Expression of TNF-α and TGF-β in oral lichen planus patients

AIM: To investigate the expression of TNF-α， TGF-β in patients with oral lichen planus (OLP) and normal oral mucosa (NOM). METHODS: An immunohistochemical technique was performed to detect TNF-α, TGF-β1 and TGF-β2 expression in 22 cases with OLP and 10 normal controls. RESULTS: In lamina propria of OLP, the expression of TNF-α and TGF-β1 were increased, whereas TGF-β2 did changed significantly compared with control group. TNF-α positive signal were mostly found in macrophages, lymphocytes. TGF-β1 positive cell was present in macrophages, endothelial cells and fibrocytes. CONCLUSION: TNF-α and TGF-β1 play an important role in the development and maintenance of OLP local inflammation.     

Relationship between SNP of TNFR gene and incidence/severity of pneumonia

AIM：To analyze the relationship between the single nucleotide polymorphism (SNP) of tumor necrosis factor receptor (TNFR) gene and the incidence and severity of pneumonia. METHODS：Total 132 Chinese individuals were enrolled in this study. There were 66 patients with pneumonia and 66 healthy subjects. The SNPs of TNFR gene including TNFR1+36A/G, TNFR1-609G/T, TNFR2+676T/G, TNFR2+1663T/G, TNFR2 +1668A/G and TNFR2 +1690C/T were genotyped by polymerase chain reaction-restriction fragment length polymorphism or gene sequencing for all subjects. Polymorphisms affecting pneumonia incidence and severity were calculated by SPSS. RESULTS：The frequencies of TNFR1-609G and T alleles in pneumonia patients were 40.9% and 59.1%, while those in healthy subjects were 53.8% and 46.2%. The frequency of TNFR1-609T in pneumonia patients was higher than that in healthy subjects (P<0.05). Besides, the frequencies of TNFR1-609G and T alleles in severe pneumonia patients were 25.0% and 75.0%, while those were 46.0% and 54.0% in non-severe pneumonia patients. The frequencies of TNFR2 +1690C and T alleles in severe pneumonia patients were 81.1% and 18.9%, while those were 61.0% and 39.0% in non-severe pneumonia patients. The frequencies of TNFR1-609T and TNFR2 +1690C in severity pneumonia subjects were higher than those in mild subjects (P<0.05). CONCLUSION：It appears that TNFR1-609T is associated with high incidence of pneumonia. TNFR1-609T and TNFR2+1690C are the risk factors of severity in pneumonia in Chinese.     

Relationship between morphologic changes in neuron or neuroglial cells and expression of TNF-α and c-Myc in cortex after focal cerebral ischemia/reperfusion in MCAO rats

AIM: To investigate the relationship between morphologic changes in neuron or neuroglial cells and expression of tumor necrosis factor α (TNF-α) and c-Myc in cortex after focal cerebral ischemia/reperfusion in MCAO rats. METHODS: The focal cerebral ischemia/reperfusion model was established by intraluminal thread occlusion of the middle cerebral artery (MCAO). The middle cerebral arteries of rats were occluded for 2 hours and reperfused for 1, 3 and 7 days. Using the techniques of immunohistochemical staining and optical microscopy, the morphologic changes in neuron or neuroglial cells were observed in the cortex of frontal or parietal lobe; the cell types which dynamicaly expressed TNF-α, c-Myc in the different period were also observed. RESULTS: The degeneration or necrosis of neuron or neuroglial cells were observed at the center of infarction, it was very serious at 3 d after reperfussion. Astrocyte and microglial cell proliferation were observed at the broder of infarction. TNF-α and c-Myc positive cells, most of which were astrocytes and microglial cells, increased significantly at 3 d after reperfusion. CONCLUSION: TNF-αand c-Myc may play an important role in the regulation of neuron or neuroglial cells after focal cerebral ischemia with reperfusion.     

Relationship between TNF-α gene polymorphism-308 and type 2 diabetes with periodontitis

AIM: To investigate the gene polymorphism-308 of tumor necrosis factor alpha (TNF-α) in the relation with the susceptibility to periodontitis combined with type 2 diabetes mellitus (DM) and its severity.METHODS: Human DNA samples were obtained from 240 DM patients with periodontitis (periodontitis group, n=120) and without periodontitis (control group, n=120). All patients were genotyped by PCR-RFLP analysis. The frequencies of genotypes and alleles were analyzed. Sulcus bleeding index (SBI) and probing depth (PD) in all patients were measured. The polymorphism-308 of TNF-α gene in the relation with the susceptibility to periodontitis combined with DM and its severity was analyzed.RESULTS: No significant difference in the frequency of genotype and allele was found between DM patients with mild periodontitis and DM patients without periodontitis (P>0.05). However, the frequencies of these genotypes and alleles in DM patients with moderate and severe periodontitis were significantly higher than those in DM patients without periodontitis (P<0.01). The findings showed that the level of TNF-α was associated with SBI and PD (P<0.01).CONCLUSION: TNF-α -308 G/A polymorphism is not associated with DM patients with mild periodontitis, whereas it may have a role in pathogenesis and prognosis of moderate and severe periodontitis combined with DM through TNF-α level.     

TNF-related apoptosis inducing ligand and its biological functions

TNF-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein in TNF family. TRAIL influences a variety of immunological functions including cellular activation, proliferation and death, upon interation with a corresponding super family of receptors. This article is about the new views on the biological functions of TRAIL.     

Aortic expression of HSP22, TNF-α and eNOS in rats with hyperlipidemia and effects of atorvastatin

AIM:To establish a rat hyperlipidemia model for studying the aortic expression of heat shock protein 22 (HSP22), tumor necrosis factor alpha (TNF-α) and endothelial nitric oxide synthase (eNOS) and the effect of atorvastatin intervention. METHODS:Hyperlipidemia model was established in SD rats. Afterwards, the rats were divided into normal control group, high fat group and high fat+atorvastatin intervention group. The expression of HSP22 and TNF-α in the rat aortas was detected by immunohistochemical assay and the expression of eNOS was assessed by Western blotting. RESULTS:No detectable expression of HSP22 and TNF-α in the normal control group was observed. However, the expression of HSP22 and TNF-α was positive in the high fat group and the atorvastatin intervention group. The mean densities of HSP22 and TNF-α positive particles were significant lower in the atorvastatin intervention group as compared with high fat group (both P＜0.05). The expression of eNOS protein in the high fat group and atorvastatin intervention group was significantly lower than that in normal control group (P<0.01). However, no marked difference of eNOS protein expression between high fat group and atorvastatins intervention group was observed. CONCLUSION: The expression of HSP22 and TNF-α in the rat aortas is increased in the hyperlipidemia rat model. This effect can be restored by atorvastatin treatment. The expression of eNOS in the rat aortas is decreased in the hyperlipidemia rat model, but this tendency could not be attenuated by atorvastatin.     

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 .     

Relationship between levels of IL-2, TNF-α and nitric oxide in aqueous humor after intraocular lens implantation

AIM: To investigate the relationship between the level of interleukin-2 (IL-2), tumour necrosis factor-α (TNF-α) and nitric oxide (NO) in aqueous humor after intraocular lens implantation. METHODS: New Zealand rabbits were divided randomly into three groups: (1) control group; (2) extracapsular cataract extraction group (ECCE); (3) extracapsular cataract extraction and posterior chamber intraocular lens implantation group (ECCE+IOL). The inflammation in all experimental rabbit eyes was observed via zoom-photo slit-lamp microscope on 1, 3, 7, 14 d and 30 d postoperation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) counting and classifying and for determining IL-2, TNF-α and NO₂^-/NO₃^- contents. RESULTS: (1) The level of IL-2 and TNF-α and NO₂^-/ NO₃^- in aqueous humor of ECCE+IOL group were higher than that in ECCE and control at 1 to 14 days postoperation, respectively, it increased to peak value at 3 to 7 days postoperation and decreased gradually two weeks postoperation; (2) The changes in IL-2, TNF-α and NO₂^-/NO₃^- in each group were basically similer; (3) The changes of IL-2 and TNF-α level were closely related with NO content in aqueous humor (r=0.69, P<0.01 and r=0.98, P<0.01). CONCLUSION: IL-2, TNF-α and NO play an important role in intraocular inflammation intraocular lens implantation.     

Lipopolysaccharide stimulates TNF-α and endothelin-1secretion from cultured rat Kupffer cells

AIM: To investigate lipopolysaccharide (LPS) stimulated cytokine secretion from normal rat Kupffer cells in vitro. METHODS: Kupffer cells were isolated from wistar rats liver and cultured. Tumor necrosis factor -α (TNF-α) and endothelin-1 (ET-1) secreted by LPS stimulated Kupffer cells were detected. RESULTS: LPS had an stimulative effect on Kupffer cell activity. LPS in definite concentrations promoted Kupffer cell secretion. CONCLUSION: LPS promotes Kupffer cell secretion, which may be associated with liver injury induced by LPS.     

Expression of TNF-α mRNA in hypertrophic myocardium by pressure overload in rats

AIM: To observe the change of TNF-α mRNA in hypertrophic cardiac myocytes induced by pressure overload in rats and the effect of captopril. METHODS: Serum and heart were collected 42 days after the cardiac hypertrophy model made by pressure overload by abdomen aorta-constriction (AC). Hypertrophic parameter and the concentration of TNF-α in serum and left ventricle were determined by ELISA. TNF-α mRNA in cardiac myocytes was determined by in situ hybridization and analyze by ELIA image analysis system. The orientation of TNF-α mRNA in cardiac myocytes was also observed. RESULTS: Left ventricle hypertrophy was observed 42 days after operation. TNF-α mRNA in AC group elevated 98% compared to sham-operated group and descended 64.14% by captopril (P<0.01), but did not descend to the normal level. The expression of TNF-α mRNA showed mostly in myocardial matrix by in situ hybridization. The level of expression was very low in sham-operation group and markedly enhanced after aorta-constriction, but it was decreased when treated by captopril. CONCLUSION: Endogenous TNF-α acts as an important adjustive factor in the pressure overload-induced cardiac hypertrophy and TNF-α mRNA increased in myocardial matrix may be activated by renin-angiotension system.     

Activation of α7nAChR promotes wound healing in diabetic mice by suppressing TNF-α expression

AIM: To explore the role of α7 nicotinic acetylcholine receptor (α7nAChR)-specific agonist PNU-282987 in promoting wound healing in diabetic mice by suppressing the expression of tumor necrosis factor α （TNF-α）.METHODS: A model of incised wound was established in diabetic mice or normoglycaemic mice (control). Skin samples were taken on 1 d, 3 d, 5 d, 10 d, 14 d and 21 d post-injury (5 mice in each posttraumatic interval). The numbers of macrophages and fibroblasts, the expression of TNF-α and the deposition of collagen were detected by the methods of immunohistochemistry, Western blotting and Masson staining, respectively. After incised wound was performed in the diabetic mice, PNU-282987 was applied by intraperitoneal injection at suitable posttraumatic interval. The above indexes were investigated again.RESULTS: Compared with control group, the diabetic mice presented delayed wound healing. In diabe-tic mice, the infiltration of macrophages and the expression of TNF-α were significantly reduced in the early phase during wound healing, while they were significantly increased from 5 d post-injury. Besides, from 5 d to 21 d post-injury, the wounds in diabetic mice showed decreased number of fibroblasts and deposition of collagen. From 5 d post-injury, PNU-282987 was applied to diabetic mice, which significantly down-regulated the expression of TNF-α, and increased the number of fibroblasts and the content of collagen in the wounds, eventually promoted wound healing.CONCLUSION: Inflammatory reactions delay wound healing in diabetic mice. Activation of α7nAChR promotes wound healing in diabetic mice by suppressing the expression of TNF-α.     

Characteristics in the secretion of liver TNF-α induced by infusion of LPS into veins in goats

AIM: To study the role of liver in immune regulation in experimental endotoxemia. METHODS: 17 castrated male goats were subjected to simultaneously installing catheters in jugular, hepatic and portal veins by surgery. Four days later, lipopolysaccharide (LPS) was infused in term of three groups as followings: In group ①, LPS of 20 EU (endotoxin unit, EU)·kg^-1 was infused into portal vein; In group ②, LPS of 20 EU·kg^-1 was infused into jugular vein and LPS of 1 500 EU·kg^-1 infused into jugular vein in group ③. Before and after infusion, blood samples were collected from the three veins through the catheters for 8 h.The plasma levels of TNF-α were measured by RIA. RESULTS: In group ①, the plasma TNF-α levels of hepatic and portal vein rose to peak value at 5 h, but that of the jugular vein did not changed. In group ②, the plasma TNF-α levels in hepatic vein rose to peak value at 3 h. The TNF-α levels of jugular vein rose to peak value at 1 h and the one in portal vein enhanced continuously between 0-8 h. In group ③, the plasma TNF-α levels in jugular, hepatic and portal vein rose to significant peaks at 1 h simultaneously. CONCLUSION: During experimental endotoxemia,liver showed different dynamic characteristics in TNF-α secretion according to the pathway and doses of LPS delivery.     

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 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.     

TNF-α exacerbates cholesterol accumulation via down-regulating LXRα promoter activity in HepG2 cells

AIM: To investigate the exacerbating effect of tumor necrosis factor alpha (TNF-α) on lipid accumulation in HepG2 cells by inhibiting liver X receptor alpha (LXRα) signaling pathway. METHODS: Luciferase reporter plasmid driven by the LXRα promoter (pGL3-Basic-LXRα-P) was constructed and transfected into HepG2 cells to detect the LXRα promoter activity. HepG2 cells were incubated with serum-free medium (control), 20 μg/L TNF-α (TNF-α), 100 mg/L LDL (LDL) and 20 μg/L TNF-α plus 100 mg/L LDL (LDL+TNF-α), respectively. The effects of TNF-α on cholesterol accumulation were examined by oil red O staining and quantitative intracellular cholesterol assay. The expression of LXRα, ABCA1 and ABCG1 at mRNA and protein levels was examined by real-time PCR and Western blotting. RESULTS: The pGL3-Basic-LXRα-P was constructed successfully. TNF-α decreased the activity of LXRα promoter in the absence or presence of LDL. Inflammatory stress inhibited the expression of LXRα, ABCA1and ABCG1 at mRNA and protein levels. The cholesterol efflux was increased after loading of LDL, while TNF-α decreased intracellular cholesterol efflux. The results of oil red O staining and quantitative intracellular cholesterol assay demonstrated that inflammatory stress increased cholesterol levels in HepG2 cells. CONCLUSION: TNF-α exacerbates the cholesterol accumulation in hepatic cells via inhibiting LXRα promoter activity and gene expression.     

Effect of oleanolic acid on expression of TNF-α and collagen in silicotic rats in vivo

AIM: To observe the effect of oleanolic acid (OA) on the expression of Tumor necrosis factor-α (TNF-α) and collagen in silicotic rats in vivo and its possible mechanism. METHODS: Male Wistar rats were divided into 4 groups according to the randomized block design: control group, model group, OA group and solvent control group (20 rats in each group). Except control group, the rats in other groups were induced by intratracheal instillation of silicon di-oxide (SiO₂; 250 mg/kg). The rats in OA group were intragastrically administered with OA (60 mg/kg) from the second day of giving SiO₂. The rats in solvent control group were gavaged daily with 0.6% sodium carboxymethyl cellulose solution (10 mL/kg). The rats in control group were given normal saline under the same condition for 56 consecutive days. All rats were killed at the 7th, 14th, 28th and 56th days. The lung coefficient was detected and the morphological changes were observed. The serum contents of TNF-α were detected by ELISA. The content of total collagen in the lung tissue was measured. The protein level of nuclear factor-κB (NF-κB) in the lung tissue was determined by immunohistochemical method. RESULTS: (1) According to the morphological changes, the silicosis model was successfully established. Compared with control group, the lung coefficient and total collagen increased obviously in model group and solvent control group. The lung coefficient and total collagen content in OA group at each time point reduced compared with those in model group and solvent group, and increased compared with those in control group at the corresponding time points. (2) The serum contents of TNF-α in model group and solvent control group significantly increased, peaking at the 14th day, slightly decreasing afterward, and showing statistically significant difference at each time point compared with those in control group. No significant difference between model group and solvent group at different time points was observed. OA had inhibitory effect on the contents of TNF-α compared with model group and solvent group at the corresponding time points. (3) NF-κB in model group and solvent control group significantly increased, peaking at the 28th day, and showing statistically significant difference at each time point compared with those in control group. The NF-κB expression in OA group was similar to model group, but significantly decreased compared with control group at each time point. CONCLUSION: OA inhibits the expression of TNF-α and collagen and attenuates the silicosis fibrosis, which may be related to the NF-κB pathway.     

SC58125 regulates TNF-α induced apoptosis in human colonic carcinoma cell line HT29 by inhibiting IκBα degrades

AIM:To investigate whether SC58125 synergizes with TNF-α to induce HT-29 cell apoptosis and study the possible molecular mechanism. METHODS:By using MTT,Hoechst 33342 staining and agarose gel electrophoresis,the effect of SC58125/TNF-α on the proliferation and apoptosis of HT-29 cell line was examined. The activity of caspase-3,the expression of IκBα,the phosphorylation level of IκBα,and the activation of NF-κB were measured after treatment with SC58125 by electrophoretic mobility shift assay and Western blotting.RESULTS:Both SC58125 and TNF-α exhibited cytotoxicity. The combination of the two reagents significantly reduced HT-29 cell viability in a dose-dependent manner. SC58125 and TNF-α co-treated cells showed induced nuclear condensation and fragmentation,and led to oligonucleosomal cleavage of genomic DNA,which was accompanied by the induction of caspase activity. IκBα levels were substantially decreased by the treatment of TNF-α. The degradation of IκBα was almost completely inhibited when SC58125 was added. NF-κB was activated in HT-29 cells after treatment with TNF-α,whereas pretreatment of HT-29 cells with SC58125 for 2 h,TNF-α induced NF-κB DNA binding was profoundly suppressed. CONCLUSION:SC58125 synergizes with TNF-α to inhibit cell growth and induces apoptosis in HT-29 cells,which may be mediated by activating caspase-3 and preventing degradation of IκBα.     

Effects of Fas,NF-κB and caspases on microvascular endothelial cell apoptosis induced by TNFα

AIM:To study the apoptotic effect of TNFα on rat pulmonary microvascular endothelial cells(PMVEC)and the role of Fas, NF-κB in its mechanism. METHODS: Apoptosis of PMVEC was analyzed and quantitated with TUNEL, flow cytometer. Northern blot was applied to assess the influence of TNFα on PMVEC Fas expression. Fas antibody was used to investigate the apoptotic effect of Fas on PMVEC. Activation of caspase-8 was examined by Western blot. Expression of caspase-3 was analyzed with histo-immunochemical staining. RESULTS:Growth curve showed that TNFα suppressed endothelial cell proliferation in a dose-dependent manner. After treatment with 5×10⁶U/LTNFα, apoptotic rate was 14.0%±3.1% detected with TUNEL, and 13.1% with flow cytometer. When the cells were co-cultured with TNFα and APDC, an NF-κB inhibitor, less cells were viable and more cells were positively stained with TUNEL. Fas expression in PMVEC was elevated after TNFα treatment. Co-culturing with Anti-Fas antibody aggravated PMVEC apoptosis. Caspase-8 activity and caspase-3 expression was elevated. Caspase-3 inhibitor significantly ameliorated PMVEC apoptosis. CONCLUSION: Large dose of TNFα(5×10⁶U/L) can induce apoptosis in rat PMVEC. NF-κB has an anti-apoptotic effect in PMVEC. TNFα up-regulates Fas expression in PMVEC, and the latter takes a part in apoptosis. Caspase-8 and caspase-3 are involved in PMVEC apoptosis induced by TNFα.