ObjectiveTo investigate the regulatory effect of resveratrol (RES) on the extracellular matrix (ECM) expression of nucleus pulposus cells (NPC), and its relative molecular mechanism.MethodsTen patients receiving discectomy were collected, of which 5 patients were young with spinal burst fracture, classified as control group; the rest 5 patients were senile with lumbar disc herniation, classified as degenerative group. The nucleus pulposus tissue of 2 groups were collected, the in situexpression of β-catenin was detected by immunohistochemistry, and the protein expressions of collagen type Ⅱ and Aggrecan were detected by Western blot. The NPC were isolated and cultured from degenerative nucleus pulposus tissues. RES treated the third-passage NPC with (group B) or without IL-1β (group C), to further determine the protein expressions of collagen type Ⅱ and Aggrecan by Western blot, the unstimulated cells were set up as blank control group (group A). Moreover, NPC treated with small interfering RNA (siRNA) targeted silent SIRT1 or β-catenin were used to determine the protein and gene expressions of β-catenin and SIRT1 by Western blot and real-time fluorescence quantitative PCR. In addition, the third-passage NPC treated with complete medium (group 1), IL-1β (group 2), RES+IL-1β (group 3), and SIRT1-siRNA+RES+IL-1β (group 4) for 24 hours were used to detect the nuclear translocation of β-catenin by cell immunofluorescence staining. Finally, the third-passage NPC treated with complete medium (group Ⅰ), IL-1β (group Ⅱ), IL-1β+β-catenin-siRNA (group Ⅲ), IL-1β+RES (group Ⅳ), and IL-1β+RES+SIRT1-siRNA (group Ⅴ) for 24 hours were used to detect the protein expressions of collagen type Ⅱ and Aggrecan by Western blot.ResultsImmunohistochemical staining and Western blot detection showed that when compared with control group, the cell proportion of expression of β-catenin were significantly increased in degenerative group (t=4.616, P=0.010); the protein expression of β-catenin was also significantly increased and the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased (P<0.05). In cytology experiments, the protein expression of β-catenin in group B was significantly higher than that in groups A and C, and the protein expressions of collagen type Ⅱ and Aggrecan in group B were significantly lower than those in groups A and C (P<0.05). After transfection of siRNA, the protein expressions of SIRT1 and β-catenin significantly decreased (P<0.05). The results of cell immunofluorescence staining further confirmed that when compared with group 3, after the SIRT1 was silenced by siRNA in group 4, the attenuated nuclear translocation of β-catenin by RES treatment was aggravated. Western blot results showed that the protein expressions of collagen type Ⅱ and Aggrecan in group Ⅱ were significantly lower than those in group Ⅰ(P<0.05); after transfection of β-catenin-siRNA in group Ⅲ, the degradation of ECM by IL-1β was obviously inhibited, the protein expressions of collagen type Ⅱ and Aggrecan were significantly increased when compared with group Ⅱ (P<0.05); after transfection of SIRT1-siRNA in group Ⅴ, the protective effect of RES on the degradation of ECM was inhibited, the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased when compared with group Ⅳ (P<0.05).ConclusionRES regulates the ECM expression of NPC via Wnt/β-catenin signaling pathway, which provide a new idea for intervertebral disc degeneration disease treatment.
ObjectiveTo understand the research status of phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway in the thyroid cancer (TC), as well as its role in the occurrence, cell differentiation, invasion, and metastasis of the TC, so as to find potential targets for treatment of TC. MethodThe literature about the research of PI3K/AKT signaling pathway in the TC was searched and summarized. ResultsThe PI3K/AKT signaling pathway was abnormally activated directly or indirectly in the TC, resulting in inhibition of cell apoptosis, malignant proliferation, accelerated cycle progression, invasion, and metastasis, etc., which promoted the occurrence and development of the TC. There were also some tumor suppressor genes, microRNAs, long chain non-coding RNAs, etc., which indirectly inhibited the activation of PI3K/AKT signaling pathway, or directly acted on it inhibiting its activity to inhibit the occurrence and development of the TC. ConclusionsFor the TC, some proteins, genes, microRNAs, and long chain non-coding RNAs directly or indirectly activate the PI3K/AKT signaling pathway through different targets to promote the occurrence and development of TC. At the same time, many targets inhibit the activation of the PI3K/AKT signaling pathway, which inhibits the malignant proliferation, invasion, and metastasis of TC. At present, there have been studies trying to use PI3K/AKT signaling pathway as a breakthrough for the treatment of TC. In-depth exploration of the role of PI3K/AKT signaling pathway in different TC is of great significance to find new targets for the treatment of TC.
ObjectiveTo investigate effects of high expression of miR-499a-5p on lung injury in rats with acute respiratory distress syndrome (ARDS) by targeting matrix metallopeptidase-16 (MMP-16).MethodsThe experiment set up sham operation group, model group, miR-499a-5p mimic group, MMP-16 group, miR-499a-5p mimic+MMP-16 group, D-ribofuranosylbenzimidazole (DRB, Nrf2 signaling pathway inhibitor) group, miR-499a-5p mimic+DRB group. A rat model of ARDS was constructed by cecal puncture. One hour before surgery, the transfection complex (50 μL) was injected into the trachea with a micro-syringe. DRB (5 mg/kg) was intraperitoneally injected 30 min before surgery. The expression levels of miR-499a-5p and MMP-16 in lung tissue were detected by RT-qPCR; Alveolar type Ⅱ epithelial cells of model group rats were separated and MMP-16 3 'UTR WT and MUT luciferase report plasmid were transfected into alveolar type Ⅱ epithelial cells with miR-499 respectively to verify the targeting relationship between miR-499 and MMP-16; the targeted relationship was verified by the dual luciferase reporter gene; lung injury was observed by hematoxylin-eosin staining; The level of inflammatory factors in bronchoalveolar lavage fluid (BALF) and the level of oxidative stress in lung tissue were detected by enzyme-linked immunosorbent assay; The expression levels of NAD(P)H: quinone oxidoreductase 1 (NQO1), heme oxygenase (HO)-1, and nuclear factor-erythroid 2-related factor 2 (Nrf2) proteins in lung tissues were analyzed by Western blotting.ResultsmiR-499a-5p was down-regulated in the lungs of ARDS model rats (P<0.01), while MMP-16 was highly expressed (P<0.01); miR-499a-5p and MMP-16 3'UTR regions had binding sites, and miR-499a-5p directly targeted negative regulation of MMP-16 expression (P<0.01); overexpression of miR-499a-5p significantly reduced the right lung wet-to-dry weight ratio in the ARDS rats (P<0.05), reduced lung tissue damage (P<0.01), and reduced tumor necrosis factor α, interleukin (IL)-1β and IL-6 levels in BALF (P<0.01), decreased malondialdehyde and myeloperoxidase levels in lung tissue, increased total anti-oxidant capacity (P<0.01), and up-regulated NQO1, HO-1, Nrf2 protein expression in lung tissue (P<0.01). However, this phenomenon was significantly reversed after the addition of MMP-16 and DRB.ConclusionOverexpression of miR-499a-5p attenuates lung injury in rats with ARDS by targeting negative regulation of MMP-16 via activating the Nrf2 signaling pathway.
ObjectiveTo understand the molecular mechanisms and targeted therapy research progress of gastric cancer associated with the RTK/RAS signaling pathway, in order to provide reference for treatment of gastric cancer. MethodThe related literatures about the molecular mechanism and targeted therapy of RTK/RAS signaling pathway related gastric cancer at home and abroad in recent years were reviewed. ResultsTargeted therapy had been widely applied in the treatment of gastric cancer associated with the RTK/RAS signaling pathway, showing good efficacy and significantly prolonging patients’ survival time, further deepening the understanding of the molecular mechanisms of gastric cancer. Targeted therapies for gastric cancer associated with the RTK/RAS signaling pathway focused on human epidermal growth factor receptor 2 (HER-2), epidermal growth factor receptor, fibroblast growth factor receptor 2, cellular-mesenchymalepithelial transition factor and Kirsten ratsarcoma viral oncogene homolog associated targets. Currently, there were many drugs targeting HER-2 target, while research on other targets mostly remains in the clinical trial stage, and showing promising prospects. ConclusionTargeted therapy can benefit most patients with gastric cancer, but the drug resistance and multi-drug combination therapy are still difficult problems that we need to overcome in the future.
ObjectiveTo investigate the expression pattern and significance of Sonic Hedgehog (Shh) signaling pathway by observing whether the Shh signaling pathway components express in the adult rat after spinal cord injury (SCI). MethodsSixty-four healthy male Sprague-Dawley rats were randomly divided into normal group (group A, 8 rats), sham group (group B, 8 rats), and SCI group (group C, 48 rats). In group A, the rats served as controls without any treatment; a decompressive laminectomy was performed on T7-9 levels without SCI in group B; and modified Allen's method was used to make SCI model in group C. Basso Beattie Bresnahan (BBB) scale was used to assess the hind limb motor function at 12 hours, 1 day, 3 days, 7 days, 14 days, and 21 days after SCI; the immunofluorescence staining, real-time PCR, and Western blot were performed to detect the mRNA and protein expression levels of Shh and Glioma-associated oncogene homolog-1 (Gli-1) in SCI zone. ResultsThe BBB score slowly increased with time in group C, but the scores at each time point in group C were significantly lower than those in group A and group B (P<0.05). The results of immunofluorescence staining showed that Shh and Gli-1 rapidly increased after SCI in astrocytes. Real-time PCR and Western blot showed that the relative expression levels of Shh and Gli-1 mRNA and protein were gradually increased in group C and reached a maximum at 7 days. In addition, the relative expression levels of Shh and Gli-1 mRNA and protein in group C were significantly higher than those in group A and group B (P<0.05). On the other hand, compared with group A, the expression of Gli-1 protein was reduced in the cytoplasm but increased in nucleus in group C. ConclusionAstrocytes synthesize and secrete Shh and Gli-1 signaling molecules after SCI, both Shh and Gli-1 significantly up-regulate and exhibit dynamic changes, which suggests Shh signaling pathway may be involved in nerve cell regeneration after SCI.
ObjectiveTo explore the role of joint regulation of Wnt and bone morphogenetic protein (BMP) signaling pathways in the differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes.MethodsHiPSCs were cultured and observed under inverted phase contrast microscope. Immunofluorescence staining was used to observe the expressions of hiPSCs pluripotent markers (OCT3/4, NANOG, and TRA-1-60). HiPSCs were passaged which were taken for subsequent experiments within the 35th passage. When the fusion degree of hiPSCs was close to 100%, the CHIR99021 (Wnt pathway activator) was added on the 0th day of differentiation. Different concentrations of IWP4 (inhibitor of Wnt production) were added on the 3rd day of differentiation, and the best concentration of IWP4 was added at different time points. The optimal concentration and the best effective period of IWP4 were obtained by detecting the expression of troponin T (TNNT2) mRNA by real-time fluorescence quantitative PCR. Then, on the basis of adding CHIR99021 and IWP4, different concentrations of BMP-4 were added on the 5th day of differentiation, and the best concentration of BMP-4 was added at different time points. The optimal concentration and best effective period of BMP-4 were obtained by detecting the expression of TNNT2 mRNA. Finally, hiPSCs were divided into three groups: Wnt group, BMP group, and Wnt+BMP group. On the basis of adding CHIR99021 on the 0th day of differentiation, IWP4, BMP-4, and IWP4+BMP-4 were added into Wnt group, BMP group, and Wnt+BMP group respectively according to the screening results. Cells were collected on the 7th and the 15th days of differentiation. The expressions of myocardial precursor cell markers [ISL LIM homeobox 1 (ISL1), NK2 homeobox 5 (NKX2-5)] and cardiomyocyte specific markers [myocyte enhancer factor 2C (MEF2C), myosin light chain 2 (MYL2), MYL7, and TNNT2] were detected by real-time fluorescent quantitative PCR. Cells were collected on the 28th day of differentiation, and the expression of cardiac troponin T (cTnT) was detected by flow cytometry and immunofluorescence staining.ResultsThe results of cell mophology and immunoflurescence staining showed that the OCT3/4, NANOG, and TRA-1-60 were highly expressed in hiPSCs, which suggested that hiPSCs had characteristics of pluripotency. The optimal concentration of IWP4 was 10.0 μmol/L (P<0.05) and the best effective period was the 3rd day (P<0.05) in inducing hiPSCs to differentiate into cardiomyocytes. The optimal concentration of BMP-4 was 20.0 ng/mL (P<0.05) and the best effective period was the 3rd day (P<0.05). The relative expressions of ISL1, NKX2-5, MEF2C, MYL2, MYL7, and TNNT2 mRNAs, the positive expression ratio of cTnT detected by flow cytometry, and sarcomere structure detected by immunofluorescence staining of Wnt+BMP group were superior to those of Wnt group (P<0.05).ConclusionJoint regulation of Wnt and BMP signaling pathways can improve the differentiation efficiency of hiPSCs into cardiomyocytes.
After the articular cartilage injury, the metabolic level is increased during the progressive degeneration, the chondrocytes secrete a variety of inflammatory factors, and the original cell phenotype is gradually changed. For a long time, a large number of researchers have done a lot of researches to promote anabolism of chondrocytes and to maintain the stability of chondrocyte phenotype. There are many molecular signaling pathways involved in the process of promoting cartilage repair. This review focuses on the key signaling molecules in articular cartilage repair, such as transforming growth factor-beta and bone morphogenetic protein, and reveals their roles in the process of cartilage injury and repair, so that researchers in related fields can understand the molecular mechanism of cartilage injury and repair widely and deeply. Based on this, they may find promising targets and biological methods for the treatment of cartilage injury.
Objective To investigate the effect of picroside Ⅱ (PIC Ⅱ) on the pyroptosis and thioredoxin-interacting protein (TXNIP)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signaling pathway in alveolar epithelial cells of severe pneumonia rats. Methods A severe pneumonia rat model was constructed and all experimental rats were divided into a control group, a severe pneumonia group, low, medium, and high dose PIC Ⅱ groups (PIC Ⅱ-L, PIC Ⅱ-M, PIC Ⅱ-H groups), and a high-dose PIC Ⅱ+TXNIP/NLRP3 pathway activator trimethylamine oxide group (PIC Ⅱ-H+TMAO group). The levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were detected by ELISA; Wright’s staining was applied to detect eosinophil count (EOS), lymphocyte count (LYM), and neutrophil count (NEU) in the sediment of alveolar lavage fluid. Hematoxylin-eosin staining was used to observe the pathological changes of lung tissue. The expressions of cysteine aspartate protease 1 (Caspase-1) and dermatin D (GSDMD) were detected by immunohistochemistry. The expressions of TXNIP, NLRP3 and apoptosis-associated microprotein (ASC) were detected by Western blot. Results Compared with the control group, the severe pneumonia group had severe lung tissue injury, obvious inflammatory cell infiltration, and increased expressions of TNF-α, IL-1β, IL-6, EOS, LYM, NEU, Caspase-1, GSDMD, TXNIP, NLRP3 and ASC (all P<0.05). Compared with the severe pneumonia group, lung tissue injury in PIC Ⅱ-L, PIC Ⅱ-M and PIC Ⅱ-H groups was reduced successively, and inflammatory cell infiltration was gradually reduced. The expressions of TNF-α, IL-1β, IL-6, EOS, LYM, NEU, Caspase-1, GSDMD, TXNIP, NLRP3 and ASC were decreased successively (all P<0.05). Compared with the PIC Ⅱ-H group, the PIC Ⅱ-H+TMAO group showed increased lung tissue damage and obviously increased inflammatory cell infiltration, the expression of TNF-α, IL-1β, IL-6, EOS, LYM, NEU, Caspase-1, GSDMD, TXNIP, NLRP3, and ASC were obviously increased (all P<0.05). Conclusion PIC Ⅱ inhibits pyroptosis of alveolar epithelial cells in severe pneumonia rats by inhibiting the TXNIP/NLRP3 pathway.
Objective To investigate the role of Notch signaling pathway in pancreas development and pancreatic cancer. Methods The related literatures were reviewed and analyzed. Results Notch signaling played a role early in development by maintaining pancreatic epithelial cells in a progenitor state and delaying their differentiation until timely appropriate. Notch signaling was reactivated in the initiation and progression of pancreatic cancer. Conclusion Notch signaling pathway plays an important role in the pancreas development. Sustained Notch signaling activity promotes the progression of pancreatic cancer, and may be one of major factors in the initiation of pancreatic cancer.
Objective To explore the role of sulforaphane (SFN) on the regulation of airway mucin 5AC mucin expression in the airway epithelial cell line (A549), and the underlying mechanism. Methods The experiments were performed in culture of PMA and H2O2 induced A549 in vitro. A549 cells divided into three groups: the control group, the model group (PMA and H2O2) and the intervention groups (SFN+PMA; SFN+H2O2). The intervention group's cells were pretreated with SFN for 30 mins before exposure to stimuli (PMA or H2O2). The MUC5AC, Nrf2 and the antioxidant gene HO-1, NQO1, GCLC mRNA levels were analyzed by real time-PCR, and protein production was assayed by western blot. Results Compared with the control group, expression of MUC5AC mucin was increased after being stimulated by PMA or H2O2 (P<0.05), but it was significantly inhibited by SFN (P<0.05). SFN induced the expression of Nrf2 gene and the antioxidant gene HO-1 (compared with the control and model groups,P<0.05). Conclusion Sulforaphane involves the airway mucous hypersecretion induced by PMA and H2O2, and Nrf2/HO-1 signaling pathway may play an important role in mucin MUC5AC regulation.