Protective effect of luteolin on acute lung injury induced by extracorporeal circulation and its mechanism / 中国胸心血管外科临床杂志

@#Objective    To investigate the protective effect of atomized inhalation of nano-luteolin preparation on acute lung injury caused by extracorporeal circulation, and to explore the anti-inflammatory mechanism of luteolin, so as to provide study basis for clinical application. Methods    Thirty male SD rats aged 5-6 weeks and weighting 160-190 g, were randomly divided into a preoperative baseline (BL) group, arteriovenous partial diversion (ECC) group, luteolin atomization pretreatment for 1 h group, 2 h group, and 3 h group by random number method, with 6 rats in each group. In the BL group, lung tissue samples were collected directly without any treatment. The ECC group received mechanical ventilation, and the whole body was heparinized after the jugular arteriovenous intubation. The flow was transferred for 30 minutes, followed by observation for 60 minutes, then lung tissue samples were collected. Subjects in the 1 h, 2 h and 3 h groups were placed in a small animal atomizer 1 h, 2 h and 3 h before flow transfer respectively, and the subsequent operation was the same as that in the ECC group. The inflammatory level of lung tissue was detected to evaluate the degree of pathological injury of lung tissue. Western blotting (WB) was used to detect the contents of p65, IKKα, IKKβ and IKKγ in the cytoplasm of lung tissue samples of each group. Results    Compared with the ECC group, the levels of IL-6 and TNF-α in lung tissues and the degree of pathological injury in the 1 h, 2 h and 3 h groups decreased, and the difference between the 3 h group and the ECC group was statistically different (P<0.05). WB results showed that compared with the ECC group, the levels of p65 in lung tissue of the 1 h, 2 h and 3 h groups decreased; the levels of IKKβ in the lung tissue increased in the 1 h, 2 h and 3 h groups, and the difference of the 3 h group was statistically different from the ECC group (P<0.05). Conclusion    Luteolin has a protective effect on acute lung injury induced by ECC, and atomization 3 h in advance has the best protective effect on lung. The mechanism plays a protective role in ECC-induced acute lung injury, may be through inhibition of IKKβ phosphorylation, thereby inhibiting the classical NF-κB signaling pathway.

The Mechanism of Neuroprotective Effects of Puerarin for the Treatment of Acute Spinal Ischemia-reperfusion Injury in Rats / 中国医科大学学报

Objective To explore the mechanism of neuropmtective effects of puerarin for the treatment of acute spinal ischenia-reperfusion injury in a rat model.Methods Acute spinal ischemia-reperfusion injury was induced via aortic occlusion in 28 male Sprague-Dawley rats.The animals were randomly divided into four groups,as follows:group negative contrast (NC sham operation),group positive control group (IR+ S ischemia/reperfusion + saline),group puerarin (IR+P ischemia/reperfusion + puerarin),group mscovitine (IR+R ischemia/reperfusion + roscovitine).The motor function,spinal infarction volume,apoptosis indices,and CDK5 and P25 activities were examined.Results Spinal ischemia-reperfusion caused the injury of the spines and was associated with motor deficit,elevation of CDK5 and P25 activities,and increase in the spinal apoptosis and spinal infarction volume.Puerarin improved motor function and decreased apoptosis,spinal infarction volume,and CDK5 and P25 activities.Conclusion The findings of the present study indicated that puerarin treatment-mediated reduction of spinal injury was associated with the inhibition of CDK5 and P25,and that the inhibition was one among the neuroprotective mechanisms of puerarin against acute ischemia/reperfusioninduced spinal injury in rats.

Expression and significance of IKKεin the specimens and cells of epithelial ovarian cancer / 中华妇产科杂志

Objective To examine the expressions of IKKε protein in the specimens and cells of epithelial ovarian cancer and investigate the effect of IKKε inhibitor on cell proliferation and apoptosis. Methods (1) A total of 118 cases of patients with the median age of 59 who have accepted surgical treatment due to ovarian cancer in the First Affiliated Hospital of Dalian Medical University from January 2006 to April 2013 were selected. Twenty cases of patients with the median age of 55 who have accepted hysterectomy and salpingo-oophorectomy due to uterine leiomyoma during the same period were selected as the control. The expressions of IKKε protein were detected by immunohistochemistry in normal ovarian tissues and epithelial ovarian cancer specimens,and the relationship between the expressions of IKKε and the clinical features of patients was analyzed. IKKε protein was determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OV4, OV5, OV8, and CAOV3 treated with or without IKKε inhibitor. The cellular proliferation and apoptosis of ovarian cancer cells after 48 hours treatment of IKKε inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results (1) The immunohistochemical results showed that IKKε was highly expressed in epithelial ovarian cancer specimens with the expression rate 66.1% (78/118), compared with normal ovarian tissue with the expression rate 35.0% (7/20), which exhibited statistically significant difference (χ2=6.993, P=0.008). The expression of IKKε protein was correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, histological grade, the level of CA125 in preoperative serum and distribution of the tumor (P0.05). (2) IKKε was widely overexpressed in different levels in SKOV3, OV2008, C13, A2780S, A2780CP, OV4, OV5, OV8, and CAOV3 cells, and the expression of IKKε decreased as the increase of the concentration of IKKε inhibitor (0.1 and 0.5 μmol/L) in OV2008, C13, A2780S, and A2780CP cells after 48 hours treatment. Different concentrations of IKKε inhibitor (0.05, 0.1, 0.5, 1, 5, 10, and 25 μmol/L) significantly inhibited the proliferation of OV2008, C13, A2780S, A2780CP, and SKOV3 cells in a concentration-dependent manner (P<0.05), and the half maximal inhibitory concentration (IC50) was 0.43, 0.86, 0.10, 0.19, and 0.24 μmol/L, respectively. The cell apoptotic rate of OV2008, C13, A2780S, A2780CP, and SKOV3 cells was significantly increased after 48 hours treatment of IKKεinhibitor with the concentration of 0.1 and 0.5 μmol/L (P<0.05). Conclusions The IKKε protein in epithelial ovarian cancer specimens and cells is overexpressed. IKKε inhibitor could inhibit cellular proliferation and induce apoptosis in a concentration-dependent manner. Together, the result indicated that IKKε may be a candidate target for the treatment of ovarian cancer in future.

NF-kappaB-Mediated Regulation of Osteoclastogenesis

Osteoclasts are multinucleated cells formed mainly on bone surfaces in response to cytokines by fusion of bone marrow-derived myeloid lineage precursors that circulate in the blood. Major advances in understanding of the molecular mechanisms regulating osteoclast formation and functions have been made in the past 20 years since the discovery that their formation requires nuclear factor-kappa B (NF-kappaB) signaling and that this is activated in response to the essential osteoclastogenic cytokine, receptor activator of NF-kappaB ligand (RANKL), which also controls osteoclast activation to resorb (degrade) bone. These studies have revealed that RANKL and some pro-inflammatory cytokines, including tumor necrosis factor, activate NF-kappaB and downstream signaling, including c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and inhibition of repressors of NFATc1 signaling, to positively regulate osteoclast formation and functions. However, these cytokines also activate NF-kappaB signaling that can limit osteoclast formation through the NF-kappaB signaling proteins, TRAF3 and p100, and the suppressors of c-Fos/NFATc1 signaling, IRF8, and RBP-J. This paper reviews current understanding of how NF-kappaB signaling is involved in the positive and negative regulation of cytokine-mediated osteoclast formation and activation.

Clinical, Biochemical, and Genetic Characterization of Glycogen Storage Type IX in a Child with Asymptomatic Hepatomegaly / 대한소아소화기영양학회지

Glycogen storage disease type IX (GSD IX) is caused by a defect in phosphorylase b kinase (PhK) that results from mutations in the PHKA2, PHKB, and PHKG2 genes. Patients usually manifest recurrent ketotic hypoglycemia with growth delay, but some may present simple hepatomegaly. Although GSD IX is one of the most common causes of GSDs, its biochemical and genetic diagnosis has been problematic due to its rarity, phenotypic overlap with other types of GSDs, and genetic heterogeneities. In our report, a 22-month-old boy with GSD IX is described. No other manifestations were evident except for hepatomegaly. His growth and development also have been proceeding normally. Diagnosed was made by histologic examination, an enzyme assay, and genetic testing with known c.3210_3212del (p.Arg1070del) mutation in PHKA2 gene.

Effect of IKK16 on proliferation and apoptosis of human glioblastoma cell line U 87 / 重庆医学

Objective To investigate the effecof Iκkinases(IKK) inhibitoIKK16 on the proliferation and apoptosiof hu-man glioblastomcell line U87 and itpossible mechanism .MethodHuman glioblastomcell line U87 wacultured in vitro and divided into 4 group:blank group(withouintervention treatmen) ,control group(1% dimethyl sulfoxide) ,low dose IKK group (70 nmol/L IKK16) and high dose IKK group(200 nmol/L IKK16) .The Mtassay waused to detecthe effecof IKK16 on the proliferation of U87 cellathe low concentration and the high concentration .Western blowaused to investigate the effecof IKK16 on the expression of p65 ,cyclinD1 ,caspase-3 and bcl-2 .ResultThe U87 proliferation rate had no statistical difference be-tween the blank group and the control group(P＞0 .05) .Compared with the control group ,IKK16 could significantly suppresthe proliferation of U87 cell(P＜0 .05) .Athe same time ,IKK16 significantly reduced the expression of p65 ,CyclinD1 and Bcl-2 ,and increased the expression of caspase-3(P＜0 .05) with dose-dependenmanne.Conclusion IKK16 could suppresthe proliferation of human glioblastomcell line U87 and promote the apoptosi.

Role of spinal IKK2/NF-κB signaling pathway in maintenance of bone cancer pain in rats / 中华麻醉学杂志

Objective To evaluate the role of spinal IKK2/NF-κB pathway in the maintenance of bone cancer pain (BCP) in rats.Methods Twenty-eight unmated adult female Sprague-Dawley rats,weighing 160-200 g,were randomly divided into 4 groups (n =7 each) using a random number table:sham operation group (group S),BCP group (group BP),BCP + normal saline group (group BN),and BCP + BMS345541 group (group BB).BCP was induced by injecting Walker 256 mammary gland cancer cell suspension 5 μl (4 × 105 cells/ml) into the bone marrow of the right tibia of rats anesthetized with chloral hydrate in BP,BN and BB groups,while the equal volume of normal saline was injected in group S.On 10-12 days after operation,selective IKK2 inhibitor BMS345541 (50 μg/10 μl) was intrathecally injected once a day in group BB,and the equal volume of normal saline (10μl) was given once a day in group BN.The mechanical paw withdrawal threshold (MWT) was measured before intra-tibia injection (T0),on 7 days after intra-tibia injection (T1),at 1 h before drug administration and 1,2,4,12 and 24 h after drug administration on day 10 after operation,and at 4 h after drug administration on day 12 after operation (T2-8).The rats were sacrificed after MWT was measured at Ts and L4-6 segments of the spinal cord were removed for determination of phosphorylated NF-κB (p-NF-κB) expression (using Western blot analysis).Results Compared with group S,MWT was significantly decreased at T1-2,and the expression of p-NF-κB was up-regulated in BP,BN,and BB groups.Compared with group BP,MWT was significantly increased at T4-6,and the expression of p-NF-κB in the spinal cord was down-regulated in group BB.Conclusion Spinal IKK2/NF-κB signaling pathway is involved in the maintenance of bone cancer pain in rats.

Effect of liver I kappa B kinase during hepatic ischemia reperfusion in rats / 中国医师杂志

Objective To explore the effect of I kappa B kinase (IKK) on liver during hepatic ischemia reperfusion (HIR) in rats.Methods Wister rats were divided randomly into HIR group in which hepatic reperfusion was given after 60 minutes of ischemia by interruption of the arterial and portal venous blood supply to the left lobes and middle lobes of the liver; HIR + PDTC treatment group in which PDTC (120 mg/kg) were injected via the dorsum vein of penis before ischemia reperfusion; and sham control group in which midline laparotomy was performed without vascular occlusion and treatment.Expression levels of IKK were measured with In situ hybridization(ISH).The NF-κB activities were determined with EMSA.Expression levels of TNF-α were measured with immunohistochemistry (IH).Serum levels of ALT were measured.Results Expression level of IKK was increased markedly from 0 to 12h and peaked 6h after reperfusion in HIR group.NF-κB was activated 0 ～ 12h after reperfusion and activities of NF-κB were maximal 6h after reperfusion in HIR group rats compared with sham control group.Expression level of TNFα was increased markedly from 0 to 12h and peaked 6h after reperfusion in HIR group.Serum levels of ALT were increased significantly after reperfusion in H1R group.Expression level of IKK was lowered markedly in HIR + PDTC group from 0 to 12h after reperfusion.NF-κB activities were significantly lower in HIR +PDTC group than in HIR group from 0 to 12h after reperfusion.Expression level of TNF-α was lowered markedly in HIR + PDTC group as compared with HIR group from 0 to 12h after reperfusion.Serum level of ALT was decreased significantly after reperfusion in HIR + PDTC group as compared with HIR group.Conclusion HIR can activate IKK-β which promotes the activation of NF-κB,then NF-κB results in upregulation transcription of TNF-α gene which gives rise to the release of other inflammatory cytokines and triggers uncontrolled inflammatory response,and induces hepatic injury.Blocking IKK-NF-κB pathway may be an effective approach to checking the generation and development of ALI,PDTC plays important prophylaxis and treatment roles in hepatic injury after HIR.

Effects of gypenosides on nuclear factor κB and p38 mitogen activated protein kinase signaling pathways in photodamaged skin of mice / 中华皮肤科杂志

Objective To observe the effects of gypenosides (GP) on nuclear factor κB (NF-κB) and p38 mitogen activated protein kinase (p38MAPK) signaling pathways in photodamaged skin of mice,and to explore the mechanisms underlying the protective effects of GP against photodamage.Methods Eighty Balb/C female mice were randomly divided into 8 groups: blank control group receiving no treatment,ultraviolet B (UVB) model group receiving UVB irradiation for 60 seconds,GP group Ⅰ receiving topical GP treatment followed by UVB irradiation,GP group Ⅱ receiving UVB irradiation followed by topical GP treatment,VitE group Ⅰ receiving topical VitE treatment followed by UVB irradiation,VitE group Ⅱ receiving UVB irradiation followed by topical VitE treatment,matrix group Ⅰ receiving topical matrix treatment followed by UVB irradiation,matrix group Ⅱ receiving UVB irradiation followed by topical matrix treatment.UVB irradiation lasted 60 seconds at one time,and was given once every other day for 7 times to establish a skin model of photodamage.The interval between irradiation and topical treatment was 30 minutes in all the groups except the control group and UVB model group.After the last treatment,mice were sacrificed.Western blot was performed to measure the protein expressions of inhibitor of NF-κB(IκB),inhibitor of NF-κB kinase (IKK),p38MAPK as well as phosphorylated p38MAPK (pp38) in skin tissue from the mice.Results No expressions of IκB or IKK were observed in the blank control group.The expression level of IκB was 0.40 ± 0.07 in UVB model group,significantly lower than that in GP group Ⅰ (1.63 ± 0.85,P ＜ 0.05) and GP group Ⅱ (0.90 ± 0.40,P ＜ 0.05),whereas the level of IKK protein was higher in UVB model group than in the GP group Ⅰ and Ⅱ (2.01 ± 1.75vs.0.23 ± 0.12 and 0.45 ± 0.29,both P ＜ 0.05).No significant difference was observed in the expression of IκB or IKK proteins between the GP group Ⅰ,Ⅱ,VitE group Ⅰ and Ⅱ or in the expression of p38MAPK between the 8 groups.The phosphorylated p38MAPK expression in UVB model group was significantly higher than that in GP group Ⅰ and Ⅱ (0.835 ± 0.049 vs.0.425 ± 0.054 and 0.571 ± 0.090,both P＜ 0.05),but similar to that in VitE groups.Conclusions UVB can activate NF-κB and phosphorylated p38MAPK signaling pathways; GP 1.5％ cream can inhibit UVB-induced activation of NF-κB and p38MAPK pathways,which may be one of the mechanisms underlying its protective effects against inflammation and photodamage.

Heat shock protein 90 regulates IkappaB kinase complex and NF-kappaB activation in angiotensin II-induced cardiac cell hypertrophy

Heat shock protein 90 (HSP90), one of the most abundant proteins in the cardiac cells is essential for cell survival. Previous studies have shown that angiotensin II induces cardiac cell hypertrophy. However, the role of HSP90 in the angiotensin II-induced cardiac hypertrophy is unclear. In this study, we showed that HSP90 regulated angiotensin II-induced hypertrophy via maintenance of the IkappaB kinase (IKK) complex stability in cardiac cells. An HSP90 inhibitor, geldanamycin (GA), significantly suppressed angiotensin II-induced [3H]leucine incorporation and atrial natriuretic factor expression in cardiac cells. GA also inhibited the NF-kappaB activation induced by angiotensin II. Importantly, treatment with GA caused a degradation of IKKalpha/beta; on the other hand, a proteasome-specific inhibitor restored the level of IKKalpha/beta. We also found that GA prevented HSP90-IKKs complex induced by angiotensin II in cardiac cells. The small interfering RNA (siRNA)-mediated knockdown of HSP90 expression significantly inhibited angiotensin II-induced cell hypertrophy and NF-kappaB activation. These results suggest that angiotensin II-induced cardiac hypertrophy requires HSP90 that regulates the stability and complex of IKK.

Cysteine-179 of I kappa B kinase beta plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines

I kappa B kinase beta (IKK beta) subunit of IKK complex is essential for the activation of NF-kappa B in response to various proinflammatory signals. Cys-179 in the activation loop of IKK beta is known to be the target site for IKK inhibitors such as cyclopentenone prostaglandins, arsenite, and antirheumatic gold compounds. Here we show that a mutant IKK beta in which Cys-179 is substituted with alanine had decreased activity when it was expressed in HEK-293 cells, and TNF stimulation did not restore the activity. Phosphorylation of activation loop serines (Ser-177 and Ser-181) which is required for IKK beta activation was reduced in the IKK beta (C179A) mutant. The activity of IKK beta (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-kappa B inducing kinase (NIK) and MAPK/extracellular signal-regulated kinase kinase kinase 1(MEKK1) or when the serine residues were replaced with phospho-mimetic glutamate. The IKK beta (C179A) mutant was normal in dimer formation, while its activity abnormally responded to the change in the concentration of substrate ATP in reaction mixture. Our results suggest that Cys-179 of IKK beta plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.

Experimental Study on the Preventive Mechanism of Anisodamine on Multiple Organ Dysfunction Syndrome / 中国医师杂志

Objective To investigate the preventive mechanism of Anisodamine (654-2) on multiple organ dysfunction syndrome (MODS) of rabbits. Methods Rabbit model of MODS induced by hemorrhagic shock and endotoxin was used in this study. Twenty-four rabbits were randomly divided into the control group (C group) , hemorrhagic shock plus endotoxin group (M group) and 654-2 treatment group (T group). The expression of IKK-? of pulmonary alveolar macrophage (PAM) and kuffer cell(KC), the NF-?B activity of nuclear protein extracted from PAM and KC and the concentration of tumor necrosis factor-? (TNF-?) in the culture supernatant were measured by in situ hybridization (ISH), electrophoretic mobility shift assay (EMSA) and enzyme linked immune absorbent analysis(ELISA), respectively. Then the blood air, biochemical and pathological changes in visceral organs were examined in each groups. Results In PAM and KC of M group, The expression of IKK-? mRNA [(0 15?0 03);(0 17?0 04)], the activity of NF-?B [(1 49?0 30);(1 72?0 36)] and the secretion level of TNF-? [( 279 74?25 91);(300 05?30 86)ng/L] were significantly higher than those of control group (P

Expression and Significance of I?B kinase-? mRNA in the Lung Tissues of Rabbits Following Hemorrhagic Shock / 中国医师杂志

Objective To investigate the changes and significance of I?B kinase-?(I?K-?) in the lung tissues of rabbits with hemorrhagic shock.Methods The expressions of I?K-? and NF-?B in the lung tissues and the concentration of tumor necrosis factor-?(TNF-?) in the plasma were measured by in situ hybridization(ISH),immunohitochemistry and enzyme linked immune adsorbent analysis(ELISA), respectively. And the pathological changes were examined with light microscope in lung tissues.Results In hemorrhagic shock group,the expressions of I?K-?(0 1685?0 0164)and NF-?B( 0 1469?0 0083)in lung tissues , the level of TNF-?(636 72?100 23) in the plasma were obviously higher than those of normal group [I?K-?(0 0427?0 0241),NF-?B(0 0358?0 0048),TNF-?(199 51?35 69)ng/L](all P

THE CHANGES IN NF-?B SIGNAL PATHWAYS IN PULMONARY ALVEOLAR MACROPHAGES (PAM) STIMULATED BY LPS / 解放军医学杂志

Objective To investigate changes in NF ?B signal pathway in PAM stimulated by lipopolysaccharide(LPS) in vitro , and to explore the molecular pathological mechanism of acute lung injury(ALI). Methods After PAM were stimulated by LPS, the changes in expression of IKK mRNA, activation of NF ?B, degradation of I?B, and secretion of TNF ? in PAM were measured at 0, 15min, 30min, 1h, 2h, and 4h by in situ hybridization, electrophoretic mobility shift assay (EMSA), and enzyme linked immune absorbing analysis (ELISA), respectively. Results The expression of IKK ? mRNA was increased 15min after LPS stimulation and reached the peak at 30 min, then returned to the base line after 1 hour. The changes in I?B ? mRNA were opposite. The activity of NF ?B was increased 15min after LPS stimulation, peaking at 1 hours, and returned to the pre stimulation level after 2 hours. The content of TNF ? was increased initially at 30min, reached the peak at 1 hour, and gradually returned to the pre stimulation level in 2～4 hour. Conclusion The transduction pathway of activation of IKK ? degradation of I?B/activation of NF ?B/synthesization of TNF ? might play a critical role in the molecular pathological mechanism of LPS induced ALI.

Function of I?B kinase links inflammation and cancer / 中国病理生理杂志

It has long been acknowledged that there is a link between inflammation and cancer, but its molecular mechanism remains unclear. A key player in inflammation is nuclear transcription factor NF-?B, that activity is triggered in response to infectious agents and proinflammatory cytokines via the I?B kinase. In parenchyma cells, inflammation through I?B kinase/NF-?B pathway suppresses apoptosis, accelerates cell cycle, then promote tumorigenesis. In mesenchyma cells inflammation through I?B kinase/NF-?B pathway produces cytokines and chemokines that may serve as tumor growth factors. To sum up, I?B kinase/NF -?B pathway represents a critical molecular link between inflammation and cancer.