Study On The Mechanism Of Lactobacillus Plantarum NJAU-01 To Relieve Oxidative Stress In Mice

Excessive reactive oxygen species(ROS)can cause serious damage to the human body and may cause cell dysfunction,cell poisoning,body aging and chronic diseases.Studies have shown that lactic acid bacteria(LAB)have excellent antioxidant activity and play a critical role in defense and protection against oxidative stress in vivo.Lactobacillus plantarum NJAU-01 was previously screened from Jinhua ham in our laboratory,with prior fermentation performance and antioxidant capacity,which was evidenced in scavenging free radical in vitro and cell model.However,it is necessary to further verify the antioxidant effect of NJAU-01 in vivo.In this paper,the role of antioxidant effect of NJAU-01 was proved by constructing D-gal aging model mice.The mechanism of NJAU-01 alleviating oxidative stress in liver of mice was elucidated by proteomics,and the effect of NJAU-01 on intestinal microflora was further explored by 16S rDNA sequencing technology.It was expected to reveal the mechanism of NJAU-01 alleviating oxidative stress in mice from the aspects of antioxidant enzymes,protein expression and intestinal microbial diversity.This will provide a theoretical basis for the development and utilization of antioxidant probiotics.The main research contents were as follows:1.Effect of Lactobacillus plantarum NJAU-01 on antioxidant activity in ICR mice.Mice were assigned to CK group(injection and intragastric administration of sterile normal normal saline),D-gal group(injection with 50 g/L D-gal,intragastric administration normal saline),CK+medium group(injection with sterile normal saline,intragastric administration of 108 CFU/mL NJAU-01),positive control of Vc group(injection with 50 g/L D-gal,intragastric administration of 0.001 g/mL VC),and low,medium and high dose NJAU-01 groups(injection with 50 g/L D-gal,intragastric administration with 107,108,109 CFU/mL NJAU-01 respectively).The entire experiment lasted six weeks and then killed for parameter detection.The results showed that the body weight of mice in D-gal group was significantly lower than that of other groups(P<0.05),and the indexes of heart and spleen were 0.72±0.13 and 0.27±0.02,which were significantly higher than CK group(P<0.05).The microstructure of the hepatic lobules in D-gal group was damaged and infiltrated by inflammatory cells.However,the pathological features of the liver were significantly alleviated in the VC group,low-dose,medium-dose and high-dose groups.The total antioxidant capacity and antioxidant enzyme activities including SOD,GSH-Px,CAT in heart and liver of low,medium and high dose groups were significantly higher than D-gal group(P<0.05).qRT-PCR showed that the expression of antioxidant enzyme mRNA level was significantly higher than D-gal group(P<0.05).Simultaneously,the content of lipid oxidation product MDA in low,medium and high dose groups was significantly decreased(P<0.05).In conclusion,NJAU-01 can effectively exert antioxidant effect in vivo and alleviate oxidative stress injury caused by D-gal treatment.2.Proteomic study of L.plantarum NJAU-01 alleviating liver oxidative stress in ICR mice.SDS-PAGE and semi-quantitative analysis showed that the relative intensity of liver proteins in CK group,D-gal group and medium dose group have significant variation(P<0.05)Expression of mouse liver proteins and significant metabolic pathways were explored by tandem mass spectrometry(TMT)proteomics.The results showed that a total of 683 differential proteins among three groups were obtained(P<0.05),which were related to cytoskeleton,carbohydrate metabolism,mitochondrial metabolism,lipid metabolism,transcriptional regulation and protein transport expression.Furthermore,CCR4-NOT complex subunit 10 protein and protein 6 containing RAS associated domain were significantly down-regulated in D-gal group(P<0.05),while carnitine acyl transferase(CROT)was significantly up-regulated in medium dose group(P<0.05).These proteins can maintain cell homeostasis and exert antioxidant effects.The genes of corresponding differential proteins including Cnot10,Crot,Rex26,and Gstm5 were verified from the qRT-PCR result.and their gene expression were consistent with the results of proteomic identification.GO analysis found that differential proteins are mainly distributed in cell,the cell membrane and organelles,protein complex,have the function of combination,catalytic function,regulation function,transcriptional regulation,transportation,antioxidant functions,etc.,involving in the body cell metabolism process,biological process,signal transmission and the immune system.KEGG gene annotation analysis showed significant pathways of differential proteins including MAPK signaling pathway,PI3K-Akt signaling pathway,NF-κB signaling pathway and other antioxidant related metabolic pathways.These results suggest that NJAU-01 may regulate the protein expression in response to oxidative stress and protect the body from oxidative damage.3.Effect of L.plantarum NJAU-01 on intestinal microflora in ICR mice.The changes of species distribution and abundance of intestinal microflora in CK group,D-gal aging model group and medium dose group were compared by 16S rDNA sequencing technology,in order to exploring the regulation effect of NJAU-01 on intestinal microflora.The results showed that the F/B value of the medium dose group was lower than that of the D-gal group.In the D-gal group,the abundance of disease-related bacteria such as Clostridium of Firmicutes,Proteobacteria and Epsilonbacteraeota of Proteobacteria,Tannerellaceae of Chlamydia,Saccharimonadaceae of Spirochetes,and Campylobacteria of Campylobacteria were increased in the D-gal group.In addition,the proportion of Firmicutes in the medium-dose NJAU-01 group were increased,compared to D-gal group.The abundance of beneficial bacteria such as Baculoides,Lactobacillus,Lactobacillus garneri and Turicibacter of Firmicutes,Myridae,Iglesiaceae and Actinomyces of Actinomycetes in the medium-dose NJAU-01 group was increased.The results suggest that NJAU-01 play an potentially protective role in alleviating oxidative stress by regulating the composition of intestinal flora.