Effect Of Active Peptide Components Of Silkworm Pupa On Mitochondrial Apoptosis Pathway In MGC-803 Cells

Silkworm pupa is the main by-product of traditional silk industry in China.Silkworm pupa annual output exceeds 200 thousand tons,silkworm pupa protein content is as high as 60%-71.9%?dry weight?,and it is a very high quality protein resource.A large number of studies have shown that:after protease hydrolysis of silkworm pupa protein,a variety of bioactive short chain peptides can be obtained.In this study,the silkworm pupa protein was as raw material,using the in vitro enzymatic technology to preparate silkworm pupa protein hydrolysates.On this basis,we studied the inhibitory effects of active peptides on cell morphology,cell cycle and mitochondrial apoptotic pathway in vitro,and explored the mechanism of active peptides on mitochondrial apoptotic pathway preliminarily.In addition,membrane ultrafiltration,G-25 dextran gel chromatography and semi preparative HPLC were used to separate and purify highly active peptides,and the peptides were identified by MS1 and MS2 spectrometry.The specific research content and results and conclusions are as follows:?1?The active peptide components of silkworm pupa have significant effects on the morphology of MGC-803 cellsMorphological changes of MGC-803 cells stimulated by SPPH 3 were observed by ordinary optical microscope,fluorescence microscope,laser scanning confocal microscope and transmission electron microscope.The results showed that normal spindle cells gradually became round,the contractions of the pseudo foot,the nuclear crinkle,and the decrease of the refractive index of the cells under ordinary optical microscope.Under the fluorescence microscope,the cells in the normal group had no fluorescent staining,but the characteristics of necrotic cells and cells in the early stage of apoptosis in the experimental group were obvious.Under the confocal laser scanning microscope,the cell microtubules gradually lost their regular bundle structure,and their coherence gradually became worse.Tubulin degenerated and dispersed in cells,and the cytoskeleton gradually collapsed and disintegrated.Under transmission electron microscopy,the chromatin of apoptotic cells was concentrated,mitochondria were edema,endoplasmic reticulum dilated,autophagic began to increase,and apoptotic body appeared.?2?The active peptide components of silkworm pupa have significant effects on the mitochondrial apoptosis pathway of MGC-803 cellsThe SPPH 3 apoptotic effect on MGC-803 cells was taken as the breakthrough point.The cell apoptosis rate and cell cycle were measured by flow cytometry.The expression level of mRNA was analyzed by RT-PCR,and the protein expression level was detected by Western Blot,explaining the signaling pathway of SPPH 3 in the regulation of mitochondrial apoptosis from the metabolic point of view.The results showed that the apoptosis rate of MGC-803 cells stimulated by SPPH 3?0.31mg/mL?was 64.80%,and the rate of apoptosis was positively correlated with the concentration.The up regulation of drug concentration?1.25mg/mL?and the rate of apoptosis reached 93.63%.The blocking effect of SPPH 3 on the cell cycle was mainly in the G₀/G₁ phase:the G₀/G₁ phase was 45.17%?p<0.05?,and the S phase decreased to21.25%.After stimulating cells by SPPH 3,it could up-regulate the expression of Bax and down-regulate Bcl-2 protein,promote the release of Cyt C,activate the linkage reaction of Caspase family gene,then the mitochondrial apoptotic pathway was activated.?3?Purification and structural identification of SPPHsThe protease of silkworm pupa protein was hydrolysated by Novozymes 37071alkaline protease.The relative molecular weight of SPPHs was mainly in the range of1-3k Da,and its content was about 49.63%.Taking DPPH free radical scavenging capacity,FRAP iron reduction capacity and ABTS total antioxidant capacity index as evaluation indexes,combined with MGC-803 cell proliferation inhibition rate?IR?,a comprehensive analysis was made.The results showed that all the 9 groups of SPPHs had different antioxidant activity,and the DPPH radical scavenging ability was highly correlated with IR?r=0.80?;The SPPH 3?DH=25%,Mw<3K Da?could be determined to have the best biological activity with the scavenging ability of DPPH free radical as acriterion.In addition,the molecular weight of SPPH 3 was measured by HPLC-Q-TOF-MS,and the molecular weight of the target peptide was 679.45 Da.