Research On The Effect Of Ricin On Autophagy Of Macrophage Cell Line Raw264.7

Ricin is one of the most poisonous toxicity in the natural toxin, possessingstrongly cell toxity. It was reported that high dose of ricin can suppress proteinsynthesised, low dose of ricin can induce cytokine damaging, lipide over oxidationdamaging and can also induce cell apoptosis. However, autophagy exists ineukaryotic organism generally, has most important role in cell growth, celldifferentiation and can maintain cell-self steady state. When organism was lack ofNutrition or in oxidative stress, the level of autophagy increase. However, there is noreport that the effects of ricin on autophagy and it mechanism over the world.Objective：This study was to investigate the effects of ricin on Raw264.7cell autophagy.Methods：1The effects of ricin on Raw264.7cell autophagyWe detected the survival rate of Raw264.7cell by the method of MTT, detectedexpression levels of LC3protein, detected the expression of LC3, Beclin-1, P62,mTOR, p-mTOR protein by the method of western blot, detected the autophagyintensity by the method of flow cytometry, and detected the ultramicro-cellularity bytransmission electron microscope.2The effects of ricin and3MA on Raw264.7cell autophagyWe detected the survival rate of Raw264.7cell by the method of MTT whenthe cell was treated with both ricin and3MA and detected the expression levels ofLC3by the method of western blot.3The mechanism that ricin effects on Raw264.7cell autophagy We detected the expression of p38MAPK and p-p38MAPK protein.Results:1The effects of ricin on Raw264.7cell autophagyBy the method of MTT we detected the survival rate of Raw264.7cell. Resultswere showed that RT inhibits Raw264.7cells growth in a time and dose dependentmanner. The median lethal dose of ricin may be100ng/mL when it treated Raw264.7cell for24h.By immune-fluorescence we detected the expression levels of LC3protein.Results were showed that the expression levels of LC3protein of positive controlwere significantly incremental, and expression levels of LC3protein of which weretreated with ricin for1h,2h,4h were significantly increased, as well.We detected the expression of LC3, Beclin-1, P62, mTOR, p-mTOR protein bythe method of western blot. The results were showed that all the expression levels ofprotein were increased except mTOR. The expression levels of LC3protein wereincreased, and the protein expression levels treated with ricin at2hours were thehighest. The expression levels of Beclin-1and P62protein were increased at first,and then decreased. There was no significant variance about the expression levels ofmTOR protein, but the expression levels of p-mTOR were increased compared withblank.We detected the autophagy intensity by the method of flow cytometry. Theresults were showed that autophagy intensity was increased when treated with ricin.We detected the ultramicro-cellularity by transmission electron microscope. Thefigures were showed that cell volμme had turned larger, and organelles more activity,and the amount of autophagy vacuoles were increased than the blank.2The effects of ricin and3MA on Raw264.7cell autophagyWe detected the survival rate of Raw264.7cell by the method of MTT whenthe cell was treated with both ricin and3MA. The results were showed cell deathrate was increased.We detected the expression levels of LC3by the method of western blot when autophagy was suppressed. The results were showed that the expression levels ofLC3were decreased.3The effect of p38MAPK signaling pathway on ricin effects on Raw264.7cellautophagyWe detected the effects that P38MAPK signaling pathway on autophagy by themethod of western blot. The results were showed that there was no significantdiversity on the expression of p38MAPK protein, and the expression levels of p-P38MAPK protein were increased.Conclusions:Low dose of ricin could induce Raw264.7cell line emerging autophagy, theamount of autophagy vacuoles was increased, and the expression levels of autophagyrelated protein were increased. Under the condition of3MA inhibitor and ricincombination, rate of cell death was increased. It was meant autophagy was played aprotective role on cell, can protect cell from death. And the cell signaling pathwaywas regulated by the p38MAPK pathway in the process of emerging autophagy.