Objective To observe the human mononuclear cell releasing TNF-α and the activation of Caspase-3 during apoptosis after stimulated by Co2+ and Cr3+, to discuss the mechanism of artificial joint wear production metal ion on aseptic loosening. Methods CoCl2 powder and CrCl3 powder were dissolved by asepsis inject water, preparing solution for10 mg/L and 500 mg/L, respectively. Mononuclear cells were acquired from peripheral blood, 4 × 106/culture dish. According to the difference of culture solution, the cells were divided into 3 groups. Group A: mononuclear cell was culture with normal sal ine as control; group B: mononuclear cell was cultured with CoCl2 solution; group C: mononuclear cell was cultured with CrCl3 solution. The production of TNF-α was assessed by ELISA, the activation of Caspase-3 was measured by colorimetric assay and the apoptotic cell was detected by TUNEL assays at 12, 24 and 48 hours after co-cultured respectively. Results The concentration of TNF-α and the expression of Caspase-3 in groups B and C were higher than those in group A (P lt; 0.05), and reached the peak level at 24, 48 hours, respetively. The TUNEL positive cells were detected in the all groups, nucleus was pyknotic and darker-staining, cell body was crinkle and cell membrane was integrity. There were significant differences in the apoptosis rate between groups B, C and group A (P lt; 0.05). And the activation of Caspase-3 increased and had the positive correlation with the apoptosis rate (r=0.765). Conclusion Co2+ and Cr3+ ions can stimulate human mononuclear cell to release TNF-α and induce human mononuclear cell apoptosis, which result in periprosethetic osteolysis and related to activation of Caspase-3.
ObjectiveTo investigate the expression of tumor necrosis factor α(TNF-α ) in isolated rat heart at different time points after myocardial hypoxia/reoxygenation. MethodsThe isolated langendorff perfused rat heart model was established. Forty-eight SD rats were randomly divided into four groups: a sham group, hypoxia/reoxygenation groups including a H/R 0.5 h group, a 1 h group and a 2 h group. The heart rate(HR), the 1eft ventricular development pressure(LVDP), maximal rates of increase/decrease of the left ventricular pressure(±dp/dtmax) were continuously recorded. The concentrations of TNF-α and creatine kinase-MB(CK-MB) in myocardium, mRNA expression of TNF-α in myocardium were tested. Ultra structure of myocardium was observed under electron microscope. ResultsThe levels of LVDP, ±dp/dtmax, and HR of hypoxia/reoxygenation group were significantly lower than those in the sham group(P<0.05).The levels of TNF-α and CK-MB and the expressions of TNF-α at mRNA level in the hypoxia/reoxygenation group were higher than those in the sham group(P<0.05).There were significant differences in the above parameters among the H/R 0.5 h group, the 1 h group, the 2 h group(P<0.05).The concentrations of TNF-α and CK-MB, the mRNA expression of TNF-α were higher in the I/R 2 h group than those in the other two groups. ConclusionThe high expression of TNF-α in myocardium after myocardial hypoxia/reoxygenation in rats is related to the degree of myocardium damage and may lead to myocardial injury.
ObjectiveTo investigate the association between TNF-α gene -308G/A polymorphism and the risk of coronary atherosclerotic heart disease (CHD) in Chinese population.MethodsWe searched PubMed, Web of Science, CNKI, WanFang Data and VIP Databases from inception to February 2017, to collect case-control studies about the association between TNF-α gene -308G/A polymorphism and risk of CHD in Chinese population. Two reviewers independently screened literature, extracted data and assessed the risk of bias of the included studies. Meta-analysis was performed by Stata 12.0 software.ResultsA total of 10 case-control studies were included. The results of meta-analysis showed a significant association between the TNF-α gene -308G/A polymorphism and CHD risk in Chinese population (A vs. G: OR=1.13, 95%CI 1.02 to 1.26, P=0.020; AA vs. GA/GG: OR=1.47, 95%CI 1.02 to 2.12, P=0.038; AA vs. GG: OR=1.50, 95%CI 1.04 to 2.16, P=0.029).ConclusionThe current evidence shows that the TNF-α gene -308G/A polymorphism may be associated with CHD risk in Chinese population and A allele may be a risk factor. Due to the limited quantity and quality of included studies, more high quality studies are needed to verify the above conclusion.
Objective To summarize results of the correlation of tumor necrosis factor-α (TNF-α) promoter –308A/G polymorphism with systemic lupus erythematosus (SLE) susceptibility in Chinese populations. Methods We collected all the publications about the correlation between TNF-α promoter –308A/G polymorphism and SLE in Chinese populations by searching PubMed, EBSCO, CBM, CNKI and Wanfang Data before the date of March 20, 2010. Meta-analysis was performed for checking the difference between two groups about genotypes such as AA versus GG, GA versus GG, AA versus GG+GA, GA+AA versus GG, and A allele versus G allele. Results A total of 8 studies involving 731 SLE patients and 901 healthy people were included. The meta-analysis of total populations showed that, there was no significant correlation between A allele and increased SLE risk (OR=1.42, 95%CI 0.97 to 2.09, P=0.07); the meta-analyses of populations in different regions showed there was no significant correlation of A allele and increased SLE risk in Chinese Taiwan populations (OR=1.04, 95%CI 0.77 to 1.40, P=0.82). Moreover, there was no significant difference between SLE group and control group in the genotypes of AA versus GG, GA versus GG, AA versus GG+GA, and GA+AA versus GG.Conclusion This meta-analysis dosen’t demonstrate the correlation between TNF-α promoter–308A/G polymorphism and SLE in Chinese populations.
急性肺损伤(acute lung injury,ALI)是临床常见的呼吸系统急危重症,其发病机制复杂,病死率高。肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)是ALI发生、发展过程中非常重要的细胞因子。本文就TNF-α在ALI中的作用和以抗TNF-α为靶点治疗ALI的相关研究进行综述。
Objective To investigate the possibility of gene therapy of osteolysis around artificial joint prosthesis by constructing the recombinant adenovirus which can silence tumor necrosis factor α (TNF-α). Methods The primer of small interfering RNA (siRNA) coding sequence of silent TNF-α was designed and amplified, and then RAPAD adenovirus packaging system was used to load the sequence to adenovirus, and the recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP which lacked both E1 and E3 regions was constructed. Then 64 female BABL/C mice (weighing, 20-25 g) were randomly divided into 4 groups (n=16): blank control (group A), positive control (group B), simple adenovirus (group C), and treatment group (group D). The prosthetic-model was established in group A, and the prosthetic-loosening-model in groups B, C, and D. At 2 weeks after modeling, PBS solution was injected first, and then the same solution was injected 24 hours later in group A; titanium particle solution was injected, and then PBS solution, Ad5 E1-CMVeGFP (1 × 109 PFU/mL), and Ad5-TNF-α-siRNA-CMVeGFP (1 × 109 PFU/mL) were injected, respectively in groups B, C, and D 24 hours later, every 2 weeks over a 10-week period. The general condition of mice was observed after operation. The tissues were harvested for histological observation, and the expression of TNF-α was detected by Western blot at 12 weeks after operation. Results The positive clones were achieved by enzyme digestion and confirmed by DNA sequencing after loading the target genes into adenovirus vector, and then HEK293 cells were successfully transfected by recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP. All mice survived to the completion of the experiment. Histological observation showed that there were few inflammatory cells and osteoclasts in group A, with a good bone formation; there were a large number of inflammatory cells and osteoclasts in groups B and C, with obvious bone destruction; inflammatory cells and osteoclasts in group D was less than those in groups B and C, with no obvious bone destruction. Significant difference was found in the limiting membrane thickness and the number of osteoclasts (group A lt; group D lt; group B lt; group C, P lt; 0.05). Western blot showed that the TNF-α expression levels were 0.235 ± 0.022, 0.561 ± 0.031, 0.731 ± 0.037, and 0.329 ± 0.025 in groups A, B, C, and D respectively, showing significant difference among 4 groups (P lt; 0.05). Conclusion The recombinant adenovirus for silencing TNF-α is successfully constructed, which can effectively inhibit osteolysis by silencing TNF-α expression in the tissues around prosthesis in mice.
Objective To investigate the effect of heme oxygenase 1 (HO-1) on the apoptosis of human degenerated nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α), and explore its possible molecular mechanism. Methods The intervertebral disc tissues were derived from patients with lumbar intervertebral disc herniation. Then, the NP cells were cultured in vitro and the third generation of NP cells were used for subsequent experiments. Cell counting kit 8 (CCK-8) method was used to observe the proliferative effect of TNF-α on the NP cells in vitro at the concentration of 10, 20, 50, 100, and 200 ng/mL. The most apropriate concentration was selected according to the result of CCK-8. The NP cells were cultured with basal medium (control group), TNF-α (TNF-α group), TNF-α and CoPP 10 μmol/L (CoPP group), and TNF-α and ZnPP 15 μmol/L (ZnPP group), respectively. After cultured, the cell poptosis was detected by Hoechst staining and flow cytometry; the expression of cleaved Caspase-3, epithelial membrane protein 1 (EMP-1), HO-1, and p-P65 proteins were detected by Western blot. In order to further explore the potential molecular mechanisms of HO-1 for cell apoptosis, the NP cells were cultured with TNF-α (TNF-α stimulated group), TNF-α and pyrrolidine dithiocarbamate (PDTC) 5 μmol/L (TNF-α+PDTC stimulated group), respectively. Then the cell apoptosis rate was measured by flow cytometry at 24 hours after cultured. Results The optimal concentration of TNF-α was 100 ng/mL. Hoechst staining showed that a few apoptotic cells could be observed in control group and CoPP group; the apoptosis-like nucleis were observed in TNF-α group and ZnPP group, which was the most significant in ZnPP group. Flow cytometry showed that the cell apoptosis rates of TNF-α group, CoPP group, and ZnPP group were significantly increased when compared with the control group (P<0.05). Compared with TNF-α group, the cell apoptosis rate in CoPP group decreased (P<0.05), while in ZnPP group it increased (P<0.05). Western blot showed that the expression of HO-1 protein in TNF-α group was decreased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were increased when compared with the control group (P<0.05). Compared with TNF-α group, the expression of HO-1 protein in CoPP group increased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were reduced (P<0.05); the expression of HO-1 protein in ZnPP group decreased (P<0.05), the expressions of cleaved Caspase-3 and EMP-1 proteins increased (P<0.05), and the expression of p-P65 protein was not significantly changed (P>0.05). Compared with TNF-α stimulated group, the cell apoptosis rate in TNF-α+PDTC stimulated group was significantly reduced (t=3.076, P=0.031). Conclusion HO-1 can inhibit the apoptosis of degerated NP cells induced by TNF-α, and its mechanism effect is by inhibiting the nuclear factor кB signaling pathway.
ObjectiveTo investigate the role of Wnt5a in the mechanism of radiculopathy and the relation between Wnt5a and tumor necrosis factorα(TNF-α) by observing the change of the expression of Wnt5a in the rat model of chronic compression of dorsal root ganglia (CCD). MethodsA total of 192 adult male Sprague Dawley rats were allocated into 4 groups: shame group (group A, n=48), CCD group (group B, n=48), CCD+sal ine group (group C, n=48), and CCD+etanercept group (group D, n=48). An L-shaped needle (about 3.5 mm in length, 0.6 mm in diameter) was inserted into the L5 intervertebral foramen, and the dorsal root ganglia (DRG) was compressed by the needle to prepare the CCD model in groups B, C, and D, and then normal sal ine (5.5 mg/kg) or etanercept was injected intraperitoneally in groups C and D. The intervertebral foramen was exposed in group A. The mechanical pain threshold of the posterior paw was tested by the von Frey filaments at 1, 3, 5, and 7 days after operation; the expressions of Wnt5a protein and mRNA were detected at 3 and 7 days after operation by immunohistochemical staining and RT-PCR, respectively. ResultsThe mechanical pain threshold of groups B and C was significantly lower than that of groups A and D, and in group D than in group A (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The Wnt5a positive cells and the mRNA expression of Wnt5a at 7 days were significantly more than those at 3 days in groups B, C, and D (P < 0.05). The Wnt5a positive cells and the mRNA expression of Wnt5a in groups B and C were significantly more than in groups A and D, and in group D than in group A (P < 0.05), but no significant difference was shown between groups B and C (P > 0.05). ConclusionThe expression of Wnt5a in the DRG is increased after CCD. The expression of Wnt5a in the DRG is decreased after the administration of the inhibitor of TNF-α.
ObjectiveTo investigate the relationship between alumina ceramic particles and aseptic loosening of the joint prosthesis and the effect of lanthanum chloride on the secretion of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) of macrophage RAW264.7 induced by alumina ceramic particles. MethodsRAW264.7 cells were cultured in vitro and divided randomly into 4 groups according to different culture solutions:blank control group (group A),1 mg/mL alumina ceramic particles (group B),1 mg/mL alumina ceramic particles and 10 μmol/L lanthanum chloride (group C),and 10 μmol/L lanthanum chloride (group D).The cell growth was detected by MTT,and ELISA,RT-PCR,and Western blot were used to test the expressions of IL-1β,TNF-α,and nuclear factor κB (NF-κB). ResultsThere was no significant difference in cell growth among all groups by MTT (F=2.180,P=0.142).RT-PCR results showed that the expressions of IL-1β,TNF-α,and NF-κB mRNA in group B were significantly higher than those in the other 3 groups (P<0.05); the expressions in group D were significantly lower than those in group A (P<0.05).ELISA results showed that the contents of IL-1β and TNF-α in group B were significantly higher than those in the other 3 groups (P<0.05); the contents in group D were significantly lower than those in group A (P<0.05).Western blot analysis revealed that the expression of NF-κB protein in group B was significantly higher than that in the other 3 groups (P<0.05). ConclusionAlumina ceramic particles can stimulate the secretion of IL-1β and TNF-α of macrophage,and lanthanum chloride can inhibit the secretion of IL-1β and TNF-α of macrophage.
ObjectiveTo explore the effects of interleukin 10 (IL-10) gene modified bone marrow mesenchymal stem cells (BMSCs) on the expression of inflammatory cytokines and neuronal apoptosis in rats after cerebral ischemia reperfusion injury.MethodsBMSCs were cultured by whole bone marrow adherence screening method. The properties of BMSCs were identified by immunocytochemical methods. BMSCs at passage 3 were transfected with recombinant adenovirus IL-10 gene (AdIL-10-BMSCs). The model of middle cerebral artery occlusion was made in 40 adult male Sprague Dawley rats by thread embolism method. The rats were randomly divided into 4 groups (n=10). At 3 hours after modelling, the rats of groups A, B, C, and D received tail intravenous injection of 1 mL L-DMEM medium containing 10% FBS, 61.78 ng IL-10, 1 mL BMSCs suspension (2×106 cells/mL), and 1 mL AdIL-10-BMSCs cell suspension (2×106 cells/mL), respectively. The cells were labelled with BrdU before cell transplantation in groups C and D. At 7 days after reperfusion, the brain tissue was harvested to detect the expression of OX42 by immunohistochemical assay, to determine the concentration of tumor necrosis factor α (TNF-α) and IL-1β by ELISA, and to detect the apoptosis by TUNEL assay. BrdU labelled cells were observed by immunofluorescence staining in groups C and D.ResultsBrdU labelled positive cells with green fluorescence were observed in the brain tissue of groups C and D, which mainly distributed in the striatum, cerebral cortex, and subcortex around the infarction area. The number of OX42 positive cells was significantly less in groups B, C, and D than group A (P<0.05), and in group D than groups B and C (P<0.05). Compared with the other 3 groups, the contents of TNF-α and IL-1β significantly decreased in group D (P<0.05). TUNEL assay showed that the apoptotic cells (TUNEL positive cells) were mainly seen in the striatum and fronto parietal subcortical tissues (equivalent to ischemic penumbra). The number of TUNEL positive cells in group D was significantly less than that in groups A, B, and C (P<0.05).ConclusionAdIL-10-BMSCs can inhibit secretion of TNF-α and IL-1β from microglial cells and inhibit the nerve cell apoptosis around infarct brain tissue, which might contribute to its protective role upon cerebral ischemia reperfusion injury.