Study On Apoptosis Of Sf9 Cells Induced By Three Kinds Of Microbial Fermentation Filtrates

Microbial Biochemical Pesticides including agricultural antibiotics and toxoid biological agents can be used to control crop pests and weeds.The agricultural antibiotic insecticides mainly bind on the insect nerve receptors,destroying the nervous system,such as Spinosad,Avermectin,Ivermectin and Nanchangmycin.The toxin insecticides mainly influence the immune system of insects,affecting normal metabolism of insect haemocytes,causing changes in the number of isoenzymes,apoptotic proteases,antimicrobial peptides and other substances in cells,resulting in cell metabolism disorders,eventually leading to death.Studies on the pathogenicity of microbial secondary metabolites to insects is mostly focused on the individual,and the results may be unstable because of individual varation,which increases the difficulty in identifying the toxic components in the fermentation filtrate.On the contrary,the results are more stable by using commercialized cell lines.It is easier to obtain cell lines,which provides a convenient approach for us to investigating the effectiveness of new drugs on cells from different species,as well as improving the efficiency of pharmaceutical development and production.In this paper,Spodoptera frugiperda ovary cells(Sf9)and three microbial fermentation filtrates were used to study the morphological and quantitative change of the cells,by ordinary optical microscope and cell viability assay(CCK-8 method).Then,we used AO/EB method,Annexin V-FITC/PI method and flow cytometry to detect apoptotic rate.The results showed that the three test microbial fermentation filtrates induced significant apoptosis of Sf9 cells.Followed,we used real-time quantitative PCR(mRNA expression level)to detect the expression of apoptosis-related genes in Sf9 cells,results showed that the three microbial fermentation filtrates induced the apoptosis of Sf9 cells,which indicated that the different components of the fermentation filtrate could activate different apoptotic pathways,and the different regulatory factors were activated in different pathways.1.Proliferation inhibition of Sf9 cells by three microbial fermentation filtrateSf9 cell line is commonly used for in vitro expression and virulence research.It is stable and easy to culture.In addition,the size of Sf9 cells is moderate with the diameter of 15-25 ?m,so it is easy for morphological observation under the ordinary optical microscope.Further more,external stimuli could produce typical changes like apoptosis in Sf9 cells.Therefore,we selected Sf9 cell line as our experimental material.When the cells are stimulated by external stimuli,the extracellular signal is transfered into cells,where the DNA repair is affected,cell cycle is blocked and cell apoptosis is activated,which will prevent cell proliferation leading to cell death eventually.After treatments of three microbial fermentation filtrate,the morphological and quantitative changes of Sf9 cells could be observed under the microscope.The activity of Sf9 cells was assayed by CCK-8 kit,which could indirectly reflect the inhibitory effect of the fermentation filtrate on cell proliferation.The three microbial strains selected for this experiment were Serratia marcescens(CS2101),Beauveria bassiana(NJBb210)and Metarhizium pingshaense.Pre-experiments showed that the optimum concentration gradient of S.marcescens fermentation filtrate was from 6.600 to 9.240 mg/mL;the concentration gradient of B.bassiana was from 0.12 to 1.92 mg/mL;and the concentration gradient of M.pingshaense fermentation filtrate is from 0.0024 to 0.0120 mg/mL.Within the concentration range,the cell viability decreased while the concentration increased(cell concentration was about 5.0×106 cells/mL).The inhibitory effect of three microbial fermentation filtrate on the proliferation of Sf9 cells was analyzed with SPSS software.IC50 and its confidence interval(95%)were 8.15(7.780-8.644)mg/mL(S.marcescens),0.907(0.500-3.075)mg/mL(B.bassiana)and 0.009(0.006-0.021)mg/mL(M.pingshaense)respectively.The results showed that the inhibitory effect of M.pingshaense fermentation filtrate on Sf9 cells was significantly higher than those of S.marcescens and B.bassiana,and Sf9 cells showed less sensibility to the fermentation filtrate of S.marcescens.In addition,the morphological characteristics of the cells were observed with an ordinary optical microscope.It was found that the number of cells was significantly decreased when the concentration of S.marcescens fermentation filtrate increased.Vacuoles,surface bulge and orderly arranged around the edge of the cell,as well as membrane shrinkaged,size waned and chromatin condensation appeared at low doses of filtrate,and a lot of cell debris was observed in the whole vision at high doses of filtrate.After treatment with B.bassiana and M.pingshaense,cell growth was significantly inhibited,at low doses,cells were shrunk and the chromatin were granulated.The cell shrinkage was severe at high doses,eventually leaving only the cell stamp on the bottom of the plate with black points.Compared with B.bassiana,the cells treated with M.pingshaense appeared mostly shrinkage,deformation,retraction and formation of apoptotic bodies as well.2.Study on apoptosis induced by three microbial fermentation filtrates in Sf9 cellMicrobial fermentation filtrate including mostly toxins and proteases,usually affects cells.For example,a hemolysin extracted from S.marcescens secondary metabolites can lyze blood cells.As an important part in the immune system,once blood cells are destroyed,insects lose the barrier,eventually leading to metabolic disorders and death.In addition,many other stimuli could be the apoptosis signals such as increased intracelluar calcium.According to the previous proliferation inhibitory assays,two treatment concentrations were chosen:low does(LD)and high does(HD)of the three microbial fermentation filtrates,for S.marcescens,they were respectively 7.920 mg/mL(IC40)and 9.240 mg/mL(IC70);for B.bassiana,they were respectively 0.48 mg/mL(IC40)and 1.92 mg/mL(IC70);for M.pingshaense,they were respectively 0.0072 ?L/mL(IC40)and 0.0120 ?L/mL(IC70).Followed by assays with AO/EB kit,Annexin V-FITC/PI kit and apoptosis detection with flow cytometry,all these were taken under the treatments at LD and HD respectively.The results showed significant differences of the apoptotic rate at HD compared with CK and LD.Changes of celluar nucleus were clearly observe under the fluorescence microscope,which were stained by AO/EB double dyes.Apoptotic cells were stained by EB in red and the red fluorescence strengthened while the concentrations of fementation filtrate increased;at the same time,healthy cells were stained by AO in green,the green fluorescence weakened while the concentrations of fementation filtrate increased.In this experiment,the apoptotic cells were counted under the fluorescence microscope,and the apoptotic rates were calculated(93.50±2.36)%,(72.33±10.49)%and(75.50±6.06)%at HD treatments of S.marcescens,B.bassiana and M.pingshaense fermentation filtrate,respectively.The assay with Annexin V-FITC/PI double dyes and apoptosis detection by flow cytometry also showed significant difference of apoptotic rate at HD compared with CK and LD.Similar results were observed with the assays by AO/EB kit under the fluorescence microscope,in which late apoptotic cells were stained by PI in red and healthy cells were stained by Annexin V-FITC in green.Red fluorescence strengthened and green fluorescence weakened while the concentrations of fementation filtrate increased.And in this experiment,the apoptotic rates at HD treatments of S.marcescens,B.bassiana and M.pingshaense fermentation filtrates,were respectively(46.03±1.92)%,(71.73±2.76)%and(76.95±3.05)%.Seven genes related to apoptosis including SfDronc,SfDredd,Sf-caspase-1(Caspl),Sf-caspase-9(Casp9),Sf-caspase-2(Casp2),SfIAP and Sfp53 were selected according to the Sf9 apoptotic typical pathways and all mRNA sequences were searched in the library of NCBI.The mRNA level expression of seven apoptotic genes were then detected with qRT-PCR.The results showed that the mRNA expression of the 7 genes were all significantly up-regulated,with Casp9 enhanced by 7.2-fold,more than others,under the treatment with HD of S.marcescens fermentation filtrate.All genes showed significant difference under treatments with LD of S.marcescens fermentation filtrate as well.Under the treatment with HD of B.bassiana fermented filtrate,the mRNA expressions of SfDredd and Casp2 showed no significant difference while the other 5 genes were significantly up-regulated among which Casp9 was up-regulated the most by 3.9-fold.But all genes showed no significant differences at LD.With HD treatment of M.pingshaense fermented filtrate,the mRNA expression of SfDronc showed no significant difference while the other 6 genes showed significant up-regulation,among which Casp2 enhanced to 4.6-fold,higher than the others.And Casp2,Caspl,SfIAP and Sfp53 were also significantly up-regulated at LD,while the others showed no significant difference.