Micronutrients at Supplemental Levels, Tight Junctions and Epithelial Barrier Function: A Narrative Review.

Disease modifiers, whether from cancer, sepsis, systemic inflammation, or microbial pathogens, all appear to induce epithelial barrier leak, with induced changes of the Tight Junctional (TJ) complex being pivotal to the process. This leak-and the ensuant breakdown of compartmentation-plays a central role in disease morbidity on many levels. Accumulation of lung water in the luminal compartment of airways was a major driver of morbidity and mortality in COVID-19 and is an excellent example of the phenomenon. Increasing awareness of the ability of micronutrients to improve basal barrier function and reduce barrier compromise in pathophysiology may prove to be a low-cost, safe, and easily administered prophylactic and/or therapeutic option amenable to large populations. The growing appreciation of the clinical utility of supplemental doses of Vitamin D in COVID-19 is but one example. This narrative review is intended to propose a general theory on how and why micronutrients-at levels above normal dietary intake-successfully remodel TJs and improve barrier function. It discusses the key difference between dietary/Recommended Daily Allowance (RDA) levels of micronutrients versus supplemental levels, and why the latter are needed in disease situations. It advances a hypothesis for why signal transduction regulation of barrier function may require these higher supplemental doses to achieve the TJ remodeling and other barrier element changes that are clinically beneficial.

Ginseng Oligopeptides Improve the Intestinal Physiology and Promote the Antioxidant Capacity of the Gut-on-a-Chip Model.

During ageing, the permeability of the intestinal barrier increases, the integrity of the intestinal barrier decreases, and the physiology of intestinal cells changes. Furthermore, intestinal inflammation and excessive oxidative stress are both likely to cause systemic diseases. Ginseng oligopeptides have a positive significant effect in terms of improving human health and delaying ageing, but their role in the ageing of the intestine has not been studied much. In our experiment, we constructed a gut-on-a-chip model and induced senescence of the chip with H2O2 so as to explore the effects of ginseng oligopeptides on the senescent intestine. The experimental results showed that ginseng oligopeptides had no obvious effects on the integrity of the intestine, including the TEER value and the expression of tight junction proteins. However, ginseng oligopeptides might have other positive effects, such as inhibiting excessive cell proliferation, promoting mucin secretion, and increasing the antioxidant capacity of the intestine, to improve intestinal health.

Sodium butyrate ameliorated diabetic nephropathy-associated tubulointerstitial inflammation by modulating the tight junctions of renal tubular epithelial cells.

As one of the most significant pathological changes of diabetic nephropathy (DN), tubulointerstitial fibrosis (TIF) had a close relationship with tubulointerstitial inflammation (TI), and the occurrence of TI could have resulted from the disrupted tight junctions (TJs) of renal tubular epithelial cells (RTECs). Studies have demonstrated that sodium butyrate (NaB), a typical short chain fatty acid (SCFA), played an important regulatory role in intestinal TJs and inflammation. In this study, our in vivo and in vitro results showed that accompanied by TI, renal tubular TJs were gradually disrupted in the process of DN-related TIF. In HG and LPS co-cultured HK-2 cells and db/db mice, NaB treatment regained the TJs of RTECs via the sphingosine 1-phosphate receptor-1 (S1PR1)/AMPK signaling pathway, relieving inflammation. Small interfering RNA of S1PR1, S1PR1 antagonist W146 and agonist SEW2871, and AMPK agonist AICAR were all used to further confirm the essential role of the S1PR1/AMPK signaling pathway in NaB's TJ protection in RTECs in vitro. Finally, NaB administration not only improved the renal function and TIF, but also relieved the TI of db/db mice. These findings suggested that the use of NaB might be a potential adjuvant treatment strategy for DN-associated TIF, and this protective effect was linked to the TJ modulation of RTECs via the S1PR1/AMPK signaling pathway, leading to the improvement of TI.

Elucidation the mechanism of the active ingredient imperatorin promoting drug absorption in cell model.

Imperatorin (IMP) is the main bioactive furanocoumarin of Angelicae dahuricae radix, which is a well-known traditional Chinese medicine. The purpose of this study was to elucidate the role of IMP in promoting absorption and the possible mechanism on the compatible drugs of Angelicae dahuricae radix. The influence of IMP on drugs' intestinal absorption was conducted by the Caco-2 cell model. The mechanism was studied by investigating the transcellular transport mode of IMP and its influence on P-glycoprotein (P-gp)-mediated efflux, protein expression of P-gp and tight junction, and cell membrane potential. The result showed IMP promoted the uptake of osthole, daidzein, ferulic acid, and puerarin and improved the transport of ferulic acid and puerarin in Caco-2 cells. The absorption-promoting mechanism of IMP might involve the reduction of the cell membrane potential, decrease of P-gp-mediated drug efflux and inhibition of the P-gp expression level in the cellular pathway, and the loosening of the tight junction protein by the downregulation of the expression levels of occludin and claudin-1 in the paracellular pathway. This study provides new insights into the understanding of the improved bioavailability of Angelicae dahuricae radix with its compatible drugs.

Effects of pectin's degree of methyl esterification on TLR2-mediated IL-8 secretion and tight junction gene expression in intestinal epithelial cells: influence of soluble TLR2.

This study investigates the anti-inflammatory effects of pectins with different degrees of methyl esterification (DM) on intestinal epithelial cells (IECs) expressing low and high levels of TLR2. It also studies the influence of soluble TLR2 (sTLR2) which may be enhanced in patients with inflammatory bowel syndrome on the inflammation-attenuating effects of pectins. Also, it examines the impact of pectins on tight junction gene expression in IECs. Lemon pectins with DM18 and DM88 were characterized, and their effects on TLR2-1-induced IL8 gene expression and secretion were investigated in low-TLR2 expressing Caco-2 and high-TLR2 expressing DLD-1 cells. The results demonstrate that both DM18 and DM88 pectins can counteract TLR2-1-induced IL-8 expression and secretion, with more pronounced effects observed in DLD-1 cells expressing high levels of TLR2. Furthermore, the presence of sTLR2 does not interfere with the attenuating effects of low DM18 pectin and may even support its anti-inflammatory effects in Caco-2 cells. The impact of pectins and sTLR2 on tight junction gene expression also demonstrates cell-type-dependent effects. Overall, these findings suggest that low DM pectins possess potent anti-inflammatory properties and may influence tight junction gene expression in IECs, thereby contributing to the maintenance of gut homeostasis.

Evaluating Prophylactic Effect of Bovine Colostrum on Intestinal Barrier Function in Zonulin Transgenic Mice: A Transcriptomic Study.

The intestinal barrier comprises a single layer of epithelial cells tightly joined to form a physical barrier. Disruption or compromise of the intestinal barrier can lead to the inadvertent activation of immune cells, potentially causing an increased risk of chronic inflammation in various tissues. Recent research has suggested that specific dietary components may influence the function of the intestinal barrier, potentially offering a means to prevent or mitigate inflammatory disorders. However, the precise mechanism underlying these effects remains unclear. Bovine colostrum (BC), the first milk from cows after calving, is a natural source of nutrients with immunomodulatory, anti-inflammatory, and gut-barrier fortifying properties. This novel study sought to investigate the transcriptome in BC-treated Zonulin transgenic mice (Ztm), characterized by dysbiotic microbiota, intestinal hyperpermeability, and mild hyperactivity, applying RNA sequencing. Seventy-five tissue samples from the duodenum, colon, and brain of Ztm and wild-type (WT) mice were dissected, processed, and RNA sequenced. The expression profiles were analyzed and integrated to identify differentially expressed genes (DEGs) and differentially expressed transcripts (DETs). These were then further examined using bioinformatics tools. RNA-seq analysis identified 1298 DEGs and 20,952 DETs in the paired (Ztm treatment vs. Ztm control) and reference (WT controls) groups. Of these, 733 DEGs and 10,476 DETs were upregulated, while 565 DEGs and 6097 DETs were downregulated. BC-treated Ztm female mice showed significant upregulation of cingulin (Cgn) and claudin 12 (Cldn12) duodenum and protein interactions, as well as molecular pathways and interactions pertaining to tight junctions, while BC-treated Ztm males displayed an upregulation of transcripts like occludin (Ocln) and Rho/Rac guanine nucleotide exchange factor 2 (Arhgf2) and cellular structures and interfaces, protein-protein interactions, and organization and response mechanisms. This comprehensive analysis reveals the influence of BC treatment on tight junctions (TJs) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway gene expressions. The present study is the first to analyze intestinal and brain samples from BC-treated Ztm mice applying high-throughput RNA sequencing. This study revealed molecular interaction in intestinal barrier function and identified hub genes and their functional pathways and biological processes in response to BC treatment in Ztm mice. Further research is needed to validate these findings and explore their implications for dietary interventions aimed at improving intestinal barrier integrity and function. The MGH Institutional Animal Care and Use Committee authorized the animal study (2013N000013).

EPs® 7630 Stimulates Tissue Repair Mechanisms and Modifies Tight Junction Protein Expression in Human Airway Epithelial Cells.

Airway epithelium repair after infection consists of wound repair, re-synthesis of the extracellular matrix (ECM), and tight junction proteins. In humans, EPs® 7630 obtained from Pelargonium sidoides roots reduces the severity and duration of acute respiratory tract infections. The effect of EPs® 7630 on tissue repair of rhinovirus-16 (RV-16) infected and control human airway epithelial cells was assessed for: (i) epithelial cell proliferation by manual cell counts, (ii) epithelial wound repair by "scratch assay", (iii) ECM composition by Western-blotting and cell-based ELISA, and (iv) epithelial tight junction proteins by Western-blotting. EPs® 7630 stimulated cell proliferation through cAMP, CREB, and p38 MAPK. EPs® 7630 significantly improved wound repair. Pro-inflammatory collagen type-I expression was reduced by EPs® 7630, while fibronectin was increased. Virus-binding tight junction proteins desmoglein2, desmocollin2, ZO-1, claudin1, and claudin4 were downregulated by EPs® 7630. The RV16-induced shift of the ECM towards the pro-inflammatory type was prevented by EPs® 7630. Most of the effects of EPs® 7630 on tissue repair and regeneration were sensitive to inhibition of cAMP-induced signaling. The data suggest that EPs® 7630-dependent modification of epithelial cell metabolism and function might underlie the faster recovery time from viral infections, as reported by others in clinical studies.

Butyrate restores the fat/lean mass ratio balance and energy metabolism and reinforces the tight junction-mediated intestinal epithelial barrier in prediabetic mice independently of its anti-inflammatory and epigenetic actions.

Tissue/cellular actions of butyrate on energy metabolism and intestinal barrier in normal metabolic conditions or prediabetes are still unclear. In this work, we investigated the beneficial effect of dietary supplementation with sodium butyrate on energy metabolism, body mass composition, and intestinal epithelial barrier mediated by tight junction (TJ) in chow diet-fed normal and high-fat diet (HF)-fed prediabetic mice, considering the well-known butyrate action as an epigenetic and inflammatory regulator. Butyrate significantly reduced the fat/lean mass ratio, slightly ameliorated dyslipidemia, restored oral glucose tolerance, and increased basal energy expenditure in prediabetic HF-fed mice but had no effect on control animals. Such effects were observed in the absence of significant alterations in the hypothalamic expression of orexigenic and anorexigenic genes and motor activity. Also, butyrate suppressed the whitening effect of HF on brown adipose tissue but did not affect cell bioenergetics in immortalized UCP1-positive adipocytes in vitro. Butyrate reinforced the intestinal epithelial barrier in HF-fed mice and in Caco-2 monolayers, which involved higher trafficking of TJ proteins to the cell-cell contact region of the intestinal epithelia, without affecting TJ gene expression or the acetylation level of histones H3 and H4 in vivo. All metabolic and intestinal effects of butyrate in prediabetic mice occurred in the absence of detectable changes in systemic or local inflammation, or alterations in endotoxemia markers. Butyrate has no effect on chow diet-fed mice but, in the context of HF-induced prediabetes, it prevents metabolic and intestinal dysfunctions independently of its anti-inflammatory and epigenetic actions.

The early impact of diets enriched with saturated and unsaturated fatty acids on intestinal inflammation and tight junctions.

The gut has been suggested as the first organ to be affected by unbalanced diets contributing to the obesogenic process. This study aimed to test a short time-course exposition model to a known pro- or anti-inflammatory enriched fatty diet to understand the early gut alterations. Male mice were exposed to the chow diet (CT), high-fat (HF) diet, or a high-fat diet partially replaced on flaxseed oil (FS), rich in omega-3 (ω3), for 14 days. HF and FS increased the total body weight mass compared with the CT group, but FS reduced the epididymal fat depot compared to HF. The bioinformatics from mice and human databases showed the Zo1-Ocln-Cldn7 tight junctions as the main protein-triad. In the ileum, the HF diet has increased IL1ß transcript and IL1ß, TNFα, and CD11b proteins, but reduced the tight junctions (Zo1, Ocln, and Cld7) compared to the CT group. Despite the FS diet being partially efficient in protecting the ileum against inflammation, the tight junctions were increased, compared to the HF group. The GPR120 and GPR40 receptors were unaffected by diets, but GPR120 was colocalized on the surface of ileum macrophages. The short period of a high-fat diet was enough to start the obesogenic process, ileum inflammation, and reduce the tight junctions. Flaxseed oil did not protect efficiently against dysmetabolism. Still, it increased the tight junctions, even without alteration on inflammatory parameters, suggesting the protection against gut permeability during early obesity development.

Dietary Leucine Improves Fish Intestinal Barrier Function by Increasing Humoral Immunity, Antioxidant Capacity, and Tight Junction.

This study attempted to evaluate the possible impact and mechanism of leucine (Leu) on fish intestinal barrier function. One hundred and five hybrid Pelteobagrus vachelli ♀ × Leiocassis longirostris ♂ catfish were fed with six diets in graded levels of Leu 10.0 (control group), 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 g/kg diet for 56 days. Results showed that the intestinal activities of LZM, ACP, and AKP and contents of C3, C4, and IgM had positive linear and/or quadratic responses to dietary Leu levels. The mRNA expressions of itnl1, itnl2, c-LZM, g-LZM, and ß-defensin increased linearly and/or quadratically (p < 0.05). The ROS, PC, and MDA contents had a negative linear and/or quadratic response, but GSH content and ASA, AHR, T-SOD, and GR activities had positive quadratic responses to dietary Leu levels (p < 0.05). No significant differences on the CAT and GPX activities were detected among treatments (p > 0.05). Increasing dietary Leu level linearly and/or quadratically increased the mRNA expressions of CuZnSOD, CAT, and GPX1α. The GST mRNA expression decreased linearly while the GCLC and Nrf2 mRNA expressions were not significantly affected by different dietary Leu levels. The Nrf2 protein level quadratically increased, whereas the Keap1 mRNA expression and protein level decreased quadratically (p < 0.05). The translational levels of ZO-1 and occludin increased linearly. No significant differences were indicated in Claudin-2 mRNA expression and protein level. The transcriptional levels of Beclin1, ULK1b, ATG5, ATG7, ATG9a, ATG4b, LC3b, and P62 and translational levels of ULK1, LC3Ⅱ/Ⅰ, and P62 linearly and quadratically decreased. The Beclin1 protein level was quadratically decreased with increasing dietary Leu levels. These results suggested that dietary Leu could improve fish intestinal barrier function by increasing humoral immunity, antioxidative capacities, and tight junction protein levels.

Efficacy of Qifu Lizhong enema prescription on intestinal mucosal tight junction function modulation of ulcerative colitis rat model.

OBJECTIVE: To investigate the efficacy and mechanism of Qifu Lizhong enema prescription(, QFLZ) on intervening ulcerative colitis (UC) rat model with TCM spleen and kidney insufficiency syndrome. METHODS: Seventy-two male Sprague-Dawley rats were randomly assigned to six groups: normal model, mesalazine, and QFLZ high, medium, and low dose groups, each with 12 rats. After 3 d of adaptation feeding, all groups except the normal group were induced using rhubarb decoction in combination with trinitrobenzene sulfonic acid (TNBS)/55 % ethanol to establish a UC rat model. Following successful modeling, the normal and model groups received daily saline enema, while the Chinese medicine and Western medicine groups received daily QFLZ and Mesalazine enema for 2 weeks respectively. The disease activity index score, hematoxylin and eosin staining, immunohistochemistry, and Western blotting were used to determine the expression of claudin 1, claudin 2, zonula occludens-1 protein (ZO-1), and F-actin proteins in each rat colon tissue following treatment. RESULTS: QFLZ significantly alleviated the structural disorganization in the form of epithelial glands in the intestinal mucosa of rats with UC and retarded the progression of the disease. The intestinal mucosal epithelial cells of UC rats showed decreased expression of claudin 1, ZO-1, F-actin ( 0.05), claudin 2 appeared elevated ( 0.05), which resulted in impaired TJ. Treatment with QFLZ resulted in elevated expression of claudin 1 ( 0.05), ZO-1 ( 0.05) and F-actin ( 0.05) and decreased expression of claudin 2 ( 0.05), which allowed for repair of the intestinal mucosal TJ, which in turn served as a treatment for UC. CONCLUSIONS: The mechanism of repairing TJ function and repairing the intestinal mucosal barrier by QFLZ may be associated with up-regulation of claudin 1, ZO-1, and F-actin levels, and down-regulation of claudin 2 expression level.

EGCG alleviated Mn exposure-caused carp kidney damage via trpm2-NLRP3-TNF-α-JNK pathway: Oxidative stress, inflammation, and tight junction dysfunction.

Manganese (Mn), an essential trace metal element in organisms. However, with extensive use of Mn in industry and agriculture, Mn becomes a heavy metal pollutant in water. (-)-epigallocatechin gallate (EGCG), an tea polyphenols, can alleviate metal toxicity. Kidney is an important detoxifying organ, but toxic mechanism of Mn to kidneys is unclear, which needs further research. Carp is an Asian important economical species for fisheries and a biological model for studying environmental toxicology. Thus, we established excess Mn and EGCG-supplemented carp model to explore molecular mechanism of EGCG alleviating Mn-caused carp kidney damage. In this experiment, we set a control group (the Con group), a Mn treatment group (the Mn group, 90 mg/L Mn), a EGCG supplement group (the EG group, 75 mg/kg EGCG), and a combined group (the Mn + EG group, 90 mg/L Mn and 75 mg/kg EGCG). Transcriptome, qRT-PCR, kit, and morphology method results indicated that excess Mn caused oxidative stress, inflammatory damage, and tight junction dysfunction in carp kidneys. Excess Mn-triggered oxidative stress caused tight junction dysfunction via trpm2-NLRP3-TNF-α-JNK pathway and inflammation. EGCG reversed the harm of Mn to fish through the above mechanism. The findings of this study provided the evidence of EGCG-alleviated Mn poisoning and offered new ideas for reducing heavy metal environmental pollution risk.

Baicalin Reduces Immune Cell Infiltration by Inhibiting Inflammation and Protecting Tight Junctions in Ischemic Stroke Injury.

Ischemic stroke is a serious health hazard that lacks effective treatment strategies. This study aims to investigate baicalin's effect on tight junctions and immune cell infiltration after ischemic stroke injury. Rat brain microvascular endothelial cells (BMECs) were treated with OGD/R to establish an in vitro model. Caspase-3, Bax, Bcl-2, zonula occludens-1 (ZO-1), occludin, claudin-5, tumor necrosis factor (TNF)-[Formula: see text], interleukin (IL)-6, inducible nitric oxide synthase (iNOS), Toll-like receptor (TLR) 2, TLR4, and nuclear factor-kappa B (NF-[Formula: see text]B) expressions were detected using qRT-PCR and western blotting. ZO-1, TNF-[Formula: see text], iNOS, IL6, CD31, and ZO-1 expressions were examined using immunofluorescence. A tube formation assay was performed to measure angiogenesis. An ischemia-reperfusion model in rats was established by middle cerebral artery occlusion. The infarct volume was observed using 2,3,5-triphenyltetrazolium chloride staining. TNF-[Formula: see text], iNOS, and IL6 levels in the serum were tested using ELISA. Flow cytometry was performed to examine immune cell inflammatory infiltration. Baicalin had no significant effect on the proliferation of normal BMECs. Baicalin inhibited apoptosis, protected against tight junction injury, and alleviated the inflammatory response in OGD/R-induced BMECs and IR rats, with the highest dose (25[Formula: see text][Formula: see text]g/mL) exerting a superior effect. Baicalin decreased the neurological function score, infarct volume, and brain water content, relieved brain morphological changes, and inhibited immune cell infiltration in vivo. In conclusion, baicalin could reduce BMECs apoptosis, protect tight junctions, and resist immune cell infiltration, thereby alleviating ischemic stroke. Our findings potentially provide a novel treatment strategy for ischemic stroke.

Selenium Deficiency Promotes the Expression of LncRNA-MORC3, Activating NLRP3-Caspase-1/IL-1ß Signaling to Induce Inflammatory Damage and Disrupt Tight Junctions in Piglets.

Selenium (Se), as a trace element, is widely found in animals in the form of selenomethionine, which can provide nutrition to the body and has anti-inflammatory effects to prevent inflammatory damage in animals. In the past decade, there have been many studies on piglet diseases caused by selenium deficiency; however, under Se deficiency, the relationship between LncRNA-MORC3, inflammatory injury, and tight junctions in piglets has not yet been studied. We established piglet selenium deficiency models divided into three groups and obtained small intestinal tissues after 35 days of feeding. Small intestinal epithelial IPEC-J2 cells were divided into three groups, and samples were collected after 24 h of culture for qPCR and Western blot experiments. First, we found that Se deficiency led to an increase in LncRNA-MORC3 expression in piglets in vivo and in vitro. We found that the binding site of NLRP3 on LncRNA-MORC3 and the expression trends of both were the same: Se deficiency increased the secretion of NLRP3 and the expression levels of the inflammatory factors Caspase-1, ASC, IL-1ß, IL-17, IL-6, IL-10, and TNF-α, which are related to the NLRP3-Caspase-1/IL-1ß signaling pathway. At the same time, Se deficiency decreased the expression levels of the tight junction factors ZO-1, Z0-2, Occludin, E-cadherin, and ZEB-1. This result showed that the tight junctions were disrupted. Herein, we demonstrated that Se deficiency promotes the expression of both LncRNA-MORC3 and inflammatory factors in piglets to activate the NLRP3-Caspase-1/IL-1ß signaling pathway and disrupt tight junctions. Ultimately, these factors lead to inflammatory damage in piglet small intestinal tissues.

Comparison of the Modulating Effect of Anthocyanin-Rich Sour Cherry Extract on Occludin and ZO-1 on Caco-2 and HUVEC Cultures.

Increased permeability of the epithelial and endothelial cell layers results in the onset of pathogenic mechanisms. In both cell types, cell-cell connections play a regulatory role in altering membrane permeability. The aim of this study was to investigate the modulating effect of anthocyanin-rich extract (AC) on TJ proteins in inflammatory Caco-2 and HUVEC monolayers. Distribution of Occludin and zonula occludens-1 (ZO-1) were investigated by immunohistochemical staining and the protein levels were measured by flow cytometry. The mRNA expression was determined by quantitative real-time PCR. The transepithelial electrical resistance (TEER) values were measured during a permeability assay on HUVEC cell culture. As a result of inflammatory induction by TNF-α, redistribution of proteins was observed in Caco-2 cell culture, which was reduced by AC treatment. In HUVEC cell culture, the decrease in protein and mRNA expression was more dominant during inflammatory induction, which was compensated for by the AC treatment. Overall, AC positively affected the expression of the examined cell-binding structures forming the membrane on both cell types.

Micronutrient Improvement of Epithelial Barrier Function in Various Disease States: A Case for Adjuvant Therapy.

The published literature makes a very strong case that a wide range of disease morbidity associates with and may in part be due to epithelial barrier leak. An equally large body of published literature substantiates that a diverse group of micronutrients can reduce barrier leak across a wide array of epithelial tissue types, stemming from both cell culture as well as animal and human tissue models. Conversely, micronutrient deficiencies can exacerbate both barrier leak and morbidity. Focusing on zinc, Vitamin A and Vitamin D, this review shows that at concentrations above RDA levels but well below toxicity limits, these micronutrients can induce cell- and tissue-specific molecular-level changes in tight junctional complexes (and by other mechanisms) that reduce barrier leak. An opportunity now exists in critical care-but also medical prophylactic and therapeutic care in general-to consider implementation of select micronutrients at elevated dosages as adjuvant therapeutics in a variety of disease management. This consideration is particularly pointed amidst the COVID-19 pandemic.

Effect of Houpo-Mahuang Decoction on aggravated asthma induced by cigarette smoke and the expression of TRPA1 and tight junctions in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cigarette smoke (CS) is a common environmental irritant and a risk factor for asthma, as it induces as well as aggravates asthmatic attacks. The injured airway epithelial tight junctions (TJs) aggravate asthma. CS can aggravate asthma by activating the transient receptor potential ankyrin A1 (TRPA1) channel and enhancing TJs destruction. Houpo Mahuang decoction (HPMHD) is a classic traditional Chinese prescription for the treatment of asthma. However, its underlying action mechanism is unclear. AIM OF THE STUDY: The present study aimed to evaluate the effect of HPMHD on the asthma phenotype and the regulation of TRPA1 and TJs in a CS-induced mouse model of aggravated asthma. MATERIALS AND METHODS: Under optimized chromatographic and mass spectrometry conditions, the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) technique was used to detect and analyze the major chemical components of HPMHD. C57BL/6 female mice were randomly divided into seven groups, viz, normal saline (NS) group, ovalbumin (OVA) + CS group, dexamethasone group, HPMHD high-dose group and low-dose groups, n-butanol extract group, and ethyl acetate extract group, with 10 mice in each group. OVA sensitization and challenge, and CS exposure were used to establish the aggravated asthma model. As the main indices to evaluate the protective effect of HPMHD, the eosinophils count in peripheral blood, percentages of inflammatory cells classified and the levels of interleukin (IL)-4, IL-5, IL-13 in the bronchoalveolar lavage fluid (BALF), airway responsiveness enhanced pause (Penh), and changes in lung histopathology were determined and compared among the groups. The mRNA and protein expression of TRPA1 and TJs in lung tissue was also examined. RESULTS: Using UPLC-QTOF-MS, the chemical components of HPMHD, including ephedrine, pseudoephedrine, laetrile, and amygdalin amide, were identified by 51 signal peaks. Compared with those in the NS group, the eosinophil number in the peripheral blood and the eosinophils and neutrophils percentages in BALF of the OVA + CS group were remarkably increased. Following the inhalation of 50 µl of acetylcholine chloride (ACH) at doses of 25 and 50 mg/mL, the Penh increased significantly (p < 0.01). Moreover, in the OVA + CS group, hematoxylin and eosin (H&E) staining of lung tissue showed a significant number of infiltrated inflammatory cells, increased mucus secretion in the lumen, damaged bronchial mucosa, increased thickness of tracheal wall, and increased score of lung damage (p < 0.01). The IL-4/5/13 levels were also remarkably increased (p < 0.01). The protein as well as gene expression of both ZO-1 and occludin decreased markedly in the lung tissue, while the expression of TRPA1 and claudin-2 was increased (p < 0.05, p < 0.01). Next, the OVA + CS group and the treatment groups were compared. The inflammatory cells, Penh value, and levels of IL-4/5/13 were significantly reduced, and less lung injury was observed in the treatment groups. The gene and protein levels of TRPA1 and TJs were corrected (p < 0.05, p < 0.01); the effects on the HPMHD high-dose and ethyl acetate extract groups were particularly remarkable. CONCLUSIONS: HPMHD reduced airway hyperresponsiveness, inflammatory cell recruitment and Th2 cytokine secretion in CS-induced aggravated asthma mice, in a manner potentially dependent on regulation of the expression of TRPA1 and TJ proteins. Both the n-butanol and ethyl acetate extracts contained the active ingredients, especially the ethyl acetate extract.

Direct inhibition of the first PDZ domain of ZO-1 by glycyrrhizin is a possible mechanism of tight junction opening of Caco-2 cells.

Glycyrrhizin (GL) is known to exhibit a variety of useful pharmacological activities, including anti-inflammation, anti-hepatotoxicity, and enhancement of intestinal drug absorption. GL has been reported to modify the assembly of actin filaments, thereby modulating tight junction (TJ) integrity, but the detailed molecular mechanisms of this remain unclear. In this study, we first found that GL binds to the first PDZ domain of zonula occludens-1 (ZO-1(PDZ1)) through NMR experiments. The structure of the GL-ZO-1(PDZ1) complex was then constructed using HADDOCK with the transferred nuclear Overhauser effect-based inter-hydrogen distance constraints as well as restrictions on the interfacial residues identified from 1H-15N HSQC spectral changes. We identified the relevant interactions between the glucuronate-2 moiety of GL and the carboxylate binding loop of the ligand binding site of ZO-1(PDZ1). We further examined the interaction of ZO-1(PDZ1) with glycyrrhetinic acid and with GA-3-monoglucuronide and observed a much lower affinity for each than for that with GL, with good agreement with the model. The other contacts found in the model were examined by using an amino acid substitution mutant of ZO-1(PDZ1). Finally, we reproduced the experiments reported by Sakai et al. in which high-dose GL prolonged the TJ-opening mediated with sodium deoxycholate as indicated by reduced transepithelial electrical resistance.

Gualou Xiebai Decoction ameliorates increased Caco-2 monolayer permeability induced by bile acids via tight junction regulation, oxidative stress suppression and apoptosis reduction.

Gualou Xiebai Decoction (GXD), a classic prescription, is widely used to dealing with inflammatory diseases in China for thousands of years. Abnormal metabolic state of bile acids (BAs) is confirmed to cause intestinal epithelial barrier dysfunction. In preliminary work, we observed that GXD could decrease intestinal permeability in hyperlipidemia mice. The present study aimed to explore the protective effect of GXD on intestinal mucosa in vitro. Caco-2 cell monolayer permeability among different groups was determined by measuring the concentrations of FITC-dextran in the lower compartments and transepithelial electrical resistance (TEER). Meanwhile, mRNA and protein expressions of tight junctions (TJs) were investigated. Generation of intracellular reactive oxygen species (ROS) and the ratio of cell apoptosis induced by BAs were assessed by fluorescence probe and flow cytometry. GXD was shown to keep the cell monolayer in low permeable status, increase TEER and mRNA and protein expressions of occludin (Ocln) and zonula occluden 2 (ZO2) remarkably in cells challenged with cholic acid (CA), deoxycholic acid (DCA) and glycocholic acid (GCA). However, no significant effects were uncovered against the pathological effects of taurocholic acid (TCA). Meanwhile, generation of ROS and increased levels of apoptotic cells caused by CA, DCA and GCA were dramatically decreased by GXD, which were not observed on TCA. GXD could significantly attenuate intestinal barrier dysfunction induced by BAs via TJs regulation, oxidative stress suppression and cell apoptosis decrease, but such effects and behind mechanisms differed among different kinds of BAs.

Nano-Bio Selenium Synthesized by Bacillus subtilis Modulates Broiler Performance, Intestinal Morphology and Microbiota, and Expression of Tight Junction's Proteins.

A green and ecofriendly bio-based synthesis of nano selenium particles was performed using the Bacillus subtilis and the products were characterized by field emission scanning electron microscope (FESEM), dynamic light scattering (DLS) and transmission electron microscopy (TEM) methods. Dietary treatments included a control diet nonsupplemented with selenium and control diet supplemented with different sources of selenium (sodium selenite, organic Se, and nano-bio Se), resulting in a total of 4 treatments with 6 replicates of 10 chicks. Broilers were assessed for performance measures, ileum morphometry, and microbial population and jejunum tight junction proteins' relative expression. The particle size of the synthesized selenium nanoparticles ranges 40 to 150 nm, with crystalline spherical shape. Inclusion of selenium increased body weight (BW) and improved FCR compared to the control diet (P < 0.05). Among the selenium sources, the highest BW were achieved in chicks fed sodium selenite or nano-bio Se. Selenium supplementation meaningfully (P < - 0.01) changed ileum morphology and reduced ileum microbiota. Inclusion of selenium increased the relative weight of the carcass, breast, and thigh and reduced the relative weight of the liver and bursa of Fabricius on day 42 (P < 0.01). The relative length of duodenum, jejunum, and ileum were increased on day 14 but reduced on day 42 by inclusion of selenium (P < 0.05). Supplementation of selenium increased (P < 0.01) the expression of claudin-1, occludin, and zonula occluden-1 and reduced (P < 0.01) the expression of claudin-5 and zonula occluden-2 on day 28. Inclusion of nano-bio selenium increased (P < 0.05) the expression of occludin, zonula occluden-1, and zonula occluden-2 and reduced (P < 0.05) the expression of claudin-5 compared to the organic selenium and sodium selenite on day 42. In conclusion, this data suggest feasibility of the biosynthesis of selenium nanoparticles by Bacillus subtilis. Additionally, the data reported herein demonstrate that nano-bio selenium can effectively improve performance and intestinal integrity compared to the common organic and inorganic sources of selenium.