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 study the effect of signal-selective parathyroid hormone (PTH) analogue peptide on Wnt signal ing factors in osteoblasts isolated from neonatal mouse, and provide theoretical basis for the mechanism of PTH’s function in bone metabolism. Methods Osteoblasts were isolated from calvaria of 2-3-day-old C57BL neonatal mouse and identified by alkal ine phosphatase (ALP) staining, and Alizarin red staining. The cells at passage 1 were divided into 4 groups: control group, PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group. Then the medium was changed to α-MEM supplemented with 1%FBS. After 12 hours, trifluoroacetic acid or three peptides [(10 nmol/L PTH (1-34), 10 nmol/L G1R19 (1-34), and 100 nmol/L G1R19 (1-28)] were added into the culture medium. After 4 hours, the cells were washed gently ithcold PBS 3 times before total RNA was isolated. The expressions of Wnt related genes were measured by quantitative eal-time PCR. Results Most of the cells were polygonal and triangular; the cells were positive for ALP staining with blue cytoplasm at 14 days and the Al izarin red staining showed the formation of red mineral ized nodules in the special mineral ization induction medium at 28 days. The expressions of osteocalcin mRNA and Wnt5b mRNA in PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group were significantly higher than those in control group (P lt; 0.05); the expression of Wnt2 mRNA was significantly lower than that in control group (P lt; 0.05); the expression of β-catenin mRNA in PTH (1-34) group was significantly higher than that in control group (P lt; 0.05); the expression of Wnt7b mRNA in PTH (1-34) group and G1R19 (1- 34) group was higher than that in control group, and the G1R19 (1-34) group was higher than PTH (1-34) group and G1R19 (1-28) group (P lt; 0.05). Conclusion In the Wnt-related factors, PTH (1-34) and G1R19 (1-34) affect mainly canonical Wnt signal factors, but the G1R19 (1-28) chiefly acts on non-canonical Wnt signal factors.
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.
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.
Continuous activation of Janus kinase (JAK)- signal transduction and activator of transcription (STAT) signaling pathway is prevalent in leukemia cells, and it has been found that this pathway plays an important role in acute leukemia (AL). JAK2/JAK1 gene mutations are found in both acute myelocytic leukemia and acute lymphoblastic leukemia and may have implications for the treatment and overall prognosis of the disease. Among the STAT family members, STAT3 and STAT5 proved to be key factors in AL. These gene mutations may provide new targets and new ideas for the treatment of AL. This article provides a review of the research progress of JAK-STAT signaling pathway, related gene mutations and AL.
Objective To explore the relationships between the polymorphisms of inhibitor genes WIF1 and DKK1 in WNT signaling pathway and susceptibility to tuberculosis, clinical characteristics and laboratory indexes. Methods From December 2014 to November 2016, 475 tuberculosis patients and 370 healthy controls of West China Hospital of Sichuan University were enrolled in the study, and the clinical data of the subjects were collected. High-throughput genotyping technique was used to detect the polymorphisms of WIF1 rs58635985 and DKK1 rs11001548 in WNT signaling pathway. The allele frequency distribution, genotype, genetic model, clinical features and laboratory indexes of two single nucleotide polymorphisms were analyzed by χ2 test and logistic regression analysis. Results There was no significant difference in the allele frequency distribution (P=0.275, 0.949), genotype (P=0.214, 0.659) or genetic models: additive model (P=0.214, 0.659), dominant model (P=0.414, 0.827), recessive model (P=0.227, 0.658) of rs58635985 and rs11001548 between the tuberculosis group and the healthy control group. Subgroup analysis showed no significant difference in allele and genotype distribution between rs58635985 and rs11001548 (pulmonary tuberculosis group vs. healthy control group: P>0.05; pulmonary tuberculosis groupvs. extra-pulmonary tuberculosis group: P>0.05). There was no significant difference in the clinical features (fever, night sweat, fatigue,etc.) or laboratory indexes (complete blood count, erythrocyte sedimentation rate, TB-DNA, etc.) (P>0.05). Conclusions There is no association between rs58635985 of WIF1 gene or rs11001548 of DKK1 gene and genetic susceptibility, clinical characteristics and laboratory indexes in Han population in Western China. To expand the sample size for verification and analysis in different populations is necessary.
ObjectiveTo investigate the effects of telmisartan on the proliferation, migration and apoptosis of non-small cell lung cancer A549 and the mechanism of regulating Wnt signaling pathway.MethodsNon-small cell lung cancer cell line A549 was cultured in vitro. Cell counting kit-8 (CCK-8) assay was used to detect the effect of telmisartan at different concentrations on the proliferative activity of A549 cells. The survival fraction of A549 treated with different concentrations of telmisartan was determined by colony-formation assay. The effect of telmisartan at different concentrations on the migration ability of A549 cells was examined in the wounding healing assay. Hoechst staining was used to detect the effects of telmisartan at different concentrations on the apoptosis of A549. Western bloting was used to detect the expressions of β-actin, proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, Wnt-3a, Beta-catenin (β-catenin), serine protein kinase 3β (p-GSK-3β), glycogen synthase kinase-3β (GSK-3β) and c-myc.ResultsDifferent concentrations of telmisartan treatment inhibited the proliferation activity, colony-formation rate and migration of A549 cells, and reduced the expression of PCNA in a concentration-dependent manner. Telmisartan treatment promoted the apoptosis of A549 cells, significantly increased the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl-2. The expression levels of Wnt-3a, β-catenin, p-GSK-3β, and c-myc in A549 cells increased after treatment with telmisartan, while the expression levels of GSK-3β decreased.ConclusionTelmisartan may play a role in the proliferation, migration and apoptosis of non-small cell lung cancer A549 cells, and inhibiting the Wnt/β-catenin signaling pathway may be one of the mechanisms.
ObjectiveTo investigate the expression of Yes-associated protein (YAP) screened by bioinformatics in rats with myocardial-ischemia reperfusion injury and establish the base for further research. MethodsThe difference of gene spectrum of rats with myocardial-ischemia reperfusion injury was analyzed by bioinformatics technique. The related signaling pathways and key genes were screened by KOBAS2.0 and KEGG. Eighteen Sprague Dawley rats were randomly divided into three groups: normal group (n=6), sham operation group (n=6) and myocardial-ischemia reperfusion injury group (n=6). The expression of target gene was detected by immunochemistry, quantitive reverse transcription polymerase chain reaction and western blotting. ResultsA total of 345 differentially expressed genes were found by bioinformatics, among which 181 were up-regulated and 164 were down-regulated. The differential genes were mainly enriched in Wnt, HIPPO, MAPK, Jak-STAT and other signaling pathways. We focused on HIPPO pathway and found that the expression of YAP increased significantly in myocardial-ischemia reperfusion injury group, compared with the normal group and sham operation group (P＜0.05). ConclusionsThe expression of YAP of HIPPO signal pathway is increased in rats with myocardial-ischemia reperfusion injury.
Objective To explore the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and the combination of bFGF and EGF in the neural differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and the role of Wnt/β-catenin signaling pathway in this process. MethodsThe identified 4th-generation hBMSCs were divided into five groups according to different induction conditions, namely control group (group A), EGF induction group (group B), bFGF induction group (group C), EGF and bFGF combined induction group (group D), and EGF, bFGF, and Dickkopf-related protein 1 (DKK-1) combined induction group (group E). After 7 days of continuous induction, the cell morphology was observed by inverted fluorescence phase contrast microscopy, levels of genes that were related to neural cells [Nestin, neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)] and key components of the Wnt/β-catenin signaling pathway (β-catenin and Cyclin D1) were detected by RT-PCR, and the levels of proteins that were related to neural cells (Nestin and GFAP) as well as genes that were involved in Wnt/β-catenin signaling pathway [β-catenin, phosphorylation β-catenin (P-β-catenin), Cytoplasmic β-catenin, and Nuclear β-catenin] were explored by cellular immunofluorescence staining and Western blot. ResultsWhen compared to groups A and B, the typical neuro-like cell changes were observed in groups C-E, and most obviously in group D. RT-PCR showed that the relative expressions of Nestin, NSE, and MAP-2 genes in groups C-E, the relative expressions of GFAP gene in groups D and E, the relative expression of NSE gene in group B, the relative expressions of β-catenin gene in groups C and D, and the relative expressions of Cyclin D1 gene in groups B-D significantly increased when compared with group A (P<0.05). Compared with group E, the relative expressions of Nestin, NSE, MAP-2, GFAP, β-catenin, and CyclinD1 genes significantly increased in group D (P<0.05); compared with group C, the relative expression of Nestin gene in group D significantly decreased (P<0.05), while NSE, MAP-2, and GFAP genes significantly increased (P<0.05). The cellular immunofluorescence staining showed that the ratio of NSE- and GFAP-positive cells significantly increased in groups C-E than in group A, in group D than in groups C and E (P<0.05). Western blot assay showed that the relative expression of NSE protein was significantly higher in groups C and D than in group A and in group D than in groups C and E (P<0.05). In addition, the relative expression of GFAP protein was significantly higher in groups C-E than in group A and in group D than in group E (P<0.05). Besides, the relative expressions of β-catenin, Cytoplasmic β-catenin, Nuclear β-catenin, and the ratio of Nuclear β-catenin to Cytoplasmic β-catenin were significantly higher in groups C and D than in group A and in group D than in group E (P<0.05), whereas the relative expression of P-β-catenin protein was significantly lower in groups C and D than in group A and in group D than in group E (P<0.05). Conclusion Different from EGF, bFGF can induce neural differentiation of hBMSCs. In addition, EGF can enhance the hBMSCs neural differentiation of bFGF, while the Wnt/β-catenin signaling pathway may play a positive regulatory role in these processes.
The classical Hippo pathway leads to the phosphorylation of downstream effector molecules Hippo-Yes-associated protein (Yap) and transcriptional coactivator PDZ-binding motif (Taz) serine sites through a kinase response, thereby promoting cell proliferation, controlling cell polarity, changing cytoskeleton, it plays an important regulatory role in various pathophysiological processes such as epithelial-mesenchymal transition and inhibition of cell contact. Studies have shown that Yap/Taz can affect the progression of vitreoretinal diseases, opening up new prospects for the pathogenesis and clinical treatment of diabetic retinopathy, proliferative vitreoretinopathy, and retinal ischemia-reperfusion injury. Exploring the molecular mechanism of Yap/Taz provides a possible therapeutic target for future research in the treatment of retinal fibrosis diseases such as diabetic retinopathy and proliferative vitreoretinopathy. At the same time, regulating the activity of local Yap/Taz in the retina will also become an effective therapeutic target for damage-repair in retinal ischemia-reperfusion injury. However, Yap inhibitors have potential retinal toxicity and are still in the preclinical development stage. Further research on the mechanism of action and clinical safety of Yap inhibitors will provide new methods for the treatment of retinal diseases.