Protective effect of panaxadiols against brain injury induced by LPS through inhibiting NOS activity and p38 expression

AIM: To explore the protective effect of panaxadiols (PDS) on brain injury induced by endotoxin and its mechanism. METHODS: Rats were divided into control,LPS,LPS+dexamethasone (DEX) and LPS+PDS group, respectively. NOS activity, NO content and phosphorylated p38 expression in brain cortex were assayed 4 h after intravenous injection of LPS. RESULTS: NOS activity, NO content and phosphorylated p38 expression in brain cortex in LPS group were obviously higher than those in LPS group. NOS activity, NO content and phosphorylated p38 expression in brain cortex in LPS+DEX and LPS +PDS groups were obviously lower than those in LPS group. CONCLUSION: The protective effects of PDS against brain injury induced endotoxin may be related to decreasing NOS activity, NO content in the brain tissue, and this process is involved in p38MAPKs signal transduction.     

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.     

Curcumin inhibits Porphyromonas gingivalis LPS-induced inflammatory response in human gingival fibroblasts by up-regulating miR-124

AIM To investigate the effects of curcumin （Cur） on the inflammatory response of human gingival fibroblasts （HGFs） induced by Porphyromonas gingivalis （Pg） lipopolysaccharide （LPS） and the role of microRNA-124 （miR-124） in this process. METHODS The HGFs were divided into control group， LPS group （10 mg/L LPS） and LPS+Cur （20， 40 and 80 μmol/L） groups （10 mg/L LPS+corresponding dose of Cur）. After treatment for 24 h， CCK-8 assay was used to measure the cell viability. ELISA was used to measure the levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in the supernatant. The level of miR-124 in the cells was detected by RT-qPCR. The protein levels of nuclear factor kappa B （NF-κB） p-p65 in cytoplasm and nucleus were determined by Western blot， and the nuclear translocation of NF-κB p-p65 was evaluated by laser confocal microscopy. After transfection with mimic-NC or miR-124 mimic， the expression of miR-124 and NF-κB p-p65 protein in the cytoplasm and nucleus of the cells were also detected. RESULTS The cell viability， the level of miR-124 in the cells and NF-κB p-p65 protein level in cytoplasm of LPS group were lower than those in control group （P<0.05）， while the levels of IL-1β and TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were higher than those in control group （P<0.05）. The cell viability， the level of miR-124 in cells and NF-κB p-p65 protein level in the cytoplasm of LPS+Cur （40 and 80 μmol/L） groups were higher than those in LPS group （P<0.05）， while the level of TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were lower than those in LPS group （P<0.05）. The level of IL-1β in the supernatant of LPS+80 μmol/L Cur group was lower than that in LPS group （P<0.05）. The levels of miR-124 and NF-κB p-p65 protein level in the cytoplasm of miR-124 mimic group were higher than those in LPS group and mimic-NC group （P<0.05）， while the level of NF-κB p-p65 proteinlevel in the nucleus was lower than that in LPS group and mimic-NC group （P<0.05）. CONCLUSION Curcumin inhibits the inflammatory response of HGFs induced by Pg LPS， which may be achieved by up-regulating miR-124 and then inhibiting the nuclear translocation of NF-κB p-p65.     

Geniposide attenuates cognitive dysfunction in sleep-deprived rats by inhibiting TLR4/NF-κB signaling pathway

AIM To investigate the effects of geniposide （Gen） on Toll like receptor 4/nuclear factor-κB （TLR4/NF-κB） signaling pathway and cognitive dysfunction in sleep deprived rats. METHODS Wistar rats （n=120） were randomly divided into normal control （NC） group， model （M） group， low-dose （5 g·kg^-1·d^-1） Gen （Gen-L） group， medium-dose （10 g·kg^-1·d^-1） Gen （Gen-M） group， high-dose （20 g·kg^-1·d^-1） Gen （Gen-H） group and Gen-H+LPS （0.4 mg·kg^-1·d^-1， tail vein injection） group. After 7 days of intervention， the sleep deprivation model of rats in M group， Gen-L， Gen-M， Gen-H and Gen-H+LPS group was established by improved small platform water environment. The escape latency of Morris water maze experiment and the behavior correct rate of Y maze experiment were measured. The serum levels of S100B and neuron-specific enolase （NSE）， and the levels of interleukin-1β （IL-1β）， IL-6 and tumor necrosis factor-α （TNF-α） in hippocampus were detected by ELISA. The mRNA levels of TLR4 and NF-κB p65 were detected by RT-qPCR， and the protein levels of TLR4 and NF-κB p65 were determined by Western blot. RESULTS Compared with NC group， the escape latency， the serum levels of S100B and NSE， the hippocampal levels of IL-1β， IL-6 and TNF-α， and the mRNA and protein expression of TLR4 and NF-κB p65 were increased significantly in M group （P<0.01）， and the behavior correct rate was decreased significantly （P<0.01）. Compared with M group， the escape latency， the hippocampal levels of IL-1β， IL-6 and TNF-α， and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were decreased significantly in Gen-L， Gen-M and Gen-H groups （P<0.01）， and the behavior correct rate was increased in turn （P<0.01）. Compared with Gen-H group， the escape latency， the serum levels of S100B and NSE， the hippocampal levels of IL-1β， IL-6 and TNF-α， and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were increased significantly in Gen-H+LPS group （P<0.01）， and the behavior correct rate was decreased significantly （P<0.01）. CONCLUSION Geniposide may inhibit the TLR4/NF-κB p65 signaling pathway to effectively improve cognitive function in sleep-deprived rats and reduce hippocampus inflammation.     

Relationship of p38MAPK,NF-κB and HO-1 in LPS-induced acute lung injury in rats

AIM: To investigate the relationship of p38 mitogen-activated protein kinases (p38MAPK), nuclear factor-kappa B (NF-κB) and heme oxygenase-1 (HO-1) in acute lung injury (ALI) in rats.METHODS: Forty male Wistar rats were divided into 5 groups (n=8) at random: control group or normal saline group (NS group), lipopolysaccharide group (LPS group), Hemin (inducer of HO-1)+LPS group, ZnPPIX (inhibitor of HO-1)+LPS group and SB203580 (inhibitor of p38MAPK)+LPS group (SB+LPS group). Six hours after endotracheal instillation of LPS or NS, the ratio of neutrophils and the protein contents in bronchoalveolar lavage fluid (BALF) of right lung, the ratio of wet/dry weight (W/D) of the superior lobe of right lung, and arterial blood gas analysis (ABG) were examined. The protein levels of p38MAPK and NF-κB in the lower lobe of right lung were detected by Western blotting. The protein expression of HO-1 in the middle lobe of right lung was measured by the method of immunohistochemisty. The structure of the lung was evaluated under light microscope. RESULTS: Compared with NS group, the ratio of neutrophils and protein contents in BALF, the ratio of W/D, the protein levels of HO-1, p38MAPK and NF-κB were obviously higher, and arterial oxygen pressure (PaO₂), partial pressure of carbon dioxide in artery (PaCO₂) and bicarbonate content (HCO^-₃) were significantly lower in LPS group, Hemin+LPS group, ZnPPIX+LPS group and SB+LPS group (P<0.05 or P<0.01). The ratio of neutrophils and proteins in BALF, the ratio of W/D, the protein levels of p38MAPK and NF-κB were significantly lower, the protein level of HO-1 was obviously higher in Hemin+LPS group and SB+LPS group than those in LPS group (P<0.05), while the changes of the parameters in ZnPPIX+LPS group were in a contrary manner (P<0.05). No significant difference of the parameters between Hemin+LPS group and SB+LPS group (P>0.05) was found. The structures of the lung tissues in LPS group were severely damaged and even severer damages were observed in ZnPPIX+LPS group. The structural changes of the lung tissues in Hemin+LPS group and SB+LPS group were slighter. CONCLUSION: p38MAPK/NF-κB and HO-1 are inhibited by each other and the effects of them are independent on the acute lung injury.     

Effects of PDS on TLR4 mRNA expression in cortex in endotoxic shock rats

AIM: To explore the molecular mechanism of brain tissue injury induced by endotoxin. METHODS: Rats were divided into LPS, LPS+DEX, LPS+PDS and control group, respectively. NOS activity, NO content and TLR4 mRNA expression were assayed 4 h after intravenous injection of LPS. RESULTS: NOS activity, NO content and TLR4 mRNA expression in LPS+DEX and LPS +PDS groups were obviously lower than those in LPS group. CONCLUSION: PDS may provide protective effects on the central nervous system by down-regulating TLR4 expression, reducing NOS activity and NO content in the brain tissue.     

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.     

Poly adenosine diphosphate-ribose polymerase regulates the expression of nuclear factor-κB and related inflammatory factors in rat hippocampus after epilepsy

AIM: To investigate the time course of nuclear factor-κB (NF-κB) and the effects of 3-aminobenzamide (3-AB) on the expressions of NF-κB, interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) in hippocampus after seizures. METHODS: Epilepsy were induced by kainic acid through cerebral ventricular injection. Western blotting was used to detect NF-κB p65 expression in nucleus at various experiment groups. Moreover, mRNA and protein expressions of IL-1β and COX-2 in different experiment groups were determined by RT-PCR and Western blotting analysis. RESULTS: NF-κB p65 immunoreactivity began to increase in the nuclear fraction at 2 h (P<0.05), kept rising at 12 h (P<0.05) and returned to control level at 24 h after epilepsy seizures. Furthermore, 3-AB sharply decreased the accumulation of NF-κB p65 in nucleus (P<0.05). In addition, 3-AB significantly decreased the mRNA and protein expressions of IL-1β and COX-2 which obviously increased in hippocampus at 6 h after epilepsy seizures (P<0.05). CONCLUSION: Seizures triggers NF-κB nucleus translocation and promotes the expressions of IL-1β and COX-2 in hippocampus. In addition, poly (adenosine diphosphate-ribose) polymerase inhibition by 3-AB suppresses NF-κB associated inflammatory pathway in epileptic rat hippocampus.     

Panaxadiol saponins and dexamethasone have similar actions on LPS-induced AKI in mice

AIM：To investigate whether the panaxadiol saponins (PDS) and dexamethasone (DEX) have similar effects on lipopolysaccharide (LPS)-induced acute kidney injury (AKI). METHODS：C57BL/6 mice were randomly divided into 4 groups: the control mice received intraperitoneal injection of normal saline; in LPS group, the mice were subjected to intraperitoneal injection of LPS (10 mg/kg); in PDS + LPS group and DEX + LPS group, the mice were injected intraperitoneally with PDS (25.0 mg/kg) and DEX(2.5 mg/kg) 1 h before LPS injection, respectively. The blood was collected from the hearts, and the kidneys were collected for the biochemical and Western blotting analysis 12 h after LPS injection. RESULTS：LPS induced AKI, evidenced by markedly increased blood urea nitrogen (BUN) and creatinine (CREA) contents compared with control group (P<0.01). However, serum contents of CREA and BUN obviously reduced in PDS + LPS group and DEX + LPS groups compared with LPS group (P<0.05). Both PDS and DEX decreased the production of TNF-α and IL-6 by inhibiting renal NF-κB signaling activation. PDS and DEX also down-regulated the expression of inducible nitric oxide synthase, up-regulated the expression of manganese superoxide dismutase and reduced oxidative stress in the kidneys of LPS-challenged mice. In addition, treatment with PDS and DEX significantly increased the nuclear glucocorticoid receptor in the kidneys of LPS-treated mice. CONCLUSION：PDS and DEX have inhibitory effects on LPS-induced AKI mice. However, it is unclear whether PDS reduces LPS-induced AKI via direct action on glucocorticoid receptor.     

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…     

Expression and activation of NF-κB and carcinogenesis of cervical cancer

AIM:To evaluate the significance of NF-κB p65 protein expression in the development of human cervical squmous cell cancer.METHODS:Immunohistochemical analysis was done in 125 casas of paraffin-embedded cervical tissue specimens of different histological grades (32 LSILs,33 HSILs,38 SCCs and 22 normal) to evaluate the expression of RelA.Western blotting was used to analyze the level of NF-κB p65 protein.RESULTS:① By using immunohistochemical analysis,RelA was mainly localized in cytosol in normal cervical tissue and low-grade squamous intraepithelial lesions,whereas in high-grade lesions and squamous cell carcinomas,RelA translocated into the nucleus.② By Western blotting analysis,RelA was detected in the cytosolic extracts in normal or LSILs.In cancer tissues,the expression of RelA increased in nuclear extracts while their expression in the cytosolic extracts was relatively less.CONCLUSIONS:Constitutive activation of NF-κB p65 may lead to oncogenesis.NF-κB p65 may be a new target for the treatment of human cervical squmous cancer.     

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.     

The expression of NF-κB and ICAM-1 in rat brain of experimental intracerebral hemorrhage and cerebral microvascular endothelial cells injured by hydrogen peroxide

AIM: To discuss the possible mechanism of the inflammation after intracerebral hemorrhage (ICH) and the relationship of nuclear factor-kappa B (NF-κB) and intercellular adhesion molecule-1 (ICAM-1). METHODS: The expression of NF-κB and ICAM-1 were detected by immunohistochemistry, in situ hybridization, immunocytochemistry and Western blotting techniques in rat brain of experimental ICH and cerebral microvascular endothelial cells (RCMECs) injured by hydrogen peroxide. RESULTS: The expression of NF-κB p65 and ICAM-1 were up-regulated in rat brain after ICH. The ICAM-1 reached the peak at 1 day while the NF-κB at 4th day. NF-κB p65 expressed remarkably in cultured RCMECs immediately after injured by hydrogen peroxide, while ICAM-1 expressed remarkably　2 hours later. PDTC, an inhibitor of NF-κB, down-regulated the expression of NF-κB and ICAM-1. CONCLUSION: NF-κB induces the expression of ICAM-1 in RCMECs injured by reactive oxygen species (ROS).     

Nontypeable haemophilus influenzae induces IL-8 expression in alveolar epithelial cells in a p38 MAPK and NF-κB dependent manner

AIM:To investigate the key molecular mechanism of inflammatory response in alveolar epithelial cells induced by nontypeable haemophilus influenzae (NTHi). METHODS: A549 cells were co-cultured with NTHi (multiplicity of infection, MOI: 10) and harvested 15 min and 30 min after stimulation. The phosphorylation of p38 mitogen activated protein kinase (p38 MAPK) in A549 cells was detected by Western blotting. The intracellular expression of nuclear factor-κB (NF-κB) p65 was examined by flow cytometry 4 h after stimulation. A549 cells were preincubated with p38 inhibitor (SB203580) or NF-κB inhibitor (PDTC) for 1 h and then stimulated with NTHi for 24 h. The level of interleukin 8 (IL-8) in the supernatants was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The phosphorylation of p38 MAPK was rapidly induced by NTHi stimulation. The expression of NF-κB p65 in A549 cells after NTHi stimulation was significantly up-regulated compared with control group (P<0.05). The level of IL-8 in the supernatants was increased 24 h after bacterial stimulation compared with control group (P<0.05). Blockage of p38 MAPK or NF-κB remarkably decreased IL-8 secretion in A549 cells (P<0.05). CONCLUSION:NTHi induces inflammatory response in alveolar epithelial cells in a p38 MAPK and NF-κB dependent manner.     

Effects of erigeron breviscapine on nuclear factor-κB expression following lung ischemia-reperfusion injury

AIM: To investigate the effects of erigeron breviscapine on nuclear factor-κB (NF-κB) expression following lung ischemia-reperfusion (I/R) injury in rats. METHODS: Thirty-two male Sprague-Dawley rats were randomized into four groups with 8 animals in each group: sham operation group (I), I/R group (II), erigeron 25 mg/kg group (III) and erigeron 50 mg/kg group (IV). A lung I/R injury rat model was established in situ. I/R injury consisted of 45 min of lung cross-clamping followed by 2 h of reperfusion; sham operation animals had a thoracotomy only. The wet-to-dry weight ratio (W /D), myeloperoxidase (MPO) of lung tissue, the content of nuclear NF-κB p65 were detected by immunohistochemical staining and Western blotting. The histopathological changes of lung tissue were observed under light microscopy. Electron microscopic evaluation was done on randomly selected lungs of two rats in each group at the end of the experiment. RESULTS: Compared to sham operation group, W /D and MPO in the I/R group increased significantly after reperfusion, and the expression of NF-κB in nucleus was up-regulated. As compared with I/R group, the level of NF-κB decreased in group III and IV. Also the changes of W /D and MPO were ameliorated as compared with group II. There was significant difference between group III and IV. CONCLUSION: Erigeron breviscapine reduced I/R lung injury through suppressing the activation of NF-κB and subsequent neutrophils accumulation.     

Angiotensin II-induced matrix metalloproteinase-9 expression mediated by NF-κB pathway in human THP-1 cells

AIM: The present study was undertaken to investigate the effect of angiotensin II (AngⅡ) on expression of MMP-9 in THP-1 macrophages. METHODS: Macrophages converted from THP-1 monocytes by incubating with PMA (0.1 μmol/L) for 48 h were divided into PMA group; PMA+AngⅡ group (10-7mol/L, 1 h); PMA+AngⅡ+PDTC group (10 μmol/L, 30 min) and PDTC group. Western blotting was used to detect the MMP-9 and phosphorylation of NF-κB p65, and the expression of MMP-9 mRNA in THP-1 macrophages was measured by RT-PCR.RESULTS: Compared to control group, the expression of MMP-9 (1.06±0.11, P<0.05) and phosphorylation of NF-κB p65 (1.02±0.10, P<0.05) in THP-1 macrophages were expressed when treated with AngⅡ (10-7mol/L); and the expression of MMP-9 mRNA were upregulated (1.22±0.08, P<0.05). However, NF-κB inhibitor PDTC reduced the NF-κB p65 (0.99±0.12, P<0.01) and MMP-9 (1.04±0.14, P<0.01) expressions and decreased the expression of MMP-9 mRNA (0.90±0.06，P<0.01). CONCLUSION: NF-κB signaling pathway contributes to the expression of MMP-9 in THP-1 macrophage induced by AngⅡ.     

Effect of the NF-κB inhibitor pyrrolidine dithiocarbamate on the apoptosis and proliferation of mesangial cells in the rat with anti-thymocyte serum nephritis

AIM: To explore the effect of pyrrolidine dithiocarbamate (PDTC), the specific inhibitor of NF-κB, on anti-thymocyte serum nephritis (ATSN) in rats. METHODS: The rat model of ATSN was reproduced with rabbit anti-thymocyte serum (ATS). The rats were divided into ATSN group, ATSN+PDTC group and control group. The expression of NF-κB p65 and the apoptosis, lysis as well as proliferation of mesangial cells (MC) were examined by immunohistochemical staining, Tdt-mediated X-dUTP nick end labeling (TUNEL), light microscope and electron microscope at 40 minutes, 24 hours and 7 days after injection of ATS or normal serum. RESULTS: The expression of glomerular NF-κB p65 in the ATSN group was observed with significant difference compared to controls at 40 min (P<0.01), it was elevated more at 24 hours, and was significantly increased at day 7, but the expression of NF-κB p65 in ATSN+PDTC group was less than that in ATSN group. The proliferation of glomerular MC and the secretion of extracellular matrix (ECM) in ATSN group were less than those in ATSN+PDTC group on day 7. PDTC had little role in the pathologic changes of rats in ATSN group in early stage (40 min and 24 hours), but affected the MC proliferation. CONCLUSION: PDTC, an inhibitor of NF-κB, suppresses glomerular MC proliferation in rats with ATSN.     

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.     

Polydatin downregulates TLR4 expression in lung ischemia reperfusion injury in rabbits

AIM: To explore the influence of polydatin (PD) on Toll-like receptor 4 (TLR4) signal transduction pathway during lung ischemia reperfusion injury in rabbits. METHODS: Rabbit lung model of ischemia reperfusion (IR) injury was constituted in vivo. Thirty rabbits were divided into groups randomly: control (C), IR and PD group, respectively. The concentration of endotoxin (ET) in plasma was analyzed by end-point chromogenic assay. The protein expressions of TLR4, nuclear factor (NF)-κB p65 and heat shock protein 70 (HSP70) were measured by immunohistochemistry. The intracellular adhesion molecule-1 (ICAM-1) mRNA expression was detected by in situ hybridization histochemistry. The ultrastructural changes were observed by electron microscope. RESULTS: No significant difference of ET concentration in plasma between groups (all P>0.05) was observed. The protein expressions of TLR-4, NF-κB p65, HSP70 and ICAM-1mRNA in IR group were significantly increased as compared to C group and PD group, while those expressions in PD group were evidently higher than those in C group (all P<0.01). The lung pathological injuries in PD group were obviously alleviated as compared to IR group under electron microscope. CONCLUSION: It suggests that lung ischemia reperfusion releases endogenous ligands of TLR4 as HSP70, then activates NF-κB, promotes the release of mediators of inflammation such as ICAM-1. PD might have a protective effect on lung ischemia reperfusion injury by regulating TLR4 signal transduction pathway.     

Effect of nuclear factor-κB on proliferation of airway smooth muscle cells regulated by protein kinase C in asthmatic rats

AIM: To investigate the effect of protein kinase C (PKC)- nuclear factor-κB (NF-κB) signal pathway on proliferation of airway smooth muscle cells (ASMCs) in asthmatic rats.METHODS: (1) 16 Wistar rats were divided into asthmatic group (8 rats) and control group (8 rats).ASMCs from asthmatic group and control group were treated with PKC agonist PMA and NF-κB inhibitor PDTC.The proliferation of ASMCs was examined by cell cycle analysis,MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining,respectively.NF-κB activity was detected by NF-κB p65 immunofluorescence staining and electrophoretic mobility shift assay (EMSA),respectively.RESULTS: The percentage of S phase,A value,the positive expression rate of PCNA,the positive expression rate of NF-κB p65 and EMSA value in asthmatic ASMCs treated with PMA were higher than those in asthmatic ASMCs without treatment (P<0.05).After asthmatic ASMCs previously treated with PDTC,then with PMA,the above figures were lower than those in asthmatic ASMCs only treated with PMA and without treatment (P<0.05).The above figures in asthmatic ASMCs only treated with PDTC were lower than those in asthmatic ASMCs without treatment (P<0.05).CONCLUSION: NF-κB may contribute to the proliferation of ASMCs in asthmatic rat,in which PKC－NF-κB signal pathway is involved.