Oxidative Stress Injury Of HepG2 Cells Mediated By Procyanidins From Lotus(Nelumbo Nucifera Gaertn.) Seedpod Induced Reactive Oxygen Species Accumulation And Its Molecular Mechanisms

Hepatoma is one of the most prevalent malignancies and is the third leading cause of cancer-related death worldwide,with an estimated incidence more than 600,000 new cases annually.It is still a serious threat to human health.Numerous studies have demonstrated that oxidative stress can be used as an effective means of anti-tumor,and can induce tumor cells death through multiple-targeting and multiple-path.Our previous work indicated that lotus seedpod procyanidins（LSPCs）has a variety of chemical components and biological effects,such as antioxidant activity,anti-cancer activity,radiation resistance,etc.In addition,LSPCs could stimulate reactive oxygen species（ROS）accumulation and induce oxidative stress in HepG2 cells.However,the ROS species,anti-tumor pathways and mechanisms of ROS were not well understood.Consequently,to elucidate the anti-tumor mechanism of LSPCs induced by oxidative stress in HepG2 cells.This paper was devoted to study the effects of LSPCs induced ROS accumulation on mitochondria function,calcium homeostasis and proteome in HepG2 cells and then revealed the anti-tumor mechanisms of oxidative stress.The main contents and conclusions are as follows:1.Effects of LSPCs induced ROS accumulation on mitochondria function in HepG2 cells.（1）The species,distribution and sources of ROS.H₂O₂-sensitive fluorescent probes and O·2-specific fluorescent probes,mitochondrial specific fluorescent probes and mitochondrial respiratory chain complexes（MRCC）inhibitors were used to detect the species,distribution and sources of ROS.The results showed that LSPCs stimulate ROS accumulation in HepG2 cells.The ROS is not a single species,there are at least H₂O₂ and O·2-.Meanwhile MRCC I and III are the major sources of ROS generation.（2）Effects of LSPCs induced ROS accumulation on mitochondria function.After12 h of incubation with 25,50 and 100 μg/m L LSPCs.The levels of mitochondrial membrane potentia,ATP and MPTP were 0.86-0.49,0.83-0.45 and 0.52-0.87 times of control group respectively.LSPCs reduced the activities of four mitochondrial enzymes-GSH,SOD,SDH and CAT in a concentration-dependent manner.The activities of four mitochondrial enzymes were 0.91-0.57,0.77-0.4,0.81-0.35 and0.83-0.3 times of control group respectively.The contents of MDA were 0.91-0.57 times of control group.The activities of MRCC I and MRCC III were 0.80-0.37 and0.78-0.33 times of control group respectively.The protein expressions of MRCC I（subunit NDUFS1）and MRCC III（subunit UQCRC1）were 0.84-0.37 and 0.86-0.35 times of control group respectively.LSPCs caused a dose-dependent release of Cyt-c from mitochondria into the cytosol in HepG2 cells.However,these phenomena were effectively attenuated in N-acetylcysteine（NAC）pretreatment.Taken together our results demonstrated that LSPCs can triggers ROS accumulation and thereby initiates a stress response leading to mitochondrial injury and the injury can be effectively attenuated by NAC pretreatment.2.Effects of LSPCs induced ROS accumulation on calcium homeostasis in HepG2 cells.The levels of ROS and Ca²⁺generally peaked after exposure to 50 μg/m L of LSPCs for 12 h and 24 h,and they were respectively 1.22 and 1.39 times of control group.Intracellular Ca²⁺ is derived from intracellular calcium stores and extracellular calcium influx.After 1,3,6,12,24 and 36 h of incubation with 25,50,75,100 and 150 μg/m L LSPCs,the correlation coefficients ROS and Ca²⁺ were 0.862,0.937,0.968,0.940 and0.931,respectively.Taken together our results demonstrated that LSPCs could induce ROS accumulation and Ca²⁺overload in HepG2 cells.There was a significant positive correlation between ROS and Ca²⁺.3.Effects of LSPCs induced ROS accumulation on proteome in HepG2 cells.Proteomics method were used to analyze and identify the protein profile of HepG2 cells pre-and post-treatment with LSPCs.The results showed that 22 proteins expressed differentially,in which 17 proteins were down-regulated and 5 proteins disappeared in the LSPCs group.These differential proteins were mainly associated with stress reaction,oxidative damage,cytoskeleton,cell apoptosis,cell migration,cell differentiation and proliferation,signal transduction,etc.Taken together our results demonstrated that oxidative stress induced by LSPCs in HepG2 cells may inhibit the proliferation of tumor cells through suppressing the levels of conresponding proteins expression.