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Piceatannol inhibits prostate cancer cell proliferation, migration and invasion

Zhangchun LI ; Po LI ; Chaonan DENG ; Heng LUO

Chinese Journal of Pathophysiology.2017;33(6):1130-1133. doi:10.3969/j.issn.1000-4718.2017.06.028

AIM:To investigate the effect of piceatannol on the viability, and the abilities of migration and invasion in the prostate cancer cells.METHODS:DU145 cells were treated with piceatannol at different doses (0, 5, 10, 20, 40 and 80 μmol/L) for different time (12, 24, 36 and 48 h) as indicated.The cell viability was assessed by CCK-8 assay.The migration and invasion abilities of the cells were analyzed by wound healing assay and Transwell assay, respectively.The protein levels of p-JAK2 and p-STAT3 were detected by Western blot.RESULTS:Piceatannol dose-dependently decreased the cell viability.After treatment with piceatannol, the abilities of migration and invasion of the cells were significantly inhibited.Moreover, treatment with piceatannol resulted in marked decreases in the protein levels of p-JAK2 and p-STAT3.CONCLUSION:Piceatannol inhibits the viability, migration and invasion of the prostate cancer cells via regulating the JAK2/STAT3 signaling pathway.

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Effects of cannabinoid HU210 on experimental acute pancreatitis and possible relationship with Toll-like receptor 4 signaling pathway

Ruiqin ZHANG ; Sisi LIN ; Min LI ; Li SHEN ; Kun LI ; Yongyu LI

Chinese Journal of Pathophysiology.2017;33(6):1112-1118. doi:10.3969/j.issn.1000-4718.2017.06.025

AIM:Using Toll-like receptor 4 gene knockout (tlr4-/-) mice and the wild-type (WT) mice with the same C57BL/10J genetic background, the effects of HU210, a cannabis preparation, on caerulein (CAE)-induced acute pancreatitis (AP) and the potential mechanisms were investigated.METHODS:WT or tlr4-/-mice were randomly divided into AP group, AP+HU210 group and control group.AP was induced by intraperitoneal injection of CAE (50 μg·kg-1·h-1) for a total of 6 times and lipopolysaccharide (LPS) at 10 mg/kg 6 h after the first injection of CAE.HU210 (50 μg/kg) was given 30 min before and 4 h after the first injection of CAE in AP+HU210 group.The animals in control group were given normal saline instead of CAE and LPS in the same way.The mice were sacrificed 3 h after the last injection.The blood, the pancreas, the lungs and the intestinal Peyer's patches were harvested.RESULTS:Compared with control group, the pancreatic pathological score and P38 protein expression, plasma amylase activity and inflammatory mediator levels, and lung MPO activity were significant increased (P<0.05) in both WT and tlr4-/-mice with AP.Compared with the WT mice with AP, the tlr4-/-mice with AP showed significantly low levels of IL-6, TNF-α and MCP-1 in the plasma, low expression levels of pancreatic P38 and p-P38 protein (P<0.05), and mild alterations of CD3+ T-lymphocytes, CD4+ T-lymphocytes and the ratio of CD4+/CD8+ (P<0.05).The administration of HU210 attenuated the pancreatic pathological changes and the lung MPO activity in both stains of mice with AP (P<0.05).However, the inhi-bitory effects of HU210 on the increased amylase activity in the plasma and the increased protein levels of pancreatic P38 and p-P38 were remarkable (P<0.05) in WT mice instead of in tlr4-/-mice.CONCLUSION:TLR4 is mainly involved in AP-related systemic inflammatory response and its mechanism may be dependent on TLR4-P38 MAPK signaling pathway.The intervention of HU210 in AP plays a protective role mainly by inhibiting the infiltration of inflammatory cells, and the relationship with TLR4 signaling pathway is not obvious.

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Rolipram represses MRP8/14-induced pro-inflammatory cytokine releases in RAW264.7 cells through NF-κB activation

Chen YANG ; Jun WANG ; Lin HUANG ; Haihua LUO ; Yong JIANG ; Aihua LIU

Chinese Journal of Pathophysiology.2017;33(6):1098-1103. doi:10.3969/j.issn.1000-4718.2017.06.023

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.

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Change of ACE2 level in serum during development of coronary heart disease

Juan CHEN ; Yubi LIN ; Gengsheng YIN ; Zicheng LI ; Wanqun CHEN ; Juan HU ; Linlin TAN ; Shaoling XU ; Dongling ZHENG ; Yongquan PAN

Chinese Journal of Pathophysiology.2017;33(6):1086-1090. doi:10.3969/j.issn.1000-4718.2017.06.021

AIM:To analyze the correlation between serum angiotensin-converting enzyme 2 (ACE2) levels and different stages of coronary heart disease (CHD), and to explore the change of serum ACE2 level during the development of CHD.METHODS:The control group included 85 non-CHD samples, and 174 CHD samples were divided into light stenosis (ls-CHD, stenosis degree <50%) group, moderate stenosis (ms-CHD, stenosis degree 50%~75%) group and severe stenosis (ss-CHD, stenosis degree ≥75%) group.The ACE2 level in each serum sample was detected by ELISA.The relationship between the ACE2 level and the development of coronary heart disease was explored by statistical analysis of serum ACE2 levels in different stages of CHD.RESULTS:The serum ACE2 levels in ls-CHD group, ms-CHD group and ss-CHD group were all higher than that in control group.The more severe the coronary artery stenosis existed, the higher the ACE2 level was observed.The serum ACE2 level in the males was higher than that in the females.In a single sex, the serum ACE2 levels in ls-CHD group, ms-CHD group and ss-CHD group were higher than that in control group with significant differences.Regression analysis found that sex, diabetes and CHD were associated with the serum ACE2 levels.Among them, sex and CHD were the independent factors to affect serum ACE2 levels.CONCLUSION:The serum ACE2 level of males was higher than that of females.Compared with the non-CHD samples, the serum ACE2 level of CHD patients was higher than that of the non-CHD samples.During the development of coronary heart disease, the serum ACE2 level increased constantly.

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Influences of semaphorin 3A over-expression on H2O2-induced injury in human umbilical vein endothelial cells

Haifang WANG ; Xiangrong ZHAO ; Xueping HUO ; Jingying SUN ; Xianglong WU ; Yinbo NIU ; Jun HU ; Qinshe LIU

Chinese Journal of Pathophysiology.2017;33(6):1080-1085. doi:10.3969/j.issn.1000-4718.2017.06.020

AIM:To explore the influences of semaphorin 3A (Sema 3A) on hydrogen peroxide (H2O2)-induced injury in human umbilical vein endothelial cells (HUVECs).METHODS:Sema 3A over-expression vectors were constructed and transfected into the HUVECs by Lipofectamine 2000, and the over-expression effect was verified by qPCR and Western blot.The HUVECs in different groups were treated with or without 200 μmol/L H2O2 for 4 h.The levels of inflammatory cytokines were measured by qPCR.The levels of lactic dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by corresponding colorimetry.The cell viability was measured by MTT assay.The cell apoptosis was analyzed by flow cytometry.The levels of apoptosis-related proteins cleaved caspase-3 and Bcl-2 were determined by Western blot.RESULTS:H2O2 induced inflammatory cytokine secretion, increased the levels of LDH and MDA, decreased SOD activity and cell viability, and increased cell apoptosis in the HUVECs.Over-expression of Sema 3A enhanced the above processes.No injury effect of Sema 3A over-expression on HUVECs without H2O2 treatment was observed, indicating that the injury effects of Sema 3A on HUVECs depended on H2O2.CONCLUSION:Sema 3A markedly enhances H2O2-induced injury in the HUVECs, which depends on H2O2.Sema 3A may promote oxidative stress-caused endothelial cell injury.

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Resveratrol attenuates apoptosis of endothelial progenitor cells induced by tert-butyl hydroperoxide

Hao ZHENG ; Zhanlu LI ; Xiaohua SHEN ; Guosheng FU

Chinese Journal of Pathophysiology.2017;33(6):1073-1079. doi:10.3969/j.issn.1000-4718.2017.06.019

AIM:To investigate the effect of resveratrol on the apoptosis of endothelial progenitor cells (EPC) induced by tert-butyl hydroperoxide (tBHP) and the underlying mechanisms.METHODS:Total mononuclear cells were isolated from peripheral blood by density gradient centrifugation, and the cells were cultured on fibronectin-coated culture dishes.After 4 d of culture, attached cells were divided into control group, tBHP group, and resveratrol plus tBHP group (pretreated with resveratrol for 24 h and then cultured with 100 μmol/L tBHP for 6 h).The proliferation and migration abilities of the EPC were assessed by MTT assay, BrdU incorporation assay and modified Boyden chamber assay.The proportion of apoptotic EPC was determined by flow cytometry after staining with fluorescein isothiocyanate-conjugated Annexin V and propodium iodide.The level of reactive oxygen species (ROS) was determined by H2DCF-DA method.Caspase-3 activity was assay using a caspase-3 colorimetric assay kit.The protein levels of cleaved caspase-3, Bcl-2 and Bax were determined by Western blot.RESULTS:Resveratrol decreased EPC apoptosis induced by tBHP in a dose-dependent manner.Moreover, resveratrol increased the proliferation and migration abilities of the EPC.Resveratrol decreased intracellular ROS level, caspase-3 activity and the protein level of cleaved caspase-3.Resveratrol also decreased protein expression of Bax and increased protein expression of Bcl-2.CONCLUSION:Resveratrol attenuates EPC apoptosis induced by oxidative stress, and its mechanisms may be related to protecting the mitochondrial function.

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CARMA3 gene knockdown in HCT116 cells inhibits cell growth, migration and invasion

Fang LIU ; Wansong LIN ; Shuping CHEN ; Yunbin YE

Chinese Journal of Pathophysiology.2017;33(6):1021-1030. doi:10.3969/j.issn.1000-4718.2017.06.011

AIM:To study the effcts of caspase recruitment domain membrane-associated guanylate kinase protein 3 (CARMA3) knockdown on the growth, migration and invasion of human colonic carcinoma HCT116 cells and to analyze the mechanism.METHODS:A colonic carcinoma cell line with CARMA3 over-expression was selected.The CARMA3 gene in the HCT116 cells was knocked down by lentivirus technique.After screening by puromycin, the stably-transfected HCT116-shCARMA3 cell line was constructed.CARMA3 expression at mRNA and protein levels was detected by real-time PCR and Western blot,respectively.The cell proliferation was analyzed by WST-1 assay and RTCA S16 system.The colony formation ability was measured by colony-forming assay.The cell cycle was analyzed by flow cytometry.The cell morphological changes were observed under microscope.The abilities of migration and invasion in vitro were observed by wound healing assay and Transwell assay.The changes of related molecules were determined by Western blot to explore the mechanism.RESULTS:The expression of CARMA3 at mRNA and protein levels in the HCT116 cells was the highest in the 4 colonic carcinoma cell lines.HCT116-shCARMA3 cells with stably-silenced CARMA3 gene were successfully established.Among them, HCT116-shCARMA3-93 cells showed the greatest inhibition of CARMA3 at mRNA and protein levels.Therefore,HCT116-shCARMA3-93 cells were chosen as the cell model.Compared with control group, the morphological changes of the HCT116-shCARMA3-93 cells had epithelial-mesenchymal transition (EMT) reversion.The abilities of proliferation, colony formation, migration and invasion in the HCT116-shCARMA3-93 cells were obviously suppressed (P<0.01).G0 /G1 phase proportion was increased and S phase proportion was correspondingly decreased (P<0.05).Bcl10 and NF-κB were down-regulated, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1)showed no change.Cyclin D1 was decreased obviously and cyclin A declined slightly.Metastasis-related mar-kers matrix metalloproteinase (MMP)-2 and MMP-9 were reduced,MMP-7 remained unchanged, while tissue inhibitor of metalloproteinase(TIMP)-1 and TIMP-2 were up-regulated.Furthermore, EMT-associated molecule E-cadherin was increased, while N-cadherin, Snail, Slug and Twist were decreased to some extent.CONCLUSION:CARMA3 has an impact on the growth,migration and invasion of colonic carcinoma cell line, which is possibly related to NF-κB signaling pathway to change cell cycle and metastasis-related markers and to regulate EMT.

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Effects of icarⅡn on viability, migration and invasion of prostate cancer cells

Wenhua ZHANG ; Wenchao ZHANG ; Yuandong YU

Chinese Journal of Pathophysiology.2017;33(6):1017-1020. doi:10.3969/j.issn.1000-4718.2017.06.010

AIM:To investigate the effects of icarⅡn on the viability, migration and invasion of the prostate cancer lines Du145 and PC3.METHODS:Du145 cells and PC3 cells were treated with icarⅡn at different concentrations (0, 5, 10, 20, 40 or 80 μmol/L), and the cell viability was measured by CCK-8 assay.The cell migration and invasion abilities were detected by Transwell assay.The protein expression of Notch-1, matrix metalloproteinase (MMP)-2, MMP-9 and hairy/enhancer of split-1 (Hes-1) was determined by Western blot.RESULTS:The results of MTT assay revealed that icarⅡn inhibited the viabilitiy of Du145 cells and PC3 cells in a dose-dependent manner.The maximal effect was at dose of 40 μmol/L.IcarⅡn treatment significantly decreased the abilities of migration and invasion of Du145 cells and PC-3 cells.Moreover, the protein expression of Notch-1, MMP-2, MMP-9 and Hes-1 was dramatically reduced after icarⅡn treatment.CONCLUSION:IcarⅡn inhibits prostate cancer cell viability, migration and invasion by decreasing the protein expression of Notch-1, MMP-2, MMP-9 and Hes-1.

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Inhibitory effect of fenbendazole on proliferation of human chronic myelogenous leukemia K562 cells

Licai HE ; Liuzhi SHI ; Rui GONG ; Zhuanyun DU ; Haihua GU ; Jianxin Lü

Chinese Journal of Pathophysiology.2017;33(6):1012-1016. doi:10.3969/j.issn.1000-4718.2017.06.009

AIM:To investigate the effect of fenbendazole (FBZ) on the proliferation of human chronic myelogenous leukemia (CML) cell line K562.METHODS:The CCK-8 assay was used to detect the effect of FBZ on viability of the K562 cells and normal peripheral blood mononuclear cells (PBMC).The cell growth was measured by the method of Trypan blue exclusion.The cell cycle was analyzed by flow cytometry.The cell cycle-related proteins were detected by Western blot.RESULTS:The growth of K562 was significantly inhibited by FBZ.However, it elicited little cytotoxic effect on PBMC.Furthermore, FBZ induced G2/M phase arrest and mitotic catastrophe in the K562 cells based on the changes of nuclear morphology, DNA content, mitotic marker analysis and the number of polykaryocytes.CONCLUSION:Fenbendazole significantly inhibits the proliferation of K562 cells and induces cell cycle arrest at G2/M phase by the regulation of cell cycle-related proteins.

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Changes of Ski expression levels in rat activated astrocytes

Xin ZHAO ; Jiangli KOU ; Yongqiang GUO ; Yanchuan PU ; Kaisheng ZHOU ; Wei NAN ; Jing WANG ; Yamin WU ; Haihong ZHANG

Chinese Journal of Pathophysiology.2017;33(6):968-974. doi:10.3969/j.issn.1000-4718.2017.06.002

AIM:To explore the time-dependent change of Ski protein expression in normal and activated astrocytes in rats.METHODS:The astrocytes were obtained from rat cerebral cortex and cultured in vitro.The astrocytes were treated with LPS and scratch injury for activation.Western blot analysis was used to determine glial fibrillary acidic protein (GFAP) and Ski protein levels in activated astrocytes at a series of time points.The indirect immunofluorescence staining method was performed to detect the location of Ski protein in the astrocytes.RESULTS:The protein of GFAP was naturally expressed in the astrocytes, beginning to increase after treated with LPS and scratch injury.Little protein expression of Ski in the normal astrocytes was observed.The Ski protein expression began to increase after treated with 1 mg/L LPS, peaked at 4 d (P<0.05) and then deceased, but was stills higher than that in the normal cells.The protein expression level of Ski after scratch injury was highly consistent with above mentioned.Ski was mainly observed in the nucleus of the normal cells and the cells treated with LPS for 6 d, while it was observed in the cytoplasm 2 and 4 d after treated with LPS.CONCLUSION:The protein of Ski is expressed in the astrocytes, and the expression level is increased in activated astrocytes,mainly located in the nucelus.Ski may plays an essential roles in the processes of activation and proliferation of astrocytes.

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