Exploring the effects of naringin on oxidative stress-impaired osteogenic differentiation via the Wnt/ß-catenin and PI3K/Akt pathways.

This study aimed to explore naringin's potential to promote the osteogenic differentiation of MC3T3-E1 under oxidative stress. It delved into Nar's connection with the Wnt/ß-catenin and PI3K/Akt signaling pathways. Initially, 2911 OP-related genes were analyzed, revealing close ties with the PI3K/Akt and Wnt pathways alongside oxidative stress. Nar's potential targets-ESR1, HSP90AA1, and ESR2-were identified through various databases and molecular docking studies confirmed Nar's affinity with ESR1 and HSP90AA1. Experiments established optimal concentrations for Nar and H2O2. H2O2 at 0.3 mmol/L damaged MC3T3-E1 cells, alleviated by 0.1 µmol/L Nar. Successful establishment of oxidative stress models was confirmed by DCFH-DA probe and NO detection. Nar exhibited the ability to enhance osteogenic differentiation, counteracting oxidative damage. It notably increased osteoblast-related protein expression in MC3T3-E1 cells under oxidative stress. The study found Nar's positive influence on GSK-3ß phosphorylation, ß-catenin accumulation, and pathway-related protein expression, all critical in promoting osteogenic differentiation. The research concluded that Nar effectively promotes osteogenic differentiation in MC3T3-E1 cells under oxidative stress. It achieved this by activating the Wnt/ß-catenin and PI3K/Akt pathways, facilitating GSK-3ß phosphorylation, and enhancing ß-catenin accumulation, pivotal in osteogenesis.

Paneth cell ablation increases the small intestinal injury during acute necrotizing pancreatitis in rats.

The present work aimed to investigate the role of Paneth cells in small intestinal injury during acute necrotizing pancreatitis (ANP) using rat models established by injection of dithizone, a metal chelator of zinc with the ability to selectively ablate Paneth cells. Sprague­Dawley rats were randomly divided into four groups: Sham­operated group, ANP group (3.5% sodium taurocholate solution, 1 ml/kg body weight), dithizone group (100 mg/kg of body weight) and ANP + dithizone group (sodium taurocholate solution was administered 6 h after dithizone injection). Each group was further divided into five subgroups (6, 12, 24, 36 and 48 h) based on the time period between induction of the model and sample collection. The present results suggested the number of Paneth cells was gradually decreased in the ANP group in a time­dependent manner. Most of the Paneth cells were ablated in the ANP + dithizone group at 6 h, but a subset of Paneth cells recovered after 24­48 h. Compared with the ANP group, combination of dithizone and ANP significantly induced more severe histopathological injuries in the pancreas and distal ileum, with higher Schmidt and Chiu's scores, respectively. Additionally, increased expression levels of tumor necrosis factor­α (TNF­α), interleukin (IL)­1ß and IL­17A were detected in the ileum, causing an increase in intestinal permeability, as assessed by a decrease in the expression level of the intestinal tight junction protein occludin and high plasma levels of diamine oxidase and D­lactate. The increase in intestinal permeability led to the translocation of bacteria to the bloodstream, triggering systemic inflammation, as assessed by the increased plasma levels of TNF­α, IL­1ß and IL­17A, reducing the survival rates of rats, which was 66.7% and 83.3% in the ANP + dithizone and the ANP group, respectively. The increase in intestinal endoplasmic reticulum stress, as assessed by high expression levels of binding­immunoglobulin protein and activating transcription factor 6, may be one mechanism associated with Paneth cells loss and intestinal barrier impairment during ANP. Collectively, the present study suggested that the absence of Paneth cells may be an important factor involved in intestinal injury, promoting the progression of ANP.

Commensal Escherichia coli Aggravates Acute Necrotizing Pancreatitis through Targeting of Intestinal Epithelial Cells.

An increase of Escherichia-Shigella was previously reported in acute necrotizing pancreatitis (ANP). We investigated whether Escherichia coli MG1655, an Escherichia commensal organism, increased intestinal injury and aggravated ANP in rats. ANP was induced by retrograde injection of 3.5% sodium taurocholate into the biliopancreatic duct. Using gut microbiota-depleted rats, we demonstrated that gut microbiota was involved in the pancreatic injury and intestinal barrier dysfunction in ANP. Using 16S rRNA gene sequencing and quantitative PCR, we found intestinal dysbiosis and a significant increase of E. coli MG1655 in ANP. Afterward, administration of E. coli MG1655 by gavage to gut microbiota-depleted rats with ANP was performed. We observed that after ANP induction, E. coli MG1655-monocolonized rats presented more severe injury in the pancreas and intestinal barrier function than gut microbiota-depleted rats. Furthermore, Toll-like receptor 4 (TLR4)/MyD88/p38 mitogen-activated protein (MAPK) and endoplasmic reticulum stress (ERS) activation in intestinal epithelial cells were also increased more significantly in the MG1655-monocolonized ANP rats. In vitro, the rat ileal epithelial cell line IEC-18 displayed aggravated tumor necrosis factor alpha-induced inflammation and loss of tight-junction proteins in coculture with E. coli MG1655, as well as TLR4, MyD88, and Bip upregulation. In conclusion, our study shows that commensal E. coli MG1655 increases TLR4/MyD88/p38 MAPK and ERS signaling-induced intestinal epithelial injury and aggravates ANP in rats. Our study also describes the harmful potential of commensal E. coli in ANP.IMPORTANCE This study describes the harmful potential of commensal E. coli in ANP, which has not been demonstrated in previous studies. Our work provides new insights into gut bacterium-ANP cross talk, suggesting that nonpathogenic commensals could also exhibit adverse effects in the context of diseases.

Dysbiosis of Intestinal Microbiota and Decreased Antimicrobial Peptide Level in Paneth Cells during Hypertriglyceridemia-Related Acute Necrotizing Pancreatitis in Rats.

Hypertriglyceridemia (HTG) aggravates the course of acute pancreatitis (AP). Intestinal barrier dysfunction is implicated in the pathogenesis of AP during which dysbiosis of intestinal microbiota contributes to the dysfunction in intestinal barrier. However, few studies focus on the changes in intestine during HTG-related acute necrotizing pancreatitis (ANP). Here, we investigated the changes in intestinal microbiota and Paneth cell antimicrobial peptides (AMPs) in HTG-related ANP (HANP) in rats. Rats fed a high-fat diet to induce HTG and ANP was induced by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct. Rats were sacrificed at 24 and 48 h, respectively. Pancreatic and ileal injuries were evaluated by histological scores. Intestinal barrier function was assessed by plasma diamine oxidase activity and D-lactate level. Systemic and intestinal inflammation was evaluated by tumor necrosis factor alpha (TNFα), interleukin (IL)-1ß, and IL-17A expression. 16S rRNA high throughput sequencing was used to investigate changes in intestinal microbiota diversity and structure. AMPs (α-defensin5 and lysozyme) expression was measured by real-time polymerase chain reaction (PCR) and immunofluorescence. The results showed that compared with those of normal-lipid ANP (NANP) groups, the HANP groups had more severe histopathological injuries in pancreas and distal ileum, aggravated intestinal barrier dysfunction and increased TNFα, IL-1ß, and IL-17A expression in plasma and distal ileum. Principal component analysis showed structural segregation between the HANP and NANP group. α-Diversity estimators in the HANP group revealed decreased microbiota diversity compared with that in NANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. In the HANP group, at phyla level, Candidatus_Saccharibacteria and Tenericutes decreased significantly, whereas Actinobacteria increased. At genus level, Allobaculum, Bifidobacterium, and Parasutterella increased significantly, while Alloprevotella, Anaerotruncus, Candidatus_Saccharimonas, Christensenellaceae_R-7_group, Rikenellaceae_RC9_gut_group, Ruminiclostridium_5, Ruminococcaceae_UCG-005, and Ruminococcaceae_UCG-014 decreased. Compared with those in the NANP rats, mRNA expression of lysozyme and α-defensin5 and protein expression of lysozyme decreased significantly in the HANP rats. Moreover, in the NANP rats and the HANP rats, Allobaculum abundance was inversely correlated with lysozyme expression, while Anaerotruncus abundance was positively correlated with it by Spearman test. In conclusion, intestinal microbiota dysbiosis and decreased AMPs of Paneth cells might participate in the pathogenesis of intestinal barrier dysfunction in HANP.

Dysbiosis of intestinal microbiota and decrease in paneth cell antimicrobial peptide level during acute necrotizing pancreatitis in rats.

OBJECTIVES: Intestinal barrier dysfunction plays an important role in acute necrotizing pancreatitis (ANP) and intestinal microbiota dysbiosis was involved in intestinal barrier failure. Paneth cells protect intestinal barrier and are associated with intestinal microbiota. Here, we investigated changes in intestinal microbiota and antimicrobial peptides of Paneth cells in ileum during ANP. METHODS: Rats with ANP were established by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct and sacrificed at 24h and 48h, respectively. Injuries of pancreas and distal ileum were evaluated by histopathological score. Intestinal barrier function was assessed by plasma diamine oxidase activity (DAO) and D-lactate. Systemic and intestinal inflammation was evaluated by TNFα, IL-1ß and IL-17A concentration by ELISA, respectively. 16S rRNA high throughput sequencing on fecal samples was used to investigate the changes in intestinal microbiota in the ANP group at 48h. Lysozyme and α-defensin5 were measured by real-time PCR, western blot and immunofluoresence. RESULTS: ANP rats had more severe histopathological injuries in pancreas and distal ileum, injured intestinal barrier and increased expression of TNFα, IL-1ß and IL-17A in plasma and distal ileum compared with those of the sham-operated (SO) group. Principal component analysis (PCA) showed structural segregation between the SO and ANP groups. Operational taxonomic unit (OTU) number and ACE index revealed decreased microbiota diversity in the ANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. At phyla level, Saccharibacteria and Tenericutes decreased significantly. At genus level, Escherichia-Shigella and Phascolarctobacterium increased significantly, while Candidatus_Saccharimonas, Prevotellaceae_UCG-001, Lachnospiraceae_UCG-001, Ruminiclostridium_5 and Ruminococcaceae_UCG-008 decreased significantly. Lysozyme and α-defensin5 mRNA expression levels decreased significantly in ANP group at 48h. Protein expression of lysozyme decreased in ANP groups at 24h and 48h. Meanwhile, the relative abundance of Escherichia-Shigella correlated inversely with the decrease in lysozyme. CONCLUSION: The disorder in intestinal microbiota and decreases of Paneth cell antimicrobial peptides might participate in the pathogenesis of intestinal barrier dysfunction during ANP.

Mucosal adherent bacterial dysbiosis in patients with colorectal adenomas.

Recent reports have suggested that the gut microbiota is involved in the progression of colorectal cancer (CRC). The composition of gut microbiota in CRC precursors has not been adequately described. To characterize the structure of adherent microbiota in this disease, we conducted pyrosequencing-based analysis of 16S rRNA genes to determine the bacterial profile of normal colons (healthy controls) and colorectal adenomas (CRC precursors). Adenoma mucosal biopsy samples and adjacent normal colonic mucosa from 31 patients with adenomas and 20 healthy volunteers were profiled using the Illumina MiSeq platform. Principal coordinate analysis (PCoA) showed structural segregation between colorectal adenomatous tissue and control tissue. Alpha diversity estimations revealed higher microbiota diversity in samples from patients with adenomas. Taxonomic analysis illustrated that abundance of eight phyla (Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Cyanobacteria, Candidate-division TM7, and Tenericutes) was significantly different. In addition, Lactococcus and Pseudomonas were enriched in preneoplastic tissue, whereas Enterococcus, Bacillus, and Solibacillus were reduced. However, both PCoA and cluster tree analyses showed similar microbiota structure between adenomatous and adjacent non-adenoma tissues. These present findings provide preliminary experimental evidence supporting that colorectal preneoplastic lesion may be the most important factor leading to alterations in bacterial community composition.

[MiR-183 Regulates Proliferation of SW1990 Pancreatic Cancer Cell Line by Targeting PDCD4].

OBJECTIVES: To investigate the effect of miR-183 on the cell proliferation in SW1990 pancreatic cancer cell line by targeting programmed cell death factor 4(PDCD4). METHODS: The SW1990 pancreatic cancer cells were transfected with miR-183 mimics and inhibitors at different concentrations, the alteration of PDCD4 levels was observed at specific concentrations by qPCR and Western blot. The cellular proliferation of transfected cells was determined by MTT assay. The distribution of cell cycle and apoptosis was examined by flow cytometry (FCM) and Hoechst 33258 staining. The expression of B-cell lymphoma(bcl-2) was evaluated by Western blot. RESULTS: The miR-183 mimic and inhibitor (at concentrations of 50 nmol/L or 150 nmol/L) showed significantly increasing or decreasing effects on the levels ofmiR-183 respectively. The expression of PDCD4 was downregulated in the cells transfected with miR-183 mimics, while significantly upregulated in the cells treated with miR-183 inhibitors. Western blot showed that miR-183 inhibitors resulted in a marked decrease in the expression levels of bcl-2. The growth of SW1990 cells was obviously inhibited after anti-miR-183 treatment, while an increase of apoptosis cells proportion and cell cycle G0/G1 arrest were observed after miR-183 inhibitors transfection. CONCLUSIONS: The miR-183 inhibitors could restrain cell proliferation, promote cell apoptosis and increase G0/G1 arrest in SW1990 pancreatic cancer cells, which may be possibly through targeting PDCD4.

miR-183 induces cell proliferation, migration, and invasion by regulating PDCD4 expression in the SW1990 pancreatic cancer cell line.

The aim of this study was to investigate the function of miR-183 in the SW1990 cancer cell line, and the mechanisms regulating these processes. miRNAs are known to play important roles in cancer cell development. However, the pattern and biological role of miR-183 in pancreatic cancer remain largely unknown. Here, we have reported the reduction in pancreatic cancer cell growth in vitro by miR-183 intervention, by inducing apoptosis and decreasing the Bcl-2 expression. Moreover, miR-183 was observed to enhance pancreatic cancer cell migration and invasion, whereas inhibition of miR-183 caused an opposite effect. miR-183 inhibition was shown to increase E-cadherin expression and decrease N-cadherin expression. These regulatory actions play an important role in the cancer epithelial-mesenchymal transition (EMT). Mechanistically, we demonstrated that the overexpression of miR-183 decreased the expression of PDCD4 (programmed cell death 4) mRNA and protein, and vice versa. This helped to identify PDCD4 as the target genes in pancreatic cancer. In conclusion, our analyses indicated miR-183 to be an important contributor to cell migration. This could also be used as a potential therapeutic target for pancreatic cancer treatment.

An Edwardsiella tarda mutant lacking UDP-glucose dehydrogenase shows pleiotropic phenotypes, attenuated virulence, and potential as a vaccine candidate.

Edwarsiella tarda is highly resistant to the action of cationic antimicrobial peptides (CAMPs). However, the mechanism underlying CAMP resistance is not clear. The enzyme UDP-glucose dehydrogenase (Ugd) that converts UDP-glucose into UDP-glucuronic acid may be important for this resistance. In this study, a ugd gene was identified in E. tarda and its functional role was analyzed using an in-frame deletion mutant Δugd and the complemented strain ugd+. The lipopolysaccharide (LPS) produced by Δugd consisted of a truncated core oligosaccharide (OS) with no O-antigen attached. The ugd mutant was extremely sensitive to CAMPs, presumably because of alterations in LPS structure. The mutant also exhibited enhanced autoaggregation and biofilm formation and reduced hemolytic activity. Using different infection models we found that Δugd was impaired in survival within macrophages and displayed significantly attenuated virulence and an impaired ability to persist within the host. The expression of ugd was induced by polymyxin B and under the control of PhoP and RcsB, two response regulators of the bacterial two-component systems that we identified previously. Moreover, vaccination of turbot (Scophthalmus maximus) with Δugd by intraperitoneal injection elicited significant protection against the wild-type E. tarda strain, suggesting that Δugd may be promising as a potential vaccine candidate against edwardsiellosis.