Effects of Clostridium butyricum on Protein Synthesis of Rumen Explants in Dairy Cows
This experiment takes Clostridium butyricum as the research object and utilizes rumen explants for in vitro studies.The purpose was to evaluate the effects of Clostridium butyricum on protein synthesis in rumen explants through molecular analysis and amino acid profiling analysis.Experiment 1:mucosal tissue from the rumen of three healthy Holstein calves was collected.Through pathological observation,as well as detection of the expression level of calcium adhesion protein E and the vitality of rumen explant cells,the optimal culture time was determined.Experiment 2:a total of 200 μL of living bacteria solution of Clostridium butyricum at concentrations of3.0×107,3.0×108,3.0×109,and 3.0×1010 CFU/mL,inactivated bacteria solution,and the corresponding inactivated bacteria culture supernatant were added to the culture medium of rumen explants.Each group was set with three repetitions.Western blot and real-time quantitative PCR(RT-qPCR)were used to detect the effects of Clostridium butyricum on the key proteins and genes of the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target protein complex of rapamycin 1(mTORC1)protein syn-thesis pathway in explants.The differences in the contents of 17 free amino acids in the living bacteria culture supernatant and inactivated bacteria culture supernatant were also examined.The results showed as follows:1)the optimal culture time for rumen explants was 24 h.2)Western blot results showed that when the living bac-teria solution concentration of Clostridium butyricum was 3.0×108 CFU/mL,the protein expression levels of phosphorylated ribosomal 70S small subunit S6 kinase(p-P70S6K),phosphorylated eukaryotic initiation fac-tor 4E-binding protein 1(p-4EBP1),and phosphorylated mammalian target of rapamycin(p-mTOR)were the highest.At living bacteria solution concentrations of 3.0×108 and 3.0×109 CFU/mL,the protein expression levels of p-PI3K and p-AKT were significantly higher than those in the control group(P＜0.01).Phosphoryla-ted programmed cell death factor 4(p-PDCD4),which mainly inhibited protein synthesis,showed significant-ly lower protein expression levels in the groups with Clostridium butyricum living bacteria solution concentra-tions of 3.0×108 and 3.0×109 CFU/mL compared with the control group(P＜0.01).The protein expression levels of p-PI3K,p-AKT,p-mTOR,p-P70S6K,and p-4EBP1 were highest at the inactivated bacteria solu-tion concentration of 3.0×109 CFU/mL,all significantly higher than those in the control group(P＜0.01).3)Western blot results indicated that there were significant differences in the protein expression levels of p-mTOR,p-P70S6K,p-4EBP1,and p-PDCD4 in the living bacteria group compared wiht the control group(P＜0.01).In the inactivated bacteria group,the protein expression levels of p-AKT and p-PDCD4 showed significant or extremely significant differences compared with the control group(P＜0.05 or P＜0.01).Com-pared with the inactivated bacteria group,the protein expression levels of p-P70S6K,p-4EBP1,and p-PDCD4 in the living bacteria group were significantly higher(P＜0.05).RT-qPCR results showed that the relative ex-pression levels of AKT,mTOR,PI3K,P70S6K,4EBP1,and PDCD4 genes in the living bacteria group were significantly different from the control group(P＜0.01).The relative expression levels of P70S6K,AKT,mTOR,4EBP1,and PDCD4 genes in the inactivated bacteria group also showed significant or extremely sig-nificant differences compared to the control group(P＜0.05 or P＜0.01).4)Compared with the control group and the inactivated bacteria culture supernatant group,the contents of asparagine(Asp),threonine(Thr),al-anine(Ala),valine(Val),isoleucine(Ile),leucine(Leu),and cysteine(Cys)in the living bacteria cul-ture supernatant were significantly different(P＜0.05).In contrast,the contents of glutamic acid(Glu),gly-cine(Gly),methionine(Met),tyrosine(Tyr),lysine(Lys),and proline(Pro)in the inactivated bacteria culture supernatant were significantly different compared with the control group and the living bacteria culture supernatant group(P＜0.05).In summary,for Clostridium butyricum,both live and inactivated bacteria can promote protein synthesis in rumen tissue explants by changing the expression levels of related factors in the PI3K/AKT/mTORC1 pathway.[Chinese Journal of Animal Nutrition,2024,36(9):5709-5721]
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