Objective To investigate the effect and mechanism of epigallocatechin-3-gallate (EGCG) on restenosis of the vein graft. Methods Totally 90 Sprague-Dawley rats were randomly divided a the control group, a vein graft group and an EGCG+vein graft group. At week 1, 2 and 4, the intimal and tunica thickness of the venous graft wall was evaluated by hematoxylin-eosin staining, and the expression of Ki-67 was assessed by immunohistochemistry analysis, and then the expression of hairy and enhancer of split-1 (HES1) was measured by Western blot assay. Results At week 2, the intimal thickness (46.76±4.89 μmvs. 8.93±0.82 μm, 46.76±4.89 μmvs. 34.24±3.57 μm), tunica thickness (47.28±4.37vs. 16.33±1.52 μm, 47.28±4.37vs. 36.27±3.29 μm), positive cell rate of Ki-67 (21.59%±2.29%vs. 1.12%±0.22%, 21.59%±2.29%vs. 15.38%±1.30%), expression of HES1 respectively increased in the experimental group than those in the control group and the EGCG+vein graft group (P<0.05, respectively). At week 4, the intimal thickness (66.38±6.23 μmvs. 8.29±0.79 μm, 66.38±6.23 μmvs. 48.39±4.23 μm), tunica thickness (63.27±6.18 μmvs. 15.29±1.49 μm, 63.27±6.18 μmvs. 44.63±4.49 μm), positive cell rate of Ki-67 (33.19%±3.03%vs. 1.09%±0.19%, 33.19%±3.03%vs. 24.37%±2.73%), expression of HES1 increased in the experimental group than those in the control group and EGCG+vein graft group (P<0.05, respectively). Conclusion EGCG may inhibite restenosis of vein graft by inhibiting Notch signal pathway.
Objective To investigate the regulating effect of Notch-1 on retinal progenitor cells (RPC) differentiating into retinal ganglion cells (RGC). Methods RPC of 14-day embryonic SD rats were induced and differentiated in the culture medium with Notch-1 antisense oligonucleotides (experimental group) or missense oligonucleotides (control group) for 14 days. The condition of growth and differentiation of the cells were observed daily under the phase-contrast microscope. RGC were identified by Thy1.1 immunocytochemistry methods and the cellular number was counted. Results RPC in both of the two groups differentiated into various retinal cells, including Thy1.1-positive RGC. The percentage of RGC derived from RPC was 31.19%plusmn;6.90% in experimental group and 16.57%plusmn;4.31% in control group, and the difference was statistically significant (t=9.84，Plt;0.001). Conclusion Notch-1 may down-regulate the differentiation of RPC, and the inhibition of Notch-1 may promote RPC differentiating into RGC. (Chin J Ocul Fundus Dis, 2007, 23: 101-103)
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 of Notch1, 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 of Notch1, Dll4 and VEGFR2 in all PDR membranes, regardless of the injection of the ranibizumab. The levels of Notch1, 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 of Notch1, 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 of Notch1, 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. Notch1 and Dll4 play a regulatory rule in the neovascularization in PDR, the acting way may be correlated with VEGFR2.
ObjectiveTo investigate effect of Notch pathway regulating by inhibiting expression of forkhead box protein A1 (FOXA1) on proliferation and invasion of colon cancer SW480 cells. MethodsThe colon cancer tissues and their corresponding paracancerous tissues of 45 patients with colon cancer admitted to the First Affiliated Hospital of Henan University of Science and Technology from June 2019 to February 2021 were selected. The immunohistochemistry and real-time fluorescent quantitative PCR (qRT-PCR) methods were used to detect the expressions of FOXA1 protein and mRNA in the tissues, respectively. In addition, SW480 cells were divided into control group (untreated), shRNA-NC group (transfected with shRNA-NC), sh-FOXA1 group (transfected with sh-FOXA1), sh-FOXA1+sodium valproate group (Add 8 mmol/L Notch pathway activator sodium valproate after transfection with sh-FOXA1). Then the qRT-PCR, MTT, clone formation test, and Transwell methods were used to detect the expressions of FOXA1 mRNA, proliferation, clonogenic ability, invasion and migration of cells in each group. Western blot method was used to detect the proliferation (c-Myc, cyclinD1), invasion and migration [matrix metalloproteinase (MMP）9, MMP2], epithelial-mesenchymal transition (Vimentin, N-cadherin, E-cadherin) and Notch pathway (Notch-1, Hes-1) related protein expressions of cells in each group. Results① In the clinical cases, the expression levels of FOXA1 protein and mRNA in the colon cancer tissues were higher than those in the corresponding paracancerous tissues (protein: 0.085±0.028 vs. 0.034±0.010, t=11.036, P<0.001; mRNA: 1.62±0.34 vs. 1.00±0.09, t=11.671, P<0.001). ② In the cell experiment, compared with the control group and shRNA-NC group, the cell survival rate, and numbers of cloned cells, invasion and migrating cells were significantly reduced (P<0.05), correspondingly, the related proteins expression levels of c-Myc, cyclinD1, MMP9, MMP2, Vimentin, N-cadherin, Notch-1, Hes-1 were significantly reduced (P<0.05) and the protein expression level of E-cadherin was significantly increased (P<0.05) in the sh-FOXA1 group, which were reversed after adding the Notch pathway activator sodium valproate (P<0.05). ConclusionFOXA1 highly expresses in colon cancer tissues and colon cancer cells and it might promote the proliferation, invasion and migration of SW480 cells by activating the Notch pathway.
Objective To observe the effects of fine particulate matter (PM2.5) on airway remodeling and Notch signaling pathway in mice with bronchial asthma, and explore the possible mechanism of its influence on airway remodeling in asthmatic mice. Methods Forty eight-week-old SPF female BALB/c mice were divided into a healthy control group, a healthy PM2.5 group, an asthma group and an asthma PM2.5 group by random number table, with 10 mice in each group. The asthma group and the asthma PM2.5 group were sensitized with ovalbumin to establish asthma mouse model, and the healthy PM2.5 group and the asthma PM2.5 group received aerosol inhalation of PM2.5 (510 μg/m3) after each provocation. After modeling, lung function was measured in each group. Hematoxylin and eosin staining and Masson staining were performed on the lung tissue sections of mice. Image analysis software was used to determine the circumference of the bronchial basement membrane, the total area of the bronchial wall, the area of bronchial smooth muscle and the area of collagen deposition. The expressions of Notch1, Hes1, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) and type Ⅰ collagen (Col-Ⅰ) were detected by immunohistochemistry and western blotting. The content of hydroxyproline (HYP) in lung tissue was determined by alkaline water method. Results The total airway wall area, airway smooth muscle area and collagen deposition area in the asthma group [(365.81±46.10), (132.80±20.14), (221.82±25.20) μm2/μm] were significantly higher than those in the healthy control group [(187.70±14.80), (89.73±8.49), (123.91±16.88) μm2/μm] (P<0.01). The healthy PM2.5 group [(244.62±42.86), (116.40±20.40), (174.91±57.41) μm2/μm] and the asthma PM2.5 group [(447.70±76.14), (236.14±36.35), (294.89±75.96) μm2/μm] were higher than those in the control group (all P<0.01). The expressions of Notch1, Hes1, α-SMA, TGF-β1 and Col-Ⅰ were strongly positive in the lung tissues of the asthmatic mice, but weak in the healthy control group. After PM2.5 intervention, compared with the control group, the expression intensity of the above molecules increased. Notch1 receptor and downstream Hes1 protein in the asthma group (0.86±0.10, 1.02±0.06) were significantly higher than those in the healthy control group (0.26±0.07, 0.56±0.09) (all P<0.01). The healthy PM2.5 group (0.44±0.06, 0.77±0.07) and asthma PM2.5 group (1.33±0.23, 1.25±0.18) were higher than the control group (all P<0.01). Airway remodeling related molecules α-SMA, TGF-β1 and Col-Ⅰ protein in the asthma group (0.60±0.04, 0.52±0.09, 0.36±0.04) were significantly higher than those in the healthy control group (0.31±0.03, 0.22±0.04, 0.23±0.04) (all P<0.01). The health PM2.5 group (0.49±0.02, 0.30±0.03, 0.28±0.03) and the asthma PM2.5 group (0.88±0.09, 0.62±0.03, 0.49±0.07) were higher than the control group (P<0.05 or P<0.01), respectively. The content of HYP in lung tissue of the asthma group (57.71±7.60) μg/100mg was significantly higher than that of healthy control group (40.53±5.73) μg/100mg. The healthy PM2.5 group (53.92±6.82) μg/100mg and asthma PM2.5 group (70.96±4.44) μg/100mg were higher than the control group (P<0.01), respectively. In asthma group and asthma PM2.5 group, the expression of Notch1 and Hes1 protein was positively correlated with the total airway wall area, airway smooth muscle area, collagen deposition area, α-SMA, TGF-β1, Col-Ⅰ and HYP (all P<0.01). Conclusion PM2.5 can promote early airway remodeling in asthma, and the activation of Notch signaling pathway may be involved in the promoting effect of PM2.5 on early airway remodeling.
Objective To explore the therapeutic effect of basic fibroblast growth factor (bFGF) on spinal cord injury (SCI) in rats and the influence of Notch/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods A total of 40 10-week-old male Sprague Dawley (SD) rats were selected to establish T10-segment SCI model by a free falling object. Among them, 32 successful models were randomly divided into model group and bFGF group, with 16 in each group. Another 16 SD rats were selected as sham-operation group, with only T10 processes, dura mater, and spinal cord exposed. After modeling, the rats in bFGF group were intraperitoneally injected with 100 μg/kg bFGF (once a day for 28 days), and the rats in model group and sham-operation group were injected with normal saline in the same way. The survival of rats in each group were observed after modeling. Basso-Beattie-Bresnahan (BBB) scores were performed before modeling and at immediate, 14 days, and 28 days after modeling to evaluate the functional recovery of hind limbs. Then, the spinal cord tissue at the site of injury was taken at 28 days and stained with HE, Nissl, and propidium iodide (PI) to observe the pathological changes, neuronal survival (number of Nissl bodies) and apoptosis (number of PI red stained cells) of the spinal cord tissue; immunohistochemical staining and ELISA were used to detect the levels of astrocyte activation markers [glial fibrillary acidic protein (GFAP)] and inflammatory factors [interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ)] in tissues, respectively. Western blot was used to detect the expressions of Notch/STAT3 signaling pathway related proteins [Notch, STAT3, phosphoryl-STAT3 (p-STAT3), bone morphogenetic protein 2 (BMP-2)] in tissues. Results All rats survived until the experiment was completed. At immediate after modeling, the BBB scores in model group and bFGF group significantly decreased when compared to sham-operation group (P<0.05). At 14 and 28 days after modeling, the BBB scores in model group significantly decreased when compared to sham-operation group (P<0.05); the bFGF group showed an increase compared to model group (P<0.05). Compared with before modeling, the BBB scores of model group and bFGF group decreased at immediate after modeling, and gradually increased at 14 and 28 days, the differences between different time points were significant (P<0.05). The structure of spinal cord tissue in sham-operation group was normal; in model group, there were more necrotic lesions in the spinal cord tissue and fewer Nissl bodies with normal structures; the number of necrotic lesions in the spinal cord tissue of the bFGF group significantly reduced compared to the model group, and some normally structured Nissl bodies were visible. Compared with sham-operation group, the number of Nissl bodies in spinal cord tissue significantly decreased, the number of PI red stained cells, GFAP, IL-1β, TNF-α, IFN-γ, Notch, p-STAT3 /STAT3, BMP-2 protein expression levels significantly increased in model group (P<0.05). The above indexes in bFGF group significantly improved when compared with model group (P<0.05). Conclusion bFGF can improve motor function and pathological injury repair of spinal cord tissue in SCI rats, improve neuronal survival, and inhibit neuronal apoptosis, excessive activation of astrocytes in spinal cord tissue and inflammatory response, the mechanism of which may be related to the decreased activity of Notch/STAT3 signaling pathway.
Objective To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance (A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point (P<0.05), andA value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group (P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences (P<0.05). There was no significant difference between 7 ATU/mL group and control group (P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group (P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences (P<0.05). Conclusion Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.
Objective To investigate the expressions and clinical significance of Notch-2 protein and Numb protein in papillary thyroid carcinoma (PTC). Methods PTC tissues and its para-cancerous tissues of 50 patients with PTC who treated in The Affiliated Hospital of Inner Mongolia University for Nationalities from Mar. 2014 to Mar. 2017 were retrospectively collectied, to detect the expressions of Notch-2 protein and Numb protein by immunohistochenmical method. Results ① Expressions of Notch-2 protein and Numb protein in PTC tissues and para-cancerous tissues: the positive-expression rate of Notch-2 protein in PTC tissues was 82.00% (41/50), which was higher than that of para-cancerous tissues〔18.00% (9/50)〕, the difference was statistically significant (χ2=40.960, P<0.001). The positive-expression rate of Numb protein in PTC tissues was 66.00% (33/50), which was higher than that of para-cancerous tissues 〔0 (0/50) 〕, the difference was statistically significant (χ2=49.254, P<0.001). ② The relationship between expression of Notch-2 protein and expression of Numb protein in PTC tissues: there was a positive correlation between expressions of Notch-2 protein and Numb protein in PTC tissues (rs=0.323, P=0.022). ③ The relationship between expressions of Notch-2 protein and Numb protein in PTC tissues and clinicopathological features of the PTC patients: the expression of Notch-2 protein in PTC tissues was not significantly correlated with gender, age, tumor diameter, capsule infiltration, cervical lymph node metastasis, and TNM staging (P>0.05). The expression of Numb protein in PTC tissues was not significantly correlated with gender, age, tumor diameter, and capsule infiltration (P>0.05), but was significantly correlated with cervical lymph node metastasis and TNM staging (P<0.05), the positive rates of Numb protein in patients of staging Ⅲ+Ⅳ group and cervical lymph node metastasis group were lower than those of patients in staging Ⅰ+Ⅱ group and non-cervical lymph node metastasis group. Conclusion The positive-expression rate of Notch-2 protein and Numb protein in PTC tissues are higher than those of para-cancerous tissues, and there is a positive correlation between them in PTC tissues, suggesting that there may be a synergistic effect in the course of PTC progression.
Objective To investigate the heterotopic odontogenesis ability ofDelta1 gene transfected human dental pulp stem cell (DPSC) and nanohydroxyapatite/collagen (nHAC) composite scaffold. Methods The cultured human DPSC was transfected with Delta1-enhanced green fluorescent protein recombinant retrovirus supernatant，and was selected by puromycin to obtain the positive cell clone. The experimental group contained the Delta1 transfected DPSC; however, the control group did not contain the Delta1 transfected DPSC but contained DPSC transfected with vectors only. The cells were seeded into the nHAC carriers and were cultured in the odonto-inductive medium. The growth of the transduced cells in the carriers was observed by the fluorescent phase contrast microscope and the scanning electron microscope (SEM). The cell-carrier composites were subcutaneously transplanted into the Delta1 transfected 8 nude mice (female, 8 weeks old). Eight weeks after operation,the composites were taken out and tested with the histological and the immunohistological methods.Results Green fluorescence was observed inthe cells in the experimental group, which were grown in the carriers by the fluorescent phase contrast microscope. Observed by SEM, great amounts of transduced DPSC were observed along the scaffold materials, even filling the porous structures of nHAC and secreting a lot of extracellular matrix. However, in the control group, much fewer cells were found in the carriers. All the 4 Delta1 transduced DPSC-nHAC composites produced dentin-like structures that lined the surfaces of some nHAC porous structures. The odontoblast-like cells extended the cytoplasmic processes into the dentinal matrix, which was interfaced with a pulp-like interstitial tissue infiltrated with the blood vessels. Dentin sialophosphoprotein was expressed in the odontoblast-like cells when immunohisochemistry was performed. The morphology of the control composite was a typical one of the fibrous connective tissue,and only a little dentin-like structure was found in 2 of the 8 control transplants. Conclution DPSC can be used as the recipient cell of the Delta1 gene for expression and secretion of the Delta1 protein. The composites of the transfected cells and nHAC can induce heterotopic odontogenesis, which indicates that Delta1 is a novel candidate for the gene enhanced dentinpulp composite engineering.
Objective To investigate the mechanism of bone morphogenetic protein-4 (BMP4) in promoting the recovery of small intestinal mucosal barrier function during the recovery period of small intestine ischemia-reperfusion (I/R) injury. Methods Twenty-eight C57BL/6J male mice aged 6–8 weeks were randomly selected and assigned to small intestine I/R group (n=24) and sham operation (SO) group (n=4) by random number table method. Small intestine I/R injury models of 24 mice were established, then 4 mice were randomly selected at 6, 12, 24 and 48 h after I/R established modeling and killed to observe the morphological changes of small intestinal mucosa and detect the expression of BMP4 mRNA in the jejunal epithelial cells, the other 8 mice were allocated for the experimental observation at the recovery period of small intestine I/R injury (24 h after I/R was selected as the observation time point of recovery period of small intestine I/R injury according to the pre-experimental results). Twelve mice were randomly divided into I/R-24 h-BMP4 group (recombinant human BMP4 protein was injected intraperitoneally), I/R-24 h-NS (normal saline) group (NS was injected intraperitoneally), and I/R-24 h-blank group (did nothing), 4 mice in each group. Then the small intestinal transmembrane electrical impedance (TER) was measured by Ussing chamber. The expressions of BMP4 protein and tight junction proteins (occludin and ZO-1), Notch signaling pathway proteins (Notch1 and Jagged1), and Smad6 protein were detected by Western blot. Results At 24 h after I/R injury, the injuries of villous epithelium, edema, and a small part of villi were alleviated. The BMP4 mRNA expressions at 6, 12, 24 and 48 h after I/R injury in the small intestinal epithelial cells were increased as compared with the SO group. Compared with the I/R-24 h-NS group and the I/R-24 h-blank group, the TER was increased, and the expression levels of occludin, ZO-1, p-Smad6, Notch1, Jagged1 were increased in the I/R-24 h-BMP4 group. Conclusion From the preliminary results of this study, during recovery period of small intestine I/R injury, the expression of BMP4 in small intestinal epithelial cells is increased, permeability of jejunal mucosal barrier is increased, which might promote the recovery of small intestinal mucosal barrier function by activating the Notch signaling pathway (Notch1 and Jagged1), Smad classic signaling pathway, and promoting the increase of tight junction protein expression (occludin and ZO-1).