoptosis in rat hippocampal neurons.Methods Sprague-Dawley rats at 18 days of gestation were anesthetized.The fetal rats were obtained under the sterile condition and decapitated.The hippocampal neurons were isolated and primarily cultured for 5 days,and were seeded in 6-well plates (2 ml/well) or in 96-well plates (100μl/well) at a density of 5 × 105/ml.The cells were randomly divided into 4 groups (n =18 each):control group (group C),fibroblast growth factor (FGF-2,an ERK agonist) group (group F),ketamine group (group K) and FGF-2 + ketamine group (group FK).The cells were cultured in the plain culture medium in group C.FGF-2 50 ng/ml was added to the culture medium in group F.Ketamine was added to the culture medium in group K.FGF-2 50 ng/ml was added to the culture medium at 20 min before ketamine 100 μmol/L was added in group FK.The phosphorylation of ERK in hippocampal neurons was detected by Western blot at 10 min after treatment.At 24 h after treatment,the neuronal apoptosis was detected by Hoechst33342/PI staining,and the cell survival rate was detected by MTT assay.The apoptosis rate was calculated.Results Compared with group C,the phosphorylation of ERK in hippocampal neurons and the cell survival rate was significantly decreased and the apoptosis rate was increased in K and FK groups (P ＜ 0.05).There was no significant difference in the parameters mentioned above between F and C groups (P ＞ 0.05).The phosphorylation of ERK in hippocampal neurons and the cell survival rat was significantly higher and the apoptosis rate was lower in group FK than in group K (P ＜0.05).Conclusion Ketamine induces apoptosis in rat hippocampal neurons by inhibiting activation of ERK in hippocampal neurons.