Biological Characteristics And Regulation Mechanism Of Lactobacillus Plantarum SAL In Response To Oxidative Stress

BackgroundThe lack of effective tumor treatments is a major problem threatening human health nowdays.Clinically,surgical resection,radiotherapy,and chemotherapy are the main methods in inhibiting tumor occurrence and metastasis.Inducing oxidative stress of tumor cells is the main mechanism of radiotherapy and chemotherapy.High-energy radiation and chemical drugs can act on malignant tumors,produce large amount of reactive oxygen free radicals（ROS）,and lead to the degeneration of biological macromolecules,such as DNA and protein and lipid peroxidation in cells and tissues.This process will eventually activate apoptosis signaling pathways that accelerate apoptosis and autophagy in tumor cells.For patients who have received radiotherapy and chemotherapy for a long time,the accumulated ROS toxicity and side effects can increase oxidative stress interference and affect the gastrointestinal microecosystem.Studies have shown that micro-ecological regulators produced by probiotics can improve the mucosal defense of the damaged gastrointestinal ecosystem,reduce the colonization of pathogenic bacteria,and maintain the stability of the gastrointestinal micro-ecological system.However,there is still a lack of probiotics that can not only regulate the gastrointestinal ecosystem,but also resist the oxidative stress.ObjectiveAnalyze the biological activity of Lactobacillus plantarum SAL.Screen the key genes of antioxidant stress of L.plantarum SAL,verify the reliability of the key genes,and explore the molecular mechanisms and metabolic pathways of antioxidant stress of L.plantarum SAL.Method1.The biological characteristics of L.plantarum SALMRS medium is prepared by adding 0-8 g/100 m L Na Cl.0.1%-0.5%bile salt,artificial gastric juice containing 1 g/100 m L pepsin（p H 1.5-2.5）,and artificial intestinal juice containing 1 g/100 m L trypsin were added to the MRS medium accordingly.The number of viable bacteria cultured at the end of logarithmic growth was counted in the high-salt and bile salt tolerance test.The number of viable bacteria cultured for 1,2,and4 hours in the artificial gastric juice tolerance test,and the number of viable bacteria cultured for 2,4,and 6 hours in the artificial intestinal fluid tolerance test were counted.The double agar sandwich method combined with seed drop method was used to detect bacteriostatic activity of L.plantarum SAL against 6 indicator bacteria.2.The regulatory mechanism of L.plantarum SAL in response to oxidative stressTo establish L.plantarum SAL antioxidant stress model,different concentration of H₂O₂ culture conditions were set up.The differentially expressed gene（DEGs）under the oxidative stress model of L.plantarum SAL were screened by RNA-seq high-throughput sequencing technology.Bioinformatics technology was used to explore the oxidative stress mechanism of L.plantarum SAL.GO and KEGG enrichment analysis were conducted on DEGs.STRING online tool and Cytascape software were utilized to build protein interaction network.Parameters in the Centiscape plug-in and MCODE plug-in were set to screen the hub genes,bottleneck genes,and core genes that meet the standards.Venn online tool was used to obtain the intersection of these three kinds of genes and finally obtain the key genes of antioxidant stress regulation.3.Functional analysis of L.plantarum SAL gad B geneOptimize the L.plantarum SAL gad B gene sequence and construct the prokaryotic expression system using Escherichia coli p Smart-I-gadb.IPTG was used to induce the expression of target protein and Ni column affinity chromatography was used to adsorb and purify 6×His labeled SUMO-GAD fusion protein.The activity of the fusion protease and the antioxidant capacity of BL21（DE3）/p Smart-I-gad B recombinant strain were detected by precolumn PITC derivatization high performance liquid chromatography.4.Effect of L-glutamic acid on antioxidant stress of L.plantarum SALSet L-glutamic acid culture solution with a concentration of 0-3%to detect the optimal L-glutamic acid concentration required for L.plantarum SAL culture.The effect of GAD protein expression on the antioxidant stress of L.plantarum SAL was indirectly verified by adding L-glutamic acid.Conclusion1.L.plantarum SAL has good prebiotic properties.Under the stress conditions such as high salt and artificial gastric juice,the growth condition of L.plantarum SAL is better than that of L.plantarum WCFS1.Under the stress conditions such as bile salt and artificial intestinal juice,the growth condition is equal to that of the classical strain,but the bacteriostatic activity is slightly lower than that of the classical strain.2.The oxidative stress model of L.plantarum SAL stimulated by 5 m M H₂O₂ for0.5 h was established.After transcriptome sequencing,a total of 313 DEGs were obtained,including 49 up-regulated genes and 164 down-regulated genes.In GO database,DEGs mainly annotate RNA transcription process,ribosomal protein composition,cell structure and molecular activity.In the KEGG pathway database,DEGs mainly annotate the ribosomal pathway,fatty acid biosynthesis,fatty acid metabolism and other pathways.The key genes gad B and pur A were obtained through Cytoscape visual network screening.Both gad B and pur A expressions were up-regulated and correlated with alanine,asparaginic acid,glutamic acid metabolic pathways,and bacterial metabolic pathways.q PCR confirmed the higher expression of gad B gene in bacterial cells,which was consistent with the transcriptome sequencing results.3.The prokaryotic expression system of BL21（DE3）/p Smart-I-gadb was successfully constructed.After 0.8 m M IPTG induction at 37℃for 4 hours,the optimized expression of SUMO-GAD recombinant fusion protein was obtained.It was about 66.91k Da in size and mainly expressed in the form of inclusion body.The purified recombinant protein was obtained by Ni column affinity chromatography.The protein expression is about 0.202 mg/L,and the enzyme activity is about 11.76 U/mg.4.Adding 1.5%L-glutamic acid to MRS medium can enhance the antioxidant stress of L.plantarum SAL.