An Exploration of the Antioxidative and Anti-Inflammatory Role of Lactiplantibacillus plantarum 106 via Improving Mitochondrial Function

In this present study, bioinformatics analysis and the experimental validation method were used to systematically explore the antioxidant activity and anti-inflammatory effect of Lactiplantibacillus plantarum A106, which was isolated from traditional Chinese pickles, on lipopolysaccharide (LPS)-induced RAW264.7 macrophages. L. plantarum A106 had a good scavenging ability for DPPH, ABTS, and hydroxyl radicals. Furthermore, L. plantarum A106 could increase the activity of RAW264.7 macrophages; raise the SOD and GSH levels, with or without LPS sensitization; or decrease the MDA, TNF–α, and IL–6 levels. In order to deeply seek the antioxidant and anti-inflammatory role and mechanism, bioinformatic analysis, including GO, KEGG, and GSEA analysis, was used to conduct an in-depth analysis, and the results showed that the LPS treatment of RAW264.7 macrophages significantly upregulated inflammatory-related genes and revealed an enrichment in the inflammatory signaling pathways. Additionally, a network analysis via the Cytoscape software (version 3.9.1) identified key central genes and found that LPS also disturbed apoptosis and mitochondrial function. Based on the above bioinformatics analysis, the effects of L. plantarum A106 on inflammation-related gene expression, mitochondrial function, apoptosis, etc., were detected. The results indicated that L. plantarum A106 restored the declined expression levels of crucial genes like TNF–α and IL–6; mitochondrial membrane potential; and apoptosis and the expression of apoptosis-related genes, Bcl–2, Caspase–3, and Bax. These results suggest that L. plantarum A106 exerts antioxidant activity and anti-inflammatory effects through regulating inflammatory and apoptosis-related gene expression, restoring the mitochondrial membrane potential.


Introduction
Lactic acid bacteria (LAB), a class of probiotics, not only maintain the ecological balance of the intestinal microbiota but also exhibit biologically active properties that have antitumor, immunomodulatory, and antioxidant effects.These properties have contributed to extensive research and application in the food and health product industries.Recently, LAB have been widely recognized for their crucial role in modulating inflammatory responses, including the regulation of cytokine production, the improvement of the intestinal barrier function, and the increase of immune cell activity [1,2].In one study, L. reuteri LM1071 inhibited the production of inflammatory mediators like NO, pro-inflammatory factors, and COX proteins, thus demonstrating anti-inflammatory potential [3].In another, L. plantarum DP189 downregulated pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in neurodegenerative diseases, thereby inhibiting inflammatory responses and modulating the gut microbiota [4].Another study, which used a D-galactose-induced senescent mouse model to explore the antioxidant effects of lactobacillus on hepatotoxicity, found Foods 2024, 13,1981 3 of 18 superoxide dismutase (SOD), reactive oxygen species (ROS), mitochondrial membrane potential, and apoptosis kits were purchased from Beyotime Biotechnology (Beijing, China).

Culture of L. plantarum A106 and Preparation of Different Components
The L. plantarum A106 that was stored at −80 • C was quickly dissolved in a 37 • C water bath and inoculated into the MRS liquid medium with a 3% inoculum.It was incubated at 37 • C for 24 h and subsequently subcultured to 2-3 generations for further experiments.
With respect to the preparation of the culture supernatant and bacterial cells, the L. plantarum A106 was put in a shaking incubator (37 • C, 150 rpm) for 18-24 h, then a centrifuge at 5610× g for 20 min at 4 • C, before the supernatant was collected and filtered through a 0.22 µm filter membrane.The bacterial cells were washed three times with PBS (0.01 mol/L at a pH of 7.4) and adjusted to 1 × 10 9 CFU/mL.
With respect to the preparation of the cell-free extract, the lysozyme (1.5 mg/mL) was added to intact L. plantarum A106 with a concentration of 1 × 10 9 CFU/mL, then put in a water bath at 37 • C for 30 min, accompanied by an ultrasonic cell crusher in an ice bath (250 W, 5 s of operation, 10 s of interval, and a duration of 30 min).Afterward, the mixture was centrifuged at 5610× g/min for 10 min to remove any bacterial debris and to obtain the cell-free extracts.

Effectiveness of Different Fractions of L. plantarum A106 on Free Radical Scavenging 2.4.1. DPPH Scavenging Rate
The DPPH scavenging rate was tested according to the method reported by Xu et al. (2019), making appropriate modifications [15].Specifically, 1 mg of DPPH was dissolved in 25 mL of anhydrous ethanol.Subsequently, 500 µL of different components were thoroughly mixed with 500 µL of DPPH-anhydrous ethanol solution.There was centrifugation at 4909× g for 10 min, before the supernatant was discarded, and the absorbance was tested at 517 nm.The DPPH scavenging rate was calculated according to the following formula: DPPH clearance (%) = [1 − (A 2 − A 1 )/A 0 ] × 100 where A 0 is the absorbance of the ultrapure water and DPPH; A 1 is the absorbance of the sample and ultrapure water; and A 2 is the absorbance of the sample and DPPH.

ABTS + Scavenging Rate
The ABTS + scavenging rate was tested according to the method reported by Li et al. (2015), with minor modifications [16].The ABTS reaction solution was prepared according to the instructions.In total, 300 µL of various fractions were separately mixed with 300 µL of the ABTS reaction solution.The mixture was shook for 30 s and then kept in dark conditions for 6 min, followed by centrifuging at 5610× g for 5 min.The supernatant was transferred to a 96-well plate to read at 734 nm.The ABTS + scavenging rate was calculated according to the following formula: ABTS + scavenging rate (%) = (1 − A 1 /A 0 ) × 100 where A 0 is the absorbance of the ABTS working solution and A 1 is the absorbance of the sample.

Hydroxyl Radical Scavenging Rate
The hydroxyl radical scavenging rate was detected according to the method described by Yang et al. (2017), with appropriate modifications [17].Firstly, 0.5 mL of 1,10-o-diazophenanthrene (1.875 mmol/L), 1 mL of PBS (0.01 mmol/L at a pH of 7.4), and 0.5 mL of ferrous sulphate (1.875 mmol/L) were added to a 5 mL Eppendorf tube and mixed thoroughly.Then, 0.5 mL of the samples and 0.5 mL of H 2 O 2 (0.1%) were added, and the mixture was vortexed thoroughly and incubated in a water bath at 37 • C for 1 h.Following this, the samples were centrifuged for 10 min at 5610× g and the supernatant was transferred to a 96-well plate to read at 536 nm.The hydroxyl radical scavenging rate was calculated according to the following formula: where A s is the absorbance of the samples and H 2 O 2 ; A n is the absorbance of the PBS; and A b is the absorbance of the H 2 O 2 .

Cell Viability Assay
The RAW264.6 macrophages were cultivated in a cell culture incubator at 37 • C with 5% CO 2 using the DMEM/high glucose medium supplemented with 10% fetal bovine serum.After subculturing for 2-3 generations, the cells were digested with trypsin, centrifuged, resuspended in the complete medium, and adjusted to a concentration of 1 × 10 6 cells/mL, before being inoculated in a 96-well plate with a 200 µL cell suspension per well and cultured for 24 h.In the experimental group, 50 µL of bacterial suspension was added to each well at a different ratio: 1:100 (1 × 10 4 CFU/mL) or 1:1000 (1 × 10 3 CFU/mL) relative to the cell concentration using the complete medium.The control group only used the complete medium.Afterwards, 1 µg/mL of LPS was added to the experimental group 4 h before the end of the cultivation; meanwhile, 20 µL of CCK-8 solution was added to each well.The cells were then incubated at 37 • C for 1.5, 2, 2.5, and 3 h.The absorbance values at 450 nm were measured at various incubation periods.The cell viability was determined using the following formula: where A 0 is the absorbance of the positive group, A 1 is the absorbance of the control group, and A 2 is the absorbance of the experimental group.

Measurement of the Levels of the Cytokines, TNF-α and IL-4
The cells were harvested and centrifuged, and the culture supernatant was collected.The cytokines TNF-α and IL-4 in the supernatant of the culture that underwent different treatments were detected using commercial ELISA kits (BioLegend, San Diego, CA, USA) according to the manufacturer's instruction.

Download of Microarray Datasets and Extraction of Differentially Expressed Genes (DEGs) in Microarray Datasets
The dataset GSE186886, downloaded from the GEO database (http://www.ncbi.nlm.nih.gov/geo/), included the control group and LPS-induced RAW264.7 group.
The "limma" (version 3.58.1)R package was employed to identify the differentially expressed genes 1 (DEGs1) between the LPS-induced group and the control group in the GSE186886 dataset, where p < 0.05 and |log2 (fold change, FC)| > 1 were set as thresholds.Subsequently, "ggplot2" (version 3.5.0)was utilized to construct a volcano plot illustrating the expression of the DEGs.

Functional Enrichment Analysis
Both the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis of the DEGs2 (differentially expressed genes in DEGs1 with |log2 (fold change, FC)| > 2) were conducted using the DAVID (https://davidbioinformatics.nih.gov/)online platform, with p-values < 0.05 and q-values < 0. The results were visualized using the "ggplot2" R package.
In our study, we conducted an enrichment analysis of the DEGs2 targeting the KEGG pathways, GO terms, and Reactome pathways using the "clusterProfiler" (version 4.10.1) in R. The gene set enrichment analysis (GSEA) method was employed to associate the DEGs2 with KEGG pathways, GO terms, and Reactome pathways, considering p < 0.05 as significant.For the visualization of these enrichment outcomes, the "ggplot2" package in R was utilized.

Protein-Protein Interaction (PPI) Network Analysis
To elucidate the functional interactions among the screened potential proteins, the identified DEGs2 were imported into the STRING database (https://string-db.org/) to construct a protein-protein interaction (PPI) network, where the confidence score threshold was set to 0. Subsequently, the PPI network was imported into the Cytoscape software (version 3.9.1),where the significant modules were depicted using the CytoNCA (version 2.1.6)and CytoHubba (version 0.1) plugin.

Gene Ontology and Pathway Enrichment Analysis Using Metascape
A Metascape analysis offers the advantage of integrating multiple gene list analysis and enrichment tools within a single platform, thereby providing comprehensive, interactive visualizations for a deeper understanding of biological functions and pathways.To deeply explore the biological functions and pathways associated with the genes related to the mitochondrial pathways identified in the Gene Ontology Cellular Component (GO CC) analysis, an enrichment analysis was conducted using Metascape (https://metascape.org).Finally, Metascape's interactive visualization tools were used to present the results.

Determination of SOD, GSH, and MDA Content
The RAW264.7 macrophages were digested, centrifuged, and resuspended.The cell concentration was adjusted to 1 × 10 6 cells/mL and then inoculated into a 6-well plate with 2 mL per well.In the experimental group, 50 µL of bacterial suspension was added to each well (see the treatment of the L. plantarum A106 in Section 2.5).The control group only used the complete medium.The LPS was added at a final concentration of 1 µg/mL to the experimental group 4 h before the end of the cultivation.The cells were lysed using a cell lysis buffer (Beyotime) and then collected and centrifuged, and the supernatant was taken for the determination of the SOD, GSH, and MDA contents using a total SOD assay kit with WST-8, a GSH assay kit with DTNB, and an MDA assay kit (Beyotime), according to the instructions of each kit.

Determination of ROS Level
The cells cultivated in the 6-well plate underwent the same treatment as described in Section 2.12.The levels of ROS kit (Beyotime) following the provided instructions.The cells were collected, centrifuged, and resuspended before the levels of ROS were analyzed using flow cytometry (CytoFLEX, Beckman Coulter, Indiana, IN, USA).

Determination of Mitochondrial Membrane Potential (MMP)
The evaluation of the MMP is an important indicator to assess the normal function of the mitochondria.The MMP was measured using an enhanced mitochondrial membrane potential assay kit with JC-1 according to the instructions of operation (Beyotime).Firstly, the cellular states and staining conditions were observed under the fluorescence microscope.Then, the cells were collected, centrifuged, and resuspended, and the intensity of the fluorescence was assayed through a flow cytometer (Beckman Coulter, CytoFLEX).

Apoptosis Assay
Generally, the loss of MPP is a sign of cellular apoptosis.In this study, cellular apoptosis was also determined using an Annexin V-FITC apoptosis detection kit, according to the protocol of the manufacturer (Beyotime).Briefly, the cells were seeded in a 6-well plate, then collected, centrifuged, and resuspended in 195 µL of Annexin V-FITC binding buffer at the end of the different treatments.This was followed by the addition of 5 µL of Annexin V-FITC and 10 µL of PI (propidium iodide) staining solution, before incubation at room temperature for 10-20 min in the dark and subsequent storage in an ice water bath for analysis in a flow cytometer.

Statistical Analysis
The statistical analysis was conducted using Origen 2019b and SPSS Statistics 25 software using a one-way ANOVA, where p < 0.05 was considered as statistical significance.All the experiments were expressed as the mean ± SEM.

Effect of Culture Supernatant, Bacterial Cells, and Cell-Free Extracts on the Scavenging Rate of Different Radicals In Vitro
The supernatant, bacterial cells, and cell-free extracts of the L. plantarum A106 culture were prepared separately and tested for the scavenging rates of DPPH, ABTS + and hydroxyl radicals.It can be seen from Figure 1 that the DPPH scavenging rate of the L. plantarum A106 culture supernatant was significantly higher than those of the bacterial suspension and the cell-free extract group (p < 0.01).Furthermore, the DPPH scavenging rate of the bacterial suspension was also obviously higher than that of the cell-free extract group (p < 0.01).Vitamin C (Vc) was the positive group (Figure 1A).Moreover, the results of the ABTS + radical scavenging rate also indicated a similar trend to the DPPH scavenging rate.(Figure 1B).Interestingly, the hydroxyl radical scavenging rate showed the same trend as the DPPH, with the only difference being that the cell-free extract group had a higher hydroxyl radical scavenging rate than the bacterial suspension group (Figure 1C).

Effect of L. plantarum A106 on the Cell Viability of LPS-Induced RAW264.7 Macrophages
The cell viability was determined with the CCK-8 assay kit.As shown in Figure 1D, the L. plantarum A106 demonstrated no cytotoxicity towards RAW264.7 macrophages.Additionally, the cell viability of the L. plantarum A106-treated group (1:100 or 1:1000) was significantly higher (p < 0.05) than that of the control group at 1.5 h.Furthermore, there were no significant differences among all groups after cultivating for 2 h; as a result, 2 h was determined to be the ideal duration for the CCK-8 treatment.After a 2 h CCK-8 treatment, there was a noteworthy reduction in the cell viability of the LPS-treated group when compared to the control group.However, the treatment with L. plantarum A106 (1:100 or 1:1000) significantly impeded the decrease in cell viability (p < 0.05) (Figure 1E).

Effect of L. plantarum A106 on the Cell Viability of LPS-Induced RAW 264.7 Macrophages
The cell viability was determined with the CCK-8 assay kit.As shown in Figure 1D, the L. plantarum A106 demonstrated no cytotoxicity towards RAW264.7 macrophages.Additionally, the cell viability of the L. plantarum A106-treated group (1:100 or 1:1000) was significantly higher (p < 0.05) than that of the control group at 1.5 h.Furthermore, there were no significant differences among all groups after cultivating for 2 h; as a result, 2 h was determined to be the ideal duration for the CCK-8 treatment.After a 2 h CCK-8 treatment, there was a noteworthy reduction in the cell viability of the LPS-treated group when compared to the control group.However, the treatment with L. plantarum A106 (1:100 or 1:1000) significantly impeded the decrease in cell viability (p < 0.05) (Figure 1E).

Effect of L. plantarum A106 Treatment on the LPS-Induced Secretion of Inflammatory Factors
As shown in Figure 1F,G, compared with the control group, the levels of the proinflammatory factor TNF−α were significantly increased (p < 0.01) and of the anti-inflammatory factor IL−4 were significantly reduced in the RAW264.7 macrophages that were stimulated by LPS (p < 0.05).In contrast, the treatment with the L. plantarum A106 significantly reduced the TNF-α levels (p < 0.01) but obviously increased the IL−4 levels (p < 0.05).

Selection and Functional Enrichment Analysis of DEGs
Among the 2465 differentially expressed genes (DEGs1) identified from the GSE186886 dataset, 519 genes were upregulated and 1946 genes were downregulated.The selection criteria were p < 0.05 and |log2 (fold change, FC)| > 1.All DEGs1 are presented in a volcano plot (Figure 2A), with red indicating the upregulated genes, blue indicating the downregulated genes, and gray representing the genes that did not show significant expression changes in the dataset.

Effect of L. plantarum A106 Treatment on the LPS-Induced Secretion of Inflammatory Factors
As shown in Figure 1F,G, compared with the control group, the levels of the proinflammatory factor TNF-α were significantly increased (p < 0.01) and of the anti-inflammatory factor IL-4 were significantly reduced in the RAW264.7 macrophages that were stimulated by LPS (p < 0.05).In contrast, the treatment with the L. plantarum A106 significantly reduced the TNF-α levels (p < 0.01) but obviously increased the IL-4 levels (p < 0.05).

Selection and Functional Enrichment Analysis of DEGs
Among the 2465 differentially expressed genes (DEGs1) identified from the GSE186886 dataset, 519 genes were upregulated and 1946 genes were downregulated.The selection criteria were p < 0.05 and |log2 (fold change, FC)| > 1.All DEGs1 are presented in a volcano plot (Figure 2A), with red indicating the upregulated genes, blue indicating the downregulated genes, and gray representing the genes that did not show significant expression changes in the dataset.
To further investigate the functions of the DEGs, the GO and KEGG analyses were performed on the DEGs2 (differentially expressed genes from DEGs1 with |log2 (fold change, FC)| > 2) using DAVID, and the top 20 enriched entries were visualized using bubble charts.As shown in Figure 2D, the KEGG enrichment analysis presented several pathways that were significantly enriched with inflammatory responses, including the cytokine-cytokine receptor interaction; the TNF signaling pathway and IL-17 signaling pathway; the NOD-like receptor signaling pathway; and the NF-kappa B signaling pathway.As depicted in Figure 2C, the BP (biological process) category was predominantly composed of the innate immune response, the positive regulation of transcription from the RNA polymerase II promoter, the inflammatory response, and the immune system process.The MF (molecular function) category (Figure 2B) comprised protein binding, identical protein binding, hydrolase activity, protein homodimerization activity, and cytokine activity.To further investigate the functions of the DEGs, the GO and KEGG analyses were performed on the DEGs2 (differentially expressed genes from DEGs1 with |log2 (fold change, FC)| > 2) using DAVID, and the top 20 enriched entries were visualized using bubble charts.As shown in Figure 2D, the KEGG enrichment analysis presented several pathways that were significantly enriched with inflammatory responses, including the cytokine-cytokine receptor interaction; the TNF signaling pathway and IL-17 signaling pathway; the NOD-like receptor signaling pathway; and the NF-kappa B signaling pathway.As depicted in Figure 2C, the BP (biological process) category was predominantly composed of the innate immune response, the positive regulation of transcription from the RNA polymerase II promoter, the inflammatory response, and the immune system process.The MF (molecular function) category (Figure 2B) comprised protein binding, identical protein binding, hydrolase activity, protein homodimerization activity, and cytokine activity.

GSEA Analysis of Biological Processes and Functions
The gene set enrichment analysis (GSEA) was conducted to delve deeper into the biological functions and pathways associated with DEGs2, because the results of the GO analysis showed that DEGs2 were mainly enriched in extracellular space and the extracellular region (Figure 3C).In terms of biological processes (Figure 3A), the main enrichment was related to the response to IL−1, monocyte chemotaxis, and granulocyte migration.Similarly, at the molecular function level (Figure 3B), there was found to be high enrichment of the cytokine activity, cytokine receptor binding, and chemokine activity.The

GSEA Analysis of Biological Processes and Functions
The gene set enrichment analysis (GSEA) was conducted to delve deeper into the biological functions and pathways associated with DEGs2, because the results of the GO analysis showed that DEGs2 were mainly enriched in extracellular space and the extracellular region (Figure 3C).In terms of biological processes (Figure 3A), the main enrichment was related to the response to IL-1, monocyte chemotaxis, and granulocyte migration.Similarly, at the molecular function level (Figure 3B), there was found to be high enrichment of the cytokine activity, cytokine receptor binding, and chemokine activity.The pathway analysis based on KEGG and Reactome highlighted the role of related pro-inflammatory cytokines (Figure 3D,E).Therefore, these results suggest that the LPS treatment of RAW264.7 macrophages may contribute to the development of inflammatory responses.

Construction of Protein-Protein Interaction Network, Selection of Hub Genes, and Metascape Enrichment Analysis
After uploading the selected DEGs2 to the STRING online database with a confidence threshold of >0.7 and visualizing the results in Cytoscape software (version 3.9.1),any isolated nodes were removed to establish a protein-protein interaction (PPI) network consisting of 164 nodes and 667 edges.Subsequently, the betweenness centrality of the nodes was assessed using the CytoNCA plugin, where darker node colors indicated higher betweenness centrality values (Figure 4A).In addition, the top 10 genes were identified in Cytohubba using the maximal clique centrality (MCC) method (Figure 4B), where the node color represented connectivity (red for higher degrees and yellow for lower degrees).Integrating the outcomes of the betweenness centrality and maximal clique centrality analyses, we propose that TNF, IL-6, Cxcl10, and IL-1β potentially play pivotal roles in modulating LPS-induced infections.
pathway analysis based on KEGG and Reactome highlighted the role of related pro-inflammatory cytokines (Figure 3D,E).Therefore, these results suggest that the LPS treatment of RAW264.7 macrophages may contribute to the development of inflammatory responses.

Construction of Protein-Protein Interaction Network, Selection of Hub Genes, and Metascape Enrichment Analysis
After uploading the selected DEGs2 to the STRING online database with a confidence threshold of >0.7 and visualizing the results in Cytoscape software (version 3.9.1),any isolated nodes were removed to establish a protein-protein interaction (PPI) network consisting of 164 nodes and 667 edges.Subsequently, the betweenness centrality of the nodes was assessed using the CytoNCA plugin, where darker node colors indicated higher betweenness centrality values (Figure 4A).In addition, the top 10 genes were identified in Cytohubba using the maximal clique centrality (MCC) method (Figure 4B), where the node color represented connectivity (red for higher degrees and yellow for lower degrees).Integrating the outcomes of the betweenness centrality and maximal clique centrality analyses, we propose that TNF, IL−6, Cxcl10, and IL−1β potentially play pivotal roles in modulating LPS-induced infections.According to the CC (cellular component) analysis (Figure 4C), the above findings were mainly associated with organelles such as the nucleus and mitochondrion.Given that the role of mitochondria in cellular functionality is critical, because they serve as the cell's powerhouses by producing ATP via respiration and regulating cellular metabolism and apoptosis, we conducted an enrichment analysis on 50 DEGs associated with mitochondria identified in the CC (cellular component) using the Metascape online platform (https://metascape.org).The results primarily indicate enrichment in the regulation of cellular respiration, the regulation of mitochondrial membrane potential, and the respiratory electron transport chain (Figure 4D).According to the CC (cellular component) analysis (Figure 4C), the above findings were mainly associated with organelles such as the nucleus and mitochondrion.Given that the role of mitochondria in cellular functionality is critical, because they serve as the cell's powerhouses by producing ATP via respiration and regulating cellular metabolism and apoptosis, we conducted an enrichment analysis on 50 DEGs associated with mitochondria identified in the CC (cellular component) using the Metascape online platform (https://metascape.org).The results primarily indicate enrichment in the regulation of cellular respiration, the regulation of mitochondrial membrane potential, and the respiratory electron transport chain (Figure 4D).

Effect of L. plantarum A106 on LPS-Induced SOD, GSH, and MDA Activities
RAW264.7 macrophages were used to establish a LPS-induced oxidative stress model to further explore the antioxidant capacity of L. plantarum A 106.The results are shown in Figure 6.Under the stimulation of LPS, the SOD and GSH viability of RAW264.7 macrophages was significantly reduced, and the MDA viability was significantly increased compared to the control (p < 0.01) (Figure 6A-C).However, the addition of L. plantarum A106 significantly increased the SOD and GSH viability (p < 0.01) and significantly reduced the MDA viability (p < 0.05).The above results indicated that treatment with L. plantarum A106 might slow down the LPS-induced oxidative stress in RAW264.7 macrophages.

Effect of L. plantarum A106 on LPS-Induced ROS Levels
The results of the ROS levels are shown in Figure 6D.The ROS level was significantly elevated in the RAW264.7 macrophages stimulated by LPS compared to the control group, while the addition of L. plantarum A106 significantly reduced the ROS levels (p < 0.01), and the high dose group (1:100) was more effective than the low dose (1:1000).This result sug- 3.8.Effect of L. plantarum A106 on LPS-Induced SOD, GSH, and MDA Activities RAW264.7 macrophages were used to establish a LPS-induced oxidative stress model to further explore the antioxidant capacity of L. plantarum A 106.The results are shown in Figure 6.Under the stimulation of LPS, the SOD and GSH viability of RAW264.7 macrophages was significantly reduced, and the MDA viability was significantly increased compared to the control (p < 0.01) (Figure 6A-C).However, the addition of L. plantarum A106 significantly increased the SOD and GSH viability (p < 0.01) and significantly reduced the MDA viability (p < 0.05).The above results indicated that treatment with L. plantarum A106 might slow down the LPS-induced oxidative stress in RAW264.7 macrophages.

Effect of L. plantarum A106 on LPS-Induced ROS Levels
The results of the ROS levels are shown in Figure 6D.The ROS level was significantly elevated in the RAW264.7 macrophages stimulated by LPS compared to the control group, while the addition of L. plantarum A106 significantly reduced the ROS levels (p < 0.01), and the high dose group (1:100) was more effective than the low dose (1:1000).This result suggests that L. plantarum A106 had the potential to effectively reverse the increase in the ROS levels induced by LPS and alleviate the oxidative damage in LPS-induced RAW264.7 macrophages.

Effects of L. plantarum A106 on LPS-Induced Mitochondrial Membrane Potential (MMP)
The above analysis revealed that lipopolysaccharide (LPS) might influence the mitochondrial membrane potential (Figure 4D).Therefore, fluorescence microscopy (Olympus, 50X) was used to preliminarily observe the cells in each treatment group.As shown in Figure 7A, more cells were with green fluorescence in the LPS-induced RAW264.7 macrophages compared to the control group, indicating a loss of MMP; subsequently, the green fluorescence weakened visually after treatment with the L. plantarum A106, but it was difficult to calculate the exact ratio between the red and green fluorescence.Therefore, flow cytometry was employed to detect the precise fluorescence change.As shown in Figure 7B,C, the red/green fluorescence intensity ratio obviously decreased in the LPSinduced RAW264.7 macrophages (p < 0.01) but remarkably increased after the treatment with L. plantarum A106 (p < 0.05).

Effects of L. plantarum A106 on LPS-Induced Mitochondrial Membrane Potential (MMP)
The above analysis revealed that lipopolysaccharide (LPS) might influence the mitochondrial membrane potential (Figure 4D).Therefore, fluorescence microscopy (Olympus, 50X) was used to preliminarily observe the cells in each treatment group.As shown in Figure 7A, more cells were with green fluorescence in the LPS-induced RAW264.7 macrophages compared to the control group, indicating a loss of MMP; subsequently, the green fluorescence weakened visually after treatment with the L. plantarum A106, but it was difficult to calculate the exact ratio between the red and green fluorescence.Therefore, flow cytometry was employed to detect the precise fluorescence change.As shown in Figure 7B,C, the red/green fluorescence intensity ratio obviously decreased in the LPS-induced RAW264.7 macrophages (p < 0.01) but remarkably increased after the treatment with L. plantarum A106 (p < 0.05).

Effects of L. plantarum A106 on LPS-Induced Apoptosis
The loss of mitochondrial membrane potential is often a precursor to apoptosis, leading to the activation of downstream caspases and resulting in cell apoptosis.In this study, flow cytometry was used to detect the apoptotic status of the cells in each treatment group.The results, as shown in Figure 8A,B, indicate that stimulation with LPS significantly increased the percentage of apoptotic cells compared to the control, and the addition of L. plantarum A106 significantly decreased the percentage of apoptotic cells (p < 0.01).Furthermore, there was no significant difference between the LPS-induced group with a low dose of L. plantarum A106 and with a high dose of L. plantarum A106.
of the mitochondrial membrane potential in different groups; and (C) a comparative histogram of the levels of mitochondrial membrane potential in different groups.The data are expressed as the mean ± SEM; and the p value was determined by a one-way ANOVA followed by multiple comparisons.* denotes a significant difference at p < 0.05; & denotes a significant difference at p < 0.01; and 106 represents L. plantarum A106.

Effects of L. plantarum A106 on LPS-Induced Apoptosis
The loss of mitochondrial membrane potential is often a precursor to apoptosis, leading to the activation of downstream caspases and resulting in cell apoptosis.In this study, flow cytometry was used to detect the apoptotic status of the cells in each treatment group.The results, as shown in Figure 8A,B, indicate that stimulation with LPS significantly increased the percentage of apoptotic cells compared to the control, and the addition of L. plantarum A106 significantly decreased the percentage of apoptotic cells (p < 0.01).Furthermore, there was no significant difference between the LPS-induced group with a low dose of L. plantarum A106 and with a high dose of L. plantarum A106.Additionally, we assessed the impact of various treatments on the expression of apoptosis-related genes (Caspase−3, Bcl−2, and Bax) through a qPCR.As shown in Figure Additionally, we assessed the impact of various treatments on the expression of apoptosisrelated genes (Caspase-3, Bcl-2, and Bax) through a qPCR.As shown in Figure 8C,D, compared with the control group, the Bcl-2 gene expression was significantly reduced (p < 0.01), but the Caspase-3 and Bax gene expressions were significantly increased in the RAW264.7 macrophages stimulated by LPS (p < 0.01), while the addition of L. plantarum A106 significantly upregulated the Bcl-2 expression (p < 0.01) but significantly downregulated the Caspase-3 and Bax expressions (p < 0.01).

Discussion
More and more studies have provided reliable evidence for the biological activities of LAB, identifying their antioxidant, antitumor, and immunomodulatory properties.LAB have also been considered as a potential natural antioxidant [19,20].However, there has been the lack of a comprehensive and precise research method to deeply explore the anti-inflammatory and antioxidative effects of LAB.Therefore, this study combined bioinformatics analysis and experiment validation to investigate the potential anti-inflammatory and antioxidative effects of L. plantarum A106 and its underlying mechanisms.
DPPH, ABTS + , and hydroxyl radicals are electronic reduction products of a class of oxygen in organisms and common reactive oxygen species.Thus, the antioxidant activity of LAB can be measured with high ratios of DPPH, ABTS + , and hydroxyl radical scavenging.Tang et al. (2017) assessed the antioxidant activity exhibited by the fermented product of L. plantarum MA2, which was isolated from the traditional kefir grains in Tibet, and demonstrated that the DPPH radical scavenging rates of both the MA2 fermentation supernatant and the intact cells were superior to those of a cell-free extract [21].Interestingly, this results were similar to our results.Specifically, in this study, L. plantarum A106 supernatant or cell-free extracts also had a higher DPPH radical scavenging rate.Moreover, the scavenging rates of the ABTS + and hydroxyl radicals also presented similar results to the DPPH radical rate, indicating that L. plantarum A106 has a strong antioxidant activity.
It has also been reported that LAB have an immunological regulation effect [22,23].Li et al. (2021) found that L. plantarum KSFY06 upregulated the anti-inflammatory factor IL-10 and downregulated the pro-inflammatory factors IL-6 and TNF-α to prevent the inflammatory response of an LPS-induced acute liver injury in mice [24].Prolonged inflammatory processes increased the ROS production and caused oxidative stress; as a result, anti-inflammation might also mitigate the oxidative damage to cells [25].In this study, the treatment with L. plantarum A106 upregulated the anti-inflammatory factor IL-4 and downregulated the gene expression of the pro-inflammatory factor TNF-α.However, further study was needed to explore the anti-inflammatory and antioxidative mechanisms of L. plantarum A106.
Subsequently, bioinformatics analysis was performed on the GSE186886 dataset, identifying 2465 differentially expressed genes (DEGs), including significantly upregulated inflammatory-related genes Csf3, Ccl5, and Ccl.Further KEGG enrichment analysis of the DEGs2 (genes from DEGs1 with |log2 (fold change, FC)| > 2) revealed key inflammatory pathways including cytokine-cytokine receptor interaction, a TNF signaling pathway, and an IL-17 signaling pathway [26].The GO BP and GO MF analyses additionally indicated enrichment in the innate immune response, inflammatory response, and cytokine activity [27].These findings suggest the potential impact of LPS on the inflammatory signaling pathways.
The gene set enrichment analysis (GSEA) results underscored the significant roles of these genes in biological functions and pathways.Specifically, these DEGs were notably enriched in cytokine-cytokine interactions, the IL-17 signaling pathway, and cytokine signaling in the immune system through the KEGG and Reactome pathway analyses, highlighting their importance in inflammatory signal transduction.Moreover, the GO analysis revealed that these genes predominantly clustered in extracellular spaces and regions, as well as in the biological processes and molecular functions associated with the innate immune and inflammatory responses.These findings further unveiled the mechanism behind LPS's promotion of inflammatory responses in RAW264.7 macrophages.These insights provide a deeper understanding of the LPS-induced inflammatory mechanisms.
By integrating the analyses of the betweenness centrality and maximal clique centrality (MCC) through the use of the Cytoscape software (version 3.9.1),we identified several key hub genes: TNF, IL-6, Cxcl10, and IL-1β [28].These genes were likely to play critical roles in the inflammatory signaling induced by LPS [29].Furthermore, the enrichment analysis of the mitochondrial-related differentially expressed genes (DEGs) using Metscape suggested that LPS perturbed cellular respiration, mitochondrial membrane potential, the respiratory electron transport chain, and the process of apoptosis [30].These findings reveal that while LPS promotes inflammatory responses, it also disrupts the cellular energy metabolism and redox reactions, affecting cellular homeostasis.
TNF and IL-1β are pro-inflammatory cytokines that play pivotal roles in the initiation and perpetuation of inflammation.They were involved in the upregulation of inflammatory pathways, enhancing the production of other cytokines and recruiting immune cells to sites of infection or injury, thereby amplifying the inflammatory response [31].IL-6 was a multifunctional cytokine that not only facilitated inflammation but also possessed immunoregulatory functions [32].L. helveticus SBT2171 significantly reduced the LPS-induced expression and secretion of IL-6 and IL-1β cytokines in peritoneal macrophages, leading to anti-inflammatory effects [33].Cxcl10, a chemokine induced by IFN-γ, promoted the migration of immune cells to inflamed tissues during the LPS-induced inflammation, further exacerbating the inflammatory milieu [34].Consistent with these findings, our bioinformatics analysis also identified these four molecules as hub genes in a macrophage model treated with LPS.Additionally, our qPCR results demonstrated an increase in the expression of these genes following the LPS stimulation.Compared to the LPS-treated group, the addition of L. plantarum A106 significantly downregulated the expression of these genes, suggesting that these cytokines may be potential targets for the therapeutic intervention of inflammatory diseases with L. plantarum A106.
Through a meta-analysis of mitochondrial-related DEGs, we discovered that LPS may significantly affect cellular respiration and the mitochondrial electron transport chain.Cellular respiration, a crucial process in cellular energy metabolism, is accompanied by the production of reactive oxygen species (ROS), particularly within the mitochondrial electron transport chain.ROS include free radicals such as superoxide anions (O2-) and non-radical forms like hydrogen peroxide (H 2 O 2 ) [35].Excessive ROS can lead to oxidative stress, damaging proteins, lipids, and nucleic acids, thereby triggering various diseases [36].To counteract the potential harm caused by ROS, cells have developed a complex antioxidant system, comprising enzymes like superoxide dismutase (SOD), catalase, and glutathione peroxidase, as well as non-enzymatic antioxidants such as glutathione and vitamin C [37].These antioxidants neutralize the ROS and maintain the redox balance within the cell [38].In this study, we found that the plant-derived L. plantarum A106 could reduce the ROS and MDA levels in RAW264.7 macrophages and enhance the SOD and GSH activities, thus boosting the cellular antioxidant capacity and ameliorating the LPS-induced oxidative stress in RAW264.7 macrophages.This result was consistent with the result that L. plantarum DP189 significantly reversed the MPTP-induced decrease in SOD and GSH-Px activity, increased the MDA activity, and improved the antioxidant capacity in Parkinsonian mice [3].
The bioinformatics analysis suggested that LPS might interfere with the regulation of the mitochondrial membrane potential, a critical indicator of mitochondrial health and function and the driving force behind ATP synthesis via oxidative phosphorylation.Disruption of this potential was often a precursor to mitochondrial apoptosis, with changes triggering the release of pro-apoptotic factors such as cytochrome c, leading to the activation of downstream caspases and resulting in cell apoptosis [39,40].In our study, we employed JC-1 staining to observe a significant decrease in the mitochondrial membrane potential (MMP) induced by LPS, suggesting an increase in the mitochondrial pathway apoptosis.Subsequently, through flow cytometry and qPCR assays, we detected an increase in the apoptosis rates and an elevated expression of the anti-apoptotic gene Bcl-2, alongside a reduced expression of the pro-apoptotic genes Caspase-3 and Bax following the LPS treatment of RAW264.7 macrophages.The treatment of RAW264.7 macrophages with L. plantarum A106 reversed the decline in the MMP and increased the apoptosis rates caused by LPS.This experimental result was consistent with Li et al. (2022b), who reported that dandelion extract reduced the apoptosis rate of RAW264.7 macrophages and attenuated the LPS-induced inflammatory response [41].

Figure 1 .
Figure 1.Scavenging rates of DPPH (A), ABTS+ (B), and hydroxyl radicals (C) of different fractions derived from L. plantarum A106 (D) Changes of cell viability depending on different treatment time (E) The effect of L. plantarum A106 treatment on the cell viability of LPS-induced RAW 264.7 macrophages; and the effect of L. plantarum A106 treatment on the levels of TNF−α (F) and IL−4 (G) in LPSinduced RAW264.7 macrophages.The data are expressed as the mean ± SEM; and the p value was determined by a one-way ANOVA followed by multiple comparisons.* denotes a significant difference at p < 0.05, and & denotes a significant difference at p < 0.01.Different letters indicated significant differences (p < 0.05).

Figure 1 .
Figure 1.Scavenging rates of DPPH (A), ABTS+ (B), and hydroxyl radicals (C) of different fractions derived from L. plantarum A106 (D) Changes of cell viability depending on different treatment time (E).The effect of L. plantarum A106 treatment on the cell viability of LPS-induced RAW264.7 macrophages; and the effect of L. plantarum A106 treatment on the levels of TNF-α (F) and IL-4 (G) in LPS-induced RAW264.7 macrophages.The data are expressed as the mean ± SEM; and the p value was determined by a one-way ANOVA followed by multiple comparisons.* denotes a significant difference at p < 0.05, and & denotes a significant difference at p < 0.01.Different letters indicated significant differences (p < 0.05).

Figure 3 .
Figure 3. GSEA enrichment analysis.(A) The significant GSEA sets for the GO-BP pathways; (B) the significant GSEA sets for the GO-MF pathways; (C) the significant GSEA sets and heatmap for the GO-CC pathways; (D) the significant GSEA sets for the Reactome pathways; and (E) the significant GSEA sets for the KEGG pathways.

Figure 3 .
Figure 3. GSEA enrichment analysis.(A) The significant GSEA sets for the GO-BP pathways; (B) the significant GSEA sets for the GO-MF pathways; (C) the significant GSEA sets and heatmap for the GO-CC pathways; (D) the significant GSEA sets for the Reactome pathways; and (E) the significant GSEA sets for the KEGG pathways.

Figure 4 .
Figure 4. PPI and mitochondrial Metascape analysis.(A) Top 20 key genes featured by CytoNCA; (B) a key module with 10 genes, the key gene screened by the MCC method; (C) the GO:CC enrichment results (top 20); and (D) the Metascape results of mitochondrial DEGs.

Figure 4 .
Figure 4. PPI and mitochondrial Metascape analysis.(A) Top 20 key genes featured by CytoNCA; (B) a key module with 10 genes, the key gene screened by the MCC method; (C) the GO:CC enrichment results (top 20); and (D) the Metascape results of mitochondrial DEGs.

Foods 2024 , 19 Figure 5 .
Figure 5.Effect of the L. plantarum A106 treatment on the relative levels of the expression of TNF−α mRNA (A), IL−1β mRNA (B), IL−6 mRNA (C), Cxcl10 mRNA (D), Csf3 mRNA (E), Ccl2 mRNA (F), and Ccl5 mRNA (G) in the LPS-induced oxidative stress in RAW264.7 macrophages.The data are expressed as the mean ± SEM; and the p value was determined by a one-way ANOVA followed by multiple comparisons.& denotes significant difference at p < 0.01; and 106 represents L. plantarum A106.

Figure 5 .
Figure 5.Effect of the L. plantarum A106 treatment on the relative levels of the expression of TNF-α mRNA (A), IL-1β mRNA (B), IL-6 mRNA (C), Cxcl10 mRNA (D), Csf3 mRNA (E), Ccl2 mRNA (F), and Ccl5 mRNA (G) in the LPS-induced oxidative stress in RAW264.7 macrophages.The data are expressed as the mean ± SEM; and the p value was determined by a one-way ANOVA followed by multiple comparisons.& denotes significant difference at p < 0.01; and 106 represents L. plantarum A106.

Figure 6 .
Figure 6.Effect of L. plantarum A106 treatment on the SOD (A), GSH (B), and MDA (C) activity and the cellular ROS levels of the LPS-induced oxidative stress in RAW264.7 macrophages; (D) ROS levels analyzed by flow cytometry; (E) overview of ROS levels in different groups; and (F) comparative histogram of the ROS levels in different groups.The data are expressed as the mean ± SEM; and the p value was determined by a one-way ANOVA followed by multiple comparisons.* denotes a significant difference at p < 0.05; & denotes a significant difference at p < 0.01; and 106 represents L. plantarum A106.

Figure 7 .Figure 7 .
Figure 7. Effect of L. plantarum A106 treatment on the mitochondrial membrane potential of LPSinduced oxidative stress in RAW264.7 macrophages.(A) Changes of mitochondrial membrane potential in different groups under the fluorescence microscope; (B) the flow cytometric analysis

Figure 8 .
Figure 8.Effect of L. plantarum A106 treatment on cell apoptosis and apoptosis-related gene expression in LPS-induced oxidative stress in RAW264.7 macrophages.(A) Flow cytometry analysis of apoptosis in different groups; (B) combined analysis of the flow cytometry results among different groups; (C) Caspase−3 mRNA expression level; (D) Bax mRNA expression level; and (E) Bcl−2 mRNA expression level in LPS-induced oxidative stress in RAW264.7 macrophages.The data are expressed as the mean ± SEM; the p value was determined by a one-way ANOVA followed by multiple comparisons; and & denotes a significant difference at p < 0.01.* denotes a significant difference at p < 0.05.

Figure 8 .
Figure 8.Effect of L. plantarum A106 treatment on cell apoptosis and apoptosis-related gene expression in LPS-induced oxidative stress in RAW264.7 macrophages.(A) Flow cytometry analysis of apoptosis in different groups; (B) combined analysis of the flow cytometry results among different groups; (C) Caspase-3 mRNA expression level; (D) Bax mRNA expression level; and (E) Bcl-2 mRNA expression level in LPS-induced oxidative stress in RAW264.7 macrophages.The data are expressed as the mean ± SEM; the p value was determined by a one-way ANOVA followed by multiple comparisons; and & denotes a significant difference at p < 0.01.* denotes a significant difference at p < 0.05.