Anti-Depressant-Like Effect of Sinomenine on Chronic Unpredictable Mild Stress-Induced Depression in a Mouse Model.

BACKGROUND Sinomenine (SIN) is an extract of the Chinese medicinal herb Sinomenium acutum; it has various pharmacological properties, including immunosuppression and anti-inflammation. The present study aimed to investigate whether SIN has an anti-depressant-like effect in a mouse model of depression induced by chronic unpredictable mild stress (CUMS), and to explore the underlying molecular mechanisms. MATERIAL AND METHODS A mouse model of depression was established and treated with different concentrations of SIN (30, 100, or 300 mg/kg). Then, behavioral tests, including sucrose preference test (SPT), forced swimming test (FST), and the tail suspension test (TST), were performed. The levels of norepinephrine (NE), 5-hydroxytryptamine (5-HT), and proinflammatory cytokines (interleukin-1ß [IL-1ß] interleukin-6 [IL-6], and tumor necrosis factor-α [TNF-α]) in the hippocampus of mice were detected by ELISA assay. The levels of p-p38, p-p65, NLRP3, ASC, and caspase-1 were measured by Western blot or/and qRT-PCR. RESULTS The results showed that SIN significantly relieved CUMSinduced depressive-like behaviors. Compared with the model mice, SIN treatment significantly increased the sucrose preference of the mice, and the immobility time in the forced swimming and the tail suspension test were shortened. In addition, SIN decreased CUMS-induced reduction in the concentrations of NE and 5-HT in the hippocampus of mice. SIN reduced CUMS-induced increases in the levels of IL-1ß, IL-6, and TNF-α in the hippocampus of mice. Furthermore, activation of the p38MAPK-NF-κB pathway and the nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome induced by CUMS were inhibited by SIN treatment. CONCLUSIONS In conclusion, our results indicate the antidepressantlike effects of SIN on chronic unpredictable mild stress-induced depression in a mouse model.

Membrane-anchored CCL20 augments HIV Env-specific mucosal immune responses.

BACKGROUND: Induction of broad immune responses at mucosal site remains a primary goal for most vaccines against mucosal pathogens. Abundance of evidence indicates that the co-delivery of mucosal adjuvants, including cytokines, is necessary to induce effective mucosal immunity. In the present study, we set out to evaluate the role of a chemokine, CCL20, as an effective mucosal adjuvant for HIV vaccine. METHODS: To evaluate the role of CCL20 as a potent adjuvant for HIV vaccine, we examined its effects on antigen-specific antibody responses, level of antibody-secreting cells, cytokine production and intestinal homing of plasma cells in vaccine immunized mice. RESULTS: CCL20-incorporated VLP administered by mucosal route (intranasal (n = 10, p = 0.0085) or intravaginal (n = 10, p = 0.0091)) showed much higher potency in inducing Env-specific IgA antibody response than those administered by intramuscular route (n = 10). For intranasal administration, the HIV Env-specific IFN-γ(751 pg/ml), IL-4 (566 pg/ml), IL-5 (811 pg/ml) production and IgA-secreting plasma cells (62 IgA-secreting plasma cells/106 cells) in mucosal lamina propria were significantly augmented in CCL20-incorporated VLP immunized mice as compared to those immunized with Env only VLPs (p = 0.0332, 0.0398, 0.033, 0.0302 for IFN-γ, IL-4, IL-5, and IgA-secreting plasma cells, respectively). Further, anti-CCL20 mAb partially suppressed homing of Env-specific IgA ASCs into small intestine in mice immunized with CCL20-incorporated VLP by intranasal (62 decreased to 16 IgA- secreting plasma cells/106 cells, p = 0.0341) or intravaginal (52 decreased to 13 IgA- secreting plasma cells/106 cells, p = 0.0332) routes. CONCLUSION: Our data indicated that the VLP-incorporated CCL20 can enhance HIV Env-specific immune responses in mice, especially those occurring in the mucosal sites. We also found that i.m. prime followed by mucosal boost is critical and required for CCL20 to exert its full function as an effective mucosal adjuvant. Therefore, co-incorporation of CCL20 into Env VLPs when combined with mucosal administration could represent a novel and promising HIV vaccine candidate.

[Role of Toll-like receptor 2/4-nuclear factor-κB signaling pathway in invasion of Mycobacterium tuberculosis to mouse dendritic cells].

OBJECTIVE: To investigate the mechanism of Mycobacterium tuberculosis invasion to mouse dendritic cells (DC). METHODS: Mycobacterium tuberculosis strain H37Rv was co-cultured with mouse DC2.4 cells.The mRNA expression of Toll-like receptor 2/4(TLR2/4) in DC2.4 cells was detected by fluorescent quantitative real-time PCR and the protein expression of nuclear factor κB(NF-κB) was assessed by Western blotting.The extracellular concentration of tumor necrosis factor α(TNF-α) was measured by ELISA methods during Mycobacterium Tuberculosis invasion.Indirect immunofluorescent staining and flow cytometry assay were used to detect the expression of CD80 and CD86 on DC2.4 cells before and after invasion. RESULTS: The invasion of Mycobacterium tuberculosis in DC2.4 cells was observed after 2 h of co-incubation.The rates of invasion were (37.9±5.6)%,(51.2±7.6)%,(57.2±8.9)% and(63.9±6.8)% at 6,8,10 and 12 h after co-incubation,respectively.The mRNA expression level of TLR2 /4 was significantly increased at 6 h but decreased at 10 h after co-incubation.The expressions of NF-κB p65 and TNF-α were higher in DC2.4 cells after being invaded by 6,8,and 10 h and then gradually decreased.CD80 and CD86 expression were increased on DC2.4 at 6 h after co-incubation. CONCLUSION: Invasion of Mycobacterium tuberculosis strain H37Rv to DC might enhance its antigen-presenting function through activation of TLR2/4-NF-kB signaling pathway.

[Role of phospholipase C in cytoskeleton rearrangements of dendritic cells invaded by Mycobacterium tuberculosis].

OBJECTIVE: To investigate the role of phospholipase C(PLC) in cytoskeleton rearrangement of mouse dendritic cells invaded by Mycobacterium tuberculosis. METHODS: Mouse dendritic DC2.4 cells were co-cultured with Mycobacterium tuberculosis H37Rv. F-actin of DC2.4 cells were strained with phalloidin-TRITC, the microtubule was stained with anti-ß-tubulin monoclonal antibody and FITC-conjugated AffiniPure anti-mouse IgG. The changes of cytoskeleton in DC2.4 cells induced by Mycobacterium tuberculosis H37Rv were determined by fluorescence microscopy and the rates of F-actin rearrangements were calculated. The expressions of PLC in cytoplasm and cytomembrane of DC2.4 cells were measured by ELISA. DC2.4 cells were pretreated with PLC inhibitor U73122, then F-actin rearrangements induced by invasion of Mycobacterium tuberculosis were observed. RESULTS: Bacterial invasion was observed while DC2.4 cells were co-incubated with Mycobacterium tuberculosis H37Rv for 2 h. The rates of invasion were (26.1 ± 4.5)%, (39.9 ± 5.6)%, (51.2 ±5.9)%, (57.9 ± 6.1)% and (63.9 ± 6.8)% at 4, 6, 8, 10 and 12 h of co-culture, respectively; while those were (13.6 ± 3.1)%, (14.2 ± 3.9)%, (15.1 ± 4.3)%, (16.8 ± 4.0)% and (18.3 ± 5.2)% after blocked by PLC, respectively. The rates of the F-actin rearrangements at 2, 4, 6, 8, 10 and 12 h after DC2.4 cells were invaded by H37Rv were (26.9 ± 1.5)%, (59.3 ± 2.8)%, (72.7 ± 4.8)%, (78.2 ± 5.9)%, (63.3 ± 2.9)% and (43.2 ± 2.6)%, respectively; while those were (18.5 ± 1.2)%, (22.3 ± 1.7)%, (3.6 ± 2.5)%, (24.8 ± 2.3)%, (22.3 ± 1.3)% and (23.8 ± 1.8)% after blocked by PLC, respectively. There were no changes of the microtubule observed in DC2.4 cells at the same time points. The rates of the F-actin rearrangements before blocked by PLC were higher than those after PLC blockade at 4, 6, 8 and 10 h (P <0.05). The expressions of PLC in cytomembrane in DC2.4 cells increased after 2 h and reached its highest level at 8 h. The PLC inhibitor U73122 inhibited the expressions of PCL in cytomembrane of DC2.4 cells, but not in cytoplasm. CONCLUSION: Mycobacterium tuberculosis can provoke to F-actin rearrangements through PLC molecule, which would further lead to Mycobacterium tuberculosis invasion of DC2.4 cells.

Genome sequence of the human-pathogenetic bacterium Vibrio vulnificus B2.

Vibrio vulnificus, which can lead to rapidly expanding cellulitis or septicemia, is present in the marine environment. Here, we present the draft genome sequence of strain B2, which was isolated from a septicemia patient in 2010.

[Effects of snakegourd root polysaccharide on apoptosis of MCF-7 cells].

OBJECTIVE: To investigate the effects of snakegourd root polysaccharide on apoptosis of human breast cancer cells (MCF-7 cells). METHODS: Colorimetric MTT assay was used to measure the inhibition of snakegourd root polysaccharide on MCF-7 cells. The morphological changes of MCF-7 cells were observed by fluorescence microscope after DAPI staining and transmission electron microscope. The apoptosis of MCF-7 cells was examined by DNA agarose gel electrophoresis analysis of DNA fragmentation amd flow cytometry. The activity of Caspase-3 and Caspase-8 was detected by colorimetric assay. RESULTS: Polysaccharide of snakegourd root significantly inhibited MCF-7 cells in a dose-and time-dependent manner. The nuclear condensation and marginalization were observed by DAPI staining and transmission electron microscope. The characteristic ladder of apoptosis in DNA electrophoresis was detected in MCF-7 cells treated with 10.0 µmol/L polysaccharide of snakegourd root at d 2. The activities of Caspase-3 and Caspase-8 were increased in a time-dependent manner. The rates of apoptosis in MCF-7 cells were (5.2 ±1.3)%, (13.1 ±4.7)%, (27.6 ±6.8)% and (43.8 ±9.8)% treated with 1.0,5.0,10.0 and 20.0 µmol/L snakegourd root polysaccharide at d 2,respectively. The maximal activities of intracellular Caspase-3 and Caspase-8 were (2.32 ±0.12)U/µg and (1.92 ±0.11)U/µg at d 2 and d 1, respectively when MCF-7 cells were treated with 10.0 µmol/L. CONCLUSION: The polysaccharide of snakegourd root can induce the apoptosis of MCF-7 cells,which is associated with the activation of intracellular Caspase-3 and Caspase-8.

[Effects of Mycobacterium tuberculosis on apoptosis of mouse dendritic cells and activation of caspase-3, caspase-8].

OBJECTIVE: To investigate the effects of Mycobacterium tuberculosis on apoptosis of mouse dendritic cells (DC 2. 4) and the activation of caspase-3, caspase-8. METHODS: Mycobacterium tuberculosis H37Rv strain was co-cultured with DC 2. 4 cells. The morphological changes of DC 2. 4 cells were observed with fluorescence microscope after DAPI staining and transmission electron microscope. The apoptosis of DC 2. 4 cells were examined by DNA agarose gel electrophoresis. The activities of caspase-3 and caspase-8 were detected by colorimetric assay. RESULTS: Bacterial invasion was observed while DC 2. 4 cells and H37Rv were co-cultured for 2 h; and the rates of invasion were (16.1 ± 4.3)%, (35.8 ± 5.1)%, (50.2 ± 5.7)%, (58.3 ± 6.2)% and(65.9 ± 6.9)% at 4, 6, 8,10, 12 h, respectively. The phenomenon of nuclear condensation and marginalization were shown by DAPI staining and transmission electron microscope in DC 2. 4 cells at 6 h of co-cultivation with H37Rv. The characteristic bands of apoptosis by DNA electrophoresis were detected. The activities of caspase-3 and caspase-8 were increased in a time-dependent manner. The rates of DC 2. 4 cell apoptosis were (6.4 ± 2.5)%, (11.8 ± 5.3)% and (31.1 ± 8.7)% at 6 h,12 h and 24 h after co-cultivation with H37Rv, respectively. The maximal activities of intracellular caspase-3 and caspase-8 at 10 h and 6 h were (2.01 ± 0.09) U/µg and (2.40 ± 0.07)U/µg, respectively, which was significantly different compared with the control groups(P<0.05). The activation of caspase-8 was earlier than that of caspase-3. CONCLUSION: Mycobacterium tuberculosis can induce the apoptosis of DC 2. 4 cells, which is associated with the activation of intracellular caspase-3 and caspase-8.

[Immunocompetence effects of polysaccharide of snakegourd root on human peripheral blood mononuclear cells in vitro].

OBJECTIVE: To establish the method of promoting human peripheral blood mononuclear cell proliferation by polysaccharide of snakegourd root and identify the effects of polysaccharide of snakegourd root on lymphocyte proliferation, T lymphocyte subsets and the different levels of TNF-alpha and IL-6. METHOD: The polysaccharide of snakegourd root preparations were purified with dialysis and ethanol precipitation. The healthy human PBMC were used as the target cells for screening potency of the drugs. MTT colorimetry was established to examine the levels of lymphocyte proliferation on human PBMC by polysaccharide of snakegourd root in vitro. The percents of lymphocyte subsets (CD3+, CD4+, CD8+ T lymphocyte) and the different levels of TNF-a and IL-6 in PBMC were analysed by FCM and ELISA, respectively. RESULT: 1.0-50.0 mmol x L(-1) of polysaccharides of snakegourd root showed the significant effects of promoting proliferation of human PBMC (P < 0.05). The percents of CD3+, CD4+, CD8+ T lymphocytes in PBMC treated with 5.0 and 10.0 mmol x L(-1) of polysaccharides of snakegourd root were significantly higher than those of the control group (P < 0.05). The levels of TNF-alpha and IL-6 were significantly higher than those of the control group after 1.0, 5.0, 10.0 mmol x L(-1) of polysaccharides of snakegourd root stimulation on the human PBMC at 8 hours (P < 0.05). CONCLUSION: The significant effects on promoting lymphocyte proliferation and activation of the polysaccharide of snakegourd root are confirmed in this study. The percents of lymphocyte subsets are increased in different degrees by the polysaccharide of snakegourd root. The high levels of TNF-alpha and IL-6 are secreted after the polysaccharides of snakegourd root stimulation on the human PBMC, which lays a foundation for further elucidating the immunocompetence effects and mechanism of the polysaccharide of snakegourd root.

[Cytoskeleton rearrangements and induced apoptosis in human umbilical venous endothelial cell and WISH cell lines during the invasion of Staphylococcus aureus].

OBJECTIVE: To investigate the changes of cytoskeleton and induced apoptosis in human umbilical venous endothelial cells (HUVEC) and WISH cells during the invasion of Staphylococcus aureus. METHODS: S. aureus suspension was collected routinely and used to infect HUVEC and WISH cells for 10, 30, 60 and 120 min respectively. The cell-invading ability of S. aureus was observed by microscope and the rearrangement of cytoskeleton of these cells observed under fluorescent microscope. DAPI fluorescent staining and DNA agarose electrophoresis were performed to analyze the apoptosis in HUVEC cells induced by S. aureus. RESULTS: There was bacterial invasion after staphylococcus aureus was co-incubated with HUVEC and WISH cells for 10 min. The rates of infection were (54.9 +/- 2.4)% and (56.1 +/- 2.4)% at 60 min respectively. The ratios of F-actin rearrangements at 10 min, 30 min, 60 min and 120 min after the invasion of HUVEC and WISH cells with S. aureus were different at (54.7 +/- 2.8)%, (63.0 +/- 2.9)%, (71.0 +/- 2.6)%, (39.5 +/- 2.7)% and (63.3 +/- 2.6)%, (65.0 +/- 2.9)%, (77.0 +/- 2.4)% and (44.0 +/- 1.8)% respectively. The ratios of F-actin rearrangements at 10 min, 30 min and 60 min were higher than those at 120 min (P < 0.05). There was no change of microtubule observed in both cells at the same time. The apoptotic appearance was observed after the invasion of HUVEC cells with s. aureus at 60 min. CONCLUSION: S. aureus may invade the HUVEC and WISH cells through F-actin rearrangement. Apoptosis is induced in HUVEC cells at 60 min.

Structure-property relationship of regenerated spider silk protein nano/microfibrous scaffold fabricated by electrospinning.

The regenerated Araneus ventricosus spider dragline silk protein fibrous scaffold with moderate strength and flexibility was fabricated by electrospinning and post treatment with 90 vol % acetone. The effect of collection method on the morphology of regenerated spider silk protein (RSSP) fibrous scaffold, the effects of the post treatment solvents and their concentrations on the molecular conformation, crystallinity and mechanical properties were studied. The results show that the morphology was affected by the solvent used in the coagulation bath. The molecular conformation, crystallinity and mechanical property of this scaffold were strongly affected by the kind of post treatment solvent and slightly influenced by its concentration when it was higher than 50 vol %. The degradation rate of this scaffold was very slow and resulting in little pH change of the degradation medium within 5 months. PC 12 cells were cultured on the electrospun RSSP fibrous scaffold and in its extraction fluid to examine the changes of PC 12 cells after different times of culture. The results show that the electrospun RSSP fibrous scaffold had good biocompatibility with PC 12 cells.

Multi-porous electroactive poly(L-lactic acid)/polypyrrole composite micro/nano fibrous scaffolds promote neurite outgrowth in PC12 cells.

In this study, poly(L-lactic acid)/ammonium persulfate doped-polypyrrole composite fibrous scaffolds with moderate conductivity were produced by combining electrospinning with in situ polymerization. PC12 cells were cultured on these fibrous scaffolds and their growth following electrical stimulation (0-20.0 µA stimulus intensity, for 1-4 days) was observed using inverted light microscopy, and scanning electron microscopy coupled with the MTT cell viability test. The results demonstrated that the poly(L-lactic acid)/ammonium persulfate doped-polypyrrole fibrous scaffold was a dual multi-porous micro/nano fibrous scaffold. An electrical stimulation with a current intensity 5.0-10.0 µA for about 2 days enhanced neuronal growth and neurite outgrowth, while a high current intensity (over 15.0 µA) suppressed them. These results indicate that electrical stimulation with a moderate current intensity for an optimum time frame can promote neuronal growth and neurite outgrowth in an intensity- and time-dependent manner.

[Expression of vascular growth factors in intestinal tissues in colorectal carcinoma patients with schistosomiasis japonica].

OBJECTIVE: To investigate the differences of mRNA quantitation and protein expression of vascular growth factors including platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) in intestinal tissues in colorectal carcinoma patients with and without schistosomiasis. METHODS: Thirty colorectal carcinoma patients with schistosomiasis and 30 colorectal carcinoma patients without schistosomiasis were included in this study. The mRNA quantitation and protein expression of PD-ECGF and VEGF in the normal tissue, peri-carcinoma tissue as well as carcinoma tissue obtained from surgical specimens were detected by qRT-PCR and Western blot. RESULTS: The mRNA relative quantitations of PD-ECGF in normal tissue, peri-carcinoma tissue and carcinoma tissue in the colorectal carcinoma patients with schistosomiasis were 1.726, 1.766 and 2.729 times to those in the colorectal carcinoma patients without schistosomiasis, respectively. The corresponding ones of VEGF were 2.138, 1.831 and 3.376 times, respectively. The protein expression levels of PD-ECGF and VEGF in normal tissue, peri-carcinoma tissue and carcinoma tissue were higher in the colorectal carcinoma patients with schistosomiasis than in the colorectal carcinoma patients without schistosomiasis. CONCLUSIONS: The expressions of vascular growth factors including PD-ECGF and VEGF are higher in the colorectal carcinoma patients with schistosomiasis than in the colorectal carcinoma patients without schistosomiasis. Therefore, schistosomiasis may be one of the risk factors of colorectal cancer.

Localization of Vibrio vulnificus infection in dendritic cells and its effects on the cytoskeleton.

BACKGROUND: Vibrio vulnificus (Vv) is an estuarine bacterium that can cause primary septicemia as well as serious wound infections. However, little is known about the mechanisms by which Vv infects dendritic cells (DCs) and its effects on cytoskeleton. In this study, we aimed to investigate the invasion, internalization, and the organelles damage of the cultured dendritic cells (a DC 2.4 strain) during Vv infection. METHODS: The study model was the cultured DCs infected by a Vv 1.758 strain. Electron microscopy was used to observe the localization of bacteria at the different time points of infection, cell morphology, and the process of organelles changes. The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined under a fluorescence microscope. RESULTS: The Vv were pinocytosised into the DC cells through double-sides, and localized at 1 - 2 mm of the inner side membrane. It took 1.3, 1.9, and 3.4 hours to reach the infection ratio of 25%, 50%, and 75%, respectively. Using electron microscopy, the DCs had been observed to have developed chromatin aggregation within 4.0 hours, and significant cytoskeleton structure disruption was noted within 6.0 hours. CONCLUSION: The high lethality of Vv infection may be associated with the direct disruption of the DCs cytoskeleton structure.

Antisense expression of PKCalpha improved sensitivity of SGC7901/VCR cells to doxorubicin.

AIM: To explore whether antisense blocking of protein kinase C alpha (PKCalpha) would reverse multi-drug resistance (MDR) in the vincristine (VCR)-resistant human gastric cancer cell line SGC7901/VCR. METHODS: SGC7901/VCR cells expressing antisense PKCalpha, SGC7901/VCR/aPKC, were established by transfection with a recombinant plasmid reversely inserted with PKCalpha cDNA. Empty vector (PCI-neo)-transfected cell clones, SGC7901/VCR/neo, served as the control. Western blot method was used to detect PKCalpha content in SGC7901, SGC7901/VCR, SGC7901/VCR/neo and SGC7901/VCR/aPKC cells, using PKCalpha-specific antibody. The sensitivity of SGC7901, SGC7901/VCR, SGC7901/VCR/neo and SGC7901/VCR/aPKC cells to doxorubicin (DOX) in vitro was determined by MTT assay. The uptake of DOX in these cells was detected with fluorescence spectrophotometer. RESULTS: Western blot analysis showed that the PKCalpha protein level was about 8.7-fold higher in SGC7901/VCR cells than that in SGC7901 cells, whereas the protein expression of PKCalpha was reduced by 78% in SGC7901/VCR/aPKC cells when compared with the SGC7901/VCR cells. SGC7901/VCR/aPKC cells had a 4.2-fold increase in DOX cytotoxicity, accompanied by a 1.7-fold increase of DOX accumulation in comparison with SGC7901/VCR cells. CONCLUSION: PKCalpha positively regulates MDR in SGC7901 cells, and inhibition of PKCalpha can partially attenuate MDR in human gastric cancer cells.