Traditional Chinese Medicine Formula Intervenes On Muscle Fiber Type Transformation And Regulates Meat Quality Of Pork

Based on the difference of meat quality and muscle fiber content from finishing pigs which administrated with different level of traditional Chinese medicine formula,mRNA-mi RNAs combined with high-throughput sequencing were used to screen out genes and miRNAs associated with the regulation of muscle fiber type transformation,and then their functions were analysed in order to explore the mechanism of muscle fiber type regulation in this thesis.This study was divided into the following five parts:Part one:Composition,component analysis of traditional Chinese medicine formula and its effects on growth performance,weight and index of internal organs in ratsThe traditional Chinese medicine formula contained the following:25%of Radix Astragali（root）,20%of Radix Glycyrrhizae（root）,15%of Cortex Eucommiae Leaf（bark and leaf）,10%of Rhizoma Atractylodis Macrocephalae（rootstalk）,10%of Rhizoma Zingiberis（rootstalk）,10%of Fructus Crataegi（fruit）,5%of Fructus Rosae Laevigatae（fruit）,3%of Fructus Ligustri Lucidi（fruit）and 2%of Pericarpium Citri Reticulatae（pericarp）.The results showed that:（1）The main active ingredient of traditional Chinese medicine formula consists of formononetin,liquiritin,eucommiol,6-gingerol,ursolic acid,atractylenoide,glycyrrhizicacid,chlorogenicacid,salidroside,nobiletinand specnuezhenide;（2）Death was not found after rats were perfused with traditional Chinese medicine formula.Weight,daily feed intake,weight gain and feed:gain ratio as well as weight and index of internal organs were not significantly changed by dietary supplementation with traditional Chinese medicine formula（P>0.05）.The second part:Effects of traditional Chinese medicine formula on growth performance,carcass traits and meat quality of finishing pigsIn total,ninety 146-day-old castrated（Pietrain×Duroc,♂）×（Landrace×Yorkshire,♀）（PDLY）finishing pigs with an average initial body weight（BW）of 84.05±0.86 kg were assigned randomly to the following three dietary treatments:the control group,basal diet;basal diet plus 0.25%traditional Chinese medicine formula and basal diet plus 0.5%traditional Chinese medicine formula.Five replications were performed per treatment,with six pigs per pen.The study lasted for 35d.The results showed that:（1）There was no significant difference of dietary supplementation with traditional Chinese medicine formula on average daily gain,average daily feed intake,feed to gain ratio and carcass traits in fattening pigs（P>0.05）;（2）Meat color a*value in longissimus dorsi was significantly increased by 37.08%by addition of 0.25%traditional Chinese medicine formula,compared with the control group（P<0.05）;Meat color and marbling scores in longissimus dorsi from pigs fed 0.25%traditional Chinese medicine formula were significantly higher than those in the control group（P<0.05）;MDA content in longissimus dorsi was significantly decreased by 24.49%and 48.57%by dietary supplementation with 0.25%and 0.5%traditional Chinese medicine formula,respectively（P<0.05）;（3）Intramuscular fat content in longissimus dorsi from pigs consumed 0.25%and 0.5%traditional Chinese medicine formula were increased by 57.41%and 43.83%,respectively（P<0.05）;（4）Dietary supplemented with 0.25%traditional Chinese medicine formula significantly reduced heptadecanoic acid（C17:0）and linoleic acid（C18:2n-6）in longissimus dorsi of pigs（P<0.05）;Longissimus dorsi oleic acid（C18:1）from pigs fed 0.5%traditional Chinese medicine formula was significantly higher than that of the control group（P<0.05）;Eicosenoic acid（C20:1）in longissimus dorsi from pigs fed 0.25%traditional Chinese medicine formula was significantly higher than those in the other two groups（P<0.05）;Aspartic acid level in longissimus dorsi from pigs consumed 0.25%traditional Chinese medicine formula was significantly increased（P<0.05）;Dietary supplementation with 0.5%traditional Chinese medicine formula significantly reduced longissimus dorsi hypoxanthine by 33.89%,and significantly increased proline and sweet amino acids content in longissimus dorsi（P<0.05）.The third part:Effects of traditional Chinese medicine formula on muscle fiber types and its relationship with meat qualityTo explore whether traditional Chinese medicine formula changes pork quality by directly or indirectly changing muscle fiber types,this experiment was conducted to investigate effects of different levels of traditional Chinese medicine formula on muscle fiber types and its relationship with meat quality in fattening pig.The test results showed that:（1）Dietary addition of 0.25%traditional Chinese medicine formula in fattening pigs significantly increased area percentage of muscle fiber type I in longissimus dorsi by 2.35%（P<0.05）;Area proportion of muscle fiber type IIb+IIx in longissimus dorsi from pigs fed0.5%traditional Chinese medicine formula was significantly higher than that of pigs fed0.25%traditional Chinese medicine formula（P<0.05）;（2）Dietary inclusion of traditional Chinese medicine formula significantly increased MyHC I mRNA expression abundance in longissimus dorsi（P<0.05）;（3）Area percentage of muscle fiber type I and type IIa and I/II ratio were significantly negative correlated with brightness value of longissimus dorsi（r=-0.52,-0.58,-0.53,P<0.05）;Brightness value was significantly positive correlated with fiber type IIb+IIx area proportion（r=0.63,P<0.05）;Red value was positive correlated with fiber type I area percentage and I/II（r=0.57,0.58,P<0.05）;Inosinic acid content was positive correlated with fiber type IIa area percentage（r=0.64,P<0.05）;MyHC I mRNA expression was positive related with intramuscular fat and sweet amino acid content in longissimus dorsi（r=0.71 and 0.79,P<0.01）,and was negative correlated with hypoxanthine content（r=-0.52,P<0.05）;The cross-sectional area of fiber type IIb+IIx was positively correlated with pH_45min（r=0.59,P<0.05）;（4）A similar pattern was observed from enzyme histochemical staining and RT-qPCR in determining muscle fiber;Enzyme histochemical staining method was more intuitive.The fourth part:mRNA and miRNA expression profile analysis and screening key regulatory factors of muscle fiber typesThe results of mRNA and miRNA transcriptome showed that:（1）We observed that29647 genes co-expressed and 75 genes differentially expressed in longissimus dorsi from pigs fed the basal diet and 0.25%traditional Chinese medicine formula.（2）Differentially expressed genes in longissimus dorsi from pigs fed the basal diet and 0.25%traditional Chinese medicine formula were enriched in muscle growth,muscle contraction and energy metabolism associated pathways using GO.KEGG enriched skeletal muscle energy metabolism and transfer process,glycolysis and Wnt-protein binding pathways,PPAR pathway,pyruvate metabolism and TCA cycle,which was directly or indirectly related with muscle contraction and muscle fiber type.（3）Genes involved in the process of traditional Chinese medicine formula on muscle fiber types regulation were screened out from DGE,including MYH7,PPAR,MAPK6,MEF2C,MSTN,FoxO1,MyoD1 and CaMK.（4）We obtained 1842 mature miRNAs and 1663 pre-miRNAs from the miRNAs sequencing,including 1128 novle mature miRNAs and 41 novel pre-miRNAs.We found that 48 and116 miRNAs differentially expressed,and 706 miRNAs co-expressed in longissimus dorsi from pigs fed the basal diet and 0.25%traditional Chinese medicine formula,respectively.（5）MTOR pathway,MAPK pathway,TCA cycle and inositol phosphate metabolism pathways associated with protein synthesis and muscle fiber type,energy metabolism,cell apoptosis and cell structure were enriched by KEGG.（6）MiRNAs involved in the process of traditional Chinese medicine formula on muscle fiber types regulation were screened out from miRNAs sequencing,including ssc-miR-423-5p and ssc-miR-27a.（7）Slow muscle fiber gene MYH7 was directly or indirectly targeted by 24 miRNAs;Binding energies between miR-423-5p,miR-320 and miR-27a and their targets were relatively low.The fifth part:Slow muscle fiber regulation pathway of miR-27aMiR-27a was performed as an example to elucidate the mechanism of miRNA on muscle fiber type regulation in this experiment.The reasons for choosing miR-27a was presented in the Chapter sixth of this thesis.The results showed that:（1）An opposite expression trend of miR-27a,MYH7 and PGC-1αwas found before and after porcine skeletal muscle satellite cells differentiation.（2）MiR-27a regulated gene and marker protein of slow muscle fiber,which was confirmed by myoblast transfected with miR-27a mimics/inhibitor.（3）We further inferred that miR-27a may regulate slow muscle fiber through PGC-1α-MEF2C pathway from the results of PGC-1α,MEF2C and mitochondrial genes（NRF-1,mtTFA,COX IV and Cytc）expression and SDH activity.（4）Results from siRNA interference of myoblast endogenous PGC-1αindicated that PGC-1αwas a direct target of miR-27a.（5）Dual luciferase reporter gene experiment showed that miR-27a could bind to the predicted target site sequences of PGC-1α.Therefore,miR-27a regulated PGC-1αexpression via inhibiting PGC-1α.In summary,we digged out genes,miRNAs and signaling pathways involved in the regulation of muscle fiber types based on improvements of traditional Chinese medicine formula on meat quality such as meat color and muscle fiber types and its relationship with meat quality,and expounded the regulation of muscle fiber type transformation by miR-27a may via PGC-1α-MEF2C-MYH7 pathway.