Effects Of Naja Naja Atra Venom (NNAV) On The Proliferation And Apoptosis Of A Human Keratinocyte Cell Line HaCaT

Objecfive:In this study,the effects of Naja Naja Atra Venom (NNAV）onproliferation, apoptosis and the possible apoptosis mechanism of a humankeratinocyte cell line HaCaT were investigated,which could providetheoretical basis for the next in vivo study and clinical trial to explorethe possibility of topical application in the treatment of psoriasis.Methods：①HaCaT cells were cultured and subcultured.②Cell countingkit-8was used to detect the best time and concentration of NNAV on theproliferation inhibition effect of HaCaT cells.③Morphologic appearanceof normal and apoptotic HaCaT cells were observed by inverted opticalmicroscope.④The apoptosis of the cells co-cultured with differentconcentrations of NNAV and different time was detected with Annexin V-FITCand PI double labeling by inverted fluorescence phase contrast microscopeand flow cytometry.⑤Analyzed the changes of caspase-3,8,9activitieswith spectrophotometry.Results:①Morphological observation under inverted microscope: HaCaTcells were characterized by typical epithelioid and arrayed into acobblestone-like structure.②HaCaT cells were incubated with1、2、4、8、16and32μg/ml of NNAV for24h、48h和72h separately.In CCK-8results,proliferation of HaCaT cells was inhibited comparing with thecontrol group,the difference was statistically significant (P<0.05).③Non-viable apoptotic cells were double labeled by Annexin V-FITC andPI when observed by inverted fluorescence phase contrast microscope,whichpresented characteristic morphological changes of apoptosis in the samefield of microscope when observed with light microscope.The apoptoticcell counts in every field of microscopy at high magnification increasedin a dose-dependent manner. The apoptotic cells were mainly non-viableapoptotic cells.The apoptosis ratio increased as the concentration getting higher and the time getting longer,the differences wasstatistically significant (P<0.05).④The levels of caspase-3andcaspase-9activities significantly increased as the concentration levelrose,with statistically significant differences(P<0.05),while therewere no significant changes in the activities of caspase-8(P>0.05).Conclusions：1.Naja Naja Atra Venom can inhitited proliferation of HaCaTcells in vitro.2.Naja Naja Atra Venom can induce the apoptosis and necrosis of HaCaTcells,and non-viable apoptotic cells account for a big part.3.The activities of caspase-3,9increased notably when HaCaT cells wereinduced to apoptosis by NNAV.This study suggested that the molecularpathway of apoptosis of HaCaT cells induced by Naja Naja Atra Venom wasconcerned with mitochondria.