Studies On Innate Immune Response Due To Sodium Azide-Induced Mitochondria Damage

Mitochondrion is a vital organelle in eukaryotic cells.Normally,mitochondrion can maintain homeostasis through regulating fission and fusion.while it is easily damaged when irritated by physical,chemical,biological and other adverse factors,which can even lead to mitochondrial diseases.However,whether mitochondrial damage will activate innate immune response remains unknown.The purpose of this study is to find out the relation of innate immune system and mitochondrial dysfunction through the model of mitochondrial damage induced by sodium azide.The basic methods are as follows:(1)Building mitochondrial damaged cell model induced by sodium azide which can be verified by measuring the characters of mitochondrial damage including ROS,ATP and mitochondrial membrane potential;(2)Using sodium azide to treat isolated mitochondria and detecting the change of copy number of mtDNA by Real-time PCR;(3)Using the latest CRISPR/Cas9 gene knockout technology to construct recombinant plasmids containing PX459-m-ko-cGAS,PX459-m-ko-STING and PX459-m-ko-IRF3 in order to filtrate L929 cGAS-/-,L929 STING-/-and L929IRF3-/-knockout cell strains;(4)Using sodium azide to stimulate wild-type L929 cells,L929 cGAS-/-,L929 STING-/-and L929 IRF3-/-knockout cell strains separately,and detecting the expression of IFN? by Real-time PCR;(5)Using sodium azide to stimulate wild-type L929 cells,the changes of protein expression in AMPK and p-AMPK are detected by Western Blot.(6)Using AMPK inhibitor Compound C to treat L929 cells in advance and then using sodium azide to stimulate it again,detecting the expression of IFN? by Real-time PCR.The results of the study are as follows:(1)Building mitochondrial damaged cell model successfully;(2)Using sodium azide to treat isolated mitochondria leads to the release of mtDNA;(3)Using sodium azide to stimulate wild-type L929 cells can induce the production of IFN? while in the knockout cells including L929 cGAS-/-,L929 STING-/-and L929 IRF3-/-,the production of IFNP decreased obviously;(4)Using sodium azide to stimulate wild-type L929 cell,an increase in the protein expression of p-AMPK is obvious;(5)Using Compound C to treat L929 cell in advance before the stimulation of sodium azide leads to an obvious decrease of mRNA in IFN?.