Enterococcus faecium HDRsEf1 Represses CYP3A29 Expression in the Intestine through the TLR1/2-induced A20 to attenuate the NF-κB/RXR-α Signaling

Enterococcus faecium HDRsEf1 Represses CYP3A29 Expression in the Intestine through the TLR1/2-induced A20 to attenuate the NF-κB/RXR-α Signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enterococcus faecium HDRsEf1 Represses CYP3A29 Expression in the Intestine through the TLR1/2-induced A20 to attenuate the NF-κB/RXR-α Signaling Yinghui Gong, Yucheng He, Yue Li, Ying Wang, Xiue Jin, Deshi Shi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4192677/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract CYP3A29, like human CYP3A4 is crucial for drug metabolism in pigs and some probiotics can regulate the expression of CYP3A in mammals. Here, we show that Enterococcus faecium HDRsEf1 significantly reduces CYP3A29 expression in pig intestinal tissues and epithelial cells, dependent on cell-cell contact. In IPEC-J2 cells, HDRsEf1 decreased the CYP3A29 promoter activity, RXR-α expression and mitigated the RXR-α or PXR-increased CYP3A29 expression. Both RXR-α/PXR over-expression synergistically increased CYP3A29 expression while RXR-α or PXR silencing reduced CYP3A29 expression. Co-immunoprecipitation revealed that RXR-α directly interacted with PXR. HDRsEf1, like a NF-kB inhibitor, significantly decreased the NF-kBp65 activation, RXR-α and CYP3A29 expression, which were abrogated by RXR-α silencing. HDRsEf1 increased A20 expression dependent on TLR1/2 expression. Therefore, HDRsEf1 inhibits the expression of CYP3A29 through the TLR1/2-induced A20 to attenuate the NF-κB/RXR-α signaling in pig intestinal tissues. Our findings suggest potential risks in the clinical application of probiotics. probiotic cytochrome P450 CYP3A29 RXR-α NF-κB TLR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Drug metabolism in the intestine or intestinal first-pass metabolism can cause low bioavailability of some oral drugs, which depends on the contents of relevant enzymes in the gastrointestinal lumen and epithelium as well as bacterial enzymes ( 1 , 2 ). Probiotics can modulate gut microbiota and regulate immune responses, benefiting patients with intestinal diseases, diabetes, tumors and obesity ( 3 ). Probiotics have been widely used in humans and animals, and probiotics may alter the expression of intestinal drug-metabolizing enzymes (DMEs) to change the bioavailability of some oral drugs. However, the effect of probiotics on the expression of intestinal DMEs and their mechanisms have not been clarified. Accordingly, it is important to understand the effect and mechanisms of probiotics on the expression of intestinal DMEs. Mammalian cytochrome P450s (CYP450s) are crucial for the oxidative metabolism of xenobiotics, including therapeutic drugs, environmental carcinogens and toxins( 4 ). CYP3A4, one of the DMEs in humans, can metabolize > 50% of clinical drugs ( 5 ). Pigs have a high similarity in physiology and anatomy to humans and become an important animal model for evaluating new drugs ( 6 , 7 ). Porcine CYP3A29 accounts for 30% of total CYP proteins and the major CYP3A activity in pig liver microsomes ( 8 ). Pig CYP3A29 and human CYP3A4 have similar tissue distribution, pharmacokinetic characteristics and regulatory mechanisms, and pig CYP3A29 is a suitable model for research of human CYP3A4 ( 9 , 10 ). The nuclear receptors (NRs), such as retinoid X receptor alpha (RXR-α), pregnane X receptor (PXR), vitamin D receptor (VDR) and the constitutive androstane receptor (CAR), are important for regulating the expression of CYP450s( 11 ). The RXR-α can directly interact with PXR, VDR or CAR to form heterodimers and bind to the CYP450s gene promoter to regulate their expression( 12 ). For example, the VDR/RXR-α heterodimer can bind to the CLEM4-ER6 motif to regulate the expression of CYP3A4( 13 ), and the CAR/RXRα and PXR/RXR-α heterodimers can interact with pER6 and dXREM to regulate the expression of CYP3A4, respectively ( 14 , 15 ). Our previous study has shown that the PXR/RXR-α can bind to the CYP3A29 promoter to induce its expression in porcine liver HepLi cells ( 16 ). Apparently, RXR-α expression is critical for the expression of CYP3A in the digestive system. The nuclear factor-kappa B (NF-κB) signaling is crucial for inflammation, immunity, cell proliferation and apoptosis( 17 ). It can regulate the expression of CYP450s by directly binding to the CYP450 promoters, or indirectly regulate the expression of NRs ( 11 , 16 , 18 ). In addition, the NF-κB can also enhance the activity of CYP450 at the post-transcriptional level by stabilizing CYP450 proteins ( 19 ). Interestingly, previous studies have revealed that some probiotics can inhibit the NF-κB activity ( 20 , 21 ). However, whether and how probiotics could modulate the expression of CYP450s, such as CYP3A29 in pig intestinal tissues, have not been clarified. Toll-like receptors (TLRs) act as a type of pattern recognition receptors (PRR) in the innate immune system and TLRs can recognize microbial components to initiate immune responses. TLR2 can directly interact with TLR1 or TLR6 to form a heterodimer and recognize conserved molecular patterns (such as peptidoglycan, lipoteichoic acid, etc.) on the cell wall of Gram-positive bacteria( 22 , 23 ). Engagement of TLR2 by the cell wall components of some probiotics can up-regulate the expression of some NF-κB inhibitors, such as A20 (tumor necrosis factor-α-induced protein 3), IRAK-M and Tollip, to inhibit inflammation( 20 , 21 ). Actually, A20 is a deubiquitinating enzyme and can inhibit the NF-kB activation ( 24 ). Our previous study indicates that Enterococcus faecium HDRsEf1 enhances the expression of occluding, the intestinal tight junction protein, by activating the TLR2 ( 25 ). Accordingly, we hypothesize that HDRsEf1 can modulate the expression of CYP3A29 in pig intestinal tissues by modulating the NB-κB signaling. In this study, we explored the effect of HDRsEf1 on CYP3A29 expression and potential mechanisms in pig intestinal tissues and epithelial IPEC-J2 cells. We found that HDRsEf1 decreased the expression of CYP3A29 in pig intestinal tissues by activating the TLR2 to up-regulate A20 expression, inhibiting the NF-κB activation to attenuate RXR-α expression. Results HDRsEf1 reduces CYP3A29 expression in vivo and in vitro Previous reports have shown that probiotics can regulate the expression of CYP450s in the intestinal tract of animals ( 26 , 27 ). Because CYP3A29 is an important enzyme in the intestine of pigs, we examined whether HDRsEf1 could modulate the CYP3A29 expression in pig intestinal tissues. After fed with HDRsEf1-contained diet for 30 days, we found that the relative levels of CYP3A29 expression in the jejunum tissues of the probiotic group of pigs were significantly lower than that in the control group (Figure, 1A). Furthermore, treatment with 10 8 CFU/mL of HDRsEf1 for 6 h did not alter the viability of IPEC-J2, but treatment with HDRsEf1 for a longer time period obviously decreased the viability of IPEC-J2 cells (Figure. 1B). In addition, treatment with 10 6 , 10 7 and 10 8 CFU/mL of HDRsEf1for 2–6 h down-regulated CYP3A29 mRNA transcripts in IPEC-J2 cells in a dose- and time-dependent manner (Figure. 1C). Moreover, treatment with 10 8 CFU/mL of HDRsEf1 for 2–6 h in a transwell system revealed that HDRsEf1 inhibited CYP3A29 expression in IPEC-J2 cells only when IPEC-J2 cells were co-cultured with HDRsEf1 in the same wells, but not in the separated transwells. Hence, HDRsEf1 down-regulated the expression of intestinal CYP3A29 in vivo and in vitro , dependent on cell-cell contact. HDRsEf1 down-regulates the expression of CYP3A29 at the transcriptional level To investigate the effect of HDRsEf1 on the transcription of CYP3A29, we performed a transcriptional inhibition assay. We found that treatment with DRB, an inhibitor of RNA synthesis, completely abolished the HDRsEf1-down-regulated CYP3A29 transcription in IPEC-J2 cells (Figure. 2A). We further cloned the potential sequences of the CYP3A29 promoter of Sus scrofa into the pGL3-BASIC (Figure. 2B). After transfection, we performed luciferase reporter assays and found that all sequences exhibited varying levels of promoter activities and the highest levels of promoter activity were from the pGL3-3A29-371-transfected cells (Figure. 2B). More importantly, HDRsEf1 treatment significantly mitigated the pGL3-3A29-371 and pGL3-3A29-2007 (the longest sequence)-control luciferase expression following co-culture with IPEC-J2 cells, but not in separated transwell culture (Figure. 2C and D). Together, such data indicated that HDRsEf1 inhibited the transcription activity of the CYP3A29 promoter, dependent on cell-cell contact. HDRsEf1 down-regulates CYP3A29 expression via RXR-α NRs are crucial for the expression of CYPP450s ( 28 ). We found that HDRsEf1 treatment significantly decreased the relative levels of RXR-α, but not PXR, CAR and VDR mRNA transcripts and protein expression (Figure. 3A) and particularly reduced nuclear RXR-α protein levels in IPEC-J2 cells (Figure. 3B). Given that RXR-α often interacts with PXR, VDR and CAR to form heterodimers and regulate the expression of CYP450s ( 12 ) we further tested whether altered their expression could modulate the HDRsEf1-inhibited CYP3A29 expression in IPEC-J2 cells. We found that RXR-α or PXR over-expression increased CYP3A29 expression while HDRsEf1 treatment abrogated or significantly reduced RXR-α, PXR and CYP3A29 expression in IPEC-J2 cells (Figure. 3C and 3D). In contrast, RXR-α or PXR silencing by specific siRNA also significantly decreased the relative levels of CYP3A29 expression in IPEC-J2 cells (Figure. 3E and 3F). However, altered VDR or CAR expression did not affect the expression of CYP3A29 in IPEC-J2 cells (Figure. S1). Interestingly, both RXR-α and PXR over-expression further significantly increased the activity of pGL-3A29-2007 (Figure. 3G) and CYP3A29 protein expression (Fig. 3 H), compared to RXR-α or PXR over-expression in IPEC-J2 cells. Co-IP assay revealed that RXR-α directly interacted with PXR in IPEC-J2 cells (Figure. 3I). Collectively, HDRsEf1 down-regulated CYP3A29 expression, dependent on inhibiting RXR-α expression and its interaction with PXR in IPEC-J2 cells. HDRsEf1 down-regulates RXR-α expression by attenuating the NF-κB signaling The NF-κB activation can significantly up-regulate the expression of RXR-α ( 29 ) while it can be inhibited by some probiotics ( 20 , 30 ). Therefore, we speculated that the down-regulated RXR-α expression by HDRsEf1 may stem from its down-regulation on the NF-κB activation. We found that HDRsEf1 treatment significantly decreased the NF-κ Bp65-driven luciferase expression in IPEC-J2 cells (Figure. 4A). Furthermore, HDRsEf1 treatment significantly reduced NF-kBp65, RXR-α and CYP3A29 expression and NF-kBp65 phosphorylation in IPEC-J2 cells (Figure. 4B-D). A similar pattern of inhibition on RXR-α and CYP3A29 expression and NF-kB activation was achieved by treatment with BAY-7082, an inhibitor of NF-kB, in IPEC-J2 cells (Figure. 4B and 4D). In addition, although the over-expressed NF-kBp65 masked endogenous NF-kBp65 detection in an automated imaging condition the NF-kBp65 over-expression significantly increased RXR-α and CYP3A29 expression, which were significantly mitigated by HDRsEf1 treatment in IPEC-J2 cells (Figure. 4E and 4F). Interestingly, HDRsEf1 failed to modulate significantly the relative levels of CYP3A29 expression in the RXR-α-silenced IPEC-J2 cells (Figure. 4G, 4H and 4I). Thus, HDRsEf1 attenuated the NF-kBp65 activation to inhibit the CYP3A29 and RXR-α expression in IPEC-J2 cells. HDRsEf1 induces A20 to inhibit CYP3A29 expression, dependent on TLR1/2 Engagement of TLRs by Gram-positive bacteria can regulate the NF-κB activity in animal intestinal tissues ( 31 ). Finally, we examined whether and how any of the TLRs participated in the HDRsEf1-decreased CYP3A29 expression in IPEC-J2 cells. We detected TLR1, 2, 4, 5 and 6 gene mRNA transcripts, which were effectively silenced by their specific siRNA in IPEC-J2 cells (Figure. 5A). HDRsEf1 treatment significantly decreased CYP3A29 mRNA transcripts in the TLR4, 5 and 6-silenced and wild-type IPEC-J2 cells, but not in the TLR1 or 2-silenced cells (Figure. 5B). Such data suggest that the down-regulated CYP3A29 expression by HDRsEf1 may depend on the sufficient expression of TLR1/2 in IPEC-J2 cells. Given that engagement of TLRs by some Grams-positive bacteria can enhance the expression of some NF-κB inhibitors, attenuating the NF-kB activation ( 30 ) we further determined the effect of HDRsEf1 on the expression of A20, IRAK-M and Tollip in IPEC-J2 cells. We found that HDRsEf1 treatment significantly increased A20, but not IRAK-M and Tollip, mRNA transcripts and protein expression in IPEC-J2 cells (Figure. 5C). The enhanced A20 mRNA transcripts by HDRsEf1 were abrogated by TLR1 or TLR2 silencing in IPEC-J2 cells, indicating that HDRsEf1 increased A20 expression in a TLR1/2-dependent manner (Figure. 5D). Actually, A20 silencing by specific siRNA increased CYP3A29 and RXR-α expression and abrogated the HDRsEf1-reduced CYP3A29 and RXR-α expression in IPEC-J2 cells (Fig. 5 E and 5 F). In contrast, A20 over-expression decreased CYP3A29 and RXR-α expression (Fig. 5 E and 5 F). Therefore, HDRsEf1 up-regulated A20 to down-regulate CYP3A29 expression in IPEC-J2 cells in a TLR1/2-dependent manner. Discussion Although many people believe that probiotics are highly safe for animals and humans a few people understand the effects of probiotics on the expression of DMEs in the intestine due to intestinal first-pass metabolism. Actually, probiotics can modulate the expression of intestinal DMEs ( 26 , 27 ), but the mechanisms underlying the action of probiotics remain unclear. Our results indicated that HDRsEf1 down-regulated the expression of CYP3A29 in pig intestinal tissues through the TLR1/2-induced A20 expression to attenuate the NF-κB/RXRα pathway. TLRs can recognize bacterial components ( 32 ). TLR2 can interact with TLR1 or TLR6 to form a heterodimer ( 22 ), and recognize Gram-positive bacterial cell wall components, such as peptidoglycan and lipoteichoic acid ( 33 , 34 ). In this study, we found that HDRsEf1 enhanced A20 expression in pig intestinal epithelia cells, dependent on TLR1/2. Given that A20 is an inhibitor of the NF-kB signalling by degrading TRAF-6( 35 ) the enhanced A20 expression by HDRsEf1 should contribute to its inhibition on the NF-kBp65 activation and CYP3A29 expression in IPEC-J2 cells. In fact, we found that HDRsEf1, like the NF-kB inhibitor of BAY11-7082, significantly attenuated the NF-kB activation and RXR-α expression in IPEC-J2 cells. Such data extended previous observations that probiotics, such as Lactobacillus amylovorus and paracasei, can bind to TLR2 to regulate the expression of NF-κB inhibitors, including A20, SOCS1 and SOCS3 in macrophages ( 20 ) , ( 21 ) and support the notion that Gram-positive bacteria can negatively regulate inflammation through the TLR1/2 to induce the expression of the NF-kB inhibitors ( 29 , 30 ). Furthermore, we found that HDRsEf1 significantly reduced the expression of RXR-α and CYP3A29 in IPEC-J2 cells, which were abrogated by NF-kBp65 overexpression, suggesting that the enhanced NF-kBp65 activity may up-regulate RXR-α expression and subsequent CYP3A29 expression. These, together with the fact that HDRsEf1 failed to decrease CYP3A29 expression in the RXR-α-silenced IPEC-J2 cells, indicated that the NF-κBp65 signaling enhanced the CYP3A29 expression indirectly by enhancing RXR-α expression in intestinal tissues. Previous reports indicate that Staphylococcus aureus -derived LTA can reduce RXR-α expression in mouse liver by inhibiting the JNK and NF-κB pathways ( 33 ), while IL-1β enhances RXR-α expression by activating the NF-kB signaling in gastric carcinoma tissues ( 29 ). Apparently, the NF-kB signaling positively regulates the expression of RXRα, which can attenuate the inhibition of NF-kB activation ( 36 ), maintaining the balance of intracellular inflammatory and anti-inflammatory responses. Interestingly, we found that HDRsEf1 did not modulate PXR expression in IPEC-J2 cells. These data were in disagreement with previous reports that the phosphorylated NF-κBp65 can down-regulate PXR expression and inhibit the formation of PXR/RXR-α heterodimers and CYP3A4 transcription in HepG2 cells ( 37 ). The discrepancy suggests that the activated NF-κBp65 may have different regulatory roles in the expression of NRs in different types of cells. Given that the activated NF-κB usually regulates the transcription of the NRs ( 16 ) , ( 38 ) we are interested in further investigating how the activated NF-κBp65 regulates RXR-α expression in pig intestinal tissues. In this study, we found that HDRsEf1 significantly reduced RXRα expression, particularly for nuclear RXRα protein levels, but did not affect the expression of other NRs tested in IPEC-J2 cells. Furthermore, RXRα or PXR overexpression and silencing significantly modulated CYP3A29 expression in IPEC-J2 cells and both RXR-α and PXR over-expression synergistically enhanced the CYP3A29 promoter activity. In addition, RXR-α directly interacted with PXR in IPEC-J2 cells. Together, such data suggest that RXR-α and PXR may form heterodimer for the CYP3A29 transcription in IPEC-J2 cells, consistent with our findings in HepLi cells( 16 ). However, we found that altered VDR or CAR expression did not affect the CYP3A29 expression in IPEC-J2 cells, implicating that the VDR/RXR-α and CAR/RXR-α might not be involved in up-regulating CYP3A29 expression although they are crucial for CYP3A4 transcription in human intestinal cells and HepG2( 13 , 15 ). Therefore, the NRs may have interspecies difference in regulating the transcription of CYP3As even if there are organ variants in the same species. We found that HDRsEf1 down-regulated the expression of CYP3A29 in IPEC-J2 in a time- and dose-dependent manner. Although HDRsEf1 supernatants have been reported to mitigate the ETEC K88ac-up-regulated IL-8 expression( 39 ), we found that HDRsEf1 inhibited the CYP3A20 expression in IPEC-J2 cells, dependent on cell-cell contact. Our findings were consistent with a recent report that Lactobacillus helveticus SBT2171 cell wall components inhibit inflammatory responses in peritoneal macrophage ( 20 ). The difference between our and those with ETEC K88ac may stem from the presence of more pathogen-associated molecular patterns on the cell wall of Gram-positive bacteria. We will continually explore which cell wall components of HDRsEf1 can bind to TLR1/2 on intestinal epithelial cells to inhibit CYP3A29 expression. Our data indicated that HDRsEf1 inhibited the CYP3A29 expression in pig intestinal tissues and IPEC-J2 cells in a dose-, time- and cell-cell contact-dependent manner. HDRsEf1 through TLR1/2-induced A20 expression attenuated the NF-kBp65 activation to reduce RXR-α expression and limit the formation of RXR-α/PXR heterodimer, inhibiting CYP3A29 transcription in intestinal epithelial cells (Figure. 6). Our findings may provide new insights into the mechanisms by which probiotics regulate the expression of CYP450s and highlight the possible risks of probiotics for oral drug overdose in the clinic. Materials and methods Special chemicals, and reagents The special reagents included Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin and Lipofectamine 2000 transfection reagent (Gibco, Life Technologies, Grand Island, NY, USA); 5,6-Dichlororibosidyl-benzimidazole (DRB) and BAY11-7082 (Sigma-Aldrich, St. Louis, MO, USA); monoclonal antibodies against CYP3A29, PXR, RXR-α, CAR and VDR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), NF-κBp65, Phospho-NF-κB p65 (Cell Signaling Technology, Beverly, MA, USA), β-actin and HRP-conjugated secondary antibodies (ABclonal Technology, Wuhan, China); and the dual-luciferase reporter assay system (Promega, Madison, WI, USA). Bacterial preparations, cell culture and treatment Enterococcus faecium HDRsEf1 is novel probiotic strain (CCTCC NO: M2011031) ( 25 ). The HDRsEf1 bacteria were grown in De Man Rogosa Sharpe (MRS) media (Difco, Sparks, USA) at 37°C for 18 h, and collected by centrifugation. After being washed twice with phosphate buffered saline (PBS), the bacteria were re-suspended in antibiotic-free DMEM at 1×10 8 colony forming units (CFU) ml-1. Porcine small intestinal epithelial IPEC-J2 cells were obtained from BeNa Culture Collection (Beijing, China), and cultured in 10% FBS DMEM. To determine the effect of HDRsEf1, IPEC-J2 cells (2×10 5 cells/well) were challenged in triplicate with vehicle or 10 6 , 10 7 , or 10 8 CFU/mL HDRsEf1 for 2, 4 or 6 h, respectively. Furthermore, IPEC-J2 cells (2×10 5 cells) were cultured in the bottom chambers of 12-well transwell plates (0.2-µm-pore-size, Corning, NY, USA) and HDRsEf1 cells (10 8 CFU/well)were cultured in the upper chamber to determine the cell-cell contact independence. Their cell viability was quantified by Cell Counting Kit-8 assay (Biosharp, Hefei, China) and CYP3A29 expression were examined by qRT-PCR and Western blot. In addition, IPEC-J2 cells were treated with 10 8 CFU/mL HDRsEf1 in the presence or absence of 5 µM DRB and/or 10 µM BAY11-7082 and the relative levels of CYP3A29 mRNA transcripts were quantified. Animals and procedures Female (Landrace×Large White) pigs at an age of 30 days with similar body weights were obtained from COFCO and randomly fed with the control corn and soybean meal-based diet ( 40 ) or the control diet containing HDRsEf1 (10 6 CFU/g) through a feeder for 30 days. Some pigs (4 each group) were randomly selected, anesthetized and sacrificed by jugular puncture ( 41 ). Their segmented jejunal tissues at the approximately middle intestine were collected for subsequent experiments. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University. Quantitative Real-Time PCR (qRT-PCR) The relative levels of target gene to the control β-actin mRNA transcripts were determined by qRT-PCR using specific primers (Table S1 ) and iQ™ SYBR Green PCR Supermix in the Bio-Rad CXF real-time PCR detection system (Bio-Rad, CA, USA) after we extracted total RNA from individual groups of cells with the TRIzol reagent (Invitrogen) and reversely transcribed (RT) into cDNA using the Superscript reverse transcriptase (Takara, Otsu, Japan). The data were analyzed by 2-ΔΔCt method. Western blotting We extracted the jejunal tissue proteins as our previously described (Yan et al., 2018) and the proteins from individual groups of cells after lyzing IPEC-J2 cells with RIPA Buffer (Pierce, Rockford, IL, USA). Similarly, we also extracted nuclear and cytoplasmic proteins from individual groups of cells using specific extraction kit (EpiZyme, Shanghai, China). The protein samples (20 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis on 10% gels and transferred onto polyvinylidene fluoride membranes. After being blocked with 5% non-fat dry milk in TBST, the membrane was incubated with primary antibodies at 4℃ overnight and reacted with secondary antibodies, followed by visualizing using ECL chemiluminescence system (Bio-Rad). Transfection and luciferase assay IPEC-J2 cells were maintained in Opti-MEM medium until 80% of confluence and transfected with pGL3-CYP3A29 that contained the CYP3A29 promoter or the NF-kBp65 promoter using Lipofectamine 2000 for 18 h. Furthermore, IPEC-J2 cells were co-transfected with pGL3-3A29-2007 and plasmid for Renilla luciferase expression for 18 h. The cells were treated with, or without, 108 CFU/mL HDRsEf1 for 6 h and the CYP3A29 promoter activity was measured using the Dual-Luciferase Reporter Assay System. In addition, IPEC-J2 cells were transfected with a control or plasmid for RXR-α, PXR, VDR or CDR over-expression for 18 h and treated with, or without, 108 CFU/mL HDRsEf1 for 6 h. The impact of RXR-α, PXR, VDR or CDR over-expression on CYP3A29 expression was determined. RNA Interference IPEC-J2 cells were cultured in 12-well plates overnight and transfected with 40 nM control scramble or gene-specific siRNA using Lipofectamine 2000 for 36 h. The cells were treated with, or without, 10 8 CFU/mL HDRsEf1 for 6 h. The efficacy of specific gene silencing and its impact on the expression of other genes were quantified by qRT-PCR and Western blot. Co-immunoprecipitation (Co-IP) The physical association between PXR and RXR-α was characterized by Co-IP using SureBeads™ Starter Kit Protein G (Bio-Rad) (Xie et al., 2019). Briefly, RXR-α, PXR and specific antibody IgG or control isotype IgG (10 µg each) were reacted and the formed immunocomplex was precipitated with the microbeads, followed by eluting the proteins with laemmli buffer for subsequent Western blot analysis. Statistical analysis Data are present as mean ± S.D. Comparison was performed by Student’s t-test, one-way ANOVA, or two-way ANOVA. Significant difference was defined when a P-value of < 0.05. Declarations Conflict of interest The authors have no fnancial confict of interest. Author Contribution Y.G., Y.H. and X.W. designed the research. Y.G., Y.H., Y.L., and X.J. performed the research. Y.G., Y.W., D.S., and X.W. analyzed the data. Y.G., Y.H., D.S., and X.W wrote the paper with the help of all authors. All authors read and approved the final version of the manuscript. Acknowledgements This work was supported by the National Key Research and Development Program of China (2017YFD0501000) and the Fundamental Research Funds for Central Universities (2662019PY061). Data availability The data that support the fndings of this study are available from the corresponding author on reasonable request. References van Herwaarden AE, van Waterschoot RA, Schinkel AH.2009. How important is intestinal cytochrome P450 3A metabolism? Trends Pharmacol Sci 30:223-7. van Waterschoot RA, Rooswinkel RW, Wagenaar E, van der Kruijssen CM, van Herwaarden AE, Schinkel AH.2009. Intestinal cytochrome P450 3A plays an important role in the regulation of detoxifying systems in the liver. Faseb j 23:224-31. Marchesi JR, Adams DH, Fava F, Hermes GD, Hirschfield GM, Hold G, Quraishi MN, Kinross J, Smidt H, Tuohy KM, Thomas LV, Zoetendal EG, Hart A.2016. The gut microbiota and host health: a new clinical frontier. Gut 65:330-9. Guengerich FP.1997. 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Regulation of gene expression of hepatic drug metabolizing enzymes and transporters by the Toll-like receptor 2 ligand, lipoteichoic acid. Arch Biochem Biophys 481:123-30. Asong J, Wolfert MA, Maiti KK, Miller D, Boons GJ.2009. Binding and Cellular Activation Studies Reveal That Toll-like Receptor 2 Can Differentially Recognize Peptidoglycan from Gram-positive and Gram-negative Bacteria. J Biol Chem 284:8643-53. Shembade N, Ma A, Harhaj EW.2010. Inhibition of NF-kappaB signaling by A20 through disruption of ubiquitin enzyme complexes. Science 327:1135-9. Ning RB, Zhu J, Chai DJ, Xu CS, Xie H, Lin XY, Zeng JZ, Lin JX.2013. RXR agonists inhibit high glucose-induced upregulation of inflammation by suppressing activation of the NADPH oxidase-nuclear factor-kappaB pathway in human endothelial cells. Genet Mol Res 12:6692-707. Gu X, Ke S, Liu D, Sheng T, Thomas PE, Rabson AB, Gallo MA, Xie W, Tian Y.2006. Role of NF-kappaB in regulation of PXR-mediated gene expression: a mechanism for the suppression of cytochrome P-450 3A4 by proinflammatory agents. J Biol Chem 281:17882-9. Vogel CF, Khan EM, Leung PS, Gershwin ME, Chang WL, Wu D, Haarmann-Stemmann T, Hoffmann A, Denison MS.2014. Cross-talk between aryl hydrocarbon receptor and the inflammatory response: a role for nuclear factor-kappaB. J Biol Chem 289:1866-75. Tian Z, Liu X, Dai R, Xiao Y, Wang X, Bi D, Shi D.2016. Enterococcus faecium HDRsEf1 Protects the Intestinal Epithelium and Attenuates ETEC-Induced IL-8 Secretion in Enterocytes. 2016:7474306. Hu J, Ma L, Nie Y, Chen J, Zheng W, Wang X, Xie C, Zheng Z, Wang Z, Yang T, Shi M, Chen L, Hou Q, Niu Y, Xu X, Zhu Y, Zhang Y, Wei H, Yan X.2018. A Microbiota-Derived Bacteriocin Targets the Host to Confer Diarrhea Resistance in Early-Weaned Piglets. Cell Host Microbe 24:817-832.e8. Hu J, Chen L, Zheng W, Shi M, Liu L, Xie C, Wang X, Niu Y, Hou Q, Xu X, Xu B, Tang Y, Zhou S, Yan Y, Yang T, Ma L, Yan X.2018. Lactobacillus frumenti Facilitates Intestinal Epithelial Barrier Function Maintenance in Early-Weaned Piglets. Front Microbiol 9:897. Additional Declarations No competing interests reported. Supplementary Files FigureS1.tif Figure S1. IPEC-J2 cells were transfected with control or pcDNA-VDR (A) or pcDNA-CAR (B) for 18 h and treated with 10 8 CFU/mL HDRsEf1 for 6 h. Furthermore, IPEC-J2 cells were transfected with control or VDR-specific (C) or CAR-specific siRNA (D) for 36 h. The relative levels of CYP3A29, VDR and CAR expression were quantified by qRT-PCR and Western blotting. Data are representative images or expressed as the mean ± SD of each group from three separate experiments. *p<0.05; **p<0.01; ***p<0.001. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4192677","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285965447,"identity":"a3a68d81-29f1-4d17-924a-8a640eeb0bdd","order_by":0,"name":"Yinghui Gong","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yinghui","middleName":"","lastName":"Gong","suffix":""},{"id":285965448,"identity":"83a613dc-68b1-4b77-b92e-3b41a1da8f19","order_by":1,"name":"Yucheng He","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yucheng","middleName":"","lastName":"He","suffix":""},{"id":285965449,"identity":"0b2e0611-b070-4b02-af82-1ee4aa3326e5","order_by":2,"name":"Yue Li","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Li","suffix":""},{"id":285965450,"identity":"63aa1705-4f3c-4edc-ad6c-ac01e33392e4","order_by":3,"name":"Ying Wang","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Wang","suffix":""},{"id":285965451,"identity":"6be841ba-b4fa-4c9b-9e85-f4b4a59e964c","order_by":4,"name":"Xiue Jin","email":"","orcid":"","institution":"Hubei Provincial Institute of Veterinary Drug Control","correspondingAuthor":false,"prefix":"","firstName":"Xiue","middleName":"","lastName":"Jin","suffix":""},{"id":285965452,"identity":"b058acb6-f83b-42c6-8d8c-a5ec42ff4bd5","order_by":5,"name":"Deshi Shi","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Deshi","middleName":"","lastName":"Shi","suffix":""},{"id":285965453,"identity":"22aef5a7-ca20-421f-a6eb-9bad3a0036fe","order_by":6,"name":"Xiliang Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYFAC5obDfwxsgDRzAzNUyICAFsbGBzwVaUAtjMRraTbgOXMYxCBSi8GNxDYJybbz0fztQC0Ff+wSG9ibt0kw1NzBreXMwTYJw7bbuTMOA7XM4ElObOA5VibBcOwZTi1mxxvbJBKBWhpAWngkmBMbJHLMJBgbDuPWcpixTeJg27nc+WAtBvWJDfJvCGg53ths2HDmQO4GsJaEw0BbePBrsT9zsPExQ0Vy7kaglsM8B44bt/GkFVskHMOtRXJG8oHDDAZ2ufPOHz74mOdPtWw/++GNNz7U4NaCAg6ACDYQkUCchlEwCkbBKBgFOAAAiAhYML1hctsAAAAASUVORK5CYII=","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xiliang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-03-30 14:51:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4192677/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4192677/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54039034,"identity":"88acb4ed-3879-4b6e-9660-91ce8ed2a581","added_by":"auto","created_at":"2024-04-03 17:19:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":610097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDRsEf1 reduces CYP3A29 gene expression\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e(A) Western blot analysis of CYP3A29 protein expression in pig jejunum tissues (n = 4 per group). (B) CCK-8 assay analysis of IPEC-J2 cell viability after treatment with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 2, 4, 6, 8, 10 and 12 h. (C) Quantitative RT-PCR analysis of CYP3A29 mRNA transcripts in IPEC-J2 cells after treatment with the indicated doses of HDRsEf1 for varying periods. (D) HDRsEf1 decreased CYP3A29 expression in IPEC-J2 cells in a cell-cell contact-dependent manner. IPEC-J2 cells were co-cultured with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 in the same wells or separately cultured in transwell plates (separate) for the indicated time periods. The relative levels of CYP3A29 mRNA transcripts and protein expression in individual groups of cells were quantified by qRT-PCR and Western blotting. Data are representative images or expressed as the mean ± SD of each group from three separate experiments. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4192677/v1/f27525664886fe8a5141aac1.jpg"},{"id":54040071,"identity":"1d1f0ab7-f9b2-4e86-9fd3-170d9f3ac86f","added_by":"auto","created_at":"2024-04-03 17:27:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":479550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDRsEf1 down-regulates the CYP3A29 transcription in IPEC-J2 cells. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eIPEC-J2 cells were treated with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h in the absence or presence of 5 μM DRB and the relative levels of CYP3A29 mRNA transcripts were quantified by qRT-PCR. (B) IPEC-J2 cells were transiently transfected with the indicated plasmids containing the sequences of the CYP3A29 promoter and one day later, their promoter activities were determined by dual luciferase reporter assays. (C, D) IPEC-J2 cells were transiently transfected with pGL3-3A29-371 (C) and pGL3-3A29-2007 (D), and 18 h later, the transfected cells were co-cultured or separated culture with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h, followed by detecting luciferase activity.\u003c/p\u003e\n\u003cp\u003eData are expressed as the mean ± SD of each group from three separate experiments.*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4192677/v1/f55a061868209638879b91f7.jpg"},{"id":54039038,"identity":"ea183d4b-ffe1-4fa3-b3d3-ee51d23f074f","added_by":"auto","created_at":"2024-04-03 17:19:31","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1046933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDRsEf1 down-regulates CYP3A29 expression by reducing RXR-α expression in IPEC-J2 cells. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eIPEC-J2 cells were treated with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h, and the relative levels of RXR-α, PXR, VDR and CAR mRNA transcripts and protein expression were quantified by qRT-PCR and Western blotting. (B) IPEC-J2 cells were treated with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h and their cytoplasmic and nuclear levels of RXR-α were quantified by Western blotting. (C, D) IPEC-J2 cells were transfected with plasmid for RXR-α (C) or PXR (D) over-expression or control vector for 18 h and treated with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h. The relative levels of CYP3A29, RXR-α or PXR mRNA transcripts and protein expression were quantified by qRT-PCR and Western blotting. (E, F) IPEC-J2 cells were transfected with control or\u003cstrong\u003e \u003c/strong\u003eRXR-α-specific (E) or PXR-specific siRNA (F) for 36 h. The relative levels of CYP3A29, RXR-α or PXR mRNA transcripts and protein expression were quantified by qRT-PCR and Western blotting. (G) IPEC-J2 cells were co-transfected with the pGL3-3A29-2007, the plasmid for Renilla luciferase expression, and pcDNA-RXR-α and/or pcDNA-PXR. The luciferase activity in each group of cells was determined by dual luciferase reporter assays. (H) IPEC-J2 cells were transfected with plasmid for RXR-αand/or PXR over-expression or control vector for 24 h, CYP3A29 protein expression were quantified by Western blotting. (I) Co-IP analysis of RXR-α and/or PXR interaction. Data are representative images or expressed as the mean ± SD of each group from three separate experiments. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4192677/v1/71ea40f7f0416f159bf96d5b.jpg"},{"id":54039042,"identity":"3ab0650d-6e90-40b2-af62-9d0210afbb5b","added_by":"auto","created_at":"2024-04-03 17:19:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":883175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDRsEf1 down-regulates RXR-α expression by inhibiting the NF-κB activation.\u003c/strong\u003e (A) IPEC-J2 cells were transfected with the plasmids containing the NF-κB promoter-driven luciferase and Renilla luciferase expression for 24 h. The cells were treated with, or without, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1, and 6 h later, their luciferase activity was examined. (B, D) IPEC-J2 cells were treated with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 and/or 10 μM BAY11-7082 for 6 h, and the relative levels of CYP3A29, RXR-α, NF-kBp65 expression and NF-kBp65 phosphorylation were quantified by Western blotting (B) and qRT-PCR (D). (C) Quantification of phosphorylated NF-kBp65 protein levels standardized to NF-kBp65. (E, F) IPEC-J2 cells were transfected with the plasmid for NF-κBp65 overexpression or control vector for 18 h, and treated with, or without, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h.\u0026nbsp; The relative levels of CYP3A29, RXR-α, NF-kBp65 expression and NF-kBp-p65 phosphorylation were quantified by Western blotting (E) and qRT-PCR (F). (G, H) IPEC-J2 cells were transfected with 20 μM\u003cstrong\u003e \u003c/strong\u003econtrol or\u003cstrong\u003e \u003c/strong\u003eRXR-α-specific siRNA for 30 h, and treated with, or without, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h. The relative levels of CYP3A29 expression were quantified by qRT-PCR and Western blotting. (I) Quantification of CYP3A29 protein levels standardized to β-actin. Data are representative images or expressed as the mean ± SD of each group from three separate experiments. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4192677/v1/4e8ced5f90731d6449c02ca5.jpg"},{"id":54039041,"identity":"b205e761-34d5-4e8b-86a4-78b3a1e857f3","added_by":"auto","created_at":"2024-04-03 17:19:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":842878,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDRsEf1 induces A20 and inhibits CYP3A29 expression in IPEC-J2 cells, dependent on TLR1/2. \u003c/strong\u003e(A) IPEC-J2 cells were transfected with control or TLR 1/2/4/5/6 specific siRNA for 36 h. The RNA interference efficiency was determined by qRT-PCR. (B) IPEC-J2 cells were transfected with control or TLR1/2/4/5/6 specific siRNA for 30 h, and treated with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h. The relative levels of CYP3A29 mRNA transcripts were quantified by qRT-PCR. (C) IPEC-J2 cells were treated with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h and the relative levels of A20, IRAK-M and Tollip mRNA transcripts were quantified by qRT-PCR. (D) IPEC-J2 cells were transfected with control or TLR1/2 specific siRNAs for 30 h, and treated with, or without, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h. The relative levels of CYP3A29 mRNA transcripts were quantified by qRT-PCR. (E, F) IPEC-J2 cells were transfected with 20 μM control or A20-specific siRNA for 30 h, and treated with, or without, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h. The relative levels of CYP3A29 expression were quantified by qRT-PCR and Western blotting. (G, H) IPEC-J2 cells were transfected with the plasmid for A20 over-expression or control vector for 24 h. The relative levels of CYP3A29, RXR-α and A20 expression were quantified by qRT-PCR and Western blotting. Data are expressed as the mean ± SD of each group from three separate experiments. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4192677/v1/e3687c4fb7d531473f22567b.jpg"},{"id":54039040,"identity":"afa6ba5e-7740-4c99-970e-472202fd1f42","added_by":"auto","created_at":"2024-04-03 17:19:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1022976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the possible mechanisms underlying the suppression of HDRsEf1 on CYP3A29 expression in the intestinal tissues. \u003c/strong\u003eHDRsEf1 binds to TLR1/2 to up-regulate A20 expression, which inhibits the NF-κB activation by degrading TRIF6 and down-regulates RXR-α expression to reduce the RXR-α/PXR heterodimer formation and CYP3A29 expression.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4192677/v1/62518997010412e2f695c61d.jpg"},{"id":54530480,"identity":"5add009b-0c6e-4013-96e9-0294ac4358d1","added_by":"auto","created_at":"2024-04-12 00:07:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1177640,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4192677/v1/f56c8940-0ffd-46bb-83c4-ad624371c2f1.pdf"},{"id":54039036,"identity":"4c1fd582-62fa-4ec9-b7f5-d4b1ed147496","added_by":"auto","created_at":"2024-04-03 17:19:31","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":690856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1.\u003c/strong\u003e IPEC-J2 cells were transfected with control or pcDNA-VDR (A) or pcDNA-CAR (B) for 18 h and treated with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL HDRsEf1 for 6 h. Furthermore, IPEC-J2 cells were transfected with control or VDR-specific (C) or CAR-specific siRNA (D) for 36 h. The relative levels of CYP3A29, VDR and CAR expression were quantified by qRT-PCR and Western blotting. Data are representative images or expressed as the mean ± SD of each group from three separate experiments. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4192677/v1/6c53252d64113b7ba2236f61.tif"},{"id":54039035,"identity":"23db88c5-3905-43f3-8332-11162ad11d78","added_by":"auto","created_at":"2024-04-03 17:19:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18388,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4192677/v1/0eeb1ff451b4b3e15416b57e.docx"},{"id":54039039,"identity":"09cc0267-da92-42c1-9b43-c1c81f292031","added_by":"auto","created_at":"2024-04-03 17:19:31","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15713,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4192677/v1/96ffd1563098ebf3d20dbd1b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEnterococcus faecium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e HDRsEf1 Represses CYP3A29 Expression in the Intestine through the TLR1/2-induced A20 to attenuate the NF-κB/RXR-α Signaling\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDrug metabolism in the intestine or intestinal first-pass metabolism can cause low bioavailability of some oral drugs, which depends on the contents of relevant enzymes in the gastrointestinal lumen and epithelium as well as bacterial enzymes (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Probiotics can modulate gut microbiota and regulate immune responses, benefiting patients with intestinal diseases, diabetes, tumors and obesity (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Probiotics have been widely used in humans and animals, and probiotics may alter the expression of intestinal drug-metabolizing enzymes (DMEs) to change the bioavailability of some oral drugs. However, the effect of probiotics on the expression of intestinal DMEs and their mechanisms have not been clarified. Accordingly, it is important to understand the effect and mechanisms of probiotics on the expression of intestinal DMEs.\u003c/p\u003e \u003cp\u003eMammalian cytochrome P450s (CYP450s) are crucial for the oxidative metabolism of xenobiotics, including therapeutic drugs, environmental carcinogens and toxins(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). CYP3A4, one of the DMEs in humans, can metabolize\u0026thinsp;\u0026gt;\u0026thinsp;50% of clinical drugs (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Pigs have a high similarity in physiology and anatomy to humans and become an important animal model for evaluating new drugs (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Porcine CYP3A29 accounts for 30% of total CYP proteins and the major CYP3A activity in pig liver microsomes (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Pig CYP3A29 and human CYP3A4 have similar tissue distribution, pharmacokinetic characteristics and regulatory mechanisms, and pig CYP3A29 is a suitable model for research of human CYP3A4 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe nuclear receptors (NRs), such as retinoid X receptor alpha (RXR-α), pregnane X receptor (PXR), vitamin D receptor (VDR) and the constitutive androstane receptor (CAR), are important for regulating the expression of CYP450s(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The RXR-α can directly interact with PXR, VDR or CAR to form heterodimers and bind to the CYP450s gene promoter to regulate their expression(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). For example, the VDR/RXR-α heterodimer can bind to the CLEM4-ER6 motif to regulate the expression of CYP3A4(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), and the CAR/RXRα and PXR/RXR-α heterodimers can interact with pER6 and dXREM to regulate the expression of CYP3A4, respectively (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Our previous study has shown that the PXR/RXR-α can bind to the CYP3A29 promoter to induce its expression in porcine liver HepLi cells (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Apparently, RXR-α expression is critical for the expression of CYP3A in the digestive system.\u003c/p\u003e \u003cp\u003eThe nuclear factor-kappa B (NF-κB) signaling is crucial for inflammation, immunity, cell proliferation and apoptosis(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). It can regulate the expression of CYP450s by directly binding to the CYP450 promoters, or indirectly regulate the expression of NRs (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In addition, the NF-κB can also enhance the activity of CYP450 at the post-transcriptional level by stabilizing CYP450 proteins (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Interestingly, previous studies have revealed that some probiotics can inhibit the NF-κB activity (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). However, whether and how probiotics could modulate the expression of CYP450s, such as CYP3A29 in pig intestinal tissues, have not been clarified.\u003c/p\u003e \u003cp\u003eToll-like receptors (TLRs) act as a type of pattern recognition receptors (PRR) in the innate immune system and TLRs can recognize microbial components to initiate immune responses. TLR2 can directly interact with TLR1 or TLR6 to form a heterodimer and recognize conserved molecular patterns (such as peptidoglycan, lipoteichoic acid, etc.) on the cell wall of Gram-positive bacteria(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Engagement of TLR2 by the cell wall components of some probiotics can up-regulate the expression of some NF-κB inhibitors, such as A20 (tumor necrosis factor-α-induced protein 3), IRAK-M and Tollip, to inhibit inflammation(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Actually, A20 is a deubiquitinating enzyme and can inhibit the NF-kB activation (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Our previous study indicates that \u003cem\u003eEnterococcus faecium\u003c/em\u003e HDRsEf1 enhances the expression of occluding, the intestinal tight junction protein, by activating the TLR2 (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Accordingly, we hypothesize that HDRsEf1 can modulate the expression of CYP3A29 in pig intestinal tissues by modulating the NB-κB signaling.\u003c/p\u003e \u003cp\u003eIn this study, we explored the effect of HDRsEf1 on CYP3A29 expression and potential mechanisms in pig intestinal tissues and epithelial IPEC-J2 cells. We found that HDRsEf1 decreased the expression of CYP3A29 in pig intestinal tissues by activating the TLR2 to up-regulate A20 expression, inhibiting the NF-κB activation to attenuate RXR-α expression.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eHDRsEf1 reduces CYP3A29 expression\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrevious reports have shown that probiotics can regulate the expression of CYP450s in the intestinal tract of animals (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Because CYP3A29 is an important enzyme in the intestine of pigs, we examined whether HDRsEf1 could modulate the CYP3A29 expression in pig intestinal tissues. After fed with HDRsEf1-contained diet for 30 days, we found that the relative levels of CYP3A29 expression in the jejunum tissues of the probiotic group of pigs were significantly lower than that in the control group (Figure, 1A). Furthermore, treatment with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 6 h did not alter the viability of IPEC-J2, but treatment with HDRsEf1 for a longer time period obviously decreased the viability of IPEC-J2 cells (Figure. 1B). In addition, treatment with 10\u003csup\u003e6\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e and 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1for 2\u0026ndash;6 h down-regulated CYP3A29 mRNA transcripts in IPEC-J2 cells in a dose- and time-dependent manner (Figure. 1C). Moreover, treatment with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL of HDRsEf1 for 2\u0026ndash;6 h in a transwell system revealed that HDRsEf1 inhibited CYP3A29 expression in IPEC-J2 cells only when IPEC-J2 cells were co-cultured with HDRsEf1 in the same wells, but not in the separated transwells. Hence, HDRsEf1 down-regulated the expression of intestinal CYP3A29 \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, dependent on cell-cell contact.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHDRsEf1 down-regulates the expression of CYP3A29 at the transcriptional level\u003c/h2\u003e \u003cp\u003eTo investigate the effect of HDRsEf1 on the transcription of CYP3A29, we performed a transcriptional inhibition assay. We found that treatment with DRB, an inhibitor of RNA synthesis, completely abolished the HDRsEf1-down-regulated CYP3A29 transcription in IPEC-J2 cells (Figure. 2A). We further cloned the potential sequences of the CYP3A29 promoter of \u003cem\u003eSus scrofa\u003c/em\u003e into the pGL3-BASIC (Figure. 2B). After transfection, we performed luciferase reporter assays and found that all sequences exhibited varying levels of promoter activities and the highest levels of promoter activity were from the pGL3-3A29-371-transfected cells (Figure. 2B). More importantly, HDRsEf1 treatment significantly mitigated the pGL3-3A29-371 and pGL3-3A29-2007 (the longest sequence)-control luciferase expression following co-culture with IPEC-J2 cells, but not in separated transwell culture (Figure. 2C and D). Together, such data indicated that HDRsEf1 inhibited the transcription activity of the CYP3A29 promoter, dependent on cell-cell contact.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHDRsEf1 down-regulates CYP3A29 expression via RXR-α\u003c/h2\u003e \u003cp\u003eNRs are crucial for the expression of CYPP450s (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). We found that HDRsEf1 treatment significantly decreased the relative levels of RXR-α, but not PXR, CAR and VDR mRNA transcripts and protein expression (Figure. 3A) and particularly reduced nuclear RXR-α protein levels in IPEC-J2 cells (Figure. 3B). Given that RXR-α often interacts with PXR, VDR and CAR to form heterodimers and regulate the expression of CYP450s (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) we further tested whether altered their expression could modulate the HDRsEf1-inhibited CYP3A29 expression in IPEC-J2 cells. We found that RXR-α or PXR over-expression increased CYP3A29 expression while HDRsEf1 treatment abrogated or significantly reduced RXR-α, PXR and CYP3A29 expression in IPEC-J2 cells (Figure. 3C and 3D). In contrast, RXR-α or PXR silencing by specific siRNA also significantly decreased the relative levels of CYP3A29 expression in IPEC-J2 cells (Figure. 3E and 3F). However, altered VDR or CAR expression did not affect the expression of CYP3A29 in IPEC-J2 cells (Figure. S1). Interestingly, both RXR-α and PXR over-expression further significantly increased the activity of pGL-3A29-2007 (Figure. 3G) and CYP3A29 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), compared to RXR-α or PXR over-expression in IPEC-J2 cells. Co-IP assay revealed that RXR-α directly interacted with PXR in IPEC-J2 cells (Figure. 3I). Collectively, HDRsEf1 down-regulated CYP3A29 expression, dependent on inhibiting RXR-α expression and its interaction with PXR in IPEC-J2 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHDRsEf1 down-regulates RXR-α expression by attenuating the NF-κB signaling\u003c/h2\u003e \u003cp\u003eThe NF-κB activation can significantly up-regulate the expression of RXR-α (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) while it can be inhibited by some probiotics (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Therefore, we speculated that the down-regulated RXR-α expression by HDRsEf1 may stem from its down-regulation on the NF-κB activation. We found that HDRsEf1 treatment significantly decreased the NF-κ Bp65-driven luciferase expression in IPEC-J2 cells (Figure. 4A). Furthermore, HDRsEf1 treatment significantly reduced NF-kBp65, RXR-α and CYP3A29 expression and NF-kBp65 phosphorylation in IPEC-J2 cells (Figure. 4B-D). A similar pattern of inhibition on RXR-α and CYP3A29 expression and NF-kB activation was achieved by treatment with BAY-7082, an inhibitor of NF-kB, in IPEC-J2 cells (Figure. 4B and 4D). In addition, although the over-expressed NF-kBp65 masked endogenous NF-kBp65 detection in an automated imaging condition the NF-kBp65 over-expression significantly increased RXR-α and CYP3A29 expression, which were significantly mitigated by HDRsEf1 treatment in IPEC-J2 cells (Figure. 4E and 4F). Interestingly, HDRsEf1 failed to modulate significantly the relative levels of CYP3A29 expression in the RXR-α-silenced IPEC-J2 cells (Figure. 4G, 4H and 4I). Thus, HDRsEf1 attenuated the NF-kBp65 activation to inhibit the CYP3A29 and RXR-α expression in IPEC-J2 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHDRsEf1 induces A20 to inhibit CYP3A29 expression, dependent on TLR1/2\u003c/h2\u003e \u003cp\u003eEngagement of TLRs by Gram-positive bacteria can regulate the NF-κB activity in animal intestinal tissues (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Finally, we examined whether and how any of the TLRs participated in the HDRsEf1-decreased CYP3A29 expression in IPEC-J2 cells. We detected TLR1, 2, 4, 5 and 6 gene mRNA transcripts, which were effectively silenced by their specific siRNA in IPEC-J2 cells (Figure. 5A). HDRsEf1 treatment significantly decreased CYP3A29 mRNA transcripts in the TLR4, 5 and 6-silenced and wild-type IPEC-J2 cells, but not in the TLR1 or 2-silenced cells (Figure. 5B). Such data suggest that the down-regulated CYP3A29 expression by HDRsEf1 may depend on the sufficient expression of TLR1/2 in IPEC-J2 cells.\u003c/p\u003e \u003cp\u003eGiven that engagement of TLRs by some Grams-positive bacteria can enhance the expression of some NF-κB inhibitors, attenuating the NF-kB activation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) we further determined the effect of HDRsEf1 on the expression of A20, IRAK-M and Tollip in IPEC-J2 cells. We found that HDRsEf1 treatment significantly increased A20, but not IRAK-M and Tollip, mRNA transcripts and protein expression in IPEC-J2 cells (Figure. 5C). The enhanced A20 mRNA transcripts by HDRsEf1 were abrogated by TLR1 or TLR2 silencing in IPEC-J2 cells, indicating that HDRsEf1 increased A20 expression in a TLR1/2-dependent manner (Figure. 5D). Actually, A20 silencing by specific siRNA increased CYP3A29 and RXR-α expression and abrogated the HDRsEf1-reduced CYP3A29 and RXR-α expression in IPEC-J2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In contrast, A20 over-expression decreased CYP3A29 and RXR-α expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Therefore, HDRsEf1 up-regulated A20 to down-regulate CYP3A29 expression in IPEC-J2 cells in a TLR1/2-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough many people believe that probiotics are highly safe for animals and humans a few people understand the effects of probiotics on the expression of DMEs in the intestine due to intestinal first-pass metabolism. Actually, probiotics can modulate the expression of intestinal DMEs (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), but the mechanisms underlying the action of probiotics remain unclear. Our results indicated that HDRsEf1 down-regulated the expression of CYP3A29 in pig intestinal tissues through the TLR1/2-induced A20 expression to attenuate the NF-κB/RXRα pathway.\u003c/p\u003e \u003cp\u003eTLRs can recognize bacterial components (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). TLR2 can interact with TLR1 or TLR6 to form a heterodimer (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), and recognize Gram-positive bacterial cell wall components, such as peptidoglycan and lipoteichoic acid (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In this study, we found that HDRsEf1 enhanced A20 expression in pig intestinal epithelia cells, dependent on TLR1/2. Given that A20 is an inhibitor of the NF-kB signalling by degrading TRAF-6(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) the enhanced A20 expression by HDRsEf1 should contribute to its inhibition on the NF-kBp65 activation and CYP3A29 expression in IPEC-J2 cells. In fact, we found that HDRsEf1, like the NF-kB inhibitor of BAY11-7082, significantly attenuated the NF-kB activation and RXR-α expression in IPEC-J2 cells. Such data extended previous observations that probiotics, such as Lactobacillus amylovorus and paracasei, can bind to TLR2 to regulate the expression of NF-κB inhibitors, including A20, SOCS1 and SOCS3 in macrophages (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003csup\u003e,\u003c/sup\u003e(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and support the notion that Gram-positive bacteria can negatively regulate inflammation through the TLR1/2 to induce the expression of the NF-kB inhibitors (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, we found that HDRsEf1 significantly reduced the expression of RXR-α and CYP3A29 in IPEC-J2 cells, which were abrogated by NF-kBp65 overexpression, suggesting that the enhanced NF-kBp65 activity may up-regulate RXR-α expression and subsequent CYP3A29 expression. These, together with the fact that HDRsEf1 failed to decrease CYP3A29 expression in the RXR-α-silenced IPEC-J2 cells, indicated that the NF-κBp65 signaling enhanced the CYP3A29 expression indirectly by enhancing RXR-α expression in intestinal tissues. Previous reports indicate that \u003cem\u003eStaphylococcus aureus\u003c/em\u003e-derived LTA can reduce RXR-α expression in mouse liver by inhibiting the JNK and NF-κB pathways (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), while IL-1β enhances RXR-α expression by activating the NF-kB signaling in gastric carcinoma tissues (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Apparently, the NF-kB signaling positively regulates the expression of RXRα, which can attenuate the inhibition of NF-kB activation (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), maintaining the balance of intracellular inflammatory and anti-inflammatory responses. Interestingly, we found that HDRsEf1 did not modulate PXR expression in IPEC-J2 cells. These data were in disagreement with previous reports that the phosphorylated NF-κBp65 can down-regulate PXR expression and inhibit the formation of PXR/RXR-α heterodimers and CYP3A4 transcription in HepG2 cells (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The discrepancy suggests that the activated NF-κBp65 may have different regulatory roles in the expression of NRs in different types of cells. Given that the activated NF-κB usually regulates the transcription of the NRs (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003csup\u003e,\u003c/sup\u003e(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) we are interested in further investigating how the activated NF-κBp65 regulates RXR-α expression in pig intestinal tissues.\u003c/p\u003e \u003cp\u003eIn this study, we found that HDRsEf1 significantly reduced RXRα expression, particularly for nuclear RXRα protein levels, but did not affect the expression of other NRs tested in IPEC-J2 cells. Furthermore, RXRα or PXR overexpression and silencing significantly modulated CYP3A29 expression in IPEC-J2 cells and both RXR-α and PXR over-expression synergistically enhanced the CYP3A29 promoter activity. In addition, RXR-α directly interacted with PXR in IPEC-J2 cells. Together, such data suggest that RXR-α and PXR may form heterodimer for the CYP3A29 transcription in IPEC-J2 cells, consistent with our findings in HepLi cells(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, we found that altered VDR or CAR expression did not affect the CYP3A29 expression in IPEC-J2 cells, implicating that the VDR/RXR-α and CAR/RXR-α might not be involved in up-regulating CYP3A29 expression although they are crucial for CYP3A4 transcription in human intestinal cells and HepG2(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Therefore, the NRs may have interspecies difference in regulating the transcription of CYP3As even if there are organ variants in the same species.\u003c/p\u003e \u003cp\u003eWe found that HDRsEf1 down-regulated the expression of CYP3A29 in IPEC-J2 in a time- and dose-dependent manner. Although HDRsEf1 supernatants have been reported to mitigate the ETEC K88ac-up-regulated IL-8 expression(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), we found that HDRsEf1 inhibited the CYP3A20 expression in IPEC-J2 cells, dependent on cell-cell contact. Our findings were consistent with a recent report that \u003cem\u003eLactobacillus helveticus\u003c/em\u003e SBT2171 cell wall components inhibit inflammatory responses in peritoneal macrophage (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The difference between our and those with ETEC K88ac may stem from the presence of more pathogen-associated molecular patterns on the cell wall of Gram-positive bacteria. We will continually explore which cell wall components of HDRsEf1 can bind to TLR1/2 on intestinal epithelial cells to inhibit CYP3A29 expression.\u003c/p\u003e \u003cp\u003eOur data indicated that HDRsEf1 inhibited the CYP3A29 expression in pig intestinal tissues and IPEC-J2 cells in a dose-, time- and cell-cell contact-dependent manner. HDRsEf1 through TLR1/2-induced A20 expression attenuated the NF-kBp65 activation to reduce RXR-α expression and limit the formation of RXR-α/PXR heterodimer, inhibiting CYP3A29 transcription in intestinal epithelial cells (Figure. 6). Our findings may provide new insights into the mechanisms by which probiotics regulate the expression of CYP450s and highlight the possible risks of probiotics for oral drug overdose in the clinic.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSpecial chemicals, and reagents\u003c/h2\u003e \u003cp\u003eThe special reagents included Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin and Lipofectamine 2000 transfection reagent (Gibco, Life Technologies, Grand Island, NY, USA); 5,6-Dichlororibosidyl-benzimidazole (DRB) and BAY11-7082 (Sigma-Aldrich, St. Louis, MO, USA); monoclonal antibodies against CYP3A29, PXR, RXR-α, CAR and VDR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), NF-κBp65, Phospho-NF-κB p65 (Cell Signaling Technology, Beverly, MA, USA), β-actin and HRP-conjugated secondary antibodies (ABclonal Technology, Wuhan, China); and the dual-luciferase reporter assay system (Promega, Madison, WI, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBacterial preparations, cell culture and treatment\u003c/h2\u003e \u003cp\u003eEnterococcus faecium HDRsEf1 is novel probiotic strain (CCTCC NO: M2011031) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The HDRsEf1 bacteria were grown in De Man Rogosa Sharpe (MRS) media (Difco, Sparks, USA) at 37\u0026deg;C for 18 h, and collected by centrifugation. After being washed twice with phosphate buffered saline (PBS), the bacteria were re-suspended in antibiotic-free DMEM at 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e colony forming units (CFU) ml-1.\u003c/p\u003e \u003cp\u003ePorcine small intestinal epithelial IPEC-J2 cells were obtained from BeNa Culture Collection (Beijing, China), and cultured in 10% FBS DMEM. To determine the effect of HDRsEf1, IPEC-J2 cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were challenged in triplicate with vehicle or 10\u003csup\u003e6\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e, or 10\u003csup\u003e8\u003c/sup\u003e CFU/mL HDRsEf1 for 2, 4 or 6 h, respectively. Furthermore, IPEC-J2 cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells) were cultured in the bottom chambers of 12-well transwell plates (0.2-\u0026micro;m-pore-size, Corning, NY, USA) and HDRsEf1 cells (10\u003csup\u003e8\u003c/sup\u003e CFU/well)were cultured in the upper chamber to determine the cell-cell contact independence. Their cell viability was quantified by Cell Counting Kit-8 assay (Biosharp, Hefei, China) and CYP3A29 expression were examined by qRT-PCR and Western blot. In addition, IPEC-J2 cells were treated with 10\u003csup\u003e8\u003c/sup\u003e CFU/mL HDRsEf1 in the presence or absence of 5 \u0026micro;M DRB and/or 10 \u0026micro;M BAY11-7082 and the relative levels of CYP3A29 mRNA transcripts were quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and procedures\u003c/h2\u003e \u003cp\u003eFemale (Landrace\u0026times;Large White) pigs at an age of 30 days with similar body weights were obtained from COFCO and randomly fed with the control corn and soybean meal-based diet (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) or the control diet containing HDRsEf1 (10\u003csup\u003e6\u003c/sup\u003e CFU/g) through a feeder for 30 days. Some pigs (4 each group) were randomly selected, anesthetized and sacrificed by jugular puncture (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Their segmented jejunal tissues at the approximately middle intestine were collected for subsequent experiments. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-Time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eThe relative levels of target gene to the control β-actin mRNA transcripts were determined by qRT-PCR using specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and iQ\u0026trade; SYBR Green PCR Supermix in the Bio-Rad CXF real-time PCR detection system (Bio-Rad, CA, USA) after we extracted total RNA from individual groups of cells with the TRIzol reagent (Invitrogen) and reversely transcribed (RT) into cDNA using the Superscript reverse transcriptase (Takara, Otsu, Japan). The data were analyzed by 2-ΔΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eWe extracted the jejunal tissue proteins as our previously described (Yan et al., 2018) and the proteins from individual groups of cells after lyzing IPEC-J2 cells with RIPA Buffer (Pierce, Rockford, IL, USA). Similarly, we also extracted nuclear and cytoplasmic proteins from individual groups of cells using specific extraction kit (EpiZyme, Shanghai, China). The protein samples (20 \u0026micro;g/lane) were separated by SDS-polyacrylamide gel electrophoresis on 10% gels and transferred onto polyvinylidene fluoride membranes. After being blocked with 5% non-fat dry milk in TBST, the membrane was incubated with primary antibodies at 4℃ overnight and reacted with secondary antibodies, followed by visualizing using ECL chemiluminescence system (Bio-Rad).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTransfection and luciferase assay\u003c/h2\u003e \u003cp\u003eIPEC-J2 cells were maintained in Opti-MEM medium until 80% of confluence and transfected with pGL3-CYP3A29 that contained the CYP3A29 promoter or the NF-kBp65 promoter using Lipofectamine 2000 for 18 h. Furthermore, IPEC-J2 cells were co-transfected with pGL3-3A29-2007 and plasmid for Renilla luciferase expression for 18 h. The cells were treated with, or without, 108 CFU/mL HDRsEf1 for 6 h and the CYP3A29 promoter activity was measured using the Dual-Luciferase Reporter Assay System. In addition, IPEC-J2 cells were transfected with a control or plasmid for RXR-α, PXR, VDR or CDR over-expression for 18 h and treated with, or without, 108 CFU/mL HDRsEf1 for 6 h. The impact of RXR-α, PXR, VDR or CDR over-expression on CYP3A29 expression was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA Interference\u003c/h2\u003e \u003cp\u003eIPEC-J2 cells were cultured in 12-well plates overnight and transfected with 40 nM control scramble or gene-specific siRNA using Lipofectamine 2000 for 36 h. The cells were treated with, or without, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL HDRsEf1 for 6 h. The efficacy of specific gene silencing and its impact on the expression of other genes were quantified by qRT-PCR and Western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eThe physical association between PXR and RXR-α was characterized by Co-IP using SureBeads\u0026trade; Starter Kit Protein G (Bio-Rad) (Xie et al., 2019). Briefly, RXR-α, PXR and specific antibody IgG or control isotype IgG (10 \u0026micro;g each) were reacted and the formed immunocomplex was precipitated with the microbeads, followed by eluting the proteins with laemmli buffer for subsequent Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are present as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. Comparison was performed by Student\u0026rsquo;s t-test, one-way ANOVA, or two-way ANOVA. Significant difference was defined when a P-value of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors have no fnancial confict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.G., Y.H. and X.W. designed the research. Y.G., Y.H., Y.L., and X.J. performed the research. Y.G., Y.W., D.S., and X.W. analyzed the data. Y.G., Y.H., D.S., and X.W wrote the paper with the help of all authors. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2017YFD0501000) and the Fundamental Research Funds for Central Universities (2662019PY061).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data that support the fndings of this study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003evan Herwaarden AE, van Waterschoot RA, Schinkel AH.2009. How important is intestinal cytochrome P450 3A metabolism? Trends Pharmacol Sci 30:223-7.\u003c/li\u003e\n\u003cli\u003evan Waterschoot RA, Rooswinkel RW, Wagenaar E, van der Kruijssen CM, van Herwaarden AE, Schinkel AH.2009. Intestinal cytochrome P450 3A plays an important role in the regulation of detoxifying systems in the liver. 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A Microbiota-Derived Bacteriocin Targets the Host to Confer Diarrhea Resistance in Early-Weaned Piglets. Cell Host Microbe 24:817-832.e8.\u003c/li\u003e\n\u003cli\u003eHu J, Chen L, Zheng W, Shi M, Liu L, Xie C, Wang X, Niu Y, Hou Q, Xu X, Xu B, Tang Y, Zhou S, Yan Y, Yang T, Ma L, Yan X.2018. Lactobacillus frumenti Facilitates Intestinal Epithelial Barrier Function Maintenance in Early-Weaned Piglets. Front Microbiol 9:897.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"probiotic, cytochrome P450, CYP3A29, RXR-α, NF-κB, TLR","lastPublishedDoi":"10.21203/rs.3.rs-4192677/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4192677/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCYP3A29, like human CYP3A4 is crucial for drug metabolism in pigs and some probiotics can regulate the expression of CYP3A in mammals. Here, we show that \u003cem\u003eEnterococcus faecium\u003c/em\u003e HDRsEf1 significantly reduces CYP3A29 expression in pig intestinal tissues and epithelial cells, dependent on cell-cell contact. In IPEC-J2 cells, HDRsEf1 decreased the CYP3A29 promoter activity, RXR-α expression and mitigated the RXR-α or PXR-increased CYP3A29 expression. Both RXR-α/PXR over-expression synergistically increased CYP3A29 expression while RXR-α or PXR silencing reduced CYP3A29 expression. Co-immunoprecipitation revealed that RXR-α directly interacted with PXR. HDRsEf1, like a NF-kB inhibitor, significantly decreased the NF-kBp65 activation, RXR-α and CYP3A29 expression, which were abrogated by RXR-α silencing. HDRsEf1 increased A20 expression dependent on TLR1/2 expression. Therefore, HDRsEf1 inhibits the expression of CYP3A29 through the TLR1/2-induced A20 to attenuate the NF-κB/RXR-α signaling in pig intestinal tissues. Our findings suggest potential risks in the clinical application of probiotics.\u003c/p\u003e","manuscriptTitle":"Enterococcus faecium HDRsEf1 Represses CYP3A29 Expression in the Intestine through the TLR1/2-induced A20 to attenuate the NF-κB/RXR-α Signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-03 17:19:26","doi":"10.21203/rs.3.rs-4192677/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d0f33ad-329e-4096-9692-f87fdb971ab7","owner":[],"postedDate":"April 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-11T23:59:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-03 17:19:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4192677","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4192677","identity":"rs-4192677","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}