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. 2023 Sep 13;89(10):e00608-23. doi: 10.1128/aem.00608-23

Anti-inflammatory effect of Lactiplantibacillus plantarum T1 cell-free supernatants through suppression of oxidative stress and NF-κB- and MAPK-signaling pathways

Rui Hao 1,2, Qianqian Liu 1, Lu Wang 1, Wenwen Jian 1, Yu Cheng 3, Qiuyue Zhang 3, Kim Hayer 4, Raja Kamarudin Raja Idris 4, Yi Zhang 3, He Lu 1, Zeng Tu 1,✉
Editor: Danilo Ercolini5
PMCID: PMC10617582  PMID: 37702501

ABSTRACT

Lactiplantibacillus plantarum T1 is an isolated probiotic lactic acid bacterium (LAB) from pickled vegetables in Chongqing, China. In this study, we evaluated the anti-inflammatory activity and the underlying mechanisms of L. plantarum T1 cell-free supernatant (CFS) on lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages in vitro. Reverse transcription quantitative PCR (RT-qPCR), immunofluorescence, Griess methods, and western blotting were utilized to assess the anti-inflammatory cytokines and antioxidative effect of L. plantarum T1 CFS. Our results showed that L. plantarum T1 CFS pretreatment significantly reduced pro-inflammatory cytokine levels, including nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor, interleukin (IL)−1β, and IL-6, as well as reactive oxygen species. Interestingly, L. plantarum T1 CFS unregulated the antioxidant indicators, including superoxide dismutase, catalase, and glutathione in RAW264.7 cells. Furthermore, L. plantarum T1 CFS activated the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathway. This study showed the excellent antioxidant and anti-inflammatory properties of L. plantarum T1 through multiple pathways, highlighting its potential for further research and application as a probiotic strain.

IMPORTANCE

L. plantarum T1 stood out in a series of acid and bile salt tolerance and bacterial inhibition tests as a probiotic isolated from paocai, which provides many health benefits to the host by inhibiting the growth of harmful pathogenic microorganisms and suppressing excessive levels of oxidative stress and inflammation. Not all LAB have good probiotic functions and are used in various applications. The anti-inflammatory antioxidant potential and mechanisms of L. plantarum T1 CFS have not been described and reported. By using RT-qPCR, Griess method, and western blotting, we showed that L. plantarum T1 CFS had anti-inflammatory and antioxidant effects. Griess assay, TBA assay, WST-8 assay, immunofluorescence assay, RT-qPCR, and western blotting data revealed that its anti-inflammatory and antioxidant mechanisms were associated with oxidative stress and NF-κB and MAPK signaling pathways. The anti-inflammatory and antioxidant effects of L. plantarum T1 CFS in paocai generates opportunities for probiotic product development.

KEYWORDS: Lactiplantibacillus plantarum, RAW264.7 macrophages, oxidative stress, NF-κB-signaling pathway, MAPK-signaling pathway

INTRODUCTION

Probiotics are live microorganisms that provide benefits to their host (1, 2). Lactic acid bacteria (LAB) are a well-known type of probiotic bacteria, including the genera such as Lactobacillus, Limosilactobacillus, Lacticaseibacillus, and Lactiplantibacillus. LAB are natural commensals of the human gastrointestinal tract and they play a vital role in maintaining the stability of gut microbiota. LAB also regulates human nutrition and immunity through the intestinal flora (3). More importantly, LAB can produce high levels of lactic acid and other metabolites, such as bacteriocins and short-chain fatty acids, which possess antiinflammatory properties (4, 5).

Inflammation serves as an important initial host defense mechanism. However, excessive and prolonged inflammation can result in harmful diseases such as arthritis and cancer (6–9). Macrophages, which secrete pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)−1β, and IL-6 are fundamental inflammatory cells that have a role in the initial stage of the inflammatory responses (10, 11). Lipopolysaccharide (LPS), a constituent of the outer membrane of gram-negative cells, has demonstrated its capacity to stimulate the release of inflammatory cytokines, which, in turn, trigger downstream inflammation-related signaling pathways such as nuclear transcription factor κB (NF-κB) and its upstream regulators, mitogen-activated protein kinases (MAPKs) (12). Generally, macrophage cells that are stimulated by LPS are recognized as one of the standard inflammation models to explore the modulation of inflammation by drugs, chemicals, and LAB.

Lactiplantibacillus plantarum, Lactobacillus acidophilus, and Lacticaseibacillus casei are commonly isolated as beneficial LAB from traditional Chinese paocai (13, 14). These LAB can produce acids that enhance food flavor, suppress pathogenic microorganisms (15), and regulate the balance of intestinal flora (16, 17). LAB exhibited various probiotic properties that vary depending on their origin, location, and climate. L. plantarum T1, which exhibits high potential as a probiotic, was isolated from paocai in Chongqing, China. The objective of the study was to explore the antiinflammatory potential and its underlying mechanism in LPS-induced RAW264.7 macrophage cells.

MATERIALS AND METHODS

Materials

L. plantarum T1 was isolated, purified, and identified from traditional Chinese paocai and is currently stored in the Laboratory of Pathogen Biology, Chongqing Medical University. The Man-Rogosa-Sharpe (MRS) broth medium was sourced from Qingdao Haibo Biotechnology Co., Ltd (Haibo, China). The agar powder, phosphate buffer (PBS), and the 5% BSA blocking solution were purchased from Solarbao Technology Co., Ltd (Solarbao, China). LPS was acquired from Shanghai Yuanye Bio-Technology Co., Ltd (Cat. No. S11060, Yuanye, China), DMEM medium, 0.25% trypsin-EDTA, 1% penicillin-streptomycin (PS), and TRIZOL were purchased from Gibco (Gibco, USA). Fetal bovine serum (FBS) was obtained from Israel BI Company (BI, USA). NO detection kit, reactive oxygen species (ROS) detection kit, MDA detection kit, total superoxide dismutase (SOD) detection kit, CAT detection kit, GSH detection kit, RIPA lysate, and protease inhibitors were all purchased from Beyotime (Beyotime, China). The reverse transcription kit and SYBR Green were purchased from Takara in Japan. iNOS and COX-2 horseradish peroxidase-labeled secondary antibodies were obtained from Sangong Company (Sangong, China), while p-ERK 1/2, ERK 1/2, p-JNK1/2/3, and JNK1/2/3 antibodies were purchased from Beyotime (Beyotime, China). p38, p-p38, and p-IκBα antibodies were purchased from Affinity Company (Affinitye, China), and IκBα and GAPDH antibodies were acquired from Wuhan Sanying Company (Sanying, China).

Preparation of cell-free supernatant

L. plantarum T1 was grown in MRS broth with glucose at 37°C. To assess its ability to alleviate inflammation, a pure culture of L. plantarum T1 was obtained after three rounds of passages, and was cultured for 36 h in MRS broth with glucose once the strain reached a steady state. Subsequently, cell-free supernatant (CFS) was obtained from bacterial cultures by centrifugation at 12,000 × g for 5 min, followed by filtration through a 0.22 µm cell strainer (Axygen, USA). The CFS concentration was adjusted based on cell number. The bacterial cell pellets were suspended in PBS. The absorbance was measured at 600 nm (OD600) using a spectrophotometer (Nano-500, Allsheng, HZ, China) to determine the cell number as colony-forming unit (CFU)/mL. The CFS concentration was then adjusted and defined as 109 (9 Log), 108 (8 Log), 107 (7 Log), and 106 (6 Log) CFU/mL for the study (18–20).

Cell viability assay

The cell viability was assessed by a CCK-8 assay (21). RAW264.7 cells (5 × 103 cells/well) were seeded into 96-well plates and cultured overnight. The cells were pretreated with four concentrations of L. plantarum T1 CFS (6, 7, 8, and 9 Log CFU/mL), and the plates were incubated at 37°C for 24 h. Following two washes with PBS, fresh medium was added, and CCK-8 reagent (Cell Counting Kit-8, Bioground, China) was added to each well. After further incubation at 37°C for 2 h, absorbance was measured at 450 nm (OD450).

Cell culture and experiment design

RAW264.7 macrophage cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) with 10% FBS at 37°C and 1% PS (100 U/mL penicillin and 100 µg/mL streptomycin, Gibco, USA) in a humidified incubator with 5% CO2 and subcultured at 80%–90% confluency.

The study was divided into six groups: control group (DMEM medium), LPS-induced inflammation model group (0.5 µg/mL), three experimental groups treated with L. plantarum T1 CFS with different concentrations (6, 7, and 8 Log CFU/mL), and a dexamethasone (DEX) anti-inflammatory positive control group (50 µg/mL). To assess the anti-inflammatory and antioxidant effects of L. plantarum T1 CFS, RAW264.7 cells were plated in six-well plates at 1 × 106 cells/well and 96-well plates at 5 × 103 cells/well. The cells were cultured overnight at 37°C and pretreated for 4 h using L. plantarum T1 CFS and DEX, and then stimulated with or without LPS (0.5 µg/mL) for 20 h. Detection of MAPKs and NF-κB pathways was done after 4 h of LPS stimulation.

RNA extraction and RT-qPCR analysis

RAW264.7 macrophages (1 × 106 cells/well) were seeded in six-well plates and cultured for 24 h. After pretreatment with L. plantarum T1 CFS and DEX for 4 h, the cells were either stimulated with LPS for 20 h or left untreated. Cells were then rinsed twice with ice-cold PBS, and total RNA was extracted using a TRIzol reagent kit (Gibco, USA) according to the manufacturer’s instructions. Reverse transcription was performed using PrimeScript RT reagent Kit with gDNA Eraser kit (Takara, Japan) to generate cDNA. RT-qPCR was conducted using a CFX96 Touch RT-qPCR instrument (Bio-Rad, USA) under the following conditions: initial denaturation at 95°C for 30 s, followed by 39 cycles of PCR reaction at 95°C for 5 s, 60°C for 30 s, and a final step at 95°C for 10 s with melt curve analysis. The RT-qPCR primer sequences are listed in Table 1. The relative mRNA level was normalized with GAPDH or β-actin as an internal reference gene, and the expression level of the target genes was calculated using the 2-△△ct method (22).

TABLE 1.

The RT-qPCR primer sequences of genes used in the study

Gene Primer sequence (5′→3′)
IL-1β Sense 5′-GAA ATG CCA CCT TTT GAC AGT G-3′
Anti-sense 5′-TGG ATG CTC TCA TCA GGA CAG-3′
IL-6 Sense 5′-TCC AGT TGC CTT CTT GGG AC-3′
Anti-sense 5′-AGA CAG GTC TGT TGG GAG TG-3′
TNF-α Sense 5′-AGC CGA TGG GTT GTA CCT TG-3′
Anti-sense 5′-ATA GCA AAT CGG CTG ACG GT-3′
iNOS Sense 5′-TGG AGC CAG TTG TGG ATT GTC-3′
Anti-sense 5′-GGT CGT AAT GTC CAG GAA GTA G-3′
COX-2 Sense 5′-CAC TAC ATC CTG ACC CAC TT-3′
Anti-sense 5′-ATG CTC CTG CTT GAG TAT GT-3′
GAPDH Sense 5′-CAA GGT CAC CAT GAC AAC TTT G −3’
Anti-sense 5′-GTC CAC CAC CCT GTT GCT ATAG-3′
β-actin Sense 5′-TGT TAC CAA CTG GGA CGA CA-3′
Anti-sense 5′-CTG GGT CAT CTT TTC ACG GT-3′
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Measurement of nitric oxide production

Nitric oxide (NO) release from RAW264.7 cells was measured by the Griess method according to a previous report (23). Briefly, RAW264.7 cells were inoculated into six-well plates (1 × 106 cells/well) and cultured overnight. The cells were stimulated with LPS (0.5 µg/mL), L. plantarum T1 CFS samples, and DEX. The supernatant was collected and centrifuged at 4,000 g for 5 min. The standard substance (concentration was 0, 1, 2, 5, 10, 20, 40, 60, and 100 µM) was added to the 96-well plate. Each well was added 50 µL supernatant and 50 µL of both Griess reagent I and II. The absorbance was determined at 540 nm using a microplate reader (Multiskan FC, Thermo, USA).

Measurements of intracellular ROS production

The effects of L. plantarum T1 CFS on intracellular ROS levels in LPS-stimulated RAW264.7 cells were detected using DCFH-DA as a probe for labeling following the instruction from company. The ROS concentrations in each group were determined by flow cytometry, and the results were expressed as a percentage of fluorescence intensity.

Detection of MDA, SOD, CAT, and GSH level

RAW264.7 cells (1 × 106 cells/well) were seeded in six-well plates. After treatment with LPS, L. plantarum T1 CFS, and DEX, Cellular lipid peroxidation product (Malondialdehyde, MDA) were detected through TBA methods. The status of the antioxidants such as SOD, catalase (CAT), and intracellular reduced glutathione (GSH) in cells were assessed separately using WST-8, Catalase Assay Kit, and GSH Kit according to the instructions. The absorbance was measured at 450 nm with a microplate reader, and the corresponding activity was calculated by a formula.

Protein extraction and western blotting

The whole protein in RAW264.7 cells was extracted using RIPA lysis buffer containing 1% phosphatase inhibitor and 1% protease inhibitor. The supernatant was collected after centrifugation at 12,000 × g for 15 min at 4°C, and the protein concentration was determined using the bicinchoninic acid (BCA) method. Western blotting was performed as previously reported (24). Briefly, electrophoresis was performed on a sodium dodecyl sulfate-polyacrylamide gel with a concentration of 12.5%, and 15 µg of the protein was loaded onto the gel. The protein separated by electrophoresis was transferred onto the PVDF membrane, blocked with 5% skimmed milk at room temperature in PBS-Tween buffer for 1 h, and then incubated with the primary antibodies (iNOS 1:500, COX-2 1:800, GAPDH 1:2,000, IκBα 1:1,000, p-IκBα 1:1,000, ERK1/2 1:1,000, p-ERK1/2 1:1,000, p38 1: 1,000, p-p38 1:2,000, JNK1/2/3 1:1,000, p-JNK1/2/3 1:1,000) overnight. The strips of the membrane were then incubated with HRP-labeled anti-rabbit or mouse immunoglobulin secondary antibodies (iNOS 1:8,000, COX-2 1:5,000, GAPDH 1:20,000, IκBα 1:10,000, p-IκBα 1:10,000, ERK1/2 1:10,000, p-ERK1/2 1:10,000, p38 1: 10,000, p-p38 1:20,000, JNK1/2/3 1:10,000, p-JNK1/2/3 1:10,000) for 1.5 h. An ECL luminescent solution (BG0001, Bioground, CQ, China) was used for visualizing the protein bands. Grayscale images of the strips were analyzed using ImageJ software. GAPDH was used as an internal reference.

Statistical analysis

The results are presented as the mean ± standard deviation (SD) from independent experiments conducted in triplicates. Statistical analysis was performed using GraphPad Prism 9.0. Results were analyzed statistically using one-way ANOVA. P ≤ 0.05 was considered statistically significant.

RESULTS

Isolation, identification, and screening of L. plantarum T1 from paocai

L. plantarum T1 was found to be a facultative anaerobic bacterium that forms round, creamy white, raised, smooth, and moist surface colonies with neat edges. These gram-positive non-sporulating bacilli can appear straight or curved under the light microscope. L. plantarum T1 can produce the phenomenon of soluble calcium circles with blue margins in a modified MRS solid medium containing 2% CaCO3 (Fig. 1). Additionally, our preliminary observations have indicated that L. plantarum T1 CFS exhibits excellent acid tolerance, bile salt tolerance, and antibacterial effects (Fig. S1; Table S1 and S2). (The conservation number of L. plantarum T1 is CCTCC M 2022082, and its sequence has been uploaded to NCBI under the sequence number ON063305.)

Fig 1.

Fig 1

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Morphological characteristics and phylogenetic analysis of L. plantarum T1 based on 16S rDNA sequences. (A) L. plantarum T1 produces a calcium-soluble circle while separation of 2% CaCO3 in MRS solid medium. (B) The soluble calcium circles with blue margins of L. plantarum T1 colonies in the modified MRS solid medium. (C) Gram staining diagram of L. plantarum T1. (D) The phylogenetic tree of L. plantarum T1 isolates based on 16S rDNA sequence (ON063305. L. plantarum strain T1).

L. plantarum T1 CFS inhibits the production of pro-inflammatory cytokines

We constructed an LPS-stimulated RAW264.7 inflammation model and conducted cell viability assays (refer to Fig. S2 and S3 for details). CFS (6, 7, and 8 Log CFU/mL) did not affect cell viability and were used in the following evaluation of anti-inflammation effect. The DEX was used as a positive control. Intriguingly, our findings suggest that L. plantarum T1 CFS treatment resulted in a dose-dependent decrease in the expression of IL-1β, IL-6, and TNF-α pro-inflammatory cytokines compared to the LPS-stimulated inflammation group (Fig. 2A through C). Furthermore, TNF-α and IL-6 mRNA levels were similar to those of the DEX group after treatment with 8 Log CFU/mL CFS.

Fig 2.

Fig 2

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L. plantarum T1 CFS inhibits the production of pro-inflammatory cytokines in RAW264.7 cells. The relative mRNA expression of pro-inflammatory cytokines (A) IL-1β, (B) IL-6, and (C) TNF-α was assessed using RT-qPCR. Fluorescence level of IL-1β (D) was presented by fluorescence microscopy (original magnification, × 200). Cells were pretreated for 4 h with three different concentrations of L. plantarum T1 CFS before stimulation of LPS (0.5 µg/mL) for 20 h. The data are presented as mean  ± SD (n = 3). ####P < 0.0001: vs. control (DMEM) group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001: vs. LPS group; %%P < 0.01, %%%P < 0.001: vs. 6 Log CFU/mL L. plantarum T1 CFS group; &&&&P < 0.0001: vs. DEX group.

We also utilized immunofluorescence to detect IL-1β fluorescence levels in four groups (DMEM, LPS, 8 Log CFU/mL L. plantarum T1 CFS, DEX). As shown in Fig. 2B, the inflammation group exhibited a higher IL-1β fluorescence intensity compared to the control group, whereas the fluorescence intensity of IL-1β decreased (Fig. 2D) after treatment with L. plantarum T1 CFS at 8 Log CFU/mL. Overall, these results indicate that L. plantarum T1 CFS has an inhibitory effect on pro-inflammatory cytokine secretion of LPS-stimulated RAW264.7 cells in a dose-dependent manner.

L. plantarum T1 CFS inhibits COX-2 and iNOS expression

To evaluate the potential effect of L. plantarum T1 CFS on COX-2 and iNOS, RAW264.7 cells were treated with the CFS for 24 h. The expression levels of COX-2 and iNOS were analyzed by measuring mRNA and protein levels (Fig. 3). The result showed that L. plantarum T1 CFS significantly reduced the expression of COX-2 and iNOS induced by LPS at the protein and mRNA levels. Therefore, L. plantarum T1 CFS could be considered as a potential antiinflammatory agent by reducing the expression of COX-2 and iNOS.

Fig 3.

Fig 3

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L. plantarum T1 CFS inhibits LPS-stimulated NO, COX-2, and iNOS expression in RAW264.7 cells. (A) L. plantarum T1 CFS inhibits LPS-induced COX-2 and iNOS mRNA expressions. (B) NO production. (C and D) The qualitative and quantitative analysis of L. plantarum T1 CFS inhibits LPS-induced COX-2 and iNOS proteins expressions. Cells were pretreated for 4 h with various concentration of L. plantarum T1 CFS before stimulation of LPS (0.5 µg/mL) for 20 h. The data are presented as mean ± SD (n = 3). ##P < 0.01, ####P < 0.0001: vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001: vs. LPS group; %P < 0.05, %%P < 0.01: vs. 6 Log CFU/mL L. plantarum T1 CFS group; &P < 0.05, &&&&P < 0.0001: vs. DEX group.

Next, the downstream factor of iNOS, NO, was analyzed by the Griess method. Results showed that L. plantarum T1 CFS dose-dependently inhibited LPS-induced NO production, indicating that L. plantarum T1 CFS inhibits NO production by regulating iNOS expression, further inhibiting the inflammation response.

L. plantarum T1 CFS inhibits LPS-induced oxidative stress levels

To evaluate antioxidative effect of L. plantarum T1 CFS, the levels of lipid peroxidation end product (MDA) and the antioxidants (SOD, CAT, and GSH) were detected (Fig. 4). The results showed that L. plantarum T1 CFS reduced MDA levels and improved the levels of SOD, CAT, and GSH activity in a dose-dependent manner. Finally, the effect of L. plantarum T1 CFS on ROS production was detected by flow cytometry (Fig. 5). Our results showed that L. plantarum T1 CFS significantly reduced the ROS release, indicating that L. plantarum T1 CFS could improve the LPS-induced inflammatory response by increasing the activity of the antioxidant enzyme SOD, inhibiting MDA production, and downregulating ROS release.

Fig 4.

Fig 4

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L. plantarum T1 CFS supplementation improved the antioxidant capacity of LPS-stimulated RAW264.7 cells. (A) The maleic dialdehyde (MDA) of RAW264.7 cells in each group. (B) The total superoxide dismutase (SOD) of RAW264.7 cells in each group. (C) The catalase (CAT) of RAW264.7 cells in each group. (D) The total glutathione (GSH) of RAW264.7 cells in each group. Cells were pretreated for 4 h with three different concentrations of L. plantarum T1 CFS before stimulation of LPS (0.5 µg/mL) for 20 h. The data are presented as mean ± SD (n = 3). ####P < 0.0001: vs. control group; ****P < 0.0001: vs. LPS group; %%%%P < 0.0001: vs. 6 Log CFU/mL L. plantarum T1 CFS group; &&P < 0.01, &&&&P < 0.0001: vs. DEX group.

Fig 5.

Fig 5

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L. plantarum T1 CFS inhibits LPS-stimulated ROS production in RAW264.7 cells. (A) The qualitative analysis of intracellular ROS formation. (B) The quantitative analysis of intracellular ROS formation. Cells were pretreated for 4 h with various concentration of L. plantarum T1 CFS before stimulation of LPS (0.5 µg/mL) for 20 h. DCHA-DA was used as a fluorescent probe, and detected by flow cytometry. The data are presented as mean ± SD (n = 3). ####P < 0.0001: vs. control group; ****P < 0.0001: vs. LPS group; %%%%P < 0.0001: vs. 6 Log CFU/mL L. plantarum T1 CFS group; &&&&P < 0.0001: vs. DEX group.

L. plantarum T1 CFS inhibits LPS-induced inflammatory effects through NF-κB and MAPK pathways in RAW264.7 cells

NF-κB and MAPK pathways are crucial in the cellular inflammatory and immune responses. The expression of essential proteins p-IκBα and IκBα in the NF-κB pathway were determined (Fig. 6). The p-IκBα relative expression was increased, whereas IκBα was decreased in LPS-stimulated RAW264.7 cells. Addition of L. plantarum T1 CFS reversed the expression pattern of p-I-IκBα and IκBα (Fig. 6A and B). Furthermore, the ratio of p-IκBα/IκBα expression gradually decreased with increasing L. plantarum T1 CFS concentration after LPS stimulation (Fig. 6B). Those results demonstrated that L. plantarum T1 CFS could relieve LPS-induced inflammatory responses by reversing the activation of p-IκB in the NF-κB pathway.

Fig 6.

Fig 6

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L. plantarum T1 CFS inhibits LPS-induced inflammatory effects through NF-κB pathways in RAW264.7 cells. (A) Western blot detection of p-IκBα and IκBα proteins. (B) Grayscale analysis of p-IκBα and IκBα protein bands. Cells were pretreated for 4 h with various concentrations of L. plantarum T1 CFS before stimulation of LPS (0.5 µg/mL) for 4 h. GAPDH acted as the control protein. The data are presented as mean + SD (n = 3). ####P < 0.0001: vs. control group; ****P < 0.0001: vs. LPS group; %%%%P < 0.0001: vs. 6 Log CFU/mL L. plantarum T1 CFS group.

To further explore the molecular mechanism of the anti-inflammatory effect of L. plantarum T1 CFS, we detected the expression levels of MAPK family proteins (JNK, ERK, and p38) in the MAPK-signaling pathway in RAW264.7 macrophages after L. plantarum T1 CFS addition. L. plantarum T1 CFS inhibited LPS-induced phosphorylation of JNK, ERK, and p38, particularly p-JNK1/2/3, p-ERK1/2, and p-p38, showing a clear tendency of concentration-dependent inhibition (Fig. 7). Moreover, treatment with high concentrations of L. plantarum T1 CFS (8Log CFU/mL) exhibited a similar inhibitory effect as DEX on LPS-induced inflammatory response, especially p-ERK1/2. These results suggest that L. plantarum T1 CFS relieves the inflammatory response by inhibiting phosphorylated proteins p-p38, p-ERK1/2, and p-JNK1/2/3 in the MAPK-signaling pathway activated by LPS.

Fig 7.

Fig 7

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L. plantarum T1 CFS inhibits LPS-induced inflammatory effects through MAPK pathways in RAW264.7 cells. (A) Western blot was used to detect the expression level of MAPK-signaling pathway-related proteins. (B–D) The level of MAPK-signaling pathway-related protein bands was evaluated with grayscale analysis. Cells were pretreated for 4 h with various concentrations of L. plantarum T1 CFS before LPS stimulation (0.5 µg/mL) for 4 h. GAPDH was used as the control protein. The data are presented as mean ± SD (n = 3). ####P < 0.0001: vs. control group; ****P < 0.0001: vs. LPS group; %%%%P < 0.0001: vs. 6Log CFU/mL L. plantarum T1 CFS group; &&&&P < 0.0001: vs. DEX group.

DISCUSSION

Probiotic LAB has been shown to have a positive impact on the gastrointestinal tract, lactose intolerance, and inflammation, among others (25–29). In this study, we investigated the antiinflammatory and antioxidative properties of a newly isolated LAB, L. plantarum T1 CFS, isolated from paocai. Our results indicate that L. plantarum T1 CFS is effective in reducing inflammation and antioxidant stress, as demonstrated by attenuate MAPK and NF-κB activation in LPS-stimulated RAW264.7 macrophage (Fig. 8). These results suggested that L. plantarum T1 could be a new probiotic bacterium.

Fig 8.

Fig 8

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Diagram of the proposed anti-inflammatory mechanism of L. plantarum T1 CFS. L. plantarum T1 CFS alleviates the inflammation through suppression of oxidative stress, NF-κB- and MAPK-signaling pathway in LPS-stimulated RAW264.7 macrophages.

Moderation of inflammation is an important aspect of maintaining host health (30). Studies have shown that probiotics can exert anti-inflammatory effect by inhibiting the pro-inflammatory cytokines production. Examples include Lacticaseibacillus paracasei KW3110, which can mitigate damage to mitochondria resulting from inflammatory responses by reducing the production of IL-1β (31), and L. plantarum CLP-0611, which ameliorated colitis in mice by inhibiting the expression of TNF-α, IL-1β, and IL-6 (32). Similarly, our study demonstrates that L. plantarum T1 CFS can negatively regulate pro-inflammatory cytokines to improve the inflammatory response caused by LPS.

LAB can also reduce inflammation through a variety of mechanisms, including the regulation of iNOS and COX-2 expression, oxidative stress levels, and NF-κB- and MAPK-signaling pathways. Elevated ROS levels, for example, can lead to excessive oxidative stress, lipid peroxidation, and tissue damage in the body (33, 34). This can contribute to bacterial translocation, intestinal destabilization, and an increased risk of developing inflammatory diseases (35–38). iNOS and COX-2 are necessary enzymes that regulate ROS and reactive nitrogen species, and their levels are elevated during inflammation and in certain pathological conditions such as tumors. Probiotic LAB has been shown to alleviate inflammatory responses by downregulating the expression of iNOS and COX-2. For instance, L. acidophilus NCFM can inhibit NO and PGE2 and counteract the endotoxin-induced inflammatory response by downregulating iNOS and COX-2 expression levels (39). Similarly, Lactobacillus crispatus JCM 2009 produces surface proteins that have anti-inflammatory property by inhibiting NO (40). In the study, we observed that L. plantarum T1 CFS reduced both mRNA and protein expression for iNOS and COX-2 in a macrophage inflammation model while reducing NO production in macrophages in response to endotoxin attack. Additionally, L. plantarum T1 CFS can impact antioxidant components such as MDA, SOD, CAT, and GSH. MDA is a key indicator of membrane lipid peroxidation and can interfere with several normal physiological and biochemical processes (41). Antioxidant enzymes such as SOD, CAT, and GSH can eliminate harmful substances produced during metabolic processes in physiological conditions, such as protection of catalase from O2- damage (42, 43). Our results indicate that L. plantarum T1 CFS can promote the production of these antioxidants and inhibit lipid peroxidation levels, thereby reducing LPS-induced inflammation. Several studies have made similar findings. For example, L. plantarum-CQPC11 reduced OVA-induced oxidative stress by increasing the lung’s GSH-Px, SOD, and catalase activities (44), and L. plantarum DP189 reduced damage in mice by increasing SOD and GSH-Px while decreasing MDA and ROS levels (45).

NF-κB is an essential transcriptional regulator of the immune response and a key downstream pathway for endotoxin-induced signaling. When stimulated by oxidants or inflammatory cytokines, NF-κB dissociates from its inhibitory protein, IκB, and IκB is transferred into the nucleus to initiate gene transcription (46). In our study, we found that L. plantarum T1 CFS inhibited the expression of P-IκBα, prevented the IκBα dissociation, and downregulated the LPS-induced NF-κB hyperactivity while concurrently reducing inflammation levels in RAW264.7 macrophages. Other research supported our finding. For example, L. plantarum Y15 reduced LPS production, inhibited NF-κB activation, and release short-chain fatty acids that alleviating type 2 diabetes in mice (47). The probiotic Enterococcus faecalis H81 alleviated Staphylococcus aureus-induced mastitis in mice by inhibiting the activation of the NF-κB-signaling pathway (48). Therefore, targeting the regulation of the NF-κB-signaling pathway could be an effective approach to treating inflammatory diseases and preventing tumors.

MAPK includes ERK1/2, p38, and JNK, which are important in regulating various physiological activities such as cell growth, development, apoptosis, oxidative stress, and inflammation. Inhibition of p-p38 in the MAPK-signaling pathway has been reported to effectively reduce inflammation by inhibiting the expression of iNOS and COX-2 proteins (49). Previous studies have found that the L. acidophilus surface protein NCFM exerts anti-inflammatory effects by inhibiting JNK and ERK phosphorylation in the MAPK-signaling pathway (38). LGG can also reduce intestinal inflammation by modulating the MAPK/NF-κB-signaling pathway (50). Similarly, this study found that L. plantarum T1 CFS regulates the expression of phosphorylated proteins p-ERK1/2, p-JNK1/2/3, and p-p38 in the MAPK pathway, thus alleviated inflammatory response.

Conclusion

In summary, our study demonstrated that L. plantarum T1 CFS isolated from paocai has excellent antiinflammatory property and an antioxidative effect in RAW264.7 macrophages (Fig. 8). The results suggest that this LAB strain may be a promising probiotic bacterium for improving gastrointestinal health and reducing inflammation, although more in vivo studies are needed.

ACKNOWLEDGMENTS

This research was supported by Chongqing Natural Science Foundation [no. cstc2021jcyj-msxmX0158], Education Teaching Reform Research Project of Chongqing (223115), and Education and Teaching Research Project of Chongqing Medical University (JY210301).

Contributor Information

Zeng Tu, Email: tuzeng@cqmu.edu.cn.

Danilo Ercolini, Universita degli Studi di Napoli, Portici, Italy.

DATA AVAILABILITY

The data that support the findings of this study are openly available in the National Center for Biotechnology Information (NCBI) repository under accession ON063305.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.00608-23.

Fig. S1 to S3, Tables S1 and S2. aem.00608-23-s0001.docx.

Potential prebiotic properties of L. plantarum T1; build inflammation models; Effects of L. plantarum T1 CFS on cell viability.

Click here for additional data file. (857.6KB, docx)
DOI: 10.1128/aem.00608-23.SuF1

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 to S3, Tables S1 and S2. aem.00608-23-s0001.docx.

Potential prebiotic properties of L. plantarum T1; build inflammation models; Effects of L. plantarum T1 CFS on cell viability.

Click here for additional data file. (857.6KB, docx)
DOI: 10.1128/aem.00608-23.SuF1

Data Availability Statement

The data that support the findings of this study are openly available in the National Center for Biotechnology Information (NCBI) repository under accession ON063305.

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

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