| Object: The oviducts of Rana dybowskii Guenther and Rana amurensis Boulenger were subjected to quantitative proteomic study by TMT quantitative technology,and the functions of the screened differential proteins were analyzed using bioinformatics to study the biological processes and metabolic pathways they are involved in,etc.The extraction process of Oviductus Ranae was optimized by response surface methodology,and the polypeptide of Oviductus Ranae was prepared by compound enzymatic hydrolysis method.The antioxidant effect of Oviductus Ranae was studied,and the related mechanism of action of Oviductus Ranae on the alcoholic liver injury was explored through in vitro activity and effect,to improve the bioavailability of Oviductus Ranae and provide a new idea for the development of Oviductus Ranae.Method:(1)To screen out the differential expression proteins of Rana dybowskii Guenther and Rana amurensis Boulenger by using the tandem mass tag(TMT)technique.The enrichment analysis was also performed using GO enrichment and bioinformatics analysis of KEGG pathway analysis to screen out differentially expressed genes from the more enriched pathways,analyze the differences between the two Rana and provide a basis for the selection of raw materials for the Oviducts in subsequent experiments.(2)The protein concentration was used as the index,and the best extraction process of Oviductus Ranae was optimized by designing response surface experiments with the results of single-factor examination.ORPH was prepared by enzymatic digestion,and the protein content of ORPH was determined by the Bradford method,and the protein distribution of ORPH was detected by SDS-PAGE gel electrophoresis for screening the enzymatic digestion process.(3)The scavenging effect of ORPH on hydroxyl free radical and DPPH free radical was tested to verify its antioxidant ability.The effect of ORPH on different cell lines was detected by the CCK-8 method.The damage concentration and treatment time of ethanol were screened by the CCK-8 method,and the effect of ORPH on the injury of L-02 hepatocytes induced by ethanol was detected.Flow cytometry was used to detect cell apoptosis and cell cycle changes before and after ORPH administration,Hoechst staining was used to detect cell apoptosis,and JC-1 staining was used to detect mitochondrial membrane potential.The changes in ROS production were detected by flow cytometry.The expressions of apoptotic proteins caspase-3,Bcl2/Bax,CytC,mitogen-activated protein kinase(MAPK)signaling pathway JNK,p38,and cell pyroptosis related proteins in L-02 hepatocytes were detected by Western blot.Result:(1)Through identification,4919.0 proteins were found,and 4549.0 proteins contained quantitative information.If the change of differential expression was 2 times as the standard change threshold of HLVSDL,the amount of up-regulated protein was 509,and down-regulated protein was 519.KEGG pathway results were concentrated mainly including pyruvate metabolism,glycolysis,carbon metabolism,etc.In glycolysis pathway,ALDH2 gene was identified to be down-regulated,and was highly expressed in Rana dybowskii.(2)Through response surface software analysis,the optimal extraction process of Oviductus Ranae was obtained as follows:liquid-solid ratio of 1:140,time of 14 h,pH value of 4.5,and protein yield of 51.30%.By evaluating the changes of protein distribution and protein content after enzymatic hydrolysis,the optimal conditions for ORPH preparation were determined as pepsin and alkaline protease combined methods.SDS-PAGE electrophoresis was used to detect the molecular weight distribution of different enzymatic hydrolysis methods.The ORPH molecular weight prepared by the compound enzymatic hydrolysis method was mainly below 35 kD,and the molecular weight decreased significantly,and the protein content did not decrease.The effect of proteolysis was good.(3)The antioxidant test of ORPH showed that the scavenging rate of hydroxyl radical and DPPH in ORPH reached 30%-40%,which was higher than that of unhydrolyzed Oviductus Ranae.CCK-8 assay showed that ORPH had no toxic effect on cells.L-02 hepatocyte injury was induced by 400 m M ethanol within 12 h(p<0.001).The model of alcoholic hepatocyte injury was established successfully.Compared with the model group,L-02 hepatocytes treated with ORPH for 24 h had increased cell survival rate,improved cell cycle arrest,decreased apoptosis rate,increased mitochondrial membrane potential,and decreased ROS production.In the model group,the expressions of apoptosis-related proteins of Bax,CytC and Caspase-3 were significantly increased,MAPK signaling pathway-related proteins JNK and p38 were up-regulated(p<0.05),and the expressions of caustic death-related proteins Caspase-1,GSDMD,and IL-1β were significantly increased,while Bcl-2 was significantly decreased(p<0.01).Compared with the model group,the protein expressions of Bax,CytC,and Caspase-3 after ORPH treatment was significantly decreased(p<0.01),the protein expression of Bcl-2 was increased(p<0.01),and the expression of MAPK signaling pathway-related proteins and pyrotic death-related proteins were decreased(p<0.01).Conclusion:Combining TMT quantitative technology and bioinformatics,the differential proteins of Rana Dybowskii and Rana amurensis were analyzed,and the high expression of AKDH2 gene in the glycolytic pathway was found in Rana Dybowskii,which improved the basis for the selection of raw materials and the study of a bioactive metabolic pathway in subsequent experiments.The extraction process of Oviductus Ranae was optimized by response surface methodology.ORPH was extracted and prepared by combined enzymatic hydrolysis method and its antioxidant activity was verified.Through experiments in vitro activity,the results show that the ORPH can inhibit ethanol L-02 liver cells caused by oxidative stress-induced liver cell damage,improve the recovery of mitochondrial membrane potential,mitochondria-mediated apoptosis pathway of protein expression,and inhibit the MAPK signaling pathways related proteins,pyrolytic pathways related protein expression,thus reduce ethanol-induced L-02 hepatocytes mitochondrial dysfunction and inflammatory response and reduce oxidative stress,for the treatment of the alcoholic liver injury. |
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