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ObjectiveTo explore the effect of Buzhong Yiqitang on the immune imbalance of helper T cell 17 （Th17）/regulatory T cell （Treg） and Notch1 signaling pathway in mice with autoimmune thyroiditis （AIT）. MethodA total of 60 8-week-old NOD.H-2h4 mice were randomly divided into the normal group， model group， western medicine group （selenium yeast tablet， 32.5 mg·kg-1）， and low-dose （4.78 g·kg-1·d-1）， middle-dose （9.56 g·kg-1·d-1）， and high-dose （19 g·kg-1·d-1） Buzhong Yiqitang groups， with 10 mice in each group. The normal group was fed with distilled water， and the other groups were fed with water containing 0.05% sodium iodide for eight weeks. After the animal model of AIT was formed spontaneously， the mice were killed under anesthesia after intragastric administration for eight weeks. Serum anti-thyroglobulin antibodies （TGAb）， thyroid-stimulating hormone （TSH）， free triiodothyronine （FT3）， and free thyroid hormone （FT4） were detected by enzyme-linked immunosorbent assay （ELISA）， and thyroid tissue changes were observed by hematoxylin-eosin （HE） staining. The mRNA and protein expressions of retinoid-related orphan receptor-γt （RORγt）， interleukin （IL）-17， forkhead box P3 （FoxP3）， IL-10， Notch1， and hair division-related enhancer 1 （Hes1） in thyroid tissue were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the normal group， the thyroid structure of the model group was severely damaged， and lymphocytes were infiltrated obviously. The levels of serum TGAb， FT3， and FT4 contents were significantly increased， and TSH content was significantly decreased （P<0.01）. The mRNA and protein expression levels of RORγt， IL-17， Notch1， and Hes1 were significantly increased， while those of FoxP3 and IL10 were significantly decreased in the model group （P<0.01）. Compared with the model group， thyroid structural damage and lymphocyte infiltration were improved in the treatment groups， and serum TGAb， FT3， and FT4 contents were significantly decreased. TSH content was increased， and mRNA and protein expression levels of RORγt， IL-17， Notch1， and Hes1 were decreased. mRNA and protein expression levels of FoxP3 and IL-10 were increased to different degrees （P<0.05， P<0.01）， and the middle-dose Buzhong Yiqitang group had the most significant intervention effect. ConclusionBuzhong Yiqitang can alleviate the thyroid structural damage in AIT mice， and its mechanism may be related to improving the abnormal differentiation of Th17/Treg immune cells and inhibiting the activation of the Notch1 signaling pathway.
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ObjectiveTo observe the effect of modified Erchentang on the expression of key molecules in the Jagged1/Notch1/Hes1 signaling pathway in lung tissues of rats with chronic obstructive pulmonary disease （COPD） and explore its anti-inflammatory effect and molecular mechanism on COPD through the Jagged1/Notch1/Hes1 signaling pathway. MethodSixty SD rats were randomly divided into normal group， model group， low-， medium-， and high-dose modified Erchentang groups （5， 10， 20 g·kg-1）， and γ-secretase inhibitor DAPT group （0.02 g·kg-1）， with 10 rats in each group. The COPD model was induced in rats by cigarette smoking combined with intratracheal instillation of lipopolysaccharide （LPS）. Rats were treated with corresponding drugs by gavage， while those in the normal group and the model group were treated with the same amount of normal saline by gavage. The serum levels of Notch1， soluble intercellular adhesion molecule-1 （sICAM-1）， activated leukocyte cell adhesion molecule （ALCAM）， and soluble vascular adhesion molecule-1 （sVCAM-1） were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA expression of Jagged1， Notch1， and Hes1 was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression of Jagged1， Notch1， Notch1 intracellular domain （NICD1）， and Hes1 in lung tissues of rats was detected by immunohistochemistry （IHC）. ResultCompared with the normal group， the model group showed increased serum content of Notch1， sICAM-1， ALCAM， and sVCAM-1 （P<0.01）， increased mRNA expression of Jagged1， Notch1， and Hes1 in lung tissues （P<0.01）， and increased protein expression of Jagged1， Notch1， NICD1， and Hes1 （P<0.01）. Compared with the model group， the medium- and high-dose modified Erchentang groups and the DAPT group showed decreased serum content of Notch1， sICAM-1， ALCAM， and sVCAM-1 （P<0.05， P<0.05）， down-regulated mRNA expression of Jagged1， Notch1， and Hes1 （P<0.05， P<0.01）， and reduced protein expression of Jagged1， Notch1， NICD1， and Hes1（P<0.05， P<0.01）. ConclusionModified Erchentang may inhibit the inflammatory response in the lung of COPD rats， and its mechanism may be related to the resistance of inflammatory injury in the lung by decreasing the mRNA expression of Jagged1， Notch1， and Hes1 and inhibiting the release of Notch1， sICAM-1， ALCAM， and sVCAM-1.
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OBJECTIVE@#To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification.@*METHODS@#Primary annulus fibroblasts from SD rats were isolated and subcultured in vitro. The calcification-inducing factors bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (b-FGF) were added to separate groups to induce calcification, which were referred to as the BMP-2 group and the b-FGF group, respectively. A control group was also set up, which was cultured in normal medium. Subsequently, cell morphology and fluorescence identification, alizarin red staining, ELISA, and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to determine the effect of calcification induction. Cell grouping was performed again, including the control group, the calcification group (adding the inducer BMP-2), the calcification + LPS group(adding the inducer BMP-2 and the Notch1 pathway activator LPS), and the calcification + DAPT group (adding the inducer BMP-2 and the Notch1 pathway inhibitor DAPT). Alizarin red staining and flow cytometry were used to detect cell apoptosis, ELISA was used to detect the content of osteogenic factors, and Western blot was used to detect the expression of BMP-2, b-FGF, and Notch1 proteins.@*RESULTS@#The induction factor screening results showed that the number of mineralized nodules in fibroannulus cells in BMP-2 group and b-FGF group was significantly increased, and the increase was greater in the BMP-2 group Meanwhile, ELISA and Western blot results showed that BMP-2, b-FGF and mRNA expression levels of BMP-2, b-FGF and Notch1 in the induced group were significantly increased (P<0.01). The results of the mechanism of Notch1 signaling pathway affecting lumbar disc calcification showed that compared to calcified group, the number of fibroannulus cell mineralization nodules, apoptosis rate, BMP-2, b-FGF content, the expression levels of BMP-2, b-FGF, and Notch1 proteins were further increased significantly However, the number of mineralization nodules, apoptosis rate, BMP-2 and b-FGF levels, BMP-2, b-FGF and Notch1 protein expression levels were decreased in the calcified +DAPT group (P<0.05 or P<0.01).@*CONCLUSION@#Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.
Subject(s)
Animals , Rats , Bone Morphogenetic Protein 2/metabolism , Calcinosis , Cell Differentiation , Cells, Cultured , Lipopolysaccharides , Osteogenesis , Rats, Sprague-Dawley , Receptor, Notch1/genetics , Signal TransductionABSTRACT
Objective:To investigate the inhibitory effect and underlying mechanism of gamma-secretase inhibitor blocking Notch1 signaling on retinal neovascularization caused by oxygen-induced retinopathy (OIR) in mice.Methods:To establish the OIR model, 7-day-old pups of C57BL/6J mice were exposed to 75% oxygen together with their mother until postnatal day (P)12.On P12, the mice were transferred to room air.All the mice were randomly divided into three groups, OIR group as control group, OIR+ DAPT group and OIR+ DMSO group receiving 1 μl intravitreal injection of gamma-secretase inhibitor (DAPT, 10 mmol/L) and 1∶20 DMSO dilution respectively.The right eye was taken as experimental eye.The mice were euthanized on P17 and the eyes were harvested to obtain retinas for further investigation.The total proteins were extracted from the retinas.The relative expression levels of Notch1 signal pathway and its downstream Hes1, the markers of M1 phenotype inducible nitric oxide synthase (iNOS) and M2 phenotype arginase-1 (Arg-1) microglia were measured by western blot.Retinal flat mounts were made and the retinal vessels were stained with isolectin B4 (IB4) to investigate the relative retinal neovascularization areas which was calculated as the ratio of neovascularization area/retinal area.The mumber of the neovascular endothelium cells beyond the inner limiting membrane was observed by hematoxylin-eosin staining.The use and care of animals complied with ARVO statement.This study protocol was approved by the Animal Ethics Committee of Guangdong Provincial People's Hospital (No.KY-Z-2021-2015-01).Results:The relative protein expression levels of Notch1 and Hes1 in OIR+ DAPT group, OIR group, and OIR+ DMSO group were 0.68±0.06 and 0.70±0.08, 1.00±0.00 and 1.00±0.00, 1.03±0.08 and 1.02±0.07, respectively, with statistically significant differences among them ( F=70.62, 53.65; both at P<0.01). Compared with the OIR group and OIR+ DMSO group, the expressions of Notch1 and Hes1 were significantly reduced in OIR+ DAPT group (all at P<0.01). The relative protein expression levels of iNOS and Arg-1 in OIR+ DAPT group, OIR group, and OIR+ DMSO group were 0.74±0.07 and 1.49±0.12, 1.00±0.00 and 1.00±0.00, 1.04±0.10 and 0.94±0.07, respectively, showing statistically significant differences ( F=31.63, 89.32; both at P<0.01). Compared with OIR group and OIR+ DMSO group, the expression of iNOS in OIR+ DAPT group was significantly reduced, and the expression of Arg-1 was significantly increased (all at P<0.01). The relative neovascularization area and the number of neovascular endothelium cells beyond the inner limiting membrane in OIR+ DAPT group, OIR group, and OIR+ DMSO group were (8.82±2.71)% and 38.17±3.29, (22.32±5.34)% and 60.83±5.11, (20.27±3.36)% and 58.67±4.75, respectively, showing statistically significant differences ( F=33.72, 39.44; both at P<0.01). The relative neovascularization area and the number of neovascular endothelium cells in OIR+ DAPT group were significantly reduced in comparison with OIR group and OIR+ DMSO group (all at P<0.01). Conclusions:Intravitreal injection of DAPT can inhibit the retinal neovascularization in OIR mice through blocking Notch1 signaling activation and promoting retinal microglia polarization from M1 to M2 phenotype.
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Objective To investigate the effect of compound Fufangteng mixture-containing serum on the proliferation of bone marrow mesenchymal stem cell (BMSC) and its mechanism. Methods Rat BMSC were isolated, cultured and purified in vitro by direct adherence method. Cell morphology was observed. Surface markers were identified by flow cytometry. The rats were treated with compound Fufangteng mixture at a dose of 3 mL/(kg·d) by gavage for 14 d, and then the drug-containing serum was collected. BMSC were divided into the blank control group, drug-containing serum group, Notch1 small interfering ribonucleic acid (siRNA) group and Notch1 siRNA+drug-containing serum group. The proliferation rate of BMSC was detected and the relative expression levels of Notch1 signaling pathway-associated messenger ribonucleic acid (mRNA) and proteins were measured in each group. Results Microscopic observation showed that the first generation BMSC were seen in the long spindle shape, and grown in the parallel or spiral pattern. The third generation BMSC positively expressed CD90 and CD44, whereas were negative for CD45. Compared with the blank control group, the proliferation rate of BMSC in the drug-containing serum group and Notch1 siRNA+ drug-containing serum group was significantly increased, whereas that of BMSC was significantly decreased in the Notch1 siRNA group (all P < 0.05). Compared with the Notch1 siRNA group, the proliferation rate of BMSC was significantly increased in the Notch1 siRNA+drug-containing serum group (P < 0.05). Compared with the blank control group, the relative expression levels of Hey1 and Delta-like ligand (DLL)1 mRNA and proteins were significantly up-regulated in the drug-containing serum group, whereas those were significantly down-regulated in the Notch1 siRNA group and Notch1 siRNA+drug-containing serum group (all P < 0.05). Compared with the Notch1 siRNA group, the relative expression levels of Hey1 and DLL1 mRNA and proteins were significantly up-regulated in the Notch1 siRNA+drug-containing serum group (all P < 0.05). Conclusions Compound Fufangteng mixture-containing serum may promote the proliferation of rat BMSC, and its mechanism is probably associated with the activation of Notch1 signaling pathway.
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Objective:To investigate the effect and mechanism of NACK knockdown on the proliferation and apoptosis of T-cell acute lymphoblastic leukemia (T-ALL) Jurkat cells. Methods:Lentivirus transfection technology was used to transfect Jurkat cells and knock down NACK gene.Real time fluorescent quantitative PCR and Western blot were used to detect the silencing efficiency of NACK gene.CCK-8 method and flow cytometry were used to detect the effects of NACK knockdown on the proliferation and apoptosis of Jurkat cells.The expressions of protein related with Notch1 pathway, such as Hes1 and c-Myc, were detected by Western blot. Results:After NACK-shRNA was successfully transfected into Jurkat cells by lentiviral vector, the expression of NACK mRNA and protein was reduced signi-ficantly ( P<0.05). Compared with the negative control group and the blank control group, the CCK-8 method showed that the cell proliferation in the experimental group was significantly inhibited [The inhibition rates of cell proliferation in the experimental group, negative control group and blank control group were (37.27±4.48)%, (4.25±2.10)% and (2.43±1.40)%, respectively]( F=132.640, P<0.05), and the flow cytometry test showed that the apoptosis in the experimental group increased significantly [The apoptosis rates of experimental group, negative control group and blank control group were (26.38±3.03)%, (6.07±2.61)% and (3.40±1.98)%, respectively]( F=90.534, P<0.05). Western blot results confirmed that the expression of Notch1 pathway-related proteins Hes1 and c-Myc was down-regulated compared with the negative control group and the blank control group, and the difference was statistically significant ( P<0.05). Conclusions:Targeting silent NACK can down-regulate the expression of Notch1 pathway-related proteins, which leads to the inhibition of Jurkat cell proliferation and increased apoptosis, thereby exerting its anti-T-ALL effect.
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Adult olfactory neurogenesis plays critical roles in maintaining olfactory functions. Newly-generated neurons in the subventricular zone migrate to the olfactory bulb (OB) and determine olfactory discrimination, but the mechanisms underlying the regulation of olfactory neurogenesis remain unclear. Our previous study indicated the potential of APPL2 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 2) as a modulating factor for neurogenesis in the adult olfactory system. In the present study, we report how APPL2 affects neurogenesis in the OB and thereby mediates olfactory discrimination by using both in vitro neural stem cells (NSCs) and an in vivo animal model-APPL2 transgenic (Tg) mice. In the in vitro study, we found that APPL2 overexpression resulted in NSCs switching from neuronal differentiation to gliogenesis while APPL2 knockdown promoted neurogenesis. In the in vivo study, APPL2 Tg mice had a higher population of glial cells and dampened neuronal production in the olfactory system, including the corpus callosum, OB, and rostral migratory stream. Adult APPL2 Tg mice displayed impaired performance in olfactory discrimination tests compared with wild-type mice. Furthermore, we found that an interaction of APPL2 with Notch1 contributed to the roles of APPL2 in modulating the neurogenic lineage-switching and olfactory behaviors. In conclusion, APPL2 controls olfactory discrimination by switching the fate choice of NSCs via interaction with Notch1 signaling.
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Adult olfactory neurogenesis plays critical roles in maintaining olfactory functions. Newly-generated neurons in the subventricular zone migrate to the olfactory bulb (OB) and determine olfactory discrimination, but the mechanisms underlying the regulation of olfactory neurogenesis remain unclear. Our previous study indicated the potential of APPL2 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 2) as a modulating factor for neurogenesis in the adult olfactory system. In the present study, we report how APPL2 affects neurogenesis in the OB and thereby mediates olfactory discrimination by using both in vitro neural stem cells (NSCs) and an in vivo animal model-APPL2 transgenic (Tg) mice. In the in vitro study, we found that APPL2 overexpression resulted in NSCs switching from neuronal differentiation to gliogenesis while APPL2 knockdown promoted neurogenesis. In the in vivo study, APPL2 Tg mice had a higher population of glial cells and dampened neuronal production in the olfactory system, including the corpus callosum, OB, and rostral migratory stream. Adult APPL2 Tg mice displayed impaired performance in olfactory discrimination tests compared with wild-type mice. Furthermore, we found that an interaction of APPL2 with Notch1 contributed to the roles of APPL2 in modulating the neurogenic lineage-switching and olfactory behaviors. In conclusion, APPL2 controls olfactory discrimination by switching the fate choice of NSCs via interaction with Notch1 signaling.
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Objective: To investigate the mechanism of Nocth1 in regulating epithelial-mesenchymal transition (EMT) in breast cancer. Methods: Jagged1 or shRNA was applied to regulate Notch signaling expression. EMT process and migration and invasion in breast cancer were determined. Moreover, Slug luciferase reporter assay was used to investigate the effect of Notch1 on Slug expression. And Slug expression was regulated by shRNA. The effects of Slug on Notch1 mediated breast cancer EMT process and migration and invasion were determined. Results: Jagged1 activated Notch signaling and promoted EMT process and migration and invasion in breast cancer. Overexpression of Notch1 could induce Slug promoter activation, and Slug overexpression could reverse the inhibition of Notch1 shRNA on breast cancer's EMT process and migration and invasion. Conclusion: Notch1 regulates EMT process, migration and invasion in breast cancer through targeting the Slug promoter.
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OBJECTIVE@#To investigate the effects and mechanisms of irbesartan on myocardial injury in diabetic rats, and to analyze the changes of Notch1 signaling pathway in it.@*METHODS@#Thirty rats were randomly divided into four groups:normal control group (CON, =6), high calorie group (HC, =6) and diabetes mellitus group (DM, =9), irbesartan + diabetes group (Ir + DM, =9). After modeling 8 weeks later, the body weight ratio and left ventricular weight index were measured and the serum levels of triglyceride (TG) and total cholesterol (TC) were measured by automatic biochemical analyzer. The changes of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in myocardium of rats were determined by the kit and the expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2 assaciated X protein (Bax) protein in myocardium were detected by immunohistochemistry. The expressions of Notch1, Hes-1 and jagged-1 in myocardium of rats were detected by Western blot.@*RESULTS@#Compared with CON group, the levels of heart weight/body weight (H/B), left ventricular weight index(LVWI) and fasting blood glucose(FBG) in HC group were not significantly changed, while the levels of blood lipids, MDA and Bax were increased significantly, and the expressions of SOD, Bcl-2 and Notch1, Hes-1 and Jagged-1 were decreased. Compared with HC group, the levels of H/B, LVWI, FBG, MDA and Bax in DM group were increased significantly, and the levels of SOD, Bcl-2 and Notch1, Hes-1 and Jagged-1 were decreased. The expression of H/B, LVWI, Notch1, Hes-1 and Jagged-1 in Ir+DM group were increased, but there was no significant difference between the other indexes. The H/B and LVWI in Ir + DM group were significantly lower than those in DM group, the levels of blood lipid and blood glucose did not change significantly, but the incidence of oxidative stress and apoptosis was reduced. While Notch1, Hes-1, Jagged -1 protein expressions were increased.@*CONCLUSIONS@#Diabetes can induce myocardial injury, and irbesartan has myocardial protective effects through activation of Notch1.
Subject(s)
Animals , Rats , Diabetes Mellitus, Experimental , Irbesartan , Myocardium , Rats, Sprague-Dawley , Receptor, Notch1 , Signal TransductionABSTRACT
Objective: To investigate the reversal effect of curcumin (Cur) on the resistance of human esophageal cancer Eca-1 09/vincristine (VCR) cells to VCR, and to explore its possible mechanism. Methods: MTT method was used to detect the effect of different concentrations of Cur on the proliferation of Eca-1 09/VCR cells and the reversal effect of Cur (25 μmol/L) on the resistance of Eca-109/VCR cells to VCR. The Eca-109/VCR cells were treated with Cur (25 μmol/L) alone, VCR (2 μg/mL) alone or Cur (25 μmol/L) combined with VCR (2 μg/mL) for 24 h, respectively. Then the apoptosis was analyzed by FCM and Hoechst 33258 fluorescence staining. The expression levels of Notch1, Jagged1, and hairy and enhancer of split 1 (Hes1) mRNAs were detected by real-time fuorescent quantitative PCR. The expressions of Notch1, Jagged1, Hes1 and P-glycoprotein (P-gp) proteins were measured by Western blotting. Results: The proliferation inhibitory rate of Eca-1 09/VCR cells was (8.82±0.80) % after the treatment with 25 μmol/L Cur. After the treatment with Cur (25 μmol/L) and various concentrations of VCR for 24 h, the half maximal inhibitory concentration (IC50) of VCR on Eca-1 09/VCR cells is decreased from (7.70±0.63) μg/mL to (2.55± 0.12) μg/mL. The reversal fold of Cur was 3.02. After the treatment with Cur (25 μmol/L) and VCR (2 μg/mL) for 24 h, the apoptosis rate of Eca-1 09/VCR cells was significantly higher than those in Cur (25 μmol/L) group and VCR (2 μg/mL) group (both P < 0.05), the expression levels of Notch1, Jagged1 and Hes1 mRNAs were significantly lower than those in Cur (25 μmol/L) group and VCR (2 μg/mL) group (all P < 0.01), and the expressions of Notch1, Jagged1, Hes1 and P-gp proteins were significantly down-regulated in Eca-1 09/VCR cells (all P < 0.05). Conclusion: Cur can obviously reverse the resistance of esophageal cancer Eca-109/VCR cells to VCR. The mechanism may be related to the inhibition of Notch1 signaling pathway and the down-regulation of P-gp expression.
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Objective:To investigate the effect of inhibiting of HOXB7 gene expression on proliferation and apoptosis of colon cancer cells.Methods:The synthetic negative control siRNA (negative control group) and HOXB7-siRNA (HOXB7 transfection group) were transfected into human colon cancer SW480 cells by LipofectamineTM2000 liposome mediated method,untreated cells as blank group.RT-PCR and Western blot were used to detect the mRNA and protein expression of HOXB7 after transfected 48 h respectively;cell proliferation was detected by CCK8 assay after transfected 24,48,72,96 h;cells apoptosis was detected by flow cytometry after transfected 48 h;the expression of apoptosis related proteins Bcl-2,Bax and Notch1 signaling pathway Notch1 and Hes1 were detected by Western blot.Results:The mRNA and protein expression of HOXB7 in HOXB7 transfected group was significantly lower than that in blank group(P<0.05);OD value was no statistical significance in the three groups after transfected 24 h(P>0.05), while after transfected 48,72,96 h,compared with the control group,OD value in HOXB7 group was significantly decreased(P<0.05);compared with the blank group,the apoptosis rate in HOXB7 transfection group increased significantly,and the expression of Bcl-2, Notch1 and Hes 1 proteins was down regulated,and the expression of Bax protein was up-regulated(P<0.05).Conclusion:RNA inter-ference in the expression of HOXB7 gene in colon cancer can inhibit the proliferation of cancer cells and induce apoptosis by inhibiting of the Notch1 signaling pathway.
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Objective:To investigate the effect of downregulation of SLP-2 gene expression on proliferation and apoptosis of brain glioma cells.Methods:The targeting siRNA sequence of SLP-2 transfected U251 human glioma cells,blank group (without any cell treatment)and negative control group (cell transfected nonsense siRNA sequence)were set,Western blot was used to detect the expression of SLP-2,Bcl-2,Bax,Notch1,Hes1 protein after transfected 48 h;cell proliferation was detected by CCKg;cell apoptosis was detected by flow cytometry.Results:The protein expression of SLP-2 in negative control group had no significant difference with the control group (P＞0.05),and the expression of SLP-2 protein in SLP-2 knock down group was significantly lower than control group (P＜0.05);cell survival rate,apoptosis rate,mRNA expression of IL-6,TNF-α and Bcl-2,Bax,Notch1,Hes1 protein expression in the negative control group and the blank group had no significant difference (P＞0.05),cell survival rate,mRNA expression of IL-6 and TNF-α and the expression of Bcl-2,Notch1,Hes1 protein in SLP-2 knock down group was significantly lower than control group,the apoptosis rate and the expression of Bax protein was significantly higher than control group (P＞0.05).Conclusion:Downregulation of SLP-2 gene expression can significantly inhibit the proliferation and induce apoptosis of glioma cells,down regulate the expression of inflammatory cytokines IL-6 and TNF-α,and the mechanism is related to the inhibition of Notch1 signaling pathway.
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AIM:To investigate the effect of propofol on the viability,invasion ability and apoptosis of colorec-tal cancer cells.METHODS:Propofol at 10,25,50 and 100 μmol/L was used to treat LoVo cells for 72 h,and propofol at 100 μmol/L was used to treat the LoVo cells for 12,24,48 and 72 h.The cell viability was measured by CCK-8 assay. The invasion ability of the LoVo cells treated with propofol at 100 μmol/L for 72 h was detected by Transwell assay.The cell cycle distribution and cell apoptotic rate were analyzed by flow cytometry.The protein levels of matrix metalloproteinase (MMP)-2,MMP-9,cleaved caspase-3,Notch1 and hairy and enhancer of split 1(Hes1)were determined by Western blot.RESULTS:Propofol inhibited LoVo cell viability.The cell invasion ability,S stage cells,and the protein levels of MMP-2,MMP-9,Notch1 and Hes1 in propofol group were significantly lower than those in control group,and the apoptotic rate,G0/G1cells and the protein level of cleaved caspase-3 were significantly higher than those in control group(P<0.01).CONCLUSION:Propofol inhibits the viability and invasion ability of colorectal cancer LoVo cells,blocks cell cy-cle and induces apoptosis.The mechanism is related to down-regulation of Notch1 signaling pathway.
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<p><b>OBJECTIVE</b>To examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis.</p><p><b>METHODS</b>The osteoclastogenesis model was builded by co-culturing human breast tumor MDA-MB-231 and mouse RAW264.7 macrophages cells. RANKL (50 ng/mL) and macrophage-colony stimulating factor (50 ng/mL) were added to this system, followed by treatment with brucine (0.02, 0.04 and 0.08 mmol/L), or 10 μmol/L zoledronic acid as positive control. The migration and bone resorption were measured by transwell assay and in vitro bone resorption assay. The protein expressions of Jagged1 and Notch1 were investigated by Western blot. The expressions of transforming growth factor-β1 (TGF-β1), nuclear factor-kappa B (NF-κB) and Hes1 were determined by enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>Compared with the model group, brucine led to a dose-dependent decrease on migration of MDA-MB-231 cells, inhibited RANKL-induced osteoclastogenesis and bone resorption of RAW264.7 cells (P<0.01). Furthermore, brucine decreased the protein levels of Jagged1 and Notch1 in MDA-MB-231 cells and RAW264.7 cells co-cultured system as well as the expressions of TGF-β1, NF-κB and Hes1 (P<0.05 or P<0.01).</p><p><b>CONCLUSION</b>Brucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.</p>
Subject(s)
Animals , Female , Humans , Mice , Bone Neoplasms , Metabolism , Breast Neoplasms , Drug Therapy , Metabolism , Pathology , Cell Differentiation , Cells, Cultured , Jagged-1 Protein , Metabolism , Macrophages , Physiology , Osteoclasts , Physiology , Receptor, Notch1 , Metabolism , Signal Transduction , Strychnine , Pharmacology , Therapeutic UsesABSTRACT
Notch-1 signaling pathway is responsible for cell differentiation, development, proliferation and apoptosis. Recent studies show that Notch-1 signaling pathway is also involved in cancer progression, including cell invasion, motility and cancer metastasis. Activation of Notch-1 signaling pathway can directly or indirectly induce cell proliferation and migration. In tumor cells, activation of Notch-1 facilitates epithelial-mesenchymal transition (EMT), keeps its mesenchyme characteristics and induces cell adhesion. Notch-1 signaling pathway also cross-talks with other pathways to regulate cell fate, such as PI3K/Akt, NF-κB pathways. The evidence shows that aberrant Notch-1 activation has been found in different solid tumors, which participates in regulating tumor metastasis. In this review, the Notch structure and function, Notch-1 signal and tumorigenesis, tumor metastasis via Notch-1 signaling, and Notch-1 signaling targeted-therapy were comprehensively summarized. To clarify the roles of Notch-1 signal pathway in tumor metastasis and its regulatory mechanisms as well as the current treatment strategies for Notch-1 signal pathway will provide references for studies on pathomechanism and clinical treatment of cancers.
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Objective To construct the eukaryotic expression vector of Notch1intracellular domain,p3XFLAG?CMV7?NICD1,so as to prepare for the further research and exploration of effect of Notch1 on promoting epithelial?mesenchymal transition of human lens epithelial cells. Methods The cDNA fragment was reversely transcribed by RT?PCR from total RNA extracted from the SRA01/04 cells and was encoded with the specific am?plification?targeted NICD1was obtained from the SRA01/04 cells,then the cDNA fragment was inserted into p3XFLAG?CMV7 to transcribe Esche?richia coli DH5α. And the recombinant plasmid was extracted after bacterial screening by LB plating medium and confirmed by the restriction endo?nuclease digestion and DNA sequencing. Results The target gene obtained had the same molecular size as predicted. It was indicated that recom?bined p3XFLAG?CMV7 plasmid contained correct recombinant human Notch1 sequences and p3XFLAG?CMV7?NICD1 was constructed success?fully. The western blotting showed protein NICD1 expressed in SRA01/04 cells transfected with p3XFLAG?CMV7?NICD1. Conclusion The suc?cessful construction of p3XFLAG?CMV7?NICD1 will provide a foundation for a further study studies in on the effect relationship of Notch signaling pathway and in posterior capsular opacification(PCO)after cataract extraction.