ABSTRACT
Although microRNA-155 (miR-155) is considered a pro-inflammatory mediator, cumulative evidence indicates that it also has anti-inflammatory effects in macrophages and dendritic cells. In this study, we identified the dramatic expression changes of more than half of potential miR-155-targeted genes upon lipopolysaccharide (LPS) stimulation; 223 genes were down-regulated and 85 genes were up-regulated, including suppressor of cytokine signaling 1 (
ABSTRACT
Objective To study the changes of immune function of endotoxin tolerant dendritic cell (ETDC) and to observe its effect on sepsis in mouse model.Methods ETDC were prepared by pretreated bone marrow dendritic cells derived from BALB/c mice with lipopolysaccharide stimulation.The cells were collected and the expressions of surface markers including major histocompatibility complex ( MHC)Ⅱ, CD86 and CD11c were detected by flow cytometry.The proliferation rate of T lymphocytes was evaluated by cell counting kit-8 and the concentrations of cytokines in the supernatant were detected by enzyme linked immuno sorbent assay.Afterwards, 36 mice were randomly assigned into 4 groups.The blank control group did not receive any treatment, the sham-operated group underwent simple incision suture, the sepsis group and ETDC reinfusion group underwent cecal ligation and puncture to establish sepsis.Before sepsis model establishment, 0.9% sodium chloride solution or suspension of ETDC and 0.9%sodium chloride were reinfused by tail vein.The serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), tumor necrosis factor (TNF)-α, interleukin(IL)-6 and IL-10, and the proportion of help T cell ( Th) 17/regulatory T cell ( Treg) in spleen of each group were detected.The pathological manifestations of liver, kidney and ileum in each group were observed.T test and χ2 test were used for comparisons between groups.Results The results of flow cytometry showed that MHCⅡ, CD86 and CD11c of ETDC were 70.4%, 43.1%, and 73.1%, respectively, which were significantly lower than those of mature dendritic cell (mDC) (96.1%, 89.5%, and 84.6%, respectively) (χ2 ＝56.47, 83.78, and 23.29, respectively, all P＜0.01).The concentrations of IL-10, TNF-αand IL-6 in the supernatant of ETDC were (978.04 ±56.70), (980.34 ±111.96) and (12 743.03 ±865.81) ng/L, respectively, and those of mDC were (741.35 ±99.23), (1 703.11 ±117.00) and (19 052.28 ±1 145.84) ng/L, respectively.The differences were all statistically significant (t＝5.073, 10.93, and 10.76, respectively, all P＜0.01).The proliferation rates of T lymphocytes co-cultured with ETDC in 1∶5 and 1∶10 ratio group were (676.95 ±85.99)%and (514.00 ±106.39)%, respectively, which were lower than those of the mDC group (956.25 ±127.12)%and (772.07 ±214.08)%, respectively.The pathological injuries of liver, kidney and ileum in ETDC treatment group were significantly lighter than those in sepsis group.Serum ALT and AST levels in the ETDC reinfusion group were (299.71 ±36.91) and (690.39 ±154.92) U/L, respectively, and TNF-α, IL-6 and IL-10 were (0.067 ±0.005), (0.428 ±0.051) and (0.058 ±0.005) ng/L, respectively.Serum ALT and AST levels in the sepsis group were (620.67 ±69.27) and (1 430.17 ±134.05) U/L, respectively, and TNF-α, IL-6 and IL-10 were (0.085 ±0.007), (0.774 ±0.088) and (0.036 ±0.005) ng/L, respectively.The differences were all statistically significant (t＝11.60, 10.96, 5.991, 8.657, and 8.04, respectively, all P ＜0.01).The proportion of Treg/Th17 in the ETDC reinfusion group was 23.4%, and that in the sepsis group was 60.8%(χ2 ＝28.69, P＜0.01).Conclusion The reinfusion of ETDC has a protective effect on sepsis in mouse model, which may play a negative immune regulatory role by regulating the differentiation of T cells.
ABSTRACT
[Objective]To investigate the effect of pellino-1 gene overexpression by lentivirus vector on the ubiquitin of tumor necrosis factor receptor-associated factors 3 (TRAF3) and the activation of mitogen-activated protein kinases (MAPK) signaling pathway in endotoxin-tolerant kupffer cells (KCs).[Methods]Isolated KCs of C57BL/6 mouse were randomly divided into two groups:control group,which transfected with control lentivirus vector for 48 h,then pretreated with 10 ng/mL lipopolysaccharide (LPS)for 24 h,and next treated with 300 ng/mL LPS;overexpression group,which transfected with pellino-1 gene overexpression lentivirus vector for 48 h,then pretreated with 10 ng/mL lipopolysaccharide(LPS)for 24 h,and next treated with 300 ng/mL LPS. The protein expressions of pellino-1,K48-Ub,TRAF3,p38,p-p38,c-Jun N-terminal kinase(JNK)and p-JNK were analyzed by Western blot. The level of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)in superna-tants were measured by enzyme linked immunosorbent assay(ELISA).[Results]Compared with control group,the protein expres-sions of pellino-1,K48-Ub,p-JNK and p-p38 and the levels of IL-1β(P < 0.05),TNF-α(P < 0.05)in supernatants was in-creased in overexpression group,while the protein levels of TRAF3 and the levels of IL-10(P < 0.05)in supernatants were de-creased.[Conclusion]Overexpression of pellino-1 can promote TRAF3 K48 ubiquitination degradation,decrease the protein expres-sion of TRAF3,activate the downstream MAPK signaling pathway,and thus impair the formulation of endotoxin tolerance.
ABSTRACT
Objective To further determine the inflammation?associated cytokine changes during endotoxin(LPS)tolerance. Methods Kun?ming mice were divided into groups. The endotoxin tolerance mouse model was established with repeated injection of LPS. Serum levels of tumor ne?crosis factor?α(TNF?α),interleukin?10(IL?10)and IL?17 were determined by ELISA assay. Results The serum concentrations of TNF?α,IL?10 and IL?17 were 2 261.49±1 090.28 pg/mL,7 780.42±2 320.04 pg/mL,and 27.28±13.76 pg/mL in the LPS control group,which were increased significantly compared to the normal healthy control group. The serum concentrations of TNF?α,IL?10 and IL?17 were 1 174.19±450.91 pg/mL, 1 293.26±520.85 pg/mL,and 22.26±11.44 pg/mL in the endotoxin tolerance group. Levels of TNF?αand IL?17 were further decreased by dexameth?asone treatment during LPS tolerance inducing,while the IL?10 level did not change significantly. Conclusion Compared to the status of endotoxin inflammation,levels of TNF?αand IL?10 decreased significantly,while levels of IL?17 were not significantly changed in the status of endotoxin toler?ance. Dexamethasone could further suppress TNF?αand IL?17 levels during endotoxin tolerance inducing.
ABSTRACT
Objective To study the effect of Xuebijing, a Chinese traditional medicine injection, on the THP-1 cells challenged by endotoxin, and to explore whether it induces endotoxin tolerance. Methods The THP-1 cells were pretreated with Xuebijing in different concentrations (10, 25, 50 mg/mL) and times (4, 12, 24 hours), and then challenged by endotoxin. The level of TNF-α in culture supernatant was detected by ELISA assay, and the expression of TLR4 and IRAK-M mRNA were detected by Real time-PCR technique. Results There was no significant difference in TNF-α level among all the groups (pretreated with different concentrations of Xuebijing for different time) (P>0.05). Only in 50 mg/mL Xuebijing group, TLR4 mRNA was 1.547-fold increase in 24 h than in 4 h group (P<0.05). Only when pretreated for 24 h, IRAK-M mRNA was 1.349-fold increase in 50 mg/mL Xuebijing group than in control group (P<0.05). However,there was no significant difference among other groups (P>0.05). Conclusions Xuebijing can not block the release of TNF-α from the THP-1 cells challenged by endotoxin;and it does not induce endotoxin tolerance. When pretreated with high concentration of Xuebijing for long time, the expression of both TLR4 and IRAK-M mRNA is up-regulated, but its significance is not yet clear.
ABSTRACT
Human monocyte leukemia cell line THP-1 was stimulated with lipopolysaccharide (LPS) to simulate the sepsis model and the expression of human glucocorticoid receptor-α (GR-α) mRNA in montocytes with endotoxin tolerance was investigated. THP-1 cells were cultured in serum-free medium, randomly divided into groups A, B, C, D and E, and stimulated with 0, 10, 10,100, 0 ng/mL LPS for 24 h followed with 100, 100, 10, 100, 0 ng/mL LPS for another 24 h respectively. The expression of GR-α mRNA was detected by semi-quantitative reverse transcriptional polymerase chain reaction. Tumor necrosis factor-α (TNF-α) was determined by enzyme linked immunosorbent assay (ELISA). The results showed that the A values of GR-α/β-actin in groups A,B, C, D and E was 0. 607±0. 006, 0. 368±0. 005, 0. 484±0. 008, 0. 509±0. 004 and 0. 564± 0. 014 respectively with the difference being significant among the groups (P＜0. 05). The GR-α mRNA expression was negatively correlated with the TNF-α expression (P＜0.01). It was concluded that the down-regulation of the expression of GR-α mRNA in endotoxin tolerance THP-1 cells might play an important role in the development of endotoxin tolerance in THP-1 cells.
ABSTRACT
Objective To investigate the endotoxemia initiated systemic and pulmonary inflammation and acute lung injury in endotoxin tolerant rats MethodsEndotoxi n tolerance (ET) models of SD rats were induced by four daily intraperitoneal in jections of 0 6 mg?kg -1 ?d -1 Escherichia coli LPS (serotype 055:B5).Normal control (NC) rats received intraperitoneal injections of the sa me volume saline On the fifth day,rats were injected with high dose of LPS (6 mg/kg) to induce endotoxemia and lung inflammation Blood,left bronchoalveolar lavage fluid (BALF) and right lung tissue were collected before and 2,6,24,48 ,72 hours after the high dose injection of LPS (six rats for each time point) Cytological examination of blood and BALF and histopathological examination wer e performed Bromine methylphenol green was adopted for measurement of serum alb umin BALF albumin was measured by en z yme-linked immunosorbent assay (ELISA) and adjusted by the ratio to serum album in to evaluate the permeability of pulmonary microvascular Results The symptomes such as less activity,accelerated respiratory rate and weight loss in NC rats was not found in ET rats after the high dose injectio n of LPS BALF albumin as well as the ratio of BALF albumin to serum albumin in c reasedt 2 hours after injection of 6 mg/kg LPS and reached their zenith at 6 hou rs in NC rats,while no increase in ET rats In NC rats the blood white cell dif f erentiating shifted from lymphocyte to PMN,and PMN percentage of BALF also incr eased from (0 443?0 345)% to (8 000?2 896)% with its peak at 24 hours a fter the injection (P
ABSTRACT
BACKGROUND: It is well known that when macrophages are stimulated with endotoxin, they produce a wide variety of cytokine mediators, including TNF-α and IL-1β. However, there is an alterationnin the macrophages responsiveness when they are challenged with repeated bouts of endotoxin, termed 'endotoxin tolerance' which is regarded as a self-protective phenomenon from continuous stimulation. In this study, endotoxin tolerance in the peripheral blood monocytes of sepsis patients was evaluated. METHODS: Fourteen patients with organism-documented sepsis were included. The severity of illness was evaluated by APACHE IIscore. Peripheral blood monocytes were isolated from the patients and diluted to 1×105/well. After stimulation with endotoxin(LPS of E. coli O114:B4, 100 ng/ml), they were incubated at 37℃ in 5% CO2 incubator for 24 hours. Supernatant was collected for the measurement of TNF-αand IL-1β with ELISA method. Peripheral blood monocytes of seven healthy volunteers were used as control. RESULTS: The APACHE IIscore(mean±SD) of the patients at the time of blood sampling was 12.2±5.7. The primary infection foci were urinary tract infection, pneumonia, subacute bacterial endocarditis, and catheter related infection, etc. The causative organisms were gram negative rods(10 cases), gram positive cocci(6 cases) with two cases of mixed infection. Serum TNF-α could be measured in 4 cases with 29.9±27.7 pg/ml. Serum IL-1β was measureable in only one patient. The TNF-α level of supernatant of cultured peripheral blood monocytes was 2,703±2,066 pg/ml in patients and 2,102±1,914 pg/ml in controls. The IL-1β level of supernatant was 884±1,050 pg/ml in patients and 575±558 pg/ml in controls. There was no difference of TNF-α and IL-1β level between patients and controls. CONCLUSION: We cannot prove the phenomenon of endotoxin tolerance in this study. Future study needs to be focused on the more severe sepsis patients who were taken for sampling earlier. Addition of serum to the culture medium could be an another valuable option for the success of this study.