Aucubin alleviates methotrexate-induced enteritis in rats by inducing autophagy.

One of the toxic side effects of methotrexate (MTX) is enteritis. Aucubin, an iridoid glycoside derived from traditional medicinal herbs, has been proven to have anti-inflammation, anti-apoptosis and anti-oxidation properties. This work explored the effect and mechanism of aucubin in treating MTX-induced enteritis in a rat model. Two doses of aucubin (5 and 10 mg/kg) were adopted for the assessment of its pharmacological activity. We observed that in rats with MTX-induced enteritis, the body weight and small intestinal weight decreased. The intestine barrier was injured, as reflected by pathological examinations and an increase in D-lactate and diamine oxidase concentration in serum. Intestinal inflammation was shown by the observation of macrophages in the intestine and the concentrations of inflammatory cytokines tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum. The NLR family pyrin domain containing 3 (NLRP3) inflammasome was shown to be activated by the enhancement of NLRP3, cleaved-caspase 1, IL-18 and IL-1ß. Moreover, autophagy was reflected by transmission electron microscopy as slightly induced, along with changes in autophagy-related markers microtubule-associated protein 1 light chain 3 (LC3) and Beclin1. Remarkably, aucubin treatment attenuated the MTX-induced disease activity index increase, intestinal damage, inflammatory response and NLRP3 inflammasome activation, but provoked autophagy. Rapamycin, an autophagy activator, showed similar therapeutic effects to aucubin on MTX-induced enteritis. However, 3-methyladenine, an autophagy inhibitor, reversed the protective effects of aucubin. These findings prompted the hypothesis that aucubin alleviates MTX-induced enteritis by aggravating autophagy. This study might provide evidence for further investigation on the therapeutic role of aucubin in MTX-resulted enteritis.

Corynoline ameliorates dextran sulfate sodium-induced colitis in mice by modulating Nrf2/NF-κB pathway.

OBJECTIVE: Corynoline is an active substance extracted from Corydalis bungeana Turcz and exerts a therapeutic effect in multiple diseases by alleviating inflammatory response. The present study sought to elucidate the role of corynoline in ulcerative colitis (UC). METHODS: The experimental colitis models were induced in BALB/c mice via receiving a drinking water supplemented with 3.5% (I) dextran sulfate sodium (DSS) ad libitum for 7 days. RESULTS: Corynoline administration inhibited body weight loss, colon shortening, disease activity index and colonic pathomorphological changes in DSS-treated mice. Besides, corynoline down-regulated the levels of pro-inflammatory interleukin (IL)-1ß, IL-6 and tumor necrosis factor Alpha (TNF-α), as well as decreased myeloperoxidase (MPO) activity in the colon of DSS-treated mice. In addition, severe oxidative stress in the colonic tissues of DSS-treated was mitigated by corynoline treatment. However, these beneficial effects were reversed by a specific nuclear factor E2-related factor 2 (Nrf2) inhibitor ML385 intervention. Further evidence confirmed that corynoline promoted Nrf2 nuclear migration and heme oxygenase-1 gene expression in the colonic tissues of UC mice. Besides, corynoline treatment restrained colonic nuclear factor-kappa B (NF-κB) activation as proved by the decrease in phosphorylation and nuclear translocation of NF-κB. CONCLUSIONS: Corynoline ameliorates DSS-induced mouse colitis, which may provide a promising therapeutic strategy for UC treatment.

Growth performance and intestinal morphology of Hyline chickens fed diets with different diet particle sizes.

The effect of feed particle size on the growth performance and intestinal morphology of Hyline chickens was studied. Sixty one-day-old chickens were randomly assigned into two groups with five replicates per group and six chickens per replicate. The control group (CG) was fed pelleted feed and the experimental group (EG) was fed crumbled feed. The geometric mean diameter (GMD) of the pelleted and crumbled feed was approximately 1.2 and 0.8 mm respectively. The trial was carried out from day 15 to day 69 of the lives of the chickens. During the experimental period, the average daily feed intake (ADFI), average daily gain (ADG) and feed conversion rate (FCR) were calculated. At 42 and 69 days of age, fifteen chickens from each group were slaughtered for the intestinal morphology analyses. The morphologies of various segments of the intestine were observed by scanning electron microscopy (SEM). The results showed that from 15 to 42 days of age, the FCR of the CG was higher than that of the EG, but the final body weight was lower in the CG than in the EG (p < 0.05). The villi heights in the duodenum, jejunum and ileum were shorter in the CG than in the EG (p < 0.05). The villi tips in the duodenum and jejunum of the CG were broken, but they were protected in the EG. At 69 days of age, the BW and the whole intestinal villi height of the CG were greater than those of the EG (p < 0.05). The villi in the duodenum and jejunum in the CG were better than 42 days, but the tips of the duodenal villi in the EG were slightly broken. In conclusion, feeding chickens crumbled feed from 15 to 42 days of age and then feeding them pelleted feed was beneficial to the growth performance of the Hyline chickens, as the pelleted feed would break the intestinal villi of young chickens, but the intestinal villi become stronger with the chicken growth.

Molecular mechanism of honeysuckle + forsythia in treatment of acute lung injury based on network pharmacology.

The pathogenesis of acute lung injury (ALI) is complex and it is a common critical illness in clinical practice, seriously threatening the lives of critically ill patients, for which no specific molecular marker exists and there is a lack of effective methods for the treatment of ALI. The present study aimed to investigate the mechanism of action of honeysuckle and forsythia in treatment of acute lung injury (ALI) based on network pharmacology and in vitro modeling. The active ingredients and targets of honeysuckle and forsythia were predicted using traditional Chinese medicine systems pharmacology, PubChem and Swiss Target Prediction databases, and the Cytoscape 3.7.2 software was used to construct a drug-component-potential target network. The potential targets were imported into the Search tool for recurring instances of neighboring genes) database to obtain protein-protein interactions and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Targets analysis using Database for Annotation, Visualization, and Integrated Discovery. AutoDock Vina 1.1.2 software was used for docking between key active ingredients and the target proteins to analyze the binding ability of the active ingredients to the primary targets in honeysuckle and forsythia. A total of 64 male BALB/c mice were randomly divided into control, model, positive drug (Lianhua-Qingwen capsule), honeysuckle, forsythia, honeysuckle + forsythia high-, medium- and low-dose groups. Lipopolysaccharide (LPS) was used to induced an ALI model. The lung tissues of the mice were stained with hematoxylin-eosin and the serum levels of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured 4 h after the LPS administration. Reverse transcription-quantitative PCR and western blotting were used to detect NF-κB mRNA and protein expression, respectively. Active ingredients of honeysuckle and forsythia acted on 265 common targets in ALI, which regulated NF-κB, tumor necrosis factor-α (TNF-α) and PI3K-AKT signaling pathway, HIF-1 signalling pathway to slow the inflammatory response in treatment of ALI. In the positive drug group, honeysuckle, forsythia group, honeysuckle + forsythia high-, medium- and low-dose groups, lung tissue damage were significantly decrease compared with the model group, and inflammatory cell infiltration was reduced. Compared with the model group, honeysuckle + forsythia groups experienced decreased damage caused by the LPS and inflammation in the lung tissues and significantly decreased TNF-α and NF-κB and MDA concentration and significantly increased the SOD and GSH-Px activities. The mechanism of the effect of honeysuckle and forsythia on ALI may be mediated by inhibition of TNF-α and NF-κB expression and the activation of antioxidant mechanisms to decrease production of pro-inflammatory cytokines in lung tissue, thus treating ALI.

Cynaroside ameliorates methotrexate-induced enteritis in rats through inhibiting NLRP3 inflammasome activation.

Introduction: Cynaroside exhibits various biological properties, including anti-inflammatory, antiviral, antitumor, and cardioprotective effects. However, its involvement in methotrexate (MTX)-induced intestinal inflammation remains inadequately understood. Thus, we investigated the impact of cynaroside on MTX-induced intestinal inflammation and its potential mechanisms. Methods: To assess the protective potential of cynaroside against intestinal inflammation, Sprague-Dawley rats were subjected to a regimen of 7 mg/kg MTX for 3 days, followed by treatment with cynaroside at varying doses (10, 20, or 40 mg/kg). Histopathological evaluations were conducted alongside measurements of inflammatory mediators to elucidate the involvement of the NLRP3 inflammasome in alleviating intestinal inflammation. Results: Administration of 7 mg/kg MTX resulted in decreased daily food intake, increased weight loss, and elevated disease activity index in rats. Conversely, treatment with cynaroside at 20 or 40 mg/kg ameliorated the reductions in body weight and daily food intake and suppressed the MTX-induced elevation in the disease activity index. Notably, cynaroside administration at 20 or 40 mg/kg attenuated inflammatory cell infiltration, augmented goblet cell numbers and lowered serum levels of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-18, as well as the CD68-positive cell rate in the intestines of MTX-induced rats. Furthermore, cynaroside downregulated the expression levels of NLRP3, cleaved caspase 1, and cleaved IL-1ß in MTX-induced rats. Discussion: Collectively, our findings indicated that cymaroside alleviates intestinal inflammatory injury by inhibiting the activation of NLRP3 inflammasome in MTX-induced rats.

Maternal effects drive intestinal development beginning in the embryonic period on the basis of maternal immune and microbial transfer in chickens.

BACKGROUND: Nutrition drives immunity and health in animals, and maternal immunity benefits offspring. In our previous study, a nutritional intervention strategy was found to promote the immunity of hens, which subsequently improved immunity and growth in offspring chicks. Maternal effects clearly exist, but how are mothers' immune advantages transferred to their offspring, and how do they benefit them? RESULTS: Here, we traced the beneficial effects back to the process of egg formation in the reproductive system, and we focused on the embryonic intestinal transcriptome and development, as well as on maternal microbial transfer in offspring. We found that maternal nutritional intervention benefits maternal immunity, egg hatching, and offspring growth. The results of protein and gene quantitative assays showed that the transfer of immune factors into egg whites and yolks depends on maternal levels. Histological observations indicated that the promotion of offspring intestinal development begins in the embryonic period. Microbiota analyses suggested that maternal microbes transfer to the embryonic gut from the magnum to the egg white. Transcriptome analyses revealed that offspring embryonic intestinal transcriptome shifts are related to development and immunity. Moreover, correlation analyses showed that the embryonic gut microbiota is correlated with the intestinal transcriptome and development. CONCLUSIONS: This study suggests that maternal immunity positively influences offspring intestinal immunity establishment and intestinal development beginning in the embryonic period. Adaptive maternal effects might be accomplished via the transfer of relatively large amounts of maternal immune factors and by shaping of the reproductive system microbiota by strong maternal immunity. Moreover, reproductive system microbes may be useful resources for the promotion of animal health. Video Abstract.

Procyanidin A1 alleviates DSS-induced ulcerative colitis via regulating AMPK/mTOR/p70S6K-mediated autophagy.

Ulcerative colitis (UC) is a recurrent chronic inflammatory disease. The symptom of UC is mainly diarrhea including bloody stools. Increasing evidence has suggested that procyanidin A1 (PCA1) exerts an anti-inflammatory effect in several diseases. However, the role of PCA1 in UC is still a mystery. In our study, we explored the effect of PCA1 in dextran sulfate sodium (DSS)-induced UC mice and lipopolysaccharide (LPS)-stimulated HT-29 and IEC-6 cells. Then, cell proliferation, apoptosis, the production of proinflammatory cytokines, and autophagy-related markers were determined. Furthermore, the AMPK/mTOR/p70S6K signaling pathway was assayed by Western blot assay. In in vivo study, we found that PCA1 administration alleviated DSS-induced UC, as evidenced by reducing weight loss, clinical scores, colon weight/length ratio, histological damage, proinflammatory cytokines, and apoptosis. Moreover, we showed that the expression of Beclin-1 and LC3II/I ratio was increased, whereas the level of p62 was decreased after PCA1 treatment in vivo. Meanwhile, the reduced AMP/ATP ratio, enhanced expression of p-AMPK, and decreased p-p70S6K and p-mTOR levels indicate the activation of AMPK/mTOR/p70S6K signaling pathway. In in vitro study, PCA1 promoted cell proliferation and inhibited cell apoptosis in LPS-stimulated HT-29 and IEC-6 cells. Pro-inflammatory cytokines and autophagy-related factors exhibited the same trend as in in vivo results. Mechanically, PCA1 activated the AMPK/mTOR/p70S6K signaling pathway. The treatment with an AMPK inhibitor compound C significantly reversed the anti-inflammatory effect of PCA1 in LPS-stimulated cells. Taken together, these data indicated that PCA1 alleviated UC through induction of AMPK/mTOR/p70S6K-mediated autophagy.

Albiflorin ameliorates inflammation and oxidative stress by regulating the NF-κB/NLRP3 pathway in Methotrexate-induced enteritis.

Methotrexate (MTX) treats various diseases but also damages intestinal barrier and leads to enteritis. Albiflorin (ALB) has a variety of pharmacological effects, including antioxidant, anti-inflammation and anti-apoptosis. In the present study, we evaluated the therapeutic effect of ALB on MTX-induced enteritis and investigated the possible mechanisms involved. Male SD rats were intraperitoneally injected with 7 mg/kg MTX for three consecutive days to establish the enteritis model. ALB (20 or 40 mg/kg/day) was intragastrically administrated since two days prior MTX treatment and lasted for six days. We found that ALB treatment increased body weight and intestinal weight of rats with MTX injection. The disease activity index (DAI) score was also decreased after ALB administration. In histological examination, ALB treatment attenuated inflammatory cells infiltration and promoted survival of goblet cells. In detection of inflammatory-associated factors, ALB treatment decreased CD68+ cells infiltration, inhibited myeloperoxidase activity, and suppressed intercellular cell adhesion molecule-1 and cyclooxygenase-2 expression. Additionally, ALB reduced malondialdehyde, glutathione levels, inhibited superoxide dismutase activity and suppressed reactive oxygen species production. Moreover, ALB treatment effectively inhibited NLRP3, as well as caspase 1 p20 and interleukin (IL)-1ß and 18 expression. Finally, nuclear factor-κB (NF-κB) p65 phosphorylation and nuclear translocation were also demonstrated to be blocked upon ALB treatment. In conclusion, our findings indicated that ALB alleviated MTX-induced enteritis via inhibiting the NF-κB/NLRP3 pathway.

Forsythiaside A alleviates methotrexate-induced intestinal mucositis in rats by modulating the NLRP3 signaling pathways.

Most chemotherapeutic drugs can kill the tumor cells, but also cause a vast damage to body, such as intestinal mucositis (IM). The present study was design to find out the effect of Forsythiaside A (FTA) on chemotherapeutic-induced IM in rats. Briefly, for 3 consecutive days, male Sprague-Dawley rats were treated with 7 mg / kg methotrexate (MTX) to establish IM and simultaneously administered with 40 or 80 mg / kg FTA for 7 days. Our results showed that the final body weight and daily food intake were increased, and the disease activity index was reduced in the MTX group after FTA treatment. The MTX group showed the pathological alterations like the inflammatory cells infiltration, the mucosal layer destruction, glands expansion, intestinal villi structure disorder and goblet cells reduction, while we found that 80 mg / kg FTA treatment displayed evident reversal effects. ELISA further suggested that TNF-α, IL-1ß and IL-18 levels in serum in MTX-induced rats were reduced after 80 mg / kg FTA treatment. Moreover, FTA decreased the number of leukocytes, neutrophils and lymphocytes in peripheral blood. Western blot and immunofluorescence results indicated that the expression levels of NLRP3, cleaved caspase 1, cleaved IL-1ß and CD68 positive rate were down-regulated in MTX-induced rats after 80 mg / kg FTA intervention. The findings of the current study suggested that FTA effectively inhibited MTX-induced IM in rats by attenuating the activation of the NLRP3 signaling pathways.

ß-Carotene prevents weaning-induced intestinal inflammation by modulating gut microbiota in piglets.

OBJECTIVE: Weaning is an important stage in the life of young mammals, which is associated with intestinal inflammation, gut microbiota disorders, and even death. ß-Carotene displays anti-inflammatory and antioxidant activities, which can prevent the development of inflammatory diseases. However, whether ß-carotene can affect intestinal microbiota remains unclear. METHODS: Twenty-four piglets were distributed into four groups: the normal suckling group (Con), the weaning group (WG), the weaning+ß-carotene (40 mg/kg) group (LCBC), and the weaning+ß-carotene (80 mg/kg) group (HCBC). The serum, jejunum, colon, and faeces were collected separately from each group. The effects of ß-carotene on the phenotype, overall structure, and composition of gut microbiota were assessed in weaning piglets. RESULTS: The results showed that ß-carotene improved the growth performance, intestinal morphology and relieved inflammation. Furthermore, ß-carotene significantly decreased the species from phyla Bacteroidetes and the genus Prevotella, and Blautia, and increased the species from the phyla Firmicutes and the genera p-75-a5, and Parabacteroides compared to the WG group. Spearman's correlation analysis showed that Prevotella and Blautia were positively correlated, and Parabacteroides and Synergistes were negatively correlated with the levels of interleukin-1ß (IL-1ß), IL-6, and tumour necrosis factor-α (TNF-α), while p-75-a5 showed negative correlation with IL-6 in serum samples from piglets. CONCLUSION: These findings indicate that ß-carotene could alleviate weaning-induced intestinal inflammation by modulating gut microbiota in piglets. Prevotella may be a potential target of ß-carotene in alleviating the weaning-induced intestinal inflammation in piglets.

Echinacea polysaccharide attenuates lipopolysaccharide­induced acute kidney injury via inhibiting inflammation, oxidative stress and the MAPK signaling pathway.

Acute kidney injury (AKI) is often accompanied by inflammation. Echinacea polysaccharide (EP) is an active ingredient that has been demonstrated to possess anti­oxidative, anti­inflammatory, antimicrobial and immunomodulatory functions. However, the role of EP in AKI has not been examined. The present study investigated the effects of EP on lipopolysaccharide (LPS)­induced AKI. Western blotting, immunohistochemistry and immunofluorescence analyses were performed to detect protein expression levels. Administration of EP significantly attenuated LPS­induced renal tissue injury, along with a decrease in blood urea nitrogen and creatinine levels. EP decreased the levels of inducible nitric oxide synthase and cyclo­oxygenase­2 in LPS­treated mice. Furthermore, LPS­induced inflammation was inhibited by EP in renal tissues and HBZY­1 cells, as demonstrated by the downregulation of tumor necrosis factor­α, interleukin (IL)­1ß, IL­6, nitric oxide and prostaglandin E2 levels. Similarly, EP administration decreased oxidative stress (OS) via decreasing reactive oxygen species, malondialdehyde and oxidized glutathione levels, and increasing superoxide dismutase, catalase, glutathione reductase and reduced glutathione activity. Notably, EP induced a marked decrease in the expression levels of phospho­extracellular signal­regulated protein kinase (p­ERK), phospho­c­Jun N­terminal kinase (p­JNK) and p­p38 in vivo and in vitro. In addition, in LPS­treated HBZY­1 cells, EP enhanced cell viability and inhibited nuclear translocation of p­ERK, p­JNK and p­p38. Overall, the present findings demonstrated that EP alleviated LPS­induced AKI via the suppression of inflammation, OS and the mitogen­activated protein kinase signaling pathway, providing insight into potential avenues for the treatment of AKI.

Strictosamide alleviates the inflammation in an acute ulcerative colitis (UC) model.

The ulcerative colitis (UC) is a typical inflammatory bowel disease (IBD) causing great damages, while strictosamide (STR) is a natural alkaloid that possesses strong anti-inflammatory property in infection and inflammation-related diseases. Our study is aimed at evaluating the anti-inflammatory activity of STR in the course of UC. Briefly, male Balb/c mice were treated with 3.5% dextran sulfate sodium (DSS) for 6 consecutive days to establish an acute model of UC, and the administration of gradient concentrations of STR was subsequently performed. Accordingly, colonic pathological alterations including the reduced ratio of colon weight/length, decreased disease activity index (DAI), and attenuated H&E damage were found in UC mice after STR treatment. Based on the analyses of real-time PCR and western blot, downregulation of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) was also determined in the colonic tissue of UC mice after the treatment of STR. ELISA and immunohistochemical staining further suggest the relief of inflammation in UC mice with decreased expressions of MPO and iNOS after STR treatment. In addition, STR was also validated to significantly inhibit NF-κB signaling in UC mice by western blot and Electrophoretic Mobility Shift Assay (EMSA). Meanwhile, restricted inflammation was also determined in STR-treated IEC6 and HT-29 cells. The utilization of PDTC, an inhibitor of NF-κB, further demonstrated that STR ameliorated the inflammation by inhibiting the NF-κB signaling in vitro. In summary, our study suggests that STR could be a potential candidate for IBD therapy.

Echinacea polysaccharide alleviates LPS-induced lung injury via inhibiting inflammation, apoptosis and activation of the TLR4/NF-κB signal pathway.

Lung injury is a common critical life-threatening syndrome. Inflammation is a key factor in the pathogenesis of lung injury. It is reported that Echinacea Polysaccharides (EP) has anti-inflammatory activity. However, the effect of EP on lung injury remains unclear. In our study, murine model of lung injury was induced with 2.5 mg/kg LPS before administration of 5 mg/kg or 10 mg/kg EP. EP ameliorated LPS-induced lung pathological damage, along with reduction in lung wet/dry weight ratio and myeloperoxidase activity. EP decreased the number of leukocytes, eosinophils, neutrophils, lymphocytes and macrophages in bronchoalveolar lavage fluid, and the release of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in LPS-treated lung. EP suppressed LPS-induced apoptosis along with down-regulation of Bcl2-associated X (Bax) and cleaved caspase-3 (CC3), and elevated B-cell lymphoma-2 (Bcl-2). Besides, RAW 264.7 cells were treated with EP 100 µg/ml for 1 h and then incubated with 1 µg/ml LPS for 24 h. TNF-α, IL-6 and IL-1ß levels were lowered by treatment of EP in LPS-treated RAW 264.7 cells. Moreover, EP down-regulated the expression of toll-like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), p-IκBα, nuclear factor kappa-B (NF-κB), p-NF-κB, and up-regulated the inhibitor of NF-κB (IκBα) in vivo and in vitro following LPS induction, which is consistent with the effect of TAK-242. In conclusion, EP may alleviate LPS-induced lung injury via inhibiting inflammation, apoptosis and activation of TLR4/NF-κB signal pathway.