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 investigate the effect of echinococcus granulosus protoscolices on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into fibroblasts.MethodsFemur bone marrow of 4-week-old C57BL/6 mice was taken and BMSCs were isolated and cultured by adherent culture. Echinococcus granulosus protoscolices was extracted from the liver of sheep infected with echinococcus granulosus. The experiment was divided into two groups. The experimental group was co-cultured with the 3rd generation BMSCs and the echinococcus granulosus protoscolices, and the control group was the 3rd generation BMSCs. Before and after co-culture, the morphology of BMSCs and the activity of echinococcus granulosus protoscolices were observed by inverted microscope. After cultured for 1, 3, 5, and 7 days, the mRNA expressions of transforming growth factor β1 (TGF-β1), collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescent quantitative PCR, the protein expressions of TGF-β1, collagen type Ⅰ, collagen type Ⅲ, Smad7, and phosphorylated Smad2/3 were detected by Western blot, and the contents of collagen type Ⅰ and collagen type Ⅲ in the supernatant of the two groups were detected by ELISA.ResultsAfter 7 days of co-culture, the morphology of BMSCs changed into fusiform and irregular triangle, which was closer to the mouse fibroblasts. The relative mRNA expressions of TGF-β1, collagen type Ⅰ, and collagen type Ⅲ in the experimental group were significantly higher than those in the control group; the relative protein expressions of TGF-β1, collagen type Ⅰ, collagen type Ⅲ, and phosphorylated Smad2/3 in the experimental group were significantly higher than those in the control group, and the relative protein expression of Smad7 in the experimental group was significantly lower than that in the control group; the contents of collagen type Ⅰ and collagen type Ⅲ in the supernatant of the experimental group were significantly higher than those in the control group. The differences between the two groups were significant (P<0.05).ConclusionEchinococcus granulosus protoscolices may promote the secretion of collagen type Ⅰ, collagen type Ⅲ, and TGF-β1 by TGF-β1/Smad signal pathway, which can promote the fibrosis of BMSCs that related to the formation of fibrocystic wall by echinococcosis.
Rheumatoid arthritis (RA) is a chronic autoimmune disease remarkably characterized by synovitis of joints, whose pathogenesis is complicated and not yet fully elucidated. A variety of cells, cytokines and intercellular signaling pathways are involved in the occurrence and development of RA. The mitogen activation protein kinase (MAPK) signaling pathway is closely related to the pathogenesis of RA, and plays an important role in the formation of pannus, synovitis, and bone destruction. This paper reviews the research progress of MAPK signaling pathway in RA from the aspects of the interaction of MAPK signaling pathway with a variety of key cells and cytokines in the pathogenesis of RA, in order to provide a direction and theoretical basis for anti-RA drug therapy research.
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.
Objective To summarize the regulations of Hedgehog signal ing pathway on the prol iferation and multidifferentiation of mesenchymal stem cells (MSCs). Methods The related l iterature in recent years concerning the regulations of Hedgehog signal ing pathway on the biological characteristics of MSCs was reviewed and analyzed. Results Hedgehog signal ing pathway promoted the prol iferation of MSCs, and played a major role in the induction of osteogenic and chondrogenic differentiations, but it inhibited the adi pocytic differentiation. Conclusion The regulations of Hedgehog signal ing pathway in MSCs multidifferentiation and prol iferation could be used as the new therapeutic targets of tissue ischemia, osteoporosis, achondroplasia, obesity, and so on.
ObjectiveTo investigate the role of PI3K/AKT/mTOR signaling pathway in skeletal muscle atrophy in rats with chronic obstructive pulmonary diseases(COPD). MethodsPassive cigarette smoking was used to establish COPD model.The protein expression of PI3K, total mTOR, phosphorylated-mTOR, total GSK-3β, phosphorylated-GSK-3β, total 4E-BP1, phosphorylated-4E-BP1, total p70S6K1 and phosphorylated-p70S6K1 in extensor digitorum longus of rats were measured by Western blot. ResultsThe protein expression of PI3K was not significantly different between two groups(P > 0.05).Compared with the control group, the protein expression of total mTOR, phosphorylated-mTOR, total GSK-3β, and phosphorylated-GSK-3βincreased significantly in the COPD group(P < 0.05).The protein expression of total 4E-BP1 and total p70S6K1 were not significantly different between two groups(P > 0.05).While the protein expression of phosphorylated-4E-BP1 and phosphorylated-p70S6K1 significantly increased in the COPD group(P < 0.05). ConclusionThe protein expressions of PI3K/AKT/mTOR signaling pathway in extensor digitorum longus increased significantly in COPD rats, suggesting that the activity of PI3K/AKT/mTOR signaling pathway increased, which may be one of the compensatory mechanism of skeletal muscle atrophy in COPD.
Objective To investigate the role of mitochondrial autophagy mediated by PINK1 (homologous phosphatase tensin induced kinase 1) /Parkin (Parkinson’s protein) signaling pathway in severe pneumonia of rats. Methods Twenty rats were randomly divided into control group and model group (severe pneumonia model), with 10 rats in each group, to explore the effects of severe pneumonia on lung function and pathology in rats. Then, 30 rats were randomly divided into control group, model group and mdivi-1 (mitochondrial autophagy inhibitor) group, with 10 rats in each group, to further explore the effects of severe pneumonia on mitochondrial autophagy indicators of rats. ResultsCompared with the control group, the resting ventilation volume [(3.44±0.22) vs. (1.58±0.18) mL/min] and airway resistance ratio (77.48±3.84 vs. 47.76±5.54) in the model group were decreased (P<0.05). In the model group, the lung tissue was injured and a large number of inflammatory cells were infiltrated. The protein and mRNA expression levels of Parkin, PINK1 and microtubule-associated protein1 light chain 3 in lung tissues of model group were increased (P<0.05). Compared with model group, the ratio of resting ventilator-to-airway resistance in mdivi-1 group increased (P<0.05). The injury and inflammatory infiltration of lung tissue were improved in mdivi-1 group. The expression levels of Parkin, PINK1 and microtubule-associated protein1 light chain 3 protein and mRNA in lung tissues of mdivi-1 group were decreased (P<0.05). Conclusion Mdivi-1 can improve the abnormal lung function structure in rats with severe pneumonia, and the mechanism may be related to mitochondrial autophagy mediated by PINK1/Parkin signaling pathway.
ObjectiveTo summarize the latest research progress of graphene and its derivatives (GDs) in bone repair. MethodsThe relevant research literature at home and abroad in recent years was extensively accessed. The properties of GDs in bone repair materials, including mechanical properties, electrical conductivity, and antibacterial properties, were systematically summarized, and the unique advantages of GDs in material preparation, functionalization, and application, as well as the contributions and challenges to bone tissue engineering, were discussed. ResultsThe application of GDs in bone repair materials has broad prospects, and the functionalization and modification technology effectively improve the osteogenic activity and material properties of GDs. GDs can induce osteogenic differentiation of stem cells through specific signaling pathways and promote osteogenic activity through immunomodulatory mechanisms. In addition, the parameters of GDs have significant effects on the cytotoxicity and degradation behavior.ConclusionGDs has great potential in the field of bone repair because of its excellent physical and chemical properties and biological properties. However, the cytotoxicity, biodegradability, and functionalization strategies of GDs still need to be further studied in order to achieve a wider application in the field of bone tissue engineering.
ObjectiveTo investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment. MethodsThe 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen typeⅡ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide. ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P < 0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P < 0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P < 0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen typeⅡand ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P < 0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen typeⅡprotein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P < 0.05); RCCS 1 group expressed high levels of collagen typeⅡand ANCN proteins at all stages (P < 0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups. ConclusionUnder the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.
ObjectiveTo explore the suppression of Wnt-1 pathway on non-small cell lung cancer (NSCLC) by establishing a NSCLC nude mice model of transplanting tumor in Xuanwei county. MethodsThere were 21 mice with tumor weight from 16-18 g and we divided them into a blank group (n=7), a control group (n=7), and an experiment group (n=7). The blank group were injected with saline, the control group were injected with docetaxel, and the experimental group were injected with Wnt-1 antibody. The mice were executed and the tumor specimens were obtained after six injections. We compared the volumes of the specimens and the inhibition rates of tumor among the three groups. ResultsThere was a statistical difference in volume between the blank group and the experiment group as well as the control group on the 21th and 27th day (P=0.002,P=0.000). The experiment within mice's body showed that both docetaxel and Wnt-1 antibody could inhibit NSCLC from growing, and the inhibition effect of docetaxel was stronger. ConclusionThe interdiction of Wnt-1 pathway is functional to restrain the growth of tumor. The docetaxel and Wnt-1 antibody have a positive effect on the treatment of NSCLC.