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Objective:To explore the possible effects of Bushen Huoxue Formula (the kidney tonifying and blood activating prescription) on the proliferation and extracellular matrix synthesis of nucleus pulposus cells by regulating the circ_0036763/miR-583 axis.Methods:Real time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of circ0036763 and miR-583 in normal and intervertebral disc degeneration (IDD) nucleus pulposus cells; IDD nucleus pulposus cells were divided into pcDNA group, pcDNA circ_0036763 group, pcDNA circ_0036763+ mimic NC group, and pcDNA circ_0036763+ miR-583 mimic group. qRT-PCR was used to detect the expression levels of circ_0036763 and miR-583 in nucleus pulposus cells in each group, methyl thiazolyl tetrazolium (MTT) was used to detect cell proliferation (A value), and Western blot was used to detect the expression of proliferating cell nuclear antigen (PCNA), collagen Ⅰ, and collagen Ⅱ proteins in nucleus pulposus cells, The dual luciferase assay reported experimental validation of the targeting relationship between circ_0036763 and miR-583. 27 mice were divided into sham surgery group, IDD group, and kidney tonifying and blood activating formula group. IDD models were established in all groups except for the sham surgery group. After successful modeling, the sham surgery group and IDD group were given physiological saline by gavage, while the kidney tonifying and blood activating formula group was given 1.5 g/ml of kidney tonifying and blood activating formula by gavage for 3 consecutive weeks. QRT-PCR was used to detect the expression levels of circ0036763 and miR-583 in the nucleus pulposus cells of mice in each group, MTT was used to detect cell proliferation, and Western blot was used to detect the expression of PCNA, collagen Ⅰ, and collagen Ⅱ proteins.Results:The expression level of circ_0036763 in IDD nucleus pulposus cells decreased, while the expression level of miR-583 increased (all P<0.05); Overexpression of circ_0036763 can promote proliferation and extracellular matrix synthesis of nucleus pulposus cells (all P<0.05); Circ_0036763 targets miR-583 and upregulates miR-583 reversible overexpression. Circ_0036763 enhances the proliferation and extracellular matrix synthesis ability of IDD nucleus pulposus cells. Compared with the sham surgery group, the IDD group showed an increase in collagen Ⅰ protein expression and miR-583 expression levels (all P<0.05), while the cell A value, PCNA and collagen Ⅱ protein expression, and circ_0036763 expression levels decreased (all P<0.05); Compared with the IDD group, the Kidney Tonifying and Blood Activating Formula group showed a decrease in collagen Ⅰ protein expression and miR-583 expression levels (all P<0.05), while the cell A value, PCNA and collagen Ⅱ protein expression, and circ_0036763 expression levels increased (all P<0.05). Conclusions:The kidney tonifying and blood activating formula (Bushen Huoxue) may induce proliferation and extracellular matrix synthesis of nucleus pulposus cells by regulating the circ_0036763/miR-583 axis.
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Objective To explore the proliferative changes and clinical significance of osteoclasts (OC) in various stages of osteoarthritis (OA). Methods Twenty healthy adult male SD rats were made the model by modified Hulht procedure,the left knee served as the control group and the right knee as the OA model group. The total knee joint (n=5) was collected at postoperative 1, 2,4,8 weeks, fixed at 4 ℃ 4% poly formaldehyde (PFA) liquid, embedded by paraffin for conducting sections, and stained by TRAP,toluidine blue(TB) and safranine O (Saf O)fast staining. Then the cartilage morphology change was observed and OC positive cells number with TRAP staining were semi-quantitatively detected,the OA cartilage destruction progression was evaluated by Mankin's method and SPSS17.0 statistics software was used to conduct statistical analysis. Results OC in the two groups showed the transient change of rapidly increasing and then decreasing. The OC number at 1 week in the control group (left knee) was (65.20±4.12) cells/mm2 ,and was gradually reduced at 2,4 weeks,which were (47.20±4.31) cells/mm and (26.20±3. 87) cells/mm2 ,which at 8 weeks was almost invisible, the number of cells was (7.00 ± 2.28) cells/mm2 ;the OC number at 1 week in the OA model group (right knee) was (70.40 ± 5.46) cells/mm2 ,increased to (86.20± 5.42)cells /mm2 at 2 weeks, reduced to (38. 0 ± 3.16) cells/mm2 at 4 weeks , was almost invisible at 8 weeks, the number of cells was (6.21 ± 2.93 ) cells/mm2 . The OC number at 2,4 weeks in the model group was significantly higher than that in the control group, the difference had statistical significance (P<0.05). Conclusion Large numbers of osteoclasts are proliferated in the early and middle stages of rat knee osteoarthritis, which indicating that OC might be involved in the formation of osteoarthritis.
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Objective To explore the effect of particulate matter (PM) 2.5 on the expression of pigment epithelium-derived factor (PEDF) protein in bronchial epithelial cells (BEAS-2B). Methods Human BEAS-2B were subcultivated, followed by low, medium and high concentrations of PM2.5 (25μg/ml, 50μg/ml, 100μg/ml) stimulation for 24 hours. The expression of PEDF protein in supernatant was ana-lyzed by enzyme-linked immunosorbent assay (ELISA), and the expression in BEAS-2B cells was detected by Western blotting. Results Compared with the control group, the expression of PEDF protein in supernatant and BEAS-2B cells induced by PM2.5 (25 μg/ml) in-creased, but no significance was found (t=-0.730, t=-1.840, P>0.05), and the expression induced by PM2.5 with the concentrations of 50μg/ml and 100μg/ml significantly increased (t>5.798, P<0.05). Conclusion PM2.5 with the concentrations of 50μg/ml and 100μg/ml could increase the expression of PEDF protein in a concentration-dependent manner both in supernatant and BEAS-2B cells.