Antagonistic Effect Of Vitamin C Against Colistin Sulfate-induced Injury In PC12Cells

Although the effectiveness of colistin against most Gram-negative bacteria, especially Pseudomonas aeruginosa and Acinetobacter baumannii. has not been doubted. Since1980s colistin was strictly limited in the treatment of patients with cystic fibrosis who suffered from pulmonary infections due to multidrug-resistant bacteria, because of its nephrotoxicity and neurotoxicity. In the past decade, the emergence of Gram-negative bacteria that are resistant to almost all classes of available antibiotics except polymyxins and the shortage of new antibiotics with activity against them has led to the reuse of polymyxins colistin. However, neurotoxicity is still one of the major adverse effects limiting its clinical uses. It has been showed that Vitamin C was effective on protecting from colistin-induced nephrotoxicity mediated by oxidative stress. However, the effects of ascorbic acid on neurotoxicity induced by colistin are unknown. In the present study, an in vitro neurotoxicity model was established with PC12cells, we observe the neuroprotective effects of ascorbic acid on PC12cells against injury induced by colistin. and further elucidate its probable mechanisms, aiming at reducing neurotoxicity of colistin and providing theoretical basis and practical foundation in clinic.To confirm the toxic dose and time for subsequent experiments, after exposure to different concentrations of colistin (62.5.125,250and500μgmL-1) for different periods (2,6,12,24and48h), the cells were taken to MTT assay. An in vitro neurotoxicity model was established with PC12cells exposed to125μg·mL-1colistin sulfate for24h.0.1.1and10μmol·mL-1of vitamin C were added to the culture medium respectively, after incubation for24h, the morphological changes were observed under a microscope; cell viability was measured by MTT assay; colorimetry was taken to determine the content of lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS), glutathione (GSH) and the activities superoxide dismutase (superoxide dismutase, SOD) activity; we also use EL1SA assay to test intracyfoplasmic cytochrome C (Cyt-c) and DNA fragmentation, real-time quantitative PCR to analysis the expression of apoptotic gene caspase-9and caspase-3. The results showed that:(1) Result of inverted microscope observation showed that with the concentration of vitamin C increased, the growth state of cell trended to the normal group, the number of cell increased, soma satiated, both adhension and refraction of cell enhanced.(2) The result of MTT assay showed1and10μmol·mL-1vitamin C can significantly improve cell viability.(3) LDH assay showed vitamin C can weaken the damage of the cell membrane damage induced by colistin sulfate.(4) Vitamin C can reduce the content of ROS, raise the level of GSH and enhance activity of SOD to elevate cellular antioxidant capacity.(5) Vitamin C decreases the release of cyt-c, reduces the level of DNA fragmentation, inhibits the expression of apoptotic gene caspase-9and-3in dose-dependent manners.In summary, vitamin C can maintain normal morphology, improve cell viability, reduce oxidative stress and suppress apoptosis mediated by the mitochondrial pathway in colistin sulfate-injured PC12cells. Taken together, ascorbic acid effectively protects PC12cells against colistin sulfate-induced neurotoxicity and suggests a neuroprotective effect on colistin insult.