Background: Pseudomonas aeruginosa (PA) is one of the most common gram-negative opportunistic pathogens in nosocomial infection. The susceptible population is mainly patients with low immunity, such as pulmonary cystic fibrosis, chronic obstructive pulmonary disease, tumor, bronchiectasis and burn.Macrophages from monocytes are the main innate immune cells recruited to the site of inflammation. After infection leads to local inflammation, these macrophages recruited to the inflammatory site play a key role in the occurrence, diffusion and regression of inflammation. The proliferation ability of macrophages is reduced, which weakens the ability of macrophages to phagocytize pathogens, resulting in persistent infection and lung tissue injury.KLF6 plays an important role in the regulation of gene expression in inflammatory response, and it has been reported that there is a functional relationship between KLF6 and inducible nitric oxide synthase (iNOS). Our previous studies found that PA can induce the expression of KLF6 in lung tissue cells at the animal level, and may mediate apoptosis of lung tissue cells by regulating iNOS. In this study, the effect of PA supernatant on RAW264.7 cells apoptosis and the expression of KLF6 and iNOS were observed. In order to further study the role of iNOS in the apoptosis of RAW264.7 cells induced by PA supernatant, S-methylisothiourea sulfate (SMT) was added to the infected RAW264.7 cells to detect the expression level of iNOS, the change of NO content and the apoptosis of RAW264.7 cells.
Methods: The MTT assay was used to detect the cell proliferation rate. Considering the different volume concentrations(the volume ratio of PA supernatant to complete medium) of PA supernatant, the experiment groups were divided into control group(add same volume of complete medium), 15% PA group, 30% PA group, 45% PA group, the cells in each group were treated for 12h and 24h respectively. In addition, SMT(+) group(SMT was the iNOS blocker) and SMT(-) group(without SMT) were designed for the experiment, the concentration was selected as 6μM according to the manual and preliminary results. According to MTT results, 30% PA supernatant treating for 24h was screened as the processing condition.Flow cytometry was used to detect the apoptosis rate. Hoechst 33342 staining was used to observe the nuclear morphological changes. Western blot was used to detect the expression of KLF6 and iNOS protein. The expression levels of KLF6 mRNA and iNOS mRNA were detected by real-time PCR. The NO content detection kit was used to detect the content of NO. Finally, the data analysis was conducted by using SPSS16.0 software.
Results: When the PA supernatant at different concentrations treated the cells for the same time interval, the proliferation rate increased in a concentration-dependant manner, indicating that the PA supernatant inhibited the proliferation of RAW264.7 cells in a time-concentration-dependent manner. Meanwhile,the same results were achieved in flow cytometry and staining with Hoechst 33342 detected the same conclusion. Furthermore, the expression of KLF6 and iNOS protein, KLF6 mRNA and iNOS mRNA increased significantly after treatment, and NO content was statistically determined after treatment in a concentration-dependent manner and a time-dependent manner. Finally, compared with the SMT(-) group, the apoptosis rate was significantly decreased, and the NO content decreased after treatment with PA supernatant,.
Conclusions: Our findings demonstrste tthat PA supernatant can inhibit the proliferation of RAW264.7 cells, and its inhibitory effect is concentration-time dependent. The mechanism by which PA induces apoptosis of RAW264.7 cells may be caused by the production of excess NO by KLF6 and iNOS. The reduction of iNOS expression has a protective effect on the cytotoxicity caused by PA supernatant infectied RAW264.7 cells.