Wortmannin inhibits the activity of NLRP3 in Kupffer cells through the Chemerin/CMKLR1 pathway and relieves nonalcoholic fatty liver in mice / 重庆医学

Objective To investigate the role and mechanism of inflammatory response of Kupffer cells induced by Chemerin in the progression of non-alcoholic fatty liver disease (NAFLD) in mice.Methods Wortmannin was used to treated on KCs which pre-treated with Chemerin in vitro for two hours,and treated on C57BL/6J mice which was fed with a high-fat die.Levels of cytokines in supernatant/serum were tested by enzyme-linked immunosorbent assays(ELISA);mRNA and protein levels of KCs' Chemokine-like receptor 1 (CMKLR) and nucleotide oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) in vivo and vitrowere detected by real time polymerase chain reaction(real time-PCR) and Western blot;changes of mouse weight were recorded;insulin resistance and glucose tolerance were detected;the severity of liver steatosis was evaluated by HE staining combined with NAS score.Results The levels of interleukin 1β (IL-1β) and IL-18 in the KCs and mice treated with wortmannin were significantly lower than the KCs treated with Chemerin only and mice fed with high fat diet only.The mRNA and protein levels of CMKLR1 and inflammasome 3 (NLRP3) were significantly lower in the KCs and mice treated with Wortmannin than the KCs treated with Chemerin only and mice fed with high fat diet only.In addition,changes in mouse weight,hepatic steatosis,liver function,insulin resistance and glucose tolerance were much milder in mice treated with Wortmannin than those mice fed with high fat diet.Conclusion Wortmannin alleviates liver steatosis and inflammation mediated by KCs via down-regulating the expression of CMKLR1 and NLRP3 in high fat diet fed mice.

Effect of Agonist and Inhibitor of PI3K/AKT on Inflammatory Response in Macrophages / 胃肠病学

Phosphoinositide 3-kinase/serine-threonine kinase (PI3K/AKT)has been found playing an important role in the pathogenesis of severe acute pancreatitis (SAP)in recent years,but the underlying mechanism has not been clarified.Aims:To investigate the role of PI3K/AKT in regulating the inflammatory response in SAP by evaluating the effect of insulin-like growth factor-Ⅰ (IGF-Ⅰ)and wortmannin,the agonist and inhibitor of PI3K/AKT on Toll-like receptor 4 (TLR4)signaling pathway in macrophage cell line RAW264.7.Methods:RAW264.7 cells were treated with different concentrations of lipopolysaccharide (LPS ), IGF-Ⅰ and wortmannin, respectively, and cell viability was determined by CCK-8 assay.RAW264.7 cells were divided into blank control group (no treatment),LPS group (LPS 1 μg/mL),IGF-Ⅰ group (IGF-Ⅰ 100 ng/mL +LPS 1 μg/mL),wortmannin group (wortmannin 100 nmol/L +LPS 1 μg/mL)and IGF-Ⅰ +wortmannin group (wortmannin 100 nmol/L +IGF-Ⅰ 100 ng/mL +LPS 1 μg/mL).Protein expressions of tumor necrosis factor-α(TNF-α)and interleukin-6 (IL-6)were detected by ELISA;mRNA expressions of TLR4,myeloid differentiation factor 88 (MyD88),AKT,PI3K,p38 mitogen-activated protein kinase (p38MAPK)and nuclear factor-κB (NF-κB)were determined by real-time PCR.Results:After treated with LPS,IGF-Ⅰand wortmannin, respectively,no differences in cell viability of RAW264.7 cells were found between different concentrations groups (P>0.05).Protein expressions of TNF-αand IL-6 in LPS,IGF-Ⅰ,wortmannin and IGF-Ⅰ +wortmannin groups were significantly higher than those in blank control group (P<0.05 ).Protein expressions of TNF-αand IL-6 in wortmannin group were significantly lower than those in LPS and IGF-Ⅰ groups (P<0.05),and those in IGF-Ⅰ+wortmannin group were significantly lower than those in IGF-Ⅰ group (P<0.05).In LPS group,mRNA expressions of AKT and PI3K as well as TLR4 and its downstream molecules MyD88,p38MAPK and NF-κB were significantly higher than those in blank control group (P <0.05 ).Expressions of all above-mentioned mRNAs in IGF-Ⅰ group were further increased and significantly higher than those in LPS group (P<0.05).Expressions of all above-mentioned mRNAs in wortmannin group were significantly lower than those in LPS and IGF-Ⅰ groups (P<0.05 ),and those in IGF-Ⅰ+wortmannin group were significantly higher than those in wortmannin group (P<0.05),but significantly lower than those in IGF-Ⅰ group (P<0.05).Conclusions:PI3K/AKT might regulate TLR4 signaling pathway and its downstream molecules in macrophages, thereby affects the expressions of inflammatory cytokines and being involved in the pathogenesis of inflammatory response in SAP.

Expression of phosphatidylinositol-3 kinase and effects of inhibitor Wortmannin on expression of tumor necrosis factor-α in severe acute pancreatitis associated with acute lung injury / 世界急诊医学杂志（英文）

@#BACKGROUND: Acute lung injury (ALI) is a common and serious complication of severe acute pancreatitis (SAP). The study aimed to investigate the protective effect and mechanism of phosphatidylinositol-3 kinase (PI3K) inhibitor Wortmannin in SAP associated with ALI. METHODS: Ninety rats were randomly divided into three groups: sham operation (SO) group (n=30), SAP group (n=30), and SAP+Wortmannin (SAP+W) group (n=30). SAP model was induced by retrograde injection of 4％ sodium taurocholate into the biliopancreatic duct of rats. The rate of lung water content, myeloperoxidase (MPO), matrix metalloproteinase 9 (MMP-9), protein kinase B (PKB), abdphosphorylation of protein kinase B (P-PKB) activity in the lung tissue were evaluated. RESULTS: In the SAP group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours (P<0.05); the rate of lung water content, MPO and TNF-α activity were also gradually increased, and the degree of lung lesion gradually increased (P<0.05). In the SAP+Wortmannin group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours; it was higher than that in the SO group (P<0.05), but signifi cantly lower than that in the SAP group (P<0.05). The rest indicators in the SAP+Wortmannin group were also signifi cantly decreased as compared with the SAP group (P<0.05). CONCLUSIONS: The expression of phosphatidylinositol-3 kinase/protein kinase B was elevated in severe pancreatitis rats with lung injury. This suggested that PI3K signal transduction pathway is involved in the control and release of proinfl ammatory cytokines TNF-α, which may play an important role in the pathogenesis of severe acute pancreatitis associated with lung injury. This finding indicated that Wortmannin can block the PI3K signal transduction pathway, and inhibit the release of infl ammatory factor TNF-α.

A likely role for a novel PH-domain containing protein, PEPP2, in connecting membrane and cytoskeleton

PH domains (pleckstrin homology) are well known to bind membrane phosphoinositides with different specificities and direct PH domain-containing proteins to discrete subcellular apartments with assistances of alternative binding partners. PH domain-containing proteins are found to be involved in a wide range of cellular events, including signalling, cytoskeleton rearrangement and vesicular trafficking. Here we showed that a novel PH domain-containing protein, PEPP2, displayed moderate phosphoinositide binding specificity. Full length PEPP2 associated with both plasma membrane and microtubules. The membrane-associated PEPP2 nucleated at cell-cell contacts and the leading edge of migrating cells. Overexpression of PEPP2 increased membrane microviscosity, indicating a potential role of PEPP2 in regulating function of membrane and microtubules.

Inhibir la contractilidad sin provocar necrosis: ¿sería útil en miocardiopatía hipertrófica? Modelo experimental de administración intramiocárdica de toxina botulínica y de wortmanina / Inhibiting contractility without necrosis: Would it be helpful for hypertrophic cardiomyopathy? Experimental model of intramyocardial botulinum toxin and wortmannin

En este artículo se presenta una idea original: inhibir la contracción miocárdica en forma regional y selectiva sin inducir necrosis. Se propone como una posible opción terapéutica en miocardiopatía hipertrófica asimétrica obstructiva, y se plantean 2 modelos farmacológicos basados en la administración intramiocárdica de toxina botulínica y de wortmanina.

The purpose of this paper is to introduce a new idea: local inhibition of contractility without necrosis. It's potential usefulness in hypertrophic cardiomyopathy treatment is discussed and 2 pharmacological models, administrating botulinum toxin and wortmannin directly in the myocardium are disclosed.

The reno-protective effect of a phosphoinositide 3-kinase inhibitor wortmannin on streptozotocin-induced proteinuric renal disease rats

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 +/- 0.56 mg/day, whereas wortmannin group was 1.77 +/- 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 +/- 10.82 mg/g, whereas wortmannin group was 20.27 +/- 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 +/- 0.02), podocin (87.81 +/- 0.03) and Rac1/Cdc42 (86.12 +/- 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 +/- 0.03), podocin (53.40 +/- 0.06) and Rac1/Cdc42 (54.05 +/- 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.

Effect of PI3K/Akt inhibitor wortmannin on proliferation and apoptosis of leukemia K562 cells / 中国肿瘤生物治疗杂志

Objective: To study the effect of wortmannin (WM), a PI3K/Akt inhibitor, on the proliferation and apoptosis of leukemia cells and the possible mechanism. Methods: Human leukemia cell line K562 was treated with different concentrations of WM. The proliferation of K562 cells was examined by MTT assay. DNA damage in K562 cells was examined by single cell gel electrophoresis assay, and apoptosis of K562 cells was detected by Annexin V-FITC/PI double-staining. The expressions of total Akt, phosphorate-Akt (p-Akt), and NF-κB p65 mRNA and protein were detected by RT-PCR and Western blotting, respectively. Results: WM inhibited the proliferation of K562 cells in a dose- and time-dependent manner, with the IC((50) value of 24 h being 25 nmol/L. WM also induced apoptosis of K562 cells in a dose-dependent manner. DNA damage in K562 cells was demonstrated by appearance of comet tail after treatment with WM, with the rate of DNA tail and the tail length being significantly higher than those in the control group (P<0.01). WM dose-dependently inhibited P-Akt and NF-κB p65, but not the total Akt, mRNA and protein expressions. Conclusion: WM can inhibit proliferation and induce apoptosis of K562 cells in a dose- and time-dependent manner, probably through down-regulation of phosphorate PI3K/Akt signal pathway and NF-κB expression.

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).

Regulation of Notch1 activity by Rab5 in pancreatic cancer BxPC3 cells / 基础医学与临床

Objective To elucidates the regulation of Notch1 activity by Rab5 which controls endosome fusion.Methods Rab5 expression of BxPC3 cells were inhibited by siRNA interference,the changes of Notch ICD protein were measured by Western blot.We use Wortmannin and LY294002 which inhibit the acticity of Phosphatidylinositol(PI) 3-kinases to investigate Notch ICD protein level.Results After inhibiting Rab5 expression or the activity of Phosphatidylinositol(PI) 3-kinases,Notch ICD protein level and the growth of BxPC3 cells decreased.Conclusion In BxPC3 cells,Rab5 and Phosphatidylinositol(PI) 3-kinases are important to regulate Notch1 activity and the activation of Notch is dependent on endocytosis.

Differential roles of phosphatidylinositol 3-kinase in mitogen-activated prote in kinase activation by insulin and epidermal growth factor / 中国药理学通报

Aim: To investigate the roles of phosphatidylinositol 3-kinase (PI3K) in the mitogen-activated protein kinase (MAPK) activation by insulin and epidermal growth factor (EGF). Methods: Phosphorylated MAPK, Akt (also termed protein kinase B) and total protein level of MAPK were determined by Western blotting using phospho- or non-phospho-state specific antibodies; the roles of PI3K in these signaling transduction pathways were assessed by use of PI3K specific inhibitor wortmannin. Results: Both insulin and EGF rapidly stimulated MAPK phosphorylation; wortmannin totally blocked the MAPK phosphorylation stimulated by insulin, but not by EGF. By contrast, wortmannin equally inhibited Akt phosphorylation stimulated by both insulin and EGF. Moreover, wortmannin inhibited insulin-stimulated MAPK phosphorylation in a concentration-dependent manner, and the inability of wortmannin to inhibit EGF-stimulated MAPK phosphorylation was not changed with the use of low concentrations of EGF. Conclusion: PI3K plays different roles in the MAPK activation induced by insulin and EGF.

Effects of Wortmannin combined with mitomycin C on proliferation and apoptosis of HepG-2 cells / 第三军医大学学报

Objective To investigate the effects of Wortmannin combined with mitomycin C (MMC) on the proliferation,apoptosis and expressions of related genes Cyclin D1 and Bad in liver cancer cell line HepG-2. Methods HepG-2 cells (104/ml) were treated with 0,25,50,100,200 or 400 nmol/L Wortmannin,0,1.75,3.75,7.5,15 or 30 nmol/L MMC,or combined concentrations (0,26.875,53.75,107.5,215 or 430 nmol/L) respectively. Cell suppression rate were detected by MTT assay. Nucleus apoptosis of Hep-G-2 cells were examined by Hochest 33258 fluorescent staining. Change of cell cycle and apoptosis in HepG-2 cells were detected by flow cytometry (FCM). Expressions of PI3K/Akt signaling transduction pathway related genes and proteins were tested by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Results The inhibition ratio of cell proliferation was significant higher in combined groups than in either single groups (P

Effect of PI3K inhibitor wortmannin on invasion and migration of human thyroid follicular carcinoma cell line / 解放军医学杂志

Objective To investigate the effect of phosphatidylinositol 3-kinase(PI3K) inhibitor wortmannin on migration and invasion of human thyroid follicular carcinoma cell line CGTHW-1 and its mechanism.Methods MTT colorimetric assay was used to select the optimal concentration and time of giving wortmannin.CGTHW-1 cells used in present study were divided into control group and experimental group(cultured in the medium with or without blood serum).Cells of experimental group were treated with wortmannin,and that of control group were given no treatment.The migration and invasion ability of CGTHW-1 cells was detected by scratch test and Transwell chamber assay respectively.Microfilament fluorescence staining with fluorescein isothiocyanate(FITC)-labeled phalloidin was performed to observe the morphologic changes in CGTHW-1 cells.The expressions of Rac1 protein and mRNA in CGTHW-1 cells were examined by immunocytochemistry staining and semiquantitative RT-PCR.Results When CGTHW-1 cells were cultured in serum medium,the migrating speed of the cells slowed down,and the number of cells passing through the Transwell chamber reduced significantly after being treated with wortmannin in experimental group(13.8?3.03) than in control group(41.6?6.95,P0.05).Conclusions Wortmannin can effectively depress the migration and invasion of human thyroid follicular carcinoma cells,and the depression effect may be due to cytoskeleton rearrangement induced by inhibiting the expression of Rac1(the downstream molecule of PI3K).PI3K and Rac1 are probably potential targets for human thyroid cancer treatment,which may provide a theoretical evidence for the drug treatment of thyroid cancer.

Effects of combination of octreotide and wortmannin on proliferation of gastric carcinoma cell BGC-823 / 中国实用内科杂志

Objective To investigate the effect and mechanism of octreotide with or without wortmannin on the growth of gastric carcinoma cell BGC-823.Methods From June to August of 2007,in Remin Hospital of Wuhan University,cells were exposed to octreotide for four dilution(10-5、10-4、10-3、10-2g/L)with or without wortmannin(a special inhibitor of PI3'K/Akt pathway,the dilution is 40 nmol/L).And then the cytotoxicity was assessed by determining cell survival with MTT after 24 hours,with also were exposed to octreotide for the dilution of 10-3 g/L with or without Wortmannin in 12 h,24 h,36 h,48 h,then the cell survivral rate was accounted and the results were formulated in a table,and the cell cycle was checked-out with Flow Cytometer(FCM);the expression level of p27 gene and protein was determinated with RT-PCR and Western-bloting Test.Results The five groups within one reagent treating were compared with control group,and the results had difference,but the combination of two reagents group had significant difference(P

The effect and mechanism of wortmannin on human gastric carcinoma cells / 中国普通外科杂志

Objective To explore the effect and mechanism of wortmannin on the human gastric carcinoma cells. Methods SGC7901 cells were treated with 10nmol/L,30nmol/L and 60nmol/L of wortmannin,a specific inhibitor of AKT,for different time periods. Cell viability was estimated by MTT assay. Western blot was used to detect the level of NF-?B neucleoprotein and reverse transcriptasepolymerase chain reaction(RT-PCR) was used to determine transcription of NF-?B mRNA. Results All three different concentrations of wortmannin could inhibit the growth of SGC7901 cells,and the depression effect obviously depended on time and drug dose(P0.05). As the action time of wortmannin was prolonged,neucleoprotein and mRNA expression of NF-?B significantly decreased compared with the control (P

Protective effect of wortmannin against pancreas and liver injuries induced by severe acute pancreatitis in rats / 中国普通外科杂志

Objective To observe the protective effect of pretreatment with wortmannin against pancreas and liver injuries indued by severe acute pancreatitis(SAP) in rats and investigate its mechanism.Methods Fifty-four SD rats were randomly divided into 3 groups: control group(C group),SAP group(P group) and SAP+wortmannin group(PW group)(n=18 per group). SAP model was induced by retrograde infusion of 50g/L sodium taurocholate into the biliopancreatic duct of rats,except C group in which sodium taurocholate was replaced by normal saline. Serum level of tumor necrosis factor-alpha(TNF-?)、 alanine aminotransferase(ALT)、aspartate aminotransferase(AST)、and nuclear factor-kappa B(NF-?B)in liver were detected. Histopathology of liver and pancreas was studied.Results In P group, serum levels of TNF-?、ALT、AST and NF-?B in liver were significantly elevated(P

The Effects of Wortmannin on Leukemia Cells Proliferation Cycle and BCL-2 Expression / 中国医师杂志

Objective To investigate the effects of wortmannin (WT) on leukemia cells cycle and BCL-2 protein expression. Methods 0 25?mol/L PI-3 kinase inhibitor wortmanin was used to treat K562 cells for 24 hours, and Arac was simultaneously used to induce the cell apoptosis. The specific fluoresent staining and FCM analysis were adopted to measure the cell cycle distribution and BCL-2 expression. Results Compared with the control cells, the proliferation index(PI) of the K562 cells treated by wortmannin was significantly lower, the cell number of G1 phase and the percentage of cell apoptosis increased, and the BCL-2 protein expression significantly decreased. Conclusion Wortmannin could inhibitedtheproliferationofK5 6 2cells ,andwascellcyclespecificagent (CCSA) .

Differential roles of phosphatidylinositol 3-kinase in mitogen-activated prote in kinase activation by insulin and epidermal growth factor / 中国药理学通报

Aim To investigate the roles of phosphatidylinositol 3 -kinase (PI3K) in the mitogen-activated protein kinase (MAPK) activation by in sulin and epidermal growth factor (EGF). Methods Phosphorylated MAPK, Akt (also termed protein kinase B) and total protein level of MAPK were d etermined by Western blotting using phospho- or non-phospho-state specific an tibodies;the roles of PI3K in these signaling transduction pathways were assess ed by use of PI3K specific inhibitor wortmannin.Results Both in sulin and EGF rapidly stimulated MAPK phosphorylation; wortmannin totally blocke d the MAPK phosphorylation stimulated by insulin, but not by EGF.By contrast, wo rtmannin equally inhibited Akt phosphorylation stimulated by both insulin and EG F. Moreover, wortmannin inhibited insulin-stimulated MAPK phosphorylation in a concentration-dependent manner,and the inability of wortmannin to inhibit EGF -stimulated MAPK phosphorylation was not changed with the use of low concentrat ions of EGF.Conclusion PI3K plays different roles in the MAPK a ctivation induced by insulin and EGF.

The role of the dephosphorylation of FOXO1 in the apoptosis and the chemotherapy sensitivity of Non-Hodgkin’s lymphoma cells / 中国免疫学杂志

Objective:Investigate the relation between the phosphorylation of FOXO1 and the apoptosis and the proliferation of lymphoma cells and to clarify its specific mechanism.Methods:The lymphoma cells Namalwa and Jurkat were treated with PI3K inhibitor wort mannin or etoposide or Wortmannin plus etoposide for different times-pan and at different concentration.The inhibition rates for cell growth of lymphoma cells were examined by XTT assay.Apoptosis were detected by flow cytometry.The expressions of p-Akt,p-FOXO1,FOXO1 and Bim were determined by Western blot analysis.Results:Wortmannin induced apoptosis of Jurkat cells and Namalwa cells and inhibited their survival effectively.The growth inhibition rate and the apoptosis rate of lymphoma cells induced by Wortmannin plus etoposide were higher than those induced by etoposide alone.After treated with Wortmannin,phosphorylation of FOXO1 remarkably reduced and bim markedly increased.Conclusion:The dephosphorylation of FOXO1 inhibits proliferation of Jurkat cells and Namalwa cells,promotes their apoptosis and enhanced the sensitivity of Non-Hodgkin lymphoma cells to etoposide.Bim activated by FOXO1 promotes cell apoptosis.

EFFECTS OF H-89 AND wortmannin ON THE PROMOTING EFFECTS OF forskolin AND IBMX ON THE REGENERATION OF INJURED RETINAL GANGLION CELLS / 解剖学报

Objective The present study was aimed at investigating the regeneration promoting mechanism of forskolin and IBMX on injured retinal ganglion cells(RGCs). Methods Fluorescent retrograde labeling and autologus sciatic nerve transplanting techniques were used to observe the effects of H 89 or/and wortmannin on the regeneration promoting effects of forskolin and IBMX on injured retinal ganglion cells by intravitreal injection. Results Both H 89 and wortmannin could partly block the promoting effects of forskolin and IBMX on regeneration of injured RGCs, the effects of wortmannin might be more powerful, if united, they could inhibit the effects of forskolin and IBMX completely.Conclusion The regeneration promoting effects of forskolin and IBMX on injured RGCs might be realized by PKA CREB and PI3 K Akt accesses.