Effects of sterigmatocystin on interferon-γ secretion of human peripheral blood mononuclear cellsin vitro

AIM:To explore the putative effects of sterigmatocystin (ST) on human help T lymphocyte(Th1)function. METHODS:The effects of ST on interferon-γ(IFN-γ)secretion of human peripheral blood mononuclear cells(HPBMc) in vitro were determined with ELISA method. RESULTS:The effects of ST on IFN-γ secretion of HPBMc in vitro were closely dependent on ST concentrations. ST at relatively lower concentrations (0.03125-0.12500 mg/L) showed inhibiting effects on IFN-γ secretion. While, stimulating effects could be found when ST concentration was above 0.25mg/L. The highest level was seen in ST 1 mg/L group (P＜0.05)。At concentration ranging from 0.25 mg/L to 1 mg/L, a positive dose- effects correlation was found between ST concentration and IFN-γ secretion (r=0.492, P＜0.01). Time-effects analysis from 1 h to 64 h after ST treatment (1 mg/L)showed that the effects of ST on IFN-γ secretion varied as the changes of treatment times. An inhibiting effect on IFN-γ secretion of HPBMc 4 h and 8 h after ST treatment was found (8 h, P＜0.05). As the treatment time prolonged from 16 h, IFN-γ level gradually increased (32 h, P<0.05). There was a positive correlation between treatment time of ST and IFN-γ level of HPBMc in vitro from 16 h to 64 h after ST treatment (r=0.736, P＜0.01).CONCLUSION:The effects of ST on IFN-γ secretion of HPBMc in vitro closely depended on concentration and treatment time of ST. Generally, inhibiting effects were found at relatively lower ST concentration and shorter treatment period, while stimulation effects could be seen at relatively higher ST concentration and longer ST treatment time period.     

Alteration of serum interleukin-6 and tumor necrosis factor-α after ischemic stimulation of coronary artery in PTCA

AIM: Inflammatory responses play an important role in the post- percutaneous transluminal coronary angioplasty (PTCA) restenosis and has been demonstrated occuring immediately after PTCA. Interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) are the main inflammatory cytokines. We try to compare the changes in interleukin-6(IL-6) and TNF-α after PTCA in the patients with and without collateral circulation to probe into the pathogenesis of early inflammatory response. METHODS: The extent of myocardial ischemia induced by balloon inflation was quantified by a scoring system referring to the Leaman coronary score. The IL-6、TNF-α levels of coronary heart disease group and control group before and after PTCA are calculated. RESULTS: The concentrations of IL-6 and TNF-α were (9.592±1.847) ng/L and (26.959±1.967) ng/L, respectively, and were significantly increased 4 hours after PTCA. CONCLUSION: IL-6 and TNF-α are sensitive indicators of the early inflammatory response after PTCA. Ischemia scores reflected the extent of ischemia reperfusion injury during PTCA. Collateral circulation decreased the early inflammatory response after PTCA.     

Effects of glycine on intracellular of free calcium and tumor necrosis factor-α of cardiomyocytes during hypoxia/reoxygenation

AIM: To observe the influence of glycine on intracellular free calcium, the concentration of tumor necrosis factor-α and the survival rate of myocardial cells during hypoxia/reoxygenation (H/R). METHODS: The simulated model of myocardial ischemia-reperfusion with the primary cultured cardiomyocytes of neonatal rats was established, and the cultured cardiomyocytes were divided into seven groups, control group, hypoxia/reoxygenation group, glycine (0.5 mmol/L) plus hypoxia/reoxygenation group, glycine (1.0 mmol/L) plus hypoxia/reoxygenation group, glycine (2.0 mmol/L) plus hypoxia/reoxygenation group, glycine (4.0 mmol/L) plus hypoxia/reoxygenation group, 4.0 mmol/L glycine group. RESULTS: Within certain concentration (0.5-2.0 mmol/L), the glycine could inhibit the calcium overload resulting from hypoxia/reoxygenation injury in cells in a dose-dependent manner with the optimal inhibitory effect at 2.0 mmol/L. Glycine inhibited the secretion of tumor necrosis factor-α from myocardial cells and increased the survival rate of myocardial cells. CONCLUSION: Glycine has a protective effect on hypoxia/reoxygenation myocardial cells, which may be related to inhibiting calcium overload and decreasing the production of tumor necrosis factor-α.     

Proteomic analysis of the effects of tumor necrosis factor-α on endothelial cells

AIM: To investigate the affected proteins by tumor necrosis factor (TNF)-α in endothelial cells, and further explore the potential molecular mechanism of TNF-α on endothelial cells. METHODS: Nitric oxide (NO) production in the cultured human umbilical vein endothelial cells (HUVECs) was measured by a NO assay kit. Proteomic alterations were analyzed using two-dimensional electrophoresis, and peptide mass fingerprinting with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. RESULTS: NO production in HUVECs decreased significantly after TNF-α treatement. Proteomics analysis showed 21 protein spots were changed including 9 spots that were increased and 11 spots that were decreased after TNF-α stimulation, and 1 spot was only detected in TNF-α activated cell gels. CONCLUSIONS: Decreased the expression of ecNOS by TNF-α might result in decreased NO production. Up-regulated MAP/ERK kinase 3 expression might imply that TNF-α activates the expression of adhesion molecules. Cytoskeletal protein actin is also involved in TNF-α injuried HUVECs. Proteomic analysis can find some clues for identifying new potential target of TNF-α.     

Human interleukin-1β induces A375-S2 human melanoma apoptosis through caspase pathway

AIM: To study the molecular biological mechanism and signal transduction pathway of interleukin-1β (IL-1β)-induced apoptosis in A375-S2 melanoma cells. METHODS: Photomicrocropy showed typical apoptotic changes. The cytotoxic effect of IL-1β in vitro and influences of caspases in this effect were measured by MTT assay. The cytotoxicity of cells was assessed by LDH-based assay. Degradation of DNA was detected by agarose gel electrophoresis. RESULTS: The inhibitory effect of IL-1β on A375-S2 cell growth was in a dose and time-dependent manner, and cell death rate reached more than 90% at 72 h after treatment with 10^-9mol/L IL-1β. The inhibitors of caspase-family, -1, -3, -8, -9, and -10, partially blocked cell death at early stage. LDH assay showed that major IL-1β-induced cell death was apoptosis, and in a dose and time-dependent manner. Typical apoptotic DNA ladder was observed in agarose gel electrophoresis. CONCLUSION: IL-1β induced apoptosis in melanoma A375-S2 cells by activating caspase pathway.     

Effects of tumor necrosis factor alpha on proliferation and apoptosis of HSCs

AIM:To explore the role of tumor necrosis factor alpha(TNF-α) in the pathogenesis of liver fibrosis.METHODS:The proliferation and apoptosis of hepatic stellate cells (HSCs) in vitro were detected with flow cytometry, electron microscopy and TUNEL.RESULTS:The flow cytometry analysis showed that the cell proliferation index (PI) in the TNF-α(0.5 μg/L, 2.0 μg/L, 8.0 μg/L) groups was evidently lower than that in the control group (P＜0.05). In the cell cycle distribution, the portion of G₀/G₁ phase in the TNF-α groups was significantly higher than that in the control group(P＜0.05), but the portion of S phase in the TNF-α groups was evidently lower than that in the control group(P＜0.05). These indicated that TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. The apoptotic rate in the TNF-α groups was evidently higher than that in the control group(P＜0.05). The gene expression of bcl-2 and bax was also detected with flow cytometry. The expression of bcl-2 in the TNF-α groups was evidently lower than that in the control group(P＜0.05), but the expression of bax in the TNF-α groups was significantly higher than that in the control group(P＜0.05). TUNEL analysis showed the apoptotic rate of HSCs in the TNF-α(2.0 μg/L) group was 18.7%±2.5% compared with 5.3%±1.2% in the control group(P＜0.05).CONCLUSIONS:TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. TNF-α down-regulated bcl-2 gene expression and up-regulated bax gene expression whereupon the apoptosis of HSCs was induced.     

A novel mechanism of RhoA activation induced by tumor necrosis factor-α in mouse cerebral microvascular endothelial cells

AIM: To investigate the effects of tumor necrosis factor α (TNF-α) on RhoA activity in mouse cerebral microvascular endothelial cells.METHODS: The bEnd.3 cells, a mouse brain microvascular endothelial cell line, were cultured. RhoA activity was analyzed by pull-down assay 10 min, 30 min and 60 min after TNF-α treatment. Expression of RhoA protein was determined by Western blotting 1 h, 3 h, 6 h, 12 h and 24 h after TNF-α treatment. Small interfering RNA (siRNA) targeting to p115RhoGEF or control nsRNA was transfected into bEnd.3 cells. The expression of p115RhoGEF was determined by Western blotting, and RhoA activity was detected by pull-down assay 30 min after TNF-α treatment.RESULTS: RhoA activity peaked at 30 min after TNF-α treatment(P<0.01) . TNF-α significantly increased the protein expression of RhoA at 12 h and 24 h (P<0.05). Knock-down of p115RhoGEF by siRNA in bEnd.3 cells attenuated TNF-α-induced RhoA activation (P<0.05).CONCLUSION: TNF-α up-regulates RhoA activity and expression. p115RhoGEF may play a role in TNF-α-induced activation of RhoA.     

Changes of IFN-γ, IL-6 and TNF-α levels in rats with severe pneumonia

AIM:To study the variety of cytokines in severe bacterial pneumonia of Sprague Dawleg (SD) rats. METHODS:A total of 60 SD rats were randomly divided into three groups: model Ⅰ group (n=24), model Ⅱ group (n=24), and control group (n=12). Rats in the model Ⅰ group and the model Ⅱ group were intratracheally instilled with suspension of klebsiella pneumoniae at different doses. Rats in the control group were intratracheally injected with 1 mL saline. On the 2nd, 4th and 6th day after intratracheal instillation, 1/3 rats in each group were killed to determine the concentration of IFN-γ, IL-6 and TNF-α in blood. RESULTS:The levels of IL-6 and TNF-α in model groups were higher than those in control group, while the level of IFN-γ was lower. The change of cytokines was more significant in the model Ⅱ group (severe pneumonia) than those in the model Ⅰ group. CONCLUSION:The cytokines we studied may play an important role in the pathogenesis of severe pneumonia. The change of cytokines is more significant in severe pneumonia than those in common pneumonia.     

Effect of aminoguanidine on TNF-α,IL-1β and neuronal apoptosis after focal cerebral ischemic injury in rats

AIM: To evaluate the effect of aminoguanidine (AG) on inflammatory factors and neuronal apoptosis after focal cerebral ischemic injury in rats and the possible mechanism of protective effect of AG against cerebral ischemic injury.METHODS: Thirty male SD rats (weighing 250 g-280 g) were randomly divided into three groups: (1) sham operated group (SH group,n=10),(2) ischemic groups (IS group,n=10),(3) AG group (n=10).In AG group,AG at dose of 100 mg·kg-1 was given intraperitoneally twice a day for 3 consecutive days.In IS group,normal saline was given instead of AG.Focal cerebral ischemia was produced by middle cerebral artery occlusion (MCAO) for 12 h.A nylon thread with rounded tip was inserted into left internal carotid artery cranially until resistance was felt.The distance from bifurcation of common carotid artery to the tip of the thread was about 18-19 mm.Focal cerebral ischemia was confirmed by left Horners syndrome and right side hemiplegia.In SH group,the carotid artery was exposed but no thread was inserted.The expression of tumor necrosis factor-α(TNF-α) was determined by immunochemistry and the content of interleulin-1β(IL-1β) was measured by radioimmunoassay.The expressions of Bcl-2 and Bax protein were detected by flow cytometry.RESULTS: The expression of TNF-α and the content of IL-1β were markedly increased after MCAO.Significantly increased DNA fragmentation,the indication of apoptosis,was detected after MCAO.The expression of TNF-α and the content of IL-1β were significantly lower in AG group than those in IS group.The percentage of apoptosis cells and expression of Bax protein were markedly lower in AG group than those in IS group but still significantly higher than those in SH group.The expression of Bcl-2 protein was markedly higher in AG group than that in IS group.No significant difference in the expression of Bcl-2 protein between IS and SH group was observed.CONCLUSION: AG inhibits the increase in the expression of TNF-α and the content of IL-1β,and protects neurons from apoptosis induced by focal cerebral ischemia through increasing the Bcl-2 protein expression and inhibiting the Bax protein expression.     

Angiotensin II type 1 receptor autoantibodies induces INS-1 islet β-cell apoptosis by upregulation of autophagy

AIM: To explore whether the angiotensin Ⅱ type 1 receptor autoantibodies (AT1-AA) induces islet β-cell apoptosis and whether autophagy is involved in the process. METHODS: The INS-1 cells treated with AT1-AA at 10^-6 mol/L for 24 h, and then the apoptosis was analyzed by flow cytometry, Western blot and Hoechst 33258 staining. In addition, the expression of autophagy-related proteins such as LC3 and beclin 1 were determined by Western blot. The effects of AT1-AA on the apoptosis, autophagy and viability of INS-1 cells with or without 3-methyladenine (3-MA; a common autophagy inhibitor) or telmisartan (an angiotensin Ⅱ type 1 receptor blocker) pretreatment, were detected by flow cytometry, Western blot and CCK-8 assay. RESULTS: Treatment with AT1-AA at 10^-6 mol/L for 24 h significantly reduced the cell viability (P<0.05). Compared with the negative IgG control group, the apoptotic cells increased after incubation with AT1-AA for 12 h, 24 h and 36 h, respectively (P<0.05). Moreover, the protein levels of LC3 and beclin 1 also increased gradually with the prolongation of treatment time, and the elevation of apoptosis and autophagy were blocked by telmisartan. After pretreatment with 3-MA, the apoptotic rate of the cells was obviously decreased compared with the cells treated with AT1-AA alone. CONCLUSION: AT1-AA induces the apoptosis of INS-1 islet β cells by upregulating autophagy via the angiotensin Ⅱ type 1 receptor pathway.     

Effect of propolis water extract on apoptosis of human umbilical vein endothelial cells in vitro

AIM: To explore the effect of water extract propolis (WEP) from Taishan on apoptosis of human umbilical vascular endothelial cells (HUVECs) induced in vitro by tumor necrosis factor-α (TNF-α). METHODS: HUVECs were collect by digestion-perfusion and cultivated. TNF-α at concentration of 50 μg/L was administrated to induce the apoptosis of HUVECs. After injury, HUVECs were treated with WEP at concentrations of 50, 100, and 200 mg/L, respectively, for 24 h. Apoptosis was evaluated by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and flow cytometry (FCM). RESULTS: Apoptosis index in injured group was significantly higher than that in control group, and decreased significantly after treating with WEP at concentrations of 50, 100, and 200 mg/L, respectively (P<0.01). CONCLUSION: WEP may be useful for protection of vascular endothelial cells by inhibiting apoptosis.     

Pretreatment with external trigeminal nerve electrostimulation inhibits seizures and decreases expression of IL-1β and TNF-α in hippocampus with status epilepticus induced by pentylenetetrazol in rats

AIM:To observe the effect of pretreatment with external trigeminal nerve electrostimulation (eTNS) on behavioral changes and the expression of interleukin-1β (IL-1β) and 　tumor necrosis factor α (TNF-α) in hippocampus of pentylenetetrazol (PTZ)-treated rats. METHODS:The rats were randomly divided into control group, PTZ group and eTNS group, and kindled by PTZ after administered 7 d, 14 d and 28 d of consecutive fake electrostimulation or eTNS. Subsequently, the severity and duration of seizure were quantitatively evaluated. The concentrations of IL-1β and TNF-α in hippocampus were detected by the methods of ELISA and immunohistochemisty. RESULTS:Compared with PTZ group, treatment with eTNS significantly inhibited the severity and duration of seizure (P<0.05), and significantly reduced the content of IL-1β and TNF-α in the hippocampus after status epilepticus (P<0.05 or P<0.01). CONCLUSION:Pretreatment with eTNS may provide a new approach for prevention and treatment of epileptogenesis.     

Effects of human xeroderma pigmentosum group D gene on proliferation of human vascular smooth muscle cells induced by interleukin-6

AIM: To investigate the effects of human xeroderma pigmentosum group D (XPD) gene on the proliferation of human vascular smooth muscle cells (VSMCs) induced by interleukin-6 (IL-6). METHODS: Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into VSMCs by liposome, and then these cells were incubated with IL-6 at 1×10⁵ U/L for 48 h. The cells were divided into 6 groups: blank control group; pEGFP-N2 group; pEGFP-N2/XPD group; IL-6 group; IL-6 + pEGFP-N2 group; IL-6 + pEGFP-N2/XPD group. The expression of green fluorescent protein was observed under fluorescence microscope. The cell growth was detected by MTT method. The cell cycle and apoptosis rate were examined by flow cytometre. The expression levels of XPD, Bcl-2, Bax and wild type P53 (wt-P53) were detected by RT-PCR and Western blotting.RESULTS: Green fluorescence was observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2, indicating successful transfection MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth, and reduced the positive effects of IL-6 on VSMCs growth. Flow cytometry results showed that the transfection of pEGFP-N2/XPD increased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, decreased the cell numbers in S phase, and reduced the effects that IL-6 decreased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, and increased the cell numbers in S phase. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, Bax and wt-P53, decreased the expression of Bcl-2, and reduced the effects that IL-6 decreased the expression of Bax and wt-P53, and increased the expression of Bcl-2. CONCLUSION: XPD gene inhibits VSMCs proliferation, promotes VSMCs apoptosis, and reduces the effects that IL-6 promotes VSMCs proliferation and inhibits VSMCs apoptosis. Therefore, XPD gene is likely to be potential molecular target for treatment of atherosclerosis.     

Recombinant human interleukin-10 inhibits vascular smooth muscle cell proliferation by TNF-α and PDGF-BB in vitro

AIM: To determine the effects of recombinant human interleukin-10(rhIL-10) on proliferation of vascular smooth muscle cells(VSMC) by TNF-α and PDGF-BB and neointimal hyperplasia after rat carotid arterial injury.METHODS: Rat aortic VSMC was cultured and treated with rhIL-10 with or without tumor necrosis factor-α(TNF-α) and platelet-derived growth factor-BB (PDGF-BB), respectively.Proliferation of VSMC was quantified by colormetric assay.Cell cycle analysis was performed by flow cytomertry.SD rats were treated with recombinant human IL-10(rhIL-10) for 3 days after carotid arteries injury.Neointima to media area ratio at the site of arterial injury was measured at 28 days after balloon injury.RESULTS: Compared to control,both TNF-α and PDGF-BB stimulated VSMC proliferation. rhIL-10 alone had no effect on VSMC growth.With TNF-α or PDGF-BB stimulation,rhIL-10,at dose as low as 10 μg/L,inhibited VSMC growth( P <0.05) for both cases.Cell number in G 0/G 1 phase of PDGF-BB and rhIL-10 co-treatment group was higher than those of PDGF-BB treatment alone( P <0.01) by flow cytometry analysis.The same results were observed in TNF-α and rhIL-10 co-treatment group( P <0.01).Compared with the arterial injury group,neotima/media area ratio of recombinant human IL-10 group was reduced at 45%( P <0.01).CONCLUSIONS: The anti-inflammatory cytokine rhIL-10 inhibits TNF-α and PDGF-BB-induced VSMC proliferation,respectively.These results suggest the possibility that recombinant human IL-10 as a potential therapeutic approach prevents neointimal hyperplasia.     

Preparation of recombinant human soluble TRAIL and its inducing effect on apoptosis of tumor cells by TRAIL combined with bortezomib

AIM: To construct a prokaryotic expression plasmid to produce recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and to verify the biological activity of TRAIL. METHODS: The prokaryotic expression plasmid pET-28a (+)-TRAIL_114-281 was constructed. Human soluble TRAIL was obtained through optimized inducing protein expression and purification conditions. The biological activity of TRAIL was verified by CCK-8 assay. The apoptosis-inducing effect of TRAIL alone and/or in combination with proteasome inhibitor bortezomib (Velcade, PS-341) on the tumor cell lines H460(TRAIL-sensitive) and K562(TRAIL-resistance) for 24 h was determined. The apoptotic rates of the cells were analyzed by flow cytometry with Annexin V-FITC/PI staining. The activities of caspase-8, -9 and -3 in the cells were detected by colorimetric method. The protein expression of Bax, Bcl-2 and cFLIP was measured by Western blot. The expression of DR4 and DR5 in the H460 cells and K562 cells after treated with bortezomib for 24 h was detected by flow cytometry. RESULTS: The recombinant human soluble TRAIL protein with stable bioactivity was successfully acquired, which induced apoptosis in H460 cells and K562 cells. After treatment with different concentrations of TRAIL, the apoptotic rate of H460 cells was significantly increased with the increase in the concentration of TRAIL (P<0.05), but the apoptotic rate of K562 cells was not affected by the increasing TRAIL concentration. Apoptotic rate in combination group was obviously higher than that in single group (P<0.05). In the process of apoptosis, the activities of caspase-8, -9 and -3 in H460 cells and K562 cells were both increased. The expression of Bcl-2 and cFLIP in treatment groups (especially the combination group) was decreased compared with control group. No significant change of the Bax expression level was observed. The expression of DR4 and DR5 in the H460 cells and K562 cells was significantly up-regulated after treated with bortezomib (P<0.05). CONCLUSION: Bortezomib combined with recombinant human soluble TRAIL synergistically induces apoptosis in tumor cell lines H460 and K562 through initiating intrinsic apoptotic pathways by up-regulating death receptors DR4 and DR5, and reducing the expression of antiapoptotic proteins Bcl-2 and cFLIP.