Notch signaling pathway inhibitor DAPT improves alcohol-induced neuronal differentiation impairment in zebrafish / 南方医科大学学报

OBJECTIVE@#To explore the role of the Notch signaling pathway in regulating neuronal differentiation and sensorimotor ability in a zebrafish model of fetal alcohol spectrum disorder.@*METHODS@#Zebrafish embryos treated with DMSO or 50 μmol/L DAPT (a Notch signaling pathway inhibitor) were examined for mortality rate, hatching rate, malformation rate, and body length at 15 days post fertilization (dpf). The mRNA expression levels of sox2, neurogenin1 and huc in the treated zebrafish embryos were detected using in situ hybridization and qRT-PCR, and their behavioral responses to strong light and vibration stimulation were observed. The zebrafish embryos were then exposed to DMSO, 1.5% ethanol, DAPT, or both ethanol and DAPT, and the changes in mRNA expression levels of sox2, neurogenin1, huc, and the Notch signaling pathway genes as well as behavioral responses were evaluated.@*RESULTS@#Exposure to 50 μmol/L DAPT significantly increased the mortality rate of 1 dpf zebrafish embryos (P < 0.01), decreased the hatching rate of 2 dpf embryos (P < 0.01), increased the malformation rate of 3 dpf embryos (P < 0.001), and reduced the body length of 15 dpf embryos (P < 0.05). DAPT treatment significantly downregulated sox2 mRNA expression (P < 0.01) and increased neurogenin1 (P < 0.05) and huc (P < 0.01) mRNA expressions in zebrafish embryos. The zebrafish with DAPT treatment exhibited significantly shortened movement distance (P < 0.001) and lowered movement speed (P < 0.05) in response to all the stimulation conditions. Compared with treatment with 1.5% ethanol alone, which obviously upregulated notch1a, her8a and NICD mRNA expressions in zebrafish embryos (P < 0.05), the combined treatment with ethanol and DAPT significantly increased neurogenin1 and huc mRNA expression, decreased sox2 mRNA expression (P < 0.01), and increased the moving distance and moving speed of zebrafish embryos in response to strong light stimulation (P < 0.05).@*CONCLUSION@#Ethanol exposure causes upregulation of the Notch signaling pathway and impairs neuronal differentiation and sensorimotor ability of zebrafish embryos, and these detrimental effects can be lessened by inhibiting the Notch signaling pathway.

Positioning of the fixed dose combination of Rosuvastatin + Clopidogrel + Aspirin in the Treatment of Cardiovascular Diseases

Dual anti-platelet therapy (DAPT) and statins are recommended by guidelines for the management of cardiovascular diseases (CVDs), even though the duration of treatment is guided by ischemic and bleeding risk. Clopidogrel and aspirin are the most commonly used DAPT in CVDs. Adding a statin to DAPT is helpful in reducing the thrombosis risk. Fixed-dose combination (FDC) therapy in CVD can help to address the factors of convenience, compliance, control, cost, and complication better than free drug combinations. Therefore, the FDC of rosuvastatin (10 mg or 20 mg) + clopidogrel (75 mg) + aspirin (75 mg) is likely to improve compliance in CVD patients, thereby reducing adverse cardiovascular outcomes and cost of treatment. There is lack of awareness on long term benefits of this FDC in Indian patients.

Short- and long-term follow-up of antithrombotic management patterns in patients hospitalized with acute coronary syndrome: Indian subgroup of EPICOR Asia study

Background Acute coronary syndrome (ACS) is associated with emergency hospitalizations, and there are limited real-world data on clinical outcomes in post-ACS Asian patients. This article presents data on the Indian subgroup from the Long-term Follow-up of Antithrombotic Management Patterns in Acute Coronary Syndrome Patients in Asia (EPICOR-Asia) study. Methods EPICOR included patients with ACS [ST-segment elevation myocardial infarction (STEMI), non–ST-segment elevation myocardial infarction (NSTEMI), or unstable angina (UA)]. The study had two phases: acute phase and follow-up phase. The primary objective was to describe short- and long-term antithrombotic management patterns. Results EPICOR-India enrolled 2468 patients (STEMI-1482; NSTEMI-562; and UA-424). Cardiovascular risk factors were present in 1362 (55.2%) patients. Prehospital care was received by 879 (35.6%) patients, and the median time from the symptom onset to the first medical attention was 3 h (0.08, 100.33). The most common drug regimen prescribed during the acute phase was ≥2 antiplatelet agents + anticoagulants with no glycoprotein IIb/IIIa inhibitors and at discharge were aspirin + clopidogrel. About 78.8% of patients were discharged on dual antiplatelet therapy (DAPT) and 16%, on single antiplatelet therapy (SAPT). At 23 months after discharge, 55.6% were on DAPT, while 16.4% were on SAPT. Postdischarge outcomes at 2 years included death in 165 (6.7%) patients, composite events of death, myocardial infarction (MI), or ischemic stroke in 182 (7.4%) patients, and bleeding events in seven (0.3%) patients. Conclusion This study showed a gap between international recommendations and implementation for managing ACS in Indian patients. Most of the patients prefer to undergo invasive management instead of non-invasive therapy. At the end of the 2-year follow-up, more than half of the population was receiving DAPT, with most patients on receiving a combination of aspirin and clopidogrel. The mortality along with composite events of death, MI, or ischemic stroke was highest for patients with NSTEMI.

Efficacy and safety of dual antiplatelet prolongation therapy after PCI in ACS patients guided by dapt scoring system / 解放军医学杂志

Objective To investigate the efficacy and safety of extended treatment for one year with different doses of clopidogrel plus aspirin after percutaneous coronary intervention (PCI) in patients with acute coronary syndrome (ACS) based on dual antiplatelet therapy (DAPT) scoring system. Methods From April 2016 to December 2016, 225 patients with ACS, one year after successfully undergoing PCI and with ≥2 of DAPT score were enrolled in present study, and randomly divided into 3 groups (75 each): control group, observation group A and observation group B. Patients in control group were given aspirin enteric tablet 100mg qd, in observation group A were given aspirin 100mg qd plus clopidogrel 75mg qd, and in observation group B were given aspirin 100mg qd plus clopidogrel 50mg qd. All patients were followed up for one year, the incidence of cardio-and cerebrovascular events and bleeding events were observed and compared among the 3 groups. At the same time, the ADP-induced platelet aggregation rate and the platelet function (MA) from thromboelastography (TEG) pre-and post-treatment were analyzed. Results During the 12-month observation period, the overall incidence of cardiovascular events showed statistical differences among control group, observation groups A and B (21.3% vs. 5.3% vs. 5.3%, P0.05). In control group, observation groups A and B, the incidences of mild bleeding (BARC1+2) (2.6% vs. 12.0% vs. 3.9%) and total bleeding events (2.6% vs. 13.3% vs. 3.9%) showed statistical differences (P0.05). The platelet aggregation rate and platelet function (MA) in observation groups A and B decreased significantly than that in control group one year after prolonged antiplatelet therapy (P<0.05). Conclusions Assessment of ischemia risk with DAPT scoring system in patients with ACS, prolonged clopidogrel plus aspirin therapy for those patients with DAPT score ≥2 could reduce the incidence of major cardiovascular events, and low dose clopidogrel dose not increase the risk of bleeding complications.

Effects of γ-Secretase Inhibitor DAPT on Inflammation-Induced White Matter Damage in Neonatal Mice / 中国药学杂志

OBJECTIVE: To investigate the effect of γ-secretase inhibitor DAPT in inflammation-induced brain white matter injury in neonatal mice. METHODS: Sixty C57BL/10J neonatal mice are randomly divided into control group, control+DAPT (10 mg•kg-1) group, inflammation (LPS) group, LPS+DAPT group (inflammation exposure after 10 mg•kg-1 DAPT treatment). All neonatal mice were killed and brain was removed to the following observation and detection:at P5, the mRNA expression variation of IL-1β, IL-8,TNF-α,Hes1 and NICD by Real-time PCR methods. Oligodendrocytes were identified by immunofluorescence staining. Myelin basic protein (MBP) protein expression was detected by Western blot assay. RESULTS: LPS group showed brain injury characterized by inhibition of brain development. There were significant differences in mRNA expression of IL-1β, IL-8, TNF-α, Hes1 and NICD between LPS+DAPT group and LPS group (P<0.05), and the mRNA expression of IL-1β, IL-8,TNF-α,Hes1 and NICD in inflammation-treated were significantly increased than control group (P<0.05). The results showed more expression of MBP in LPS+DAPT group compared with LPS group (P<0.05). Compared with the blank control group, which was obviously decreased after 48 h of inflammation (P<0.05).CONCLUSION: Inflammation leads to abnormal of notch signal expression in neonatal mice, and which is shows inflammation involved in brain damage.Its mechanism is probably associated with the maturation of oligodendrocytes.

The Effect of Contusion and Exhaustive Exercise on Satellite Cells Activation and Pax7/CBF1/DAPT Contents in Skeletal Muscles of Rats / 中国运动医学杂志

Objective To study the effect of contusion and exhaustive exercise on satellite cells' activation and Pax7/CBF1/DAPT contents in skeletal muscles of rats,and reveal the repair mechanism of the skeletal muscle injury.Methods Twenty-four seven-week-old male Sprague-Dawley(SD)rats were randomly divided into a control(C)group,an immediately after exhaustive exercise(E0)group,a 24 hours after exhaustive exercise(E24)group and a 48 hours after exhaustive exercise(E48)group,each of 6.Other 18 SD rats were randomly divided into 3 groups,6 rats in each group:an immediately after contusion group(D0),a 24h post-contusion group(D24)and a 48h post-contusion group(D48).All groups were killed at different time points after exhaustive exercise and the contusion respectively while the control group was at resting state,and their serum was extracted.The right gastrocnemius muscles were resected and divided into 2 parts:one was used immediately for culturing satellite cells,while the other was stored in the bridge at-80℃.All the serum and gastrocnemius muscles were tested for Pax7,CBF1 and DAPT contents using the enzyme-linked immunosorbent assay(ELISA).Moreover,three-day-old newborn rats were also slaughtered for the above experiment.Results The results of culture in vitro showed that one day after the culture began a small amount of spindle satellite cells could be seen in the newborn rats,but the satellite cells of other groups were growing slowly.However,on the third day and the fifth day,the spindle satellite cells of each group began to proliferate in quantity and reached the peak.Then all the satellite cells had a good proliferation until the seventh day after serial passage,and some patial fusions to microtubules occurred.The biggest cell number and proliferation rate was in newborn rats,but no skeletal muscle satellite cell was found in the control group.At the same time,the number and the proliferation rate of skeletal muscle satellite cells in contusion groups were significantly higher than the exhaustive exercise groups.The results of Elisa showed that the contents of CBF1 and Pax7 in skeletal muscles in exhaustive groups and contusion groups,were significantly upregulated,compared to the control group(P＜0.05).Amomg them,the CBF1 content in group D0,together with the Pax7 content in group D24 and D48,was significantly higher than that of the other groups(P＜0.05).On the other hand,the content of DAPT in skeletal muscles of exhaustive groups and contusion groups,compared to the control group,was significantly downregulated (P＜0.05).To be more specific,the value of group D0 was significantly lower than that of the other groups (P＜0.05).However,there was no significant difference in it among group E0,E24 and E48.Conclusions Contusion and exhaustive exercise can activate the skeletal muscle satellite cells to proliferate and differentiate.The number of activated skeletal muscle satellite cells in the contusion groups is significantly larger than that of exhaustive groups maybe due to the stronger stimulating by contusion.Both contusion and exhaustive exercise increase the contents of Pax7 and CBF1 in skeletal muscles,but decrease that of DAPT.The Pax7 and CBF1 may play a positive regulating role in terms of activating skeletal satellite cells and repairing skeletal muscle injury,while DAPT may play a negative regulating role.

De-Escalation of P2Y₁₂ Receptor Inhibitor Therapy after Acute Coronary Syndromes in Patients Undergoing Percutaneous Coronary Intervention

Dual antiplatelet therapy (DAPT) — a combination of a P2Y₁₂ receptor inhibitor and aspirin — has revolutionized antithrombotic treatment. Potent P2Y₁₂ inhibitors such as prasugrel and ticagrelor exhibit a strong and more consistent platelet inhibition when compared to clopidogrel. Therefore, ticagrelor and prasugrel significantly reduce ischemic events, but at an expense of an increased bleeding risk in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI). These observations have engaged intensive clinical research in alternative DAPT regimens to achieve sufficient platelet inhibition with an acceptable bleeding risk. Our review focusses on P2Y₁₂ receptor therapy de-escalation defined as a switch from a potent antiplatelet agent (ticagrelor or prasugrel) to clopidogrel. Recently, both unguided (platelet function testing independent) and guided (platelet function testing dependent) DAPT de-escalation strategies have been investigated in different clinical studies and both switching strategies could be possible options to prevent bleeding complications without increasing ischemic risk. In light of the still limited data currently available, future large-scale trials should accumulate more data on various DAPT de-escalation regimens with both ticagrelor and prasugrel in unguided and guided de-escalation approaches. In the current review we aim at summarizing and discussing the current evidence on this still emerging topic in the field of antiplatelet treatment.

Expression of Notch signaling pathway in cultured human osteoarthritic articular chondrocytes / 局解手术学杂志

Objective To explore the role of Notch signaling pathway in the matrix synthesis of osteoarthritis cartilage cell and its possi -ble mechanism.Methods Selected the femoral condylar cartilage of 8 patients who were admitted into our hospital and treated with knee joint replacement as the observation group,while the normal femoral condyle cartilage of one patient with above-knee amputation was selected as the control group.The specimens were given histological examination or cell isolation culture and detection.The cultured cells were divided into 4 groups,namely the normal cartilage cells,OA cartilage cells,OA cartilage cells with γ-secretase inhibitor DAPT,OA cartilage cells with recombinant human proteins Delta4.Then immunohistochemistry and Western blot were used for detecting the expression of Notch -1,Notch-3, Jagged-1,Jagged-2 and HES5 in chondrocytes in vitro.Results The expression of Notch signaling pathway and the phenotype of cartilage cells changed in the osteoarthritis.The expression of Notch-1,Jagged-1,Jagged-2 and HES5 were activated.γ-secretase inhibitor DAPT (20 μmol/L)could inhibit the expression of Notch-1,HES5,Jagged-1,and Notch-3,while it had no obvious effect on the expreesion of Jag-ged-2.Recombinant human proteins Delta 4(100 ng/μL)could promote the expression of Notch-1,Jagged-2,and HES5,and it had no obvi-ous effect on the expreesion of Jagged-1 and Notch-3.Conclusion The expression of Notch signaling pathway of cartilage cells changed in the osteoarthritis.DLL4 can activate the Notch signaling pathway and DAPT can inhibit the Notch signaling pathway.

Uso de terapia antiagregante dual luego de angioplastía coronaria: duración, frecuencia de sangramientos y validación de scores de riesgo de hemorragia en una cohorte chilena / Dual anti platelet therapy post PTCA: duration, bleeding risk and validation of scores for predicting risk of bleeding in a Chilean population

Resumen: Introducción: La terapia antiagregante dual (TAD) con aspirina más clopidogrel o ticagrelor es fundamental para prevenir trombosis de stent y nuevos eventos cardiovasculares (CV) en pacientes sometidos a angioplastía coronaria (AC). Sin embargo, TAD se asocia a un riesgo aumentado de hemorragias, en particular cuando su uso se prolonga. Recientemente se han creado puntajes (DAPT, PRECISE-DAPT) que buscan estimar el riesgo de sangrado en pacientes con TAD por tiempo prolongado, los que quisimos evaluar en nuestra población. Métodos: Se utilizó la base de datos prospectiva de Prevención Cardiovascular del Hospital Clínico U. Católica, seleccionando pacientes sometidos a AC el año 2015. Se realizó una encuesta telefónica estandarizada para identificar episodios de sangrado definidos según clasificación ISTH, tiempo de uso de TAD y nuevos eventos CV. Se calcularon los puntajes DAPT y PRECISE-DAPT. Se usó pruebas de t de Student, test exacto de Fisher y curva ROC, según correspondiese, considerando significativa una p<0,05. Resultados: Se incluyeron 227 pacientes (edad 64,2±12,3 años, 22,5% mujeres), de los cuales el 69,6% eran hipertensos, 28,6% diabéticos, 26,9% fumadores y 5,3% insuficientes renales crónicos. En el 63% de los pacientes la AC fue por síndrome coronario agudo, se implantaron 1,4±0,7 stents/paciente y el 37% de los pacientes recibió sólo stents metálicos. Al momento de la encuesta, el seguimiento fue de 26±3 meses. Se registró un tiempo promedio de duración de TAD de 12,6±7,4 meses, con 99,1% de los pacientes recibiendo aspirina, 93,4% clopidogrel, 6,6% ticagrelor y 9,3% anticoagulantes orales. Hubo 35 (15,4%) nuevos eventos CV (revascularización 14, infarto 12, accidente cerebrovascular 2 y muerte 7) y 31 (13,6%) episodios de sangrados (criterio ISTH). De acuerdo con el criterio TIMI de sangrado se registraron 5 (2,2%) episodios graves, 9 (3,9%) leves y 17 (7,4%) menores. En 10 (4,4%) pacientes se modificó la TAD debido al sangrado. PRECISE-DAPT se asoció de manera significativa a los episodios de sangrado (p<0,01); tener un puntaje de alto riesgo (>25) aumentó más de 3 veces el riesgo de sangrado (OR 3,1 IC 1,4-7,1, p<0,01) y una curva ROC estableció que en la población estudiada el mejor punto de corte fue de 18 puntos (C-statistic 0,69) (Figuras 1A y B). El uso de TACO aumentó el riesgo (OR 3,4 IC 1,2-9,5, p=0,02). Si bien miden distintos parámetros, los puntajes de riesgo DAPT y PRECISE-DAPT se correlacionaron significativamente en nuestra cohorte (p<0,01). Conclusiones: En esta cohorte de la vida real se demuestra que la ocurrencia de sangramientos es un evento frecuente en pacientes con TAD, similar a la tasa de nuevos eventos CV, y por tanto debe ser un factor relevante a considerar al momento de la AC y la selección de la TAD. El puntaje PRECISE-DAPT es una herramienta útil para predecir sangrados, aunque nuestros resultados sugieren que en población chilena los valores de corte pueden ser algo menores que lo previamente publicado .

Abstracts: Background: Dual antiplatelet therapy (DAT) with aspirin plus clopidogrel or ticagrelor is essential for the prevention of stent thrombosis and new cardiovascular events in patients undergoing PCI. However, DAT is associated with an increased risk of bleeding, more so when it is used for prolonged time periods. Scores (DAPT, PRECISE-DAPT) developed to predict bleeding risk were evaluated in this study. Method: The prospective Cardiovascular Prevention database at Catholic University Hospital was used to select patients who underwent PCI followed by DAT during 2015. By phone contact information on bleeding episodes - according to the ISTH classification -, new cardiovascular events and DAT duration were collected. DAPT and PRECISE- DAPT scores were calculated. Student's t test, Fisher exact test and ROC analysis were used. Significance was established at p< 0.05. Results: 277 patients were included (age 64.2±12.3 y-o, 22.5% women). Hypertension was present in 66.9%, diabetes in 28.6%, smoking habit in 26.9% and renal failure in 5.3%. The indication for PCI was acute coronary syndrome in 63%, 1.4±0.7 stents per patient were implanted and 37% of patients received bare metal stents exclusively. Follow-up extended for 26±3 months. DAT was active for 12.6±7.4 months and 9.3% of patients received oral anticoagulant therapy. There were 35 (15.4%) new cardiovascular events (14 revascularizations, 12 myocardial infarctions, 2 CVA and 7 deaths). Conversely, there were 31 (13.6%) bleeding episodes. According to the TIMI classification, bleeding episodes were severe in 2.2%, mild in 3.9% and minor in 7.4%. In 4% of patients DAT was modified due to bleeding. PRECISE-DAPT score was significantly associated to bleeding episodes (p<0.01). A high score (>25) was associated with a 3-fold risk of bleeding (OR 3.1, CI 1.4-7.1 (p<0.01). Through ROC analysis the best PRECISE-DAPT cutting point in this cohort was 18 (C=0.69). The use of oral anticoagulation increased bleeding risk (OR 3.4 CI 1.2 - 9.5, p=0.02). DAPT and PRECISE-DAPT were significantly correlated (p<0.01). Conclusion: Bleeding is a frequent complication of DAT, similar to the risk of new cardiovascular events. PRECISE-DAPT score is useful to estimate the risk of bleeding, although this study suggests that in the studied population the cutting point may be somewhat lower than previously published.

DAPT attenuates ox-LDL-induced human umbilical vein endothelial cell injury / 中国病理生理杂志

AIM:To investigate the effect of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) on the Notch signaling pathway in a model of oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cell (HUVEC) damage.METHODS:HUVECs were divided into control group, ox-LDL group, DAPT group and ox-LDL+DAPT group.The morphological changes of the HUVECs with different treatments were observed under light microscope.The viability of the HUVECs was measured by CCK-8 assay.The protein expression levels of Notch1, Notch4 and Jagged1 were determined by Western blot.RESULTS:ox-LDL induced great damage to the HUVECs, evidenced by increased cell death and debris in the culture.However, the cell damage was abolished by adding DAPT into the culture.The viability of the HUVECs was increased by co-treatment with DAPT and ox-LDL.ox-LDL treatment significantly decreased the protein expression levels of Notch1 and Jagged1, and elevated Notch4.However, these changes were totally reversed by DAPT.None of these proteins showed significant change in the HUVECs co-treated with DAPT and ox-LDL as compared with control group.CONCLUSION:ox-LDL is able to induce HUVEC damage in vitro.DAPT attenuates ox-LDL-induced damage in the HUVECs by regulating the Notch signaling pathway.

DAPT regulates Treg/Th17 cells immune balance to inhibit atherosclerosis / 中国免疫学杂志

Objective:To observe the effect of Notch signal inhibitor DAPT (γ-secretase inhibitor ) on the pathological changes of atherosclerosis mice and the immune balance of Treg/Th17.Methods:24 ApoE knockout C57BL mice were randomly divided into blank group,model group and DAPT group.The blank group were fed with normal diet ,the model group and the experimental group were fed with high fat diet.After 5 weeks of feeding,the mice in the experimental group were injected with DAPT [100 mg/(kg· d),re-suspended in DMSO],and the other two groups were injected with the equivalent amount of DMSO .After another 5 weeks,pathological changes of the mice in each group were analyzed by HE staining .ELISA was used to detect the level of IL-17 in plasma,and the propor-tion of splenic Treg/Th17 cells in each group was detected by flow cytometry .Results:HE staining results showed that the model group had obvious plaque formation and foam cell formation ,which showed that the AS model was successfully prepared .The degree of arterial disease in the DAPT group was significantly less than that in the model group .The plasma levels of IL-17 in the blank group , model group and DAPT group were(293.94±28.59),(454.05±172.68) and (335.40±89.57) pg/ml,respectively .The percentages of Treg cells in the blank group,model group and DAPT group were(3.80±0.56)%,(2.54±0.38)%and(4.73±0.64)%,respective-ly.The Th17 cell subsets of mice in the blank group , model group and DAPT group were ( 3.46 ±0.23 )%, ( 4.52 ±0.85 )% and (1.38±0.37)%,respectively .Conclusion:DAPT decreased the plasma level of IL-17 in AS mice,inhibited the differentiation of Th17 cell subsets,and promoted the differentiation of Treg ,and reduced the atherosclerosis by changing the Treg/Th17 cells immune balance.

Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

Objective To investigate the influence of Notch signaling on osteoprotegerin (OPG) expression in a human oral squamous cell carcinoma cell line. Methods Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a γ-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG mRNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software. Results Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 mRNA expression, confirming the intracellular activation of Notch signaling. OPG mRNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a γ-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 mRNA levels, and a marked increase in OPG protein expression was observed. These results implied that Notch signaling regulated OPG expression in HSC-4 cells. However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line (HSC-5) or a human head and neck squamous cell carcinoma cell line (HN22). Conclusions Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

Caracterização da inter-relação entre as vias de sinalização Notch e TLR na paracoccidioidomicose experimental / Characterization of the inter-relationship between Notch and TLR signaling pathways in experimental paracoccidioidomycosis

A paracoccidioidomicose é uma micose sistêmica de natureza profunda que afeta preferencialmente o tecido pulmonar podendo disseminar via linfo-hematogênica para outros órgãos e tecidos, sendo causada principalmente pelo Paracoccidioides brasiliensis, fungo que apresenta dimorfismo térmico. O sistema imune inato mediado por macrófagos é extremamente importante para o controle de infecções e está envolvido na indução e regulação da resposta imune/inflamatória. Estas células são capazes de reconhecer patógenos por meio de receptores de reconhecimento de padrões (PRRs), tais como receptores Toll-like (TLR). Além desses PRRs, recentemente, demonstrou-se a importância da via de sinalização Notch no sistema imune inato e na regulação da atividade dos macrófagos. Nossos dados demonstram que a cepa Pb18 do P. brasiliensis é capaz de ativar o receptor Notch1 em macrófagos J774. A ativação desse receptor concomitante com a ativação de TLR 4 (via LPS) induz a produção de IL-6, e apresenta elevada carga fúngica e menor fagocitose, o que favorece a patogenia. Ao utilizarmos um inibidor farmacológico da γ-secretase (DAPT) para inibir a ativação do receptor Notch1 em macrófagos, é possível observar diminuição da carga fúngica, diminuição de IL-6, aumento de TNF-α e aumento da fagocitose. Entretanto, a ausência do receptor TLR 4 em macrófagos derivados de medula óssea de camundongos TLR 4-/-, na presença de DAPT, percebe-se diminuição da capacidade fagocítica desses macrófagos e também diminuição da carga fúngica, evidenciando a relação entre TLR 4 e Notch1. Em adição, realizamos um tratamento em camundongos BALB/c com DAPT previamente à infecção com Pb18. Nossos resultados evidenciaram que animais com este tratamento apresentaram diminuição da carga fúngica dos pulmões, diminuição de IL-6, ativação de macrófagos e aumento de IgG, após 45 dias de infecção, indicando um perfil de cura desses animais. O mesmo tratamento foi realizado em camundongos BALB/c NUDE, seguido da infecção com Pb18. Nestes animais, verificamos que há maior produção de citocinas pró-inflamatórias no pulmão, aumento de células CD19+ e a carga fúngica dos animais tratados manteve-se similar ao dos animais não tratados, indicando que o perfil protetor observado em animais com DAPT é dependente da resposta das células T. Juntos, esses resultados evidenciam que o Pb18 é capaz de ativar o receptor Notch1 em macrófagos e utiliza a via de sinalização Notch-TLR 4 como um possível mecanismo de escape, podendo fornecer uma nova abordagem de estudo da imunidade envolvida na paracoccidioidomicose experimental

Paracoccidioidomycosis is a systemic mycosis of deep nature that primarily affects the lung and can spread via lymphatic and hematogenous to other organs and tissues. It is mainly caused by Paracoccidioides brasiliensis fungus which exhibit thermal dimorphism. The innate immune system mediated by macrophages is extremely important for the control of infection and is involved in the induction and regulation of immune/inflammatory response. These cells are able to recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR). Beyond these PRRs, the importance of Notch signaling has recently been demonstrated in the innate immune system and the regulation of macrophage activity. Our data demonstrate that the Pb18 strain of P. brasiliensis is able to activate the Notch1 receptor in J774 macrophages. Activation of this receptor with also activation of TLR 4 (via LPS) induces IL-6 production, induces phagocytosis and decreases fungal burden, which favors the pathogenesis. By using a γ-secretase pharmacological inhibitor (DAPT) for inhibiting the activation of Notch1 receptor on macrophages, it is possible to observe decreased fungal burden, less production of IL-6, and increased TNF-α and phagocytosis. However, due to the absence of TLR 4 receptor in bone marrow derived macrophages from TLR 4-/- mice, these macrophages showed decreased phagocytic ability and also reduced fungal burden in the presence of DAPT, showing a relationship between TLR 4 and Notch1. In addition, we made a treatment with DAPT in BALB/c mice prior to infection with Pb18. And our results showed that DAPT-treated animals exhibited a decrease of fungal burden in the lungs, and a decrease of IL-6. Furthermore, we observed an increase of IgG after 45 days of infection, indicating probably a healing of these animals. Same treatment was made in BALB/c NUDE mice, followed by infection with Pb18. In these animals, we observed an increased production of proinflammatory cytokines in the lung and increased CD19+ cells, but fungal burden was similar in both group (treated and untreated), which indicates that treatment with DAPT is dependent on T cell response. Taken together, these results showed that Pb18 is able to activate the Notch 1 receptor on macrophages and uses the Notch-TLR 4 signaling pathway as a possible escape mechanism, and may provide a new immunity study approach in experimental paracoccidioidomycosis

Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

Objective: To investigate the influence of Notch signaling on osteoprotegerin (OPG) expression in a human oral squamous cell carcinoma cell line. Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a γ-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG mRNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software. Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 mRNA expres-sion, confirming the intracellular activation of Notch signaling. OPG mRNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a γ-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 mRNA levels, and a marked increase in OPG protein expression was observed. These results implied that Notch signaling regulated OPG expression in HSC-4 cells. However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line (HSC-5) or a human head and neck squamous cell carcinoma cell line (HN22). Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

γ-Secretase inhibitor DAPT reduces aristolochic acid-induced phenotypic transformation by inhibiting Notch pathway in renal tubular epithelial cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect ofγ-secretase inhibitor N-[N-(3,5-difluorophen?acetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)on phenotypic transformation and matrix accu?mulation induced by aristolochic acid(AA) in renal tubular epithelial cells(NRK-52E)and explore the mechanism. METHODS NRK-52E cells were divided stochastically into normal cell control group,AA 10 mg·L-1 group and AA 10 mg·L-1+DAPT 1 and 10μmol·L-1 group. After 24 h,the mRNA expressions of Notch1,Jagged1,Numb,E-cadherin,transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),bone morphogenic protein 7 (Bmp7),typeⅠ a1 (Col1a1) and Ⅲ collagens a1 (Col3a1)were quantified by quantitative real-time RT-PCR. The protein expressions of Notch1,Jagged1,α-SMA,and Col3a1 in NRK-52E cel s were detected by immunofluorescence staining. RESULTS In NRK-52E cells,AA enhanced the expression of TGF-β1,α-SMA and Col3a1 mRNA(P<0.05),reduced the expression of E-cadherin mRNA(P<0.05),up-regulated the mRNA expression of Notch1 mRNA(P<0.01)and Jagged1(P<0.05),and down-regulated the mRNA expression of Numb mRNA(P<0.05) compared with normal cell control group,indicating that phenotypic transformation and matrix accumu?lation occurred in AA-treated NRK-52E cells,accompanied by activated Notch signaling. Treatment with DAPT inhibited Notch signaling by decreasing the expression of Notch1 and Jagged1 (P<0.05),and increasing the expression of Numb mRNA(P<0.05). Furthermore, DAPT also down-regulated the expression levels of TGF-β1,α-SMA,Col1a1 and Col3a1 mRNA(P<0.05), and up-regulated the expression level of Bmp7 and E-cadherin mRNA(P<0.05) compared with AA group,suggesting that DAPT inhibited phenotypic transformation and matrix accumulation in AA-treated NRK-52E cells. CONCLUSION AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells,which is inhibited by DAPT treatment. The possible mechanism is that DAPT suppresses the activation of Notch signaling,resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.

DAPT suppresses the proliferation of human glioma cell line SHG-44

OBJECTIVE@#To explore the suppressing effect of γ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.@*METHODS@#The SHG-44 cell was treated by DAPT with different concentration. The proliferation of cells was detected by MTT assay; cell cycle and TSC of CD133(+) were determined by flow cytometry analysis technique; the key factor in Notch signaling pathway (Notch-1, Delta-1, Hes-1) was measured by reverse transcriptase-polymerase chain reaction and western blotting.@*RESULTS@#DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05). And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner. DAPT increased the rate of cells in G0/G1 phase of SHG-44 cells, while it decreased the rate of cells in S phase. TSC of CD133(+) was significantly reduced after DAPT treated SHG-44 cells. The expression of protein and mRNA of Notch-1, Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.@*CONCLUSIONS@#DAPT can downregulate these key factor in Notch signaling pathway, reduce the TSC of CD133+ and inhibit the proliferation of SHG-44 cells.