| Objective: To investigate the Toxic Effects of Levofloxacin on Rat Annulus Fibrossus Cells.Methods: Main objective of this study was to explore the toxic effects of levofloxacin, one of typical fluoroquinolone antibiotic drug, on rat AF cells in vitro. Rat annulus fibrosus(RAF) cells were treated with levofloxacin at different concentrations(0μg/ml, 10μg/ml, 20μg/ml, 30μg/ml, 40μg/ml, 60μg/ml, 80μg/ml and 90 μg/ml) and were assessed to investigate the possible cytotoxic effects of levofloxacin. Inverted phase-contrast microscopy was used to perform the morphological observation of apoptosis of the treated cells. Western blot and Real-time quantitative RT- PCR(q PCR) was used to explore the expression of active caspase-3 and MMP-3. Flow cytometry was used to measure the apoptotic incidences.They were divided into 5 groups: pre-clinical stage of APACG group,remission period of APACG group, early stage of CPACG group, progress period of CPACG group and normal group. Heidelberg Spectralis OCT was used to measure the anterior segment biological parameters of each group. SPSS13.0 was used to compare with the anterior chamber width(ACW), angle opening distance(AOD), trabecular iris area(TISA), iris thickness(IL) and crystalline lens rise(CLR) of APACG group and CPACG group to normal group, pre-clinical stage group to remission period group, early stage group to progress period group. Above parameters were used t test and(or) independent samples nonparametric test for statistical analysis. The age of the groups were analyzed by variance analysis, gender ratio were analyzed by chi-square test.Results:(1)The first-passage RAF cells presented polygonal and/or long fusiform, with clear outline.RAF cells began to adhere to the bottom of culture flask in 24 or 48 hours。the cultured cells without levofloxacin grew well, and their shapes were fusiform or polygonous. However, the cells treated with levofloxacin at different concentration(30,60,90 μg/ml),presented shrinkage phenomenon and typical degenerative changes,such as multiple vacuoles in the cytoplasm.(2)Levofloxacin increased the percentage of early apoptotic cells in a concentration-dependent manner, as is shown in Fig. 2. After cells were treated respectively with levofloxacin at a concentration of 30,60,90μg/ml for 24 h,the apoptotic rate of RAF cells was found to be 2.70%±0.32%,6.41%±0.33%,9.35%±0.64%,12.83%±0.67%. The data indicated that levofloxacin induced apoptosis of RAF cells in a dose-dependent manner.(3)caspase-3 activity, a marker of apoptosis, was significantly increased after RAF cells were treated with levofloxacin(10 to 80 μg/ml) for 48 h in a concentration-dependent manner. The caspase-3 activity was promote to 3.1,3.9,4.5,6.4 times compared to control. This result promoted another evidence that levofloxacin could induce the apoptosis of RAF cells by enhancing the activity of active caspase-3.(4)MTS: Compared with the control group, the cell viability of different groups decreased to 95.8%, 91.5%, 88.2%, and 83.2%(at concentration of 10, 20, 40, 80 μg/ml) respectively.(5)Western blotting of caspase-3 subunit demonstrated that grey value(/β-actin) was 0.19±0.03,0.28±0.02,0.39±0.02,0.49±0.03(levofloxacin 30, 60 and 90 μg/ml for 24 h).Western blotting of MMP-3 subunit demonstrated that grey value(/β-actin) was 0.21±0.05, 0.45±0.06, 0.61±0.03, 0.81±0.03 0.04,0.78±0.06(levofloxacin 30,60 and 90 μg/ml for 24 h). q PCR to investigate(levofloxacin 30, 60 and 90 μg/ml for 24 h).(6) q PCR to investigate the expression of caspase-3:the relative ct value(/β-actin) was 0.19±0.03,0.31±0.06,0.65± the expression of MMP-3:the relative ct value(/β-actin) was 0.21±0.05,0.45±0.06,0.61±0.03,0.81±0.03(levofloxacin 30, 60 and 90 μg/ml for 24 h).Conclusions: Our study suggest that levofloxacin has cytotoxic effects on RAF cells characterized by enhancing apoptosis, reducing cell viability,promote the expression of caspaes-3 and MMP-3 and indicate a potential toxic effect of fluoroquinolones on RAF cells. |
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