Therapeutic and protective effects of high-density lipoprotein of human plasma on endotoxemia in rats

AIM: To observe the therapeutic and protective effects of high-density lipoprotein (HDL) of human plasma on rat endotoxemia.METHODS: 26 rats were divided into 3 groups randomly,the treated group received bolus intravenous endotoxin (500 U/kg),followed by a bolus HDL (75 mg/kg) intravenously as their blood pressure (BP) decreased obviously.The control group just received bolus intravenous endotoxin without HDL.The protective group received bolus intravenous HDL before endotoxin challenge.BP,survival time,concentration of tumor necrosis factor(TNF) and levels of endotoxin in plasma were determined using radioimmunoassay and limulus lysate test.RESULTS: ①BP in control and treated groups all decreased obviously (P<0.01),and the extent of BP decrease in control group lowered more than that of treated group P<0.01),while no significant BP decrease was found in protective group (P>0.05).The survival time of treated and preventive rats were prolonged significantly than that of control group (P<0.01).②No obvious changes were found on the levels of endotoxin among the three groups (P>0.05).③On 3rd time point,the concentration of plasma TNFα decreased in treated and protective rats compared with that of control rats (P>0.05). CONCLUSION:HDL of human plasma could attenuate or inhibit the decrease in BP induced by endotoxin and prolong the survival time.These results indicated that HDL has therapeutic and protective effect on rat with endotoxemia.Inhibition of TNF release might be one of mechanisms.     

Effects of interferon-γ, tumor necrosis factor-α and interleukin-1β on apoptosis of human airway smooth muscle cellsin vitro

AIM:To clarify if interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro.METHODS:Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-γ,TNF-α and IL-1β, were used separately or together in the treatment of human ASMCs. The effects of IFN-γ,TNF-α and IL-1β on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p 53, bcl- 2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS:(1)IFN-γ or IFN-γ together with TNF-α and IL-1β decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic"ladder"of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p 53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-γ(4×10⁵ U/L),TNF-α(4×10⁵ U/L)and /or IL-1β (10×10⁴ U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group (P＜0.01).CONCLUSION:Stimultaneous treatment with IFN-γ,TNF-α and /or IL-1β induced apoptosis of human ASMCs. These immune cytokines may play an important role in airway remodeling of asthma and of chronic obstructive pulmonary disease.     

Epidermal growth factor receptor pathway participates in growth promotion of chondrocytes by growth hormone from sex hormone-inhibited adolescent rats

AIM: To investigate the possibility that epidermal growth factor receptor pathway participates in the growth promotion by growth hormone (GH) of growth plate chondrocytes cultured in vitro from adolescent rats treated with gonadotropin-releasing hormone analogue (GnRHa). METHODS: The chondrocytes from tibial growth plate of 5-8 female Sprague-Dawley (SD) rats treated with GnRHa were cultured in monolayer. Specific pharmacological inhibitor of Janus kinase (JAK2 tyrphostin AG490, 1, 10, 100 nmol/L), EGFR kinase inhibitor AG1478 (0.1, 1, 10 nmol/L) and neutralizing antibodies against EGF (0.1, 1, 10 mg/L) were added before GH stimulation. The proliferation of chondrocytes was investigated by the methods of MTT and immunohistochemical staining for PCNA. Phosphorylations of ERK1/2 and EGFR were detected by Western blotting. RESULTS: GH enhanced the proliferation of chondrocytes and the levels of ERK1/2 and EGFR phosphorylation in a dose dependent manner. The effect peaked at the concentration of 100 μg/L. Pretreatment with tyrphostin AG490 and AG1478 almost completely inhibited the proliferation of chondrocytes and phosphorylation of ERK1/2 and EGFR by GH. However, the neutralizing antibodies against EGF only partially inhibited the effects of GH.CONCLUSION: GH achieves its direct effect on the promotion of cell proliferation by the activation of JAK2 pathway and the downstream end of MAPK-ERK signaling molecules. The promotion of cell proliferation can be mediated by activation of EGFR pathway. The results suggest that there is signaling cross-talk between GH and EGF-EGFR pathway.     

Knockdown of growth hormone receptor prevents growth hormone induced inflammatory cytokine production in 3T3-L1 adipocytes

AIM: To investigate the effect of growth hormone receptor (GHR) knockdown on nuclear factor-κB (NF-κB) activity and inflammatory cytokine production stimulated by growth hormone (GH) in 3T3-L1 adipocytes. METHODS: The specific siRNA for GHR was transfected into 3T3-L1 adipocytes to silence GHR expressions. The effects of GH on NF-κB activation and inflammatory cytokine production in 3T3-L1 adipocytes transfected with siRNA-GHR or siRNA-control were measured by dual-luciferase system analysis, real-time RT-PCR and ELISA. RESULTS: The protein expression of GHR was diminished after transfection with GHR specific siRNA. Dual-luciferase reporter system analysis revealed that GHR knockdown resulted in attenuation of GH-stimulated NF-κB activation in the 3T3-L1 adipocytes. GHR knockdown ameliorated the GH-induced production of inflammatory cytokines TNF-α, IL-1β, IL-6, MCP-1 and MIP-1α in the 3T3-L1 adipocytes. CONCLUSION: Knockdown of GHR might be efficacious to prevent GH-induced inflammatory responses in the 3T3-L1 adipocytes.     

Experimental study on anti-endotoxin activity of a tetrahydropyrimidine derivative,ZL-5015

AIM: To investigate the protective effect of 1, 3-dicyclopentyl-1, 2, 3, 6-tetrahydropyrimidine-4, 5-dicarboxylic acid diethyl ester (ZL-5015) on lethal endotoxin-challenged mice and to explore the underlying mechanism. METHODS: Mouse model of lethal endotoxin challenge and endotoxemia were established by intraperitoneal administration of lipopolysaccharide (LPS) at a dose of 70 mg/kg to the C57BL/6J mice. Mouse peritoneal macrophages stimulated with LPS (10 mg/L) were used as an in vitro inflammatory model. The levels of interleukin-1β (IL-1β), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was used to evaluate the mRNA expression of the cytokines. RESULTS: Prophylactic treatment of the mice with ZL-5015 (100 and 200 mg/kg, ig) slightly increased the survival rate, extended the survival time, decreased the serum levels of IL-1β and TNF-α, and increased the serum level of IL-10 in the early stage of endotoxemia as compared with model group. The results of in vitro study demonstrated that treatment of the endotoxin-stimulated mouse peritoneal macrophages with ZL-5015 (10, 20 and 40 μmol/L) inhibited the expression of IL-1β and TNF-α at both mRNA and protein levels but promoted the expression of IL-10 at both mRNA and protein levels. CONCLUSION: The tetrahydropyrimidine derivative ZL-5015 shows a moderate anti-endotoxin effect by increasing the survival rate and extending the survival time of the mice challenged by endotoxin, which may result from inhibition of the expression of pro-inflammatory cytokines such as IL-1β and TNF-α, and promotion of the expression of anti-inflammatory cytokine IL-10.     

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.     

Mechanism of systemic inflammatory response syndrome induced by EC DNA

AIM: To investigate whether the bacterial DNA participates in SIRS and its possible mechanism. METHODS: Escherichia coli genomic DNA (EC DNA) was extracted and purified from Escherichia coli 25922 by alkaline lysis method. Mortality of mice challenged with EC DNA and the changes of TNF-α and IL-6 in rat serum were observed. ANA-1 cells were cultured in vitro, after the cells were stimulated by different concentrations of EC DNA and LPS, the level of TNF-α and IL-6 in supernatant were tested. Meanwhile,expression of TLR9 and TLR4 on cell surface was measured. Activation of NF-JB was also observed. RESULTS: The lethal effect of EC DNA on mice with an obvious dose-effect relationship was observed. The death happened within 24 hours. Calf thymus DNA and DNase I-treated EC DNA did not lead to mice to die. The changes of serum TNF-α and IL-6 in rats induced by EC DNA and LPS were similar, but TNF-α peak level of EC DNA group appeared 1 hour earlier than that of LPS group. In vitro, large amount of TNF-α and IL-6 were released from ANA-1 cells stimulated by EC DNA. High expression of TLR9 and TLR4 was observed on surfaces of THP-1 cells. In particularly, LPS induced strong activation of NFκB. The results suggested other pathway possibly took part in the signal transduction inducea by EC DNA. CONCLUSION: EC DNA has the abilities to lead to death of mice, andinduces serum TNF- αand IL- 6 level to increase in rats and ANA- 1 cells to release cytokines in vitro. High expression of TLR9 and TLR4, strong activation of NF- κB may be its importantmolecular mechanism, but other pathway probably exists to play an important role.     

Effects of diosmin on changes of TNF-α, IL-1β, IL-6, IL-8 and IL-10 in serum and kidney tissues of rats with kidney ischemia and reperfusion

AIM: To observe the effects of diosmin on the production of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), IL-6, IL-8 and IL-10 in serum and kidney tissues of rats with kidney ischemia and reperfusion (I/R). METHODS: Sprague-Dawley rats (180 in total) were randomly divided into 3 groups including sham operation group (sham),I/R group and diosmin+I/R group (diosmin+I/R). At the end of the experiment, the blood and kidney tissues were obtained and TNF-α, IL-1β, IL-6, IL-8 and IL-10 were detected by ELISA. RESULTS: The levels of TNF-α, IL-1β, IL-6, IL-8 and IL-10 in serum and kidney tissues in I/R group and diosmin+I/R group were significantly higher than those in sham group (P<0.01 or P<0.05). Following the development of the pathologic process, the level of TNF-α, IL-1β, IL-6 and IL-8 was significantly increased in I/R group and diosmin+I/R groups, but the level of IL-10 was significantly decreased in I/R group and significantly increased in diosmin+I/R group. The levels of TNF-α, IL-1β, IL-6 and IL-8 in I/R group was significantly higher than those in diosmin+I/R group (except TNF-α at 1 h in diosmin+I/R group). The level of IL-10 in diosmin+I/R group was significantly higher than that in I/R group (P<0.01 or P<0.05). CONCLUSION: Diosmin not only decreases the production of TNF-α, IL-1β, IL-6 and IL-8, but also promotes the production of anti-inflammatory cytokine IL-10, suggesting that the protective effect of diosmin on kidney I/R injury was associated with anti-inflammatory mechanism.     

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 lipopolysaccharide,IL-6 and TNFα on the tissue factor expression of astrocytes

AIM:To study the effects of lipopolysaccharide(LPS), interleukin-6(IL-6)and tumor necrosis factor α (TNFα) on tissue factor(TF) expression of astrocytes. METHODS:Astrocytes were identified with anti-glial fibrillary acidic protein antibody. The TF activity of cell lysate was measured with one stage clotting assay. RESULTS:TF activity of astrocytes of LPS,IL-6,TNFα groups were obviously higher than that of the control group(P <0.05); While LPS,IL-6 and TNFα were combined with trifluoperazine or H₇, their inductive effects were inhibited. CONCLUSION:LPS,IL-6 and TNFα promoted the TF expression of astrocytes and its mechanisms may connected with Calcium/Camodulin and protein kinase C pathway.     

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.     

Factors involved in regulation of polymorphonuclear neutrophils apoptosisin vitro

AIM:This study aimed at elucidated the possibility that prevent tissue from secondary injury by regulating polymorphonuclear neutrophil (PMN) apoptosisin vitro. METHODS:Neutrophils, isolated from peripheral blood, were incubated with sodium arsenite (Ars), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), burned serum and traumatic serum, respectively. Apoptosis rate, expression of CD11b, respiratory burst and concentration of Ca²⁺ were then measured. RESULTS:The elevation of PMN apoptosis rate was Ars concentration dependent, but activated PMN became insensitive to Ars. IL-6 delayed PMN apoptosis (compared with control at 24 h,P＜0.05), inhibited CD11b expression. Burned and traumatic serum had more significant effects on PMN compared with IL-6. CONCLUSION:PMN was observed for the first time to resist spontaneous apoptosis when activated by LPS/TNF, and became insensitive to apoptosis-inducing substance Ars. IL-6, burned serum and traumatic serum could delay PMN apoptosis and recover PMN functions partly.     

Alteration of serum IL-6, IL-8, TNF-α and sICAM-1 in patients with pre-menstruation recurrent aphthous ulceration

AIM:To investigate the serum levels of interleeukin-6(IL-6), interleukin-8(IL-8), tumor necrosis factor-alpha(TNF-α) and soluble intercellular adhesion molecule-1 (sICAM-1)in female patients with pre-menstruation recurrent aphthous ulceration(RAU).METHODS:Serum levels of IL-6, IL-8, TNF-α and sICAM-1 in 21 pre-menstruation RAU patients were examined using ELISA technique, and compared to 10 healthy individuals and 22 the female RAU patients unrelated to menstrual cycle.RESULTS:The serum levels of IL-6, IL-8, TNF-α in patients with pre-menstruation RAU were not only significantly higher than that in the normal control group(P＜0.01), but also higher than that in the RAU patients without pre-menstruous recurrence (P＜0.01).The level of serum TNF-α in the RAU patients without pre-menstruous recurrence was slightly higher than that in the normal control group(P＜0.05).The level of sICAM-1 had not changed.CONCLUSION:The serum levels of IL-6, IL-8 and TNF-α increase in pre-menstruation RAU patients, which might play a role in local lesion of RAU.     

Effects of NF-κB "decoy" oligodeoxynucleotides on TNF-α and IL-6 expression in LPS-induced mouse macrophages

AIM: To study the effect of NF-κB "decoy" oligodeoxynucleotides on TNF-α and IL-6 expression in LPS-induced mouse macrophages. METHODS: Mouse macrophage cell line J774.1 cells were cultured with LPS and liposome-mediated oligodeoxynucleotides, and the levels of TNF-α and IL-6 measured in the different culture supernatant by enzyme linked immunosorbent assay. RNA was extracted from macrophages, and the mRNA expression of TNF-α and IL-6 in macrophages was observed by RT-PCR. RESULTS: NF-κB "decoy" oligodeoxynucleotides decreased the expression of TNF-α and IL-6 in LPS-induced macrophages and inhibited generation of TNF-α and IL-6. The level of TNF-α and IL-6 did not change in control group. CONCLUSIONS: NF-κB "decoy" oligodeoxynucleotides inhibit the expression of TNF-α and IL-6 in LPS-induced macrophages, which is probably due to the specific inhibition of activated NF-κB binding sites .     

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.     

Effect of recombinant plasmids encoding IL-1RII and IL-1RAcP on rat experimental autoimmune myocarditis and possible mechanism

AIM To evaluate the effects of recombinant plasmids encoding interleukin-1 type II receptor （IL-1RII） and interleukin-1 receptor accessory protein （IL-1RAcP） on rat experimental autoimmune myocarditis （EAM） and the possible mechanism. METHODS The recombinant plasmids pCAGGS-IL-1RII and pCAGGS-IL-1RAcP were constructed, and pCAGGS-SP （signal peptide） served as the control plasmid. Male Lewis rats （n=29） were divided into 4 groups： control group （rats without immunization or injection, n=5）, EAM+SP group （immunized rats injected with pCAGGS-SP, n=9）, EAM+IL-1RII group （immunized rats injected with pCAGGS-IL-1RII, n=8） and EAM+IL-1RII+IL-1RAcP group （immunized rats injected with pCAGGS-IL-1RII and pCAGGS-IL-1RAcP, n=7）. The rats were immunized to induce EAM on day 0, and injected with recombinant plasmids by hydrodynamics-based delivery on day 6. Echocardiography was performed, and the rats were killed on day 17. The ratio of heart weight to body weight （HW/BW） was evaluated, and the histopathological changes of the myocardial tissues were observed by HE staining. The mRNA expression of atrial natriuretic peptide （ANP）, brain natriuretic peptide （BNP） and inflammatory factors in the myocardial tissues was detected by RT-qPCR. Recombinant plasmids pUC19-IL-1RII-actin and pUC19-IL-1RAcP-tub were transfected into Cos7 cells, and the culture supernatants were collected and added to lipopolysaccharide （LPS）-induced H9c2 cells. The expression of inflammatory genes were detected by RT-qPCR. Recombinant plasmids pEGFP-IL-1RII-actin and pEGFP-IL-1RAcP-tub were transfected into the Cos7 cells to identify the formation of IL-1RII/IL-1RAcP heterodimer by co-immunoprecipitation （Co-IP）. RESULTS Compared with EAM+SP group, injection with plasmids effectively attenuated EAM in EAM+IL-1RII group and EAM+IL-1RII+IL-1RAcP group, as indicated by the decreases in HW/BW, left ventricular end-systolic diameter, and myocardial expression of ANP, BNP, TNF-α, IL-2, IFN-γ and TGF-β, and the increase in expression of IL-4 in the hearts. In LPS-induced H9c2 cells, compared with LPS group, the levels of TGF-β and IL-6 in the culture supernatants were significantly decreased （P<0.01）, and the level of IL-10 was significantly increased （P<0.05） in LPS+IL-1RII group and LPS+IL-1RII+IL-1RAcP group. Compared with LPS+IL-1RII group, the expression of TNF-α and IL-2 was significantly decreased （P<0.05）, and the expression of IL-13 was significantly increased in LPS+IL-1RII+IL-1RAcP group （P<0.01）. The formation of IL-1RII/IL-1RAcP heterodimer was detected by Co-IP. CONCLUSION Plasmids encoding IL-1RII and IL-1RAcP effectively attenuate EAM, and the possible mechanism may be related to the inhibition of inflammatory factor expression and the formation of IL-1RII/IL-1RAcP heterodimer.     

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.