| Objective: To further explore the changes in extracellular matrix and matrix metalloproteinases(MMP)of rat annulus fibrosus(AF)cells based on levofloxacin-induced apoptosis.Methods: Rat AF cells were primarily cultured by methods of enzyme digestion,and subcultured to next generation,AF cells of the passage 3(P3)were cultured in complete DMEM/ HIGH GLUCOSE(1×)without fetal bovine serum(FBS)and phenol red,and then treated with levofloxacin in a dose-and time-dependent manner.Dose-dependent group: AF cells were treated with levofloxacin at different concentrations(0μg/ml,10μg/ml,30μg/ml,60μg/ml,90μg/ml and 120μg/ml)for 24 h.Time-dependent group: AF cells were treated with levofloxacin(60μg/ml)for 0,8,12,24,36,48 h.Inverted phase-contrast microscopy was used to perform morphological observation of apoptotic cells.The mRNA expression levels of MMP-2 and-13 were quantified by reverse transcription and real-time quantitative polymerase chain reaction(RT-qPCR).Protein level of MMP-2 and MMP-13 were determined by western blot.Results: 1 The first-passage AF cells presented long fusiform,with clear outline.AF cells began to adhere to the bottom of culture flask in 24 or 48 hours.The cells were incubated for 8 or 9 days,during which time cells initiated logarithmic growth.the cultured cells without levofloxacin grew well,and their shapes were fusiform or polygonous.However,the cells treated with levofloxacin in a dose-and time-dependent manner,presented shrinkage phenomenon and typical degenerative changes,such as multiple vacuoles in the cytoplasm;2 caspase-3 activity,was significantly increased after AF cells were treated with levofloxacin(0 to 120μg/ml)for 24 h in a dose-dependent manner,the caspase-3 activity of the cells were 1.81、3.22、4.23、5.54、6.56 fold compared to control,data difference between groups was statistically significant(P<0.05).Treated with levofloxacin(60μg/ml)for 0 to 48 h in a time-dependent manner,the caspase-3 activity of the cells were 1.54,2.98,4.18,5.30,6.40 fold compared to control,data difference between groups was statistically significant(P<0.05);3 Cell adhesion to type Ⅰ collagen showed that: in dose-dependent group,the values of OD were 0.91,0.83,0.76,0.70,0.60 fold compared to control respectively,and data difference between groups was statistically significant(P<0.05).In time-dependent group: the values of OD were 0.91,0.82,0.75,0.67,0.61 fold compared to control respectively,and data difference between groups was statistically significant(P<0.05);4 RT-qPCR,in dose-dependent group: the 2-△△Ct value of MMP-2 and MMP-13 were 1.02±0.22,1.38±0.19,1.71±0.25,2.04±0.22,2.50±0.18,3.24±0.38 and 1.00±0.06,1.37±0.09,1.75±0.15,2.48±0.18,3.09±0.28,3.70±0.31 respectively,data difference between groups was statistically significant(P<0.05).In time-dependent group: the 2-△△Ct value of MMP-2 were 1.01±0.15,1.23±0.11,1.64±0.21,2.07±0.38,3.05±0.15,3.53±0.22;the 2-△△Ct value of MMP-13 were 1.01±0.13,1.33±0.17,1.88±0.32,2.54±0.48,3.18±0.47,3.84±0.46.In the time dependent group,whether it is MMP2 or MMP13,the control group and no statistical difference between the 8 h groups(P>0.05),statistically significant differences in other groups(P<0.05);5 Western blotting: in dose-dependent group: the grey value(/GAPDH)of MMP-2 and MMP-13 were 0.12±0.012,0.20±0.017,0.31±0.018,0.42±0.022,0.51±0.021,0.60±0.025;0.17±0.018,0.28±0.020,0.39±0.023,0.46±0.023,0.57±0.027,0.68±0.031.In time-dependent group: the grey value(/GAPDH)of MMP-2 and MMP-13 were 0.10±0.012,0.19±0.023,0.32±0.027,0.41±0.027,0.52±0.031,0.65±0.035;0.16±0.019,0.26±0.024,0.35±0.023,0.48±0.027,0.57±0.031,0.66±0.040.Whether it is in a dose dependent group or time dependent group MMP-2 and MMP-13 the difference between the groups was statistically significant(P<0.05).Conclusions: the results above suggest that the possible cytotoxic effects of levofloxacin on AF cells in vitro may be attributed to the decreased cell binding to type Ⅰ collagen and up-regulated expression of MMP-2 and MMP-13. |
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