| The modern horse industry has gradually transformed into a new industry,which one including animal husbandry,finance,culture and sports.Biomass extracted from mulberry and moraceae plants is considered to be a potential livestock feed resource,As one of the main functional components in mulberry leaves,flavonoids mainly play the role of anti-oxidation and scavenging free radicals.This study extract mulberry leaves flavonoids by different process,and use the extracted flavonoids for total flavonoids yield detection and ingredient identification,based on the mulberry flavone extract,optimum extraction process and filter optimum addition concentration which one can alleviate oxidative stress in equine skeletal muscle satellite cells,and then study the Nrf2-ARE signaling pathways mediating the mulberry leaves flavonoids to alleviate the horse skeletal muscle satellite cells,the mechanism of oxidative stress for further discussion on the mulberry leaf flavonoids of research as well as relieve horse movement in the process of oxidative stress,providing theoretical basis.Experiment 1: Extraction of Flavonoids from Mulberry Leaves,In vitro antioxidant performance test and mulberry leaf flavonoids identified:Extract flavonoids from mulberry leaves through 3 different processes,the extraction methods were ultrasound-assisted extraction,AB-8 macroporous resin purification extraction and n-butanol extraction.Finally,the three process products were microwave assisted crude extraction(CEP),AB-8 macroporous resin purification extract(RP)and n-butanol extraction product(NB-EP),The purity of the products extracted by three different processes was determined,and the final concentration of CEP was 32.20%,RP was 37.94%,and NB-EP was 40.96%,respectively,nd three kinds of flavonoids extracted from mulberry leaves were tested for anti-oxidation in vitro,and the DPPH free radical scavenging rate was detected.The results showed that the free radical scavenging rate of the CEP group was 89.30%,the PR group was 90.62% and the NB-EP group was 90.32%.Next,non-targeted metabolome sequencing was performed for the three extracts obtained from the process.The metabolites were classified according to the chemical structure of flavonoids,and the different metabolites were screened out.Among them,glycyrrhiza,myricetin and isoflavones of neopsoraleae were unique to the CEP group.The specific metabolites of NB-EP were poplicin and neoggambogic acid.Due to the different extraction processes,the flavonoid metabolites obtained from the final enrichment are also different.Experiment 2: The mitigation effect of mulberry leaf flavonoids with three different processes on oxidative stress in equine skeletal muscle satellite cells:Under a microscope for the first three kinds of technology of mulberry leaf flavonoid extracts of different concentration of protection after 24 h,600 tendency for adding hydrogen peroxide/L processing after 6 h,the preliminary observation of cell morphology,you can see the CEP group under the condition of 0.6 0.8 mg/ml concentration of cells form protective effect best,RP and Nb-EP group concentration of 0.4 to 0.6 mg/ml of cells form has a better protective effect;CCK8 and MTT kits were used to detect the cell viability according to the instructions.Finally,the optimal concentration of flavonoids extract from three different processes was screened out,which were 0.8mg/ m L in the CEP group and 0.6mg/ m L in the RP and NB-EP groups,respectively.Experiment 3: The mechanism of mulberry leaf flavonoids mediated by Nrf2-ARE signaling pathway to relieve oxidative stress regulation of equine skeletal muscle satellite cells:Design gene-specific primers related to the Nrf2-ARE pathway: Through the relative expression of related antioxidant genes and downstream regulation of antioxidant enzyme activity,the mechanism of Nrf2-ARE signaling pathway mediated by mulberry leaf flavonoids in regulating the oxidative damage of Mongolian horse skeletal muscle satellite cells is determined.The blank group of equine skeletal muscle satellite cells,the hydrogen peroxide model group,and the optimal concentration group(treatment group)of the three selected mulberry leaf flavonoids process extracts were constructed for experiments.The expression of Nrf2 gene in oxidative stress model group cells was significantly lower than that of blank group cells(P<0.01),In conclusion,0.8mg/ m L of the CEP group and 0.6mg/ m L of the RP and NB-EP groups as the optimal concentration can mediate the expression of SOD1,SOD2,GPX1,GPX3 and Trx R1 through the Nrf2-ARE signaling pathway,thereby increasing the activities of SOD and GSH-Px and antioxidant enzymes,and reducing the content of MDA,and finally alleviating the oxidative damage of horse skeletal muscle satellite cells.The result shows: The expression of Nrf2 gene in the oxidative stress model group cells was significantly lower than that of the blank group cells(P<0.01),and the downstream related genes SOD1,SOD2,GPX1,GPX3 and Trx R1 blank group cells were compared with the model group cells.Significantly up-regulated(P<0.01);When 0.8mg/ml CEP was added,the expression of Nrf2 gene was significantly higher than that of model group cells(P<0.01);except for SOD2 gene,the expression of other antioxidant-related genes was significantly higher than that of model group cells Rise(P<0.01);When the RP group was pretreated with0.6mg/ml mulberry leaf flavonoids,the expression of Nrf2 gene and the expression of downstream related genes increased significantly compared with the oxidative stress model group cells(P<0.01),and 0.6mg was added After /ml Nb-EP,the changes of Nrf2 gene and its downstream regulatory genes were the same as those in the0.6mg/ml RP group,and the expression of each gene was extremely significantly up-regulated(P<0.01).The results of antioxidant-related enzyme activities showed that compared with the model group,the blank group had significantly up-regulated GSH-Px and SOD,and the total antioxidant capacity did not rise significantly.When the concentration of CEP was 0.8 mg/ml,the RP group was 0.6 mg/ml and After Nb-EP 0.6mg/ml pre-protected equine skeletal muscle satellite cells,compared with the oxidative stress model group,the activities of related antioxidant enzymes GSH-Px and SOD were significantly increased,and MDA was significantly down-regulated(P<0.01).The results showed that compared with the oxidative stress model group,the oxygen consumption rate and in vitro acidification rate of the cells in the mulberry leaf flavonoids supplemental group were significantly increased.In summary,the results show that 0.8mg/ml CEP,0.6mg/ml RP and0.6mg/ml Nb-EP are the optimal concentrations determined respectively as the three extraction products of mulberry leaf flavonoids,which can mediate SOD1 through the Nrf2-ARE signal pathway.,SOD2,GPX1,GPX3 and Trx R1 antioxidant gene expression,thereby increasing SOD and GSH-Px,antioxidant enzyme activity,reducing MDA content,and improve the respiration of the cell’s mitochondri,o achieve the role of alleviating the oxidative damage of equine skeletal muscle satellite cells. |
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