In vitro protective effects of Lactobacillus plantarum Lac16 on Clostridium perfringens infection-associated intestinal injury in IPEC-J2 cells

Background: Clostridium perfringens causes intestinal injury through overgrowth and secretion of multiple toxins, leading to diarrhea and necrotic enteritis in animals, such as pigs. Lactobacillus plantarum (L. plantarum) Lac16 has been reported to protect broilers against C. perfringens infection. This study aimed at investigating the protective effects of Lactobacillus plantarum Lac16 on C. perfringens infection-associated intestinal injury in intestinal porcine epithelial cell line (IPEC-J2). Results: The results showed that L. plantarum Lac16 signicantly inhibit the growth and biolm formation of C. perfringens (P < 0.001). In the co-culture system, L. plantarum Lac16 signicantly suppressed colony forming units (CFU) of C. perfringens (P < 0.05), which was accompanied by a decrease in pH levels (P < 0.01). Moreover, L. plantarum Lac16 signicantly elevated the mRNA expression levels of host defense peptides (HDPs) in IPEC-J2 cells (P < 0.05), decreased C. perfringens-induced cellular cytotoxicity (P < 0.01) and adhesion to cells (P < 0.05). At the same time, L. plantarum Lac16 signicantly attenuated C. perfringens-induced damage to intestinal barrier integrity and the decrease in claudin-1 (P < 0.01) as well as zona occludens 1 (ZO-1) expressions. Preincubation with L. plantarum Lac16 signicantly suppressed mRNA expression levels of pattern recognition receptors (PRRs) (Toll-like receptor (TLR) 1, TLR2, nucleotide-binding oligomerization domain (NOD) 1) in C. perfringens-challenged IPEC-J2 cells (P < 0.01). C. perfringens signicantly elevated the phosphorylation of p38 mitogen-activated protein kinase (MAPK), JNK, and p65 nuclear factor-κB (NF-κB) medium as single bacterial strain groups, respectively. Regarding the co-culture system, 100 μL of L. plantarum and C. perfringens were both inoculated in 9.8 mL of modied RCM medium. The above cultures were incubated at 37 ℃ for 12 h, their pH values were determined, after which they were serially diluted, cultured on TSC agar and incubated at 37 ℃ for 12 h to quantitate C. perfringens populations. These experiments were done in triplicates. biolm formation abilities of C. perfringens, reducing pH levels in the environment, preventing pathogenic adhesion to epithelial cells, promoting the expressions of endogenous host defense peptides, protecting intestinal epithelial barrier integrity, and alleviating inammatory responses by attenuating p65 phosphorylation. These ndings highlight the signicance of L. plantarum Lac16 as a potential therapeutic strategy against C. perfringens infection and provide a theoretical basis for the application of L. plantarum Lac16 in animal husbandry to resist pathogen infections.

balance of intestinal microbiota [13,14]. Lactobacillus spp. have been shown to exert protective effects against C. perfringens infection in vivo and in vitro. Guo et al. [15] reported that L. acidophilus and L. fermentum inhibited the growth and α-toxin production capacity of C. perfringens in vitro. At the same time, another study showed that L. fermentum also inhibited β2-toxin secretion [16]. Furthermore, members of Lactobacillus spp., such as L. fermentumc, L. salivarius, L. plantarum, and L. acidophilus inhibit C. perfringens infection associated necrotic enteritis by improving intestinal morphology and barrier integrity, reducing lesions, ameliorating in ammation, as well as by modulating intestinal microbiota [17][18][19][20].
In our previous study, we found that L. plantarum Lac16 effectively protected broilers from C. perfringens infection [21]. However, it has not been established whether L. plantarum Lac16 can protect pig intestines in cases of C. perfringens infection. The porcine jejunal epithelial cell line, IPEC-J2, has exhibited a high speci city in pig studies and is a suitable model for investigating the interactions between bacteria and intestinal epitheliums in vitro [22,23]. Thus, this study aimed at investigating the protective effects of L. plantarum Lac16 on C. perfringens infection-associated intestinal injury in IPEC-J2 cells in vitro.

Methods
Bacterial strains and culture conditions. L. plantarum Lac16 (CCTCC, No. M2016259) was isolated in our laboratory and preserved at the China Center for Type Culture Collection. L. plantarum Lac16 was cultured in Mann-Rogosa-Sharpe (MRS) medium and incubated at 37 ℃ for 18 h. C. perfringens type A (ATCC 13124) was cultured in Reinforced clostridium medium (RCM; Hopebio, Qingdao, China) and incubated at 37 ℃ in anaerobic conditions for 18 h.
To determine bacterial concentrations, we centrifuged the overnight-incubated bacterial cultures at 5000 rpm for 5 min. After being washed three times using sterile phosphate-buffered saline (PBS, pH = 7.2), bacteria were resuspended in PBS and their concentrations determined using a standard curve. Then, they were diluted to a certain concentration and stored at 4 ℃.
Agar-diffusion method for detecting bacteriostasis of L. plantarum Lac 16 in a fermentation supernatant.
Agar-diffusion was performed as previously described by Wang et al. [24] with some modi cations. Brie y, L. plantarum broth was centrifuged at 5000 rpm for 10 min to obtain the supernatant. The supernatant was ltered through a 0.22 μm membrane to remove suspended bacteria and stored at 4 ℃. About 0.2 % (v/v) of the overnight culture of C. perfringens was added to tryptose sul te cycloserine (TSC; Hopebio, Qingdao, China) agar which cooled down to about 50 ℃. Then, the medium was well mixed and poured into plastic plates which placed oxford cups in advance. The oxford cups were removed after the medium had solidi ed. Then, 100 µL of L. plantarum Lac16 fermentation supernatants were injected into each well after which plates were placed in anaerobic gas generating packs (Hopebio, Qingdao, China) for 12 h. This bacteriostatic experiment was performed in triplicates.
Bio lm assays were performed as previously described by Jiang et al. [25] with some modi cations. L. plantarum and C. perfringens were cultured in modi ed RCM medium (glucose content was increased to 20 g/L on the original basis) and incubated at 37 ℃ for 18 h, respectively. Then, the concentration of the C. perfringens culture was adjusted to 10 7 CFU/mL using the modi ed RCM medium, after which the supernatant of L. plantarum was collected as described above.
Experimental groups were treated as: i. Sterile modi ed RCM medium (200 μL) were inoculated into 96-well culture plates and designed as the control group; ii. Resuspended C. perfringens (100 μL) or 100 μL of L. plantarum supernatant were added to 100 μL of sterile modi ed RCM medium, and designed as the CP or Lac16 fermentation supernatant group; iii. L. plantarum fermentation supernatant (100 μL) and 100 μL of resuspended C. perfringens were inoculated and designed as the experimental treatment group, which was labeled Lac16 fermentation supernatant + Cp group. The 96-well culture plate was incubated in an anaerobic environment at 37 ℃ for 12 h after which the bacterial proliferation index was read at OD 600 using SpectraMax M5 (Molecular Devices, USA). Then, bacterial cultures were removed with caution, wells were gently washed thrice using PBS and incubated with 100 μL of 1% crystal violet for 30 min. Crystal violet in the wells was removed and wells were gently washed thrice using PBS. Then, 100 μL of 95% alcohol was added into the wells to dissolve excess crystal violet and OD 590 in each well measured. The higher the optical density, the more bio lm formation. Experiments were done in triplicates.
Co-culture experiment and pH determination of cultures.
Bacterial co-culture experiments were done as previously described by Guo et al. [15] with some modi cations.
L. plantarum and C. perfringens were adjusted to 10 7 CFU/mL using the modi ed RCM medium. Ten milliliter of the modi ed RCM medium were used as the blank control. At the same time, L. plantarum or C. perfringens suspensions (100 μL) were inoculated in 9.9 mL of modi ed RCM medium as single bacterial strain groups, respectively. Regarding the co-culture system, 100 μL of L. plantarum and C. perfringens were both inoculated in 9.8 mL of modi ed RCM medium. The above cultures were incubated at 37 ℃ for 12 h, their pH values were determined, after which they were serially diluted, cultured on TSC agar and incubated at 37 ℃ for 12 h to quantitate C. perfringens populations. These experiments were done in triplicates.
Cell cultures.
Determination of expression levels of HDPs by Real-Time PCR.
IPEC-J2 cells were seeded in 12-well cell culture plates (Corning Life Science, MA, USA) at a density of 5 × 10 5 cells/well. L. plantarum cultures were centrifuged and resuspended in DMEM/F12 supplemented with 10% FBS and stored at 4 ℃. When IPEC-J2 cells reached 80% con uence, they were co-incubated in cell culture media containing different concentrations of L. plantarum Lac16 (10 6 , 10 7 , and 10 8 CFU/mL) for 6 h. After being washed three times using PBS, IPEC-J2 cells were lysed by RNAiso Plus (Takara, Dalian, China) to extract RNA.
IPEC-J2 cells were seeded in 12-well culture plates at a density of 5 × 10 5 cells/well. When cells reached 80% con uence, they were incubated with or without L. plantarum (10 7 CFU/mL) for 6 h, respectively. After being washed three times using PBS, cells were infected with C. perfringens (10 6 CFU/well) under anaerobic conditions for 1 h or 3 h, respectively. Then, cell suspensions were collected and centrifuged at 10 000 rpm/min for 5 min to remove cell debris and bacteria. The release of lactate dehydrogenase (LDH) from damaged cells was measured using the LDH kit (Nanjing Jiancheng Biological Product, Nanjing, China), according to the manufacturer's instructions. Experiments were performed in triplicates.
Bacterial adhesion assay was performed as previously described by Jiang et al. [25] with some modi cations.
Brie y, IPEC-J2 cells were seeded in 12-well cell culture plates at a density of 5 × 10 5 cells/well. At 80% con uence, cells were pre-incubated with L. plantarum (10 7 CFU/mL) for 6 h, after which C. perfringens were added into the wells (10 6 CFU/mL) and incubated for 1 h under anaerobic conditions. Cells treated with C. perfringens only were used as the controls. Then, cells were washed three times using sterile PBS to remove non-adherent C. perfringens. Two hundred microliters of 0.25 % trypsin-EDTA solution was added to the wells and digested for 15 min, then, 800 μL sterile PBS was added to each well and completely mixed. Liquids containing bacteria were serially diluted and incubated in TSC agar for 12 h to quantitate C. perfringens populations. Each assay was performed in triplicate.
At the same time, we used uorescein isothiocyanate (FITC; Solarbio, Beijing, China) labeling method to observe the adhesion effect of C. perfringens. Brie y, the concentration of C. perfringens culture, after centrifuged, was adjusted to 10 7 CFU/mL with diluted FITC-solution (200 μg/mL). Avoid light and incubate for 2 h at 37 ℃. Then the bacteria were washed with sterile PBS for three times and storaged at 4 ℃. After incubation with L. plantarum Lac16 for 6 h, the cells were washed three times with sterile PBS and co-incubated with C. perfringens (10 6 CFU/mL), which was labeled with FITC, for 1 h under anaerobic conditions and away from light. Then the cells were washed three times with sterile PBS and examined under a uorescence microscope (Nikon, Japan). All experiments were performed in triplicate.
Cell permeability to uorescein sodium.
Cell permeability to uorescein sodium was assessed as previously described by Nie et al. [26] with some modi cations. Brie y, IPEC-J2 cells were seeded in 12-well transwell inserts (Corning Life Science, MA, USA), with pore sizes of 0.4 mm and membrane areas of 1.12 cm 2 , at a concentration of 1 × 10 5 cells/mL. Since IPEC-J2 cells can develop tight junctions and differentiate into tight monolayers after 9 days of culture on transwell lters [22], we renewed the culture medium in both apical and basolateral sides of the lters every 24 h for 9 days. On the 10 th day, the culture medium on the apical side of the lters was removed and IPEC-J2 cells were treated as described in adhesion assay section. Then, 100 μg/mL uorescein sodium (Sigma-Aldrich, MO, USA), dissolved in PBS, was added to the apical inserts for 1 h, after which 200 μL of medium from each basolateral side was collected. Fluorescence intensity was determined using a SpectraMax M5 (Molecular Devices, USA) at an excitation wavelength of 495 nm and an emission wavelength of 525 nm. Then, we calculated apical to basolateral ux of uorescein sodium using the standard curve. Apparent permeability coe cient (P app ) was calculated using the formula: P app = ΔQ/Δt×(1/AC 0 ) [26]. Whereby, ΔQ/Δt is the permeability rate (μg/s), A is the diffusion area of the monolayer (cm 2 ), while C 0 is the initial concentration (μg/mL) of uorescein sodium in the transwell apical inserts. All experiments were performed in triplicates.
IPEC-J2 cells (5×10 5 cell/mL) were seeded on glass coverslips in a 12-well at-bottom culture plate for at least 9 days to form tight junctions. On the 10 th day, the monolayer reaching polarization was treated with bacteria as described in adhesion assay section. Cells were xed in 4% paraformaldehyde for 20 min at room temperature and blocked with 2.5% bovine serum albumin (BSA; Solarbio, Beijing, China) for 1 h at room temperature. Cells were incubated with rabbit polyclonal anti-ZO-1 primary antibody (Invitrogen, MA, USA) for 12 h at 4 ℃, after which they were incubated with secondary antibody Alexa uor 488 goat anti-rabbit (Abcam, Cambridge, UK) for 1 h at room temperature and away from light. Nuclei were stained with 4',6-Diamidino-2phenylindole dihydrochloride (DAPI; Beyotime, Shanghai, China). Fluorescence images were obtained through laser scanning confocal microscopy (LSM 880 with AiryScan) (Zeiss, Germany). All experiments were performed in triplicates.
The IPEC-J2 cells (5×10 5 cell/mL) were grown in 24-well plates (Corning Life Science, MA, USA) for 9 days and pretreated with bacteria as described in adhesion assay section. Then, cells were washed using PBS and xed in 70% ethanol for 10 min at room temperature. Periodic acid-Schiff staining was performed according to the manufacturer's instructions (Beyotime, Shanghai, China). Images were obtained using a light microscope (Leica, Germany). Experiments were performed in triplicate.
Quantitative Real-Time PCR.
IPEC-J2 cells were pretreated with bacteria as described in adhesion assay section. Then, cells were lysed using RNAiso Plus (Takara, Dalian, China) to extract RNA. Total RNA was reverse transcribed to cDNA using the HiScript II Q Select RT SuperMix (Vazyme, Nanjing, China). The qRT-PCR analysis was performed using StepOne Plus Real-Time PCR system (Applied Biosystems, USA) and the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). Primer sequences used in this study are shown in Table 1. All samples were run in triplicates. β-actin was selected as an endogenous control and relative gene expressions were analyzed using the 2 -ΔΔCt method [27]. Each assay was performed in triplicate. Western blot analysis.
After pretreatment with bacteria as described in adhesion assay section, IPEC-J2 cells were lysed using the using the chemiluminescent HRP substrate kit (Millipore, MA, USA). Intensities of protein bands were determined using the ImageJ software. All experiments were performed in triplicate.
Statistical Analysis.
Experimental data were analyzed by IBM SPSS Statistics 20 and presented as mean ± SD. Statistical signi cance between two groups was determined by two-tailed Student's t test, while multiple comparisons were performed by one-way ANOVA. P ≤ 0.05 was considered signi cant. Graphs were drawn using the OriginPro 2018 software.

Results
L. plantarum Lac16 and its fermentation supernatant signi cantly inhibited the growth of C. perfringens.
As shown in Fig. 1A, the positive group (100 µg/mL of ampicillin) exhibited the best bacteriostatic effect on C. perfringens, with L. plantarum Lac16 fermentation supernatant also exhibiting good bacteriostatic effects, forming a clear boundary of bacteriostatic zone. In the bio lm formation experiment (Fig. 1B), after 12 h of culture, optical density (OD 600 ) of the CP group increased to 0.837 ± 0.031, while the OD 600 of the control group was 0.090 ± 0.002, indicating that C. perfringens proliferated rapidly. However, in the group in which C. perfringens were co-incubated with the L. plantarum Lac16 fermentation supernatant, the OD 600 only increased to 0.189 ± 0.008. These results indicate that L. plantarum Lac16 fermentation supernatant could signi cantly inhibit the growth of C. perfringens (P < 0.001). Similar results were obtained from the bio lm assays (Fig. 1C). The L. plantarum Lac16 fermentation supernatant signi cantly inhibited the formation of C. perfringens bio lms when compared to the CP group (P < 0.001).
After that, we co-cultured L. plantarum Lac16 with C. perfringens and determined the corresponding pH. It was found that CFUs of C. perfringens in the co-culture group were signi cantly decreased when compared to the CP group (P < 0.05) (Fig. 1D). Although L. plantarum Lac16 and C. perfringens could reduce the pH of the medium, the pH of Lac16 and co-culture groups were lower relative to those of the CP group (P < 0.01) (Fig. 1F).
Expression levels of HDP genes, including pBD1, pBD2, pBD3 and pEP2C, were all found to be elevated after coincubation with L. plantarum Lac16 (Fig. 2). Speci cally, expression levels of pBD1 were signi cantly elevated after co-incubation with L. plantarum Lac16 at concentrations of 10 7 CFU/mL and 10 8 CFU/mL (P < 0.001) ( Fig. 2A). The best effect was obtained at the concentration of 10 7 CFU/mL. At the same time, the trend in pBD2 gene expression level was similar (Fig. 2B), except that the group with a concentration of 10 7 CFU/mL was the only one that exhibited a signi cant increase in gene expression (P < 0.05). Gene expression levels of pBD3 and pEP2C were signi cantly elevated in a concentration dependent manner after co-incubation with different concentrations of L. plantarum Lac16 (P < 0.05; Fig. 2C and D). To some extent, we found that L. plantarum Lac16 at elevated concentrations (10 8 CFU/mL) competed with cells for nutrients, therefore, we selected the medium concentration (10 7 CFU/mL) for the follow-up experiments.
Pretreatment with L. plantarum Lac16 did not increase LDH release in both groups (P > 0.05; Fig. 3A). However, when IPEC-J2 cells were infected with C. perfringens, LDH release was signi cantly elevated (P < 0.01), which was alleviated by pre-incubation with L. plantarum Lac16 (P < 0.01). However, the increased LDH release, which was associated with C. perfringens infection for 3 h, was not alleviated to normal levels after L. plantarum Lac16 pre-incubation (P < 0.001), indicating cytotoxic induction due to the infection. Therefore, for subsequent experiments, we selected IPEC-J2 cells infected with C. perfringens for 1 h as the time point for sampling.
L. plantarum Lac16 suppressed the adhesion of C. perfringens to IPEC-J2 cells.
For the bacterial adhesion assay, we de ned adhesion rate as 100 % in the CP group, in which cells were only co-incubated with C. perfringens (Fig. 3B). However, when cells were pre-incubated with L. plantarum Lac16, the adhesion rate of C. perfringens to IPEC-J2 cells decreased signi cantly (P < 0.05). Similar results were obtained in the images of uorescence labeling method (Fig. 3C). When IPEC-J2 cells were only infected with C. perfringens, the quantity of uorescent labeled pathogens that adhered to cells was signi cantly larger than that of the group pretreated with L. plantarum Lac16.
L. plantarum Lac16 attenuated C. perfringens-induced damage to intestinal barrier function.
When IPEC-J2 cells developed tight junctions and were completely differentiated into tight monolayers, they were infected with C. perfringens. At the same time, the ux of uorescein sodium signi cantly increased (P < 0.001; Fig. 4A). However, L. plantarum Lac16 pretreatment signi cantly alleviated C. perfringens-induced increase in Papp of the IPEC-J2 monolayers (P < 0.001). Then, we evaluated the effects of bacterial pretreatment on the production of mucins (Fig. 4B). After PAS staining, it was found that C. perfringens infection inhibited mucin production. Moreover, L. plantarum Lac16 pretreatment effectively inhibited C. perfringens -mediated decrease in mucin production.
To evaluate C. perfringens-induced damage to intestinal epithelial barrier functions and the corresponding protective effects of L. plantarum Lac16, we determined the expression levels of tight junction proteins in IPEC-J2 cells (Fig. 4C). Compared to the control group, there were no signi cant differences in expression levels of occludin in each group after incubation with bacteria (P > 0.05). However, C. perfringens signi cantly suppressed the expression levels of claudin-1 in IPEC-J2 cells (P < 0.01). L. plantarum Lac16 treatment signi cantly elevated the expressions of claudin-1 (P < 0.05) and alleviated the decrease in protein expressions caused by C. perfringens infection (P < 0.01). Regarding immuno uorescence images of ZO-1, we found that L. plantarum Lac16 pretreatment had no effect on protein expression levels, whereas C. perfringens infection signi cantly suppressed the expression of ZO-1 (Fig. 4D). At the same time, compared to the CP group, IPEC-J2 cells that had been co-incubated with L. plantarum Lac16 and C. perfringens exhibited increased expressions of ZO-1.
L. plantarum Lac16 alleviated the increase in mRNA expression levels of PRRs after C. perfringens infection.
mRNA expression levels of several PRRs in IPEC-J2 cells after co-incubation with L. plantarum Lac16 and C. perfringens were investigated (Fig. 5). Speci cally, when cells had been co-incubated with bacteria, mRNA expression levels of TLRs were signi cantly up-regulated (P < 0.05) (Fig. 5A-C). The most signi cant increase in mRNA expression levels of TLRs was in the group infected with C. perfringens only. However, the increased mRNA expression (TLR1 and TLR2) caused by C. perfringens infection was effectively inhibited (P < 0.01) by co-incubation with L. plantarum Lac16. The trend in mRNA expression levels of NODs was similar to that of TLRs ( Fig. 5D and E). Although both bacteria increased NOD1 gene expression (P < 0.001), preincubation with L. plantarum Lac16 signi cantly reduced the increased gene expression associated with C. perfringens infection (P < 0.01) (Fig. 5D). However, this effect was not signi cant in NOD2 gene expression (P > 0.05) (Fig.  5E).
C. perfringens induced in ammatory effects through MAPK and NF-κB signaling pathways in IPEC-J2 cells.
To investigate the potential signaling pathway that led to the release of in ammatory cytokines, phosphorylation levels of certain proteins of the MAPK and NF-κB were determined (Fig. 6). Speci cally, C. perfringens infection enhanced the phosphorylation of p38, JNK, as well as p65 when compared to the control group (P < 0.05). Furthermore, ERK phosphorylation in each group did not change signi cantly (P > 0.05).
L. plantarum Lac16 alleviated C. perfringens infection-associated increase in pro-in ammatory cytokine gene expression levels.
We determined mRNA expression levels of in ammatory cytokines in IPEC-J2 cells after co-culture with bacteria (Fig. 7). After co-incubation with L. plantarum Lac16, mRNA expression levels of pro-in ammatory cytokines, such as interleukin (IL)-6 and IL-8, were signi cantly elevated (P < 0.05; Fig. 7B and C). At the same time, gene expression levels of pro-in ammatory cytokines, such as IL-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α, were sharply elevated after C. perfringens infection (P < 0.001), which was signi cantly alleviated by preincubation with L. plantarum Lac16 (P < 0.05). L. plantarum Lac16 was very effective in alleviating the increase in C. perfringens infection-associated gene expressions of IL-8 and TNF-α (P < 0.001). Furthermore, when cells were respectively incubated with L. plantarum Lac16 or C. perfringens, gene expression levels of antiin ammatory cytokines, such as IL-10 and transforming growth factor (TGF)-β, did not change signi cantly (P > 0.05) ( Fig. 7E and F). However, mRNA expression levels of anti-in ammatory cytokines in the Lac16+CP group decreased signi cantly when compared to the CP group (P < 0.05).

Discussion
Probiotics provide bene ts to the host through various mechanisms, including producing anti-bacterial substances, competing with pathogenic microorganisms for enterocyte binding, regulating the secretion of proand anti-in ammatory cytokines, and maintaining intestinal barrier integrity [28]. There were several studies indicated that Lactobacillus spp. could resist the infection of pathogenic bacteria, including Escherichia coli, C. perfringens, and Salmonella Enteritidis [29,30,28,15]. Previously, we found that L. plantarum Lac16 exhibits protective effects against C. perfringens infection in broilers [21]. In this study, we have shown that L. plantarum Lac16 inhibits the growth of C. perfringens and attenuates C. perfringens infection-associated intestinal injury in IPEC-J2 cells.
Probiotics exert antimicrobial activities by secreting antimicrobial substances, such as bacteriocins, organic acids, and hydrogen peroxide [31]. Cell-free supernatants containing antimicrobial substances secreted by probiotics effectively inhibit the growth of various pathogens [32]. In addition, C. perfringens strains form bio lms to enhance their persistence and increase resistance to various stressors, such as oxidative and antibiotic stress [33], and this phenomenon has been proved to be involved in a large proportion of bacterial infections [34]. In the current study, we found that the fermentation supernatant of L. plantarum Lac16 inhibited the growth of C. perfringens. Furthermore, the fermentation supernatant of L. plantarum Lac16 signi cantly suppressed C. perfringens bio lm formation. The antimicrobial activity of L. plantarum is associated with the production of organic acids, which decreases environmental pH [35]. C. perfringens is a pH sensitive bacterium [36]. Acidic environments downregulate the expression of virulence factors of C. perfringens while inhibiting its growth [36,37]. These ndings indicate that L. plantarum Lac16 and its metabolites decreased environmental pH, and inhibited bio lm formation as well as the growth of C. perfringens.
HDPs, as important components of the innate immune system, play critical roles in infection resistance [38].
They are mainly secreted by intestinal epithelial cells and phagocytes in the gastrointestinal tract [24]. HDPs are involved in the maintenance of intestinal homeostasis and innate immune defenses during infection through multiple mechanisms. Speci cally, HDPs secreted by intestinal epithelial cells exert direct antimicrobial effects on invading bacterial pathogens and intestinal microbiota [39]. There were several studies indicated that Lactobacillus spp. enhance the expression of HDPs [40][41][42]. In this study, we found that different concentrations of L. plantarum Lac16 promoted the expression of endogenous HDPs in IPEC-J2 cells.
Increased secretion of endogenous HDPs improves early immune system responses to pathogenic infections and in ammation [43]. Our ndings are consistent with those of Wang et al. who concluded that L. plantarum ZLP001 upregulates the expression of HDPs, and they also concluded that L. plantarum enhances intestinal defense responses by promoting the secretion of HDPs [24].
LDH is a stable cytoplasmic enzyme that possesses oxidation-reduction activities. When cells are subjected to cell membrane damage that is caused by intracellular or extracellular stress, LDH will rapidly release into the extracellular environment [44]. Alpha toxin, which is produced by C. perfringens type A, can result in extensive degradation of the plasma membrane, leading to LDH release [45]. Elevated LDH release is a key feature of apoptosis, necrosis, and other forms of cellular damage [46,47]. We found that C. perfringens infection elevated LDH release from IPEC-J2 cells, implying that C. perfringens damaged the intestinal epithelial cells, resulting in intracellular enzyme leakage. However, when cells were pre-incubated with L. plantarum Lac16, the C. perfringens infection-associated increase in LDH release was effectively alleviated. Similar protective results have been reported [48][49][50][51].
Pathogenic adherence to host epithelial cells is an indispensable step in the occurrence of infection [52]. In the meantime, Lactobacillus could effectively prevent pathogenic adhesion to intestinal epithelial cells and play an important role in maintaining intestinal homeostasis [53]. Thus, one way for Lactobacillus exerts its antibacterial activity is by occupying the adhesion site of pathogens to intestinal epithelial cells [54]. Probiotic adhesion to intestinal epithelial cells can optimize the balance and activities of intestinal microbiota [55]. It is worth mentioning that adherence of C. perfringens strains increases toxin production [56]. In this study, we found that L. plantarum Lac16 signi cantly suppressed C. perfringens adhesion to IPEC-J2 cells, thereby resisting C. perfringens infection and protecting intestinal epithelial cells. This nding is similar to that of another study, which showed that L. rhamnosus effectively inhibited the adhesion of C. perfringens to pig intestinal mucosa [57].
Epithelial cells form a layer that acts as a physical barrier connected by tight junctions between each cell [58].
The main components of tight junction proteins are claudins, zona occludens, and occludin [59]. Tight  [70]. In the current study, we found that L. plantarum Lac16 elevated the expression levels of TLRs. Changes in expression levels of TLRs in intestinal epithelial cells regulate β-defensin expression [71], corresponding to our previous conclusion that L. plantarum Lac16 enhances the expression of endogenous HDPs. The C. perfringens challenge sharply elevated the mRNA expression levels of TLRs, including TLR1, TLR2, and TLR4, whereas preincubation with L. plantarum Lac16 attenuated this dramatic increase. When pathogens gain entry into the cytoplasm, NLRs have been shown initiate innate immune responses [72]. We found that L. plantarum Lac16 alleviated C. perfringens-induced elevations in NOD1 expression, in tandem with other studies that reported that probiotics attenuate pathogen associated elevations in PRR expression [73,28]. In our opinion, L. plantarum Lac16 activates PRR-dependent signaling pathways and strengthens the immune system to resist C. perfringens infection.
MAPK signaling pathways are signal transduction modules that transform extracellular signals into intracellular responses that regulate the processes of cell growth, differentiation, and migration [74,75]. In addition, MAPK play a crucial role in modulating the synthesis and release of in ammatory mediators during in ammatory responses [76,77]. Elevated expressions of PRRs enhance the phosphorylation of MAPK [78,79].
In this study, C. perfringens infection signi cantly elevated p38 and JNK phosphorylation in IPEC-J2 cells. However, preincubation with L. plantarum Lac16 did not signi cantly attenuate these phenomena. We indicated that C. perfringens-induced in ammatory responses are partly mediated by MAPK signaling pathways.
NF-κB, an important transcription factor, is a key factor for modulating the expression of genes and proteins involved in in ammatory responses [80]. For example, production of proin ammatory cytokines, such as TNF-α, are closely associated with activation of NF-κB [81]. Probiotics have also been shown to exert their protective mechanisms against pathogenic infections by modulating the NF-κB signaling pathways [82,83,28]. In this study, preincubation with L. plantarum Lac16 signi cantly attenuated C. perfringens-induced increase in p65 phosphorylation, implying that L. plantarum Lac16 prevents C. perfringens infection-associated excess immune responses by attenuating p65 phosphorylation.
Infections with pathogenic microbes, such as C. perfringens, often leads to signi cant in ammatory responses [84,85]. Pro-in ammatory cytokines mediate in ammatory responses to invading pathogens through multiple modulatory mechanisms, such as lymphocyte activation, neutrophil migration, and cell proliferation [86].
However, excess secretion of pro-in ammatory cytokines has deleterious effects on the host [87]. In this study, although L. plantarum Lac16 elevated the expression levels of pro-in ammatory cytokines, such as IL-6 and IL-8, preincubation with L. plantarum Lac16 signi cantly inhibited C. perfringens associated in ammatory responses. In our previous study involving broilers, we found similar protective effects, whereby L. plantarum Lac16 alleviated C. perfringens infection-associated in ammatory responses in the ileum mucosa [21]. In general, we postulate that one of the mechanisms through which L. plantarum Lac16 protects intestinal epithelial cells from C. perfringens injury is by relieving in ammation. Interestingly, when intestinal epithelial cells were incubated with L. plantarum Lac16 or C. perfringens, mRNA expression levels of anti-in ammatory cytokines were not signi cantly altered, while expression levels in the Lac16 + Cp group were signi cantly suppressed. These ndings should be con rmed in more studies.