Study On The Mechanism Of Atg 5 In TLR4 - Mediated Ricin Injury

Ricin toxin(RT) is a toxin protein separated from ricinus communis which belongs to Euphorbiaceae Ricinus plants. It has tow compounds, A chain and B chain. A chain has highly depurination affinity to the adenine A4324 of eukaryote and forms an α-sarcin/ricin loop(SRL) of the large r RNA. In this way, it functions as a poison to suppress the expression of protein. RT can induce apoptosis by causing oxidative stress and inflammatory reaction.While oxidative stress and inflammatory reaction can usually induce autophagy.Autophagy is a highly conservative survival mechanism of eukaryotic cell. It works as a recycling system to protect cell where it mediates the breakdown of proteins, organelle and pathogenic microorganism in selective/nonselective ways, which can then be used in the synthesis of new proteins. The formation of autophagosome, which requires surface protein LC3, is the label of autophagy. Autophagosome is a double membranes vesicle, which can encapsulate and transport cargo from cytoplasm. Autophagosome can usually be detected in 15 to 30 min after stimulation. Autophagy can divide as Macrophagy, Microphagy and Chaperones medicated autophagy by its generating process. The autophagy we usually talk about reference to Macrophagy. There are four stages of autophagy, include induction, formation, transportation and fusion, degradation. In induction stage, the autophagy signaling can be activated by multiple signaling crosstalks and induce Beclin-1 forming phagophore. During the formation stagy, the Atg 7 makes LC3 I turn to LC3 II, which then located to phagophore by Atg 5-Atg12. With the help of LC3 II, phagophore extends its memberence and selectively/nonselectively encapsulates cargo from cytoplasm to form autophagosome. In transportation and fusion stage, the autophagosome fuse with phagosomes and lysosomes by the locating function of multiple protein and form autolysosomes at last. Autolysosome degrades its cargo by enzymatic reaction. After that, the autolysosomes will be lysed and recycled to finish its degradation stage.Toll like receptor is one of the pattern recognition receptor. There are 13 members of Toll like receptors, TLR1-TLR10 in human, TLR1-9 and TLR11-13 in mouse. Toll like receptor signaling can be divided into My D88 signaling pathway and TRIF signaling pathway by the different adaptor protein. After the My D88 signaling pathway of Toll like receptor 4 is activated, My D88 formed myddsome complex with IRAK, followed by IRAK1 separated frome My D88, resulting in polyubiquitination of TRAF6. TRAF6 alone with UBC13 and UEV1 A, catalyzes polyubiquitination of TAK1, activates MAPK signaling and NF-κB signaling to product inflammatory factor. IRAK signaling needs TRAM to fulfill adaptor protein sorting. TRIF is activated through self oligomerization, followed by polyubiquitination of TRAF6 and TRAF3, which causes the recruitment of IKKε and TBK1 to catalyze the phosphorylation of IRF3 to induce expression of IFN-I.There is crosstalk between Toll like receptor signaling and autophagy signaling. Activated Toll like receptor can induce autophagy and also feedback by autophagy through autophagy’s cytokine control and enzyme activate ability. The adaptor protein on the downstream of TLR4, such as My D88, TRIF, TAK1 have been proved to control autophagy signaling with Beclin 1 and Bcl-2. As the marker gen of autophagy on macrophages, Atg 5 is the necessary for the formation autophagysome.Our group has found ricin can activate the TLR4 signaling of macrophages. This study focused on investigates the function of Atg 5 in the ricin damage medicated by TLR4, which can offer some information for function exploring and poisoning treatment of ricin. This study includes:(1)The autophagy induction functions of Ricin on RAW 264.7 cell.IC50 of ricin on RAW 264.7 cell was detected as 100ng/m L. The dose we chosed for inducing the autophagy by ricin is one-tenth of IC50, i.e. 10ng/m L. The flux of autophagy induced in different times had been detected by Western blot. And the model of low dose ricin causing RAW 264.7 cell autophagy is to use 10ng/m L ricin incubates 2.5×105 cell/m L RAW 264.7 cell for 2 h.(2) Influence on Atg 5 expression caused by ricin.Genetic level of Atg5 of ricin inducing autophagy on RAW 264.7 cell had been detected by PCR, which shows improved level and positive correlation with the protein level of LC3. The peak of which is 2h.(3) Influence on ricin actived TLR4 signaling of Atg 5.This study built Atg 5 silencing RAW 264.7 steady cell line and control gen silencing RAW 274.7 steady cell line by transfection of psi RNA-m Atg 5 and psi RNA-Luc GL3 plasmid to RAW 264.7 and followed antibiotics screening. The silencing rate of Atg 5 silencing RAW 264.7 steady cell line on genetic level is 49.4% and on protein level is 89.7%, which detected by PCR and Western blot.After treated RAW 264.7 with 10 ng/m L ricin for 2 h, the protein level of TLR4, My D88 and TRIF of TLR4 signaling had been detected by Western blot, and compared to normal RAW 264.7 cell. The result shows after ricin inducing autophagy, the level of TLR4 and My D88 had significantly improved, but TRIF shows no difference. This result shows ricin can activate TLR4 signaling and it’s downstream My D88 signaling, but have no influence on TRIF signaling.The protein level of TLR4, My D88 and TRIF had been detected by Western blot, and compared between Atg 5 silencing cells, normal cells. The result shows all the three proteins had raised after Atg 5 silencing.After treated Atg 5 silencing RAW 264.7 steady cell line with ricin, Western blot results of TLR4, My D88 and TRIF of shows no regularity. The TLR4 protein decreased a little while My D88 and TRIF have no significant change. After ricin inducing autophagy, TLR 4 and My D88 decreased a little while TRIF rose significantly on normal cells compared to Atg 5 silencing cells.In view of the report of autophagy can regulate the production of inflammatory and ROS activity and comprehensive consider about the result of study, function of autophagy and strychnos of ricin, and put forward a point to explain the no regularity of protein of TLR4 signaling that: Atg 5 can negative feed back medicate TLR4 activity. So Atg5 silencing cells show less content of TLR4 related proteins compared to normal cells. Accroding to this, the TLR4 related proteins should been rise after stimulate with ricin to induce autophagy. In fact, the TLR4 protein decreased a little while My D88 and TRIF protein had no significant change. This might be caused by the strychnos of ricin as main influence that inhibits the expression of protein while Atg 5 silencing infects less in enhancing TLR4 related protein expression, which can also explain TLR4 and My D88 decreased a little in Atg 5 silencing cells compared to normal cells after treated with ricin. As the expression of TRIF significantly rose, there might be other signaling can medicate this protein. But this conjecture still need more experiments to proved.This study concludes as:(1)2.5×105 cell/m L RAW 264.7 cell incubated with 10ng/m L ricin for 2 h can induced max autophagysome flow, which corresponding to dose.(2)The Atg5 experession have positive correlation with the autophagysome flow.(3)Atg 5 can negatively medicates TLR4 signaling, which will reduce inflammation response.