Effects of antioxidant and NF-κB on the induction of IL-8 in human colon cancer cell line HT-29 cells in vitro

AIM: To investigate the role of nuclear factor κB (NF-κB) in the induction of IL-8 gene by TNF-α in colon cancer cells and the effect of antioxidant on the induction of IL-8. METHODS: ELISA was used to detect the concentrations of IL-8. IL-8 mRNA was analyzed by using RT-PCR. NF-κB in the cell nuclei was detected with electrophoretic mobility shift assay. RESULTS: (1) IL-8 production and IL-8 mRNA expression induced by TNF-α was blocked by pyrrolidine dithiocarbamate (PDTC). (2) TNF-α triggered the activation and translocation of NF-κB and PDTC inhibited the activation of NF-κB induced by TNF-α. CONCLUSION: The induction of IL-8 gene and protein by TNF-α is dependent on the activation of NF-κB. Antioxidants may inhibit the induction of IL-8 gene and protein through inhibiting NF-κB activation.     

CO2 与养分交互作用对番茄幼苗养分利用的影响

以番茄(Lycopersicon esculentumMill.)‘合作906’为材料进行溶液培养试验,设2个因子:CO2和营养液浓度;CO2浓度设正常(360μL/L)和倍增(720μL/L)2个水平;营养液浓度设基本营养液(日本山崎番茄营养液),微量元素采用阿农营养液配方的1/2、1/4、1/8、1/164个水平,完全试验方案8个处理,3次重复。pH为6·0±0·2,3d更换1次营养液。移植到1·2L盆(2株/盒)中,植株在CO2生长箱(VS-3DMC)中培养,全天施放CO2,白天25℃,晚上15℃,光照为14h/d,光照强度11000lx,相对湿度60%。46d时收获,根、茎、叶经蒸馏水冲洗吸干水分后,放入纸袋105℃杀青,75…     

Effect of inhibiting nuclear factor-kappa B on TNF-α-induced expression of angiotensinogen and production of angiotensinⅡ in glomerular mesangial cells

AIM: To study the effect of inhibiting nuclear factor-kappa B (NF-κB) activity on the expression of angiotensinogen (AGT) and the production of angiotensinⅡ (AngⅡ) induced by tumor necrosis factor-α (TNF-α) in glomerular mesangial cells (MCs) of SD rats. METHODS: The MCs of SD rats were isolated and divided into three groups as follows: control; MCs treated with TNF-α, and the MCs treated with TNF-α + pyrrolidinedithiocarbamate (PDTC). The activity of nuclear factor-kappa B was measured by electrophoretic mobility shift assay. The expression of AGT was determined by RT-PCR for mRNA and Western blotting for protein. The concentration of angiotensinⅡ in supernatant was measured by RIA. RESULTS: The NF-κB activity in the MCs treated with TNF-α (20.67±9.14)×102 μg/cell was significantly higher than that in control cells ［(8.25±4.35)×102 μg/cell, P<0.01］ and the MCs treated with TNF-α+PDTC ［(7.20±4.57)×102 μg/cell, P<0.01］, and no significant difference was found between control and the MCs treated with TNF-α＋PDTC (P>0.05). The AGT mRNA level in the MCs treated with TNF-α (0.27±0.05) was higher than that in the control cells (0.20±0.05, P<0.05), and no significant difference was observed when compared with that in the MCs treated with TNF-α＋PDTC (0.22±0.06, P>0.05). The expression of AGT protein in the MCs treated with TNF-α (0.60±0.19) μg/cell was higher than that in the control ［(0.37±0.15)μg/cell, P＜0.05］ and the MCs treated with TNF-α＋PDTC ［(0.37±0.17)μg/cell, P<0.05］, and no significance was found between the MCs treated TNF-α＋PDTC and the control (P>0.05). The AngⅡ level in supernatant of cultured MCs treated with TNF-α ［(9.73±2.38)×10-5 ng·L-1/cell］ was significantly higher than that in the control ［(7.50±1.51)×10-5 ng·L-1/cell, P<0.05］ and in the MCs treated with TNF-α＋PDTC ［(6.94±1.46)×10-5 ng·L-1/cell, P<0.05］, however, the difference between the MCs treated with TNF-α＋PDTC and the control was of no significance (P>0.05). CONCLUSION: TNF-α activates the NF-κB in glomerular MCs, induces the AGT expression and the production of AngⅡ. Inhibition of NF-κB decreases the AGT expression and the production of AngⅡ. Therefore, the effects of TNF-α on AGT and AngⅡ may be mediated by NF-κB.     

NF-κB activation in keloid fibroblasts stimulated with TNF-α

AIM: To investigate NF-κB p65 activation and IκB-α expression in keloid fibroblasts (KFB) and normal skin fibroblasts (NSF) stimulated with TNF-α and to explore the underlying molecular pathogenesis of keloid formation. METHODS: Primary KFB was cultured. The location of NF-κB p65 and IκB-α in KFB and NSF at quiescent condition and the nuclear translocation of NF-κB p65 after TNF-α stimulation were observed by immunofluorescence technique. NF-κB p65 DNA binding activity was detected with TransAMTM NF-κB p65 kit. The IκB-α protein level was determined by means of Western blotting technique. RESULTS: After stimulated with TNF-α, NF-κB p65 translocated into the nucleus. NF-κB p65 DNA binding activity increased to its maximum at 1 h and was dropped to normal at 4 h. TNF-α induced most degradation of IκB-α at 15 min and became detectable in cytoplasm after 4 h. KFB showed more sensitive ability to TNF-α stimulation than NSF. CONCLUSION: NF-κB may play a role in keloid pathogenesis.     

Effects of thalidomide on the expression of NF-κB and TNF-α in experimental hepatic fibrotic rats

AIM: To study the effects of thalidomide on the expressions of nuclear factor κB (NF-κB) and tumor necrosis factor-α (TNF-α) in rat liver fibrosis.METHODS: The fibrosis of rat liver was induced by intraperitoneal injection of carbon tetrachloride thrice weekly.Meanwhile thalidomide (10 mg·kg^-1·d^-1 or 100 mg·kg^-1·d^-1) was given daily by the intragastric route for 8 weeks.Serum alanine aminotransferase (ALT),aspartate aminotransferase (AST),prealbumin (PA),hyaluronic acid (HA) and laminin (LN),and hydroxyproline (HYP) contents in the liver,NF-κB p65 and α-smooth muscle actin (α-SMA) protein in the liver,IκBα and TNF-α protein in cytoplasm and NF-κB p65 protein in nucleus and TNF-α mRNA levels in the liver were studied.RESULTS: Compared with the model group,the Knodell score,serum ALT,AST,HA,LN levels and HYP contents in liver,NF-κB p65 protein in nucleus and α-SMA protein in the liver,and TNF-α mRNA and protein in the liver of rats given high dose of thalidomide were decreased significantly (P<0.01).Meanwhile PA level and IκBα protein in cytoplasm were elevated significantly (P<0.01).CONCLUSION: Thalidomide exerts its effect on the down-regulation of NF-κB-induced TNF-α via inhibiting dissociation and degradation of IκB and prevents liver fibrosis in rats.     

uclear factor κB in LPS stimulated rat alveolar macrophages promotes TNF-α secretion

AIM: To investigate the activation of nuclear factor κB (NF-κB) induced by lipopolysaccharides (LPS) in rat alveolar macrophages (AMs) and its regulatory role in tumor necrosis factor (TNF-α) secretion. METHODS: The dynamic activity changes of NF-κB induced by LPS were determined with electrophoretic mobility shift assay (EMSA). Antisense oligonucleotides of NF-κB subunit (p65) were transfected into AMs prior to LPS stimulation. The effect of antisense oligonucleotide transfection on expressions of p65 and TNF-α in supernatant were measured with Western blotting and enzyme linked immunosorbent assay (ELISA), respectively.RESULTS: NF-κB activity increased markedly and reached its peak level at 4 h after LPS stimulation. After transfected with antisense oligonucleotides of NF-κB subunit (p65), expression of p65 and TNF-α in supernatant decreased markedly.CONCLUSION: NF-κB activity has a positive effect on regulating secretion of TNF-α in AMs induced by LPS.     

Effects of rosiglitazone on expressions of peroxisome proliferator-activated receptor gamma and TGF-β1 in rats with adriamycin nephrosis

AIM: To investigate the expression of renal peroxisome proliferator-activated receptor gamma (PPARγ) in rats with adrimycine nephrosis (ADR), and the effect of rosiglitazone on the activation of NF-κB p65 in renal tissue rats with ADR. METHODS: The rats were randomly assigned to following groups: control (CTR) group, adrimycine nephrosis (ADR) group, and ADR treated with rosiglitazone (5 mg·kg^-1·d^-1) group（RGL）. The levels of urinary protein, albumin, total cholesterol, triglyceride and renal function change in rats were measured after 12 weeks. The nuclear-translocation of cortical NF-κB p65 was detected by immunohistochemistry. The activity of cortical NF-κB p65 was measured by sandwich ELISA. The mRNA levels of cortical PPARγ and TGF-β₁ were detected by RT-PCR. The protein expressions of PPARγ and TGF-β₁ in the rat kidney tissues were detected by Western blotting. RESULTS: As compared to ADR group, the urinary protein excretion in RGL treatment group was decreased and the serum albumin levels were increased, but the serum total cholesterol and triglyceride were decreased and the renal pathological lesion was ameliorated. The activity of NF-κB p65 and the expressions of TGF-β₁ mRNA and protein were significantly decreased in rosiglitazone group, while the expression of PPARγ mRNA and protein was increased in RGL group (P<0.01). The correlation analysis was manifested: in ADR and RGL group, a negative correlation between the activity of NF-κB p65 and the expression of PPARγ in renal tissue (r=-0.8305, P<0.01) was observed. There was a negative correlation between the expression of TGF-β₁ and PPARγ in renal tissues (r=－0.7938, P<0.01). CONCLUSION: The expression of renal cortical PPARγ is up-regulated in rats with adrimycine nephrosis by rosiglitazone. Rosiglitazone inhibits the activation of renal cortical NF-κB p65 in part, so it inhibits the gene expression of renal TGF-β₁ and relieves the renal pathological lesion.     

PPAR-α involves in cardiomyocyte hypertrophy induced by high glucose and insulin

AIM: To study the role of peroxisome proliferator-activated receptor-α (PPAR-α) signal transduction pathway in cardiac hypertrophy induced by high glucose and insulin (HGI). METHODS: The cultured neonatal rat cardiomyocytes were used to observe the effect of fenofibrate (FF), a selective PPAR-α agonist, on cardiomyocyte hypertrophy induced by HGI (glucose at concentration of 25.5 mmol/L and insulin at 0.1 μmol/L). The cardiomyocyte hypertrophic responses were assayed by measuring the cell surface area, protein content, and mRNA expression of atrial natriuretic factor (ANF). The expressions of mRNA and protein were assayed by real -time PCR and Western blotting. RESULTS: In cultured cardiomyocytes, HGI induced profound change of hypertrophic morphology, the significant increase in cell surface area, protein content and ANF mRNA expression compared to those in vehicle control (P<0.01), but the expressions of PPAR-α mRNA and protein decreased significantly (P<0.05). At the same time, the expression of cyclooxygenase 2 (COX-2), one of the PPAR-α downstream effectors was obviously elevated (P<0.05). However, FF (0.1, 0.3 and 1 μmol/L) inhibited the cardiomyocyte hypertrophy induced by HGI in a concentration-dependent manner (P<0.01). FF at concentration of 0.3 μmol/L increased the expressions of PPAR-α in both mRNA and protein levels (P<0.05) and inhibited the expressions of COX-2 (P<0.05), which were abolished by MK 886 (0.3 μmol/L), a selective PPAR-α antagonist (P<0.05). CONCLUSION: PPAR-α signal transduction pathway and its downstream effector COX-2 might involve in the cardiomyocyte hypertrophy induced by HGI.     

Effects of metformin on nuclear factor-κB expression and serum high sensitivity C-reactive protein level in atherosclerosis model of rabbit

AIM: To investigate the effects of metformin on nuclear factor-κB (NF-κB),its inhibitor IκB,and the level of serum high sensitivity C-reactive protein (hs-CRP) in rabbits.METHODS: 24 New Zealand male rabbits were randomly divided into control group,atherosclerosis (AS) group and metformin (Met) group.AS group and Met group were made as models by cholesterolenriched diets feeding and vascular intimal immunologic injury.The AS model was confirmed by high frequency ultrasound.Met group were given metformin 150 mg·kg-1·d-1 for 8 weeks.At the end of experiment,serum hs-CRP and serum lipids in all three groups were detected.Immunohistochemistry and Western blotting technique were applied to detect the expression of nucleus NF-κB p65 and cytoplasma IκBα in aorta in all three groups.RESULTS: Compared to normal control group,the level of serum hs-CRP was elevated (1.27±0.43 vs 3.96±0.63,P<0.01),the expression of nucleus NF-κB p65 increased significantly (P<0.01) while the expression of IκBα reduced significantly (P<0.01).Compared to AS group,metformin significantly reduced the level of serum hs-CRP (2.79±0.40 vs 3.96±0.63,P<0.05) and the expression of nucleus NF-κB p65 (P<0.01),and increased the expression of IκBα (P<0.05).CONCLUSION: Metformin inhibits the activation of NF-κB p65 and the degradation of IκBα,and decreases the levels of serum hs-CRP in AS rabbits.These results suggest that metformin exerts direct vascular anti-inflammatory effects.It may be one important mechanism of metformins antiatherogenic properties.     

RIP1-mediated necroptosis regulates inflammatory response of renal tubular epithelial cells via NF-κB signaling pathway

AIM To investigate the effect of receptor-interacting protein 1 (RIP1)-mediated necroptosis on human kidney proximal tubular cell inflammation and its related mechanisms. METHODS Human kidney proximal tubular HK-2 cells were cultured in vitro, and stimulated with tumot tumor necrosis factor-α (TNF-α) and Z-VAD-FMK for 24 h. Lactate dehydrogenase (LDH) cytotoxicity assay was used to detect the percentage of necrosis. Western blot was used to detect the protein expression of RIP1, IKK-α and NF-κB p65. The protein levels of interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were determined by Western blot and ELISA. Real-time PCR was used to detect the mRNA expression level of NF-κB p65. Furthermore, the RIP1 inhibitor necrostatin-1 (Nec-1) and the NF-κB specific inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) were used, and the above indicators were also detected. RESULTS Compared with control group, the protein level of RIP1 was increased in TNF-α combined with Z-VAD-FMK stimulation group (T/Z group). The protein levels of IKK-α and NF-κB p65 were obviously increased, and the release of LDH was increased (P<0.01). Western blot and ELISA showed that the expression levels of IL-1β and MCP-1 were significantly increased (P<0.01). Real-time PCR showed that the mRNA expression level of NF-κB p65 was also obviously increased. After Nec-1 or PDTC stimulation (T/Z+N group or T/Z+P group), the release of LDH, and the expression levels of inflammation-related indicators IL-1β and MCP-1 were significantly decreased. The protein expression levels of IL-1β and MCP-1 were further reduced after treatment with the above 2 stimulati (T/Z+P/N group). CONCLUSION Under T/Z condition, RIP1-mediated necroptosis plays an important role in renal tubular inflammatory response, which may be partly achieved by regulating the activation of NF-κB signaling 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.     

Inhibitory effects of CCK-8 on NF-κB activities stimulated by LPS in rat PIMs

AIM: To investigate the inhibitory effects of cholecystokinin octapeptide (CCK-8) on nuclear factor-κB (NF-κB) activities stimulated by lipopolysaccharide (LPS) by using forskolin, the activator of adenylate cyclase, and PKA inhibitor H-89 in rat pulmonary interstitial macrophages (PIMs).METHODS: PIMs were isolated and purified.EMDA was applied to detect NF-κB activities and Western blotting was used to analyze the IκB-α protein level in rat PIMs.RESULTS: The NF-κB activity was not detected in normal control rat PIMs.The NF-κB activity in LPS-treated rat PIMs was obviously higher than that in control group (P<0.01).The IκB-α protein level in endochylema decreased obviously compared to control group (P<0.01).No obvious change of NF-κB activity and IκB-α protein level in CCK or Fsk treated rat PIMs was observed (P>0.05).The NF-κB activity in CCK＋LPS group and LPS＋Fsk group were obviously lower than that in LPS group (P<0.05).The IκB-α protein level was obviously higher (P<0.01).In LPS＋CCK＋H-89 group, the NF-κB activity in rat PIMs was obviously higher than that in CCK＋LPS group (P<0.01), while the IκB-α protein level decreased (P<0.01).CONCLUSIONS: The activation of cAMP-PKA signaling pathway inhibits the increase in NF-κB activity and the decrease in IκB-α protein level stimulated by LPS in rat PIMs.The anti-inflammatory effects of CCK-8 were taken effect by activating cAMP-PKA signaling pathway and further inhibiting the NF-κB activity.     

Effect of TNF-α on production and activation of caspase-3 in primary rat renal proximal tubule cells

AIM: To investigate the production and activation of caspase-3 in primary rat renal proximal tubule cells in response to tumor necrosis factor-α(TNF-α) and the implication of nuclear factor-κB (NF-κB) in the process. METHODS: Isolated rat renal proximal tubule cells (PTCs) from male adult Sprague Dawley rats were treated with TNF-α according to the indicated time courses. A specific NF-κB inhibitor, Bay11-7082, was used alone or as a pretreatment for 1 h followed by exposure to TNF-α for 24 h. The protein levels of cleaved caspase-3, caspase-3, I-κBα, phosphorylated I-κBα, and GAPDH were detected by Western blotting using specific antibodies. RESULTS: The protein level of cleaved caspase-3 relative to caspase-3 was significantly increased in the presence of TNF-α for 6 h, 12 h, and 24 h. Protein levels of caspase-3 were significantly decreased by 12 h and returned to baseline by 24 h in the presence of TNF-α. Treatment with Bay11-7082 for 25 h alone or pretreatment with Bay11-7082 for 1 h followed by addition of TNF-α for 24 h caused a remarkable reduction in both cleaved caspase-3 and caspase-3 as compared to control and TNF-α treated groups. An increase in phosphorylated I-κBα was observed from 15 min to 60 min after treatment with TNF-α at a dose of 10 μg/L in PTCs. CONCLUSION: NF-κB is not only associated with the activation of caspase-3 but also the production of caspase-3 in primary rat renal proximal tubule cells in response to TNF-α.     

Effect of rhSOD on pulmonary nuclear factor-kappa B and MIP-1α expression in meconium-induced acute lung injury

AIM: To evaluate the role and mechanisms of recombinant human superoxide dismutase (rhSOD) in meconium-induced acute lung injury (ALI) by evaluating pulmonary MIP-1α and NF-κB expression. METHODS: 24 health male Sprage-Dawley rats were randomized to 3 groups (8, each group), followed by intratracheal (IT) administration with (1) saline at 1 mL/kg (control group); (2) 20% human newborn meconium suspension at 1 mL/kg, followed by saline at 1 mL/kg (Mec/saline group); (3) 20% human newborn meconium suspension at 1mL/kg, followed by rhSOD at 20 mg/kg (Mec/rhSOD group). The animal was killed 24 h after treatment. The measurements included the bronchoalveolar lavage (BAL) cell count, RT-PCR analysis of pulmonary MIP-1α mRNA expression, Western blotting analysis of pulmonary NF-κB expression. RESULTS: Meconium-induced ALI was characterized by increased BAL cell count, increased expressions of pulmonary MIP-1α mRNA and NF-κB protein ［(4.68±1.40)×109 cells/L vs (0.53±0.19)×109 cells/L, 3.60±0.75 vs 1.56±0.33, 0.72±0.31 vs 0.23±0.12, respectively in control rats, all P<0.01］. IT administration of rhSOD early in the ALI rat significantly decreased meconium-induced BAL cell count ［(3.13±0.77)×109 cells/L vs (4.68±1.40)×109 cells/L in Mec/saline rats, P<0.01］, inhibited the expression of pulmonary MIP-1α mRNA (2.20±0.39 vs 3.60±0.75, in Mec/saline rats, P<0.01) and NF-κB protein (0.44±0.21 vs 0.72±0.31 in Mec/saline rats, P<0.05). CONCLUSION: The early IT administration of rhSOD in ALI rat following meconium aspiration protects lung from inflammatory injury through inhibiting meconium-induced pulmonary MIP-1α mRNA and NF-κB protein expression.