| Lotus seedpod belongs to mature torus of Nelumbo nucifera Gaertn., which contains rich protein, fat, polyphenols,procyanidians, etc. Most lotus seedpod had been abandoned in processing process, except when sometimes used as a traditional medicine with hemostasis function and for eliminating bruise. Procyanidins from lotus seedpod(LSPCs) are constituted by a variable number of flavan-3-ols units linked together through C4–C8(or C6) interflavanoid bonds, and the oligomeric procyanidins are considered to be the main active constituents of LSPCs. Studies shown that LSPCs have avariety of chemical composition and biological effects, such as antioxidant activity, anti-cancer activity, radiation resistance, etc. Our work indicated that LSPCs could trigger apoptosis and autophagy Hep G2 cell death by stimulating reactive oxygen species(ROS) generation with mitochondria-dependent signaling way.Consequently, we investigated the mechanism of autophagic and apoptosis death of human hepatoma G2 cells mediated by procyanidins from Lotus seedpod, and morphological analysis, determination of physiological index and protein molecular imprinting technology were applied to this paper. Meanwhile, the present study was performed to investigate the relationship between autophagy and apoptosis, and then studied the signaling pathway and molecular mechanisms on the relationship between autophagy and apoptosis.The main contents and conclusions are as follow:1. Research of procyandins from lotus seedpodinduced autophagic cell death by ROS and its molecular mechanism in Hep G2 cells.3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide(MTT) assay was performed to test cell viability and 50% inhibitory concentration(IC50) on Hep G2 cells. Autophagy was detected by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide(MDC) staining, accumulation of GFP-LC3 puncta and transmission electron microscopy images.The distribution of cell cycle was assessed by flow cytometry; Intracellular ROS level was detected by2’,7’-dichlorofluorescein diacetate(DCFH-DA) staining; Mitochondrial membraneVI potential was observed with 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl- imidacarbocyanine iodide(JC-1) staining; the autophagic molecular mechanism was evaluated by western blot.(1) LSPCs inhibited cell growth in a concentration- and time-dependent manner,with a maximum cell growth inhibition of 89.20 ± 5.10% after treatment with 100μg/m L LSPCs for 96 h. Values of 50% inhibitory concentration(IC50) of LSPCs on Hep G2 cells were between 495.51 ± 0.34 and 29.66 ± 0.27 μg/m L after various time(6- 96 h).(2) The autophagy induced by LSPCs was determined by morphological and biochemical matters in Hep G2 cells. The appropriate time and concentration(24h and50μg/m L) of LSPCs were synthesiaed to investigate the mechanism of autophagy. The characteristics of autophagy were observed. Autolysosomes/lysosomes have been shown in the fluorescence microscope and TEM containing various acid hydrolases.LSPCs induced the formation of green fluorescent protein-microtubule associated protein 1 light chain 3(GFP-LC3) puncta in Hep G2 cells stably expressing GFP-LC3,increased the accumulation of microtubule-associated proteins microtubule associated protein 1 light chain 3(LC3-II). However, LSPCs-induced autophagy was inhibited by autophagy blockers 3-Methyladenine(3-MA) autophagy inhibitor 3-MA.(3) LSPCs decreased the expression of Cyclin A and induced S cell cycle arrest in Hep G2 cells. These phenomena were not observed in the group pretreated with the autophagy inhibitor 3-MA. LSPCs also induced DNA damage in Hep G2 cells and3-MA could inhibit the DNA damage induced by LSPCs.(4) LSPCs triggered ROS generation in a dose-dependent mannerand caused the reduction of mitochondrial membrane potential in Hep G2 cells. N-acetylcysteine(NAC) pretreatment blocked LSPCs-induced autophagy and cell death in cells. LSPCs could trigger autophagy via ROS generation, which may be associated with the mitochondria-dependent signaling way.2. Research of procyandins from lotus seedpod induced apoptosis cell death by ROS and its molecular mechanism in Hep G2 cells.MTT assay was used to investigate the inhibition effect of procyanidins fromVII lotus seedpod on human hepatoma Hep G2 cell lines. The morphological changes of apoptotic nuclei observed through Hoechst 33258 staining. Apoptotic cell percentage calculated by flow cytometry. Intracellular ROS level was detected by DCFH-DA staining; We detected the DNA damage by the comet assay and mitochondrial membrane potential by JC-1 staining. Western blotting assay was used to determine apoptotic protein expression levels.(1) LSPCs induced the cell death in Hep G2 cells, while the NAC and Z-VAD could suppress LSPCs-induced cell death.(2) LSPCs induced apoptosis in Hep G2 cells. After treatment with LSPCs, the number of chromatin condensed cells increased from 3.86% to 42.76%, and the proportion of apoptotic cells increased from 5.32% to 67.05%. Treatment of Hep G2 cells with LSPC caused an increase in the protein levels of cytochrome c、caspase-3、caspase-9 and the ratio of Bax/Bcl-2. Z-VAD and NAC could inhibit the apoptosis induced by LSPCs.(3) LSPCs triggered ROS generation, which caused DNA damage and a loss of mitochondrial membrane potential, the ratio of Bax/Bcl-2 increased. Analysis of the experiment results showed that LSPCs could trigger apoptosis in Hep G2 cells via mitochondria-mediated death pathway. Z-VAD and NAC could inhibit the mitochondrial membrane potential damage and the protein expression of caspase,block the LSPCs-induced apoptosis.3. Research of the relationship between autophagy and apoptosis induced by procyandins from lotus seedpod in Hep G2 cells.MTT assay was performed to test cell viability. Autophagic effect was detected by MDC staining, accumulation of GFP-LC3 puncta at 24 h in Hep G2 cells. The influence of autophagy in apoptosis was analyzed by annexin V-FITC/PI staining and Hoechst33258 staining. The relationship between autophagy and apoptosis induced by LSPCs were evaluated by western blot. ROS-mediated Bcl-2, phosphatidylinositide 3-kinases PI3K/AKT/m TOR, c-Jun N-terminal kinase JNK and extracellular regulated protein kinases ERK signaling pathways played an important role in the regulation of autophagy and apoptosis.(1) Autophagy inhibitor 3-MA down-regulated karyopyknosis which caused by LSPCs in Hep G2 cells and reduced the apoptosis rate.Results showed that the inhibition of autophagydown-regulated apoptosis caused by LSPCs in Hep G2 cells.(2) Apoptosis inhibitor Z-VAD could block GFP-LC3 membrane fusion proteins transfer to the autophagy membrane, reduced the green fluorescence spotsand the expression of LC3-II. The results showed that Z-VAD can inhibit the LSPCs-induced autophagy in Hep G2 cells.(3) Compared with LSPCs group, pretreatment with 3-MA and NAC decreased the expression of Bax 、Bad and cleaved caspase3, and increased the expression of Bcl-2, 3-MA, NAC inhibited the expression of apoptosis protein, which suggested that mitochondrial proteins Bax, Bad, the Bcl-2 regulated the process of autophagy and apoptosis by the ROS generation.(4) Compared with LSPCs group, pretreatment with 3-MA and NAC increased the expression of p-Akt, m TOR, PI3 K and decreased the expression of LC3-II and cleaved caspase3 in Hep G2 cells. The results showed that LSPCs induced autophagy and apoptosis via down-regulated the PI3K/AKT/m TOR associated proteins in Hep G2 cells. LSPCs might regulate the autophagy and apoptosis of Hep G2 cells by regulating PI3K/Akt/m TOR pathway through the ROS generation.(5) LSPCs increased the expression of P-JNK, P-ERK, autophagy protein LC3-II and apoptosis protein Cleaved caspase3, which suggested LSPCs induced autophagy and apoptosis in Hep G2 cells by promoting the phosphorylation of JNK and ERK.JNK pathway inhibitor SP600125、ERK pathway inhibitor PD98059 and NAC could inhibit autophagy and apoptosis which caused by LSPCs. |
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