Study On The Mechanism Of Apoptosis In HepG2 Cells Induced By Simulated Gastrointestinal Digests From Preserved Eggs

Preserved eggs are the first traditional alkali-pickled food in China,and have been enjoyed by consumers and studied extensively for their nutritional tastes and anti-tumour,anti-inflammatory,antioxidant,lipid-lowering and blood pressure-lowering properties.Pre-laboratory studies have shown that preserved eggs could inhibit tumour cell growth by regulating or inducing apoptosis.Preserved eggs could inhibit the proliferation of human hepatocellular carcinomas（HepG2）and hinder their cycle progression.At the same time,they induced apoptosis in HepG2 cells by up-regulating the expression of the pro-apoptotic gene Bax and inhibiting the activation of the anti-apoptotic genes cyclooxygenase-2（COX-2）and the B-cell lymphoma-2 gene（Bcl-2）.In addition,preserved eggs could also initiate the Caspase cascade reaction by increasing the levels of Cytochrome C（Cytc）,Cysteinyl aspartate-specific proteinase-3（Caspase-3）and Caspase-9,induce apoptosis ultimately in HepG2 cells.These results suggested that preserved eggs might induce HepG2 cells apoptosis through the mitochondrial apoptosis and FAS/FAS ligand（FASL）signalling pathways,but the specific pathway causing apoptosis and the associated mechanism of action were unclear.Based on this,this thesis investigated the effect of preserved eggs on the apoptotic process of HepG2 cells,and explored the molecular mechanisms associated with its induction of apoptosis from the perspectives of oxidative stress,mitochondrial dysfunction and the FAS/FASL signalling pathway.It is expected to provide data support for the anti-tumour function of preserved eggs,and also lay the foundation for the subsequent isolation of anti-tumour active components of preserved eggs,which will help the development of functional egg products.The main research contents and results are as follows:（1）The digestive properties of preserved eggs and the antioxidant capacity of their digestion products were investigated in a dynamic human gastrointestinal-IV（DHGI-IV）digestion system.The p H of preserved egg digests（PED）was found to be 2.00 after 2 h of digestion in the DHGI-IV gastric model,which was similar to the p H of fasting gastric juice（1.60）,and the amount of chyme in the stomach was less than 10%of the initial loading of the sample.This indicated that after 2 h of digestion,the stomach was emptied largely of the preserved eggs with a lag time of 45 min.During intestinal digestion,the p H of the sample changed dynamically from 7.01 to 8.39,indicating that the preserved eggs remained alkaline after digestion.As digestion progressed,the particle size of the preserved eggs decreased significantly,from 447.7μm to 42.4μm,a reduction of approximately 9 times,and the particle size was more dispersed evenly.Preserved egg protein and fat were hydrolysed significantly,with digestibilities of 90%and 87%,respectively.Moreover,PED increased significantly the free radical scavenging capacity of ABTS,DPPH,FRAP and hydroxyl groups.The scavenging rates of DPPH,FRAP,Hydroxyl and ABTS radical increased gradually with increasing PED concentrations,with IC_50s of 3.51,2.35,1.13 and 0.9 mmol/L,respectively.These findings provide a reliable theoretical basis for later studies on the anti-tumour activity of preserved eggs.（2）Based on the significant antioxidant efficacy of PED,the anti-HepG2 cellular activity of PED was investigated in this chapter.PED was found to inhibit HepG2 cell proliferation,migration and clone formation in a dose-dependent manner,while causing cells to exhibit apoptotic morphological features,including cell crumpling and deformation,as well as nuclear fragmentation.Meanwhile,PED promoted apoptosis.The apoptosis rates of HepG2 cells were 7.15%,7.63%and 9.02%at PED concentrations of250,500 and 1000μg/m L,respectively.After treatment with PED（1000μg/m L）,ROS levels in HepG2 cells increased significantly from 44.2%in the untreated group to 99.6%（P<0.05）,thereby disrupting the intracellular redox homeostasis.PED induced apoptosis through the mitochondrial apoptotic pathway by increasing the mRNA and protein expression of the pro-apoptotic gene Bak,while decreasing the mRNA and protein expression of the anti-apoptotic gene Bcl-2 in HepG2 cells.Furthermore,PED down-regulated the intracellular expression of pro-angiogenic genes vascular endothelial growth factor（VEGF）and hypoxia-inducible factor-1a（HIF-1a）,which inhibited cellular angiogenesis and cell migration further promoted apoptosis.（3）This chapter further investigated the effect of PED on mitochondrial apoptosis in human hepatoma cells HepG2,and explored possible mechanisms in terms of oxidative stress and mitochondrial damage.The experimental results showed that PED treatment increased significantly levels of malondialdehyde（MDA）,aspartate aminotransferase（AST）,alanine aminotransferase（ALT）and glutathione（GSH）in HepG2 cells,resulting in cellular oxidative stress.At the same time,PED down-regulated Nrf2 and the downstream proteins Heme Oxygenase-1（HO-1）,Ferritin Heavy Chain（FTH1）and NAD（P）H quinone acceptor oxidoreductase 1（NAD（P）H）.oxidoreductase 1（NQO1）levels,and Nrf2 levels increased after N-acetyl-L-cysteine（NAC）intervention,suggesting that the Nrf2 pathway might be affected by oxidative stress.PED was also found to reduce adenosine triphosphate（ATP）content,Na⁺-K⁺-ATPase activity and mitochondrial copy number（mtDNA）.Also,PED down-regulated the expression of outer membrane subunit 20（TOM20）,mitochondria-encoded genes CO1,CO2 and CO3,and mitochondrial transcription factor A（Tfam）,while up-regulating the expression of the oxidative stress marker 8-hydroxydeoxyguanosine（8-OHdG）,which inhibited the mitochondrial biogenesis pathway.In addition,PED also initiated the mitochondrial apoptotic process,leading to a decrease in mitochondrial membrane potential（MMP）,causing cytochrome C translocation to the cytoplasm and cleavage of Caspase-3.In summary,PED induced apoptosis by causing oxidative stress in HepG2 cells and inhibiting the Nrf2 pathway and mitochondrial biogenesis.（4）Chapter 3 showed that PED up-regulated the expression level of Caspase-3 in HepG2 cells,suggesting that PED-induced apoptosis might involve the FAS/FASL signalling pathway.Therefore,in this chapter,a FASL overexpression plasmid was constructed by double digestion with Nhe I and Xho I,and three FASL small interfering RNA（siRNA-FASL）sequences were designed.The FASL overexpression plasmid and ri RNA-FASL were transfected into HepG2 cells by liposome transfection method,as a means to investigate the involvement of the FAS/FASL death receptor pathway in PED-induced apoptosis in HepG2 cells.The results showed that the death receptor gene FAS and its ligand FASL were expressed highly in PED-exposed HepG2 cells,but mRNA expression of TNFR1,DR4 and DR5,as well as their respective ligands TNF-αand TRAIL,was not observed.The pc DNA3.1（+）-FASL recombinant plasmid was constructed successfully by double digestion with Nhe I and Xho I and transfected in HepG2 cells,where it was found to be expressed stably in HepG2 cells.The effect of PED on the expression of FASL protein in HepG2/pc DNA3.1（+）-FASL transfected cell groups was investigated by Western Blot,using the transfected empty vector pc DNA3.1（+）as a control.It was found that the expression of FADD,Caspase-8 and Caspase-3 proteins was increased significantly in HepG2 cells transfected with FASL gene recombinant plasmid and then added to PED treatment relative to the empty plasmid group.Meanwhile,three small interfering fragments of siFASL（siFASL1,siFASL2 and siFASL3）were designed successfully,from which siFASL1 with the best knockdown effect on the FASL gene in HepG2 cells was selected,and the interference efficiency was twice as high as that of the control group.The siFASL1 was transfected into HepG2 cells followed by the addition of PED treatment,which inhibited significantly the expression of FADD,Caspase-3 and Caspase-8.In addition,pc DNA3.1（+）-FASL and siFASL1 were transfected into HepG2 cells followed by the addition of PED treatment.The former caused Bid cleavage,forming t Bid and accelerating apoptosis,while the latter attenuated this apoptosis.The results suggested that PED could cause directly apoptosis in HepG2cells by initiating the FAS/FASL-Caspase-8/-3 pathway.Bid and Caspase-3 genes link the FAS/FASL signalling pathway to the mitochondrial apoptotic pathway.In conclusion,we investigated the promotion of apoptosis in HepG2 cells by PED and its possible molecular mechanisms.The results showed that PED induced oxidative stress and mitochondrial dysfunction,and activated the FAS/FASL signaling pathway to induce apoptosis in HepG2 cells.