Mycophenolic Acid Synergizing with Lipopolysaccharide to Induce Interleukin-1β Release via Activation of Caspase-1 / 中华医学杂志(英文版)

<p><b>Background</b>The previous study showed that mycophenolic acid (MPA) synergizing with lipopolysaccharide (LPS) promoted interleukin (IL)-1β release, but the mechanism is unclear. This study aimed to investigate the mechanism of MPA synergizing with LPS to induce IL-1β release.</p><p><b>Methods</b>Undiluted human blood cells, THP-1 human myeloid leukemia mononuclear cells (THP-1) cells, or monocytes were stimulated with LPS and treated with or without MPA, and the supernatant IL-1β was detected by enzyme-linked immunosorbent assay. The mRNA levels of IL-1β were detected by real-time quantitative polymerase chain reaction. The intracellular protein levels of nuclear factor kappa B (NF-κB) phospho-p65 (p-p65), precursor interleukin-1β (pro-IL-1β), NOD-like receptor pyrin domain containing-3 (NLRP3), and cysteine aspartic acid-specific protease-1 (caspase-1) p20 in THP-1 cell were measured by Western blot.</p><p><b>Results</b>The MPA alone failed to induce IL-1β, whereas MPA synergized with LPS to increase IL-1β in a dose-dependent manner (685.00 ± 20.00 pg/ml in LPS + 5 μmol/L MPA group, P = 0.035; 742.00 ± 31.58 pg/ml in LPS + 25 μmol/L MPA group, P = 0.017; 1000.00 ± 65.59 pg/ml in LPS + 75 μmol/L MPA group, P = 0.024; versus 408.00 ± 35.50 pg/ml in LPS group). MPA alone has no effect on the IL-1β mRNA expression, LPS induced the expression of IL-1β mRNA 2761 fold, and LPS + MPA increased the IL-1β expression 3018 fold, which had the same effect with LPS group (P = 0.834). MPA did not affect the intracellular NF-κB p-p65 and pro-IL-1β protein levels but activated NLRP3 inflammasome. Ac-YVAD-cmk blocked the activation of caspase-1 and subsequently attenuated IL-1β secretion (181.00 ± 45.24 pg/ml in LPS + MPA + YVAD group vs. 588.00 ± 41.99 pg/ml in LPS + MPA group, P = 0.014).</p><p><b>Conclusions</b>Taken together, MPA synergized with LPS to induce IL-1β release via the activation of caspase-1, rather than the enhanced production of pro-IL-1β. These findings suggested that patients immunosuppressed with mycophenolate mofetil may have overly activated caspase-1 during infection, which might contribute to a more sensitive host defense response to invading germs.</p>

Effects of decitabine on proliferation and apoptosis of NB4 and K562 cells / 中国实验血液学杂志

This study was aimed to investigate the effects of decitabine (DAC) on proliferation and apoptosis of leukemia NB4 and K562 cells. The proliferation inhibition of DAC on NB4 and K562 cells was detected by Trypan blue staining. After treatment of DAC at different concentrations, the changes of cell cycle and CD11b expression was determined by flow cytometry. The cell morphological changes were observed by Wright's staining. The DNA ladder was used to detect cell apoptosis. The results indicated that DAC significantly inhibited the proliferation of NB4 and K562 cells in dose-and time-dependent manner. The median inhibitory concentration (IC50) of DAC-treated NB4 and K562 cells for 72 h was 0.113 µmol/L and 0.138 µmol/L, respectively. After treating these two cell lines with DAC at different concentration for 72 h, the cell ratio in G0/G1 phase significantly increased, while the cell ratio in S phase obviously decreased in 0.15 µmol/L DAC group (P < 0.05). The expression levels of myeloid differentiation antigen CD11b of both cell lines significantly increased in contrast to the control group (P < 0.05). The cell morphology detected by Wright's staining displayed partial differentiation and apoptosis after treating NB4 and K562 cells with DAC for 48 h. Typical apoptotic DNA ladder was observed in 0.15 µmol/L DAC group at 48 h. It is concluded that DAC can inhibit NB4 and K562 cell proliferation, induce cell differentiation and apoptosis, but more obviously for NB4 cells.

Impact of transfusion of apoptotic and necrotic thymocytes on the survival of mice with sepsis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of transfusion of apoptotic and necrotic thymocytes prior to sepsis on the survival rate of mice.</p><p><b>METHODS</b>BALB/c mice are divided into 3 groups and received intravenous injection of PBS (control), apoptotic thymocytes, or necrotic thymocytes. Three days later, cecal ligation and puncture (CLP) were performed to induce sepsis in these mice, and their survival and organ damage were observed.</p><p><b>RESULTS</b>The survival rates of mice in PBS group was 44.6% at the end of first week after CLP, and obvious lung and kidney damages were observed. A significant increase in the survival rate was found in apoptotic cell transfusion group (69.6%, P=0.012), with also lessened lung and kidney damages. The survival rate of mice in necrotic cell transfusion group was only 31.6% at 2 weeks, significantly lower than that in PBS group (P=0.035), and the lung and kidney damage was even more obvious.</p><p><b>CONCLUSION</b>Transfusion of apoptotic thymocytes 3 days before induction of sepsis can reduce organ damage and improve the survival rate of mice, while necrotic cell transfusion produces the opposite effect.</p>

Differences in the response to sepsis between C57BL/6 and BALB/c mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the responses to sepsis between C57BL/6 and BALB/c mice.</p><p><b>METHODS</b>Thirty C57BL/6 mice and 30 BALB/c mice were randomized into sham-operated group and sepsis group (n=15). Sepsis model was established by cecal ligation puncture (CLP) in the mice, and 6 h after the operation, 5 mice from each group were selected randomly for cytokine detection including IL-1beta, IL-2, IL-4, IL-5, IL-10, GM-CSF, IFN-gamma and TNF-alpha by Bio-plex. The other 10 mice in each group were used for survival analysis.</p><p><b>RESULTS</b>The survival rates of BALB/c and C57BL/6 mice were both 100% in one week after the sham operation, but lowered to 10% and 50% in one week after CLP, respectively. The survival rate of C57BL/6 mice was significantly lower than that of BALB/c mice (P<0.05). After CLP, C57BL/6 mice showed significantly greater IL-4, TNF-alpha and IL-10 production than the sham-operated mice, but the concentrations of the 8 cytokines in BALB/c mice after CLP showed no significant increment.</p><p><b>CONCLUSION</b>Compared with BALB/c mice, C57BL/6 strain mouse is more sensitive to sepsis.</p>

Effect of FK506 on cytokine secretion in whole blood / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of FK506 on cytokine secretions in whole blood from healthy individuals.</p><p><b>METHODS</b>Blood samples collected from healthy volunteers were co-cultured with different concentrations of FK506 and stimulated with PMA and IONO. The concentrations of 8 cytokines including IL-2, IL-6, IL-12, IL-17, IFN-gamma, TNF-alpha, GM-CSF and G-CSF were detected by Bio-Plex suspension system.</p><p><b>RESULTS</b>Compared with the control group, high-concentration FK506 (20 ng/ml) significantly inhibited the secretions of IL-2, IL-6, IL-12, IL-17, IFN-gamma, TNF-alpha, GM-CSF and G-CSF. At a moderate concentration (5 ng/ml), FK506 inhibited the secretion of GM-CSF significantly.</p><p><b>CONCLUSION</b>FK506 effectively inhibits the secretion of proinflammatory cytokines including IL-6, IFN-gamma and TNF-alpha and also the secretion of IL-2, IL-12, IL-17, GM-CSF and G-CSF. FK506 might play the role of immunosuppression by inhibiting the production of these cytokines by the immune cells. Monitoring the levels of these cytokines might be a potential method for evaluating the adequacy of FK506 doses administered.</p>

Necrotic cells induce and promote inflammatory responses in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of necrotic cells on the secretion of inflammatory cytokines.</p><p><b>METHODS</b>RAW264.7 macrophages and necrotic mouse thymocytes induced by heating were incubated for 18 h at a ratio of 5:1 in the absence or presence of lipopolysaccharide (LPS, 100 ng/ml). The supernatant of the cell culture was collected and the expression and secretion of the pro-inflammatory cytokines were measured using Bio-Plex suspension system.</p><p><b>RESULTS</b>The secretions of tumor necrosis factor-alpha (TNF-alpha) and interlukine-6 (IL-6) by macrophages co-cultured with the necrotic cells were significantly enhanced as compared with the control cells. The necrotic cells also significantly augmented the secretion of the pro-inflammatory cytokines induced by LPS.</p><p><b>CONCLUSION</b>Necrotic cells not only induces pro-inflammatory cytokine expression by themselves but also work synergistically with LPS to enhance the cytokine production, suggesting the important roles of necrotic cells to initiate and maintain the inflammatory responses.</p>

Effects of apoptotic lymphocytes on proinflammatory cytokine secretion by hepatic sinusoidal endothelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of apoptotic lymphocytes on the secretion of cytokines by hepatic sinusoidal endothelial cells (HSEC).</p><p><b>METHODS</b>Human HSEC cells were co-cultured for 16 h with allogenetic apoptotic lymphocytes induced by UVB irradiation. The supernatants were collected and the levels of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha were detected by Luminex technique.</p><p><b>RESULTS</b>All the cytokines were down-regulated by about 50% in HSECs after co-culture with the apoptotic lymphocytes as compared with those in the control group (P<0.05).</p><p><b>CONCLUSIONS</b>Co-culture with apoptotic lymphocytes can down-regulate the secretion of pro-inflammatory cytokines in HSECs, which may contribute to tolerogenic microenvironment in the liver.</p>

Apoptosis induction of rhesus peripheral blood lymphocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a method for inducing apoptosis of rhesus peripheral blood lymphocytes (PBLs).</p><p><b>METHODS</b>Rhesus PBLs were irradiated with X-ray, (60)Co gamma-rays and ultraviolet (UVC254 nm), respectively, and the cell apoptosis was evaluated with flow cytometry using annexin-V staining and propidium iodide staining.</p><p><b>RESULTS</b>X-ray and (60)Co gamma-ray irradiation induced only low apoptotic rates of the PBLs, and UVC resulted in the highest apoptotic rate of about 60%. UVC irradiation of the PBLs in RPMI supplemented with 10% heat-inactivated fetal calf serum for 60 min at a distance of 20 cm led to an early apoptotic rate of 58.85% and necrotic rate of 11.5%. The apoptotic rate of PBLs increased in a dose- and time-dependent fashion.</p><p><b>CONCLUSION</b>For inducing apoptosis of the rhesus PBLs, UVC can be more effective than X-ray and (60)Co gamma-ray. The highest apoptotic rate can be achieved when the rhesus PBLs in RPMI supplemented with 10% heat-inactivated fetal calf serum are exposed to UVC for 60 min at the distance of 20 cm.</p>

In vivo tracing of transferred apoptotic cell labeled using CFSE: a flow cytometry-based assay method / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an assay method for detecting the migration of transferred apoptotic cells into the recipient using flow cytometry.</p><p><b>METHODS</b>Spleen lymphocytes were isolated and labeled with an intracellular amine dye, carboxyfluorescein diacetate succinimidyl ester (CFSE), to allow discrimination. The labeled cells were induced with dexamethasone to undergo apoptosis and transferred into recipient mice via tail venous transfusion. Flow cytometry and histological examination of different tissues were performed at different time points. The stability of CFSE labeling for apoptotic cells was also tested.</p><p><b>RESULTS</b>The CFSE-labeled apoptotic cells were highly fluorescent with a positive labeling rate of (98.0+/-1.9)%. The stability of CFSE-labeling was testified, and the CFSE-labeled apoptotic cells entering different tissues at different time points were detected by flow cytometry and verified by histological examination.</p><p><b>CONCLUSION</b>Flow cytometry using CFSE labeling is reliable, sensitive, precise and convenient for apoptotic cell tracing in vivo and in vitro.</p>

The phagocytosis of apoptotic allogeneic lymphocytes by recipient liver antigen-presenting cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To track the location of the transfused apoptotic allogeneic lymphocytes and asses the process of their accumulation and phagocytosis removal as consequences on allograft tolerance in recipient mice.</p><p><b>METHODS</b>Donor spleen lymphocytes were labeled by CFSE and induced to apoptosis by dexamethasone incubation. After purification by anti-annexin V-conjugated magnetic beads isolation, apoptotic lymphocytes were transfused into recipient mice through the tail veins. Tissue samples from various organs were taken at various time points to analyze the fates of the apoptotic allogeneic lymphocytes and the phagocytosis of them by organ resident APCs.</p><p><b>RESULTS</b>Using fluorescent microscopy and flow cytometry, after the apoptotic cells were recognized and uptaken, the largest amount of labeled cells were accumulated in the livers and disappeared within not more than 12 hours. Recipient liver APCs were highly efficacious in phagocytosis of apoptotic allogeneic lymphocytes; the removal was completed within 15 minutes after incubation. LSEC, KC and LDC all phagocytosized the apoptotic allogeneic lymphocytes but with significantly different rates. Considering the numbers of those cells in a normal liver, it could be calculated that LSEC and KC had greater effects in this activity.</p><p><b>CONCLUSIONS</b>The liver deserves foremost attention for study of the mechanism of allografts tolerance induced by pre-transfusion of apoptotic donor spleen lymphocytes. LSEC and KC are the main functional APCs to the alloantigens.</p>

Protecting effects of p44/42 MAPK signal transduction pathway on hepatocytes in ischemic preconditioning / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the significance of PKC and p44/42 mitogen-activated protein kinase (MAPK) signal transduction in ischemic preconditioning (IP).</p><p><b>METHODS</b>Through liver cell IP models, PKC inhibitor and MEK inhibitor were utilized to analyze the phosphorylation level of p44/42 MAPK and cell viability was also observed. Rat liver IP models were established which were treated with various drugs. Then the phosphorylation level of p44/42 MAPK in vivo and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) concentrations were detected. And cellular structures were observed under light microscopy.</p><p><b>RESULTS</b>Similar results were obtained in vivo and in vitro IP models. Compared with the ischemia reperfusion (IR) group in vivo, the phosphorylation level of p44/42 MAPK was obviously increased in IP treated rats (q = 27.217, P < 0.01), and the cellular structure injured slightly. The concentrations of serum ALT and AST in IP group were significantly lower than those in IR group (281.0 U/L +/-35.6 U/L vs 762.8 U/L +/-130.5 U/L and 407.7 U/L +/-73.7 U/L vs 820.9 U/L +/-111.3 U/L, P < 0.01). However, opposite changes were found in PKC and MEK inhibited groups, when compared to IP group. The phosphorylation level of p44/42 MAPK was obviously decreased, the liver tissues injured evidently, and the concentrations of serum ALT and AST (645.61 U/L +/-90.4 U/L, 678.6 U/L +/-136.5U/L and 466.2 U/L +/-82.8 U/L, 732.9 U/L +/-91.1 U/L, respectively) were significantly greater than those in IP group.</p><p><b>CONCLUSION</b>These results suggest that p44/42 MAPK pathway plays a vital role in the protection of hepatocytes in ischemic preconditioning.</p>

Study the mechanisms and inducing transplantation immune tolerance of FTY720 / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the operational mechanisms and potential approach to inducing transplantation immune tolerance of FTY720.</p><p><b>METHODS</b>Mouse splenocytes were incubated with FTY720, then the DNA was extracted and analyzed using gel electrophoresis. Hearts of inbred BALB/c (H-2(d)) mice were transplanted heterotopically in C57BL/6 (H-2b) mice. Recipients were randomly divided into six groups. Group-1 (n = 6) was the nil-treated control. Groups-2, 3 and 4 were given FTY720 at the dose of 3 mg.kg(-1) by oral gavage once a day with different time courses. Group-2 (n = 14) were administrated from 3 days before transplantation (day-3) to the 11th day after the transplantation (day 11); Group-3 (n = 6) from day 0 to day 14; Group-4 (n = 6) from day-3 to day 0. Group-5 (n = 5) and 6 (n = 5) were treated with Cyclosporine A (10 mg.kg(-1)) and 40-O-(2-hydroxyethyl)-rapamycin (RAD) (3 mg.kg(-1)) respectively by daily gavage from day 3 to day 11. The long survivors (> 100 d) in Group-2 were detected with their IL-4 and IFN-gamma levels and their tolerant state was challenged with second graft: the donor type skin.</p><p><b>RESULTS</b>Apoptosis changes of the mouse splenocytes incubated with FTY720 was showed by typical DNA ladders. The median survival time (MST) of Group-1 was 8 d. MST of Group-2 was 55 d and grafts in six mice survived more than 100 d. MST of Group-3 was 16.5 d. Group-4 has a MST of 14 d with one case exceeded 100 d. MST of Group-5 and 6 were 10 d and 13 d respectively. Long survivors of Group-2 can accept donor-type skin graft and the level of IL-4 in their serum is up-regulated while IFN-gamma remained stable.</p><p><b>CONCLUSIONS</b>Pretreatment of FTY720 bring about effect on the early events of transplantation immune responses. This effect might be mediated by apoptosis induction in lymphocytes using this drug. We originally designed the regime of FTY720 monotherapy, which started pre-operationally and maintained for a short period of time, and induced stable tolerance the allo-graft in mouse.</p>