The regulation of the TACE pro-domain to tumor necrosis factor-α converting enzyme activity

AIM: To study functions of pro-domain in tumor necrosis factor-α converting enzyme (TACE) maturation and to develop an approach to interfere with inflammation processes. METHODS: The plasmid pIRES2-EGFP was used to generate the expression plasmids constructed with the signal peptide and pro-domain, full length or without the pro-domain(designated as pIRES2-EGFP/T648, pIRES2-EGFP/T2472, pIRES2-EGFP/T57-T1824,respectively). The plasmids were used to transfect the U937 cells.After the transfected cells were stimulated by LPS, the effect of expression plasmids on TNF-α secretion was detected by ELISA and flow cytometry(FCM).RESULTS: The plasmid pIRES2-EGFP/T648 inhibited 61.09% TNF-α secretion and mediated the accumulation of TNF-α on the surface of U937 cells.The sTNF-α secretion and the level of the mTNF-α in the plasmid pIRES2-EGFP/T57-T1824 transfected cells had no significant deviation compared with the pIRES2-EGFP transfected cells. The sTNF-α secretion of the cells transfected by the plasmid pIRES2-EGFP/T2472 increased and the level of the mTNF-α decreased.CONCLUSION: The pro-domain has dual effects in TACE maturation and might be applied to molecular design of anti-inflammation drug.     

Effect of rhIL-10 on IL-6 and TNF-α levels in serum and liver of lipopolysaccharide-challenged mice

AIM: To investigate the effect of recombinant human interleukin-10 (rhIL-10) on IL-6 and TNF-α levels in serum and liver of mice exposed to lipopolysaccharide (LPS). METHODS: rhIL-10 was prepared by using genetic engineering technology. Mice were intraperitoneally with 500 μg of LPS, and then were treated intravenously with various dosages of rhIL-10. The levels of IL-6 and TNF-α in hepatic tissue and serum were determined by ELISA at 12 h, 24 h, 48 h and 72 h post rhIL-10 treatment. RESULTS: rhIL-10 markedly inhibited the increase in IL-6 and TNF-α levels in hepatic tissue and serum at 12 h after rhIL-10 treatment in LPS-challenged mice, and the inhibition effect was significant at 24-48 h after rhIL-10 treatment （P＜0.05）. CONCLUSION: rhIL-10 can inhibit the increase in IL-6 and TNF-α levels induced by LPS in mice.     

Effects of lipopolysaccharide binding protein on activation of p38 signaling pathway induced by LPS in macrophages

AIM:To investigate the regulatory effects of lipopolysaccharide binding protein(LBP)on activation of p38 signaling pathway induced by lipopolysaccharide(LPS)in alveolar macrophages.METHODS:The LBP from actue phase rat serum was purified by ammonium sulphate precipitation, Bio-Rex70 resin and the MonoQ column. Rat alveolar macrophages were exposed to LPS (0.01 mg/L or 1 mg/L) the various concentrations of LBP(0 mg/L, 0.01 mg/L, 0.1 mg/L, 1 mg/L and 10 mg/L).Western blotting were used to detect phospho-p38 in alveolar macrophages. RESULTS:SDS-PAGE analysis indicated that the purified preparation of rat LBP showed homogeneity and the molecu-lar weight was 60 kD.The binding of lipopolysaccharide to mononuclear cells were enhanced by purified rat LBP.Stimu-lation of rat alveolar macrophages with LPS at concentration of 0.01 mg/L was LBP dependent.LBP at concentrations up to 1 mg/L was able to increase the activation of p38.However, when LBP concentrations were further increased to 10mg/L, the phosphorylation levers of p38 were lower as compared with that in the presence of 1 mg/L.Stimulation of ratalveolar macrophages with LPS at concentrations of 1 mg/L was LBP-independent.CONCLUSION:The activation of p38 induced by LPS at lower concentration(0.01 mg/L) was LBP-dependent, meanwhile, LPS at higher concentration(1 mg/L) was LBP-independent.     

Doxycycline upregulates autophagy to modulate the levels of inflammatory cytokines in LPS-stimulated THP-1 cells

AIM:To analyze the effect of autophagy on inflammatory response regulated by doxycycline in lipopolysaccharide (LPS)-stimulated THP-1 cells and to investigate its molecular mechanism. METHODS:A human monocyte/macrophage cell line THP-1 was stimulated with LPS to establish an cell model of inflammatory response, and the cells were treated with doxycycline. The cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8), in cell culture supernatant were measured by ELISA for evaluating the inflammatory levels. For determining the level of autophagy and its effect on inflammatory cell signaling pathways, the protein levels of LC3B, nuclear factor κB (NF-κB) and phosphorylated mammalian target of rapamycin (p-mTOR) were determined by Western blot. 3-Methyladenine (3-MA), an autophagy inhibitor, and rapamycin, an autophagy inducer, were used to study the effect of autophagy on inflammatory response regulated by doxycycline in LPS-stimulated THP-1 cells. RESULTS:The levels of TNF-α and IL-8 were increased rapidly and peaked at 12 h in LPS-stimulated THP-1 cells (P<0.05). Doxycycline significantly inhibited LPS-induced cytokine production in the THP-1 cells. Doxycycline up-regulated LPS-induced autophagy in THP-1 cells and doxycycline itself was an autophagy inducer. The protein levels of p-mTOR was up-regulated by LPS and down-regulated by doxycycline, suggesting that doxycycline induced autophagy via mTOR-dependent pathway while LPS through mTOR-independent pathway. Further studies showed that the combination of LPS, rapamycin and doxycycline inhibited the protein levels of NF-κB, and rapamycin increased the inhibitory effect of doxycycline on cytokine releases. Conversely, 3-MA, the autophagy inhibitor, attenuated the inhibitory effect of doxycycline on NF-κB and cytokine production. CONCLUSION:Autophagy is involved in the process of doxycycline modulating LPS-induced inflammatory response in the THP-1 cells.     

Combination of sorafenib and daunorubicin has antileukemic activity on K562 cells

AIM: To investigate the synergetic inhibitory effect of sorafenib and daunorubicin (DNR) on K562 and U937 cells. METHODS: The inhibitory rate of sorafenib or daunorubicin alone, and the combined inhibitory rate of sorafenib and IC10 daunorubicin were measured by MTT assay. Apoptotic rate of single drug or combination was assessed by flow cytometry (Annexin Ⅴ/PI staining) and Hoechst 33258 staining assay. p-ERK1/2 level was detected by Western blotting after the cells were treated with sorafenib, daunorubicin and U0126 or combinations. Synergistic or antagonistic effect of proliferation and apoptosis on K562 and U937 was estimated according to the Jins Method. RESULTS: Combination of sorafenib and DNR showed synergistic growth inhibition (q>1.15, P<0.01) and synergistic promotion of apoptosis (q>1.15, P<0.05) in K562 and U937 cells. The level of p-ERK1/2 in K562 cells was obviously higher than that in U937 cells (P<0.01). p-ERK1/2 expression was completely inhibited in sorafenib or U0126 treated K562 cells for 24 h. Combination of U0126 with DNR inhibited the proliferation of K562 cells synergistically. CONCLUSION: Combination of sorafenib with DNR showed synergistic cell growth inhibition and promotion of apoptosis in K562 and U937 cells. U937 cells were more sensitive to DNR than K562 cells while K562 cells were more sensitive to sorafenib. Sorafenib enhances the anti-leukemic activity of DNR in K562 and U937 cells via down-regulation of p-ERK1/2 expression.     

Influence of glycine on LBP mRNA expression induced by LPS in the liver of rats

AIM:To study the influence of glycine(GLY) on lipopolysaccharide-binding protein(LBP) mRNA expression induced by LPS.METHODS:The level of LBP mRNA expression in liver tissues of rats was examined by RT-PCR, and the effects of glycine on LBP mRNA expression in liver tissues of rats induced by LPS were investigated.RESULTS: The level of LBP mRNA expression in hepatic tissue of rats in the LPS group was significantly higher than that in the control group(P＜0.01), the level of LBP mRNA expression in the hepatic tissue of rats in the LPS+GLY group was lower than that in the LPS group(P＜0.01).CONCLUSION:LPS can induce LBP mRNA expression in the hepatic tissue of rats, glycine can inhibit LBP mRNA expression in the hepatic tissue of rats treated by LPS.     

Effect of mininally modified low density lipoprotein on the adhesive function of vascular endothelial cell and its mechanism

AIM: To observe the effect of MM-LDL (minimally modified LDL) on the interaction between human umbilical vein endothelial cell (HUVEC) and U937 monocyte-like cell line and the exporession of vascular cell adhesion-1 (VCAM-1), intercellular adhesion molecule-1(ICAM-1), P-selectin.METHODS:The adhesive percentage between HUVEC treated with MM-LDL and U937 was determined by counting and the expression of VCAM-1, ICAM-1, P-selectin were examined by ELISA. RESULTS: Treatment of HUVEC with MM-LDL (75 mg/L) for 4 hours significantly increased adhesion of U937 to HUVEC ( P <0.01) and did not induce the surface expression of VCAM-1, ICAM-1, P-selectin . Recombination tumor necrosis factor-alpha (rTNF_α) 5.0 μg/L, as a positive control, induced the expression of these adhesion molecules ( P <0.05). Prolonged (18 h) exposure to MM-LDL resulted in the expression of P-selectin, but not VCAM-1.CONCLUSION: the adhesion of monocytes to endothelial cells induced by MM-LDL is not mediated by VCAM-1, ICAM-1. P-selectin induction may be partly involved in the process.     

Effects of thymopeptide and 4 polysaccharides isolated from Chinese medicine on immune functions of spleen and tumor tissues in U14 cervical cancer-bearing mice

AIM: To identify the enhancement of relatively specific immune responses by thymopeptide and 4 polysaccharides isolated from traditional Chinese medicine in the mice bearing cervical cancer. METHODS: The model of cervical cancer-bearing mice was established by implantation of cervical cancer U14 cells into the armpit of the right forelimb of the mice. Beginning from the 2nd day of tumor implantation, 4 polysaccharides Lycium barbarum polysaccharide (LBP), Angelica sinensis polysaccharide (ASP), Ganodema lucidum polysaccharide (GLP) and Panax ginseng polysaccharide (PGP)], thymopeptide or saline solution were intragastric administered for 21 days according to the experimental design. The animals were then killed, the tumor inhibitory rate and spleen index in treatment groups were compared with those in control group. The splenic T-lymphocytes (TLC) and natural killer (NK) cells, and the content of IFN-γ and IL-10 in the tumor tissues were also determined by flow cytometry and immunohistochemical methods. RESULTS: The order of tumor inhibitory rates was: ASP < PGP < thymopeptide < LBP < GLP. The effects of GLP, LBP and thymopeptide were statistically different from that of control group. No difference of tumor inhibition between PGP/ASP groups and control group was observed. The order of spleen index was: ASP < tumor control group < thymopeptide < GLP < PGP < LBP, that of CD4/CD8 enhancement was: PGP < ASP < GLP < thymopeptide < LBP, and that of CD49b⁺ NK hyperplasia was: ASP < thymopeptide < LBP < GLP < PGP,with statistically significant differences among the groups. The changes of IFN-γ and IL-10 in the tumor tissues showed that Th1/Th2 in thymopeptide group, LBP group and GLP group shifted to Th1. No shift to Th1 in PGP group and ASP group was observed. CONCLUSION: LBP, GLP and thymopeptide show significant inhibitory effects on tumor growth in U14 cervical tumor-bearing mice and can enhance the immune functions in the animals.     

Coupling of actin with IRAK-NF-κB in bacterial lipoprotein induced cross tolerance in monocytes

AIM: Tolerance to bacterial wall components, such as bacterial lipoprotein (BLP) and lipopolysaccharide (LPS), is an adaptive host response. The current study was aimed to explore the role of actin cytoskeleton in BLP-tolerance and BLP cross-tolerance in monocytes. METHODS: Cellular model of BLP-tolerance induced by pre-exposed to low dose BLP was established in human monocyte cell line (THP-1). The concentration of proinflammatory cytokines (TNF-α, IL-1β, IL-6) was measured by ELISA. Actin was stained with FITC-phalladin. The NF-κB DNA binding activity was tested by EMSA. RESULTS: When THP-1 cells were insulted with LPS (100 μg/L) or BLP (100 μg/L), the cellular morphology changed significantly with the formation of pseudopod and the actin reorganized to form the actin-band, interleukin 1 receptor-associated kinase-1(IRAK-1) activity and NF-κB DNA binding activity upregulated, and the concentration of cytokins (TNF-α, IL-1β and IL-6) in supernatant increased. Pretreatment with BLP (10 μg/L) improved the morphologic changes and decreased pseudopod formation. IRAK-1 activity and NF-κB DNA binding activity were downregulated, and the release of cytokins (TNF-α, IL-1β and IL-6) decreased after insulted with BLP (100 μg/L) or LPS (100 μg/L). Phalladin, a specific actin cytoskeleton reorganization inhibitor, partly abolished the BLP tolerance and BLP cross-tolerance in THP-1 cells induced by 10 μg/L BLP, reflected by the upregulation of IRAK-1 activity and NF-κB DNA binding activity and the increase in cytokins (TNF-α, IL-1β and IL-6) release. CONCLUSION: Coupled with IRAK-NF-κB signal cascade, actin cytoskeleton plays an important role in BLP tolerance and BLP cross tolerance in THP-1 cells induced by pretreatment of BLP.     

Effect of monoclonal antibody to CD47 molecule on dendritic cell differentiation and function

AIM: To explore the influence of CD47 molecules on the maturation and function of cultured dendritic cells (DCs). METHODS: Monocyte cell-derived DCs were propagated in granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) plus interleukin (IL)-4, in the presence or absence of anti CD47 monoclonal antibodies (anti-CD47 mAbs). Flow cytometry was used to detect the cell surface phenotype. The concentration of IL-12P70 in supernatant was measured by ELISA technique. The antigen-presenting functions of DCs were determined in one-way mixed leukocyte reaction by Brdu-ELISA. Electrophoretic mobility shift assays (EMSA) was used to examine NF-κB activity. RESULTS: The anti-CD47 mAbs markedly suppressed the expression of CD80,CD86,CD83,CD1a,HLA-DR on the surface of DCs (P＜0.05). The data of mixed leukocyte reaction and IL-12P70 production were consistent with the flow cytometry results (P＜0.01). Nuclear extracts from the anti-CD47 mAb-treated DCs revealed a decrease in NF-κB binding activity. CONCLUSION: The anti-CD47 mAb exerts a negative effect on the maturation and function of in vitro cultured DCs via inhibiting of NF-κB binding activity.     

ARHI gene inhibits cell growth,induces G2/M phase arrest and apoptosis of acute myeloid leukemia cell line U937

AIM: To investigate the expression of aplasia rashomolog member I (ARHI) gene in acute myeloid leukemia cells (AML) and to study the effects of ARHI on the growth of AML cell line U937.METHODS: The mRNA expression of ARHI in AML cells, 293FT cells, AML primary cells and healthy volunteer blood cells were detected by RT-PCR. After transfection with the MSCV-IRES-GFP-ARHI plasmid to the U937 cells, the growth curve was analyzed by MTT assay. U937 cells were re-suspended by fresh medium and cultured for 24 h, then the cell cycle distribution and apoptotic rate were determined. RESULTS: The mRNA of ARHI was positively detectable in 293FT cells and healthy volunteer blood cells instead of AML cell line and AML primary cells. The growth curve showed that cell viability in U937 cells with high expression of ARHI (U937-ARHI) was lower than that in the control cells (U937-GFP) on 6th~8th day. The ratio of G₂/M phase and apoptotic rate in the U937-ARHI cells were increased compare with control group(P<0.05). CONCLUSION: The mRNA level of ARHI is low in AML cells. High expression of ARHI gene in U937 cells inhibits cell growth, arrests the cells at G₂/M phase and induces apoptosis.     

uclear factor κB in LPS stimulated rat alveolar macrophages promotes TNF-α secretion

AIM: To investigate the activation of nuclear factor κB (NF-κB) induced by lipopolysaccharides (LPS) in rat alveolar macrophages (AMs) and its regulatory role in tumor necrosis factor (TNF-α) secretion. METHODS: The dynamic activity changes of NF-κB induced by LPS were determined with electrophoretic mobility shift assay (EMSA). Antisense oligonucleotides of NF-κB subunit (p65) were transfected into AMs prior to LPS stimulation. The effect of antisense oligonucleotide transfection on expressions of p65 and TNF-α in supernatant were measured with Western blotting and enzyme linked immunosorbent assay (ELISA), respectively.RESULTS: NF-κB activity increased markedly and reached its peak level at 4 h after LPS stimulation. After transfected with antisense oligonucleotides of NF-κB subunit (p65), expression of p65 and TNF-α in supernatant decreased markedly.CONCLUSION: NF-κB activity has a positive effect on regulating secretion of TNF-α in AMs induced by LPS.     

Rapamycin inhibits HMGB1 expression and releases in RAW264.7 cells induced by lipopolysaccharides in vitro

AIM: To observe the mechanism that rapamycin (RPM) affects HMGB1 expression and release in RAW264.7 cells induced by lipopolysaccharides (LPS).METHODS: RAW264.7 cells were cultured in six wells plate and divided into five groups: control group, 250 μg/L LPS treatment group, 100 μg/L RPM treatment group, 50 μg/L rTNF-α treatment group and 100 μg/L TNF-α antibody treatment group. After 4 h treatment, the TNF-α level in the culture media was evaluated by ELISA assay. After 24 h, the expression of HMGB1 mRNA was measured by RT-PCR, and HMGB1 protein level in the culture media was determined by Western blotting analysis.RESULTS: TNF-α level in the culture media of RAW264.7 cells has no significant difference between RPM treatment group and control group (P>0.05). Both HMGB1 mRNA expression and HMGB1 protein level were remarkably higher in LPS treatment group than that in control group (P<0.05). RPM attenuated LPS-induced HMGB1 mRNA and HMGB1 accumulation. Compared with that in RPM treatment group, HMGB1 accumulation was increased in rTNF-α treatment group, and had no significant difference in TNF-α antibody treatment group (P>0.05).CONCLUSION: RPM inhibits HMGB1 expression not only by directly suppressing STAT3 activation, but also by indirectly reducing TNF-α level.     

Regulation of ATP-binding acssette transporter A1 on apolipoprotein E secretion from macrophages

AIM: To investigate the regulatory effect of the adenosine triphosphate binding cassette transporter A1 (ABCA1) on apolipoprotein E secretion from human THP1 macrophages.METHODS: Differentiation of THP1 macrophages from monocytes was stimulated with phorbol 12-myristate 13-acetate. The macrophages then were incubated with factors which regulate ABCA1 expression. After periods of incubation, apo E secreted in the medium and synthesized in the cell was determined with ELISA, and apo E mRNA espression was detected with Northern blot.RESULTS: An increase in apo E secretion from THP1 macrophages was observed by 8 h of incubation with 8-Br-cAMP, an activator of ABCA1 expression (P＜0.05). Glyburide, a putative ABCA1 inhibitor, and antisense oligonucleotides specifically against ABCA1 mRNA remarkably decreased apo E secretion from both THP1 macrophages and macrophage foam cells (P＜0.01,respectively). Neither apo E mRNA expression nor intracellular apo E level in THP1 macrophages was changed by inhibition of ABCA1.CONCLUSION: ABCA1 may promote the secretion of apo E from macrophages and macrophage foam cells and the effect may occur at the level of post-translation. The present results reveal a new aspect underlying antiatherogenic properties of ABCA1.     

Unfractionated heparin ameliorates lipopolysaccharide-induced expression of interleukin-8 in human endothelial cells through Toll-like receptor 4 signaling

AIM:To determine the effect of unfractionated heparin(UFH) on lipopolysaccharide(LPS)-induced expression of interleukin-8(IL-8) and the role of Toll-like receptor 4(TLR4) signaling. METHODS:Human pulmonary microvascular endothelial cells(HPMECs) were either exposed to LPS alone(10 mg/L) or in combination with 100 U/L or 10³ U/L UFH. UFH was added to the cells 15 min prior to stimulation with LPS. Those samples not receiving LPS or UFH received an equal volume of phosphate-buffered saline. The concentrations of IL-8 in the cell culture supernatants were detected by ELISA at 2, 6 and 12 h. The mRNA expression of IL-8, CD14 and TLR4 in HPMECs was detected by real-time fluorescence quantitative polymerase chain reaction at 2, 6 and 12 h. RESULTS:Compared with normal control group, the mRNA expression of IL-8 in LPS group was increased, and reached the peak at 6 h. The protein level of IL-8 reached the peak at 12 h. The mRNA expression of TLR4 in LPS group reached the peak at 6 h. They were down-regulated in UFH group. The mRNA expression of CD14 was not detected. CONCLUSION:The expression of IL-8 is obviously increased in LPS-treated HPMECs. UFH might exert its therapeutic effect through TLR4 signaling.     

Role of TRAF6 in maturation of human PBMC-derived dendritic cells

AIM: To investigate the immunological effect of tumor necrosis factor receptor-associated factor 6 (TRAF6) on the maturation of dendritic cells in vitro.METHODS: The human peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), induced by recombined human granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, were stimulated to mature with TNF-α, LPS or cocktail of cytokines (TNF-α, IL-6, IL-1β and PGE₂).The cell surface markers, T-cell stimulatory proliferation capacity and IL-12 p40 production of the DCs were determined by the methods of flow cytometry, ELISA and mixed lymphocyte reation (MLR), respectively.The mRNA expression of TRAF6 was detected by real-time PCR.RESULTS: The expression of TRAF6 was observed in all groups, which in cocktail group was the highest in the DCs with optimum state of maturation.Furthermore, TRAF6 enhanced the production of IL-12 and the ability of T-cell stimulation of mature DCs.CONCLUSION: TRAF6 might play an important role in inducing the maturation of human PBMC-derived DCs.