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Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) is one of important kinases in inflammation to phosphorylate inhibitor of nuclear factor kappa-B (IκBα) and then activate nuclear factor kappa-B (NF-κB). Inhibition of IKKβ has been a therapeutic strategy for inflammatory and autoimmune diseases. Here we report that IKKβ is constitutively activated in healthy donors and healthy Ikkβ C46A (cysteine 46 mutated to alanine) knock-in mice although they possess intensive IKKβ-IκBα-NF-κB signaling activation. These indicate that IKKβ activation probably plays homeostatic role instead of causing inflammation. Compared to Ikkβ WT littermates, lipopolysaccharides (LPS) could induce high mortality rate in Ikkβ C46A mice which is correlated to breaking the homeostasis by intensively activating p-IκBα-NF-κB signaling and inhibiting phosphorylation of 5' adenosine monophosphate-activated protein kinase (p-AMPK) expression. We then demonstrated that IKKβ kinase domain (KD) phosphorylates AMPKα1 via interacting with residues Thr183, Ser184, and Thr388, while IKKβ helix-loop-helix motifs is essential to phosphorylate IκBα according to the previous reports. Kinase assay further demonstrated that IKKβ simultaneously catalyzes phosphorylation of AMPK and IκBα to mediate homeostasis. Accordingly, activation of AMPK rather than inhibition of IKKβ could substantially rescue LPS-induced mortality in Ikkβ C46A mice by rebuilding the homeostasis. We conclude that IKKβ activates AMPK to restrict inflammation and IKKβ mediates homeostatic function in inflammation via competitively phosphorylating AMPK and IκBα.
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ObjectiveTo explore the effect and mechanism of Sangmei Zhike granule （SMZK） on airway inflammation in rats with cough variant asthma（CVA）. MethodSix-week-old male SD rats were randomly divided into normal group， model group， and SMZK （2.48 g·kg-1） group. The rats in the model group and the SMZK group received intraperitoneal injection of a mixed solution containing 10% ovalbumin （OVA） and aluminium hydroxide on the 1st and 8th days and aerosol inhalation of 1% OVA solution from the 15th day for CVA model induction. The intervention lasted for two weeks from the 15th day. At the end of animal manipulation， the lung function was detected and inflammatory cells in the peripheral blood were counted. The serum interleukin-4 （IL-4）， interleukin-5 （IL-5）， and interleukin-10 （IL-10） levels were determined. Hematoxylin-eosin （HE） staining was performed on the lungs. Western blot was used to detect the protein expression of nuclear factor kappa-B （NF-κB） and its inhibitor α（IκBα） in lung tissues. ResultCompared with the normal group， the model group showed reduced forced expiratory volume in the first 0.1 second （FEV0.1），FEV0.1/forced vital capacity （FVC），and forced expiratory flow 50% （FEF50%） （P<0.05， P<0.01）， increased white blood cells and eosinophils （P<0.01）， and up-regulated serum IL-4， IL-5， and IL-10 （P<0.01）. As revealed by HE staining， the model group displayed shed epithelial cells of the bronchus， airway stenosis， hyperplasia and expansion of mucous glands， disarrangement of layer structures， disorderly arranged cells， and extensive infiltration of inflammatory cells. The protein expression of NF-κB p65 was higher （P<0.01） and that of IκBα was lower （P<0.01） in the lung tissues of the model group than that in the normal group. Compared with the model group， the SMZK group showed increased FEV0.1，FEV0.1/FVC，and FEF50% （P<0.05）， decreased white blood cells and eosinophils in the peripheral blood （P<0.01）， and declining serum IL-4， IL-5， IL-10 （P<0.01）. HE staining demonstrated mild bronchial mucosal injury and relieved inflammatory cell infiltration， gland hyperplasia， and epithelial degeneration and necrosis in the SMZK group. The protein expression of NF-κB p65 was decreased （P<0.05） and that of IκBα was increased （P<0.05） in lung tissues of the SMZK group than that in the model group. ConclusionSMZK can improve lung function and inhibit airway inflammation in rats with CVA. The underlying mechanism may be related to the regulation of IκBα/NF-κB protein expression in the lungs.
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Aim To investigate the effects of schisandrin C on arterial inflammation and atherosclerosis of ApoE-/-mice fed with high-fat diet. Methods ApoE-/-mice were divided into high fat diet group (HFD) and high fat diet group + schisandrin group (HFD + Sch C). High fat diet and Sch C were given at the same time. After 12 weeks, mice were executed and arterial tissues were collected. RT-q PCR and Western blot were respectively used to detect the expressions of IL-6, TNF-α, ICAM-1, β-actin and the phosphorylation and expression of IκB-α in arteries. Oil red O staining and biochemical kit were respectively used to detect the lipid changes in aortic root and the blood lipid levels. Results Compared with HFD group, the area of atherosclerotic plaque in HFD + Sch C was reduced (P < 0. 05), but Sch C did not affect blood lipid levels. Compared with HFD group, the mRNA expression of TNF-α, IL-6 and ICAM-1 and the phosphorylation of IκB-α of arterial tissue in HFD + Sch Cgroup decreased (P < 0. 05). Conclusions Sch C could effectively alleviate atherosclerosis induced by high-fat diet in ApoE-/-mice, accompanied by alleviation of arterial inflammation.
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Objective:To observe the effect of Shenling Baizhusan on the protein and mRNA expression of inhibitor of nuclear factor kappa B kinase (IκK)/inhibitor of nuclear factor kappa B(IκB)/nuclear factor kappa B(NF-κB) signaling pathway in the colon of rats with ulcerative colitis (UC) of spleen deficiency and dampness stagnation type, and to explore the mechanism of Shenling Baizhusan in the treatment of UC. Method:The 48 Wistar rats were randomly divided into normal group, model group, Shenling Baizhusan group (15.6 g·kg-1) and osalazine sodium group (0.68 g·kg-1), 12 rats in each group. The model of UC with spleen deficiency and dampness stagnation was reproduced by trinitrobenzene sulfonic acid (TNBS)/ethanol enema combined with environment and diet intervention.Serum levels of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β(IL-1β) were determined by enzyme-linked immunosorbent assay（ELISA）. The expression of NF-κB p65, IκBα, IκKβ protein in colon tissue was measured by Western blot and immunohistochemical method, and the mRNA expression of NF-κB p65, IκBα and IκKβ in colon tissue of rats in each group was detected and compared by real time polymerase chain reaction (Real-time PCR). Result:Compared with normal group,the levels of TNF-α,IL-6 and IL-1β in the serum, the protein and mRNA expression of NF-κB p65, IκKβ in colon tissue of the model group was significantly higher than that of normal group (P<0.01), and the protein and mRNA expression of IκBα was significantly lower than that of normal group (P<0.01). Compared with model group,the protein and mRNA expression of NF-κB p65, IκKβ in colon tissue of the Shenling Baizhusan group and osalazine sodium group were significantly decreased (P<0.01), and the protein and mRNA expression of IκBα was significantly increased (P<0.01). Conclusion:Shenling Baizhusan can obviously down regulate the protein and mRNA expression of NF-κB p65, IκKβ,up regulate the expression of IκBα in colon tissue of UC rats with spleen deficiency and dampness stagnation. The inhibition of IκK/IκB/NF-κB signal pathway activation by Shenling Baizhusan is an important mechanism of its role in protecting intestinal mucosa.
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Nuclear factor (NF)-κB,a kind of protein transcription factor,mediates inflammation and other complex biological processes,and is a key regulator of many immune and inflammatory pathways,cell proliferation,differentiation and apoptosis.Psoriasis is an inflammatory skin disease characterized by increased levels of phosphorylated NF-κB,which leads to an increase in anti-apoptotic activity of keratinocytes and aggravation of inflammatory reactions.This review summarizes advances in NF-κB signaling pathway and its role in psoriasis.
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Objective:To observe the expression of tumor necrosis factor receptor-associated death domain (TARDD), nuclear transcription factor-κB inhibiting protein α(IκBα)IκB kinase-α (IKKα) and nuclear transcription factor (NF)-κB p65 protein in the NF-κB signaling pathway of synovial tissues of complete Freund's adjuvant (CFA) rats after treatment with Xiao Chaihutang (XCHT). Method:In animal experiments, SPF health adult female Wistar rats were used to prepare the CFA animal model of rats with rheumatoid arthritis with Freund's complete adjuvant and cattle Ⅱ collagen type. According to the random number table, the rats were randomly divided into the normal group, the model group, the low-dose XCHT group, the medium-dose XCHT group, the high-dose XCHT group, and the Tripterygium glucosides group. The drugs were given at 7 d after the model was built. Both normal group and model group were given water for injection,and low-dose XCHT group(5.94 g·kg-1),medium-dose XCHT group(11.88 g·kg-1),high-dose XCHT group(23.76 g·kg-1),Tripterygium glucosides group(0.006 3 g·kg-1) were given corresponding drugs by gavage for three times a day, 2 mL/time. The histopathology of rat ankle joint was observed, and the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in the NF-κB signaling pathway in synovial tissue of CFA rats were detected by Western blot. Result:With the increase of the dosage of XCHT, the histopathological score of the right posterior ankle joint of the experimental rats was increased. And in the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in NF-κB signaling pathway in Synovial Tissue of CFA rats, compared with the model group, the statistical results of the low-dose XCHT group showed decreased protein expressions (PPPα, IκB α, NF-κB p65 in the NF-κB signaling pathway were significantly increased (PPα, IκBα, NF-κB p65 key protein expressions in the NF-κB signaling pathway and protein expressions in low-dose XCHT group were obviously lower (PPConclusion:This study shows that as the dose of Xiao Chaihutang increases, it could effectively improve synovitis, and suppress the expressions of key proteins in the inflammatory signaling pathway of NF-κB, thereby preventing inflammation and suppressing bone erosion.
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Objective:To analyze the liver injury,and the percentage,apoptotic status,cytokine profiles of immune cell subsets in liver from IκBα transgenic mice.Methods:HE staining was performed to detect the liver injury.FACS was used to evaluate the percentage and phenotype of immune cell subsets,including T cells,NK cells and NKT cells,and Annexin V staining was used to evaluate the apoptosis rate of NK and T cells.Furthermore,real-time quantitive PCR was performed to analyze the expression of CCL2,IFN-γ,IL-2 and IL-15.Results:Liver injury was observed in IκBα transgenic mice.The percentage of T cells was lower in liver from IκBα transgenic mice than that in Wild Type(WT) mice,a similar trend was found in NK cells and NKT cells.We also found that NKp46 was inhibited in NK cells from IκBα transgenic mice,accompanied with the increased apoptosis.Also,IFN-γand CCL2 were decreased in IκBα transgenic mice.Conclusion:The percentage,phenotype and cytokine profile of immune cell subsets were affected in IκBα-transgenic mice.
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Objective:To explore the intervention effect of proteasome inhibitor MG132 in rats with collagen-induced arthritis(CIA),which resembles human rheumatoid arthritis(RA).Methods:Forty-eight female SD rats were randomly divided into three groups,including blank control group,CIA model group and MG132-treated group.There were sixteen rats in each group.Rats in CIA model group and MG132-treated model group were injected with type Ⅱ collagen to established CIA rats.21 days after the initial immunization,the rats in the MG132-treated model group were injected subcutaneously with 1 mg/kg MG132 once daily for 2 weeks.42 days after the initial immunization,the change of paw-swelling and the arthritis scores were determined.The synovial pathology examination was performed with HE staining.The 20S proteasome activity in synovial tissue was measured by fluorescence substrate assay.The expression of NF-κB/p65,IκBα in synovial tissue were analyzed by Western blot.Results:Proteasome inhibitor MG132 significantly attenuated the severity of arthritis and histopathological changes in CIA rats.Compared with the blank control group,the 20S proteasome activity was increased significantly in the CIA model group(P<0.05),and decreased after injection of MG132.Compared with CIA rats,the expression of NF-κB/p65 significantly decreased in rats treated with MG132(P<0.01).Compared with the blank control group,the expression of IκBα protein decreased in CIA model group.After injected with MG132,the protein was significantly increased(P<0.01).Conclusion:The proteasome inhibitor MG132 may attenuates the severity of arthritis and histopathological changes in CIA rats.These effects may be mediated through the inhibition of NF-κB activity.
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Objective To investigate the effects of breviscapine injection on intestinal injury induced by intestine ischemia-reperfusion(IIR). Methods 48 males SD rats with 8-week old were randomly divided into 4 groups:Sham,intestine ischemia-reperfusion(IIR),EB+IIR,TP+IIR. Breviscapine injection 20 mg/(kg·d) was given intraperitoneally in EB + IIR group. TPCA-1(12 mg/kg)was given intravenously 30 min before surgery in TP+IIR group. Rats were subjected to superior mesenteric artery occlusion consisting of 45 min of ischemia and 6 h of reperfusion;sham laparotomy served as controls. Intestine pathology was assayed by H&E staining. Con-centrations of TNF-α,IL-1β,and IL-6 in intestinal mucosa were determined by ELISA. The protein expressions of IκB-α,NF-κB ,ICAM-1of intestine tissue were assayed by western blot. Results IIR induced serious intesti-nal injury ,evidenced as poor intestine pathology ,elevation of TNF-α,IL-1β,and IL-6 levels in intestinal mu- cosa,accompanied with IκB-α/NF-κB/ICAM-1 pathway activation. However,breviscapine injection pretreatment could inhibit IκB-α/NF-κB/ICAM-1 pathway activation,leading to reduction of TNF-α,IL-1β,and IL-6 concen-trations in lung,finally attenuate ALI induced by IIR. Conclusion Breviscapine injection pretreatment could atten-uate inflammation in intestine after IIR injury via inhibiting IκB-α/NF-κB/ICAM-1signaling pathway.
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Objective To investigate the effects of breviscapine injection on intestinal injury induced by intestine ischemia-reperfusion(IIR). Methods 48 males SD rats with 8-week old were randomly divided into 4 groups:Sham,intestine ischemia-reperfusion(IIR),EB+IIR,TP+IIR. Breviscapine injection 20 mg/(kg·d) was given intraperitoneally in EB + IIR group. TPCA-1(12 mg/kg)was given intravenously 30 min before surgery in TP+IIR group. Rats were subjected to superior mesenteric artery occlusion consisting of 45 min of ischemia and 6 h of reperfusion;sham laparotomy served as controls. Intestine pathology was assayed by H&E staining. Con-centrations of TNF-α,IL-1β,and IL-6 in intestinal mucosa were determined by ELISA. The protein expressions of IκB-α,NF-κB ,ICAM-1of intestine tissue were assayed by western blot. Results IIR induced serious intesti-nal injury ,evidenced as poor intestine pathology ,elevation of TNF-α,IL-1β,and IL-6 levels in intestinal mu- cosa,accompanied with IκB-α/NF-κB/ICAM-1 pathway activation. However,breviscapine injection pretreatment could inhibit IκB-α/NF-κB/ICAM-1 pathway activation,leading to reduction of TNF-α,IL-1β,and IL-6 concen-trations in lung,finally attenuate ALI induced by IIR. Conclusion Breviscapine injection pretreatment could atten-uate inflammation in intestine after IIR injury via inhibiting IκB-α/NF-κB/ICAM-1signaling pathway.
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Aim To observe the effects of heat shock protein 70 ( Hsp70 ) activator SW02 on lipopolysaccha-ride( LPS)-induced expression of inducible nitric oxide synthase ( iNOS ) and LPS-induced production of nitric oxide ( NO ) in macrophages. Methods RAW264. 7 cells were stimulated by LPS, and were divided into DMSO,DMSO+LPS(1 mg·L-1),SW02,and SW02+LPS ( 1 mg · L-1 ) groups. The protein expression was detected by Western blot. NO concentration was measured by Griess kit. The iNOS mRNA was detected by real-time PCR. The NF-κB binding to iNOS promot-ers was measured by chromatin immunoprecipitation ( ChIP ) assays. Results SW02 significantly blocked the protein and mRNA expression of iNOS as well as the production of NO in LPS-stimulated RAW264 . 7 cells(P0. 05 , SW02 +LPS group vs DMSO+LPS group ) and nuclear translocation of NF-κB ( P > 0. 05 , SW02 + LPS group vs DMSO + LPS group) . However,SW02 reduced the NF-κB binding to iNOS promoters inside the cell( P<0. 05,SW02+LPS group vs DMSO+LPS group) . Conclusion These re-sults show that SW02 prevents iNOS expression and NO induction likely through attenuation of the NF-κB bind-ing to iNOS promoters in macrophages.
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Objective To explore the possible mechanism of action of telomerase in hepatic precancerous lesions, and the regulatory effect of a Chinese medicine prescription HU Qi Shan ( HQS) and its principal drug mistletoe alkali on the telomerase activity in rat liver tissues.Methods Rat model of hepatic precancerous lesions was established by Solt-Farber two-step protocol.The model rats were randomly divided into 5 groups, including the model group, high-dose HQS [8 g/(kg· d)] group, low-dose HQS [4 g/(kg· d)] group, and mistletoe alkali[8 mg/(kg· d)] group.γ-Glutamy-transpeptidase (γ-GT) was analyzed by immunohistochemistry.AFP was detected by immunofluorescence technique.The telomerase activity was detected using a quantitative telomerase detection kit.The expression of NF-κB P65 was detected by immunohistochemistry.The cytoplasmic protein IκB-αwas detected by western blotting.Results After treated with HQS and mistletoe alkali, the areas ofγ-GT-positive foci and number of AFP-positive cells in the liver tissus were significantly decreased than those of the model group ( P<0.05 for both) , the telomerase activity was decreased, the number of NF-κB P65-positive cells was also decreased ( P <0.05 ) , whereas the intracytoplasmic expression of IκB-αproteins was significantly increased ( P <0.05 ) .Conclusions HQS and mistletoe alkali can suppress the telomerase activity.Its possible mechanism may be through inhibition of the over-expressed apoptosis-related genes such as NF-κB P65 and increase the expression of IκB-αdecreasing the telomerase activity.
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Objective To investigate the therapeutic effects and mechanisms of curcumin derivatives C66 treatment on hepatic fibrosis .Methods Thirty three C57BL/6J mice were randomly divided into 3 groups:normal control group ,model control group and curcumin derivatives C66 treatment group .Nine mice in normal control group were fed with water and food .Hepatic fibrosis model was induced in 24 mice by intraperitoneal injection of 40% carbon tetrachloride at a dose of 4 mL/kg for the first time ,followed by 2 mL/kg twice a week for 6 weeks . At week 6 ,6 mice were randomly selected to perform pathological examination to evaluate whether the hepatic fibrosis were successfully induced .Mice with hepatic fibrosis were randomized into model control group and curcumin derivatives C66 treatment group with 9 mice in each group .From week 6 on ,mice in the treatment group were lavaged with curcumin derivatives C66 at a dosage of 10 mg ·/(kg · d) .The rest mice were administered with equivalent dosage of 0 .5% carboxymethylcellulose sodium .Serum alanine aminotransferase (ALT) ,aspartate aminotransferase (AST ) and liver hydroxyproline ( Hyp ) contents were detected , and the semi‐quantitative analysis of liver fibrosis was performed by pathological examination in hepatic tissue by hematoxylin and eosin (HE) and Masson staining .The expressions of collagen Ⅰ ,α‐smooth muscle actin (α‐SMA) mRNA and collagen Ⅰ ,α‐SMA ,nuclear factor‐kappa B p65 (NF‐κB p65) ,inhibitor kappa B alpha (IκBα) protein in each group were detected by quantitative real‐time polymerase chain reaction (RT‐PCR) and Western blot .Data were analyzed with one‐way ANOVA analysis .Results The serum levels of ALT and AST in model control group ,C66 treatment group and normal control group were (202 .71 ± 19 .66 ) U/L , (233 .42 ± 23 .97 ) U/L ;(102 .00 ± 11 .04 ) U/L , (120 .87 ± 13 .83 ) U/L ;(36 .66 ± 6 .37) U/L and (43 .33 ± 8 .08)U/L ,respectively .The differences between model and normal control group were both significant (t=23 .96 and 22 .39 ,respectively ;both P<0 .05) .The C66 treatment group showed significantly lower levels of serum ALT and AST in contrast with model control group (t =11 .56 and 10 .52 ,respectively ;both P<0 .05) .Compared to the model control group ,hepatic Hyp contents in normal control group and C66 treatment group were significantly different (t= 17 .50 , P< 0 .05;t=11 .45 ,P<0 .05) .Collagen Ⅰand α‐SMA mRNA expressions in C66 treatment group were remarkably lower in contrast with that in model control group (t= 7 .23 and 7 .95 ,respectively ;both P< 0 .05) . Protein levels of Collagen Ⅰ ,α‐SMA and NF‐κB p65 decreased in C66 treatment group ,while IκBαincreased significantly (all P<0 .05) .Conclusion The application of C66 can contribute to the regression of liver fibrosis and the mechanism may rely on the regulation of NF‐κB expression .
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This study was aimed to investigate the influence of Bi-Y uan-Shu (BYS) Oral Liquid on glucocorticoid re-ceptor (GR) and nuclear factor IκBα expression of nasal sinuses mucosa epithelium among chronic rhinosinusitis (CRS) models in order to explore its therapeutic mechanism for CRS from the anti-inflammatory reaction aspect. One hundred New Zealand rabbits were selected and randomly divided into the normal group, sham operation group, model group, BYS group, and clarithromycin group, with 20 rabbits in each group. After the CRS model was established, no intervention was given to the normal group, sham operation group, or model group. The intragastric administrations of BYS (1.5 mL·kg-1·d-1) and clarithromycin (25 mg·kg-1·d-1) were given for 14 days, respectively. The nasal sinuses mucosa was taken after the treatment. And HE stain was used to observe its pathological changes. Western Blotting was used in the detection of nasal sinuses mucosal epithelium cytoplasm GR and IκBα expression. The results showed that there were obvious nasal sinuses mucosa inflammatory cell infiltration, chronic inflammation changes, and obvious hyperplasia of glandular organs and goblet cells. Compared with the normal group, the GR expression was obviously reduced (P< 0.01). And the IκBα expression was obviously increased (P< 0.01). After the intragastric administration of BYS, the nasal sinuses mucosal epithelium was repaired with no obvious inflammatory cell infiltration, glandular organs, or goblet cells. Compared with the model group, the GR expression was obviously increased (P< 0.01). The IκBα expression was obviously decreased (P<0.01). It was concluded that BYS can promote GR expression to inhibit inflammation. Meanwhile, it can restrain IκBα expression to prevent the over inhabitation of IκBα on NF-κB. It can dynamically regulate the balance of the nasal sinuses mucosa epithelium inflammation.
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Objective To explore the role and mechanism of H2 S in severe acute pancreatitis .Methods SD rats were randomly assigned into several experimental groups :contorl group(n=6) ,SAP group(n=10) ,PAG+SAP group(n=10) ,5 mg/kg NaHS+SAP group(n=10) ,10 mg/kg NaHS+SAP group(n=10) ,20 mg/kg NaHS+SAP group(n=10) ,100 mg/kg NaHS+SAP group (n=10) ,wortmannin(W)+ SAP group(n= 10) ,5 mg/kg NaHS + W+ SAP group(n=10) and 100 mg/kg NaHS+ W+ SAP group(n=10) .These rats were intra-peritoneal injected L-Arginine to get SAP model ,and sacrificed ar 24 h hour after the first in-jection .Plasma IL-6 and MPO activity in pancreas were determined by ELISA .Expression of p-AKT and IκBαin pancreas were de-tected by Western blot ,and NF-κB activity was assessed with EMSA .Results SAP and PAG+ SAP resulted in a significant in-creasing in plasma IL-6 ,MPO activity and expression of p-AKT in pancreas ,when compared with the control group(P<0 .05) ,and NF-κB activity also increased ,and NF-κB activity in PAG+SAP group was more significant (P<0 .05);however ,IκBαexpression in SAP and PAG+SAP down-regulated obviously ,when compared with the control group(P<0 .05) ,and the expression in PAG+SAP group was significantly lower(P<0 .05) .Different dosage of NaHS reduced that the level of plasma IL-6 ,MPO activity ,ex-pression of p-AKT and NF-κB activity in pancreas changed in different degrees ,yet IκBαexpression in these groups increased gradu-ally .When compared with the SAP group ,5 mg/kg NaHS+SAP and 100 mg/kg NaHS+SAP ,plasma IL-6 ,MPO activity and ex-pression of p-AKT in pancreas significantly depressed after given wortmannin ,besides NF-κB activity also down-regualated;never-theless IκBαexpression up-regulated .Conclusion H2 S in precondition induced the development of an anti-inflammatory activity in SAP ,and in proportion with the concentration of H2 S in blood in a degree .H2 S regulated the degree of SAP injury via PI3K/AKT-NF-κB pathway .
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AIM: To seek a small interfering RNA ( siRNA ) sequence targeting rat inhibitor of nuclear factor kappa Bα ( IκBα) that can specifically and effectively suppress IκBα mRNA expression of rat ciliary muscles in vivo.METHODS:Three IκBα specific double stranded siRNAs were designed and synthesized. They were transfected into rat A7r5 cells which express IκBα gene. Flow cytometry was used to assess transfected efficiency. The mRNA and protein levels of IκBα were examined by Real Time quantitative polymerase chain reaction ( Real Time-PCR ) and western blot to screen a candidate valid sequence with the highest inhibitory rate. The Cy3 labeled non-specific control siRNA or the candidate valid siRNA was then injected into rat anterior chamber. Distribution of Cy3- siRNA in rat ciliary muscles was viewed by fluorescence microscopy, and the inhibitory effect in vivo of the valid siRNA was identified via Real Time-PCR and immunofluorescence. RESULTS: The suppression effect of the siRNA targeting the CTACGATGACTGTGTGTTT of IκBα gene was most obvious by vitro screening. By anterior chamber injection, this valid siRNA could reach rat ciliary muscles and effectively suppress IκBα gene expression with the highest inhibitory rate of 59. 0% on mRNA level at 24h after RNAi, and 52. 3% on protein level at 72h after RNAi (P<0. 01).CONCLUSION: It proves that the siRNA targeting the CTACGATGACTGTGTGTTT of IκBα gene is the valid sequence to suppress rat IκBα expression of ciliary muscles by RNAi in vivo.
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Objective To investigate the effect of curcumin on the expression of nuclear transcription factor (NF-kappa B) and its inhibitory protein (Iκ-Bα) in liver cancer cells (Hepg-2) induced by TNF-α, and explore its effect on non-alcoholic steatohepatitis. Methods The cells were divided into normal group, TNF-alpha group, and different concentrations of curcumin treatment group. MTT and Western blotting were used to assay curcumin’s effect on Hepg-2 proliferation activity and changes of NF-κB and Iκ-Bα’s in Hepg-2. Results Compared with the normal group, TNF-αgroup enhanced NF-κB and Iκ-Bαexpression, but the increase of Iκ-Bα had no statistical significance (P>0.05). Curcumin treatment group’s NF-κB expression was significantly weakened than TNF-α group, while Iκ-Bα was significantly enhanced than that of TNF-α group. Conclusion Curcumin can antagonize NF-κB inflammatory signaling pathway activation induced by TNF-α, thus reduce the inflammatory injury of the liver cells.
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Objective To explore the effects of zinc finger protein A20(A20) on the expression of CD40 and the phosphorylation of IκBαas well as the local inflammatory reaction of abdominal cavity in Sprague Dawley (SD) rats with peritoneal dialysis-related a-cute peritonitis induced by lipopolysaccharide (LPS ) .Methods 24 male SD rats were equally randomized to four groups (n= 6 , each) .Control group:injected with 4 .25% dextrose peritoneal dialysate(PDF) via abdominal cavity (90 mL/kg);LPS group :injec-ted with LPS(1 mg/kg) via abdominal cavity 4 hour later followed by PDF injection ;transfection A20 plasmid group and empty plasmid group:after transfer pGEM-T easy-A20 or pGEM-T easy plasmid via intraperitoneally using an ultrasound-microbubble-mediated system for 3 days ,then injected with LPS and PDF via abdominal cavity .The rats were killed 4 hours after PDF injection . Peritoneum tissue was stained using Masson and HE .Leucocytes count in abdominal dropsy was performed .The proteins expres-sion of A20 ,p-IκBα,IκBα,and CD40 in peritoneum tissue were analyzed by western blot ;the expression of CD40 mRNA in peritone-um tissue were determined by RT-PCR;IL-6 level in abdominal dropsy was determined by ELISA .Results LPS could induce the protein expression of A20 in rats peritoneum tissue .Compared with LPS group and empty plasmid group ,the degree of edema ,in-flammatory cells infiltration ,and the ratio of p-IκBα/IκBα,mRNA and protein expression of CD40 in rats peritoneum as well as leu-cocyte counts and IL-6 level of abdominal dropsy were also significantly decreased in transfection A20 plasmid group(P0 .05) ,but the mRNA and protein expression of CD40 were significantly higher than that of control group (P<0 .05) .Conclusion Over expression of A20 in SD rats peritoneum tissue could down-regulate the inflammatory reaction in peritonitis induced by LPS ,which might be involved in modulating the expression of associated functional protein during LPS signal pathw ay .
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The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro,as well as the roles of nuclear factor-kappa B (NF-κB) and IκBα was investigated.The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay.Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay.The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy.The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells.Triptolide at nM range induced proliferation inhibition in a dose- and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells.Besides,we observed the inhibition of NF-κB/p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol.These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.
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Objective: To construct IκBα vector carrying SAA3 promoter and to observe its effect on NF-κB activity and LPS-induced inflammation, so as to lay a foundation for treatment of sepsis. Methods: Mouse liver cells and kupffer cells were co-cultured and were divided into three groups: control group, LPS group and LPS+ gene transfer group. Twenty-four hours after LPS injection, the levels of AST, ALT, LDH, IL-6 and TNF-α were measured in the supernatants of the each group. For animal experiments: (1) Mice were divided into three groups: control group, LPS group and LPS+gene transfer group(n=10). The levels of TNF-α and IL-6 were measured in the serum and liver tissues 24 hours after intraperitoneal injection of 250ug LPS or saline. (2) Mice were also divided into two groups: LPS group and LPS+gene transfer group(n=21). Mice were injected with 150 μg LPS twice at 0 and 48 h, then the activities of NF-κB and IκBα in the liver were measured at 0,2,24,48,50,72, and 96 h after the first injection. The values at 0 h were taken as control group. (3) Mice were also divided into another two groups: LPS group and LPS+gene transfer group(n=20). The survival rates of animals were observed at 0,2,24,48,50,72, and 96 h after injection of 350 μg LPS. Results: Compared with LPS group, the levels of AST, LDH, TNF-α and IL-6 in the culture supernatants of LPS+gene transfer group were decreased, but were still higher than those in the control group(P<0.05). Compared with LPS group, the levels of TNF-α and IL-6 in the liver tissues and sera of LPS + gene transfer group were significantly decreased(P<0.05). Compared with LPS group, the activity of NF-κB in the liver tissues of LPS + gene transfer group were decreased, but was still significantly higher than that of the control group(P<0.05). Compared with LPS group, LPS+gene transfer group had higher survival rate at 72 and 96 h(P<0.05). Conclusion: IκBα gene can be expressed in the liver with SAA3 promoter, and transfection of IκBα can effectively inhibit endotoxin-induced liver and general inflammation.