Effect Of Oxidative Stress On The Liver Transcriptome Characteristic And Meat Quality Of Pigs

Stress is an important factor affecting the growth performance of pigs in modern pig production,and oxidative stress is one of the more common types of stress.Oxidative stress causes the oxidative damage of the body tissues by affecting the expression of related genes and long noncoding RNA(lncRNA)in the body of the pig,causing changes in various functions and metabolism,making it in a sub-health state and reducing the production performance.The pigs can use its own antioxidant system to eliminate the negative effects of oxidative stress;the carcass antioxidant enzymes can maintain the balance of muscle redox status after pig slaughter.Then,oxidative stress caused abnormal expression of lncRNAs and genes in pig liver tissues,and the evaluation of antioxidant properties of carcass muscles using pH value at 24 h after slaughter(the ultimate pH,pHu)was not reported.Therefore,this study first used the Landrace weaned pigs as experimental animals,and used diquat(DQ)to construct the oxidative stress model of weaned piglets to explore the expression characteristics of the liver tissue transcriptome of weaned piglets under oxidative stress conditions.Then,analyzing the relationship between pHu value,antioxidant enzyme activity and related gene expression after pig slaughter was elucidated.The main research contents and results are as follows:1.After intraperitoneal injection of 10 mg/kg body weight DQ in weaned piglets in oxidative stress group,piglets showed vomiting,decreased appetite,and clinical symptoms such as significant increase in rectal temperature.At the end of the experiment,the average body weight and average daily gain(ADG)of weaned piglets in the oxidative stress group were significantly lower than the negative control group(P < 0.05),and the activity of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX)activity and total antioxidant capacity(T-AOC)in the liver were significantly lower(P < 0.05)than that in the negative control group,and the content of malondialdeyhde(MDA)was significantly higher(P < 0.05)than that in negative control group.HE staining analysis of liver tissue showed that the liver cells in the negative control group were closely arranged and the cell structure was intact,while the cytoplasm of the liver cells in the oxidative stress group was vacuolated,the nucleus was lysed,and lymphocytes were accumulated.2.A total of about 36.44 Gb of sequencing raw data was generated by RNA-Seq sequencing in 6 libraries of oxidative stress group and negative control group weaned pigliver.82.52%-84.48% of clean reads can be compared to the pig reference genome,of which39.16%-50.61% of clean reads are located in the exon region.After rigorous screening,a total of 6897 lncRNAs were obtained,including 3033 lincRNAs.Differential expression analysis of mRNAs and lncRNAs was performed using DESeq2 software.Eight differentially expressed lncRNAs(6 up-regulated and 2 down-regulated)and 30 differentially expressed mRNAs(8 up-regulated and 22 down-regulated)were identified in oxidative stress group weaned piglets.GO enrichment analysis of differentially expressed mRNAs and lncRNAs’ target genes revealed that seven genes(GNMT,PSAT1,PCK1,PSPH,SCD,APOA4,GCK)were enriched in the oxoacid metabolism process,and two genes(ENSSSCG00000027363,ENSSSCG00000029066)were enriched in intramolecular oxidoreductase activity,and another6 genes(GNMT,ENSSSCG00000010464,SCD,APOA4,GCK,ENSSSCG00000027643)were enriched in the redox process and play an important role in oxidative stress;KEGG pathway analysis shows insulin signaling and glucose metabolism pathways were closely related to oxidative stress.3.Using qPCR to verify the accuracy of lncRNA and gene expression in RNA-Seq sequencing data,the results showed that the difference fold between lncRNA and mRNA was consistent between qPCR and RNA-Seq sequencing analysis,and there was significant correlation.At the same time,the primary liver cells of piglets were isolated and verified.The results showed that the expression of up-regulated GNMT was significantly increased in pig primary hepatocytes after DQ stimulation(P < 0.05);the expression of GCK,down-regulated gene,was significantly decreased(P < 0.05)in pig primary hepatocytes after DQ stimulation for 12 h.4.A total of 137 pigs from three pig breeds with the same feeding condition were slaughtered and used to measure the pHu,superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),total antioxidant capacity(T-AOC),malondialdehyde(MDA)content and gene expression of SOD1 and GPX4.Loins from 137 pigs of three breeds were classified based on pHu into three groups: low(L-pH: ≤ 5.50),intermediate(I-pH: 5.51-5.90)and high(H-pH: ≥ 5.91).A majority of loins(47.5%-52%)were classified in intermediate group.The results suggested that the pHu value was correlated to the activity of SOD,GSH-PX,T-AOC,MDA,and gene expression of SOD1 and GPX4 using all pigs.In addition,our results also indicated a linear relationship between the pHu value and antioxidant traits.The pHu value accounted for 23%-40% of the variation in the antioxidant traits.In summary,we successfully constructed an oxidative stress model of Landrace weaned piglets,and found that oxidative stress leads to inhibition of growth performance of weanedpiglets,decreased liver antioxidant capacity,vacuolar degeneration of liver cells,lymphocyte infiltration and eventually Inflammatory response;aberrant expression of lncRNA and mRNA in liver tissue,and these differentially expressed mRNAs and lncRNAs target genes are enriched in oxidative stress-related pathways.Secondly,it can be evaluated by using the pHu of the carcass muscles at 24 h after slaughter,which indicates that pHu can be used to evaluate the antioxidant properties of the carcass muscles.The above results not only lay a foundation for studying the molecular mechanism of DQ-induced oxidative stress and tissue damage,but also provide a theoretical basis for further study of the effects of oxidative stress on pork quality.