Toxicity Of Bisphenol A To Gill And Liver Of Cyprinus Carpio And Protective Effect Of Geniposide

Bisphenol A（BPA）is a phenolic compound synthesized from phenol and acetone under acidic conditions.It is an important material for the preparation of epoxy resin,polystyrene resin and polycarbonate,and is widely used in industrial production.The widespread use of BPA makes it ubiquitous in the environment,which has aroused public concern about the safety of its use.BPA is a lipophilic compound,which can enter animals through the food chain or direct contact,and cuases harmful effects to health.In recent years,the toxic effects of BPA on fish have attracted wide attention,but its toxic mechanism has not been fully clarified.Common carp（Cyprinus carpio）is one of the most widely distributed freshwater bony fishes in the world.It is often used in ecotoxicological studies due to its strong adaptability,cold resistance,alkali resistance,low oxygen resistance,sensitive response to toxic substances and rapid response to changes in the water environment.Geniposide is an iridoid glycoside compound extracted from the dried and mature fruits of Rubiaceae plant gardenia（Gardenia jasminoides Ellis）.As a Chinese herbal medicine that protects liver and gallbladder,geniposide has been used in the prevention and treatment of liver diseases in mammals.However,in aquatic animals,there are few reports on the protective effects of geniposide on fish liver.In this study,common carp were selected as the research object to evaluate the toxic effects of long-term exposure to different concentrations of BPA by detecting the changes of related biochemical indexes and gene expression levels in gills and liver.At the same time,transcriptome and mi RNA sequencing were performed on the liver of common carp exposed to 0.5 mg/L BPA by high-throughput sequencing technology,and the hepatotoxicity mechanism of BPA was deeply analyzed at the molecular level.Furthermore,the protective effects of geniposide at different doses on hepatotoxicity of common carp caused by BPA were also investigated,which provided a theoretical basis for the application of geniposide in the prevention and treatment of fish liver diseases.Specific research contents and main research results are as follows:1.Effects of BPA exposure on oxidative stress,ion balance and immune regulation in gills of common carp:After 30 days of exposure to different concentrations of BPA（0,0.01,0.1,0.5 and 2 mg/L）,the gill tissues of common carp were collected for biochemical analysis and gene expression detection.The results showed that catalase（CAT）,glutathione（GSH）,glutathione S transferase（GST）and total antioxidant capacity（T-AOC）levels were significantly decreased after exposed to BPA at 0.1,0.5 and 2 mg/L.The levels of superoxide dismutase（SOD）,malondialdehyde（MDA）and 8-hydroxy-deoxyguanosine（8-OHDG）were significantly increased after exposed to BPA at moderate or high concentrations（0.1,0.5 or 2 mg/L）.The q PCR results showed that high concentrations of BPA（0.5 and 2 mg/L）activated Nrf2 signaling pathway and upregulated the expression levels of nrf2 and ho-1 in response to BPA induced oxidative stress.Meanwhile,BPA exposure destroyed the cytosine ion balance,and increased the concentrations of Na⁺and Ca²⁺in 0.1,0.5 and 2 mg/L BPA exposure.The gene expression of Na⁺/K⁺ATPase was upregulated after exposed to BPA at0.1 mg/L.In addition,BPA exposure induced immune and inflammatory responses.High concentrations of BPA（0.5 and 2 mg/L）exposure significantly increased the activity of alkaline phosphatase（AKP）and upregulated the gene expression of c-lyz.Inflammation-related genes expression results showed that TLRS signaling pathway was activated by 0.5and 2 mg/L BPA,the upregulations of tlr2,tlr4,myd88,irf3,I-ifn,il-1β,il-6 and il-10indicating that BPA exposure damaged the immune regulatory function of gill tissues of common carp and induced inflammation response.2.Effects of BPA exposure on oxidative stress,lipid metabolism and immune function of in the liver of common carp:After 30 days of exposure to different concentrations of BPA（0,0.01,0.1,0.5 and 2 mg/L）,blood and liver tissues of common carp were collected for biochemical analysis and gene expression detection.The results showed that BPA exposure significantly changed the activities of glutamate pyruvate transaminase（GPT）,AKP and glutamate oxalate transaminase（GOT）in serum of common carp,indicating that BPA resulted in hepatotoxicity in common carp.Lipid metabolism data showed that 2 mg/L of BPA significantly increased seurm total cholesterol（TC）,high-density lipoprotein（HDL-C）contents,decreased the content of serum low density lipoprotein（LDL-C）;0.1,0.5,and 2mg/L of BPA significantly upregualed the genes expression of fatp1,ppar-γ,fas,atgl,hsl,ppar-α,cpt1b and acox-1,suggesting that BPA exposure induced abnormal lipid metabolism in common carp liver.Oxidative stress results showed that high concentration of BPA（0.5 or2 mg/L）exposure significantly decreased the levels of SOD,CAT and T-AOC,and increased the levels of GSH and MDA in common carp liver.The expressions of nrf2,ho-1,cyp1a and cyp1b genes were significantly upregulated,indicating that BPA exposure induced oxidative stress in the liver of common carp.Immunoresponse data showed that exposure to BPA,especially high concentrations of BPA（0.5 and 2 mg/L）activated TLRs signaling pathways and significantly upregulated the gene expressions of tlr1,tlr2,tlr4,tlr5,myd88,p65,tnf-α、il-1β、il-6,il-10.Meanwhile,the expression of hepc and hsp70 genes were upregulated,suhhesting that BPA exposure induced immune and inflammatory responses in liver of common carp.3.Effects of BPA exposure on endoplasmic reticulum stress,autophagy and apoptosis in the liver of common carp:Common carp were exposed to different concentrations of BPA（0,0.01,0.1,0.5 and 2 mg/L）for 30 days.Liver tissues were collected for biochemical index and q PCR analysis.The results show that 0.5 and 2 mg/L BPA exposure significantly increased the content of Ca²⁺in liver tissue.0.1 and 0.5 mg/L BPA exposure significantly upregulated the expressions of grp78,perk,atf4,atf6,ire1 and chop genes suggesting that BPA exposure induced endoplasmic reticulum stress in common carp liver.Meanwhile,0.1and 0.5 mg/L BPA exposure significantly downregulated the expression of mtor gene and upregulated the gene expressions of atg3,atg7,tfeb,uvrag and mcoln1,suggesting that BPA exposure activated the autophagy pathway.In addition,BPA exposure（0.1,0.5 and 2 mg/L）significantly upregulated the gene expressions of caspase-8,bax,cytc,caspase-9 and caspase-3 and promoted hepatocyte apoptosis.4.Transcriptome sequencing analysis of hepatotoxicity of common carp induced by BPA exposure:Liver tissues from BPA-treated group（0.5 mg/L）and control group were taken for transcriptome sequencing to analyze the molecular mechanism of hepatotoxicity caused by BPA.The results showed that compared with the control group,A total of 1576differentially expressed genes were screened out in the BPA-treated group,including 930upregulated genes and 646 downregulated genes.GO analysis annotated a total of 45secondary functions.KEGG enrichment analysis found that differentially expressed genes were enriched in 37 metabolic pathways,involving 159 signal pathways.The top 20 signal pathways with significant enrichment of differentially expressed genes involved in lipid synthesis,metabolism and transport,decomposition and metabolism of carbohydrate,amino acid synthesis and metabolism,cell proliferation,differentiation,autophagy and apoptosis,immune regulation and inflammatory response and many other functions.Further,q PCR verification showed that the transcriptome sequencing results were reliable,which could provide a strong basis for the analysis of the molecular mechanism of BPA induced hepatotoxicity in common carp.5.Analysis of the mechanism of common carp hepatotoxicity induced by BPA by mi RNA sequencing:Liver tissues from BPA-treated group（0.5 mg/L）and the control group were taken for mi RNA sequencing.The results showed that a total of 6418 conserved mi RNAs were detected in liver samples and the length was mainly 22nt.Family analysis of these mi RNAs revealed that they belonged to 623 families,including let-7,mir-10,mir-17,mir-30,and mir-15,etc.Among the 6418 conserved mi RNAs,5500 mi RNAs predicted 47194target genes.Compared with the control group,a total of 79 differentially expressed mi RNAs were screened in BPA-treated group,of which 27 were upregulated and 52 were downregulated.GO annotations were performed on the target genes of these 79 differentially expressed mi RNAs,and the results showed that a total of 2341 target genes were annotated into 45 secondary functions in the GO database.KEGG enrichment analysis showed that the target genes of differentially expressed mi RNAs were enriched in 47 metabolic pathways,involving 265 signaling pathways.Among them,the top 20 signal pathways with significant enrichment are involved in the occurrence of chronic hepatitis,tumor occurrence and migration,cell growth and development,cell autophagy and apoptosis,inflammatory response,protein digestion and absorption and other functions.Finally,q PCR verification showed that mi RNA sequencing results were reliable,which could provide a strong basis for the analysis of the molecular mechanism of BPA induced hepatotoxicity in common carp.6.Protective effects of geniposide on hepatotoxicity of common carp induced by BPA:Common carp were exposed to 0.5 mg/L BPA and fed with three geniposide containing diets（1.25 g/kg,2.5 g/kg,5 g/kg）for 70 days,and the blood and liver tissues of common carp were collected for detection.The results showed that the decrease of specific growth rate and increase of feed coefficient induced by BPA were significantly improved by geniposide.GPT,GOT and AKP activities were significantly reduced by 2.5 g/kg and 5 g/kg of geniposide,and the degree of liver injury was alleviated.Feeding 1.25 g/kg or 2.5 g/kg of geniposide significantly decreased the levels of TG and LDL-C in serum and the expression levels of fatp1 and ppar-αin liver tissue,increased the content of HDL-C in serum,and improved lipid metabolism disorder in the liver of common carp.Meanwhile,after treatment with geniposide,the activity of SOD and the expression levels of nrf2 and ho-1 in liver tissues were significantly increased,and the expression level of keap1 was significantly decreased,indicating that geniposide had an alleviating effect on oxidative stress induced by BPA.In addition,geniposide significantly downregulated the expressions of tlr1,myd88,il-1βand il-6,upregulated the expression of il-10,and inhibited the inflammatory response of common carp liver.In conclusion,geniposide alleviated liver injury caused by BPA to different degrees in common carp.Considering the actual production cost,geniposide with a concentration of2.5 g/kg in feed is recommended in this study as the basis for screening liver protective drugs.