LPS-induced Intestinal Barrier Dysfunction Research Articles

Regulatory effects of the p38 mitogen-activated protein kinase-myosin light chain kinase pathway on the intestinal epithelial mechanical barrier and the mechanism of modified Pulsatilla decoction in the treatment of ulcerative colitis.

To investigate the mechanism of the protective effect of modified Pulsatilla decoction (, MPD) on the mechanical barrier of the ulcerative colitis (UC) intestinal epithelium in vitro and in vivo. We established an intestinal epithelial crypt cell line-6 cell barrier injury model by using lipopolysaccharide (LPS). The model was then treated with p38 mitogen-activated protein kinase-myosin light chain kinase (p38MAPK-MLCK) pathway inhibitors, p38MAPK-MLCK pathway silencing genes (si-p38MAPK, si-NF-κB, and si-MLCK), and MPD respectively. Transepithelial electronic resistance (TEER) measurements and permeability assays were performed to assess barrier function. Immunofluorescence staining of tight junctions (TJ) was performed. In in vivo experiment, dextran sodium sulfate-induced colitis rat model was conducted to evaluate the effect of MPD and mesalazine on UC. The rats were scored using the disease activity index based on their clinical symptoms. Transmission electron microscopy and hematoxylin-eosin staining were used to examine morphological changes in UC rats. Western blotting and real-time quantitative polymerase chain reaction were performed to examine the gene and protein expression of significant differential molecules. In in vitro study, LPS-induced intestinal barrier dysfunction was inhibited by p38MAPK-MLCK pathway inhibitors and p38MAPK-MLCK pathway gene silencing. Silencing of p38MAPK-MLCK pathway genes decreased TJ expression. MPD treatment partly restored the LPS-induced decreased in TEER and increase in permeability. MPD increased the gene and protein expression of TJ, while down-regulated the LPS-induced high expression of p-p38MAPK and p-MLC. In UC model rats, MPD could ameliorate body weight loss and disease activity index, relieve colonic pathology, up-regulate TJ expression as well as decrease the expression of p-p38MAPK and p-MLC in UC rat colonic mucosal tissue. The p38MAPK-MLCK signaling pathway can affect mechanical barrier function and TJ expression in the intestinal epithelium. MPD restores TJ expression and attenuates intestinal epithelial barrier damage by suppressing the p38MAPK-MLCK pathway.

Alleviative effect of poly-β-hydroxybutyrate on lipopolysaccharide-induced oxidative stress, inflammation and cell apoptosis in Cyprinus carpio

In this study, the alleviative effects of poly-β-hydroxybutyrate (PHB) in bioflocs on oxidative stress, inflammation and apoptosis of common carp (Cyprinus carpio) induced by lipopolysaccharide (LPS) were evaluated. Common carp were irregularity divided into 5 groups and fed five diets with 0 % (CK), 2 %, 4 %, 6 % and 8 % PHB. After 8-week feeding trial, LPS challenge was executed. Results showed that appropriate level of PHB enhanced serum immune function by reversing LPS-induced the decrease of C3, C4, IgM, AKP, ACP and LZM in serum, alleviated LPS-induced intestinal barrier dysfunction by decreasing the levels of 5-HT, D-LA, ET-1 and DAO in serum, increasing ZO-1, Occludin, Claudin-3 and Claudin-7 mRNA, improving intestinal morphology. Moreover, dietary PHB reversed LPS-induced the decrease of AST and ALT in hepatopancreas, while in serum exhibited the opposite trend. Suitable level of PHB reversed LPS-induced the reduction of GSH-PX, CAT, T-SOD and T-AOC in intestines and hepatopancreas, whereas MDA showed the opposite result. PHB alleviated LPS-induced the decrease of Nrf2, HO-1, CAT, SOD and GSH-PX mRNA, the increase of Keap1 mRNA. Appropriate level of PHB alleviated LPS-induced inflammation and apoptosis by up-regulating TGF-β, IL-10 and Bcl-2 mRNA, down-regulating NF-κB, TNF-α, IL-6, Bax, Caspase-3, Caspase-8 and Caspase-9 mRNA. Furthermore, PHB inhibited activation of NLRP3 inflammasomes by reducing the levels of NLRP3, Caspase-1, ASC, IL-1β and IL-18 mRNA and protein. In addition, the increases of dietary PHB linearly and quadratically affected LPS-induced adverse effects on common carp. Summary, this study suggested that appropriate level of dietary PHB alleviated LPS-induced oxidative stress, inflammation, apoptosis and the activation of NLRP3 inflammasome in common carp. And the appropriate level of PHB in common carp diets was 4 %.

Taraxacum coreanum Nakai extract attenuates lipopolysaccharide-induced inflammatory responses and intestinal barrier dysfunction in Caco-2 cells

Ethnopharmacological relevanceTaraxacum coreanum Nakai (TC) is a dandelion native to Korea that has long been used as a medicinal herb with antioxidant and anti-inflammatory properties. Intestinal inflammation is closely associated with intestinal epithelial barrier disruption, which leads to the progression of various intestinal diseases. Aim of the studyThe aim of this study was to investigate the protective effects of TC extract on inflammatory responses and intestinal barrier dysfunction in lipopolysaccharide (LPS)-stimulated Caco-2 cells. Materials and methodsThe inhibitory effect of TC on nitric oxide (NO) and pro-inflammatory cytokines production were determined by Griess reagent and enzyme-linked immunosorbent assay, respectively. The epithelial permeability was evaluated by transepithelial electrical resistance (TEER) assay, and inflammation- and tight junction (TJ)-related protein expression were analyzed by Western blotting. In addition, the presence of ten active compounds was identified and quantified using UHPLC-ESI-MS and HPLC-DAD analyses. ResultsTreatment with TC significantly reduced NO production and pro-inflammatory cytokines production [interleukin (IL)-6 and tumor necrosis factor (TNF)-α] compared to the group treated with LPS only, particularly at 100 μg/mL. TC significantly decreased monolayer permeability as detected by TEER. In addition, the transmission of fluorescein isothiocyanate-dextran 4 across the barrier was decreased after treatment with TC. Inflammation-related proteins (inducible NO synthase, cyclooxygenase-2, TNF-α, IL-6, and IL-1β) were down-regulated after treatment with TC. In contrast, TC significantly increased the protein levels of the TJ-related protein, claudin-5. Ten phytochemicals (protocatechuic acid, chlorogenic acid, caffeic acid, scopoletin, chicoric acid, hyperoside, nicotiflorin, luteoloside, sophoricoside, and luteolin) were identified by UHPLC-ESI-MS and HPLC-DAD analysis. ConclusionOur findings suggest that ethanolic extract of TC could attenuate the LPS-induced intestinal barrier dysfunction by increasing the TJ protein and suppressing inflammatory responses.

Consumption of honey ameliorates lipopolysaccharide-induced intestinal barrier dysfunction via upregulation of tight junction proteins.

The leaky gut barrier is an important factor leading to various inflammatory gastrointestinal disorders. The nutritional value of honey and variety of its health benefits have long been recognized. This study was undertaken to assess the role of Indian mustard honey in preventing lipopolysaccharide (LPS)-induced intestinal barrier dysfunction using a combination of in vitro and in vivo experimental model systems. LPS was used to induce intestinal barrier damage in a trans-well model of Caco-2 cells (1µg/ml) and in Swiss albino mice (5mg/kg body weight). Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to analyse sugar and phenolic components in honey samples. The Caco-2 cell monolayer integrity was evaluated by transepithelial electrical resistance (TEER) and paracellular permeability assays. The histopathology of intestinal tissue was analysed by haematoxylin and eosin dual staining. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify the transcription of genes. The protein expression was analysed by immunofluorescence, western blot and ELISA-based techniques. The in vitro data showed that honey prevented LPS-induced intestinal barrier dysfunction dose dependently as was measured by TEER and paracellular flux of FITC-dextran dye. Further, the in vivo data showed a prophylactic effect of orally administered honey as it prevented the loss of intestinal barrier integrity and villus structure. The cellular localization and expression of tight junction (TJ) proteins were upregulated along with downregulation of pro-inflammatory cytokines in response to the administration of honey with LPS. The findings of this study suggest a propitious role of honey in the maintenance of TJ protein integrity, thereby preventing LPS-induced intestinal barrier disintegration.

Huangshui Polysaccharide Exerts Intestinal Barrier Protective Effects through the TLR4/MyD88/NF-κB and MAPK Signaling Pathways in Caco-2 Cells.

Several reports have demonstrated that natural polysaccharides exert protective effects on intestinal barrier function. In our previous study, we isolated a polysaccharide named HSP-W from Huangshui (HS). In the present study, the protective role of HSP-W in LPS-induced intestinal barrier dysfunction was determined by several molecular biological techniques. The results showed that HSP-W treatment alleviated the deduced TEER and increased the permeability of intestinal epithelial cells induced by LPS through inhibiting the release of inflammatory cytokines and enhancing the expression of tight junction (TJ) proteins. The underlying molecular mechanisms were elucidated by RNA-seq technique, which indicated that the differentially expressed genes (DEGs) between the LPS-treated and LPS+HSP-W-treated groups were enriched in the "MAPK" signaling pathway. Notably, the overlapping DEGs reversed by HSP-W intervention highlighted the pathways of the "Toll-like receptor" and "NF-κB" signaling pathways. The suppression of p38 and NF-κB were mediated by the inhibition of MyD88. Furthermore, HSP-W treatment prevented the translocation of NF-κB to nucleus, thus inhibiting the release of TNF-α, IL-6, and IL-1β. Overall, HSP-W has beneficial effects on LPS-induced inflammation; it protects the intestinal barrier from injury in Caco-2 cells through inhibiting the TLR4/MyD88/NF-κB and p38 MAPK signaling pathways.

Egg White Protein Ovotransferrin-Derived IRW (Ile-Arg-Trp) Inhibits LPS-Induced Barrier Integrity Dysfunction and Inflammation in Caco-2 Cells.

Tripeptide IRW derived from egg ovotransferrin was initially identified to be an inhibitor of angiotensin-converting enzyme. Later, IRW has been shown to possess various bioactivities, including anti-inflammatory activity and the ability to suppress colitis development. Nevertheless, its role in protecting intestinal barrier integrity has not been reported. This study aims to investigate the effect of IRW on inhibiting intestinal barrier dysfunction and inflammation in lipopolysaccharide (LPS)-treated Caco-2 cells. Pretreatment with IRW could mitigate the LPS-induced reduction of transepithelial electronic resistance values and decrease the paracellular permeation of differentiated Caco-2 cell monolayers. Meanwhile, IRW restored the expression level and cell surface distribution of the tight junction protein occludin. Furthermore, IRW showed LPS-neutralizing activity and could significantly inhibit LPS-induced activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. In conclusion, our study demonstrated the ability of IRW to prevent LPS-induced intestinal barrier dysfunction and prohibit inflammatory responses.

Yajieshaba prevents lipopolysaccharide-induced intestinal barrier injuryanti-inflammatory and anti-apoptosis.

To investigate the protective effect of Yajieshaba (YJSB) on the intestinal barrier dysfunction induced by lipopolysaccharide (LPS). C57BL/6 mice and rat intestinal epithelial cells were treated with LPS. Thiazolyl Blue Tetrazolium Bromide assay were used to detect cell viability. D-Lactate, diamine oxidase and myeloperoxidase and cytokines were determined by enzyme-linked immunosorbent assay. Western blot was used to detect apoptosis-related proteins and tight junction (TJ) proteins. Real-time quantitative polymerase chain reaction was used to quantify the levels of mRNA expression of cytokines. Histological analysis was performed by hematoxylin and eosin staining. An immunofluorescence staining assay was performed to determine the expression level of TJ protein. YJSB increased cell viability and decreased apoptosis, maintained intestinal permeability after LPS-induced. YJSB inhibited LPS-induced decrease of TJ protein expression, pro-inflammatory cytokine levels and neutrophil infiltration. YJSB protect against LPS-induced intestinal barrier dysfunction anti-inflammatory and anti-apoptosis, suggesting its therapeutic potential against intestinal barrier injury-related diseases.

Toll-like receptor 4 deficiency alleviates lipopolysaccharide-induced intestinal barrier dysfunction

BackgroundThe intestinal tract is considered the body’s “engine” and the most impacted organ during sepsis. In this study, we explored toll-like receptor 4 (TLR4) functions in sepsis-induced intestinal barrier dysfunction. MethodsWild-type and TLR4-knockout (KO) mice were used to establish a sepsis-induced dysfunctional intestinal barrier model via the intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). Hematoxylin and eosin staining, Transmission electron microscope, enzyme linked immunosorbent assay, western blot, quantitative real-time polymerase chain reaction, TdT-mediated dUTP nick end labeling staining, 16 S rRNA gene sequencing were used to explore differences in inflammatory cytokines, apoptosis, tight junction (TJ) protein expression, and intestinal flora diversity between groups. ResultsTLR4-deficiency reduced procalcitonin and C-reactive protein to prevent sepsis, and also inhibited inflammatory response by decreasing interleukin (IL)− 1β, IL-6 and tumor necrosis factor-α levels. Also, BAX/Bcl2 and cleaved-caspase 3 expressions were decreased in TLR4-KO mice to suppress the intestinal mucosal cell apoptosis. TJ proteins, including zonula occludens protein, Occludin and Claudin-5 were significantly increased and intestinal fatty acid binding protein, myosin light chain and myosin light chain kinase were reduced in TLR4-KO mice. Additionally, 16 S rRNA gene sequencing indicated that TLR4-deficiency improved flora diversity and altered normal and abnormal bacterial proportions. ConclusionsTLR4 deficiency alleviated LPS-induced intestinal barrier dysfunction by reducing inflammatory responses and apoptosis, impairing intestinal damage, and regulating intestinal flora disturbance.

Alisol B 23-Acetate Ameliorates Lipopolysaccharide-Induced Intestinal Barrier Dysfunction by Inhibiting TLR4-NOX1/ROS Signaling Pathway in Caco-2 Cells.

Alisol B 23-Acetate (AB23A) is a naturally occurring triterpenoid, which can be indicated in the rhizome of medicinal and dietary plants from Alisma species. Previous studies have demonstrated that AB23A could inhibit intestinal permeability by regulating tight junction (TJ)-related proteins. Even so, the AB23A protective mechanism against intestinal barrier dysfunction remains poorly understood. This investigation seeks to evaluate the AB23A protective effects on intestinal barrier dysfunction and determine the mechanisms for restoring intestinal barrier dysfunction in LPS-stimulated Caco-2 monolayers. According to our findings, AB23A attenuated the inflammation by reducing pro-inflammatory cytokines production like IL-6, TNF-α, IL-1β, and prevented the paracellular permeability by inhibiting the disruption of TJ in LPS-induced Caco-2 monolayers after treated with LPS. AB23A also inhibited LPS-induced TLR4, NOX1 overexpression and subsequent ROS generation in Caco-2 monolayers. Transfected with NOX1-specific shRNA diminished the up-regulating AB23A effect on ZO-1 and occludin expression. Moreover, transfected with shRNA of TLR4 not only enhanced ZO-1 and occludin expression but attenuated NOX1 expression and ROS generation. Therefore, AB23A ameliorates LPS-induced intestinal barrier dysfunction by inhibiting TLR4-NOX1/ROS signaling pathway in Caco-2 monolayers, suggesting that AB23A may have positive impact on maintaining the intestinal barrier’s integrity.

Naphthoquinone derivatives as P-glycoprotein inducers in inflammatory bowel disease: 2D monolayers, 3D spheroids, and in vivo models

Inflammatory bowel disease (IBD) represents a chronic inflammation of the gastrointestinal tract characterized by an overreaction of immune responses and damage at the intestinal mucosal barrier. P-glycoprotein (P-gp) plays a key role to protect the intestinal barrier from xenobiotic accumulation and suppressing excessive immune responses. Therefore, induction/activation of P-gp function could serve as a novel therapeutic target to treat IBD. This study aimed to evaluate the potential therapeutic values of naphthoquinone derivatives (NQ-1 - NQ-8) as P-gp modulators to counterbalance intestinal inflammation. The data indicate that NQ-2, NQ-3, and NQ-4 act as P-gp inducers/activators and are recognized as substrates for P-gp. The three derivatives possess anti-inflammatory effects mediated by suppression of NF-κB and HDAC6 activity in Caco2 monolayer cells. Besides, they reversed LPS-induced intestinal barrier dysfunction by enhancing the expression of P-gp and ZO-1 tight junction proteins in a Caco-2 spheroid model. NQ-2, NQ-3, and NQ-4 showed a robust inhibitory effect on IL-1β maturation in LPS-primed THP-1 cells. This effect may contribute to alleviate the inflammatory cascades associated with IBD. Distinctively, NQ-2 and NQ-3 exerted anti-NLRP3 inflammasome activity evidenced by the inhibition of CASP-1 activity and the promotion of autophagy. Both compounds induced disruptions of the microtubule network in transfected U2OS-GFP-α-tubulin cells. Treatment with NQ-2 remarkably attenuated dextran sulfate sodium (DSS)-induced colitis in rats by suppressing changes in colon length, colon mass index, and intestinal histopathology scores. Thus, 1,4-naphthoquinone derivatives such as NQ-2 may provide potential therapeutic anti-inflammatory effects for IBD patients and for other NLRP3-associated inflammatory diseases.

Pasteurized Akkermansia muciniphila Ameliorate the LPS-Induced Intestinal Barrier Dysfunction via Modulating AMPK and NF-κB through TLR2 in Caco-2 Cells.

Akkermansia muciniphila is well known for the amelioration of inflammatory responses and restoration of intestinal barrier function. The beneficial effect of A. muciniphila occurred through contacting Toll-like receptor 2 (TLR2) on intestinal epithelial cells by wall components. In this case, the downstream mechanism of pasteurized A. muciniphila stimulating TLR2 for ameliorated intestinal barrier function is worth investigating. In this study, we evaluated the effect of live and pasteurized A. muciniphila on protecting the barrier dysfunction of Caco-2 intestinal epithelial cells induced by lipopolysaccharide (LPS). We discovered that both live and pasteurized A. muciniphila could attenuate an inflammatory response and improve intestinal barrier integrity in Caco-2 monolayers. We demonstrated that A. muciniphila enhances AMP-activated protein kinase (AMPK) activation and inhibits Nuclear Factor-Kappa B (NF-κB) activation through the stimulation of TLR2. Overall, we provided a specific mechanism for the probiotic effect of A. muciniphila on the intestinal barrier function of Caco-2 cells.

Sinapic Acid Alleviated Inflammation-Induced Intestinal Epithelial Barrier Dysfunction in Lipopolysaccharide- (LPS-) Treated Caco-2 Cells.

The integrity and permeability of the intestinal epithelial barrier are important indicators of intestinal health. Impaired intestinal epithelial barrier function and increased intestinal permeability are closely linked to the onset and progression of various intestinal diseases. Sinapic acid (SA) is a phenolic acid that has anti-inflammatory, antihyperglycemic, and antioxidant activities; meanwhile, it is also effective in the protection of inflammatory bowel disease (IBD), but the specific mechanisms remain unclear. Here, we evaluated the anti-inflammatory of SA and investigated its potential therapeutic activity in LPS-induced intestinal epithelial barrier and tight junction (TJ) protein dysfunction. SA improved cell viability; attenuated epithelial permeability; restored the protein and mRNA expression of claudin-1, ZO-1, and occludin; and reversed the redistribution of the ZO-1 and claudin-1 proteins in LPS-treated Caco-2 cells. Moreover, SA reduced the inflammatory response by downregulating the activation of the TLR4/NF-κB pathway and attenuated LPS-induced intestinal barrier dysfunction by decreasing the activation of the MLCK/MLC pathway. This study demonstrated that SA has strong anti-inflammatory activity and can alleviate the occurrence of high intercellular permeability in Caco-2 cells exposed to LPS.

Propionic Acid Targets the TLR4/NF-κB Signaling Pathway and Inhibits LPS-Induced Intestinal Barrier Dysfunction: In Vitro and In Vivo Studies.

Intestinal barrier dysfunction contributes to the development of intestinal diseases. Propionic acid (PA), a metabolite generated by anaerobic fermentation of dietary fiber in the intestinal cavity, has been proved to exert anti-inflammatory effects in a variety of diseases. However, the exact role of PA in LPS-induced intestinal barrier dysfunction is still unclear. Accordingly, we examined the latent mechanism of PA and its protective role in LPS-induced intestinal barrier dysfunction by both in vitro and in vivo experiments. In vitro, we identified that PA treatment could strongly promote cell migration, inhibit activation of NLRP3 inflammasome and maintain intestinal barrier function in LPS-induced IEC-6 cells, indicating the protective effect on the intestinal barrier function of PA. Further investigation of the mechanism involved revealed that PA could suppress the activation of TLR4/NF-κB pathway. In vivo, in a LPS-induced rat model, PA-induced protective effects in intestinal barrier dysfunction could be detected. In summary, our findings clarify the role of PA in intestinal barrier dysfunction and suggest that it is promising for the treatment of LPS-related intestinal diseases.

EphrinA1/EphA2 Promotes Epithelial Hyperpermeability Involving in Lipopolysaccharide-induced Intestinal Barrier Dysfunction.

Background/AimsLipopolysaccharide (LPS) is the key factor inducing mucosal and systemic inflammation in various intestinal and parenteral diseases, which could initially disrupt the epithelial barrier function. EphrinA1/ephA2 is speculated to increase the epithelial permeability for its “repulsive interaction” between adjacent cells. This study aim to investigate the role of ephrinA1/ephA2 in LPS-induced epithelial hyperpermeability.MethodsIn vivo model challenged with oral LPS in C57BL/6 mice and in vitro model exposed to LPS in Caco2 monolayer were established. The barrier function was assessed including expression of tight junction proteins (occludin and claudin-1), transepithelial electrical resistance, and permeability to macromolecules (fluorescein isothiocyanate-labeled fluorescent dextran 4 kDa [FD4]). Moreover, the expression and phosphorylation of ephrinA1/ephA2 were quantified, and its roles in the process of epithelial barrier disruption were confirmed via stimulating ephA2 with ephrinA1-Fc chimera (ephrinA1-Fc) and inactivating ephA2 with ephA2-Fc chimera (ephA2-Fc), or ephA2 monoclonal antibody (ephA2-mab), as well as inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059.ResultsLPS induced significant barrier dysfunction with dismissed occludin and claudin-1 expression, reduced transepithelial electrical resistance and increased FD4 permeability, accompanied by upregulated ephrinA1/ephA2 pathway and phosphorylation of ephA2 receptor. Furthermore, ephA2-Fc, and ephA2-mab ameliorated LPS-induced epithelial hyperpermeability, which was also inhibited by PD98059. Additionally, ephrinA1-Fc led to apparent epithelial leakage in Caco2 monolayer by promoting the phosphorylation of ERK1/2, which could be obviously blocked by ephA2-mab and PD98059.ConclusionEphrinA1/ephA2 promotes epithelial hyperpermeability with an ERK1/2-dependent pathway, which involves in LPS-induced intestinal barrier dysfunction.

Koumine Alleviates Lipopolysaccharide-Induced Intestinal Barrier Dysfunction in IPEC-J2 Cells by Regulating Nrf2/NF-B Pathway.

Gelsemium elegans Benth. (G. elegans), a traditional Chinese medicine, has great potential as an effective growth promoter in animals, however, the mechanism of its actin remains unclear. Here, we evaluated the protective effects of koumine extract from G. elegans against lipopolysaccharide (LPS)-induced intestinal barrier dysfunction in IPEC-J2 cells through alleviation of inflammation and oxidative stress. MTT and LDH assays revealed that koumine significantly reduced LPS cytotoxicity. Transepithelial electrical resistance (TEER) and cell monolayer permeability assays showed that koumine treatment attenuated the LPS-induced intestinal barrier dysfunction with no particularly different effects in tight junction proteins such as ZO-1, claudin-1, and occludin. LPS-triggered inflammatory response was also suppressed by koumine, as evidenced by the downregulated inflammatory factors, including TNF-, IL-6, IL-1, NO, iNOS, and COX-2, which was closely connected with the inhibition of NF-B pathway for the decrease of phosphorylation of IB and NF-B and nuclear translocation of p-p65. Amount of reactive oxygen species (ROS) and MDA induced by LPS was also reduced by koumine through activation of Nrf2 pathway, and increased in the levels of Nrf2 and HO-1 degradation of keap-1 to promote anti-oxidants, including superoxide dismutase (SOD) and catalase (CAT). To summarize, koumine-reduced the oxidative stress and inflammatory reaction triggered by LPS through regulation of the Nrf2/NF-B signaling pathway and preventing intestinal barrier dysfunction.

Metformin Protects against LPS-Induced Intestinal Barrier Dysfunction by Activating AMPK Pathway.

Metformin not only regulates energy metabolism but also participates in many cellular processes. In this study, we investigated the effect of metformin on lipopolysaccharide (LPS)-induced intestinal barrier damage. We found that LPS treatment decreased the expression of tight junction proteins and caused a proinflammatory response and oxidative stress in the intestine. Interestingly, metformin treatments attenuated LPS-induced intestinal barrier damage, inflammation, and oxidative stress. We found that metformin improved the expression of intestinal tight junction proteins (ZO1, occludin, and Claudin1) that were reduced by LPS stimulation. Moreover, metformin alleviated LPS-induced NF-κB phosphorylation, promoted Nrf2 nuclear translocation, and increased the expression of the antioxidative genes (HO-1 and NQO-1), leading to reduced intestinal ROS content. Mechanistically, we found that metformin protects against LPS-induced intestinal barrier dysfunction by activating AMPK. These results reveal the potential of metformin as an effective therapy for treating intestinal diseases.

Lactobacillus reuteri ZJ617 maintains intestinal integrity via regulating tight junction, autophagy and apoptosis in mice challenged with lipopolysaccharide.

Live probiotics are effective in reducing gut permeability and inflammation. We have previously reported that Lactobacillus reuteri ZJ617 (ZJ617) with high adhesive and Lactobacillus rhamnosus GG (LGG) can ameliorate intestine inflammation induced by lipopolysaccharide (LPS). The present study was aimed at elucidating the roles of ZJ617 and LGG in alleviating the LPS-induced barrier dysfunction of ileum in mice. Six C57BL/6 mice per group were orally inoculated with ZJ617 or LGG for one week (1× 108 CFU/mouse) and intraperitoneally injected with LPS (10 mg/kg body weight) for 24 h. The results demonstrated that pretreatment with ZJ617 and LGG attenuated LPS-induced increase in intestinal permeability. The probiotics supplementation suppressed LPS-induced oxidative stress. Both ZJ617 and LGG strongly reversed the decline of occludin and claudin-3 expression induced by LPS challenge. ZJ617 relieved LPS-induced apoptosis by decreasing caspase-3 activity. Noticeably, ratio of microtubule-associated light chain 3 (LC3)-II/LC3-I and LC3 activity were elevated by LPS stimulation, whereas such increases were obviously attenuated by both of the probiotics treatment. Moreover, phosphorylated mammalian target of rapamycin (p-mTOR) was significantly inhibited by LPS, whereas complementation of ZJ617 and LGG markedly increased the expression of p-mTOR. Collectively, our results indicated that ZJ617 could protect LPS-induced intestinal barrier dysfunction via enhancing antioxidant activities and tight junction and attenuating apoptosis and autophagy via mTOR signaling pathway. These findings could serve as systematic mechanisms through which probiotics promote and maintain gut homeostasis.

Role of RhoA in amelioration of lipopolysaccharide-induced human intestinal barrier dysfunction by hydrogen-rich saline: in vitro experiment

Objective To evaluate the role of RhoA in amelioration of lipopolysaccharide (LPS)-induced human intestinal barrier dysfunction by hydrogen-rich saline. Methods Caco-2 cells were plated on transwells at a density of 2×105/ml (400 μl/well), and randomly divided into 5 groups (n=50 each) using a random number table: control group (group C), hydrogen-rich saline group (HRS group), LPS group, LPS + hydrogen-rich saline group (group LPS+ HRS), and LPS + RhoA inhibitor C3 exoenzyme group (group LPS+ C3). Cells were cultured in the medium containing 0.6 mmol/L hydrogen-rich saline in HRS and LPS+ HRS groups, or in the medium containing 100 μg/ml LPS in LPS, LPS+ HRS and LPS+ C3 groups.In group LPS+ C3, C3 exoenzyme 2.5 μg/ml was added at 1 h before addition of LPS.Five wells were selected at 0, 3, 6, 12, 24, 36 and 48 h of incubation with LPS, and the transepithelial electrical resistance (TEER) and permeability coefficient (PE) were measured.Five wells were selected at 24 h of incubation with LPS for determination of occludin and E-cadherin expression (by Western blot and immunofluorescence) and RhoA activity (using GST pull-down assay). Results Compared with group C, TEER was significantly decreased, PE and RhoA activity were increased, and the expression of occludin and E-cadherin was down-regulated at 6-48 h of incubation with LPS in LPS, LPS+ HRS and LPS+ C3 groups (P 0.05). Compared with group LPS, TEER was significantly increased, PE and RhoA activity were decreased, and the expression of occludin and E-cadherin was up-regulated at 6-48 h of incubation with LPS in LPS+ HRS and LPS+ C3 groups (P<0.05). Compared with group LPS, the distribution of occluding and E-cadherin in the cell membrane was significantly increased, the distribution in the cytoplasm was decreased, and the structures of occludin and E-cadherin partly recovered in LPS+ HRS and LPS+ C3 groups. Conclusion Hydrogen-rich saline can alleviate LPS-induced intestinal barrier dysfunction via inhibiting RhoA activation, thus up-regulating occludin and E-cadherin expression and inhibiting the disturbance of the distribution. Key words: RhoA GTP-binding protein; Hydrogen; Endotoxemia; Intestinal mucosa

Carbachol ameliorates lipopolysaccharide-induced intestinal epithelial tight junction damage by down-regulating NF-κβ and myosin light-chain kinase pathways

Carbachol is a cholinergic agonist that protects the intestines after trauma or burn injury. The present study determines the beneficial effects of carbachol and the mechanisms by which it ameliorates the lipopolysaccharide (LPS)-induced intestinal barrier breakdown. Rats were injected intraperitoneally with 10mg/kg LPS. Results showed that the gut barrier permeability was reduced, the ultrastructural disruption of tight junctions (TJs) was prevented, the redistribution of zonula occludens-1 and claudin-2 proteins was partially reversed, and the nuclear factor-kappa beta (NF-κβ) and myosin light-chain kinase (MLCK) activation in the intestinal epithelium were suppressed after carbachol administration in LPS-exposed rats. Pretreatment with the α7 nicotinic acetylcholine receptor (α7nAchR) antagonist α-bungarotoxin blocked the protective action of carbachol. These results suggested that carbachol treatment can protect LPS-induced intestinal barrier dysfunction. Carbachol exerts its beneficial effect on the amelioration of the TJ damage by inhibiting the NF-κβ and MLCK pathways in an α7nAchR-dependent manner.