Cyclophilin A Regulates Autophagy Of RAW264.7 Cells Through PI3K/Akt/mTOR Signaling

ObjectiveTo explore whether Cyclophilin A(Cy PA)could regulate the RAW264.7 cells autophagy through 3-kinase(PI3K)/proteinkinase B(Akt)/mammalian target of rapamycin(m TOR)signaling pathway.MethodsRAW264.7 cells were incubated with different concentration of Cy PA(0 ng/m L,1 ng/m L,10ng/ m L,100 ng/ m L,500 ng/ m L and 1000 ng/ m L)to look for the suitable concentration of Cy PA,Western Blot was used to detect the expression of autophagy related proteins LC3.Experiment was divided into two groups: control,Cy PA(100 ng/ m L),and cells were treated with Cy PA for 6 h to observe the influence of Cy PA on signaling ways,then we used Western Blot to detect the expression of LC3,p-ERK,pP38,p-JNK,p-PI3 K,p-Akt and p-m TOR.Then experiment was divided into four groups: control,Cy PA(100 ng/ m L),LY294002(20 μmol/L),and Cy PA+LY294002 to confirm the influence of Cy PA on PI3K/Akt/m TOR signaling,and Western Blot was employed to detect the expression of LC3,p-PI3 K,p-Akt and p-m TOR.The autophagosome was observed by fluorescence microscopy and electron microscope.Results(1)In a certain concentration range,the level of autophagy in RAW264.7 cells increased with the increasing of the concentration of Cy PA.The response was dose dependent with 100ng/m L concentration and with 6-h exposure causing maximum change in LC3 II expression(p<0.05).But when the concentration of Cy PA increases,the level of autophagy in RAW264.7 decreased(p<0.05).(2)RAW264.7 cells were treated with adenovirus m RFP-GFP-LC3 double fluorescent labeling adenovirus.The results showed that the number of yellow spots in Cy PA was significantly higher than that in control.It was also confirmed by electron microscopy that the formation of autophagosome in Cy PA was also significantly higher than the control.(3)The expression of p-PI3 K,p-Akt and p-m TOR in the Cy PA was lower than that in the control(p<0.05),but there was no significant difference in the expression of p-ERK,p-P38 and p-JNK in the two groups.(4)The expression of p-PI3 K,p-Akt and p-m TOR in Cy PA,LY294002 and Cy PA+LY294002 were lower than those in control group(p<0.05).The observation of autophagosomes in fluorescence microscope and electron microscope in LY294002,Cy PA+ LY294002 were more than the control.Conclusions(1)Cyp A induces autophagy in RAW264.7 cells in dose and time-dependent manner,with Cy PA at 100ng/m L concentration for 6h exposure causing substantial increasing expression of LC3 II.(2)Cy PA may induce autophagy via PI3K/Akt /m TOR pathway in RAW264.7 cells.