Epithelial Protein Expression Research Articles

DANCR maintained colon epithelial homeostasis by regulating the TNFα/NF-κB pathway

Epithelial homeostasis is fundamental for the physiological functions of colon tissue. Dysregulation of colon epithelial structure leads to abnormal immune responses and diseases such as inflammatory bowel disease. In this work we found long non-coding RNA DANCR was a novel regulator to colon epithelial homeostasis. Silencing DANCR resulted in decreased expression of epithelial barrier proteins and enhanced susceptibility to TNFα stimulation, which was accompanied by hyperactivation of the NF-κB pathway. Mechanistical studies revealed DANCR modulated the expression of a protein methyltransferase SET7 to suppress responses to TNFα, as well as the activity of NF-κB pathway. In summary, DANCR regulated colon epithelial homeostasis through modulating the TNFα/NF-κB axis. These findings cast light on the discovery of novel regulators to colon epithelial homeostasis and added new evidence to the physiological functions of DANCR.

Targeted lung therapy with rosmarinic acid encapsulated in PLGA microspheres for radiation-induced pulmonary fibrosis

Radiation-induced pulmonary fibrosis (RIPF) is a frequent complication among individuals affected by nuclear accidents and patients undergoing chest radiation therapy for tumors. However, the availability of effective drugs for prevention and treatment of RIPF remains limited. Rosmarinic acid (RA), which displays inhibitory properties against RIPF, suffers from poor targeting in previous drug studies. To overcome this issue and achieve optimal therapeutic effects, continuous administration over several days is usually employed. In this study, we developed PLGA microspheres with an average size of 17 μm and drug loading efficiency of 24.97 % (±0.97 %), loaded with RA (RA/PLGAMS) using the membrane emulsification method. By controlling the size of microspheres in the pulmonary capillaries, we achieved lung targeting of microspheres, and based on the release of drugs as PLGA degrades, the residence time of drugs in the body is prolonged. And it was confirmed by in vivo imaging and SEM imaging that PLGAMS could exist in the lungs for at least 7 days. Our results demonstrated successful targeting of the lungs in a mouse model and sustained release of RA encapsulated in the PLGA microspheres. Notably, under the same administration conditions, the therapeutic efficacy of PLGA-encapsulated RA surpassed that of ordinary RA. We observed decreased expression of inflammatory factors, reduced M2 macrophage polarization, and weakened epithelial mesenchymal transformation following our treatment, which might explain the radiation damage alleviating effect of RA. After treatment, it was shown that the deep inspiratory capacity of the mice improved by 57.8 %, and WB showed a decrease of 52.4 % and 28.3 % in the expression of epithelial mesenchymal transition related proteins (TGF-β1 and α-SMA). In summary, PLGA microspheres offer a promising drug delivery system and treatment strategy for radiation-induced pulmonary fibrosis.

Sijunzi decoction alleviates inflammation and intestinal epithelial barrier damage and modulates the gut microbiota in ulcerative colitis mice.

As a representative classical prescription, Sijunzi decoction has powerful therapeutic effects on spleen-stomach qi insufficiency. Ulcerative colitis (UC) is a chronic, diffuse, and non-specifically inflammatory disorder, the etiology of which still remains unclear. In the traditional Chinese medicine (TCM) perspective, splenic asthenia is the primary cause of UC. Based on this, Sijunzi decoction has been extensively used in TCM clinical practice to alleviate UC in recent years. However, the pharmacological mechanism of Sijunzi decoction in modern medicine is still not completely clear, which limits its clinical application. The purpose of this study was to investigate the Sijunzi decoction's curative effect on acute UC mice and probe into its potential pharmacological mechanism. The UC mouse model was set up by freely ingesting a 3% dextran sulfate sodium (DSS) solution. The relieving role of Sijunzi decoction on UC in mice was analyzed by evaluating the changes in clinical parameters, colon morphology, histopathology, inflammatory factor content, intestinal epithelial barrier protein expression level, and gut microbiota balance state. Finally, multivariate statistical analysis was conducted to elucidate the relationship between inflammatory factors, intestinal epithelial barrier proteins, and gut microbiota. First, the research findings revealed that Sijunzi decoction could visibly ease the clinical manifestation of UC, lower the DAI score, and attenuate colonic damage. Moreover, Sijunzi decoction could also significantly inhibit IL-6, IL-1β, and TNF-α while increasing occludin and ZO-1 expression levels. Subsequently, further studies showed that Sijunzi decoction could remodel gut microbiota homeostasis. Sijunzi decoction was beneficial in regulating the levels of Alistipes, Akkermansia, Lachnospiraceae_NK4A136_group, and other bacteria. Finally, multivariate statistical analysis demonstrated that key gut microbes were closely associated with inflammatory factors and intestinal epithelial barrier proteins. Sijunzi decoction can significantly prevent and treat UC. Its mechanism is strongly associated with the improvement of inflammation and intestinal epithelial barrier damage by regulating the gut microbiota.

Role of ferroptosis in Porphyromonas gingivalis-induced impairment of epithelial junction

ABSTRACT Objectives This study aims to clarify the effect of ferroptosis by P. gingivalis on periodontal epithelium impairment and potential mechanisms. Materials and methods The expression of epithelial junction proteins (CDH1, OCLN, ZO-1), FTL and GPX4 in healthy and periodontitis tissues was analyzed using bioinformatics analysis and validated in vivo. An in vitro model was constructed to evaluate ferroptosis by mitochondria morphology, content of iron and GSH, and level of lipid peroxidation, FTL, GPX4 and SLC7A11. The iron concentration was changed with iron chelator DFO and iron supplementation FAC. The epithelial impairment was assessed by protein expression. To investigate the mechanism, si-MYB (a negative transcription factor of SLC7A11) and GPX4 inhibitor RSL3 were employed. Results CDH1, OCLN, ZO-1 and GPX4 expression was decreased, while FTL expression was elevated in periodontitis tissues. Infected cells showed ferroptosis change of the mitochondria with higher level of lipid peroxidation, iron, FTL and lower level of GPX4, GSH, SLC7A11. FAC augmented ferroptosis and weakened epithelial junction, while DFO exhibited a counteractive effect. Silencing MYB rescued SLC7A11, GPX4 and epithelial junction proteins, which was hindered by RSL3. Conclusions Our study demonstrated that P. gingivalis weakened the oral epithelial barrier by causing ferroptosis via inhibiting SLC7A11/GSH/GPX4 axis.

Development of an Inflamed High Throughput Stem-cell-based Gut Epithelium Model to Assess the Impact of Annexin A1.

Annexin A1 (ANXA1) plays a role in maintaining intestinal hemostasis, especially following mucosal inflammation. The published data about ANXA1 was derived from experimental animal models where there is an overlapping between epithelial and immune cells. There is no in vitro gut epithelial model that can assess the direct effect of ANXA1 on the gut epithelium. We developed high-throughput stem-cell-based murine epithelial cells and bacterial lipopolysaccharides (LPS) were used to induce inflammation. The impact of ANXA1 and its functional part (Ac2-26) was evaluated in the inflamed model. Intestinal integrity was assessed by the transepithelial electrical resistance (TEER), and FITC-Dextran permeability. Epithelial junction proteins were assessed using confocal microscopy and RT-qPCR. Inflammatory cytokines were evaluated by RT-qPCR and ELISA. LPS challenge mediated a damage in the epithelial cells as shown by a drop in the TEER and an increase in FITC-dextran permeability; reduced the expression of epithelial junctional proteins (Occludin, ZO-1, and Cadherin) and increased the expression of the gut leaky protein, Claudin - 2. ANXA1 and Ac2-26 treatment reduced the previous damaging effects. In addition, ANXA1 and Ac2-26 inhibited the inflammatory responses mediated by the LPS and increased the transcription of the anti-inflammatory cytokine, IL-10. ANXA1 and Ac2-26 directly protect the epithelial integrity by affecting the expression of epithelial junction and inflammatory markers. The inflamed gut model is a reliable tool to study intestinal inflammatory diseases, and to evaluate the efficacy of potential anti-inflammatory drugs and the screening of new drugs that could be candidates for inflammatory bowel disease.

Epithelial MAPK signaling directs endothelial NRF2 signaling and IL-8 secretion in a tri-culture model of the alveolar-microvascular interface following diesel exhaust particulate (DEP) exposure

BackgroundParticulate matter 2.5 (PM2.5) deposition in the lung’s alveolar capillary region (ACR) is significantly associated with respiratory disease development, yet the molecular mechanisms are not completely understood. Adverse responses that promote respiratory disease development involve orchestrated, intercellular signaling between multiple cell types within the ACR. We investigated the molecular mechanisms elicited in response to PM2.5 deposition in the ACR, in an in vitro model that enables intercellular communication between multiple resident cell types of the ACR.MethodsAn in vitro, tri-culture model of the ACR, incorporating alveolar-like epithelial cells (NCI-H441), pulmonary fibroblasts (IMR90), and pulmonary microvascular endothelial cells (HULEC) was developed to investigate cell type-specific molecular responses to a PM2.5 exposure in an in-vivo-like model. This tri-culture in vitro model was termed the alveolar capillary region exposure (ACRE) model. Alveolar epithelial cells in the ACRE model were exposed to a suspension of diesel exhaust particulates (DEP) (20 µg/cm2) with an average diameter of 2.5 µm. Alveolar epithelial barrier formation, and transcriptional and protein expression alterations in the directly exposed alveolar epithelial and the underlying endothelial cells were investigated over a 24 h DEP exposure.ResultsAlveolar epithelial barrier formation was not perturbed by the 24 h DEP exposure. Despite no alteration in barrier formation, we demonstrate that alveolar epithelial DEP exposure induces transcriptional and protein changes in both the alveolar epithelial cells and the underlying microvascular endothelial cells. Specifically, we show that the underlying microvascular endothelial cells develop redox dysfunction and increase proinflammatory cytokine secretion. Furthermore, we demonstrate that alveolar epithelial MAPK signaling modulates the activation of NRF2 and IL-8 secretion in the underlying microvascular endothelial cells.ConclusionsEndothelial redox dysfunction and increased proinflammatory cytokine secretion are two common events in respiratory disease development. These findings highlight new, cell-type specific roles of the alveolar epithelium and microvascular endothelium in the ACR in respiratory disease development following PM2.5 exposure. Ultimately, these data expand our current understanding of respiratory disease development following particle exposures and illustrate the utility of multicellular in vitro systems for investigating respiratory tract health.

Vaginal candida infection is associated with host molecular signatures of neutrophil activation in the adjacent ectocervical mucosa in Kenyan sex workers

AbstractProblemOvergrowth of candida species in the human vaginal mucosa causes inflammation, which could render the mucosal barrier more susceptible to HIV infection. Here, we investigated whether this condition also affects the ectocervical mucosa, a potential site of HIV entry, in women at high risk of HIV infection.Method of studyRetrospective medical data and ectocervical tissue samples were obtained from a cohort of Kenyan sex workers. Among 108 women, seven had signs of vaginal candida infection by wet smear microscopy and/or the presence of characteristic discharge. Women lacking these two criteria served as controls. Host transcriptomic profiling and quantitative in situ image analysis of epithelial barrier markers and CD4+ cell distribution were performed.ResultsThe candida group had 162 differentially expressed genes out of 15 435 genes as compared with the control group. Among these 162 genes, 147 were upregulated and 15 were downregulated. Gene expression pathway analysis indicated associations with an upregulated inflammatory response, defined primarily by markers of neutrophil activation. Transcription factor analysis revealed upregulation of pathways related to RELA/REL/NFKB1, JUN and STAT1 in the candida group. In situ image analysis of ectocervical tissue samples showed no differences between groups in terms of epithelial height, expression of epithelial junction proteins (E‐cadherin, claudin‐1, zonula occludens 1, and desmoglein‐1), or epithelial CD4+ cell distribution.ConclusionsVaginal candida infection was associated with inflammation and neutrophil infiltration, but not with severe epithelial disruption or CD4+ cell infiltration, in the ectocervical mucosa.

P385 Epithelial neutrophil localization and Claudin-2 immunohistochemical "leaky gut" expression are innovative predictors of outcomes in Ulcerative Colitis patients in endoscopic remission

Abstract Background Patients with Ulcerative Colitis (UC) in clinical and endoscopic remission (ER) may still experience disease relapse, emphasizing the need to identify reliable predictors of outcome in clinical practice. Recently, neutrophil-based histological scores have highlighted the pivotal role of neutrophils in predicting disease outcome. Furthermore, the impairment of the intestinal barrier has been proposed as a crucial element in forecasting disease outcomes. Hence, this study aims to assess the predictive role of epithelial versus lamina propria neutrophil localization and barrier proteins expression in UC patients in ER. Methods UC patients in clinical remission who underwent colonoscopy between January 2020 and June 2022 at two tertiary referral centres were retrospectively enrolled. Patients in ER, defined by Mayo Endoscopic Score (MES) ≤1 and UC Endoscopic Index of Severity (UCEIS) ≤1 were included. Histological activity was assessed with Robarts Histopathology Index (RHI), Nancy Histological Index (NHI) and PICaSSO Histological Remission Index (PHRI). Neutrophil localization in the epithelium and lamina propria was accurately evaluated. Additionally, immunohistochemical expression of epithelial barrier proteins, namely Claudin-2 and JAM-A, was assessed to evaluate the integrity of intestinal barrier. Two pathologists experienced in IBD independently analysed all histological slides to ensure reliability. Kaplan-Meier were plotted to analyse survival free from adverse events such as flare-up, change/optimization of medication or hospitalization over a 12-month follow-up period. Results Out of 60 UC patients in ER (58.3% males, mean age 44.2±13.6 years), 36.7%, 38.3% and 36.7% were in histological remission according to RHI, NHI and PHRI, respectively. 25% of patients developed an adverse outcome within 12 months. Neutrophils in the epithelium significantly correlated with adverse outcomes compared with lamina propria localization (p=0.03) or their absence altogether (p<0.01). Claudin-2 showed significantly higher expression according to endoscopic and histological activity (Table 1), while no significant difference was found for JAM-A. Notably, strong and diffuse claudin-2 expression was associated with a higher rate of adverse outcomes (p<0.01), as well as the simultaneous presence of epithelial neutrophils and Claudin-2 (p=0.04). Substantial agreement between pathologists was found (K=0.67). Conclusion This multicentre retrospective study highlights the potential role of epithelial neutrophils localization and claudin-2 ‘leaky gut’ protein expression as practical clinical tools to predict outcomes in UC patients. These findings hold promise in guiding patient-tailored therapy in IBD.

P068 Recapitulation of human colonocytes in situ by human colon organoids in vitro and after orthotopic transplantation

Abstract Background The intestinal epithelium plays a central role in human health and disease, and several chronic inflammatory disorders associate with a weakened epithelial barrier. The organoid model allows the cultivation, expansion and analysis of non-immortalized intestinal epithelial cells and has been instrumental in studying epithelial behavior in homeostasis and disease. Recent advances in human organoid transplantation into mouse and human lay the base for models to study human epithelial cell behavior within the intestinal tissue context, and promise novel therapeutic approaches for diseases such as short bowel syndrome and inflammatory bowel disease. It remained unclear how organoid transplantation into the colon would affect epithelial phenotypes and protein expression, which is key to assess the suitability of this model to study human epithelial cells in situ and safety aspects upon transplantation into human recipients. Methods To address this, we employed state-of-the-art deep visual proteomics to compare human colonic epithelial stem and differentiated cells in vivo, upon transplantation in situ, and to organoids cultured in vitro. Results We find that organoids transplanted into the murine colon closely resemble human intestinal epithelial cells in vivo compared to organoids grown in vitro, indicating that organoid culture induces a transient shift in epithelial phenotypes, which is reversible upon reintroduction into the mucosa. Phenotypic differences between epithelial cells in vitro and in situ/in vivo were largely driven by hallmarks of high proliferation and lower functional differentiation in organoids, likely due to culture conditions. Conclusion Taken together, we demonstrate that transplanted epithelial cells in situ represent a physiological, relevant model for studying functional aspects of mature colonocytes compared to the organoid model. We furthermore show that the proliferative phenotype of organoids in vitro is a transient state driven by culture conditions, which is reversible upon reintroduction into the mucosal environment. This is an important consideration regarding the safety of organoid transplantation into humans as a potential therapeutic strategy.

A novel Trichinella spiralis serine proteinase disrupted gut epithelial barrier and mediated larval invasion through binding to RACK1 and activating MAPK/ERK1/2 pathway.

Gut epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism by which larval penetration of gut epithelium is not completely elucidated. Previous studies showed that proteases secreted by T. spiralis intestinal infective larvae (IIL) degraded tight junctions (TJs) proteins of gut epithelium and mediated larval invasion. A new T. spiralis serine proteinase (TsSPc) was identified in the IIL surface proteins and ES proteins, rTsSPc bound to the intestinal epithelial cell (IECs) and promoted larval invasion of IECs. The aim of this study was to characterize the interacted proteins of TsSPc and IECs, and to investigate the molecular mechanisms of TsSPc mediating larval invasion of gut mucosa. IIFT results showed natural TsSPc was detected in infected murine intestine at 6, 12 hours post infection (hpi) and 3 dpi. The results of GST pull-down, mass spectrometry (MS) and Co-IP indicated that rTsSPc bound and interacted specifically with receptor for activated protein C kinase 1 (RACK1) in Caco-2 cells. rTsSPc did not directly hydrolyze the TJs proteins. qPCR and Western blot showed that rTsSPc up-regulated RACK1 expression, activated MAPK/ERK1/2 pathway, reduced the expression levels of gut TJs (occludin and claudin-1) and adherent protein E-cad, increased the paracellular permeability and damaged the integrity of intestinal epithelial barrier. Moreover, the RACK1 inhibitor HO and ERK1/2 pathway inhibitor PD98059 abolished the rTsSPc activating ERK1/2 pathway, they also inhibited and abrogated the rTsSPc down-regulating expression of occludin, claudin-1 and E-cad in Caco-2 monolayer and infected murine intestine, impeded larval invasion and improved intestinal epithelial integrity and barrier function, reduced intestinal worm burdens and alleviated intestinal inflammation. rTsSPc bound to RACK1 receptor in gut epithelium, activated MAPK/ERK1/2 pathway, decreased the expression of gut epithelial TJs proteins and disrupted the epithelial integrity, consequently mediated T. spiralis larval invasion of gut epithelium. The results are valuable to understand T. spiralis invasion mechanism, and TsSPc might be regarded as a vaccine target against T. spiralis invasion and infection.

Effect of Vitamin D on the Proliferation and Barrier of Atrophic Vaginal Epithelial Cells.

Atrophic vaginitis is very common in postmenopausal women due to declining estrogen levels. Vitamin D plays an important role in promoting epithelial cell proliferation, migration and adhesion. We established a rat model of ovariectomy (OVX) induced atrophic vaginitis with the aim of investigating the effects of Vitamin D supplementation on the vaginal epithelial barrier. The results showed that ovariectomised rats had significantly higher vaginal pH, reduced Lactobacillus, significantly lower uterine and vaginal weights, and lower vaginal epithelial PCNA, occludin, and E-cadherin mRNA expression compared with sham-operated rats. Vitamin D supplementation could reduce the vaginal pH, promote the proliferation and keratinization of vaginal epithelial cells, enhance the expression of PCNA mRNA in vaginal tissues, and improve the vaginal and uterine atrophy. Vitamin D can also increase the expression of E-cadherin and occludin proteins in vaginal tissues, maintain the integrity of the vaginal epithelium, increase the number of Lactobacillus, and reduce pathogenic bacterial infections. In vitro experiments demonstrated that 1,25(OH)2D3 could promote the proliferation and migration of VK2/E6E7 vaginal epithelial cells and increase the expression of E-cadherin protein. In conclusion, we demonstrated that Vitamin D can regulate the expression of vaginal epithelial tight junction proteins, promotes cell proliferation, and improves vaginal atrophy due to estrogen deficiency.

Medication adjustment of afatinib and combination therapy with sitagliptin for alleviating afatinib-induced diarrhea in rats

Afatinib, as the first-line treatment for non-small cell lung cancer (NSCLC), causes severe gastrointestinal adverse reactions that greatly affect patients' quality of life and even potentially result in treatment discontinuation. Multiple dose adjustments and concomitant use of anti-diarrheal medications are commonly employed to manage diarrhea, also allowing for a recovery period between each adjustment. However, these approaches are based on empirical guidance and still have limitations. This study aims to explore reliable approaches to alleviate diarrhea by focusing on two strategies: adjusting the dosing regimen of afatinib itself and implementing combination therapy. In this study, we firstly revealed a dose-dependent relationship between afatinib-induced diarrhea and gastrointestinal epithelial damage, resulting in atrophy, reduced expression of tight junction proteins, and increased permeability. We further found that even after discontinuation of the medication, although the severity of diarrhea had improved to baseline, the tight junction proteins and permeability of the intestinal epithelium did not fully recover, and the pharmacokinetics studies showed that drug absorption significantly increased than normal. This indicated the recovery period was longer than expected and may accelerate the occurrence of subsequent episodes of diarrhea. Hence, it would be prudent to consider adjustments to the starting dose or the recovery interval. Furthermore, we initially investigated the relationship between DPP enzyme and afatinib-induced diarrhea and found a significant decrease in plasma DPP enzyme activity following afatinib-induced diarrhea. Subsequently, we conducted continuous treatment with sitagliptin and observed significant improvement in afatinib-induced diarrhea. We observed that sitagliptin can promote the production of anti-inflammatory factors, increase the expression of intestinal epithelial tight junction proteins, and improve intestinal microbiota, further validating the mechanism through the use of GLP-23-33 as GLP-2 receptor inhibitor. In conclusion, sitagliptin exhibits promising potential as a therapeutic option for managing afatinib-induced diarrhea. Taken together, our study provides valuable insights into alleviating afatinib-induced diarrhea through both afatinib medication adjustment and sitagliptin combination therapy.

Loricrin and Cytokeratin Disorganisation in Severe Forms of Periodontitis

ObjectiveThe aim of this research was to investigate the role of the cornified epithelium, the outermost layer of the oral mucosa, engineered to prevent water loss and microorganism invasion, in severe forms of periodontitis (stage III or IV, grade C). MethodsPorphyromonas gingivalis, a major periodontal disease pathogen, can affect cornified epithelial protein expression through chronic activation of signal transducer and activator of transcription 6 (Stat6). We used a mouse model, Stat6VT, that mimics this to determine the effects of barrier defect on P gingivalis–induced inflammation, bone loss, and cornified epithelial protein expression, and compared histologic and immunohistologic findings with tissues obtained from human controls and patients with stage III and IV, grade C disease. Alveolar bone loss in mice was assessed using micro-computerised tomography, and soft tissue morphology was qualitatively and semi-quantitatively assessed by histologic examination for several proteins, including loricrin, filaggrin, cytokeratin 1, cytokeratin 14, a proliferation marker, a pan-leukocyte marker, as well as morphologic signs of inflammation. Relative cytokine levels were measured in mouse plasma by cytokine array. ResultsIn the tissues from patients with periodontal disease, there were greater signs of inflammation (rete pegs, clear cells, inflammatory infiltrates) and a decrease and broadening of expression of loricrin and cytokeratin 1. Cytokeratin 14 expression was also broader and decreased in stage IV. P gingivalis–infected Stat6VT mice showed greater alveolar bone loss in 9 out of 16 examined sites, and similar patterns of disruption to human patients in expression of loricrin and cytokeratins 1 and 14. There were also increased numbers of leukocytes, decreased proliferation, and greater signs of inflammation compared with P gingivalis–infected control mice. ConclusionsOur study provides evidence that changes in epithelial organisation can exacerbate the effects of P gingivalis infection, with similarities to the most severe forms of human periodontitis.

Antibiotic-induced microbial depletion enhances murine small intestinal epithelial growth in a serotonin-dependent manner.

Regulation of small intestinal epithelial growth by endogenous and environmental factors is critical for intestinal homeostasis and recovery from insults. Depletion of the intestinal microbiome increases epithelial proliferation in small intestinal crypts, similar to the effects observed in animal models of serotonin potentiation. Based on prior evidence that the microbiome modulates serotonin activity, we hypothesized that microbial depletion-induced epithelial proliferation is dependent on host serotonin activity. A mouse model of antibiotic-induced microbial depletion (AIMD) was employed. Serotonin potentiation was achieved through either genetic knockout of the serotonin transporter (SERT) or pharmacologic SERT inhibition, and inhibition of serotonin synthesis was achieved with para-chlorophenylalanine. AIMD and serotonin potentiation increased intestinal villus height and crypt proliferation in an additive manner, but the epithelial proliferation observed after AIMD was blocked in the absence of endogenous serotonin. Using Lgr5-EGFP-reporter mice, we evaluated intestinal stem cell (ISC) quantity and proliferation. AIMD increased the number of ISCs per crypt and ISC proliferation compared to controls, and changes in ISC number and proliferation were dependent on the presence of host serotonin. Furthermore, western blotting demonstrated that AIMD reduced epithelial SERT protein expression compared to controls. In conclusion, host serotonin activity is necessary for microbial depletion-associated changes in villus height and ISC proliferation in crypts, and microbial depletion produces a functional serotonin-potentiated state through reduced SERT protein expression. These findings provide an understanding of how changes to the microbiome contribute to intestinal pathology and can be applied therapeutically.

Early Solid Diet Supplementation Influences the Proteomics of Rumen Epithelium in Goat Kids.

It is well known that solid diet supplementation in early life can significantly promote rumen development and metabolic function in young ruminants. However, the changes in the expressed proteome and related metabolism in rumen epithelium in response to a supplemented solid diet remain unclear. In this study, rumen epithelial tissue from goats in three diet regimes including milk replacer only (MRO), milk replacer supplemented concentrate (MRC), and milk replacer supplemented concentrate plus alfalfa pellets (MCA) were collected for measurement of the expression of epithelial proteins using proteomic technology (six per group). The results showed that solid diet significantly improved the growth performance of goats, enhanced the ability of rumen fermentation, and promoted the development of epithelial papilla (p < 0.05). Proteome analysis revealed the distinct difference in the expressed protein in the MRC and MCA group compared with the MRO group (42 upregulated proteins and 79 downregulated proteins in MRC; 38 upregulated proteins and 73 downregulated proteins in MCA). Functional analysis showed that solid diet supplementation activated a variety of molecular functions in the epithelium, including protein binding, ATP binding, structural constituent of muscle, etc., in the MRC and MCA groups. Meanwhile, the expression of proteins related to fatty acid metabolism, the PPAR signaling pathway, valine, leucine, and isoleucine degradation, and butanoate metabolism were upregulated, being stimulated by solid feed. In contrast, the proteins associated with carbohydrate digestion and absorption and glycosaminoglycan degradation were downregulated. In addition, the protein expression of enzymes involved in ketone body synthesis in the rumen was generally activated, which was caused by solid feed. In summary, solid feed promoted the development of rumen epithelium by changing the expression of proteins related to fatty acid metabolism, energy synthesis, and signal transduction. The ketone body synthesis pathway might be the most important activated pathway, and provides energy for rumen development.

Hepatotoxicity of polymeric proanthocyanidins is caused by translocation of bacterial lipopolysaccharides through impaired gut epithelium

Polymeric proanthocyanidins (P-PAC) induced hepatotoxicity in C57BL/6 mice. Mice were supplemented with P-PAC alone or with a mixture of probiotic bacteria (PB), Lactobacillus, Bifidobacterium, and Akkermansia muciniphila for 14 consecutive days. The liver tissues of sacrificed mice were analyzed by mass spectrometry to identify and quantify the P-PAC metabolites. Potential P-PAC metabolites, 2-hydroxyphenylacetic acid and pyrocatechol were detected in higher concentrations and 4-hydroxybenzoic acid was detected exclusively in the mice supplemented with P-PAC and PB. Supplementation with P-PAC alone or with PB caused no shift in the α-diversity of mice gut microbiota. P-PAC induced nonalcoholic steatohepatitis in mice through increasing liver exposure to intestinal bacterial lipopolysaccharides by reducing expression of gut epithelial tight junction proteins, claudin-3 and occludin. Lipopolysaccharide concentrations in the livers of mice supplemented with P-PAC were significantly high compared to the control mice. Furthermore, P-PAC downregulated the expressions of claudin-3 and claudin-4 tight junction proteins in cultured Caco-2 cell monolayers. PB biotransformed P-PAC into bioavailable metabolites and potentially reduced the toxicity of P-PAC. The toxicity of P-PAC and their synbiotics need to be critically evaluated for the safety of human consumption.

P040 Caspase-8 blockade attenuates NSAID-induced colitis and cell death via NLRP3 inflammasome and NF-κB activation

Abstract Background Non-steroidal anti-inflammatory drugs (NSAIDs) are believed to exacerbate symptoms and inflammation in patients with inflammatory bowel disease (IBD). Recent studies indicate the gut microbiota may play a role in NSAID-promoted inflammation. However, mechanism(s) underpinning NSAID-induced inflammation are not fully understood. In this study, using the piroxicam-accelerated model of colitis (PAC) in IL-10-/- mice, we examined the mechanism(s) regulating NSAID-induced colitis. Methods To establish whether microbiota plays a role in PAC, IL-10-/- mice were treated with streptomycin for 24hrs followed by feeding with 100 ppm piroxicam in their food for 9 days. In the inhibitor study, mice were injected intra-peritoneally with the caspase-8 inhibitor (Z-IETD-FMK, 10mg/kg) every other day starting before piroxicam-feeding. Colon tissue was collected on day 14 for analysis on inflammation and cell death markers by qRT-PCR, MesoScaleDiscovery (MSD)-assay, western blot, and histology. Faecal samples were collected at different time-points for 16S rRNA gene sequencing. Results A significantly higher expression of colonic inflammatory markers and a reduced expression of epithelial barrier proteins and elongated crypts were found in PAC-mice, which was independent of microbiota. Piroxicam feeding did not cause major changes in fecal microbiota diversity or composition on day 14. An activation of members of the NLRP3 inflammasome family including caspase-1 and IL-1b, Caspase-8, and the pro-apoptotic markers -cleaved caspase 3 and PARP- were associated with piroxicam-induced colitis. Inhibition of caspase-8 ameliorated NSAID-provoked colitis and apoptosis by preventing mitochondrial damage, and downregulating NF-κB and NLRP3 activation as well as reducing cleaved caspase-3 and PARP levels. Histology analysis revealed an improvement in colonic epithelial morphology associated with shorter crypts and reduced immune cell infiltration to the lamina propria. Conclusion Our data provides evidence on how NSAIDs can promote intestinal inflammation by activating caspase-8, NLRP3 inflammasome and NF-κB, resulting in induction of inflammation and apoptosis. Targeting Caspase-8 could be a potential treatment strategy for patients with NSAID-worsened symptoms and inflammation

Eribulin Treatment Promotes Re-expression of Estrogen Receptor in Endocrine Therapy-resistant Hormone Receptor-positive Breast Cancer Cells.

Hypoxia is significantly associated with the development of drug resistance, and endocrine therapy is ineffective against hormone receptor (HR)-positive breast cancer in hypoxic tumor environments. Eribulin has a unique anticancer effect in breast cancer cells and improves tumor hypoxia by vascular remodeling. Therefore, we investigated the effect of eribulin on HR-positive breast cancer cells that were resistant to endocrine blockade. We established hypoxia-resistant breast cancer cell lines by continuous culture in a hypoxic environment. Parental and hypoxia-resistant cell lines were treated with eribulin and/or tamoxifen, and estrogen receptor (ER)-, epithelial-mesenchymal transition-, and hypoxia-related gene and protein expression changes in each surviving cell line were assessed. In addition, proliferation was assessed after eribulin treatment in the parental and hypoxia-resistant cell lines. We also assessed the effect of eribulin in vivo using subcutaneous xenograft models. Hypoxia-resistant cell lines showed significantly decreased expression of epithelial and ER-related markers and exhibited a higher level of resistance to tamoxifen. Conversely, eribulin treatment increased epithelial and ER-related gene and protein expression in hypoxia-resistant cell lines and enhanced the anticancer effect of tamoxifen. In in vivo xenograft models, eribulin treatment of hypoxia- and tamoxifen-resistant tumors slightly induced the re-expression of ER. In addition, hypoxia-resistant tumors treated with eribulin tended to respond better to tamoxifen. Eribulin ameliorated the aggressive behavior caused by hypoxia and induced the re-expression of ER in hypoxia-resistant breast cancer cells. Eribulin treatment of HR-positive breast cancer may resensitize cells to hormone blockade.

Cimifugin suppresses type 2 airway inflammation by binding to SPR and regulating its protein expression in a non-enzymatic manner

BackgroundCimifugin is one of the main bioactive components of Yu-Ping-Feng-San, a well-known traditional Chinese medicine, which can effectively relieve Allergic asthma (AA) and atopic dermatitis and reduce recurrence in clinic. However, the underlying mechanism of cimifugin on AA is still unknown. PurposeIn the present study, we aimed to investigate the effect and mechanism of cimifugin on AA. Study designIn vivo and in vitro experimental studies were performed. MethodsThe effect of cimifugin on AA was demonstrated in vivo and in vitro. Sepiapterin reductase (SPR) was predicted as the most potent target of cimifugin in treating AA by reverse docking. Molecular docking and microscale thermophoresis (MST) were used to analyze the direct binding between cimifugin and SPR. Overexpression and interference of SPR were performed to verify whether targeting SPR is a key step of cimifugin in the treatment of AA. QM385, an inhibitor of SPR, was administrated in vivo and in vitro to evaluate the role of SPR in AA. Further, HPLC and cell-free direct hSPR enzyme activity assay were performed to research whether cimifugin regulated SPR by influencing the enzyme activity. Simultaneously, the inhibitors of protein degradation were used in vitro to explore the mechanism of cimifugin on SPR. ResultsWe found cimifugin effectively alleviated AA by reducing airway hyperresponsiveness, inhibiting type 2 cytokines-mediated airway inflammation, and restoring the expression of epithelial barrier proteins. Molecular docking predicted the direct binding ability of cimifugin to SPR, which was further verified by MST. Notably, the therapeutic effect of cimifugin on AA was dampened with SPR interfering, in contrast, the phenotypic features of AA were significantly alleviated with QM385 application both in vivo and in vitro. Interestingly, cimifugin showed no effect on the enzyme activity of SPR, as the level of its substrate sepiapterin was not affected with cimifugin treatment by cell-free enzyme activity assay. Furthermore, we found cimifugin could reduce SPR protein expression without affecting its mRNA expression probably through autophagosome pathway. ConclusionsTo our knowledge, we're reporting for the first time that cimifugin can suppresses type 2 airway inflammation to alleviate AA by directly binding to SPR and regulating its protein expression in a non-enzymatic manner.

Intestinal Epithelial Digestive, Transport, and Barrier Protein Expression Is Increased in Environmental Enteric Dysfunction

Environmental enteric dysfunction (EED) is characterized by malabsorption and diarrhea that result in irreversible deficits in physical and intellectual growth. We sought to define the expression of transport and tight junction proteins by quantitative analysis of duodenal biopsies from patients with EED. Biopsies from Pakistani children with confirmed EED diagnoses were compared to those from age-matched North American healthy controls, patients with celiac disease, and patients with nonceliac disease with villous atrophy or intraepithelial lymphocytosis. Expression of brush border digestive and transport proteins and paracellular (tight junction) proteins was assessed by quantitative multiplex immunofluorescence microscopy. EED was characterized by partial villous atrophy and marked intraepithelial lymphocytosis. Epithelial proliferation and enteroendocrine, tuft, and Paneth cell numbers were unchanged, but there was significant goblet cell expansion in EED biopsies. Expression of proteins involved in nutrient and water absorption and that of the basolateral Cl− transport protein NKCC1 were also increased in EED. Finally, the barrier–forming tight junction protein claudin-4 (CLDN4) was significantly upregulated in EED, particularly within villous enterocytes. In contrast, expression of CFTR, CLDN2, CLDN15, JAM-A, occludin, ZO-1, and E-cadherin was unchanged. Upregulation of a barrier–forming tight junction protein and brush border and basolateral membrane proteins that support nutrient and water transport in EED is paradoxical, as their increased expression would be expected to be correlated with increased intestinal barrier function and enhanced absorption, respectively. These data suggest that EED activates adaptive intestinal epithelial responses to enhance nutrient absorption but that these changes are insufficient to restore health.