The Study Of The Protective Effects Of Omega-3 Fatty Acid On Oxaliplatin-Induced Sinusoidal Injury And Its Related Mechanism

Introduction:More than 25% of patients with colorectal cancer(CRC)were initially diagnosed with synchronous hepatic metastases.Up to half of patients with CRC develop metachronous liver metastasis at some point in their life.One fifth of those with metastatic CRC were confined to the liver,for them,liver metastasis was the leading cause of death.Until now,surgery is still the gold standard for the treatment of paitents with CRC hepatic metastases,which may achieve a 5-year survival of around 50%.Moreover,for selective patients,curative resection combined with neoadjuvant or adjuvant therapy may further improve their progression free survival(PFS)and lower their recurrence rates.However,patients with initially resectable liver metastasis were less than 20%.For nearly 90% of patients with CRC hepatic metastases who were diagnosed with unresectable,chemotherapy,or combined with bioactive therapy,would present a survival benefit by limiting the progression of tumor.For conversional treatment,palliative treatment,neoadjuvant treatment or adjuvant treatment,chemotherapy is the essential part of them.With the increasing use of cytotoxic agnets,it has become clear that the incidence of chemotherapy-related hepatic toxicity or even chemotherapy-associated liver failure has soared over these years,leading to the decrease of both treatment response and quality of life(QOL).Among those regents with hepatic toxicity,oxaliplatin is the most respective one that featured with hepatic sinusoidal injury.Typical histological change of hepatic sinusoidal injury has shown the dilatation of Dissel’s space,leading to atrophy of hepatocytes;apoptosis of endothelial cell leading to leaky vessel wall.Severe sinusoidal injury can cause sinusoidal obstruction syndrome(SOS),which would significantly has an impact on the outcome of patients with CRC liver metastasis.The underlying mechanism of sinusoidal injury has not been completely elucidated.Possible mechanisms may be associated with four aspects: cell cycle,oxidative stress,angiogenesis,coagulation cascade.Among these mechanisms,oxidative stress is the dominant hypothesis.Accumulating evidence has demonstrated that oxaliplatin exhausted antioxidant glutathione(GSH),causing elevating levels of intracellular reactive oxygen species(ROS),which leads to direct cell damage.Oxidative stress response is referred to the imbalance between intracellular free radicals and redox signaling,which could exert detrimental effect on organisms.Keap1/Nrf2/ARE signaling pathway is one of the most pivotal regulatory mechanisms towards cellular defense and homeostasis.Simply,Nrf2 is one of important transcription factors.Under basal conditions,Nrf2 combines with Keap1(Kelch-like ECH-associated protein 1),leading to its proteasomal degradation by ubiqutylation.However,when exposed to various stimuli,such as electrophiles,Keap1,acts as a sensor of the stimuli,is covalently modified at various cysteine residues within it,causing its conformative change and preventing Nrf2 from ubiquitylation.As a result,Nrf2 is accumulating in cytoplasm and translocates to the nucleus,then heterodimerizes with s Maf proteins.Thus,the Nrf2-s Maf dimer binds to ARE region of a series of genes that involve cytoprotection,finally upregulates transcription of those genes,exerting adaptive effect on cells.ω-3 fatty acid is one of essential fatty acids for human being.The most widely available dietary source ofω-3 fatty acid is oily fish and some seed oil.ω-3 fatty acid plays an important role in body development,especially in brain development and cell membrane construction.In recent articles,the increasing benefits of ω-3 fatty acid have been found,such as prevention of cancer,immune regulation,combat oxidative stress,maintain cell homeostasis.However,rare evidence has been reported the effect ofω-3 fatty acid on hepatic sinusoidal injury.There is increasing demand for exploring the underlying mechanism.Objective:Establish animal model of oxaliplatin-induced hepatic sinusoidal injury,assess the protective effects of ω-3 fatty acid on oxaliplatin-induced sinusoidal injury,explore the relation between oxaplatin-induced sinusoidal injury and oxidative stress,analyze its underlying mechanismMaterials and Methods:1.Establishment of oxaliplatin-induced hepatic sinusoidal injury model in C57BL/6 mice:C57BL/6 mice(n=12),male,6-8 week old,weight 18-20 g.Randomly divided into two groups: Group Oxaliplatin(Group O)(n=6),Group vehicle(Group V)(n=6).Mice in Group O were treated with oxaliplatin 8mg/kg i.p.on day 1 of a weekly basis for 5 weeks;Mice in Group V only recieved saline.All mice were culled one week after the final dose of oxaliplatin.Blood and liver samples were collected for analysis.Determine severity of murine liver injury by H&E staining,serum aspartate transaminase(AST)and Alanine transaminase(ALT).Ensure the successful establishment of murine model.2.Discover the protective effects of ω-3 fatty acid on oxaliplatin-induced sinusoidal injury and its underlying mechanisms2.1 C57BL/6 mice(n=30),male,6-8 week old,weight 18-20 g.Randomly divided into three groups: Group ω-3 fatty acid +oxaliplatin(Group W)(n=12),Group Oxaliplatin(Group O),Group vehicle(Group V)(n=6).Mice in Group W were treated with oxaliplatin 8mg/kg i.p.followed 2h later by ω-3 fatty acid 2ml/kg i.p.on day 1 of a weekly basis for 5 weeks,and mice were only treated ω-3 fatty acid solution i.p.on the following two days of every week;Mice in Group O were treat with oxaliplatin i.p.by the same way with Group W,but noω-3 fatty acid supplement;Mice in Group V only recieved saline.All mice were culled one week after the final dose of oxaliplatin.Blood and liver samples were collected for analysis.Check the histological changes in liver by H&E staining and blood biochemical changes2.2 Detect the amount of GSH by Spectrophotometry2.3 Detect the vitality of Superoxide Dismutase(SOD)by Xanthine oxidase2.4 Analyze the protein expression of Keap1,Nrf2,HO-1 and NQO1 by western blotting.2.5 Assess the transcriptional expression of Nrf2 and its downstream molecules NQO1,HO-1 by PCR.Results:1.Compared with Group V,murine serum level of ALT and AST in Group O is significantly higher,H&E staining demonstrated severe liver degeneration,hepatic sinusoidal dilation.A significant decrease has been seen in SOD vitality and GSH amount.Western blotting showed decresed protein level of Nrf2’s target antioxidative proteins HO-1.PCR has shown a decreased m RNA level of HO-1.A successful animal model has been established.2.Compared with Group O,Group W had a significantly lower murine serum level of ALT and AST;H&E staining demonstrated abrogated liver degeneration or slight hepatic sinusoidal dilation.SOD vitality and GSH amount recovered as well.PCR and western blotting showed obvious increments of Nrf2 and its targeted antioxidative molecules HO-1 and NQO1,while protein level of keap1 decreased significantly.Conclusion: Oxaliplatin lowered the amount of GSH and SOD within hepatocytes,leading to oxidative stress.This may attribute to inactivation of Keap1/Nrf2/ARE signaling,causing damage on hepatic sinusoid.ω-3fatty acid exerted protective effects on oxaliplatininduced liver injury in C57BL/6 mice.The potential mechanism may be involved in Keap1/Nrf2/ARE signaling,which promotes degradation of Keap1 and translocation of Nrf2 in nucleus,upregulates the transcription of its downstream antioxidative molecules such as HO-1 and NQO1,thereby attenuating hepatic sinusoidal damage caused by oxidative stress.