Efficacy of electroacupuncture stimulating Zusanli (ST36) and Xuanzhong (GB39) on synovial angiogenesis in rats with adjuvant arthritis.

OBJECTIVE: To investigate the efficacy of electroacupuncture (EA) stimulating Zusanli (ST36) and Xuanzhong (GB39) on synovial angiogenesis in rats with adjuvant arthritis (AA). METHODS: AA models were established by bilateral injection of Freund's complete adjuvant (FCA) in male Sprague-Dawley rats. Three days after injection, rats were given EA at Zusanli (ST36) and Xuanzhong (GB39) acupoints, once every other day, for 16 d. The arthritis index score, paw volume, and hematoxylin-eosin (HE) staining was performed for each animal. Angiogenesis marker cluster of differentiation 34 (CD34) expression and synovial cell apoptosis in synovial tissue were observed. The levels of Notch1, hairy and enhancer of split homolog-1 (Hes1), transforming growth factor-beta (TGF-ß) and basic fibroblast growth factor (bFGF) were subsequently detected. RESULTS: We found that EA significantly decreased arthritis index scores, paw volume, and HE staining scores. EA could significantly inhibit the expression of CD34, promoting apoptosis of synovial cells in the joint synovial tissue of AA rats. The expression of Notch1 signaling pathway proteins and mRNAs (Notch1, Hes1, TGF-ß, and bFGF) were markedly downregulated by EA treatment. CONCLUSIONS: These results prove that EA attenuates synovial angiogenesis by inhibiting the Notch1 signaling pathway in AA rat models. Based on our findings, we propose that EA is a promising complementary and alternative therapy in rheumatoid arthritis.

Mechanism study on regulation of glioblastoma cell proliferation and apoptosis by sciadopitysin combined with CX-4945 through Notch1 pathway / 国际肿瘤学杂志

Objective:To investigate the effects and mechanism of sciadopitysin combined with CK2 inhibitor CX-4945 on proliferation and apoptosis of glioblastoma U87 cells.Methods:Glioblastoma U87 cells were cultured in vitro, and treated with 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin respectively. U87 cells were treated with 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945. U87 cells were divided into control group (without any treatment), sciadopitysin group (100.00 μmol/L of sciadopitysin), CX-4945 group (5.00 μmol/L of CX-4945), sciadopitysin combined with CX-4945 group (100.00 μmol/L of sciadopitysin plus 5.00 μmol/L of CX-4945). MTT method was used to detect cell viability, Caspase3/7 activity assay and Annexin Ⅴ/ PI double staining were used to detect cell apoptosis, and Western blotting was used to detect the expressions of Notch1 pathway related proteins ICN1, HES1 and DLL3. Results:The cell viabilities of U87 cells treated with 0, 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin were (100.00±6.30) %, (112.02±7.63) %, (140.84±6.73) %, (113.92±7.92) %, (102.60±7.12) % and (73.16±2.74) % respectively, and there was a statistically significant difference ( F=55.21, P<0.001). There were statistically significant differences in the cell viabilities of U87 cells between 0 μmol/L and 0.01, 0.10, 1.00, 100.00 μmol/L of sciadopitysin treatment ( P=0.009; P<0.001; P=0.003; P<0.001). The cell viability of U87 cells was inhibited by 100.00 μmol/L of sciadopitysin, while sciadopitysin at other low concentrations manifested as enhancement or no obvious effect. The cell viabilities of U87 cells treated with 0, 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945 were (100.00±5.53) %, (108.70±10.24) %, (93.14±2.82) %, (81.46±4.92) %, (56.92±3.99) % and (31.24±2.67) % respectively, and there was a statistically significant difference ( F=135.18, P<0.001). There were statistically significant differences in the cell viabilities of U87 cells between 0 μmol/L and 1.25, 5.00, 10.00, 20.00 μmol/L of CX-4945 treatment ( P=0.022; P<0.001; P<0.001; P<0.001). Low concentration (1.25 μmol/L) of CX-4945 enhanced the cell viability of U87 cells, however higher concentrations (5.00, 10.00, 20.00 μmol/L) of CX-4945 shown inhibitory effect. The cell viabilities of U87 cells in the control group, sciadopitysin group, CX-4945 group and sciadopitysin combined with CX-4945 group were (100.00±5.53) %, (71.96±2.10) %, (77.66±4.12) % and (42.56±4.22) % respectively, and there was a statistically significant difference ( F=160.56, P<0.001). There were statistically significant differences between the control group and each treatment groups (all P<0.001). There were statistically significant differences between the sciadopitysin combined with CX-4945 group and sciadopitysin group, CX-4945 group (both P<0.001). The Caspase3/7 activities of U87 cells in the above four groups were 2.34±0.47, 4.02±0.22, 3.67±0.32 and 5.85±0.28 respectively, and there was a statistically significant difference ( F=55.80, P<0.001). The apoptosis rates of each groups were (0.40±0.10) %, (17.37±0.57) %, (3.00±0.66) % and (33.47±0.87) % respectively, and there was a statistically significant difference ( F=1 822.18, P<0.001). Further pairwise comparison showed that there were statistically significant differences in Caspase3/7 activities and apoptosis rates between the control group and each treatment groups ( P<0.001, P=0.001, P<0.001; P<0.001, P=0.001, P<0.001). There were statistically significant differences in Caspase3/7 activities and apoptosis rates between the sciadopitysin combined with CX-4945 group and sciadopitysin group, CX-4945 group (all P<0.001). The protein expression levels of Notch 1 pathway related proteins ICN1 (0.55±0.07 vs. 1.01±0.09), HES1 (0.66±0.08 vs. 1.00±0.06) and DLL3 (0.74±0.04 vs. 1.01±0.09) in U87 cells decreased significantly after treatment with 100.00 μmol/L of sciadopitysin ( t=5.94, P=0.004; t=5.15, P=0.007; t=4.00, P=0.016) . Conclusion:Sciadopitysin can synergize with CK2 inhibitor CX-4945 to inhibit the proliferation and promote apoptosis of glioblastoma U87 cells by inhibiting Notch1 signaling pathway.

Salvia miltiorrhiza-asarum ointment combined with Chinese medical massage alleviates symptoms of osteoarthritis in a rat model through the Notch1/ matrix metalloproteinase-13 signaling pathway.

OBJECTIVE: This study investigated the effect of salvia miltiorrhiza-asarum ointment (SMAO) plus Chinese medical massage on knee osteoarthritis in a rat model. METHODS: Hulth's method was used to establish a Sprague-Dawley rat model of knee osteoarthritis (OA). The levels of matrix metalloproteinase-13 (MMP-13), collagen-II, aggrecan, interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α), and IL-6 were measured by enzyme-linked immunosorbent assays. The joint space was assessed by a Perlove X-ray system. Histopathology was examined by hematoxylin-eosin and Safranin O staining. The mRNA and protein expression levels of Notch1, MMP-13, collagen-II, and aggrecan were measured by quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively. RESULTS: SMAO plus Chinese medical massage significantly decreased the levels of MMP-13, IL-1ß, TNF-α, and IL-6, and increased serum collagen-II and aggrecan levels. Pathological injury of the knee joint was improved by SMAO treatment. mRNA and protein expression of Notch1 and MMP-13 was remarkably downregulated, but collagen-II and aggrecan levels were significantly upregulated in cartilage tissues. CONCLUSION: SMAO combined with Chinese medical massage effectively relieves OA symptoms, which may involve inhibiting inflammation through the Notch1/MMP-13 signaling pathway.

Role of Notch1/Hes1 signaling pathway in high glucose and hypoxia-reoxygenation injury to cardiomyocytes / 中华麻醉学杂志

Objective:To evaluate the role of Notch1/hairy and enhancer of split homolog1(Hes1) signaling pathway in high glucose and hypoxia-reoxygenation (H/R) injury to cardiomyocytes.Methods:H9c2 cardiomyocytes were cultured in low-glucose DMEM culture medium supplemented with 10% fetal bovine serum.The cells were divided into 6 groups ( n=12 each) using a random number table method: control group (group C), H/R group, H/R+ Jagged-1 group (group H/R+ J), high glucose group (group HG), high glucose+ H/R group (group HG+ H/R) and high glucose+ H/R+ Jagged-1 group (group HG+ H/R+ J). The cells were incubated in low-glucose culture medium for 72 h in group C. After incubated in low-glucose culture medium for 72 h, the cells were exposed to 24-h hypoxia in an incubator filled with 95% N 2-5% CO 2 at 37℃, immediately followed by 6-h reoxygenation in an incubator filled with 95% O 2-5% CO 2 at 37℃ in group H/R.In group H/R+ J, Jagged-1 (Notch1/Hes1 signaling pathway specific activator) 5μg/ml was added to low-glucose culture medium and the cells were incubated for 72h before H/R.In group HG, H9c2 cardiomyocytes were incubated in high-glucose culture medium containing 33 mmol/L glucose for 72 h. In group HG+ H/R, the cells were incubated in high-glucose medium for 72 h before H/R.In group HG+ H/R+ J, Jagged-1 5μg/ml was added to high-glucose culture medium, and the cells were incubated for 72 h before H/R.At 6 h of reoxygenation, the supernatant of the culture medium was collected for detection of the activities of superoxide dismutase (SOD) and lactic dehydrogenase (LDH), the cell viability (by CCK-8 assay) and the cell apoptosis rate (by flow cytometry) and for determination of expression of Notch1, Hes1 and c-caspase-3 (by Western blot). Results:Compared with group C, the cell survival rate and SOD activity were significantly decreased, and apoptosis rate and LDH activity were increased in H/R, H/R+ J and HG groups, expression of Notch1, Hes1 and c-caspase-3 was up-regulated in H/R and H/R+ J groups, and the expression of Notch1 and Hes1 was down-regulated and c-caspase-3 expression was up-regulated in group HG ( P<0.05). Compared with group H/R, the cell survival rate and SOD activity was significantly increased, apoptosis rate and LDH activity were decreased, expression of Notch1 and Hes1 was up-regulated, and c-caspase-3 expression was down-regulated in group H/R+ J, and the cell survival rate and SOD activity were significantly decreased, apoptosis rate and LDH activity were increased, expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG, the cell survival rate and SOD activity were significantly decreased, and apoptosis rate and LDH activity were increased in HG+ H/R and HG+ H/R+ J groups ( P<0.05), and expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG+ H/R, the cell survival rate and SOD activity were significantly increased, apoptosis rate and LDH activity were decreased, expression of Notch1 and Hes1 was up-regulated, and c-caspase-3 expression was down-regulated in group HG+ H/R+ J ( P<0.05). Compared with group H/R+ J, the cell survival rate and SOD activity were significantly decreased, apoptosis rate and LDH activity were increased, expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R+ J ( P<0.05). Conclusion:Activation of Notch1/Hes1 signaling pathway is the endogenous protective mechanism of high glucose and H/R injury to cardiomyocytes.

Notch1 in Tumor Microvascular Endothelial Cells and Tumoral miR-34a as Prognostic Markers in Locally Advanced Triple-Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is associated with poor prognosis with limited treatment options. Angiogenesis is known to be involved in the progression of TNBC, and targeting this pathway results in modest clinical benefits. In this study, we analyzed the role of tumor microvascular endothelial Notch1 (EC Notch1) and tumoral miR-34a as prognostic markers in patients with TNBC. METHODS: The expression of miR-34a was analyzed using archival tumor tissues from 114 patients with TNBC. Simultaneously, archival tumor tissues were also checked for the expression of CD34 and Notch1 by immunostaining. The ratio of Notch1-microvascular density (MVD) to CD34-MVD was defined as EC Notch1. The association between the expression of miR-34a or EC Notch1 and clinicopathological characteristics was analyzed. RESULTS: In the overall patient population, patients with low expression of EC Notch1 was associated with better overall survival (OS, p = 0.041) than those with high expression of EC Notch1. In lymph node-positive TNBC patients, high levels of miR-34a and low levels of EC Notch1 correlated significantly with higher survival benefits in terms of OS (p = 0.026), disease-free survival (p = 0.009), and metastasis-free survival (p = 0.038) relative to that in other patients. Decreased expression of EC Notch1 and increased expression of miR-34a also showed a survival benefit in locally advanced TNBC. CONCLUSION: The fact that miR-34a and EC Notch1 are associated with the angiogenesis suggests that angiogenesis may play a role in the development and progression of TNBC.

Effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells / 国际肿瘤学杂志

Objective To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells.Methods Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group,Nc-shRNA group and Notch1-shRNA group.The Nc-shRNA group was a negative control RNAi lentivirus group,and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group.The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1.The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting.Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay.Apoptosis was detected by Annexin V/7-AAD double staining.Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA),B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1.Results The results of qRT-PCR showed that the relative expression levels of Notch1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were 1.000 ± 0.000,0.937 ± 0.025,0.490 ± 0.036 and 1.000 ± 0.000,1.077 ± 0.070,0.373± 0.038,with statistically significant differences (F =359.707,P ＜0.001;F =210.455,P ＜0.001),further paired comparison,the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P ＜ 0.05).Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results.MTT assay showed that the 24 h A values of A549 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.209 ± 0.005,0.219 ± 0.009,0.159 ±0.006,48 h A values were 0.293 ± 0.004,0.302 ± 0.004,0.205 ± 0.005,72 h A values were 0.450 ± 0.003,0.430 ± 0.012,0.348 ± 0.017,with statistically significant differences (F =79.487,P＜0.001;F =508.664,P ＜0.001;F =57.156,P ＜0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 24,48,72 h (all P ＜ 0.05).The 48 h A values of SPC-A-1 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.438 ±0.022,0.412 ± 0.015,0.364 ± 0.010,72h A values were 0.540 ± 0.016,0.519 ± 0.009,0.438 ± 0.019,with statistically significant differences (F =15.667,P =0.004;F =37.299,P ＜ 0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P ＜ 0.05).The sphere sizes of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667 ± 6.506) μm,(136.667 ± 7.095) μm,(86.676 ± 7.638) μm,with statistically significant difference (F =65.940,P ＜ 0.001).The sphere sizes of the three groups in SPC-A-1 cells were (118.667 ± 6.658) μm,(128.000 ± 7.000) μm,(60.675 ± 4.509) μm,with statistically significant difference (F =105.372,P ＜0.001).Further paired comparison,the sphere size of Notch1shRNA group was significandy smaller than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).The apoptosis rates of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1cells were (0.489 ± 0.014)％,(0.633 ± 0.021)％,(1.683 ± 0.221)％ and (1.323 ± 0.194)％,(1.690 ± 0.188) ％,(3.017 ± 0.356) ％,with statistically significant differences (F =77.660,P ＜ 0.001;F=32.200,P =0.001),further paired comparison,the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).Western blotting showed that the expressions of PCNA,Bcl-2 and Hes-1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F =155.343,P ＜ 0.001;F =22.576,P =0.002;F =70.108,P＜0.001),and the expressions of PCNA,Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F =49.419,P ＜0.001;F =28.090,P =0.001;F =12.040,P =0.007).Further paired comparison,the expressions of PCNA,Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells,and the differences were statistically significant (all P ＜0.05).Conclusion Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis,which may be related to the down-regulation of its downstream gene Hes-1.

Notch1 in Tumor Microvascular Endothelial Cells and Tumoral miR-34a as Prognostic Markers in Locally Advanced Triple-Negative Breast Cancer / 한국유방암학회지

The predicting value of Notch1 expressed on peripheral blood mononuclear cell for coronary artery calcification / 中国医师杂志

Objective To investigate the predicting value of Notch1 levels expressed in peripheral blood mononuclear cell (PBMC) for coronary artery calcification.Methods 300 consecutive patients with coronary artery disease (CAD) who hospitalizing in the department of cardiology in Guangzhou first people's hospital from January 2016 to June 2017 were enrolled.All Patients received 320-slice multi-detector row computed tomography scanning and coronary artery calcium sore(CCS)were measured.Patients were divided into three groups:control group (CCS =0),Low CCS group (CCS ＜97.6) and high CCS group (CCS ≥97.6) according to the mean value of CCS (CCS =97.6).Notch1 expressed in PBMC,serum interlekin-6 (IL-6) and high sensitivity C reactive protein (hs-CRP)of patients were examined and compared among three groups.Results The levels of Notch1 in PBMC and serum IL-6,hs-CRP of patients in high CCS group were significant higher than the other two groups [Notch1:7.02 ± 0.86 vs 6.32 ± 0.78 vs 5.49 ± 0.71;IL-6:(133.66 ± 10.18) μg/L vs (127.49 ± 10.79) μg/L vs (111.62 ± 9.87) μg/L;hs-CRP:(3.98 ± 1.02) mg/L vs (3.11 ±0.95)mg/L vs (2.56 ±0.76)mg/L] (P ＜0.05).The Spearman correlation analysis showed that the levels of Notch1 in PBMC were positive correlated with the levels of serum IL-6 and hs-CRP in enrolled patients with coronary calcification (P ＜ 0.05).Multivariate logistic regression analysis showed that the levels of Notch1 in PBMC and serum IL-6 were two strong independent risk factors for severity of coronary calcification in patients with CAD (P ＜ 0.05).Conclusions Notch1 expression in PBMC of patients with CAD was valuable to predicate the severity of coronary calcification.That the Notch1 signal path regulating the inflammation conditions in patients may be one of the most important mechanisms in the formation and progress of coronary calcification.

The expression and role of Notch1/RBP-Jκ/Msx2 signaling pathway in the aorta of chronic kidney disease rats with vascular calcification / 中华肾脏病杂志

Objective To observe the expression of Notch1 signaling pathway in the aorta of chronic kidney disease (CKD) rats with vascular calcification and to explore the role of this signaling pathway activation in aortic calcification of CKD.Methods A total of 40 male SD rats were randomly divided into normal group (Nor group) and CKD with vascular calcification group (CKD+VC group).Rats in each group were sacrificed at 4 weeks,6 weeks and 8 weeks respectively after the success of modeling.Their 24-hour urine was reserved to test 24 hour urine protein (24 h Upro);blood sample was collected from abdominal aorta to test blood urea nitrogen (BUN),serum creatinine (Scr),Ca and P.The histopathology of renal was detected by HE staining.The aortic calcification was detected by alizarin red S staining.Immunohistochemistry (IHC) was used to test the protein expressions of alpha-smooth muscle actin (or-SMA),Runt-related transcription factor 2 (Runx2),Notchl,recombination signal-binding protein for immunoglobulin kappa J region (RBP-Jκ),Msh homeobox 2 (Msx2),Jagged1 and Notch1 intracellular domain (N1-ICD) in the aorta.Real time PCR was applied to detect the mRNA expressions of α-SMA,smooth muscle 22 alpha (SM22α),Runx2,alkaline phosphatase (ALP),Notch1,RBP-Jκ,Msx2 and Jagged1.Results Compared with those in Nor group,24 h Upro,BUN and Scr increased in the CKD+VC group at 4th,6th and 8th weeks (all P ＜ 0.05).Numerous continuous calcified nodules were detected in the vascular wall of the CKD+VC group,but none in Nor group.As compared with Nor group,the expression of α-SMA was low,while the expression of Runx2 was relatively high in the CKD+VC rats at each time point (all P ＜ 0.05).The expressions of Notchl,RBP-Jκ,Msx2,Jaggedl and NI-ICD in the Nor group were slightly appeared in the aortic wall,while in CKD+VC group these signal protein expressions increased relatively during the experimental period (all P ＜ 0.05).As compared with Nor group,the expressions of α-SMA and SM22α mRNA were low,yet the expressions of Runx2 and ALP mRNA were high in the CKD+VC rats at each time point (all P ＜ 0.05).The mRNA levels of Notch1,RBP-Jκ,Msx2 and Jagged1 in the CKD+VC group at each time point were significantly up-regulated as compared with the Nor group (all P ＜ 0.05).Conclusions There exist phenotypic changes in smooth muscle cells and activations of Notch1/RBP-Jκ/Msx2 signaling pathway in CKD rats with vascular calcification.It may be one of the important signal transduction pathways.

Association of notch 1 signal pathway with lymph node and distant metastases of papillary thyroid carcinoma: A meta analysis / 肿瘤

Objective: To analyze the associations of Notch 1 expression with the lymph node metastasis and distant metastasis of papillary thyroid carcinoma (PTC) by Meta analysis. Methods: Computer retrieve was conducted in PubMed (MEDLINE), Cochrane Library, EMBASE, China Journal Full-text Database (CNKI), China Biology Medicine disc (CBMdisc) to search the studies which were about the association of Notch1 signal with the lymph node and distant metastases of PTC, and published from 2010 to 2017. The literatures were screened and evaluated, then the information was extracted independently by 2 researchers according to the literature selection criteria. Meta analysis was performed using RevMan 5.3 and STATA 12.0 software. The odds ratio (OR) and 95% confidence interval (CI) were calculated. The sensitivity analysis and publication bias test were performed. Results: A total of 7 clinical case-control studies involving 743 patients with PTC were selected. Meta-analysis showed that the expression of Notch1 was significantly positively correlated with lymph node metastasis of PTC (OR = 4.68, 95% CI: 3.00-7.30), furthermore the test for overall effect showed that Z = 6.80 and P < 0.000 01. However, there was no significant correlation between Notch1 expression and the distant metastasis of PTC (OR = 1.59, 95% CI: 0.88-2.89), the test for overall effect showed that Z and P values were 1.53 and 0.1 3 respectively. Conclusion: The Notch1 signaling pathway plays a promoting role in the lymph node metastasis of PTC, which suggests that the expression of Notch1 has a certain predictive value for the clinical prognosis of PTC.

Notch signaling pathway regulates osteosarcoma stem cell characteristics by inhibiting Eph pathway / 中华骨科杂志

Objective To investigate the role of Notch signaling pathway to maintain the stem cell-like characteristics of osteosarcoma and its underlying mechanism.Methods Lentiviral NICD1 or Numb-shRNA was transduced into MG63 osteosarcoma cells to activate Notch activity in vitro.The impact of Notch on osteosarcoma stem cells were assessed by the tumor sphere formation assay and flow cytometry analysis of cell surface markers STRO-1/CD117.The expression of stem cell related genes (Sox2,Oct4) were evaluated by Western blot and qPCR.The nude mice were randomly divided into 3 groups:the NICD1 overexpression (NICD-OE) group,the DAPT group and the control (CON) group.The tumor growth was monitored for 8 weeks and the tumor volume and weight were recorded weekly.To investigate whether Notch regulates Eph pathway,Eph pathway related protein EphB,pEphB was measured by Western blot.The impact of ephrinB 1 on NICD overexpression cell were assessed by tumor sphere formation assay.The expression of Sox2 and Oct4 was evaluated by Western blot.Results NICD1 overexpression or Numb-shRNA increased the activity of Notch pathway.The Notch-activated osteosarcoma showed enhanced in vitro tumor spheroid formation capacity,increased Stro-1/CD117double positive ratio,and upregulated expression of Sox2 and Oct4 in vitro.In animal experiments,it was found that activation of Notch pathway promoted tumor formation in vivo and Notch inhibition decreased it.The primary osteosarcoma cells were obtained from mice xenograft treated with DAPT and its tumor sphere formation capacity was significantly reduced.Finally,The Notch pathway inhibits the phosphorylation of EphB,as well as the downstream signal pathway of EphB,but there is no significant change in total EphB.The activation of Eph pathway inhibited Notch induced up-regulation of tumor sphere formation and Sox2 and Oct4 expression.Conclusion Notch signaling pathway maintains the stem cell-like characteristics of osteosarcoma probably by inhibiting the Eph pathway.

[Clinical significance of S100A6 and Notch1 in multiple myeloma patients].

Objective: To investigate the expression levels of S100A6, Notch1 in multiple myeloma (MM) patients and its clinical significance. Mathods: The expression levels of S100A6, Notch1 in 28 MM cases and 20 healthy controls were determined by real time quantitative PCR (RQ-PCR) , and their relationships with clinical features and outcomes were analyzed. Immunohistochemical was used to analysis the levels of S100A6 and Notch1 in bone marrow biopsy samples and intramedullary metastases soft tissues. RQ-PCR and Western blot were used to test the changes of Notch1 mRNA and Notch1 protein in U266 MM cells after S100A6 silenced by siRNA. Results: ①The expression levels of S100A6, Notch1 in primary MM patients was 2.19±1.25, 2.98±0.64, significantly higher than those in controls (0.71±0.20, 0.58±0.39, P<0.05) and patients in platform status (0.85±0.26, 0.72±0.40, P<0.05) . 8 cases with intramedullary metastasis had significantly higher levels of S100A6 (3.36±1.23) and Notch1 (5.71±3.96) , as compared to those without extra medullary metastases. ②S100A6 expression was positive correlation with Notch1 (r=0.505, P=0.007) . ③S100A6 and Notch1 proteins were positive in plasma cells of bone marrow biopsy samples and intramedullary metastases soft tissues. ④The Notch1 mRNA and Notch1 expression decreased significantly after 48 hours treatment by S100A6 siRNA in U266 cells. Conclusion: S100A6 and Notch1 were closely associated with MM progress and intramedullary metastasis. They have significant correlation and might be as two prognostic molecular markers in MM.

Effect of Yiqihuoxue prescription on myocardial energy metabolism after myocardial infarction via cross talk of liver kinase B1-dependent Notch1 and adenosine 5'-monophosphate-activated protein kinase.

OBJECTIVE: To investigate the effect of Yiqihuoxue prescription (YQHX) from Traditional Chinese Medicine (TCM) on myocardial glucose and lipid metabolism after myocardial infarction via the cross talk between the liver kinase B1 (LKB1)-dependent Notch1 and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK). YQHX was prepared with substances with properties that benefit, to activate blood circulation based on the TCM theory. METHODS: Animal models of myocardial infarction were established by ligating Sprague Dawley rats' left anterior descending coronary arteries. The animals were randomly divided into a myocardial infarction (MI) group, a YQHX group, a perindopril group, a r-secretase inhibitor, Notch signal inhibitor (DAPT) group, a DAPT+YQHX group and a sham group. The related drugs were administered on the second day after operation, and changes in the relevant indexes were examined on weeks 1 and 4. Changes in cardiac structure and function were examined by echocardiography. The glucose and free fatty acids (FFA) were examined by ELISA. The expression of Notch, LKB1 and AMPK mRNA was examined by a real-time fluorescence quantitative method. The expression of glucose transporter 4 (GLUT4), and the expression of total acetyl-CoA carboxylase (ACC) and its phosphorylation were examined by western blotting. RESULTS: Compared with the sham group, the expression of Notch, LKB1 and AMPK mRNA in the MI group was lower. Compared with the MI group, the expression of these mRNAs in the YQHX and perindopril groups was higher, and their expression in the DAPT group was lower. At all time points, the protein expression of GLUT4 and pACC decreased in the MI group. On week 1, the expression of pACC protein was higher. In the DAPT group, the expression of pACC protein decreased. Compared with the YQHX group, the expression of pACC protein in the DAPT + YQHX group was lower. On week 4, compared with the MI group, the expression of GLUT4 protein in the YQHX group and the perindopril group was higher. The expression of GLUT4 protein in the DAPT group decreased. Compared with the YQHX group, the expression of GLUT4 protein in the DAPT+YQHX group was lower. There was no significant difference in the expression of ACC protein between the groups. CONCLUSION: YQHX promoted cross talk between the LKB1-dependent Notch1 and AMPK in myocardial tissue after myocardial infarction. Furthermore, it regulated the glucose and lipid metabolism of cardiomyocytes at different time points, thereby ameliorating the cardiac energy metabolism via different mechanisms and protecting the heart.

The Role of Notch1 Signaling in Anaplastic Thyroid Carcinoma.

PURPOSE: The Notch signaling pathway is widely expressed in normal, reactive, and neoplastic tissues; however, its role in thyroid tissues has not been fully elucidated. Therefore, this study was conducted to characterize the expression of the Notch signaling pathway in papillary thyroid cancer (PTC) cells and anaplastic thyroid cancer (ATC) cells. MATERIALS AND METHODS: Expression of activated Notch1 in ATC and PTC paraffin-embedded tissues was determined by immunohistochemistry. The small interfering RNA techniquewas employed to knock down Notch1 expression in ATC and PTC cell lines. RESULTS: The expression of activated Notch1 was higher in ATC cases than in PTC cases. Inhibition of Notch1 significantly reduced proliferation and migration of ATC cells, but not PTC cells. In addition, inhibition of Notch1 in ATC cells significantly reduced the expression of key markers of epithelial-mesenchymal transition and cancer stem cells. Conversely, changes in the expression of these proteins were not observed in PTC cells. CONCLUSION: The results of this study suggest that Notch1 expression plays different roles in tumor progression in ATC and PTC cells. We also found that Notch1 expression was significantly related to the highly invasive or proliferative activity of ATC cells.

Clinical significance of S100A6 and Notch1 in multiple myeloma patients / 中华血液学杂志

Objective@#To investigate the expression levels of S100A6, Notch1 in multiple myeloma (MM) patients and its clinical significance.@*Mathods@#The expression levels of S100A6, Notch1 in 28 MM cases and 20 healthy controls were determined by real time quantitative PCR (RQ-PCR) , and their relationships with clinical features and outcomes were analyzed. Immunohistochemical was used to analysis the levels of S100A6 and Notch1 in bone marrow biopsy samples and intramedullary metastases soft tissues. RQ-PCR and Western blot were used to test the changes of Notch1 mRNA and Notch1 protein in U266 MM cells after S100A6 silenced by siRNA.@*Results@#①The expression levels of S100A6, Notch1 in primary MM patients was 2.19±1.25, 2.98±0.64, significantly higher than those in controls (0.71±0.20, 0.58±0.39, P<0.05) and patients in platform status (0.85±0.26, 0.72±0.40, P<0.05) . 8 cases with intramedullary metastasis had significantly higher levels of S100A6 (3.36±1.23) and Notch1 (5.71±3.96) , as compared to those without extra medullary metastases. ②S100A6 expression was positive correlation with Notch1 (r=0.505, P=0.007) . ③S100A6 and Notch1 proteins were positive in plasma cells of bone marrow biopsy samples and intramedullary metastases soft tissues. ④The Notch1 mRNA and Notch1 expression decreased significantly after 48 hours treatment by S100A6 siRNA in U266 cells.@*Conclusion@#S100A6 and Notch1 were closely associated with MM progress and intramedullary metastasis. They have significant correlation and might be as two prognostic molecular markers in MM.

Endogenous neural stem cells promotes Notch-1 expression after spinal cord injury in rats / 中国医师杂志

Objective To explore the expressions and changes of endogenous neural stem cells (ENSCs) and Notch-1 after acute spinal cord injury in adult rats,and to explore its role in the nerve regeneration process.Methods The 30 Sprague Dawley (SD) female rats (3-6 months) (300-350 g) were divided into control groups (n =5) and experimental group (n =25) by digital random method.The control group only accepted lamina decompression,and the experimental group was the spinal cord injury group.Five rats were drawn when at 1 d,3 d,1 w,2 w,and 4 w,then behavior,histology,immunohistochemistry and immunofluorescence method were used to detect the proliferation and expression of endogenous neural stem cells and Notch-1 protein.Results Behavioral observation showed the experimental group rats were disfunction.Histological observation showed nerve fiber structure turbulence,edema and denaturation in the experimental group.Immunohistochemistry staining showed the Notch-1 expression every experimental group.Notch-1 positive cell peaked at 3 days.Immunofluorescence test showed,in the experimental group damage to segment the area surrounding the BrdU positive staining cells was significantly higher than the control group.Using three-dimensional quantitative study method detected in 1 w after spinal cord injury was the number of newborn cells mitosis,most about was about 75 times in the control group.Linear regression method was used to analyze the different time points after BrdU and Notch-1 protein expression positive area,the area of the results found that both into linear correlation.Conclusions The new born neurons after spinal cord injury in rats Notch-1 expression has a certain relevance.In addition,the expression of signal protein Notch-1,might be associated with early proliferation of ENSCs in rats.

Endogenous neural stem cells promotes Notch-1 expression after spinal cord injury in rats / 中国医师杂志

Objective To explore the expressions and changes of endogenous neural stem cells (ENSCs) and Notch-1 after acute spinal cord injury in adult rats,and to explore its role in the nerve regeneration process.Methods The 30 Sprague Dawley (SD) female rats (3-6 months) (300-350 g) were divided into control groups (n =5) and experimental group (n =25) by digital random method.The control group only accepted lamina decompression,and the experimental group was the spinal cord injury group.Five rats were drawn when at 1 d,3 d,1 w,2 w,and 4 w,then behavior,histology,immunohistochemistry and immunofluorescence method were used to detect the proliferation and expression of endogenous neural stem cells and Notch-1 protein.Results Behavioral observation showed the experimental group rats were disfunction.Histological observation showed nerve fiber structure turbulence,edema and denaturation in the experimental group.Immunohistochemistry staining showed the Notch-1 expression every experimental group.Notch-1 positive cell peaked at 3 days.Immunofluorescence test showed,in the experimental group damage to segment the area surrounding the BrdU positive staining cells was significantly higher than the control group.Using three-dimensional quantitative study method detected in 1 w after spinal cord injury was the number of newborn cells mitosis,most about was about 75 times in the control group.Linear regression method was used to analyze the different time points after BrdU and Notch-1 protein expression positive area,the area of the results found that both into linear correlation.Conclusions The new born neurons after spinal cord injury in rats Notch-1 expression has a certain relevance.In addition,the expression of signal protein Notch-1,might be associated with early proliferation of ENSCs in rats.

The Role of Notch1 Signaling in Anaplastic Thyroid Carcinoma / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The Notch signaling pathway is widely expressed in normal, reactive, and neoplastic tissues; however, its role in thyroid tissues has not been fully elucidated. Therefore, this study was conducted to characterize the expression of the Notch signaling pathway in papillary thyroid cancer (PTC) cells and anaplastic thyroid cancer (ATC) cells. MATERIALS AND METHODS: Expression of activated Notch1 in ATC and PTC paraffin-embedded tissues was determined by immunohistochemistry. The small interfering RNA techniquewas employed to knock down Notch1 expression in ATC and PTC cell lines. RESULTS: The expression of activated Notch1 was higher in ATC cases than in PTC cases. Inhibition of Notch1 significantly reduced proliferation and migration of ATC cells, but not PTC cells. In addition, inhibition of Notch1 in ATC cells significantly reduced the expression of key markers of epithelial-mesenchymal transition and cancer stem cells. Conversely, changes in the expression of these proteins were not observed in PTC cells. CONCLUSION: The results of this study suggest that Notch1 expression plays different roles in tumor progression in ATC and PTC cells. We also found that Notch1 expression was significantly related to the highly invasive or proliferative activity of ATC cells.

The effect of Notch1 and Delta-like ligand 4 in the neovascularization of the proliferative diabetic retinopathy / 中华眼底病杂志

Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR),and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2).Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study.The PDR patients were divided into non-injection group (30 patients,32 eyes) and injection group (27 patients,28 eyes).The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery.The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy.Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls.The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression ofNotch1,Dll4 and VEGFR2.In the meantime,the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted.Results The immunohistochemical staining revealed that there were positive expression ofNotch1,Dll4 and VEGFR2 in all PDR membranes,regardless of the injection of the ranibizumab.The levels ofNotch1,Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45,6.01,4.08;P=0.030,0.008,0.023).In injection group,the number of endothelial cells in the membranes reduced (17.17 ± 2.48) compared with that of the non-injection group (41.50± 5.57).There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05).RT-PCR showed that the differences of the mRNA expression ofNotch1,Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50,12.50,12.02;P＜0.05).The expression ofNotch1,Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients.In the PDR group,the expression of Notch1,Dll4 and VEGFR2 of non-injection group was higher than that of injection group.Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83),VEGFR2 and Notch1 (r=0.81),Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05).Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group,and it is positively correlated with the expression of the VEGFR2.Notchl and Dll4 play a regulatory rule in the neovascularization in PDR,the acting way may be correlated with VEGFR2.

Altered DNA Methylation of Long Noncoding RNA H19 in Calcific Aortic Valve Disease Promotes Mineralization by Silencing NOTCH1.

BACKGROUND: Calcific aortic valve disease is characterized by an abnormal mineralization of the aortic valve. Osteogenic activity in the aortic valve is under the control of NOTCH1, which regulates the expression of key pro-osteogenic genes such as RUNX2 and BMP2. Long noncoding RNAs (lncRNAs) may reprogram cells by altering the gene expression pattern. METHODS: Multidimensional genomic profiling was performed in human aortic valves to document the expression of lncRNAs and the DNA methylation pattern in calcific aortic valve disease. In-depth functional assays were carried out to document the impact of lncRNA on the mineralization of the aortic valve. RESULTS: We documented that lncRNA H19 (H19) was increased in calcific aortic valve disease. Hypomethylation of the promoter region was observed in mineralized aortic valves and was inversely associated with H19 expression. Knockdown and overexpression experiments showed that H19 induces a strong osteogenic phenotype by altering the NOTCH1 pathway. Gene promoter analyses showed that H19 silenced NOTCH1 by preventing the recruitment of p53 to its promoter. A knockdown of H19 in valve interstitial cells (VICs) increased the expression of NOTCH1 and decreased the level of RUNX2 and BMP2, 2 downstream targets repressed by NOTCH1. In rescue experiments, the transfection of a vector encoding for the active Notch intracellular domain prevented H19-induced mineralization of valve interstitial cells. CONCLUSIONS: These findings indicate that a dysregulation of DNA methylation in the promoter of H19 during calcific aortic valve disease is associated with a higher expression of this lncRNA, which promotes an osteogenic program by interfering with the expression of NOTCH1.