Effect Of N-acetylcysteine On The Ability Of Macrophages To Remove Vancomycin-sensitive Staphylococcus Aureus With Different Sensitivity

ObjectiveStaphylococcus aureus is a common clinical pathogen,which can cause lobar pneumonia,pericarditis,meningitis and other infectious diseases.According to the widespread use of antibiotics,there is a trend of multi-drug resistance and pan-drug resistance.N-acetylcysteine(NAC)is the precursor of glutathione and also an antioxidant containing sulfydryl groups.Reactive oxygen species(ROS)are chemical substances containing oxygen,including peroxide,superoxide,hydroxyl radical,singlet oxygen and other forms.ROS is a normal metabolite of oxygen and plays an important role in normal metabolic processes such as cell signaling.However,under the influence of factors such as infection and environmental pressure,ROS levels can sharply increase to cause serious damage to cellular structure,which is known as oxidative stress.When a cell is infected by bacteria,it will produce a variety of ROS,such as hydrogen peroxide,hydroxyl radical and superoxide.If the ROS produced is not cleared in time,the cell will have a series of pathological reactions,and the excess ROS produced will cause oxidative damage to the cell.Macrophages are the first line of defense of the innate immune system in the human body,and bronchial epithelial cells are the main defense barrier of the respiratory system.This study intends to use NAC to pretreat RAW264.7 macrophages and Beas2 b bronchial epithelial cells,and observe that after the cells are infected with vancomycin sensitive Staphylococcus aureus(VSSA)strain 11 or vancomycin moderately resistant Staphylococcus aureus(VISA)strain 11 y,the changes in the number of bacteria surviving within cells and the levels of ROS produced by cells,as well as the impact of NAC on the growth of these cells and strains.This study will provide new ideas for clinical control of drug-resistant Staphylococcus aureus infections.Methods1.MTT experiment was conducted to detect the effect of different concentrations of NAC on the growth of RAW264.7 macrophages and Beas2 b bronchial epithelial cells,and the optimal concentration of NAC was selected based on the experimental results.2.Using bacterial biofilm formation experiments to investigate the effect of different concentrations of NAC on the biofilm formation ability of strains 11 and 11 y.3.Colony formation counting experiment: Based on the MTT test results,RAW264.7 macrophages and Beas2 b bronchial epithelial cells were pretreated with appropriate concentrations of NAC.The two cells were infected with strains 11 or 11 y,respectively.After cell lysis,the number of viable bacteria in the cells was detected using agar plate colony formation counting method.4.ROS assay was used to detect the changes in ROS production by RAW264.7macrophages and Beas2 b bronchial epithelial cells after infection with strain 11 or 11 y.Results1.The MTT experiment results showed that when the concentration of NAC was between 0.5 m M and 20 m M,it had a promoting effect on the growth of RAW264.7cells,and only had a slight inhibitory effect on cell growth at 0.25 m M concentration.When the concentration of NAC is less than 2 m M,it has a promoting effect on the growth of Beas2 b cells;When the concentration of NAC is more than 5 m M,it exhibits an inhibitory effect on the growth of Beas2 b cells.2.The experimental results of bacterial biofilm formation showed that under the NAC concentrations from 0.25 m M to 20 m M,the ability of strains 11 and 11 y bacterial biofilm formation was inhibited within 4 hours.When the NAC concentration is 5 m M,the biofilm formation ability of strain 11 is the weakest;When the NAC concentration is 0.5 m M,the biofilm formation ability of strain 11 y is the weakest.3.The results of the colony formation counting experiment showed that compared with the control group,the number of strains 11 or 11 y entering RAW264.7 cells and Beas2 b cells pre-treated with NAC decreased significantly.4.The ROS experiment results showed that compared with the control group,the ROS produced by RAW264.7 cells and Beas2 b cells pre-treated with NAC significantly decreased after infecting strains 11 or 11 y.Conclusion1.NAC can inhibit the biofilm formation of strains 11 and 11 y,and decrease the infection of Staphylococcus aureus.2.NAC can significantly inhibit the survival of strains 11 or 11 y in RAW264.7macrophages and Beas2 b bronchial epithelial cells.3.NAC can inhibit the ability of RAW264.7 macrophages and Beas2 b bronchial epithelial cells to produce ROS after infection with strains 11 or 11 y.