The Alleviating Effects Of Docosahexaenoic Acid (DHA) On Acute Liver Injury And Related Physiology Mechanisms In Fish

The liver is the largest parenchymatous organ in the vertebrate body,which has various important physiological functions such as in digestion,detoxification,metabolism,excretion and immunity.However,the liver is also extremely vulnerable to damage from a variety of toxins,and in fish,impaired liver function frequently results in a number of negative effects like systemic metabolic disorders,which in turn cause the emergence of infectious diseases and syndromes,seriously jeopardizing the environmentally friendly and healthy development of aquaculture.Recently,with the rapid development of intensive aquaculture,high-energy diets have been widely used in aquaculture in order to further improve the economic efficiency and shorten the production cycle.However,long-term energy imbalance can trigger a series of metabolic syndromes in fish,especially fat deposition in the liver and impaired liver function.In addition,water pollution and antibiotic abuse further aggravate the stress on the liver of cultured fish.How to improve the liver health of cultured fish and provide high-quality aquatic products for human consumption has been the focus of attention in the aquaculture industry.Currently,exploring the protective effects of various functional feed additives on fish liver health is a hot topic in aquaculture nutrition.Docosahexaenoic acid（DHA,22:6n-3）is an n-3 highly unsaturated fatty acid（HUFAs）that has a number of advantages physiological functions.Some studies have reported its role in promoting fish growth and reproduction,but research on whether DHA can improve fish organism health and alleviate liver damage caused by liver toxins is limited.A number of stable and controlled samples of fish liver injury must to be constructed to investigate the protective effect of functional feed additives on liver injury in fish and to study the process and mechanism of liver disease development in cultured fish.Therefore,it is critical to develop fish liver injury models under experimental conditions,however,there are still relatively few fish liver injury models that perform more standardized studies.Therefore,to assess the protective effect of DHA on fish liver health,this study first developed three models of liver injury using the model organism zebrafish.Then,using two different models of liver DHA accumulation,zebrafish（Danio rerio）and the commercial fish species Nile tilapia（Oreochromis niloticus）were used to investigate whether DHA could alleviate D-galactosamine/lipopolysaccharide（D-Gal N/LPS）-induced liver injury.1.Development of the adult zebrafish model of liver injury induced by CCl4 and D-Gal N/LPSThe classical chemical liver toxicants include carbon tetrachloride（CCl₄）and D-Gal N/LPS.Currently,some studies have constructed CCl₄ intraperitoneal injection models in fish such as grass carp,tilapia and common carp,but the optimal doses for inducing liver injury obtained from different studies vary greatly,and no uniform standard yet been constructed.Furthermore,no research on the use of D-Gal N/LPS to induce liver injury in fish has been reported.Although zebrafish is increasingly being used as a model organism in disease treatment and drug screening studies,the use of adult zebrafish as an animal model for liver injury research has received little attention.In this part of the present study,three models of liver injury were developed in adult zebrafish using CCl₄ and D-Gal N/LPS,namely the CCl₄ water exposure model:0.2m M CCl₄ as water exposure for 7 days;the CCl₄ intraperitoneal injection model:injecting 15%,30%or 45%CCl₄ with 3μl/g body weight for 72 hours;and the D-Gal N/LPS feed feeding model:feeding fish with 1g/kg,4g/kg or 7g/kg dietary D-Gal N/LPS with 4%body weight per day for 4 weeks.The results showed that all the three models approaches induced liver injury,with similar pathological features including the significantly increased serum activities of glutamic-pyruvic transaminase（ALT）and glutamic-oxaloacetic transaminase（AST）,elevated liver content of malondialdehyde（MDA）,and upregulation of the gene expressions related to inflammation and apoptosis.For the three methods,0.2m M CCl₄ exposure for 7 days,or injecting 15%CCl₄ with 3μl/g body weight for 72 hours,or feeding fish with 7g/kg dietary D-Gal N/LPS with 4%body weight per day for 4 weeks,could efficiently induce the obvious liver injury in adult zebrafish.These three zebrafish liver injury models can provide valuable references for several research domains such as environment,metabolism and aquaculture.2.DHA accumulation in the liver of vtg1 deficiency zebrafish alleviates D-Gal N/LPS-induced liver injury.A previous study in our laboratory discovered that knocking out yolk proteinogen1（Vtg1）on zebrafish（vtg1 knockout）inhibited the DHA transport from liver to ovary in female zebrafish,resulting in a significant increase in DHA content in the liver of vtg1 knockout females.This provides a good model for physiological DHA accumulation in the liver for the studies in this thesis.Therefore,this part investigated the effect of hepatic DHA accumulation on D-Gal N/LPS-induced liver injury using vtg1knockout female zebrafish as a model.The experiment was included three treatment groups and were named as negative control group（CC,wt zebrafish fed control diet）,positive control group（CD,wt zebrafish fed D-Gal N/LPS diet）and experimental group（VD,vtg1 deficiency zebrafish fed D-Gal N/LPS diet）with a four-week culture period.The results of the experimental showed that feeding D-Gal N/LPS diets significantly reduced the motility of zebrafish compared with the CC group,and caused fat deposition in liver,disrupted redox homeostasis,and caused liver injury.In contrast,DHA was significantly alleviated the liver injury induced by D-Gal N/LPS in zebrafish.Further analysis showed that D-Gal N/LPS activated Toll-like receptor 4（TLR4）signaling pathway in the liver of zebrafish,which in turn caused nuclear factor NF-κB to become activated and released a number of inflammatory factors（il1β,il6,il8,il11,il12α）,ultimately resulting in liver injury.In contrast,DHA was able to alleviate the liver injury by inhibiting the activation of TLR4/NF-κB signaling.These results suggest that DHA can alleviate D-Gal N/LPS-induced liver injury in zebrafish by regulating liver lipid metabolism,redox,TLR4 signaling pathway and inflammation.3.Dietary docosahexaenoic acid reduces fat deposition and alleviates liver damage induced by D-galactosamine and lipopolysaccharides in Nile tilapia.Considering that in actual production,functional substances are usually added to feed to improve the growth performance and organism health of fish,it is more practical to supplement DHA to diets.Nile tilapia（Oreochromis niloticus）is a warm-water omnivorous fish,which farmed in several countries around the world due to its excellent growth performance and environmental adaptability,and is an important economic fish species in China’s aquaculture industry.This study investigated the role of DHA supplementation in fat deposition and liver damage caused by D-galactosamine（D-Gal N）and lipopolysaccharides（LPS）in Nile tilapia（Oreochromis niloticus）.In this part,Nile tilapia with an initial weight of 2.0±0.1 g were randomly divided into four treatment groups（75 fish/group）and randomly assigned to three tanks（25 fish/tank）.Four diets were formulated as control diet（Con）,Con supplemented with 1%DHA,2%DHA and 4%DHA diets（dietary supplement with 1%DHA,2%DHA and 4%DHA）,respectively.The diets were fed to 25 Nile tilapia for four weeks.After the four weeks,20 fish in each treatment were randomly selected and injected with a mixture of 500mg D-Gal N and 10μl LPS per ml（1μl/g weight）to induce acute liver injury,the acute liver injury experiment lasted for a total of 7 days.The results showed that the Nile tilapia fed on DHA diets decreased liver lipid content and triglyceride concentrations in serum and liver than those fed on the Con diet.Moreover,after D-Gal N/LPS injection,the fish fed on DHA diets decreased alanine aminotransferase（ALT）and aspartate transaminase（AST）activities in the serum.Interesting,liver q PCR and transcriptomic results together indicated that feeding the fish on the DHA diets improved liver health by downregulating the expression of genes related to toll-like receptor 4（TLR4）signaling pathway,inflammation,and apoptosis.This study indicates that DHA supplementation in Nile tilapia alleviates the liver damage caused by D-Gal N/LPS through increasing lipid catabolism,decreasing lipogenesis,TLR4 signaling pathway,inflammation,and apoptosis.Our study provides novel knowledge on the role of DHA in improving liver health in cultured aquatic animals for sustainable aquaculture.In summary,the present study developed three models of liver injury in adult zebrafish,which will provide important references for studying the protective effects of functional feed additives on liver injury in fish and the process and mechanism of liver disease development in cultured fish.In addition,we also provided comprehensively assessment of the mitigating effect of DHA on D-Gal N/LPS-induced acute liver injury in fish,and illustrated that DHA can protect liver health by regulating lipid metabolism,redox response and TLR4 signaling in fish.Our results support that in future practical production,DHA can be supplemented to diets to alleviates liver injury,improve organism health in cultured fish,and support environmentally friendly and sustainable development of aquaculture.