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 investigate the expression and clinical significance of Notch-1 protein in papillary thyroid carcinoma (PTC) tissues and cervical lymph node metastases. Methods Immunohistochemical method was used to detect the expression of Notch-1 protein in 69 cases of PTC tissues, along with tumor adjacent tissues and 34 cases of metastatic lymph node tissues, and to analyze its role in PTC and metastatic lymph node tissue. Results Compared with PTC tissues or cervical lymph node metastases and tumor adjacent tissues, the positive rates of expression of Notch-1 protein in PTC tissues or cervical lymph node metastases were significantly lower than that in cancer adjacent tissues (P＜0.05). The expression of Notch-1 protein was correlated with the tumor size and capsule invasion of patients with PTC. Conclusions Notch-1 protein expression is decreased in PTC tissues and metastatic lymph node tissues, suggesting that the Notch-1 protein may play an important role in the development, invasion and metastasis of PTC. There is no significant difference in the positive rates of Notch-1 protein expression in PTC tissues and metastatic lymph node tissues, it's suggested that the malignant degree of cancer cells in lymph node metastasis is not significantly increased, and the biological behavior remained relatively stable.
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.
ObjectiveTo detect the expression of Notch1, Bax, Bcl-2 genes in rat knee joint cartilage cells in a state of activation and inactivation of the Notch signaling pathway, and preliminarily study the mechanism of Notch signaling pathway on experimental rat knee osteoarthritis (OA) chondrocytes apoptosis.MethodsA total of 34 specefic-pathogen-free Sprague Dawley rats were selected, of which 32 were established the right knee OA models using Hulth method, and the other 2 were normally fed. Four weeks later, two randomly selected OA rats and the two normally fed rats were put to death, to observe the morphological changes of the right knee and ensure the OA models were successfully established by pathology examination. The remaining 30 rats were randomly divided into three groups with 10 in each. The rats were injected intra-articularly on each Tuesday and Friday, with Nocth signal pathway specific activator Jagged1 protein (25 ng/kg) in the activation group, γ-secretase inhibitor DAPT (GSI-IX) (100 ng/kg) in the inhibition group, and phosphate-buffered saline in the control group, respectively. The rats were sacrificed after 8 weeks of articular cavity injection. Taking the right knee articular cartilage speciments of femoral condyle, we observed the degeneration of articular cartilage of the three groups, observed the histomorphological changes by microscope, evaluated the Mankin scores, and used the immunohistochemistry to detect the expression of Notch1, Bax, Bcl-2 proteins.ResultsAfter the 8-week articular cavity injection, the Mankin scores in the activation group, the inhibition group, and the control group were 3.40±0.84, 6.70±0.95, 11.10±1.37, respectively, and the differences between the three groups were statistically significant (P<0.05). The positive rates of Notch1 and Bax of chondrocyte in the inhibition group were lower than those in the control group and the activation group (P<0.05), while the positive rate of Bcl-2 of chondrocyte in the inhibition group was higher than that in the control group and the activation group (P<0.05).ConclusionActivating the Notch signaling pathway may facilitate the chondrocyte apoptosis and aggravate OA by up-regulating Bax protein expression and down-regulating Bcl-2 protein expression; inhibiting the Notch signaling pathway may inhibit the chondrocyte apoptosis and relieve OA by up-regulating Bcl-2 protein expression and down-regulating Bax protein expression.
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 examine the expression of promoter CpG island methylation of Notch1 gene and explore the variable sites for DNA methylation in lung CD4 + T cells of asthmatic rat models.Methods An ovalbumin ( OVA) sensitized- challenged asthmatic rat model was established. Total T cells were isolated and CD4 + T lymphocytes were purified using magnetic beads. Twenty Wistar rats were randomly divided into a control group and an asthma group ( n = 10 in each group) . CD4 + T cells were isolated by immunomagnetic beads and identified by flow cytometry ( FCM) . Realtime PCR was employed to examine the mRNA expression of Notch1 gene in lung CD4 + T cells and the methylation level of Notch1 gene was examined by methylation-specific PCR. Results The mRNA expression of Notch1 in lung CD4 + T cells of the asthma group was 2. 254 ±0. 403 times as much as that of the control group. The total methylation level of asthma group was lower than that of the control group ( 0. 150 ±0. 108 vs. 0. 300 ±0. 667, P lt;0. 01) . Conclusion Promoter demethylation is one of the major mechanisms of Notch1 gene up-regulation in pathogenesis of asthma.
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).
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 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 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.