Preliminary Studies On Two Insect Hemolymph's Inhibition Effect On Apoptosis Induced By Actinomycin D In Spodoptera Litura Cells

Actinomycin D is the potent inhibitor of RNA synthesis, and is demonstrated to be able to induce Sf-9 cell, K562 cell, aleolar macroages (AM) cell CTLL-2 and Jurkat cell apoptosis. Actinomycin D induce Spodoptera litura (Sl-1) cell apoptosis in this study. Sl-1 cells were treated with final concentrations of 50ng/ml, 100ng/ml, 200ng/ml, 500ng/ml actinomycin D, and it was clearly observed 24 hours after treatment, the addition of actinomycin D at final concentrations of 200ng/ml, 500ng/ml to induce apoptosis, microscopic examination of SL-1 cell treated with ActD reaveal progressive blebbing and culminating in most cell destruction. The fragmentation of the cell nuclei is showed with DAPI. Agarose gel electrophoresis of the DNA extrate from cells treated with ActD shows typical DNA ladder. Control and the cells are treated with 50ng/ml, 100ng/mL 200ng/ml, 500ng/ml actinomycin D for 24 h the percentage of apoptotic cells increased from 3.34%, 10.36%, 15.66%, 31.68%, to 74.75% ,which is analyzed by flow cytometry. This suggests actinomycin D at certain concentrations could induce Sl-1 cells apoptosis, which much dependent on the concentration of does of actinomycin D.Some paper about insect hemolymph study on something :most about silkworm hemolymph to inhibit apoptosis, some about Lonomia oblique hemolymph which could extend cell culture longevity and induce high levels of Cell growth. Insect hemolymph has the three major effects :growth promotion ,nutrition, and cell longevity increase.It is demonstrated that silkworm hemolymph inhibits virus induced apoptosis,as well as apoptosis induced by various chemicals such as actinomycin D, camptothecin, staurosporine and sodium butyrate. Hemolymph of Helicoverpa armigera and Spodoptera exigua inhibit apoptosis induced by actinomycin D in this study. Hemolymph is collected from the fifth instar larvae by clipping the side of an abodominal leg after it is heat-treated at 60 °C for 6 minute. Then the collected hemolymph is heat-treated for 24 minute, chille and centrifuge, the supernatant is filter with a 0.2μm membrane filter and use for supplementing the medium.. Hemolymph of Helicoverpa armigera and Spodoptera exigua at various concentrations(with 1%, 3%, 5%) and 5% fetal bovine serum could be obviously observed to inhibit apoptosis induced by actinomycin D 24 hour, microscope shows, higher hemolymph concentration ,less apoptotic body or low percentage of apoptotic cells ,same too with DAPI and by flow cytometry .This sdudy also shows Spodoptera exigua hemolymph did effectively than Helicoverpa armigerahemolymph in inhibition of apoptosis induced by actinomycin D. The effect of Helicoverpa armigera and Spodoptera exigua hemolymph on inhibiting Sl-1 cell apoptosis induced by actinomycin D was investigated, the result is that Helicoverpa armigera and Spodoptera exigua hemolymph could effetively prevent apoptosis induced by actinomycin D.Acording to this study and some reports relating to insect hemolymph, we could conclude that it may be same to all the insect hemolymph inhibiting Apoptosis induced virus and some chemicals.