PXD101 induces apoptosis of human prostate cancer cell line PC3 by mitochondrial signal pathway / 中国病理生理杂志

AIM:To explore the mitochondrial pathway in the apoptosis of PC3 cells induced by PXD101 (also named as belinostat).METHODS:PC3 cells were treated with PXD101 at different doses or stimulated with PXD101 for different time.The effect of PXD101 on the viability of PC3 cells was measured by CCK-8 assay.The apoptotic rates and the mitochondrial membrane potential (MMP) were analyzed by flow cytometry.The protein levels of Bcl-2, Bax and cytochrome C (Cyt C) were determined by Western blot.The caspase-3 activity were tested by caspase-3 activity assay kit.RESULTS:The viability of the PC3 cells was inhibited by PXD101 in a dose-and time-dependent manner.Flow cytometric analysis showed that the apoptotic rates were increased in the cells treated with PXD101 (P<0.01).At the same time, PXD101 induced the decreases in MMP and Bcl-2, the release of Cyt C, and the increase in caspase-3 activity.CONCLUSION:PXD101 induces the apoptosis of human prostate cancer cell line PC3 by mitochondrial signal pathway.

STC2 promotes proliferation and EMT-related migration of HepG2 cells / 中国病理生理杂志

AIM:To explore the effects of stanniocalcin 2 (STC2) on the proliferation, migration and the process of epithelial-mesenchymal transition (EMT) in human hepatocellular carcinoma HepG2 cells.METHODS:The expression levels of STC2 in the hepatocellular carcinoma cell lines and normal liver cells were assessed by Western blot.Colony formation assay was used to test the effect of STC2 on the proliferation of HepG2 cells.The effects of STC2 on the expression of proliferation-related molecules at mRNA and protein levels were determined by RT-qPCR and Western blot.The effect of STC2 on the migration ability was measured by Transwell assay.The mRNA and protein levels of vimentin and E-cadherin in STC2-overexpressing and-silencing cell lines were detected by RT-qPCR and Western blot.RESULTS:Compared with the normal liver cell line, the protein expression of STC2 was up-regulated in the hepatocellular carcinoma cell lines.The results of colony formation assay indicated that STC2 promoted the proliferation of HepG2 cells.STC2 significantly regulated the proliferation-related gene expression, such as cyclin D1.The results of Transwell assay showed that STC2 enhanced the migration ability of the HepG2 cells and influenced the EMT process.CONCLUSION:STC2 promotes the proliferation of HepG2 cells and affects the expression of proliferation-related genes.STC2 influences the process of EMT and promotes the migration of HepG2 cells.

Rolipram represses MRP8/14-induced pro-inflammatory cytokine releases in RAW264.7 cells through NF-κB activation / 中国病理生理杂志

AIM:To investigate the inhibitory effect of PDE4 inhibitor rolipram on the releases of inflammatory cytokines in mouse macrophages (RAW264.7 cells) induced by myeloid-related protein 8/14 (MRP8/14), and to explore the role of nuclear factor-κB (NF-κB) in this process.METHODS:RAW264.7 cells were treated with MRP8/14.The inflammatory cytokines TNF-α and IL-6 were determined by LiquiChip and qPCR.In contrast, RAW264.7 cells were pretreated with rolipram prior to MRP8/14 exposure.After MRP8/14 challenge, the inflammatory cytokines TNF-α and IL-6 were determined by LiquiChip and qPCR.Moreover, the phosphorylation level of NF-κB P65 was determined by Western blot.The nuclear translocation of NF-κB P65 in RAW264.7 cells was detected by indirect immunofluorescence.RESULTS:The results of LiquiChip and qPCR showed that MRP8/14 enhanced the expression of TNF-α and IL-6 (P<0.01),while pretreatment with rolipram markedly down-regulated the level of inflammatory cytokines induced by MRP8/14 (P<0.05).Meanwhile, MRP8/14 significantly activated the phosphorylation of NF-κB P65, and the protein expression of p65 in the nucleus was increased,while pretreatment with rolipram suppressed the phosphorylation of NF-κB P65 induced by MRP8/14.CONCLUSION:Rolipram may significantly reduce the releases of the inflammatory cytokines induced by MRP8/14 through the NF-κB signaling.

Enteric dysbiosis-linked gut barrier disruption triggers early renal injury induced by chronic high salt feeding in mice

Chronic high-salt diet-associated renal injury is a key risk factor for the development of hypertension. However, the mechanism by which salt triggers kidney damage is poorly understood. Our study investigated how high salt (HS) intake triggers early renal injury by considering the ‘gut-kidney axis’. We fed mice 2% NaCl in drinking water continuously for 8 weeks to induce early renal injury. We found that the ‘quantitative’ and ‘qualitative’ levels of the intestinal microflora were significantly altered after chronic HS feeding, which indicated the occurrence of enteric dysbiosis. In addition, intestinal immunological gene expression was impaired in mice with HS intake. Gut permeability elevation and enteric bacterial translocation into the kidney were detected after chronic HS feeding. Gut bacteria depletion by non-absorbable antibiotic administration restored HS loading-induced gut leakiness, renal injury and systolic blood pressure elevation. The fecal microbiota from mice fed chronic HS could independently cause gut leakiness and renal injury. Our current work provides a novel insight into the mechanism of HS-induced renal injury by investigating the role of the intestine with enteric bacteria and gut permeability and clearly illustrates that chronic HS loading elicited renal injury and dysfunction that was dependent on the intestine.

NF-κB regulates apoptosis of human alveolar epithelial cells induced by MRP8/14 / 中国病理生理杂志

AIM:To investigate the effects of myeloid-related protein 8/14 ( MRP8/14 ) on the survival and apoptosis of human alveolar epithelial cell line A549, and to explore the role of nuclear factor-κB (NF-κB) in this process. METHODS:A549 cells were treated with different doses of MRP8/14 or stimulated with MRP8/14 for different time.The effect of MRP8/14 on the viability of A549 cells was determined by CCK-8 assay.The apoptotic rates were tested by flow cytometry.The nuclear translocation of NF-κB p65 was detected by Western blot and indirect immunofluorescence.Be-sides, the phosphorylation level of NF-κB p65 was determined by Western blot.NF-κB-specific inhibitor Bay 11-7082 was used to further analyze the role of NF-κB in the apoptosis induced by MRP8/14.RESULTS:The viability of the A549 cells was affected by MRP8/14 in a dose-and time-dependent manner.Flow cytometric analysis showed that the apoptotic rates were increased in the cells treated with MRP8/14 (P<0.01).In A549 cells administered with MRP8/14, NF-κB p65 was significantly phosphorylated and translocated into the nuclei, suggesting the activation of NF-κB signaling pathway. However, NF-κB-specific inhibitor Bay 11-7082 significantly attenuated the cell apoptosis induced by MRP8/14 ( P <0.01).CONCLUSION:NF-κB plays an important role in regulating the apoptosis of human alveolar epithelial cells in-duced by MRP8/14.

Effect of GDF11 on expansion of CD8 +memory stem T cells / 中国病理生理杂志

AIM:To investigate the effect of growth differentiation factor 11 ( GDF11 ) on the expansion of CD8 +memory stem T cells ( Tscm) and to further improve the effect of adoptive immunotherapy.METHODS:Healthy human peripheral blood mononuclear cells ( PBMCs) were isolated by density gradient centrifugation at first.Among the i-solated PBMCs, CD8 +T cells were further purified with MACS microbeads.The CD8 +T cells were then randomly divided into experimental groups and control group.The same volume of different concentrations of GDF11 were added into the ex-perimental groups, and the same volume of PBS solution was added into the control group.Finally, the expansion of Tscm in experimental groups and control group was measured by flow cytometry at several time points.RESULTS:GDF11 sig-nificantly increased the number of Tscm in CD8 +T cells in vitro expansion and also dramatically increased the ratio of Tscm in CD8 +T cells.Furthermore, 400 μg/L GDF11 treatment for 3 weeks was the optimal condition to induce CD8 +Tscm. CONCLUSION:GDF11 effectively increases the number and ratio of Tscm in the CD8 +T cells in cell culture growth, thereby creating a new strategy to further improve the efficiency of adoptive immunotherapy.

Post-transcriptional regulation of dual-specificity phosphatase-1 by RNA-binding protein HuR T118 in heat shock / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the post-transcriptional regulation of dual-specificity phosphatase-1 (DUSP1) by the RNA- binding protein HuR in heat shock.</p><p><b>METHODS</b>The recombinant plasmids carrying wild-type (WT) HuR or its mutants at threonine 118 were constructed and transiently transfected into NIH 3T3 cells via liposome, and the changes in the expressions of DUSP1 mRNA and protein were detected by quantitative real-time PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Heat shock caused significantly enhanced phosphorylation of HuR at the residue T118. In 3T3 cells transfected with the plasmids carrying wild-type HuR for its over-expression showed significantly up-regulated DUSP1 mRNA and protein expressions at 24 h after transfection. Over-expression of HuR(T118A) down-regulated DUSP1 mRNA and protein expressions in cells challenged with heat shock, while HuR(T118E) over-expression significantly increased DISP1 expression at both mRNA and protein levels. After heat shock, HuR(WT) translocated from the cell nucleus to the cytoplasm to form particles. HuR(T118E) was diffusely distributed in the cytoplasm before heat shock and formed particles after heat shock. HuR(T118A) did not undergo such translocation in response to heat shock challenge.</p><p><b>CONCLUSION</b>HuR regulates DUSP1 mRNA and protein expression at the post-transcriptional level to increase its expression after heat shock by enhancing the phosphorylation HuR T118.</p>

Construction of eukaryotic expression vectors for different domains of the extracellular region of RAGE and their expression in prostate cancer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct eukaryotic expression vectors for different domains (V and VC1) of the extracellular region of the receptor of advanced glycation end products (RAGE) and investigate the roles of these domains in prostate cancer.</p><p><b>METHODS</b>The coding sequence of V and VC1 domains was amplified from the plasmid pcDNA3-HA-RAGE by PCR and cloned into the pcDNA3-HA vector following routine procedures. After identification by PCR and sequencing, the vectors including V and VC1 domains were transfected into PC-3 cells. Western blotting and immunofluorescence were used to detect the expression and distribution of the expressed products in transfected PC-3 cells.</p><p><b>RESULTS</b>The expression vectors containing V and VC1 domains of RAGE were successfully constructed as confirmed by PCR and DNA sequence analysis. The V and VC1 domains of RAGE were highly expressed and showed a cytoplasmic distribution in transfected PC-3 cells.</p><p><b>CONCLUSION</b>The constructed eukaryotic expression vectors for V and VC1 domains of RAGE can be efficiently expressed in prostate cancer cells.</p>

Effects of ecoimmunonutrition on intestinal barrier and pancreas in rats with severe acute pancreatitis / 中华临床营养杂志

@#Objective To explore the effects of ecoimmunonutrition support on the intestinal barrier function and pancreas in rats with severe acute pancreatitis (SAP). Methods Totally 64 SPF rats were randomly divided into sham operation group (control group) , SAP without enteral nutrition support group (SAP group), SAP with early enteral nutrition support group (EEN group), and SAP with early ecoimmunonutrition support group (EIN group). Bacteria translocation (BT), plasma endotoxin (ET) , gut permeability, pancreas pathology score,and distant ileum pathology were determined on the 4th and 7th post-modeling day. Results The BT rate was significantly higher in SAP group, EEN group, and EIN group than in control group (P ＜ 0.05), was significantly lower in EEN group and EIN group than in SAP group (P ＜ 0.05), and was significantly lower in EIN group than in EEN group (P ＜ 0.05). ET and FD-40 levels in blood were both significantly higher in SAP group, EEN group, and EIN group than in control group (P ＜0.01)and were significantly lower in EIN group and EEN group than in SAP group (P ＜0.01); ET was significantly lower in EIN group than in control group (P ＜0.05). Pathological scores were significantly higher in SAP group, EEN group, and EIN group than in control group (P ＜0.01)and were significantly lower in EEN group and EIN group than in SAP group (P ＜ 0.01). The individual pathological scores of EIN group were not significantly different from EEN group (P ＞ 0.05), while the total score was significantly lower in EIN group than in EEN group (P ＞ 0.05). Distant iliac mucosa was significantly thicker in EIN group than in other groups. Conclusions Early enteral nutrition support protects the intestinal barrier and pancreas in rats with SAP. Ecoimmunonutrition has better nutritional effectiveness than the normal enteral nutrition.