The Investigation Of Antimicrobial Peptide From Musca Domestica Larva Induced Growth Arrest And Apoptosis In HepG2 Cells

Objective:Comparing the different influence on the HepG2 cells exerted by Musca domestica larva,purification I1,I2,I3,I4 and crude extract of Musca domestica larva antibacterial peptide.And try to conduct the antitumor experiment in vitro with a more stronger inhibitive components,then explore the potential anti-tumor mechanism.Methods:1.MTT assay was used to detect the effect of Musca domestica larva and the different components extracted from Musca domestica larva antimicrobial peptide on HepG2 cells proliferation.2.Hoechst 33258 staining was used to observe the cell nuclear morphology of HepG2 after treatment with antimicrobial peptide from Musca domestica larva.3.Laser scanning confocal microscopy and flow cytometry were used to observe the changed of intracellular [Ca2+]i levels in HepG2 cells after treated by antimicrobial peptide from Musca domestica larva.4.Flow cytometry were used to analysis the effect of antimicrobial peptide on cell cycle,apoptosis,mitochondrial membrane potential and reactive oxygen of HepG2 cells.5.Spectrophotometry was used to detecte the activation of Caspase 3 and Caspase9 of HepG2 cells after treated by antimicrobial peptide from Musca domestica larva.6.Western blotting was used to detect the expression of Cleaved Caspase 3,Cleaved Caspase 9,Bcl-2 and Bax of HepG2 cells after treated by antimicrobial peptidefrom Musca domestica larva.Results:1.Results obtained from MTT analysis showed that the proliferation of HepG2 cells was significantly inhibited by crude extract of Musca domestica larva antibacterial peptide and purification I1,I2,I4 at each time(P<0.05),as the time goes on,the inhibiting effect become more apparent.When the intervention time is higher than 48 h,the inhibition rate of the crude extracts group of Housefly antimicrobial peptide was significantly higher than the other groups(P<0.05).The Housefly antimicrobial peptide crude extraction can inhibite the proliferation of HepG2 cells within 60-480 ?g/m L in a dose dependent manner.2.The results of the HepG2 cell cycle assay showed that the crude extract of Musca domestica larva antibacterial peptide significantly increased the percentage of cells in G0/G1 phase(P<0.01),while the proportion of cells in S phase and G2/M phase were significantly decreased.3.The results of apoptosis detection showed that the crude extract of Musca domestica antibacterial peptide can induce HepG2 cellls apoptosis.From the morphological point of view,after treated by the crude extract of Musca domestica antibacterial peptide,some of the HepG2 cells behaved significantly apoptotic morphologic alterations including karyopyknosis,chromatic agglutination and nuclear fragmentation,containing bright blue patches in the nuclei;In control group,HepG2 nuclei were round and stained homogeneously.From the perspective of apoptosis rate:crude extract of 60,240 and 480 ug/mL can induce apoptosis(P<0.05),and with the increase of concentration,the rate of apoptosis was also increased,in a dose-dependent manner.4.For the fluorimetric analysis of mitochondrial membrane potential in the HepG2 cells,crude extract of Musca domestica antibacterial peptide can break the balance of mitochondrial membrane potential,promote the decrease of mitochondrial membranepotential of HepG2 cells.5.The results showed that the expression of intracellular free calcium ion were obviously improved after treatment with the crude extract of Musca domestica antibacterial peptide.Laser scanning confocal microscopy showed that compared to the control group,the intensity of [Ca2+]i fluorescent signal in HepG2 cells treated with crude extract of Musca domestica antibacterial peptide increased significantly,connecting cell morphology with the fluorescence intensity,the Calcium fluorescence intensity in the cells which turn round and shrunken were higher than that of the normal form of the cell.The result was highly consistent with that obtained by flow cytometry,the average intensity of [Ca2+]i fluorescent signal in HepG2 cells treated with crude extract of Musca domestica antibacterial peptide were significantly higher than the control group(P<0.05),with the increase of concentration,calcium peak shifted to the right obviously,the fluorescence intensity gradually increased.6.Results from ROS detection showed: contrasted withthe control group antibacterial peptides of 240 and 480 ug/mL can significantly promote the release of ROS in HepG2 cells(P<0.01).7.Measurement of caspase activity showed: Caspase-3 and Caspase-9 activity were significantly increased in the lysates of HepG2 cells treated with crude extract of Musca domestica antibacterial peptide for 48 hours compared with controls(P<0.05).8.Western blot indicated that the crude extract of Musca domestica antibacterial peptide can increase the expression level of Cleaved Caspase 3,Cleaved Caspase 9 and Bax protein,and decreased the expression of Bcl-2 protein.Conclusion:1.The crude extract and purification I1,I2,I4 of Musca domestica larva antibacterial peptide can significantly inhibite the proliferation of HepG2 cells,the most obvious of which was crude extract.2.The crude extract of Musca domestica larva antibacterial peptide can inhibite theproliferation of HepG2 cells in a dose-dependent manner,and arrest the HepG2 cell cycle in G0/G1 phase,cell proliferation may be affected by DNA replication and mitosis.3.The increase of the concentration of ROS and calcium in HepG2 cells after treated by the extraction of antibacterial peptides from Musca domestica larvae may be the upstream signaling molecules to initiate the apoptosis pathway.4.The crude extract of Musca domestica larva antibacterial peptide may induce apoptosis in HepG2 cells through mitochondrial pathway,the mechanism may be related to up-regulation of Bax,and down-regulation of Bcl-2 level.