Selected Lactobacillus Strains Inhibit Inammation in LPS-induced RAW264.7 Macrophages by Suppressing the TLR4-Mediated NF-κB and MAPKs Activation

Pathogenic bacteria, oxidative stress, immunity disorder, and inammation are involved in inammatory bowel disease (IBD) development. Lactobacillus acidophilus KLDS 1.0901, Lactobacillus helveticus KLDS 1.8701, and Lactobacillus plantarum KLDS 1.0318 isolated from Chinese fermented dairy food with various functional characteristics, such as antibacterial, antioxidant, and immunomodulatory activity. Therefore, we selected three strains as a potential application for preventing IBD. However, the anti-inammatory properties of the tested strains are unknown. The current study evaluated the anti-inammatory potential of the tested strains using the LPS-induced RAW264.7 cells model. The results showed that all tested strains could inhibit iNOS and COX-2 expression, reducing NO and PGE2 production. And all tested strains markedly reduced the production and mRNA expression of proinammatory cytokines (TNF-α, IL-1β, and IL-6). Moreover, these results may be associated with inhibiting the translocation of NF-κB into nuclear and MAPKs activation. The study can provide evidence for further in vivo studies of Lactobacillus strains to prevent IBD.


Introduction
In ammatory Bowel Diseases (IBD) has since been recognized as an emerging global public health 1 .
Although the speci c causative factors are not clear 2 , it is generally known that IBD is an in ammatory disease of the gastrointestinal tract, with some researchers speculating that the gut microbiota may play critical roles in its mitigation 3 . Also, several studies posit that the intestinal ora is not only involved in the occurrence of IBD but also could initiate the in ammatory cascade typical of IBD. When gut dysbiosis occurs as observed in IBD sufferers, the mucosal barrier function disappears, which results in the invasion of the mucosa region by pathogenic intestinal bacteria. Also, microbial antigens are activated, and the host's normal immune functions are disrupted, leading to oxidation and in ammation response 4,5 . Therefore, regulating the intestinal ora, inhibiting pathogenic bacteria, improving immunity status, reducing oxidative stress, and suppressing in ammation are essential for the prevention and treatment of IBD.
In ammation is a defensive immune response to protect the body from exogenous stimuli. However, excessive or persistent in ammation can lead to various in ammation-related diseases 6 . Macrophages, derived from monocytes, play a crucial role in innate immunity by their unique phagocytosis function 7 .
Lipopolysaccharide (LPS) comprises lipids and polysaccharides connected by a covalent bond and is the central part of the outer membrane of gram-negative bacteria. When pathogenic bacteria invasion occurs, the host and intestinal mucosa barrier are compromised, allowing LPS release into the bloodstream 8 . A combination of LPS, LPS binding protein (LBP), and CD14 forms the LPS-LBP-CD14 complex, which in turn binds to the Toll-like receptor 4 (TLR4)/ myeloid differentiation protein 2 (MD-2) on the mononuclearmacrophage membrane 9 . The TLR4 is stably expressed in cell membranes and binds to myeloid differentiation factor 88 (MyD88) through interactions with the Toll-interleukin-1 receptor (TIR) domains and activates the MyD88-dependent signaling pathway, responsible for downstream nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPKs) pathways 10,11 . The activation of NF-κB and MAPKs could in uence in ammation, immune regulation, cell proliferation, and apoptosis. In the LPSstimulated murine RAW 264.7 cells, the expression of proin ammatory mediators and cytokines, including nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor (TNF) -α, interleukin (IL) -1β, IL-6, and IL-8, as well as other immune-related genes have been reported 12 . The RAW 264.7 cell model by LPS-inducing factors is widely used to study in ammatory response 13 .
Probiotics are living microorganisms that can provide bene cial effects to the host when administered in adequate amounts, such as regulation of gut microbiota, strengthening the intestinal barrier, and inhibiting intestinal diseases 14 . In recent years, Lactobacillus strains have been a probiotic focus group for the presentment and treatment of in ammations 15,16 . For instance, L. acidophilus SMC-S095 exerted protective effects against dextran sulfate sodium (DSS)-induced murine colitis. This strain downregulated the expression of IL-23 and transforming growth factor (TGF) -β1 and the phosphorylation of signal transducer and activator of transcription (STAT) 3, resulting in decreased production levels of the proin ammatory cytokine IL-17 mediated by T helper cell (Th) 17 cells 17 . Furthermore, in LPS-stimulated RAW264.7 cells, the administration of L. helveticus NS8 can increase the production levels of IL-10 in peripheral blood mononuclear cells (PBMCs) in vivo 18 . Similarly, L. plantarum 21 signi cantly alleviated TNBS-induced colitis in rats by inhibiting TNF-α and IL-1β protein and mRNA expression in colon tissue, reducing the level of NO, and increasing the content of reduced glutathione (GSH) and IL-10 protein and mRNA expression 19 .
L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 were isolated from traditional fermented dairy food in Sinkiang (China) and identi ed using 16S rDNA sequence analysis. Our previous results showed that all tested strains exhibited high acid and bile salt-resistances and high cell adhesion. These characteristics implied that all tested strains could colonize the host intestine and confer probiotic properties. Furthermore, these strains displayed several health-promoting characteristics, such as antibacterial activity (L. acidophilus KLDS 1.0901) 20 , antioxidant activity (L. helveticus KLDS 1.8701) 21 , and immunomodulatory activity (L. plantarum KLDS 1.0318) 22 .
As mentioned earlier, pathogenic bacteria, immunity disorder, oxidative stress, and in ammation are involved in IBD development. Therefore, we selected L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 for potential IBD therapy applications. However, investigation of the anti-in ammatory properties of these three strains has not been carried out. The current study thus aimed to assess the potentials of L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 in suppressing proin ammatory gene expressions and the related-signaling pathways (NF-κB and MAPK) activation in LPS-stimulated RAW 264.7 murine cell lines. We hope that this study will illustrate molecular evidence regarding these Lactobacillus strains in preventing IBD and provide the theoretical basis for future targeted in vivo experiments.

Materials And Methods
Bacterial strain and culture conditions. L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 were isolated from traditional fermented dairy foods in Sinkiang Province (China) and preserved at the Northeast Agricultural University's (NEAU) Key Laboratory Dairy Science (KLDS) in Harbin, China. Strains were identi ed using the 16S rDNA sequence analysis and inoculated in improved Man Rogosa Sharpe (MRS) broth at 37 °C for 18 h and were sub-cultured twice prior to the experiment. The bacterial cell pellets were harvested using centrifugation (8000 rpm,10 min) at 4℃ and washed triple with sterile saline buffer (pH 7.4). The bacterial cell pellets were resuspended with high Dulbecco's modi ed Eagle's medium (DMEM) without antibiotics (100 ug/ml streptomycin and 100 U/ml penicillin).
Cells Culture. The murine macrophage RAW264.7 cell line was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in high DMEM medium including 10% fetal bovine serum (FBS) and 1% antibiotics at 37℃ (5% CO2). helveticus KLDS 1.8701, and L. plantarum KLDS1.0318 at a MOI of 10 (1.0 × 10 6 CFU/well) for 1 h, and then incubated with LPS (1ug/ml) for 24 h. Equal volumes of Griess reagent (Nanjing, China) were mixed with culture supernatants, and static for 10 min at room temperature. The absorbance was measured at 550 nm by an ultraviolet spectrometer.
Real-time quantitative polymerase chain reaction (RT-qPCR). RAW264.7 cells were preincubated in 6-well plates (1 × 10 6 cells/well). Cells were treated with the L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 (1.0 × 10 7 CFU/well) for 1 h prior to the stimulation with LPS (1 µg/ml) for 24 h. The total RNA of RAW264.7 cells was extracted using Total RNA Kit (Tiangen, China), and 2 µg of the total RNA were reverse-transcribed to synthesize cDNA using a GoScript™ Reverse Transcription Mix (Promega, USA). PCR was ampli cated and detected by GoTaq SYBR-Green qPCR Master Mix (Promega, USA) with 1 ug of cDNA. The relative expressions of target genes were analyzed using the 2 −ΔΔCt calculation method. The gene primers were rst mined from the NCBI database, then designed and synthesized by Sangon Biotech Co., Ltd (Shanghai, China) ( Table 1.). Table 1  (1:5000) for 1 h at room temperature. After washing thrice in TBST, the chemiluminescence detection system was enhanced with the ECL kit to display antibody-speci c proteins.
Statistical analysis. All data were assessed using SPSS 23.0 software (SPSS Inc., Chicago, IL, USA) and expressed as mean ± standard deviation (S.D.). The one-way analysis of variance (ANOVA) followed by Duncan's multiple range test was used to compare groups. In this study, P-values of 0.05, 0.01, and 0.001 were considered signi cant.

Results
The effect of the tested strains on cell viability. The effect of L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 on cell viability was assessed by CCK8 assay. As shown in Fig. 1, the tested strains signi cantly inhibited cell viability within MOI of 100 and 1000 compared with that of the control group. These may have been induced by competition of nutrients between three strains and RAW264.7 macrophages. However, no signi cant differences in cell viability were observed between the control group and all tested strains-treated groups when the MOI is 10. Given the above results, an MOI of 10 was applied during subsequent experiments.
Inhibition effect of the tested strains on in ammatory mediators. To evaluate the potential anti- in ammatory property of the tested strains. The concentration of in ammatory mediators (NO and PGE2) and their relative mRNA (iNOS and COX-2) expressions were detected in LPS-stimulated RAW264.7 macrophages. Compared to the control group, the production of NO was increased in the LPS group ( Fig. 2A). However, it was signi cantly reduced in the L. acidophilus KLDS 1.0901 (P < 0.001), L. helveticus KLDS 1.8701 (P < 0.05), and L. plantarum KLDS 1.0318 (P < 0.001) group compared with that in the LPS group. This result was consistent with the changes of iNOS mRNA levels (Fig. 2C). As shown in Fig. 2B, the PGE2 level of the LPS group was markedly higher in contrast to that of the control group. However, treatment with L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 decreased the production of PGE2 (P < 0.01, P < 0.05, P < 0.001). Furthermore, the expression level of COX-2 that regulated PGE2 production showed a similar trend (Fig. 2D). As aforementioned, all tested strains could decrease the concentration and gene expression of proin ammatory mediators. Furthermore, the L. plantarum KLDS 1.0318 had a more substantial inhibition effect in comparison to L. acidophilus KLDS 1.0901, followed by L. helveticus KLDS 1.8701.
Inhibition effect of the tested strains on in ammatory cytokines. In this study, we also evaluated the effect of these strains against proin ammatory cytokines by measuring the production of TNF-α, IL-1β, IL-6 and the expression of their mRNA. The TNF-α, IL-1β, and IL-6 concentration in the LPS group was signi cantly higher than that in the control group, implying that LPS treatment could increase the secretion of pro-in ammatory cytokines. However, the concentration of those proin ammatory cytokines was reduced markedly by all texted strains to the LPS group (Fig. 3A-3C). In terms of the transcription level of these cytokines, inhibition was also observed in Fig. 3D-F. Consistent with the above results, all tested strains could suppress TNF-α, IL-1β, and IL-6 mRNA expression to some extents (P < 0.001). In summary, the above results showed that all tested strains effectively inhibited the secretion of proin ammatory cytokines by down-regulating their gene transcription levels in LPS-induced RAW264.7 macrophages. Also, L. plantarum KLDS 1.0318 had the best anti-in ammatory effect.
Effect of the tested strains on TLR4 mRNA and protein expressions. To estimate the capacity of lactobacillus to inhibit LPS receptor expression, TLR4 mRNA transcription and protein expression levels were detected using RT-qPCR and western blot analysis, respectively. Compared with the control group and the LPS group, the relative expression of TLR4 mRNA increased from 1.35 ± 0.27 to 9.65 ± 0.28 by stimulating with LPS (Fig. 4A) Effect of the tested strains on NF-κB activation. The critical role of NF-κB in in ammatory responses, which can stimulate the production of in ammatory factors. Therefore, we analyzed the inhibition effect of the tested strains on the NF-κB signaling pathway activation. As shown in Fig. 5A, after treatment with LPS, the relative expression p-IκB protein in the LPS group was markedly higher than that in the control group (P < 0.001). We also observed a signi cant reduction in the p65 protein expression level, and the trend of p65 protein expression in the nucleus was opposite to that of the cytosol (Fig. 5B) Effect of the tested strains on MAPKs activation. To determine whether the MAPKs signaling pathway has a dominant position of the anti-in ammatory potential of the tested strains against the in ammation response induced by LPS, the phosphorylation level of MAP kinases (p38, JNK, and ERK) in the RAW264.7 macrophages was measured. As shown in Fig. 6, the LPS group showed remarkable increase in the phosphorylation level of p38, JNK, and ERK compared with that of the control group (P < 0.001).
However, all tested strains pretreatment restroed the changes in MAP kinase. The p-p38/p38 expression with the tested strains pretreated was signi cantly reduced by 12 Contrast KLDS 1.0318 and NF-κB /MAPKs inhibitors on in ammatory pathways. Based on the above results, NF-κB and MAPKs inhibitors were used to assess further the anti-in ammatory effect of L. plantarum KLDS 1.0318, which was most effective. As shown in Fig. 7, compared with the control group, the increase of iNOS and COX-2 protein expressions were found in the LPS group (p < 0.001). However, there was conspicuously decreased in the iNOS and COX-2 expression levels in L. plantarum KLDS 1.0318 group (P < 0.001), which showed the same trend with the BAY11-7082 (NF-kB inhibitor, 2.5 µM),SB203580 p38 inhibitor, 10 µM , SP600125 (JNK inhibitor, 10 µM), and PD98059 (ERK inhibitor, 10 µM) inhibitors group. In particular, the combination L. plantarum KLDS 1.0318 with speci c inhibitors group showed more effective suppression, compared with L. plantarum KLDS 1.0318 or speci c inhibitors alone. Furthermore, the iNOS level was more effectively suppressed by the combined treatment of L. plantarum KLDS 1.0318 with BAY11-708 or SB203580, compared with L. plantarum KLDS 1.0318 alone. Moreover, L. plantarum KLDS 1.0318 with PD98059 or BAY 11-708 showed a higher inhibitory effect than the L. plantarum KLDS 1.0318 group in terms of COX-2 expression. These ndings indicated that L. plantarum KLDS 1.0318 exhibits synergistic inhibition when combined with the speci c inhibitors.

Discussion
In ammation responses can eliminate harmful external invasion or heal tissue injury. However, excessive in ammatory responses are closely related to the development of mild or chronic disease conditions. Therefore, reducing in ammation is a critical preventive and therapeutic strategy for controlling various diseases. Furthermore, due to the limitations and side effects of traditional anti-in ammatory drugs, it is essential to develop alternative measures that are effective and without side effects. For long-term oral administration and minimizing drug toxicity, there is currently no suitable food-grade products to treat in ammations. Many research works have shown that probiotics can improve infectious and in ammatory diseases and are widely used to prevent and treat IBD. Therefore, in our present study, the anti-in ammatory potential of L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 was evaluated, and the associated cellular signaling pathways were investigated.
Drug cytotoxicity requires evaluation before development towards therapeutic applications. Therefore, we assessed the effect of L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 on the viability of RAW 264.7 macrophages. All tested strains did not show cytotoxicity at the MOI of 10. Therefore, the tested strains (MOI = 10) were further studied to illustrate the anti-in ammatory potential. NO and PGE2 are crucial biomarkers in the in ammatory response, modulated by iNOS and COX-2, respectively.
NO and PGE2 are crucial biomarkers in the in ammatory response, modulated by iNOS and COX-2, respectively 24 . Although the iNOS and COX-2 levels are not easily detected, LPS stimulated RAW264.7 cells to induce iNOS and COX-2 expression by activating NF-κB and MAPKs signal pathways to excessive NO production. At the in ammatory site, NO acts on vascular smooth muscle cells, which increases vascular permeability and accelerate the in ltration of in ammatory, pain mediators and monocytes into the in ammatory site. Besides, PGE2 is associated with fever, swelling, and pain perception 25 . Therefore, inhibiting the overexpression of iNOS and COX-2 is an essential strategy for the prevention and treatment of in ammatory diseases. In the current study, all tested strains signi cantly reduced the production of NO and PGE2 by inhibiting iNOS and COX-2 mRNA levels. These results are supported by many experimental pieces of evidence to validate claims that probiotics can ameliorate in ammation. The previous study demonstrated that Hwangryunhaedoktang weakly inhibited various in ammatory mediators (NO, PGE2, iNOS, and COX-2) induced by LPS. However, fermentation with lactobacilli signi cantly increased the inhibitory effect of the herbal compound, Hwangryunhaedoktang, on in ammatory mediator expression 26 . Especially among the tested strains, L. plantarum KLDS 1.0318 had the most potent inhibitory effect.
LPS-induced murine RAW264.7 cells produce several proin ammatory cytokines, including TNF-α, IL-1β, and IL-6. Excessive release of proin ammatory cytokines can cause acute or chronic in ammatory diseases. For example, TNF-α is a key cytokine that stimulates the production of other cytokines in in ammatory responses and promotes the immune response process. 27 However, compared to the LPS group, we discovered a remarkable reduction in the production and mRNA expression of proin ammatory cytokines (TNF-α, IL-1β, and IL-6) in the L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318-treated groups. Consistent with these ndings, L. plantarum 10hk2 isolated from fermented vegetables can reduce IL-1β, IL-6, and TNF-α levels and increase the secretion of IL-10 in LPSinduced RAW 264.7 cells 28,29 . Also, compared to the LPS group, we discovered a substantial decrease in the production and mRNA expression of proin ammatory cytokines (TNF-α, IL-1β, and IL-6) with the treatment of L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318. In agreement with this observation, the L. plantarum 10hk2 strain also reduced the production of IL-1β, IL-6, and TNF-α and increased the secretion of IL-10 in LPS-induced RAW 264.7 cells 30 . The above results established the anti-in ammatory potential of L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318.
To further identify the possible mechanisms by which these strains confer the reported bene cial properties, we investigate whether TLR4-mediated NF-κB and MAPKs signaling pathways are associated with the anti-in ammatory ability L. acidophilus KLDS1.0901, L. helveticus KLDS1.8701, and L. plantarum KLDS1.0318. TLR4 is an integral receptor for LPS to activate NF-κB and MAPK pathways. NF-κB signal plays a crucial role in gene expression in macrophages (Beinke, 2004). LPS induces degradation and ubiquitylation of IκB and NF-κB transfer into the nucleus; afterward, NF-κB regulates downstream gene transcription to promote the expression of proin ammatory cytokines (TNF-α, IL-6, and IL-1β) and in ammatory mediators (NO and PGE2) [31][32][33][34] . In this study, the three strains studied showed signi cant inhibitory effects on the nuclear translocation of NF-κB. Similarly, Lactobacillus-fermented adlay-soymilk could suppress the production of PGE2 and NO and the synthesis of IL-1β, IL-6, and TNF-α by down-regulated translocation of NF-κB p65 in the LPS-induced RAW164.7 cells 35 . Generally, the maximum phosphorylation of LPS-induced MAP kinase is achieved within 30 min after LPS treatment and then rapidly decreased. Therefore, we measured the phosphorylation changes of MAP kinase after LPS stimulation for 20 min. Also, the production of in ammatory mediators (NO and PGE2) and proin ammatory cytokines (TNF-α, IL-1β, and IL-6) was measured for 24 h. However, the data did not speci cally show the change of NF-κB and MAP kinase activity in the late status of LPS-stimulated RAW 264.7 cells. Therefore, we aimed to determine the effect of LPS treatment on NF-κB and MAPKs signaling pathways in the late stage, using strategic co-administration of L. plantarum KLDS 1.0318 with speci c inhibitors, thereby decreasing the protein level of the downstream target genes (iNOS and COX-2) 31 . The results showed that each combined treatment (L. plantarum KLDS 1.0318 + a speci c inhibitor) exhibited better inhibition on LPS-stimulated iNOS and COX-2 expressions. Speci cally, LPS-induced iNOS expression was markedly suppressed by the combinations of L. plantarum KLDS 1.0318 with BAY 11-708 and with SB203580. Moreover, LPS-stimulated COX-2 expression was signi cantly inhibited by L. plantarum KLDS 1.0318 with PD98059, and with BAY 11-708.
We investigated the anti-in ammatory potential of L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 isolated from traditional dairy products in LPS-stimulated murine RAW264.7 cells. All tested strains could inhibit in ammatory mediators (NO and PGE2) and proin ammatory cytokines (TNF-α, IL-1β, and IL-6) though down-regulating their relative gene expressions. This inhibitory function was related to reduced translocation of NF-κB into the nucleus and the phosphorylation of MAP kinase. This study provides that L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 could be functional probiotics to prevent in ammationassociated disorders. Effects of the tested strains at different MOI on the cell viability by the CCK8 assay. Data are expressed as means ± SD (n = 6). *P < 0.05 and ** P <0 .01 vs. the control group. Effects of the tested strains at different MOI on the cell viability by the CCK8 assay. Data are expressed as means ± SD (n = 6). *P < 0.05 and ** P <0 .01 vs. the control group.     Data are expressed as means ± SD (n = 3). * P < 0.05, ** P < 0.01 and *** P <0.001 vs. the LPS group. Effects of the tested strains on mRNA and protein relative expression of TLR4. Cells were with L. acidophilus KLDS1.0901, L. helveticus KLDS1.8701 and L. plantarum KLDS1.0318 for 1 h. TLR4 mRNA (A) and protein (B) were determined by RT-qPCR and western blot analysis after LPS stimulated for 12 h. Data are expressed as means ± SD (n = 3). * P < 0.05, ** P < 0.01 and *** P <0.001 vs. the LPS group. Figure 5 acidophilus KLDS1.0901, L. helveticus KLDS1.8701 and L. plantarum KLDS1.0318 for 1 h. The IκB, phosphorylation of IκB A and nuclear p65, cytoplasm p65 (B) were determined by western blot analysis.