Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway / 中国中药杂志

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1β, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

Apoptotic and cytostatic actions of maslinic acid in colorectal cancer cells through possible IKK-β inhibition

Objective: To explore the anti-cancer activity of maslinic acid against colorectal cancer (CRC) cell lines and its possible mechanism. Methods: The inhibitory effect of maslinic acid was screened against five CRC cell lines (HT-29, HCT 116, SW480, SW48, and LS 174T) via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis and cell cycle analyses were carried out using annexin V-FITC/propidium iodide staining and propidium iodide staining, respectively and subjected to fluorescence-activated cell sorting analysis. Protein expression studies of inhibitor of κB kinase-β (IKK-β), checkpoint kinase 1 (Chk1) and cyclin D1 were conducted using the JESS system. Results: Maslinic acid exhibited growth inhibitory effect in a doseand time-dependent manner in HT-29 and HCT 116 cell lines. A more prominent apoptosis induced by maslinic acid was observed in HCT 116 cell line. However, in HT-29 cell line, maslinic acid induced cell cycle arrest by inhibiting the G1-S transition, which was accompanied by the downregulation of cyclin D1. The expression of unphosphorylated IKK-β protein was increased in both (HT-29 and HCT 116) cell lines after maslinic acid treatment. Conclusions: Maslinic acid inhibits the growth of HT-29 and HCT 116 cells in a different manner, induces cell cycle arrest in HT-29 cells and causes apoptosis in HCT 116 cells partially via NF-κB pathway inhibition.

Apoptotic and cytostatic actions of maslinic acid in colorectal cancer cells through possible IKK-β inhibition

Objective: To explore the anti-cancer activity of maslinic acid against colorectal cancer (CRC) cell lines and its possible mechanism. Methods: The inhibitory effect of maslinic acid was screened against five CRC cell lines (HT-29, HCT 116, SW480, SW48, and LS 174T) via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis and cell cycle analyses were carried out using annexin Ⅴ-FITC/propidium iodide staining and propidium iodide staining, respectively and subjected to fluorescence-activated cell sorting analysis. Protein expression studies of inhibitor of κB kinase-β (IKK-β), checkpoint kinase 1 (Chk1) and cyclin D1 were conducted using the JESS system. Results: Maslinic acid exhibited growth inhibitory effect in a dose- and time-dependent manner in HT-29 and HCT 116 cell lines. A more prominent apoptosis induced by maslinic acid was observed in HCT 116 cell line. However, in HT-29 cell line, maslinic acid induced cell cycle arrest by inhibiting the G1-S transition, which was accompanied by the downregulation of cyclin D1. The expression of unphosphorylated IKK-β protein was increased in both (HT-29 and HCT 116) cell lines after maslinic acid treatment. Conclusions: Maslinic acid inhibits the growth of HT-29 and HCT 116 cells in a different manner, induces cell cycle arrest in HT-29 cells and causes apoptosis in HCT 116 cells partially via NF-κB pathway inhibition.

IKKε and TBK1 pathways and their inhibitors in tumor / 中国肿瘤生物治疗杂志

@# 非经典信号通路IKKε和TBK1与恶性肿瘤密切相关，多种因素激活IKKε和TBK1通路，可引起NF-κB途径的激活， 导致肿瘤细胞的凋亡减少、细胞周期加快，促进肿瘤发生和发展。抑制IKKε和TBK1信号通路，可增加多种细胞凋亡因子的表 达，抑制肿瘤细胞增殖，促进肿瘤细胞凋亡，同时提高化疗和放疗的敏感性。因此，阻断IKKε和TBK1信号通路可有效治疗恶性 肿瘤，已有的实验证实有多种阻断IKKε和TBK1通路的药物均具有良好的抗肿瘤作用。

Mechanism of catgut embedding at back- points for nonalcoholic steatohepatitis based on IKK/IKB/NF-κB signaling pathway / 中国针灸

OBJECTIVE@#To explore the mechanism of catgut embedding at back- points on nonalcoholic steatohepatitis (NASH) in rats based on IKK/IKB/NF-κB signaling pathway and downstream inflammatory factors.@*METHODS@#Eighty SPF SD rats were selected, among them 10 rats were selected divided into a normal group (group A), and the remaining 70 rats were fed with high-fat diet to establish NASH model. At the end of 12 weeks, 10 rats were randomly selected to verify whether the model establishment was successful. Then the remaining 60 rats were randomly divided into a model group (group B), a catgut embedding at back- points group (group C), a catgut embedding at abdominal points group (group D), an acupuncture at back- points group (group E), a sham catgut embedding group (group F) and a western medication group (group G), 10 rats in each group. The rats in the group C were treated with catgut embedding at "Ganshu" (BL 18), "Pishu" (BL 20), "Weishu" (BL 21) and "Shenshu" (BL 23); the rats in the group D were treated with catgut embedding at "Daheng" (SP 15), "Fujie" (SP 14), "Huaroumen" (ST 24) and "Tianshu" (ST 25); the rats in the group E were treated with acupuncture at the same acupoints as the group C; the rats in the group F were treated with catgut embedding at back- points but the needle did not enter subcutaneous tissue gamma; the rats in the group G were treated with intragastric administration of vitamin E capsule. All the treatment was given for 4 weeks. The rats in the group A were fed with normal diet until the end of 16 weeks without any intervention. The rats in the group B continued to be fed with high-fat diet until the end of 16 weeks. After the intervention, the liver index was calculated; the liver histomorphology was observed by HE staining; the liver function [alanine aminotransferase (ALT), gamma glutamyl transferase (γ-GGT), alkaline phosphatase (ALP)] and blood lipid [serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL)] were measured by serum biochemistry. The serum levels of TNF-α, IL-6 and IL-1βwere detected by ELISA, and the expressions of IKK-α, NF-κBp65, IL-6, IL-1β and TNF-α proteins in liver tissue were detected by Western blot. The temperature of the conception vessel and the governor vessel was measured by infrared thermography.@*RESULTS@#Compared with the group A, the obvious steatosis and inflammatory cell infiltration were observed in the group B, and the body weight, liver wet-weight and liver index were all increased (0.05), while the temperature of the governor vessel in the group C was superior to that in the group D (<0.05).@*CONCLUSION@#The catgut embedding at back- points might inhibit the activation of IKK/IKB/NF-κB signaling pathway to interrupt the inflammatory cascade, and reduce the "second hit" of inflammatory factors on liver, which could slow down NASH progress and prevent and treat NASH.

Study on the Shidan granules for chronic atrophic gastritis with dysplasia rat model based on NF-κB signaling pathway / 国际中医中药杂志

Objective To establish a rat chronic atrophic gastritis with dysplasia model and observe the effect of Shidan granules on the positive expression of NF-κB p65 in gastric tissues and the extent of IKKβprotein content in gastric tissues of chronic atrophic gastritis with dysplasia model rats. Methods Seventy SPF male Wistar rats were randomly divided into the blank group and 60 into the model group. In addition to the blank group, the modeling group was made using the MNNG-based comprehensive modeling method (150 μg/ml MNNG 5 ml/kg each stomach, containing 0.03％ ranitidine feed, free drinking 0.1％ ammonia), and then model successed at the 28th week. The remaining rats in the modeling group were randomly divided into 5 groups: the model group with intragastric administration of gavage, the western medicine group with intragastric administration of viralin, the Shidan granules group with intragastric administration of low/medium/high concentration Chinese medicine decoction. After 12 weeks of treatment, they were sacrificed and the expression of NF-κB p65 and IKKβ was measured and compared. Results Compared with the model group, the average optical density of NF-κBp65 (0.387 ± 0.011, 0.252 ± 0.022, 0.193 ± 0.003 vs. 0.442 ± 0.035) and the expression level of IKKβ (0.309 ± 0.056, 0.216 ± 0.025, 0.125 ± 0.016 vs. 0.405 ± 0.042) in the low-, middle- and high-dose Shidan granules groups significantly decreased (all Ps<0.01). Conclusions The mechanism of improving the precancerous lesions of gastric cancer by Shidan granules may be achieved by inhibiting the expression of IKK, reducing the abnormal activation of NF-κB.

Influence of IKK Inhibitor on Carotid Artery Restenosis in Rat by Balloon Catheter Injury / 沈阳医学院学报

Objective:To investigate the influence of inhibitor of kappa B kinase ( IKK) inhibitor on carotid artery restenosis in rat by balloon catheter injury. Methods: A total of 32 male Wistar rats were randomly divided into control group, injured group and med?icine group. The balloon catheter injury was performed on left common carotid artery of rat by imitating the process of angioplasty. In?jured group and control group were injected saline, medicine group were injected IKK inhibitor. After 15 days, the process of neointi?mal and media hyperplasia was observed by HE staining and the expression level of NF?κB, IKK, inhibitor of nuclear factor kappa B ( IκB) was detected by Western blot, the content of IL?6, TNF?αwas detected by radioimmunoassay, and the content of MMP?1 was determined by MMP?1 kit. Results: Arterial neointima hyperplasia hapened in 3 groups. There was significantly difference between control group and medicine group and injured group in intimal area, the expression level of NF?κB, IKK, IκB, and the content of IL?6, TNF?α and MMP?1 ( P<0?05 or 0?01) . There was no significance in these indexes between control group and medicine group ( P>0?05) . Conclusion:IKK inhibitor can reduce the carotid artery restenosis after balloon injury by inhibiting inflammatory reaction induced by IKK?IκB?NF?κB pathway.

The role of the IKK / NF-kappa B signaling pathways in the extinction of auditory fear condition / 中华行为医学与脑科学杂志

Objective To identify the role of the IKK / NF-kappa B signaling pathways in the acquisition and consolidation of extinction memory in conditioning fear.Methods Aduh SD rats were treated with nuclei buried surgery.After a week recovery from nuclei buried surgery,adult SD rats accepted auditory fear conditioning training.Before the extinction training,bilateral intra-BLA infusion of sulfasalazine(SSZ),an inhibitor of IKK/NFκ B,and Vehicle were done.The freezing time were tested on the 24h and 1w after the extinction.Results After training,all the rats showed higher percentage of freezing time than the baseline(Vehicle:pre-CS 21.16％,post-CS 71.23％,P＜0.01 ; SSZ:pre-CS 22.23％,post-CS 72.14％,P＜0.01).The percentage of freezing time had no significant difference(SSZ 47.52％,Vehicle 46.20％,P＞0.05)between Vehicle and SSZ at the end of extinction training.However,SSZ groups showed significantly higher percentage of freezing time than the control group on the 24h(Vehicle 41.03％,SSZ 60.51％,P＜0.01) and the same was found on 1 weck test(Vehicle 34.17％,SSZ 57.21％,P＜ 0.01).The rats injection with SSZ showed significant fear response for the auditory fear condition.Conclusion All above results suggest that interference of IKK/NF-κ B signaling pathway impaire the consolida tion but have no effect on the acquisition of fear extinction memory in conditioning fear.The available date could provide us some clinical guidance in the treatment of post-traumatic stress disorder and panic disorder.

PPM1B and P-IKKβ expression levels correlated inversely with rat gastrocnemius atrophy after denervation

Activated inhibitor of nuclear factor-κB kinase β (IKKβ) is necessary and sufficient for denervated skeletal muscle atrophy. Although several studies have shown that Mg2+/Mn2+-dependent protein phosphatase 1B (PPM1B) inactivated IKKβ, few studies have investigated the role of PPM1B in denervated skeletal muscle. In this study, we aim to explore the expression and significance of PPM1B and phosphorylated IKKβ (P-IKKβ) during atrophy of the denervated gastrocnemius. Thirty young adult female Wistar rats were subjected to right sciatic nerve transection and were sacrificed at 0 (control), 2, 7, 14, and 28 days after denervation surgery. The gastrocnemius was removed from both the denervated and the contralateral limb. The muscle wet weight ratio was calculated as the ratio of the wet weight of the denervated gastrocnemius to that of the contralateral gastrocnemius. RT-PCR and Western blot analysis showed that mRNA and protein levels of PPM1B were significantly lower than those of the control group at different times after the initiation of denervation, while P-IKKβ showed the opposite trends. PPM1B protein expression persistently decreased while P-IKKβ expression persistently increased for 28 days after denervation. PPM1B expression correlated negatively with P-IKKβ expression by the Spearman test, whereas decreasing PPM1B expression correlated positively with the muscle wet weight ratio. The expression levels of PPM1B and P-IKKβ were closely associated with atrophy in skeletal denervated muscle. These results suggest that PPM1B and P-IKKβ could be markers in skeletal muscle atrophy.

Interleukin-17 and Interleukin-22 Induced Proinflammatory Cytokine Production in Keratinocytes via Inhibitor of Nuclear Factor kappaB Kinase-alpha Expression

BACKGROUND: The pathogenesis of psoriasis may involve the interleukin (IL)-23 and Th17-mediated immune responses. Th17 cells secret IL-17 and IL-22, which mediates dermal inflammation and acanthosis. OBJECTIVE: As inhibitor of nuclear factor kappaB kinase-alpha (IKKalpha) has been previously identified as a primary regulator of keratinocyte differentiation and proliferation, we proposed that IL-17 and IL-22 might affect keratinocyte differentiation by changing the expression of IKKalpha. METHODS: We employed HaCaT cells maintained culture medium at a low calcium concentration (0.06 mM) and induced differentiation by switching to the high concentration (2.8 mM) media with IL-17 or IL-22, then compared the IKKalpha expression and the cell cycle. We employed reconstituted human epidermal skin (Neoderm) and mice ears for the in vivo studies. RESULTS: Elevated calcium concentration induced IKKalpha expression and terminal differentiation with cell cycle arrest in HaCaT cell cultures. Moreover, IL-17 and IL-22 treatment also induced IKKalpha in HaCaT cells and reconstituted human epidermis. IKKalpha induction was also noted, following the injection of IL-17 and IL-22 into mice ears. CONCLUSION: Although the induction of IKKalpha was accompanied by keratinocyte differentiation, IL-17 and IL-22 did not affect calcium-mediated differentiation or the cell cycle. Rather, IL-17 and IL-22 appear to contribute to the inflammation occurring via the induction of IKKalpha from keratinocytes or skin layers.

Migration and proliferation of human mesenchymai stem cells from patients with systemic lupus erythematosus mediated by IKK-β / 中华风湿病学杂志

ObjectiveTo investigate the effect of IκB kinase (IKK-β) on migration and proliferation of bone marrow mesenchymal stem cells(BMSCs) from patients with systemic lupus erythematosus (SLE).MethodsHuman bone marrow aspirates were collected from iliac of six donors and six SLE patients and cultured in vitro.Migration of BMSCs were observed by wound healing and transwell migration assays.Proliferation of BMSCs was quantified by cell counting kit-8 assay.Total RNA was extracted and reverse transcribed into complementary DNA.Real-time PCR technique was used to determine the gene expression of IKK-β at transcription level.The expression of IKK-β and phospho-IKK-β(p-IKK-β) protein were determined by Western blotting analysis.Statistical analysis was conducted with or Mann-Whitney rank test.Results① The migration rate of BMSCs from SLE patients(5.2±3.8)‰ were significantly reduced as compared with normal controls (7.0±2.9)‰(P＜0.05 ).The proliferation of BMSCs of SLE patients (0.21±0.49)was lower than that from healthy controls ( 1.00±0.35 )(P＜0.05 ).②) No difference in IKK-β3 mRNA expression between SLE ( 1.9± 1.4) subjects and normal controls (1.9±2.4) (P＞0.05).IKK-β protein expression of BMSCs from SLE patients (1.41 ±0.19) increased significantly compared with healthy controls (0.93±1.24) (P＜0.05).③ Inhibitor of IKK-β caused a significant increase in cell migration (3.3±1.6)‰ and proliferation (1.13±0.26) of BMSCs from SLE patients compared with untreated cells (2.3±1.1)‰ and (0.81±0.17),respectively (P＜0.05).ConclusionMigration and proliferation of BMSCs are significantly decreased in SLE patients.IKK-β may be involved in migration and proliferation of BMSCs.

The expression of IKB kinase in the peripheral blood and its correlation with the production of interferon-α in systemic lupus erythematosus patients / 中华风湿病学杂志

Objective To investigate the mRNA expression of IKB kinase (IKK-α) and interferon-α (IFN-α) in the peripheral blood leukocytes of patients with systemic lupus erythematosus (SLE), and to explore the role of IKK-α in the production of IFN-α in SLE patients. Methods SYBR green dye I based real-time quantitative PCR was used to detect the mRNA expression levels of IKK-α and IFN-α in the peripheral blood leucocytes of SLE patients and healthy controls. Serum levels of IFN-α were measured with ELISA method. Results IKK-α mRNA expression levels in SLE patients were significantly higher than those of normal controls (P<0.05). IKK-α mRNA expression levels in SLE patients with active disease were significantly higher than patients with stable disease (P<0.01). IFN-α mRNA expression level in SLE patients was significantly lower than that of the normal controls (P<0.01). IFN-α mRNA expression levels in SLE patients with active disease were significantly higher than patients with stable disease (P<0.01). Serum levels of IFN-α in SLE patients with active disease was significantly higher than that of the normal controls and patients with stable disease (P<0.05). The anti-dsDNA antibody correlated positively, and complement C3 correlated negatively with serum concentration of IFN-α. IKK-α mRNA expression levels in SLE patients correlated positively with serum concentration of IFN-α. Conclusion IKK-α correlates positively with serum concentration of IFN-α. The IFN-α level is significantly correlated with disease activity, This suggests that IKK-α may play an important role in the pathogenesis of SLE.

The effect of smoking and smoking cessation on the phosphorylation of IKK-β in type 2 diabetic rats / 中华内科杂志

Objective To investigate the effect of smoking and smoking cessation on the phosphorylation of IKK-β in type 2 diabetic rats. Methods Forty-two six-week-old Wistar rats were randomly divided into 4 groups: normal control(NC, n =7), diabetes control (DC, n =7), diabetes with smoking (DS, n = 14) and diabetes with smoking cessation(SC, n = 14). Rats in DS and SC groups were further assigned randomly into 8w and 12w subgroups. DS group was given passive smoking twice a day for 8 or 12 weeks, while SC group ceased passive smoking for 4 weeks after 8 or 12 weeks of smoking . Western blot method was used to detect the level of IKK-13 phosphorylation in skeletal muscle. Results Compared with the NC group,the phosphorylation of IKK-β protein in DC group was increased (0. 16±0. 05 vs 0. 30±0. 08, P < 0. 01). There was an increasing trend with the phosphorylation level of IKK-β in the DS (8w) subgroup, but there was no statistical difference between the DC group and SC(8w) subgroup (0. 40±0. 09 vs 0. 30±0. 08,0. 36±0. 10, P >0. 05). The phosphorylation level of IKK-β in DS(12w) group increased obviously, being significantly higher than that in the DC group and SC (12w) subgroup(0. 74 ± 0. 11 vs 0.30±0.08,0.35±0.07,P < 0.01). Conclusion With the prolongation of smoking duration, the phosphorylation of IKK-β in type 2 diabetic rats increased. After smoking cessation, the phosphorylation of IKK-β decreased. The phosphorylation of IKK-β may be involved in the mechanism by which smoking causes type 2 diabetes.

Prolongation of rat renal allograft survival by recipient-derived immature dendritic cells transfected with IKK2dn gene / 中华器官移植杂志

Objective To investigate the effect and mechanisms of recipient-derived immature dendritic cells(imDC) transfected with IKK2dn and loaded with donor antigen on renal allografts survival in the rats.Methods DC were cultured from recipient rats'(Lewis) bone marrow,transfected with IKK2dn and loaded with donor antigen.The expression of CD86 and MHC Ⅱ was detected,and the ability of DC stimulating lymphocyte proliferation in vitro was measured.Male Brown Norwav rats and Lewis rats were used as donors and recipients respectively.Four groups were set up(DC group,empty transfection group,transfection group and control group),receiving 1×10~7 DC,Adv-0-DC,Adv-IKK2dn-DC loaded with BN antigen,and equal volume of normal saline,respectivelv 7 davs before transplantation.In the third party donor-group,Wistar rats as donors were treated the same as DC;group before transplantation.After transplantation,the T lymphocyte proliferation in reciPients was measured and the expression of serum IL-2 and IFN-γ was detected.The survival time of recipients and the acute reiection were observed.Pathological changes were examined tO identify the grade of rejection.Results DC assessment in vivo revealed that the transfected DC could still express CD86 and MHC Ⅱ in a low level as compared with those not transfected with IKK2dn. After DC were loaded with donor's antigen,the expression of CD86 and MHC Ⅱ was up-regulated.After DC were transfected with IKK2dn before loaded with donor's antigen, the expression of CD86 and MHC Ⅱ had no significant change. When DC were loaded with donor's antigen, its allostimulatory activity of T lymphocyte proliferation was enhanced (P<0. 05). When DC were transfected with IKK2dn before loaded with donor's antigen, its allostimulatory activity of T lymphocyte proliferation was not enhanced. Compared with control groups, IKK2dn-transfected DC pulsed with BN splenocyte lysate markedly prolonged the survival of renal allografts (26. 8±1.76d, P<0.01), and elicited markedly lower proliferative responses and reduced IL-2 and IFN-γ production. The pathological grade of rejection was low in the transfection group. Conclusion Recipient-derived imDC transfected with IKK2dn and loaded with donor splenocyte lysate could prolong the renal allograft survival in rats probably by down-regulating the expression of DC costimulatory molecules and inhibiting the T_H 1 cytokine production.

Wortmannin Inhibits K562 Lukemic Cells by Regulating PI3k/Akt Channel In Vitro / 华中科技大学学报（医学）（英德文版）

ssion of total Akt showed no change. It is concluded that wortmannin can inhibit the proliferation and induce apoptosis of K562 leukemia cells possibly by down-regulating the survival signaling pathways (PI3K/Akt and NF-κB channels).

C-Terminal Serine Rich Region of IKK-beta Interacts with Bfl-1, a Homologue of Bcl-2 / 대한진단검사의학회지

BACKGROUND: Bcl-2 family proteins play a central role in regulating apoptosis. In human, over 20 members of this family have been identified to date. Bfl-1, a member of the Bcl-2 family, has been known to retard apoptosis in various cell lines. However, the function of Bfl-1 remains unclear. METHODS: In order to investigate the Bfl-1 function, we employed yeast two-hybrid system to identify the proteins which are capable of interacting with Bfl-1. The interaction of inhibitor kappaB kinase-beta (IKK-beta) and Bfl-1 was confirmed using glutathione S-transferase pull down assays. To determine which regions of IKK-beta were required for interaction with Bfl-1, we constructed 12 deletion mutants of IKK-beta and 5 deletion mutants of Bfl-1. RESULTS: Bfl-1 interacted with the C-terminal region of IKK-beta which is a subunit of IKK complex, and IKK-beta activity is very important in the NF-kappaB related pathway. In addition, the amino acids 673-745 of IKK-beta were important for Bfl-1 interactions, and amino acids 1-484 of Bfl-1, including Bcl-2 homology domains (BH1, BH2, BH3, BH4), were crucial for IKK-beta interactions. CONCLUSIONS: IKK beta C-terminus contains many serine residues as binding partner of Bfl-1. Our results suggested that Bfl-1 is involved in the NF-kappaB activation through interaction of IKK-beta and Bfl-1. Further studies need to be performed to understand functions of the IKK-beta and Bfl-1 associated with the regulation of the NF-kappaB activation pathway.

Immunohistochemical study of NF-?B signal transductionpathway in laryngeal carcinoma / 重庆医科大学学报

Objective:To explore the expression and significance of IKK, I?B and NF-?B P65 of NF-? B signal transduction pathway in laryngeal carcinoma patients.Methods:The expression of IKK?, I?B?,NF-?B P65 was examined by immunohistochemical S-P method in laryngeal carcinoma tissues of 40 cases and normal laryngeal tissues of 20 cases.Results:The expression of IKK? and NF-?B P65 in laryngeal carcinoma tissue was higher compared with normal laryngeal tissue,while the expression of I?B? was lower( P

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

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.

Molecular mechanism for berberine to improve insulin resistance in 3T3-L1 adipocytes / 中华内分泌代谢杂志

Objective To investigate the effect of berberine on insulin resistance induced by free fatty acid in 3T3-L1 adipocytes and the possible molecular mechanism.Methods 3T3-L1 adipocytes were treated with 0.5 mmol/L palmitic acid to induce insulin resistance.Berberine was used for treatment and aspirin for positive control.Glucose oxidase method was employed for measuring the glucose consumption in the medium and 2-deoxy- [~3H]-D-glucose method was used for the determination of glucose uptake.Western blot was used for the determination of IKB kinase(IKK)?SerlS1 phosphorylation,insulin receptor substrance-1(IRS-1)Ser307 phosphorylation,the protein expression of IKK?,IRS-1,phosphatidylinositol 3-kinase(PI-3K)p85 and glucose transporter 4(Glut4).Results After the treatment with 0. 5 mmol/L of palmitic acid for 24 h,glucose consumption by 3T3-L1 adipocytes was decreased by 41%,insulin-stimulated glucose transport was inhibited by 67%,IRS-1 and PI-3K p85 proteins were reduced, and phosphorylations of IKK?Ser181 and IRS-1 Ser307 were induced.The above results were reversed by adding berberine or aspirin.But Glut4 and IKK?protein abundance was not changed during this study.Conclusion Berberine significantly improves insulin resistance induced by free fatty acid in 3T3-L1 adipocytes via inhibiting IKK?serine phosphorylation.