The Researches On The Mechanisms Of Elimination Of Intracellular Pathogens By Triclosan Through Inducing Autophagy In Macrophages

Autophagy is a conserved intracellular process that delivers the cytoplasmic molecules and damaged organelles to the lysosome for degradation and therefore recycling them. Autophagy is used for substrates degradation not only in an non-selective way, but also with a selective method. The selective autophagy is an important immune response to hosts and plays an essential role in combating with pathogens. The conserved and complex process is supported by a comprehensive regulation system. More than 36 autophagy-related genes(ATG) were defined in yeast and their corresponding proteins participated in the regulation generally with multiple forms. In addition, more and more pathways were found playing roles during the process of autophagy.Triclosan is a broad-spectrum antibacterial agent, which can combat with most of gram-positive bacteria, gram-negative bacteria without spore, some fungi,plasmodium and Toxoplasma. The well known antibacterial mechanism of triclosan is that triclosan can inhibit the activity of Fatty acid synthase II(FAS II), which is a key enzyme in the process of fatty acid biosynthesis. With this mechanism, drug tolerance is rarely found in triclosan. Therefore, triclosan was added to all kinds of disinfectants and washing products for protecting us from infection. Researchers also have attempted to make triclosan be a candidate drug for systemic infections treating.Due to the broad antibacterial spectrum of triclosan and the important roles of antophagy in pathogens combating, we try to explore whether triclosan can induce autophagy and therefore enhance the capacities of pathogens killing in macrophages.RAW264.7 cells combined with mice models were used in this research to solve thefollowing problems: whether triclosan can induce autophagy, what’s the mechanisms involved in triclosan-induced autophagy and whether triclosan can improve the abilities of pathogens killing of macrophages by autophagy induction.Firstly, the fluorescence microscope was used for GFP-LC3 punta observation in triclosan-treated He La cells. The images of autophagosomes in RAW264.7 cells were collected by transmission electron microscope. Western blot was used to assay the expressions of LC3 which came from the livers or spleens of triclosan-treated mice.Fluorescence microscope was also used for the comparison of GFP-LC3 and RFP-LC3 punta in He La cells. The expressions of LC3 in triclosan or Chloroquine(CQ)-treated RAW264.7 cells were assayed by western blot. The results showed that there was an increase in the number of GFP-LC3 punta during triclosan treatment.Triclosan treatment also resulted in autophagosomes accumulation. The ratio of LC3-II/LC3-I generally increased in RAW264.7 cells, mice livers or mice spleens after triclosan treatment. Compared with GFP-LC3 puncta, more RFP-LC3 puncta were found in triclosan-treated He La cells. The results also demonstrated that the process of LC3 degradation was inhibited by CQ.Secondly, different concentrations of triclosan were used to deal with RAW264.7cells for different times, then western blot was used to assay the phosphorylation of relevant proteins, including m TOR, p70S6 K, 4E-BP1, AMPK-α, AMPK-β, ULK1,ERK, JNK and p38. Three inhibitors PD98059, SB203580, SP600125 were used to pre-treated with the RAW264.7 cells followed by the detection of LC3 expression and ERK, JNK or p38 phosphorylation. The results showed that the phosphorylation of m TOR, p70S6 K or 4E-BP1 could not be inhibited by triclosan treatment, the phosphorylation of AMPK-α, AMPK-β, ULK1, ERK, JNK and p38 could be induced by triclosan. Furthermore, SP600125 pre-treatment inhibited the phosphorylation of ERK, JNK and p38, SB203580 successfully inhibited the phosphorylation of ERK and p38, however, PD98059 only played a role in ERK phosphorylation inhibiting.Finally, the confocal laser scanning microscope was used to observe the colocalization among p62 or NDP52, LC3 puncta and pathogens in RAW264.7 cells with or without triclosan treatment. Colony counting was used to assay the abilities ofphagocytosis or killing in triclosan-treated RAW264.7 cells. Mouse models of salmonella or Candida albicans infection were established to evaluate the abilities of pathogens killing after therapy with triclosan. The results demonstrated that triclosan treatment increased the expressions of p62 and NDP52 and the ratio of the colocalization was also increased. The results of in vitro experiments showed that the abilities of phagocytosis or killing to pathogens in RAW264.7 cells were enhanced by triclosan. The results of in vivo experiments showed that triclosan also played roles in improving the abilities of combating with pathogens in mice.Overall, the results of this research revealed that triclosan induced autophagy via AMPK/ULK1 and JNK/ERK/p38 pathways rather than a m TOR-dependent pathway.Autophagy induction improved the killing abilities of macrophages. This research contributed to the abundance of antimicrobial mechanisms and pave a way for the more applications of triclosan. It also lay the foundation for setting up a new drug target.