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1.
Zhongguo Zhen Jiu ; 44(4): 449-454, 2024 Apr 12.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-38621733

RESUMO

OBJECTIVES: To observe the effects of moxibustion on intestinal barrier function and Toll-like receptor 4 (TLR4)/nuclear factor-κB p65 (NF-κB p65) signaling pathway in obese rats and explore the mechanism of moxibustion in the intervention of obesity. METHODS: Fifty-five Wistar rats of SPF grade were randomly divided into a normal group (10 rats) and a modeling group (45 rats). In the modeling group, the obesity model was established by feeding high-fat diet. Thirty successfully-modeled rats were randomized into a model group, a moxibustion group, and a placebo-control group, with 10 rats in each one. In the moxibustion group, moxibustion was applied at the site 3 cm to 5 cm far from the surface of "Zhongwan" (CV 12), with the temperature maintained at (46±1 ) ℃. In the placebo-control group, moxibustion was applied at the site 8 cm to 10 cm far from "Zhongwan" (CV 12), with the temperature maintained at (38±1) ℃. The intervention was delivered once daily for 8 weeks in the above two groups. The body mass and food intake of the rats were observed before and after intervention in each group. Using ELISA methool, the levels of serum triacylglycerol (TG), total cholesterol (TC) and lipopolysaccharide (LPS) were detected and the insulin resistance index (HOMA-IR) was calculated. HE staining was used to observe the morphology of colon tissue. The mRNA expression of zonula occludens-1 (ZO-1), Occludin, Claudin-1, TLR4 and NF-κB p65 in the colon tissue was detected by quantitative real-time PCR; and the protein expression of ZO-1, Occludin, Claudin-1, TLR4 and NF-κB p65 was detected by Western blot in the rats of each group. RESULTS: Compared with the normal group, the body mass, food intake, the level of HOMA-IR, and the serum levels of TC, TG and LPS were increased in the rats of the model group (P<0.01); those indexes in the moxibustion group were all reduced when compared with the model group and the placebo-control group respectively (P<0.01, P<0.05). Compared with the normal group, a large number of epithelial cells in the mucosa of colon tissue was damaged, shed, and the inflammatory cells were infiltrated obviously in the interstitium in the rats of the model group. When compared with the model group, in the moxibustion group, the damage of the colon tissue was recovered to various degrees and there were few infiltrated inflammatory cells in the interstitium, while, the epithelial injury of the colon tissue was slightly recovered and the infiltrated inflammatory cells in the interstitium were still seen in the placebo-control group. The mRNA and protein expressions of ZO-1, Occludin and Caudin-1 were decreased in the model group compared with those in the normal group (P<0.01). When compared with the model group and the placebo-control group, the mRNA and protein expressions of these indexes were increased in the moxibustion group (P<0.01, P<0.05). In the model group, the mRNA and protein expressions of TLR4 and NF-κB p65 were increased when compared with those in the normal group (P<0.01), and the mRNA and protein expressions of these indexes were reduced in the moxibustion group when compared with those in the model group and the placebo-control group (P<0.01). CONCLUSIONS: Moxibustion can reduce the body mass and food intake, regulate the blood lipid and improve insulin resistance in the rats of obesity. It may be related to alleviating inflammatory response through improving intestinal barrier function and modulating the intestinal TLR4/NF-κB p65 signaling pathway.


Assuntos
Resistência à Insulina , Moxibustão , Ratos , Animais , NF-kappa B/genética , NF-kappa B/metabolismo , Ratos Wistar , Receptor 4 Toll-Like/genética , Lipopolissacarídeos/metabolismo , 60435 , Ocludina/metabolismo , Claudina-1/metabolismo , Transdução de Sinais , Obesidade/genética , Obesidade/terapia , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
Vet Res ; 55(1): 49, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38594770

RESUMO

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.


Assuntos
Infecções por Flavobacteriaceae , Meningite , Doenças das Aves Domésticas , Riemerella , Animais , Barreira Hematoencefálica/metabolismo , Patos/metabolismo , Virulência , Fatores de Virulência/metabolismo , Ocludina/genética , Ocludina/metabolismo , Infecções por Flavobacteriaceae/veterinária , Riemerella/metabolismo , Meningite/veterinária , Colágeno/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
3.
J Ethnopharmacol ; 328: 117998, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38484956

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: According to ancient literature, Prunella vulgaris L. (P vulgaris) alleviates mastitis and has been used in China for many years; however, there are no relevant reports that confirm this or the mechanism of its efficacy. AIM OF THE STUDY: To explore the anti-acute mastitis effect and potential mechanism of P vulgaris extract. MATERIALS AND METHODS: First, the active ingredients and targets of P vulgaris against mastitis were predicted using network pharmacology. Next, the relevant active ingredients were enriched using macroporous resins and verified using UV and UPLC-Q-TOF-MS/MS. Lastly, a mouse model of acute mastitis was established by injecting lipopolysaccharides into the mammary gland and administering P vulgaris extract by oral gavage. The pathological changes in mammary tissue were observed by HE staining. Serum and tissue inflammatory factors were measured by ELISA method. MPO activity in mammary tissue was measured using colorimetry and MPO expression was detected by immunohistochemistry. The expression of tight junction proteins (ZO-1, claudin-3, and occludin) in mammary tissue was detected by immunofluorescence and Western blot. iNOS and COX-2 in mammary tissue were detected by Western blot. MAPK pathway and NF-κB pathway related proteins were also detected by Western blot. RESULTS: Network pharmacology predicted that phenolic acids and flavonoids in P vulgaris had anti-mastitis effects. The contents of total flavonoids and total phenolic acids in P vulgaris extract were 64.5% and 29.4%, respectively. UPLC-Q-TOF-MS/MS confirmed that P vulgaris extract contained phenolic acids and flavonoids. The results of animal experiments showed that P vulgaris extract reduced lipopolysaccharide-induced inflammatory edema, inflammatory cell infiltration, and interstitial congestion of mammary tissue. It also reduced the levels of serum and tissue inflammatory factors TNF-α, IL-6, and IL-1ß, and inhibited the activation of MPO. Furthermore, it downregulated the expression of MAPK and NF-κB pathway-related proteins. The expressions of ZO-1, occludin, and claudin-3 in mammary gland tissues were upregulated. CONCLUSIONS: P vulgaris extract can maintain the integrity of mammary connective tissue and reduce its inflammatory response to prevent acute mastitis. Its mechanism probably involves regulating NF-κB and MAPK pathways.


Assuntos
Mastite , Prunella , Humanos , Animais , Feminino , Camundongos , NF-kappa B/metabolismo , Lipopolissacarídeos/toxicidade , Lipopolissacarídeos/metabolismo , Transdução de Sinais , Leite/metabolismo , Ocludina/metabolismo , Claudina-3/metabolismo , Espectrometria de Massas em Tandem , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mastite/induzido quimicamente , Mastite/tratamento farmacológico , Mastite/metabolismo , Flavonoides/farmacologia
4.
J Ethnopharmacol ; 328: 117956, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38428658

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese herbal medicine Gegen Qinlian Decoction (GQD) has been clinically shown to be an effective treatment of ulcerative colitis (UC) in China. However, the underlying mechanism of GQD's anti-ulcerative colitis properties and its effect on gut microbiota still deserve further exploration. AIM OF THE STUDY: This study observed the regulatory effects of GQD on Th2/Th1 and Tregs/Th17 cells balance, the NOD-like receptor family pyrin domain containing 3 (NLRP3) infammasome and gut microbiota in TNBS-induced UC in BALB/c mice. MATERIALS AND METHODS: 61 main chemical compounds in the GQD were determined by UPLC-Q-TOF/MS. The UC BALB/c model was established by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and GQD was orally administered at low and high dosages of 2.96 and 11.83 g/kg/day, respectively. The anti-inflammatory effects of GQD for ulcerative colitis were evaluated by survival rate, body weight, disease activity index (DAI) score, colonic weight and index, spleen index, hematoxylin-eosin (HE) staining and histopathological scores. Flow cytometry was used to detect the percentage of CD4, Th1, Th2, Th17 and Tregs cells. The levels of Th1-/Th2-/Th17-/Tregs-related inflammatory cytokines and additional proinflammatory cytokines (IL-1ß, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iß, Occludin and Zonula occludens-1 (ZO-1) on colon tissues were detected by Western blot and RT-PCR. Transcriptome sequencing was performed using colon tissue and 16S rRNA gene sequencing was performed on intestinal contents. Fecal microbiota transplantation (FMT) was employed to assess the contribution of intestinal microbiota and its correlation with CD4 T cells and the NLRP3 inflammasome. RESULTS: GQD increased the survival rate of TNBS-induced UC in BALB/c mice, and significantly improved their body weight, DAI score, colonic weight and index, spleen index, and histological characteristics. The intestinal barrier dysfunction was repaired after GQD administration through promoting the expression of tight junction proteins (Occludin and ZO-1). GQD restored the balance of Th2/Th1 and Tregs/Th17 cells immune response of colitis mice, primarily inhibiting the increase in Th2/Th1 ratio and their transcription factor production (GATA3 and T-bet). Morever, GQD changed the secretion of Th1-/Th2-/Th17-/Tregs-related cytokines (IL-2, IL-12, IL-5, IL-13, IL-6, IL-10, and IL-17A) and reduced the expressions of IL-1ß, IL-18. Transcriptome results suggested that GQD could also remodel the immune inflammatory response of colitis by inhibiting NOD-like receptor signaling pathway, and Western blot, immunohistochemistry and RT-PCR further revealed that GQD exerted anti-inflammatory effects by inhibiting the NLRP3 inflammasome, such as down-regulating the expression of NLRP3, Caspase-1 and IL-1ß. More interestingly, GQD regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria like Helicobacter, Proteobacteria, and Mucispirillum, while the probiotic gut microbiota, such as Lactobacillus, Muribaculaceae, Ruminiclostridium_6, Akkermansia, and Ruminococcaceae_unclassified were increased. We further confirmed that GQD-treated gut microbiota was sufficient to relieve TNBS-induced colitis by FMT, involving the modulation of Th2/Th1 and Tregs/Th17 balance, inhibition of NLRP3 inflammasome activation, and enhancement of colonic barrier function. CONCLUSIONS: GQD might alleviate TNBS-induced UC via regulating Th2/Th1 and Tregs/Th17 cells Balance, inhibiting NLRP3 inflammasome and reshaping gut microbiota, which may provide a novel strategy for patients with colitis.


Assuntos
Colite Ulcerativa , Colite , Medicamentos de Ervas Chinesas , Microbioma Gastrointestinal , Humanos , Camundongos , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Medicamentos de Ervas Chinesas/efeitos adversos , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Interleucina-18/farmacologia , Interleucina-18/uso terapêutico , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células Th17 , Ocludina/metabolismo , RNA Ribossômico 16S/metabolismo , Camundongos Endogâmicos CBA , Colite/tratamento farmacológico , Citocinas/metabolismo , Trinitrobenzenos/metabolismo , Trinitrobenzenos/farmacologia , Trinitrobenzenos/uso terapêutico , Anti-Inflamatórios/farmacologia , Peso Corporal , Caspases/metabolismo , Modelos Animais de Doenças , Colo
5.
Biochem Biophys Res Commun ; 707: 149783, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38493746

RESUMO

Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.


Assuntos
Adesinas Bacterianas , Porphyromonas gingivalis , Animais , Camundongos , Humanos , Cisteína Endopeptidases Gingipaínas/metabolismo , Células CACO-2 , Porphyromonas gingivalis/fisiologia , Citosol/metabolismo , Ocludina/metabolismo , Adesinas Bacterianas/metabolismo
6.
Mol Med Rep ; 29(5)2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38488031

RESUMO

Ulcerative colitis (UC) is a chronic idiopathic inflammatory condition affecting the rectum and colon. Inflammation and compromisation of the intestinal mucosal barrier are key in UC pathogenesis. Resveratrol (Res) is a naturally occurring polyphenol that exhibits anti­inflammatory and antioxidant properties. Nuclear factor erythroid­2­related factor 2/heme oxygenase 1 (Nrf2/HO­1) pathway regulates occurrence and development of numerous types of diseases through anti­inflammatory and antioxidant activity. However, it is not clear whether Nrf2/HO­1 pathway is involved in the treatment of Res in UC. Therefore, the present study aimed to investigate whether Res modulates the Nrf2/HO­1 signaling pathway to attenuate UC in mice. Dextran sulfate sodium (DSS) was used to induce experimental UC in male C57BL/6J mice. Disease activity index (DAI) and hematoxylin eosin (H&E) staning was used to assessed the magnitude of colonic lesions in UC mice. ELISA) was utilized to quantify inflammatory cytokines (IL­6, IL­1ß, TNF­α and IL­10) in serum and colon tissues. Immunohistochemistry and Western blot were used to evaluate the expression levels of tight junction (TJ) proteins [zonula occludens (ZO)­1 and Occludin] in colon tissues. Pharmacokinetic (PK) parameters of Res were derived from TCMSP database. Networkpharmacology was employed to identify the biological function and pharmacological mechanism of Res in the process of relieving UC, and the key target was screened. The binding ability of Res and key target was verified by molecular docking. Finally, the effectiveness of key target was substantiated by Western blot. Res decreased DAI, ameliorated histopathological changes such as crypt loss, disappeatance of the mucosal epithelium, and inflammatory infiltration in mice. Additionally, Res decreased expression of pro­inflammatory cytokines IL­6, IL­1ß and TNF­α and increased anti­inflammatory factor IL­10 expression. Res also restored the decreased protein expression of ZO­1 and occludin after DSS treatment, increasing the integrity of the intestinal mucosal barrier. The PK properties of Res suggested that Res possesses the therapeutic potential for oral administration. Network pharmacology revealed that Res alleviated UC through anti­inflammatory and antioxidant pathways, and confirmed that Nrf2 has a high binding affinity with Res and is a key target of Res against UC. Western blotting demonstrated that Res treatment increased the protein levels of Nrf2 and HO­1. In conclusion, Res treatment activated the Nrf2/HO­1 pathway to decrease clinical symptoms, inflammatory responses, and intestinal mucosal barrier damage in experimental UC mice.


Assuntos
Experimentação Animal , Colite Ulcerativa , Colite , Masculino , Camundongos , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Resveratrol/farmacologia , Resveratrol/uso terapêutico , Interleucina-10/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Colo/patologia , Antioxidantes/metabolismo , Interleucina-6/metabolismo , Ocludina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Farmacologia em Rede , Simulação de Acoplamento Molecular , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Anti-Inflamatórios/uso terapêutico , Sulfato de Dextrana , Modelos Animais de Doenças , Colite/patologia
7.
Bull Exp Biol Med ; 176(4): 442-446, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38488962

RESUMO

We performed a comparative study of the effects of X-ray irradiation and bleomycin on the mRNA levels of E-cadherin and tight junction proteins (claudin-3, claudin-4, claudin-18, ZO-2, and occludin) in an alveolar epithelial cell line L2. Irradiation decreased claudin-4 levels and increased occludin levels, while the levels of other mRNAs remained unchanged. Bleomycin increased the expression levels of all proteins examined except claudin-3. Irradiation and bleomycin have different effects on the expression level of intercellular junction proteins, indicating different reactions triggered in alveolar epithelial cells and a great prospects of further comparative studies.


Assuntos
Células Epiteliais Alveolares , Junções Íntimas , Células Epiteliais Alveolares/metabolismo , Junções Íntimas/metabolismo , Ocludina/genética , Ocludina/metabolismo , Claudina-4/metabolismo , Claudina-3/metabolismo , Bleomicina/farmacologia , Bleomicina/metabolismo , Junções Intercelulares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Células Epiteliais
8.
Food Chem Toxicol ; 186: 114549, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38442786

RESUMO

The tight junctions (TJs) and barrier function of the intestinal epithelium are highly sensitive to radiation. However, polyphenols can be used to reverse the effects of radiation. Here, we investigated the effects of hesperidin (hesperetin-7-rhamnoglucoside) on X-ray-induced intestinal barrier dysfunction in human epithelial Caco-2 monolayers. To examine whether hesperidin mitigated the effects of X-ray exposure (2 Gy), cell survival was evaluated and intestinal barrier function was assessed by measuring the transepithelial flux, apparent permeability coefficient (Papp), and barrier integrity. Hesperidin improved the survival of Caco-2 cell monolayers and attenuated X-ray exposure-induced intestinal barrier dysfunction. For fluorescein transport experiments, transepithelial flux and Papp of fluorescein in control group were significantly elevated by X-ray, but were restored to near control by 10 µM hesperidin pretreatment. Further, X-ray exposure decreased the barrier integrity and TJ interruption by reducing TJ-related proteins occludin and claudin-4, whereas cell monolayers pretreated with hesperidin before X-ray exposure were reinstated to control level. It was concluded that hesperidin treatment before X-ray exposure alleviated X-ray-induced intestinal barrier dysfunction through regulation of TJ-related proteins. These results indicate that hesperidin prevents and mitigates X-ray-induced intestinal barrier dysfunction.


Assuntos
Gastroenteropatias , Hesperidina , Enteropatias , Humanos , Células CACO-2 , Hesperidina/farmacologia , Raios X , Mucosa Intestinal/metabolismo , Ocludina/metabolismo , Fluoresceínas/metabolismo , Fluoresceínas/farmacologia , Junções Íntimas , Permeabilidade
9.
Int J Nanomedicine ; 19: 1451-1467, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38371456

RESUMO

Background: Ischemic stroke (IS) causes tragic death and disability worldwide. However, effective therapeutic interventions are finite. After IS, blood-brain barrier (BBB) integrity is disrupted, resulting in deteriorating neurological function. As a novel therapeutic, extracellular vesicles (EVs) have shown ideal restorative effects on BBB integrity post-stroke; however, the definite mechanisms remain ambiguous. In the present study, we investigated the curative effects and the mechanisms of EVs derived from bone marrow mesenchymal stem cells and brain endothelial cells (BMSC-EVs and BEC-EVs) on BBB integrity after acute IS. Methods: EVs were isolated from BMSCs and BECs, and we investigated the therapeutic effect in vitro oxygen-glucose deprivation (OGD) insulted BECs model and in vivo rat middle cerebral artery occlusion (MCAo) model. The cell monolayer leakage, tight junction expression, and metalloproteinase (MMP) activity were evaluated, and rat brain infarct volume and neurological function were also analyzed. Results: The administration of two kinds of EVs not only enhanced ZO-1 and Occludin expressions but also reduced the permeability and the activity of MMP-2/9 in OGD-insulted BECs. The amelioration of the cerebral infarction, BBB leakage, neurological function deficits, and the increasing ZO-1 and Occludin levels, as well as MMP activity inhibition was observed in MCAo rats. Additionally, the increased levels of Caveolin-1, CD147, vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial growth factor A (VEGFA) in isolated brain microvessels were downregulated after EVs treatment. In vitro, the employment of Caveolin-1 and CD147 siRNA partly suppressed the expressions of VEGFR2, VEGFA and MMP-2/9 activity and reduced the leakage of OGD insulted BECs and enhanced ZO-1 and Occludin expressions. Conclusion: Our study firstly demonstrates that BEC and BMSC-EVs administrations maintain BBB integrity via the suppression of Caveolin-1/CD147/VEGFR2/MMP pathway after IS, and the efficacy of BMSC-EVs is superior to that of BEC-EVs.


Assuntos
Isquemia Encefálica , Vesículas Extracelulares , AVC Isquêmico , Ratos , Animais , Barreira Hematoencefálica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Caveolina 1/metabolismo , Ocludina/metabolismo , Células Endoteliais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Infarto da Artéria Cerebral Média , Isquemia Encefálica/metabolismo , Glucose/metabolismo , Vesículas Extracelulares/metabolismo
10.
Int J Mol Sci ; 25(3)2024 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-38338705

RESUMO

This study investigates the intricate composition and spatial distribution of tight junction complex proteins during early mouse neurulation. The analyses focused on the cranial neural tube, which gives rise to all head structures. Neurulation brings about significant changes in the neuronal and non-neuronal ectoderm at a cellular and tissue level. During this process, precise coordination of both epithelial integrity and epithelial dynamics is essential for accurate tissue morphogenesis. Tight junctions are pivotal for epithelial integrity, yet their complex composition in this context remains poorly understood. Our examination of various tight junction proteins in the forebrain region of mouse embryos revealed distinct patterns in the neuronal and non-neuronal ectoderm, as well as mesoderm-derived mesenchymal cells. While claudin-4 exhibited exclusive expression in the non-neuronal ectoderm, we demonstrated a neuronal ectoderm specific localization for claudin-12 in the developing cranial neural tube. Claudin-5 was uniquely present in mesenchymal cells. Regarding the subcellular localization, canonical tight junction localization in the apical junctions was predominant for most tight junction complex proteins. ZO-1 (zona occludens protein-1), claudin-1, claudin-4, claudin-12, and occludin were detected at the apical junction. However, claudin-1 and occludin also appeared in basolateral domains. Intriguingly, claudin-3 displayed a non-canonical localization, overlapping with a nuclear lamina marker. These findings highlight the diverse tissue and subcellular distribution of tight junction proteins and emphasize the need for their precise regulation during the dynamic processes of forebrain development. The study can thereby contribute to a better understanding of the role of tight junction complex proteins in forebrain development.


Assuntos
Proteínas de Junções Íntimas , Junções Íntimas , Camundongos , Animais , Proteínas de Junções Íntimas/metabolismo , Claudina-4/metabolismo , Claudina-1/metabolismo , Ocludina/metabolismo , Claudina-3/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Claudinas/metabolismo
11.
Front Cell Infect Microbiol ; 14: 1337439, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38390621

RESUMO

Introduction: The etiology of Escherichia coli in yaks, along with its drug resistance, results in economic losses within the yak breeding industry. The utilization of lactic acid bacteria treatment has emerged as a viable alternative to antibiotics in managing colibacillosis. Methods: To elucidate the therapeutic mechanisms of Lactobacillus against Escherichia coli-induced intestinal barrier damage in yaks, we employed yak epithelial cells as the experimental model and established a monolayer epithelial barrier using Transwell. The study encompassed four groups: a control group, a model group (exposed to E. coli O78), a low-dose Lactobacillus group (E. coli O78 + 1 × 105CFU LAB), and a high-dose Lactobacillus group (E. coli O78 + 1 × 107CFU LAB). Various techniques, including transmembrane resistance measurement, CFU counting, RT-qPCR, and Western Blot, were employed to assess indicators related to cell barrier permeability and tight junction integrity. Results: In the Model group, Escherichia coli O78 significantly compromised the permeability and tight junction integrity of the yak epithelial barrier. It resulted in decreased transmembrane resistance, elevated FD4 flux, and bacterial translocation. Furthermore, it downregulated the mRNA and protein expression of MUC2, Occludin, and ZO-1, while upregulating the mRNA expression and protein expression of FABP2 and Zonulin, thereby impairing intestinal barrier function. Contrastingly, Lactobacillus exhibited a remarkable protective effect. It substantially increased transmembrane resistance, mitigated FD4 flux, and reduced bacterial translocation. Moreover, it significantly upregulated the mRNA and protein expression of MUC2, Occludin, and ZO-1, while downregulating the mRNA and protein expression of FABP2 and Zonulin. Notably, high-dose LAB demonstrated superior regulatory effects compared to the low-dose LAB group. Discussion: In conclusion, our findings suggest that Lactobacillus holds promise in treating yak colibacillosis by enhancing mucin and tight junction protein expression. Furthermore, we propose that Lactobacillus achieves these effects through the regulation of Zonulin.


Assuntos
Infecções por Escherichia coli , Lactobacillus , Animais , Bovinos , Lactobacillus/genética , Mucosa Intestinal/microbiologia , Escherichia coli/genética , Ocludina/metabolismo , Infecções por Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , Junções Íntimas/metabolismo
12.
Theriogenology ; 219: 75-85, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38402700

RESUMO

Endometritis is a disease caused by a postpartum bacterial infection with a poor prognosis that primarily affects dairy cows. Three-dimensional organoids have been used as a model for endometritis, because they exhibit a structure comparable to that of the endometrium, demonstrating both expansibility and hormone responsiveness. These characteristics render them an ideal platform for in vitro investigations of endometrial diseases. Estradiol (E2) is an endogenous steroid hormone with demonstrated anti-inflammatory properties, and the objective of this study was to determine the mechanism by which E2 modulates the inflammatory response and the Wnt signal transduction pathway in bovine endometrial epithelial cells and organoids following E. coli infection. We present the techniques for isolating and culturing primary bovine endometrial epithelial cells (BEECs), and producing endometrial organoids. For the experiments, the endometrial epithelial cells and organoids were infected with E. coli for 1 h, followed by incubation with E2 for 12 h. The mRNA and protein expressions of the inflammation-related genes, IL-1ß, IL-6, TLR4, and NF-κB, as well as the Wnt pathway-related genes, Wnt4, ß-catenin, c-Myc, and CyclinD1, were assessed using real-time quantitative-PCR and western blotting, respectively. The CCK8 viable cell counting assay was utilized to determine the optimal concentration of the Wnt inhibitor, IWR-1. The mRNA and protein expression of Wnt pathway-related genes was assessed following IWR-1 treatment, while the expression levels of proliferation-associated genes (Ki67, PCNA) and barrier repair genes (occludin, claudin, and Zo-1) in BEECs and organoids were evaluated after E2 treatment. The results of this study show that mRNA expression of the inflammatory genes, IL-1ß, TLR4, and NF-κB (P < 0.05) decreased in BEECs following E2 treatment compared to the E. coli group. The protein expression of the IL-1ß, IL-6, TLR4 and NF-κB genes was also inhibited (P < 0.05). Similar results were observed in tests on the organoids. Our findings demonstrate that E2 significantly upregulates the expression of Wnt-related genes, including ß-catenin and c-Myc, while concurrently downregulating the expression of GSK3ß (P < 0.05). Next, we treated E. coli-infected BEECs and organoids with the Wnt inhibitor, IWR-1. Compared with E. coli and E. coli + E2, the expression of mRNA and protein from Wnt 4, ß-catenin, and CyclinD1 in E. coli + E2 and E. coli + IWR-1 was down-regulated (P < 0.05). The expression of the proliferation genes, Ki67, PCNA, and the tight junction genes, occludin, claudin1, and Zo-1, in organoids was significantly higher than that in BEECs (P < 0.05). In summary, we found strong potential for E2 mitigation of the E. coli-induced inflammatory response in BEECs and organoids, through activation of the Wnt pathway. In addition, the proliferation and repair capacity of organoids was much higher than that of BEECs.


Assuntos
Doenças dos Bovinos , Endometrite , Infecções por Escherichia coli , Feminino , Bovinos , Animais , Endometrite/veterinária , NF-kappa B/metabolismo , Via de Sinalização Wnt , Interleucina-6/metabolismo , Escherichia coli/metabolismo , Estradiol/farmacologia , Estradiol/metabolismo , Receptor 4 Toll-Like/metabolismo , beta Catenina , Antígeno Ki-67/metabolismo , Ocludina/metabolismo , Ocludina/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Endométrio/metabolismo , Células Epiteliais/metabolismo , Infecções por Escherichia coli/veterinária , Infecções por Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , Doenças dos Bovinos/metabolismo
13.
Toxicon ; 240: 107633, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38331107

RESUMO

As a global toxin invasive species, the whole herb of Ageratina adenophora (A. adenophora) contains various sesquiterpenes, which can cause various degrees of toxic reactions characterized by inflammatory damage when ingested by animals. Current studies on the toxicity of A. adenophora have focused on parenchymatous organs such as the liver and spleen, but few studies have been conducted on the intestine as the organ that is first exposed to A. adenophora and digests and absorbs its toxic components. In this study, after feeding goats with 40 % A. adenophora herb powder for 90 d, we found that the intestinal structure of goats showed pathological changes characterized, and the damage to the small intestinal segments was more severe than that of the large intestine. The MLCK/ROCK signaling pathway was activated, the cytoskeleton underwent centripetal contraction, the composition of tight junctions between intestinal epithelial cells was altered table, Occludin, Claudin-1 and Zonula occluden (ZO-1) amount was decreased, and the intestinal mechanical barrier was disrupted. The intestinal damage markers diamine oxidase (DAO) and D-lactate (D-LA) levels were elevated. In addition, we also found that intestinal bacteria translocate and enter the portal vein to colonize the liver and mesenteric lymph nodes. The expression of intestinal pro-inflammatory factors and anti-inflammatory factors was changed, the intestinal immune function was disrupted. The present study is the first to analyze the mechanism of poisoning of A. adenophora from the intestinal tract in compound-gastric animals.


Assuntos
Ageratina , Animais , Ageratina/metabolismo , Cabras , Intestinos , Ocludina/metabolismo , Transdução de Sinais , Mucosa Intestinal/metabolismo
14.
Immun Inflamm Dis ; 12(2): e1193, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38372468

RESUMO

INTRODUCTION: The intestinal tract serves as an innate barrier, safeguarding the internal milieu from microorganisms and toxins. Various intestinal inflammatory diseases have a strong association with intestinal barrier dysfunction. The primary functional cells within the intestinal tract, intestinal epithelial cells (IECs) and their tight junctions (TJs), are crucial in preserving the integrity of this mechanical barrier. Resveratrol (Res), a plant-derived phenolic compound, exhibits a range of health-promoting benefits attributed to its anti-inflammatory properties. This study aims to examine Res's efficacy in bolstering IECs barrier function. METHODS: Dextran sulfate sodium (DSS) was employed to induce barrier dysfunction in IECs. Inflammatory cytokines in supernatants (interleukin [IL]-6, IL-1ß, tumor necrotic factor [TNF]-α, and IL-10) were quantified via enzyme-linked immunosorbent assay (ELISA). Then we assessed monolayer integrity using transepithelial electrical resistance (TEER). TJ protein expression (zonula occludens [ZO]-1 and Occludin) in IECs was evaluated through immunofluorescence and Western blot analysis. Network pharmacology helped identify the biological processes, signaling pathways, and key targets involved in Res's mitigation of DSS-induced IECs barrier dysfunction. The efficacy of the primary target was further corroborated using Western blot. RESULTS: Res was shown to increase cell viability and IL-10 expression while reducing TNF-α, IL-6, and IL-1ß levels, thus mitigating the inflammatory response. It enhanced TEER values and upregulated TJ protein expression (ZO-1 and Occludin). Network pharmacology revealed that Res potentially targets the NFE2L2 (nuclear factor erythroid-2-related factor 2, Nrf2), a vital antioxidant factor. Significantly, Res augmented Nrf2 and heme oxygenase 1 (HO-1) protein levels, counteracting oxidative stress in the IECs barrier dysfunction model. CONCLUSION: Overall, our findings suggested that Res ameliorated DSS-induced IECs barrier dysfunction by activating Nrf2/HO-1 pathway, showcasing significant therapeutic potential in the early stages of colitis.


Assuntos
Interleucina-10 , Mucosa Intestinal , Humanos , Células CACO-2 , Sulfato de Dextrana/toxicidade , Heme Oxigenase-1/metabolismo , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ocludina/metabolismo , Resveratrol/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
15.
Phytomedicine ; 126: 155254, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38342016

RESUMO

BACKGROUND: The gut-brain axis (GBA) plays a central role in cerebral ischaemia-reperfusion injury (CIRI). Rhubarb, known for its purgative properties, has demonstrated protective effects against CIRI. However, it remains unclear whether this protective effect is achieved through the regulation of the GBA. AIM: This study aims to investigate the mechanism by which rhubarb extract improves CIRI by modulating the GBA pathway. METHODS: We identified the active components of rhubarb extract using LC-MS/MS. The model of middle cerebral artery occlusion (MCAO) was established to evaluate the effect of rhubarb extract. We conducted 16S rDNA sequencing and untargeted metabolomics to analyze intestinal contents. Additionally, we employed HE staining, TUNEL staining, western blot, and ELISA to assess intestinal barrier integrity. We measured the levels of inflammatory cytokines in serum via ELISA. We also examined blood-brain barrier (BBB) integrity using Evans blue (EB) penetration, transmission electron microscopy (TEM), western blot, and ELISA. Neurological function scores and TTC staining were utilized to evaluate neurological outcomes. RESULTS: We identified twenty-six active components in rhubarb. Rhubarb extract enhanced α-diversity, reduced the abundance of Enterobacteriaceae, and partially rectified metabolic disorders in CIRI rats. It also ameliorated pathological changes, increased the expressions of ZO-1, Occludin, and Claudin 1 in the colon, and reduced levels of LPS and d-lac in serum. Furthermore, it lowered the levels of IL-1ß, IL-6, IL-10, IL-17, and TNF-α in serum. Rhubarb extract mitigated BBB dysfunction, as evidenced by reduced EB penetration and improved hippocampal microstructure. It upregulated the expressions of ZO-1, Occludin, Claudin 1, while downregulating the expressions of TLR4, MyD88, and NF-κB. Similarly, rhubarb extract decreased the levels of IL-1ß, IL-6, and TNF-α in the hippocampus. Ultimately, it reduced neurological function scores and cerebral infarct volume. CONCLUSION: Rhubarb effectively treats CIRI, potentially by inhibiting harmful bacteria, correcting metabolic disorders, repairing intestinal barrier function, alleviating BBB dysfunction, and ultimately improving neurological outcomes.


Assuntos
Isquemia Encefálica , Doenças Metabólicas , Fármacos Neuroprotetores , Traumatismo por Reperfusão , Rheum , Ratos , Animais , Neuroproteção , Rheum/metabolismo , Ocludina/metabolismo , Interleucina-6 , Fator de Necrose Tumoral alfa/genética , Eixo Encéfalo-Intestino , Cromatografia Líquida , Claudina-1 , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Espectrometria de Massas em Tandem , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Azul Evans/uso terapêutico , Traumatismo por Reperfusão/metabolismo , Doenças Metabólicas/tratamento farmacológico , Infarto da Artéria Cerebral Média/tratamento farmacológico
16.
Vet Microbiol ; 291: 110013, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38364468

RESUMO

Potassium diformate (KDF) is a kind of formate, which possesses the advantages of antimicrobial activity, growth promotion and preventing diarrhea in weaned piglets. However, the researches of KDF in animal production mostly focused on apparent indexes such as growth performance and the mechanisms of KDF on intestinal health have not been reported. Thus, porcine small intestinal epithelial cells (IPEC-J2) infected with Enterohemorrhagic Escherichia coli (EHEC) was used to investigate the role of KDF on alleviating intestinal inflammation in this study. The 0.125 mg/mL KDF treated IPEC-J2 cells for 6 h and IPEC-J2 cells challenged with 5 × 107 CFU/mL EHEC for 4 h were confirmed as the optimum concentration and time for the following experiment. The subsequent experiment was divided into four groups: control group (CON), EHEC group, KDF group, KDF+EHEC group. The results showed that KDF increased the cell viability and the gene expression levels of SGLT3 and TGF-ß, while decreased the content of IL-1ß compared with the CON group. The cell viability and the gene expressions of SGLT1, SGLT3, GLUT2, Claudin-1, Occludin and TGF-ß, and the protein expression of ZO-1 in EHEC group were lower than those in CON group, whereas the gene expressions of IL-1ß, TNF, IL-8 and TLR4, and the level of phosphorylation NF-кB protein were increased. Pretreatment with KDF reduced the content of IgM and IL-1ß, the gene expressions of IL-1ß, TNF, IL-8 and TLR4 and the level of phosphorylation NF-кB protein, and increased the gene expression of TGF-ß and the protein expression of Occludin in IPEC-J2 cells infected EHEC. In conclusion, 0.125 mg/mL KDF on IPEC-J2 cells for 6 h had the beneficial effects on ameliorating the intestinal inflammation because of reduced pro-inflammatory cytokines and enhanced anti-inflammatory cytokines through regulating NF-кB signaling pathway under the EHEC challenge.


Assuntos
Escherichia coli Êntero-Hemorrágica , Doenças dos Suínos , Animais , Suínos , Ocludina/genética , Ocludina/metabolismo , Escherichia coli Êntero-Hemorrágica/metabolismo , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Receptor 4 Toll-Like , Linhagem Celular , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/veterinária , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Mucosa Intestinal , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/metabolismo
17.
Food Funct ; 15(2): 481-492, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38197139

RESUMO

Studies have shown that dietary polysaccharides, which are widely present in natural foods, have an important impact on the intestinal mucosal barrier. Dietary polysaccharides can maintain the intestinal barrier function through multiple mechanisms. The intestinal barrier is composed of mechanical, chemical, immune, and biological barriers, and dietary polysaccharides, as a bioactive component, can promote and regulate these four barriers. Dietary polysaccharides can enhance the expression of tight junction proteins and mucins such as occludin-1 and zonula occludens-1 (ZO-1) between intestinal epithelial cells, inhibit inflammatory response and oxidative stress, increase the growth of beneficial bacteria, produce beneficial metabolites such as short chain fatty acids (SCFAs), and promote the proliferation and metabolism of immune cells. Given the critical role of the intestinal mucosal system in health and disease, the protective effects of dietary polysaccharides may be potentially valuable for the prevention and treatment of gut-related diseases. Therefore, it is of great significance to further study the mechanism and application prospects of the intestinal mucosal barrier derived from plant, animal, fungal and bacterial sources.


Assuntos
Mucosa Intestinal , Polissacarídeos , Animais , Polissacarídeos/farmacologia , Polissacarídeos/metabolismo , Mucosa Intestinal/metabolismo , Células Epiteliais , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo , Ocludina/genética , Ocludina/metabolismo
18.
PLoS Negl Trop Dis ; 18(1): e0011872, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38190388

RESUMO

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDING: 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. CONCLUSIONS: 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.


Assuntos
Trichinella spiralis , Triquinelose , Humanos , Animais , Camundongos , Larva/fisiologia , Serina Proteases/genética , Células CACO-2 , Claudina-1/metabolismo , Sistema de Sinalização das MAP Quinases , Ocludina/metabolismo , Proteínas de Helminto/metabolismo , Células Epiteliais/metabolismo , Camundongos Endogâmicos BALB C , Mucosa Intestinal/metabolismo , Receptores de Quinase C Ativada/metabolismo , Proteínas de Neoplasias/genética
19.
Sci Rep ; 14(1): 393, 2024 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-38172276

RESUMO

Boron is an essential trace element with roles in growth, development, and physiological functions; however, its mechanism of action is still unclear. In this study, the regulatory roles of the PI3K/Akt signaling pathway on boron-induced changes in barrier function, proliferation, and apoptosis in rat intestinal epithelial cells were evaluated. Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein expression levels of PCNA were higher, while the proportions of cells in the G0/G1 and S phases, apoptosis rate, and caspase-3 mRNA and protein expression levels were lower in cells treated with 0.8 mmol/L boron than in control IEC-6 cells (P < 0.01 or P < 0.05). However, 40 mmol/L boron decreased ZO-1 and Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein levels of PCNA and increased the apoptosis rate and caspase-3 mRNA expression (P < 0.01 or P < 0.05). After specifically blocking PI3K and Akt signals (using LY294002 and MK-2206 2HCL), 0.8 mmol/L boron had no effects on Occludin, PCNA level, apoptosis rates, and caspase-3 levels (P < 0.05); however, the proliferation rate and PCNA levels decreased significantly (P < 0.01 or P < 0.05). The addition of 40 mmol/L boron did not affect ZO-1 and Occludin levels and did not affect the apoptosis rate or PCNA and caspase-3 levels. These results suggested that the PI3K/Akt signaling pathway mediates the effects of low-dose boron on IEC-6 cells.


Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Ratos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Caspase 3/metabolismo , Boro/farmacologia , Boro/metabolismo , Ocludina/genética , Ocludina/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proliferação de Células , Transdução de Sinais , Células Epiteliais/metabolismo , Apoptose , RNA Mensageiro/metabolismo
20.
Int J Pharm ; 652: 123854, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38280499

RESUMO

Borneol is an aromatic traditional Chinese medicine that can improve the permeability of the blood-brain barrier (BBB), enter the brain, and promote the brain tissue distribution of many other drugs. In our previous study, borneol-metformin hydrochloride water/oil/water composite submicron emulsion (B-Met-W/O/W SE) was prepared using borneol and SE to promote BBB penetration, which significantly increased the brain distribution of Met. However, the dynamic images, transport pathway (uptake and efflux), promotion of BBB permeability, and mechanisms of B-Met-W/O/W SE before and after entering cells have not been clarified. In this study, rhodamine B and coumarin-6 were selected as water-soluble and oil-soluble fluorescent probes to prepare B-Met-W/O/W dual-fluorescent SE (B-Met-W/O/W DFSE) with concentric circle imaging. B-Met-W/O/W SE can be well taken up by brain microvascular endothelial cells (BMECs). The addition of three inhibitors (chlorpromazine hydrochloride, methyl-ß-cyclodextrin, and amiloride hydrochloride) indicated that its main pathway may be clathrin-mediated and fossa protein-mediated endocytosis. Meanwhile, B-Met-W/O/W SE was obviously shown to inhibit the efflux of BMECs. Next, BMECs were cultured in the Transwell chamber to establish a BBB model, and Western blot was employed to detect the protein expressions of Occludin, Zona Occludens 1 (ZO-1), and p-glycoprotein (P-gp) after B-Met-W/O/W SE treatment. The results showed that B-Met-W/O/W SE significantly down-regulated the expression of Occludin, ZO-1, and P-gp, which increased the permeability of BBB, promoted drug entry into the brain through BBB, and inhibited BBB efflux. Furthermore, 11 differentially expressed genes (DEGs) and 7 related signaling pathways in BMECs treated with B-W/O/W SE were detected by transcriptome sequencing and verified by quantitative real-time polymerase chain reaction (qRT-PCR). These results provide a scientific experimental basis for the dynamic monitoring, transmembrane transport mode, and permeation-promoting mechanism of B-Met-W/O/W SE as a new brain-targeting drug delivery system.


Assuntos
Barreira Hematoencefálica , Canfanos , Células Endoteliais , Barreira Hematoencefálica/metabolismo , Ocludina/metabolismo , Células Endoteliais/metabolismo , Fluorescência
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