ObjectiveTo investigate the effect of the femoral tunnel angle on the femoral tunnel after anterior cruciate ligament (ACL) reconstruction in rabbits. MethodsFifty-four healthy 4-5 months old rabbits (weighing, 1.8-2.3 kg, male or female) were randomly divided into 3 groups (n=18). The ACL reconstruction models of the right knee were established in 3 experimental groups using its Achilles tendons, and the left knee served as the control group. On the coronal position, the angle between the femoral tunnel and the femoral shaft axis was 30°, 45°, and 60°. The level of tumor necrosis factor α (TNF-α) in the synovial fluid at 1, 2, and 4 weeks, the maximum load of the ligament and the rate of bone tunnel enlargement at 4, 8, and 12 weeks were detected. ResultsThe level of TNF-α significantly increased, and the maximum load of the ligament significantly decreased in the 3 experimental groups when compared with ones in the control group (P<0.05), but no significant difference was found among 3 experimental groups (P>0.05). The bone tunnel enlargement was observed in 3 experimental groups at each time point and reached the peak at 4 weeks, but no significant difference was shown among 3 groups (P>0.05). ConclusionThe 30-60° angle between the femoral tunnel and the femoral shaft axis in the coronal position has no significant effect on the femoral tunnel enlargement after ACL reconstruction in rabbits.
Acute hemorrhagic and necrotizing pancreatitis (AHNP) was induced by injection of sodium taurocholate into pancreatic and biliary duct of rats. TNFα MCAb was infused intravenously 15 minutes before pancreatitis was induced, and plasm TNFα level, serum lipase level and pancratic pathologic changes were tested.Results: the amount of ascites, serum lipase level and palsm TNFα level were significantly incresed and severe pancreatic pathologic changes was induced after AHNP, as compared with those in the control group .However, plasm TNFα level was not elevated after administration of TNFα MCAb, and the amount of ascites and pathologic damage to the pancreas were markely reduced. The animal fatality was reduced too. Conclusions: these suggest that TNFα play an important role in the pathogenesis of AHNP, and TNFα MCAb have a certain therapeutic effect on AHNP in rats.
Objective To investigate whether P12，a kind of lipopolysaccharide（LPS）-binding protein（LBP） inhibitory peptide，could suppress the binding of LPS to alveolar macrophages（AMs） in a mouse model of endotoxemia in vivo.Methods Forty mice were randomly divided into five groups，ie.a control group，an endotoxemia group，a low dose P12-treated group，a middle dose P12-treated group and a high dose P12-treated group.Mouse model of endotoxemia was established by LPS injection intraperitoneally in the endotoxemia group and P12-treated groups.P12 was instilled via the tail vein.The effects of P12 on the binding of LPS to AMs were determined by flow cytometric analysis and quantization by mean fluorescence intensity(MFI).The productions of tumor necrosis factor α(TNF-α) in serum of mice were measured by enzyme-linked immunosorbent assay(ELISA).Results MFI in AMs from low，middle and high dose P12-treated groups was 40.08%，30.76% and 24.45%，respectively，which was higher than that of the control group(4.61%)，but less than that of the endotoxemic mice(45.31%).The concentration of TNF-α in serum of low，media and high dose P12-treated mice was (112.69±19.78)pg/mL，(86.34±9.25) pg/mL，(70.48±8.48)pg/mL respectively，which was higher than that of the control group[(24.88±5.82)pg/mL]，but less than that of the endotoxemic mice[(180.17±39.14)pg/mL].Conclusion The results suggest that P12 inhibit the binding of LPS and AMs，thus reduce the proudction of TNF-α stimulated by LPS.
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 evaluate the correlation of TNF-α G308A polymorphism and rheumatic heart disease (RHD) using meta-analysis. Methods Databases including PubMed, EMbase, CNKI and WanFang Data were searched to collect case-control study on the correlation of TNF-α G308A polymorphism and RHD, published from January 1990 to June 2011. Two reviewers independently screened studies according to the inclusion and exclusion criteria, extracted data and evaluated the methodological quality of the included studies. Then meta-analysis was performed using RevMan 5.1 and SPSS 16.0. Results A total of 5 studies were included, involving 539 RHD cases and 624 controls. The results of meta-analysis according to recessive genetic model of TNF-α G308A showed that there were significant differences in RHD risk between the AA genotype carriers and the GA+GG genotype carries (OR=5.06, 95%CI 2.15 to 11.89, P=0.0002), the same as the results of meta-analysis calculated according to dominant genetic model (OR=3.14, 95%CI 1.05 to 9.38, P=0.04). Conclusion Current evidence shows that TNF-α G308A polymorphism is related to RHD, and the AA genotype carriers tend to face an increasing RHD risk. This conclusion still needs to be further proved by more high-quality and large-scale clinical trials.
Objective To investigate the effects of expression of TNFα mRNA on glucose uptake in both the liver and skeletal muscle after endotoxemia. Methods In the mice with intraperitoneal injection of lipopolysaccharide (LPS), the changes of TNFα level of plasma and uptake of 2-deoxyglucose (2-DG) in the isolated soleus muscle and hepatic tissues were determined, then the reinstatement of glucose uptake by injecting TNF-McAb for 3 days was also observed. In addition, changes of TNFα mRNA expression of liver were evaluated. Results The expression of TNFα mRNA in the liver showed markedly increased in the first 3 hours post endotoxemia and remaind high for 3 days, and the plasma TNFα level paralleled with TNFα mRNA expression of liver also was elevated. The basal uptake of 2-DG both in muscle and liver were markedly increased, but the stimulated 2-DG uptake with insulin was greatly reduced as compared with the control. In addition, these abnormalities of 2-DG uptake can be partially corrected by neutralization of the circulatory TNFα by administration of TNF-McAb. Conclusion The disorders of glucose uptake of the liver and the muscle due to the overexpression of TNFα mRNA and elevated circulatory TNFα level may be the mechanism of insulin resistance after endotoxemia.
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 effect of Vitamin C (Vit C) on the apoptosis of human nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α) and serum deprivation. MethodsThe NP cells were isolated from patients undergoing spine corrective operation by collagenase trypsin. The experiment was divided into 3 groups:Vit C group (group A), TNF-α group (group B), and serum deprivation group (group C). Group A was reassigned to A1 subgroup (basic medium), A2 subgroup (100 μg/mL Vit C), and A3 subgroup (200 μg/mL Vit C). Group B was reassigned to B0 subgroup (control group), B1 subgroup (100 ng/mL TNF-α), B2 subgroup (100 μg/mL Vit C+100 ng/mL TNF-α), and B3 subgroup (200 μg/mL Vit C+100 ng/mL TNF-α). Group C was reassigned to C0 subgroup (Control group), C1 subgroup (2% FBS), C2 subgroup (2%FBS+100 μg/mL Vit C), and C3 subgroup (2% FBS+200 μg/mL Vit C). After C1 subgroup (2% FBS), C2 subgroup (2%FBS+100 μg/mL Vit C), and C3 subgroup (2% FBS+200 μg/mL Vit C). After application of 100 μg/mL or 200 μg/mL Vit C for 24 hours, NP cells were stimulated by TNF-α and serum deprivation, then the apoptosis rate of NP cells was detected by a flow cytometry, and the gene expressions of the extracellular matrix of NP cells (collagen type Ⅰ, collagen type Ⅱ, aggrecan, and Sox9) and apoptosis related genes (p53, FAS, and Caspase 3) were detected by real-time fluoroscent quantitative PCR. ResultsGroup A:Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 of NP cells in A2 and A3 subgroups when compared with A1 subgroup (P<0.05), but there was no significant difference between A2 subgroup and A3 subgroup (P>0.05); Vit C could promote the expressions of the extracellular matrix (collagen type Ⅰ, collagen type Ⅱ, aggrecan, and Sox9) of NP cells in a concentration dependent manner (P<0.05). Group B:TNF-α significantly increased the apoptosis rate and the gene expressions of p53, FAS, and Caspase 3 in B1 subgroup when compared with B0 subgroup (P<0.05); however, Vit C significantly increased the apoptosis rate and the gene expressions in B2 subgroup, and significantly decreased them in B3 subgroup when compared with B1 subgroup (P<0.05). Group C:2% FBS significantly increased the apoptosis rate of NP cells and significantly reduced the gene expressions of p53, FAS, and Caspase 3 in C1 subgroup when compared with C0 subgroup (P<0.05); Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 in C3 subgroup, but it could significantly increase them in C2 subgroup when compared with C1 subgroup (P<0.05). ConclusionVit C can promote the synthesis and secretion of extracellular matrix of NP cells. 200 μg/mL Vit C may delay the apoptosis induced by TNF-α and serum deprivation, indicating the potential therapeutic effect of Vit C on intervertebral disc degeneration.
ObjectiveTo investigate the anti-apoptotic ability of synovium-derived mesenchymal stem cells (SMSCs) by comparing the apoptosis induced by tumor necrosis factor α (TNF-α) between SMSCs and bone marrow mesenchymal stem cells (BMSCs). MethodSMSCs and BMSCs were isolated with tissue adhering and density gradient centrifugation respectively, and cells at passages 3-5 were used in further experiments. After immunophenotype identification and differentiation induction, cells were divided into 4 groups. In the experimental groups, apoptosis of SMSCs and BMSCs were induced by 20 ng/mL TNF-α and 10 μg/mL cycloheximide, and cells were cultured in normal culture medium in the control groups. Cellular morphology were observed by inverted phase contrast microscope. After apoptosis induction for 24 hours, cell viability was determined by cell counting kit 8 assay and apoptotic index was detected by flow cytometer. Moreover, the level of Cleaved Caspase-8, 3 were determined by Western blot. ResultsBoth SMSCs and BMSCs accorded with the definition criteria of MSCs according to results of immunophenotype identification and differentiation induction. After apoptosis induction, cells became shrinking and partially floated and cellular morphologies became worse than those in the control groups. After apoptosis induction for 24 hours, cell viabilities of SMSCs and BMSCs in the control groups were both 100%, and no apoptotic cells were observed. However, cell viabilities of SMSCs and BMSCs in the experimental groups were 60.13%±8.63% and 46.55%±10.54% respectively, which were both significantly lower than those in the control groups (P<0.05) , and cell viability in the SMSCs experimental group was significantly higher than that in the BMSCs experimental group (t=3.152, P=0.006) . The apoptotic index was 36.54%±8.63% in the SMSCs experimental group and was 53.77%±11.52% in the BMSCs experimental group, both were significantly higher than the control groups (1.12%±0.24% and 1.35%±0.31%) (P<0.05) . What's more, it was significantly lower in SMSCs experimental group than that in BMSCs experimental group (t=3.785, P=0.001) . Moreover, no expression of Cleaved Caspase-8, 3 was detected in the control groups. But the levels of Cleaved Caspase-8, 3 were significantly enhanced in the experimental groups and they were lower in SMSCs than in BMSCs (t=13.870, P=0.000; t=7.309, P=0.000) . ConclusionsTNF-α induced apoptosis is lower in SMSCs than in BMSCs, which means that SMSCs may have stronger anti-apoptosis ability than BMSCs.
ObjectiveTo observe the relationship of serum tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and C-reactive protein (CRP) with obstructive sleep apnea hypopnea syndrome (OSAHS) associated pulmonary hypertension (OSAHS-PH). MethodsFrom September 2013 to October 2014, 38 OSAHS patients, 32 OSAHS-PH patients and 35 healthy subjects were enrolled from the General Hospital of Ningxia Medical University. OSAHS was diagnosed by polysomnography. The pulmonary artery systolic pressure (PASP) was measured by echocardiograph, and the diagnose criteria for pulmonary hypertension was PASP≥40 mm Hg. Serum TNF-α, IL-6, CRP and endothelin 1 (ET-1) were detected by enzyme-linked immunosorbent assay. The correlation between TNF-α, IL-6, CRP, ET-1 and PASP was analyzed. ResultsThe serum levels of TNF-α, IL-6, CRP and ET-1 were remarkably different among three groups (F=55.34, 25.05, 23.85, 34.06 respectively; all P < 0.05). The levels of TNF-α, IL-6, CRP and ET-1 in the OSAHS group were higher than those in the healthy group, and lower than those in the OSAHS-PH group (all P < 0.05). The PASP was positively correlated with the levels of the four factors (r=0.755, 0.762, 0.747, 0.759 respectively; all P < 0.01). ConclusionThe levels of serum TNF-α, IL-6 and CRP are correlated with pulmonary hypertension and they may be involved in the process of OSAHS-PH.