OBJECTIVES: To investigate the therapeutic mechanism of nodakenin for Crohn's disease (CD)-like colitis in mice. METHODS: Using a colonic organoid model with lipopolysaccharide (LPS)- and ATP-induced pyroptosis, we investigated the effects of nodakenin on pyroptosis, intestinal barrier function and inflammatory response by detecting key pyroptosis-regulating factors and assessing changes in permeability and pro-inflammatory factors. In a mouse model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis, the therapeutic effect of nodakenin was evaluated by measuring changes in body weight, DAI score, colonic histopathologies, inflammation score, intestinal barrier function and intestinal epithelial cell pyroptosis. The mechanism of nodakenin protection against pyroptosis of intestinal epithelial cells was explored using network pharmacology analysis and in vivo and in vitro experiments. RESULTS: In LPS- and ATP-induced colonic organoids, treatment with nodakenin significantly inhibited the expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11, improved intestinal FITC-dextran (FD4, 4000) permeability, and decreased the levels of IL-1β and IL-18. In the mouse model of TNBS-induced colitis, nodakenin treatment significantly alleviated weight loss, reduced DAI score, inflammatory cell infiltration and inflammation score, and decreased serum FD4 and I-FABP levels and bacteria translocation to the mesenteric lymph nodes, spleen and liver. The mice with nodakenin treatment had also lowered expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11 in the intestinal mucosa. Network pharmacology analysis suggested that the inhibitory effect of nodakenin on colitis was associated with the PI3K/Akt pathway. In both the colonic organoid model and mouse models of colitis, nodakenin effectively inhibited the activation of the PI3K/Akt pathway, and the application of IGF-1, a PI3K/Akt pathway activator, strongly attenuated the protective effect of nodakenin against intestinal epithelial cell pyroptosis and intestinal barrier dysfunction. CONCLUSIONS Nodakenin protects intestinal barrier function and alleviates CD-like colitis in mice at least partly by inhibiting PI3K/Akt signaling to reduce intestinal epithelial cell pyroptosis.
Animals ; Pyroptosis/drug effects* ; Mice ; Trinitrobenzenesulfonic Acid ; Colitis/drug therapy* ; Epithelial Cells/drug effects* ; Intestinal Mucosa/cytology* ; Disease Models, Animal ; Coumarins/pharmacology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Crohn Disease/drug therapy*

OBJECTIVES: To investigate the therapeutic effects of cimifugin on Crohn's disease (CD)-like colitis in mice and its possible mechanism. METHODS: Thirty adult male C57BL/6 mice were randomized equally into control group, 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis model group, and cimifugin treatment (daily gavage at 12.5 mg/kg) group. The therapeutic effect of cimifugin was evaluated by observing changes in body weight, disease activity index (DAI) scores, colon length, histopathological inflammation scores, and inflammatory cytokine levels in the colonic mucosa. Intestinal barrier integrity in the mice was assessed using immunofluorescence assay and Western blotting for claudin-1 and ZO-1; T-helper (Th) cell subset ratios in the mesenteric lymph nodes were analyzed with flow cytometry. Network pharmacology, KEGG enrichment analysis and molecular docking were used to predict the targets of cimifugin and analyze the key pathways and cimifugin-MAPK protein interactions, which were validated by Western blotting in the mouse models. RESULTS: In mice with TNBS-induced colitis, cimifugin treatment significantly attenuated body weight loss and colon shortening, lowered DAI and histopathological scores, decreased IFN-γ and IL-17 levels, and increased IL-4 and IL-10 levels in the colonic mucosa. Cimifugin treatment also significantly improved TNBS-induced claudin-1 dislocation and reduction of goblet cells, upregulated claudin-1 and ZO-1 expressions, reduced Th1 and Th17 cell percentages, and increased Th2 and Treg cell percentages in the colonic mucosa of the mice. KEGG analysis suggested a possible connection between the effect of cimifugin and MAPK signaling, and molecular docking showed strong binding affinity between cimifugin and MAPK core proteins. Western blotting demonstrated significantly decreased phosphorylation levels of JNK, ERK, and p38 in the colonic mucosa of cimifugin-treated mouse models. CONCLUSIONS Cimifugin alleviates TNBS-induced CD-like colitis by repairing intestinal barrier damage and restoring Th1/Th2 and Th17/Treg balance via suppressing MAPK pathway activation.
Animals ; Mice, Inbred C57BL ; Male ; Mice ; Crohn Disease/immunology* ; Colitis/immunology* ; MAP Kinase Signaling System/drug effects* ; Trinitrobenzenesulfonic Acid ; T-Lymphocytes, Helper-Inducer/drug effects* ; Intestinal Mucosa ; Disease Models, Animal

Objective To investigate the role and mechanism of afzelin(AFZ)in treating Crohn's disease-like colitis.Methods A mouse model of 2,4,6-trinitrobenzene sulfonic acid-induced colitis was established to assess the effect of AFZ on experimental colitis in vivo.A Caco-2 cell model of tumor necrosis factor(TNF)-α-induced inflammation was established to evaluate the effects of AFZ on the intestinal barrier function,intestinal epithelial cell apoptosis,and mitochondrial function in vitro.The animal and cell experiments were performed to validate the regulatory role of the adenosine monophosphate-activated protein kinase(AMPK)/silent information regulater 1(SIRT1)/peroxisome proliferator-activated receptor gamma coactivator(PGC)-1α pathway in the treatment of colitis with AFZ.Results AFZ reduced the disease activity index(P=0.003),weight loss(P<0.001),colon shortening(P<0.001),inflammation score(P=0.002),pro-inflammatory cytokine release(interleukin-6:P<0.001;TNF-α:P=0.010),and intestinal barrier permeability(fluorescein isothiocyanate dextran 4:P<0.001;intestinal-type fatty acid-binding protein:P=0.013).Meanwhile,AFZ increased the colonic transepithelial electric resistance(P=0.001),reduced bacterial translocation(P<0.001),and promoted the localization and up-regulated the expression of tight junction proteins [zonula occluden-1(P=0.005) and Claudin-1(P=0.024)].AFZ exerted a protective effect on the Caco-2 cells exposed to TNF-α in terms of intestinal epithelial cell permeability(P=0.017),transepithelial electric resistance(P=0.014),and tight junction protein[zonula occluden-1(P=0.014) and Claudin-1(P=0.006)] localization and expression.Furthermore,the cell and animal experiments confirmed that AFZ reduced the percentage of apoptosis(P<0.001,P=0.013)and the expression of cleaved-caspase 3(P=0.028,P=0.004)and Bax(P=0.004,P=0.020),and upregulated the Bcl2(P=0.020,P=0.006)level in intestinal epithelial cells.Additionally,AFZ increased the number of mitochondria,mitochondrial membrane potential,and copy number of mitochondrial DNA(P=0.007)in intestinal epithelial cells,while enhancing the activities of mitochondrial respiratory chain complex Ⅰ(P=0.005)and complex Ⅳ(P=0.001).The activation of the AMPK/SIRT1/PGC-1α pathway was involved in the protective effects of AFZ on mitochondrial function and apoptosis in intestinal epithelial cells.Conclusion AFZ alleviates mitochondrial dysfunction and apoptosis in intestinal epithelial cells by activating the AMPK/SIRT1/PGC-1α pathway,thereby ameliorating intestinal barrier dysfunction and experimental colitis.
Animals ; Colitis/drug therapy* ; Humans ; Caco-2 Cells ; Mice ; Trinitrobenzenesulfonic Acid ; Apoptosis/drug effects* ; Disease Models, Animal ; AMP-Activated Protein Kinases/metabolism* ; Sirtuin 1/metabolism*

OBJECTIVES: To investigate the effects of asperosaponin VI (AVI) on intestinal epithelial cell apoptosis and intestinal barrier function in a mouse model of Crohn's disease (CD)-like colitis and explore its mechanisms. METHODS: Male C57BL/6 mice with TNBS-induced CD-like colitis were treated with saline or AVI (daily dose 150 mg/kg) by gavage for 6 days. The changes in body weight, colon length, DAI scores, and colon pathologies of the mice were observed, and the expressions of inflammatory factors and tight injunction proteins were detected using ELISA and RT-qPCR. The effects of AVI on barrier function and apoptosis of mouse intestinal epithelial cells and TNF‑α‑treated Caco-2 cells were analyzed using immunofluorescence staining, TUNEL assay, and Western blotting. Network pharmacology, TUNEL assay, and Western blotting were performed to explore and validate the therapeutic mechanisms of AVI for CD. RESULTS: In the mouse models of CD-like colitis, AVI significantly improved body weight loss, colon shortening and DAI and tissue inflammation scores, alleviated intestinal villi and goblet cell injuries, and lowered the expressions of inflammatory factors. AVI treatment significantly reduced the loss of tight junction proteins and apoptosis in both mouse intestinal epithelial cells and TNF‑α-stimulated Caco-2 cells. KEGG enrichment pathway analysis suggested that the therapeutic effect of AVI on CD was associated with inhibition of PI3K/AKT/NF-κB pathway activation, which was confirmed by lowered expressions of p-PI3K, p-AKT, and p-p65 in AVI-treated mouse models and Caco-2 cells. In Caco-2 cells, Recilisib significantly blocked the inhibitory effect of AVI on the PI3K/AKT/NF-κB pathway and TNF-α-induced apoptosis, and AKT1 knockdown experiment confirmed the role of the PI3K/AKT pathway for mediating the activation of downstream NF-κB signaling. CONCLUSIONS AVI can improve TNBS-induced CD-like colitis in mice by reducing intestinal epithelial cell apoptosis and intestinal barrier damage via inhibiting the PI3K/AKT/NF-κB signaling pathway.
Animals ; Saponins/therapeutic use* ; Mice ; Crohn Disease/metabolism* ; Apoptosis/drug effects* ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mice, Inbred C57BL ; Male ; Humans ; Caco-2 Cells ; Phosphatidylinositol 3-Kinases/metabolism* ; NF-kappa B/metabolism* ; Colitis/drug therapy* ; Disease Models, Animal ; Epithelial Cells/drug effects* ; Trinitrobenzenesulfonic Acid ; Intestinal Mucosa/drug effects* ; Tumor Necrosis Factor-alpha/metabolism*

OBJECTIVE: To investigate the anti-inflammatory activity of Radix Panacis quinguefolii root extract (RPQE) and its therapeutic effects on inflammatory bowel disease (IBD). METHODS: The 72-hour post-fertilization zebrafish was used to generate the local and systematic inflammation models through tail-amputation and lipopolysaccharide (LPS)-induction (100 µ g/mL), respectively. The Tg(zlyz:EGFP) zebrafish was induced with 75 µ g/mL 2,4,6-trinitrobenzene sulfonic acid (TNBS) for establishing the IBD model. The tail-amputated, LPS-, and TNBS-induced models were subjected to RPQE (ethanol fraction, 10-20 µ g/mL) administration for 12 and 24 h, respectively. Anti-inflammatory activity of RPQE was evaluated by detecting migration and aggregation of leukocytes and expression of inflammation-related genes. Meanwhile, TNBS-induced fish were immersed in 0.2% (W/V) calcein for 1.5 h and RPQE for 12 h before photographing to analyze the intestinal efflux efficiency (IEE). Moreover, the expression of inflammation-related genes in these fish was detected by quantitative polymerase chain reaction. RESULTS: Subject to RPQE administration, the migration and aggregation of leukocytes were significantly alleviated in 3 zebrafish models (P<0.01). Herein, RPQE ameliorated TNBS-induced IBD with respect to a significantly reduced number of leukocytes, improved IEE, and inhibited gene expression of pro-inflammatory factors (P<0.05 or P<0.01). CONCLUSION RPQE exhibited therapeutic effects on IBD by inhibiting inflammation.
Animals ; Zebrafish ; Lipopolysaccharides ; Disease Models, Animal ; Inflammatory Bowel Diseases/metabolism* ; Inflammation/drug therapy* ; Anti-Inflammatory Agents/therapeutic use* ; Trinitrobenzenesulfonic Acid/adverse effects* ; Colitis/drug therapy*

OBJECTIVES: Liver disease is the most common extra-intestinal manifestation of ulcerative colitis (UC), but the underlying pathogenesis is still not clarified. It is well accepted that the occurrence of UC-related liver disease has close correlation with immune activation, intestinal bacterial liver translocation, inflammatory cytokine storm, and the disturbance of bile acid circulation. The occurrence of UC-related liver disease makes the therapy difficult, therefor study on the pathogenesis of UC-related liver injury is of great significance for its prevention and treatment. Glutathione (GSH) shows multiple physiological activities, such as free radical scavenging, detoxification metabolism and immune defense. The synthesis and the oxidation-reduction all contribute to GSH antioxidant function. It is reported that the deficiency in hepatic GSH antioxidant function participates in multiple liver diseases, but whether it participates in the pathogenesis of UC-related liver injury is still not clear. This study aims to investigate the feature and underlying mechanism of GSH synthesis and oxidation-reduction function during the development of UC, which will provide useful information for the pathogenesis study on UC-related liver injury. METHODS: UC model was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS)-ethanol solution (5 mg/0.8 mL per rat, 50% ethanol) via intra-colonic administration in rats, and the samples of serum, liver, and colon tissue of rats were collected at the 3rd, 5th, and 7th days post TNBS. The severity degree of colitis was evaluated by measuring the disease activity index, colonic myeloperoxidase activity, and histopathological score, and the degree of liver injury was evaluated by histopathological score and the serum content of alanine aminotransferase. Spearman correlation analysis was also conducted between the degree of colonic lesions and index of hepatic histopathological score as well as serum aspartate aminotransferase level to clarify the correlation between liver injury and colitis. To evaluate the hepatic antioxidant function of GSH in UC rats, hepatic GSH content, enzyme activity of GSH peroxidase (GSH-Px), and GSH reductase (GR) were determined in rats at the 3rd, 5th, and 7th days post TNBS, and the protein expressions of glutamine cysteine ligase (GCL), GSH synthase, GSH-Px, and GR in the liver of UC rats were also examined by Western blotting. RESULTS: Compared with the control, the disease activity index, colonic myeloperoxidase activity, and histopathological score were all significantly increased at the 3rd, 5th, and 7th days post TNBS (all P<0.01), the serum aspartate aminotransferase level and hepatic histopathologic score were also obviously elevated at the 7th day post TNBS (all P<0.05). There was a significant positive correlation between the degree of liver injury and the severity of colonic lesions (P=0.000 1). Moreover, compared with the control, hepatic GSH content and the activity of GSH-Px and GR were all significantly decreased at the 3rd and 5th days post TNBS (P<0.05 or P<0.01), and the protein expressions of GCL, GSH-Px, and GR were all obviously down-regulated at the 3rd, 5th, and 7th days post TNBS (P<0.05 or P<0.01). CONCLUSIONS There is a significant positive correlation between the degree of liver injury and the severity of colonic lesions, and the occurrence of reduced hepatic GSH synthesis and decreased GSH reduction function is obviously earlier than that of the liver injury in UC rats. The reduced hepatic expression of enzymes that responsible for GSH synthesis and reduction may contribute to the deficiency of GSH synthesis and oxidation-reduction function, indicating that the deficiency in GSH antioxidant function may participate in the pathogenesis of UC related liver injury.
Animals ; Antioxidants ; Aspartate Aminotransferases ; Colitis/chemically induced* ; Colitis, Ulcerative/metabolism* ; Colon/pathology* ; Glutathione/biosynthesis* ; Liver/metabolism* ; Peroxidase/metabolism* ; Rats ; Trinitrobenzenesulfonic Acid

Central sensitization is essential in maintaining chronic pain induced by chronic pancreatitis (CP), but cortical modulation of painful CP remains elusive. Here, we examined the role of the anterior cingulate cortex (ACC) in the pathogenesis of abdominal hyperalgesia in a rat model of CP induced by intraductal administration of trinitrobenzene sulfonic acid (TNBS). TNBS treatment resulted in long-term abdominal hyperalgesia and anxiety in rats. Morphological data indicated that painful CP induced a significant increase in FOS-expressing neurons in the nucleus tractus solitarii (NTS) and ACC, and some FOS-expressing neurons in the NTS projected to the ACC. In addition, a larger portion of ascending fibers from the NTS innervated pyramidal neurons, the neural subpopulation primarily expressing FOS under the condition of painful CP, rather than GABAergic neurons within the ACC. CP rats showed increased expression of vesicular glutamate transporter 1, and increased membrane trafficking and phosphorylation of the N-methyl-D-aspartate receptor (NMDAR) subunit NR2B and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluR1 within the ACC. Microinjection of NMDAR and AMPAR antagonists into the ACC to block excitatory synaptic transmission significantly attenuated abdominal hyperalgesia in CP rats, which was similar to the analgesic effect of endomorphins injected into the ACC. Specifically inhibiting the excitability of ACC pyramidal cells via chemogenetics reduced both hyperalgesia and comorbid anxiety, whereas activating these neurons via optogenetics failed to aggravate hyperalgesia and anxiety in CP rats. Taken together, these findings provide neurocircuit, biochemical, and behavioral evidence for involvement of the ACC in hyperalgesia and anxiety in CP rats, as well as novel insights into the cortical modulation of painful CP, and highlights the ACC as a potential target for neuromodulatory interventions in the treatment of painful CP.
Animals ; Anxiety/etiology* ; Chronic Pain/etiology* ; GABAergic Neurons ; Gyrus Cinguli/metabolism* ; Hyperalgesia/metabolism* ; Pancreatitis, Chronic/pathology* ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate/metabolism* ; Trinitrobenzenesulfonic Acid/toxicity*

Animals ; Codonopsis ; Colitis, Ulcerative/drug therapy* ; Colon ; Male ; NF-kappa B ; Rats ; Rats, Wistar ; Saponins/pharmacology* ; Trinitrobenzenesulfonic Acid

OBJECTIVE: To investigate the protective effect of procyanidin B2 (PCB2) on the intestinal barrier and against enteritis in mice with trinitrobenzene sulphonic acid (TNBS)-induced colitis and explore the possible mechanism. METHODS: A mouse model of TNBS-induced colitis was established in male Balb/c mice aged 6-8 weeks. The successfully established mouse models were randomly divided into PCB2 treatment group (=10) and model group (=10) and were treated with daily intragastric administration of PCB2 (100 mg/kg, 0.2 mL) and 0.2 mL normal saline, respectively. After 4 weeks, the disease symptoms, intestinal inflammation, intestinal mucosal cell barrier function and the changes in PI3K/AKT signaling were evaluated using HE staining, immunofluorescence assay and Western blotting. RESULTS: The disease activity index of the mice was significantly lower and the mean body weight was significantly greater in PCB2 group than in the model group in the 3rd and 4th weeks of intervention ( < 0.05). The levels of colonic inflammation and intestinal mucosal inflammatory mediators IL-1β and TNF-α were significantly lower while IL-10 was significantly higher in PCB2 group than in the model group ( < 0.05). Compared with those in the model group, the mice in PCB2 treatment group showed a significantly lower positive rate of bacterial translocation in the mesenteric lymph nodes and a lower thiocyanate-dextran permeability of the intestinal mucosa ( < 0.05). Western blotting showed that PCB2 treatment significantly increased the expressions of claudin-1 and ZO-1 ( < 0.05) and significantly lowered the expression levels of p-PI3K and p-AKT in the intestinal mucosa as compared with those in the model group ( < 0.05). CONCLUSIONS PCB2 suppresses intestinal inflammation and protects intestinal mucosal functions and structural integrity by inhibiting intestinal PI3K/AKT signaling pathway, suggesting the potential of PCB2 as a new drug for Crohn's disease.
Animals ; Biflavonoids ; Catechin ; Colitis ; chemically induced ; Colon ; Enteritis ; Intestinal Mucosa ; Male ; Mice ; Phosphatidylinositol 3-Kinases ; Proanthocyanidins ; Trinitrobenzenesulfonic Acid

Although the etiology of inflammatory bowel disease is still uncertain, increasing evidence indicates that the excessive activation of NLRP3 inflammasome plays a major role. Norisoboldine (NOR), an alkaloid isolated from Radix Linderae, has previously been demonstrated to inhibit inflammation and IL-1β production. The present study was to examine the effect of NOR on colitis and the underlying mechanism related to NLRP3 inflammasome activation. Our results showed that NOR alleviated colitis symptom in mice induced by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). Moreover, it significantly reduced expressions of cleaved IL-1β, NLRP3 and cleaved Caspase-1 but not ASC in colons of mice. In THP-1 cells, NOR suppressed the expressions of NLRP3, cleaved Caspase-1 and cleaved IL-1β but not ASC induced by lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Furthermore, NOR could activate aryl hydrocarbon receptor (AhR) in THP-1 cells, inducing CYP1A1 mRNA expression, and promoting dissociation of AhR/HSP90 complexes, association of AhR and ARNT, AhR nuclear translocation, XRE reporter activity and binding activity of AhR/ARNT/XRE. Both siAhR and α-naphthoflavone (α-NF) markedly diminished the inhibition of NOR on NLRP3 inflammasome activation. In addition, NOR elevated Nrf2 level and reduced ROS level in LPS- and ATP-stimulated THP-1 cells, which was reversed by either siAhR or α-NF treatment. Finally, correlations between activation of AhR and attenuation of colitis, inhibition of NLRP3 inflammasome activation and up-regulation of Nrf2 level in colons were validated in mice with TNBS-induced colitis. Taken together, NOR ameliorated TNBS-induced colitis in mice through inhibiting NLRP3 inflammasome activation via regulating AhR/Nrf2/ROS signaling pathway.
Alkaloids ; administration & dosage ; Animals ; Colitis ; chemically induced ; drug therapy ; genetics ; immunology ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Inflammasomes ; drug effects ; immunology ; Interleukin-1beta ; genetics ; immunology ; Lindera ; chemistry ; Male ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; genetics ; immunology ; Receptors, Aryl Hydrocarbon ; agonists ; genetics ; metabolism ; Trinitrobenzenesulfonic Acid ; adverse effects