Gut microbiota dynamics and fecal SCFAs after colonoscopy: accelerating microbiome stabilization by Clostridium butyricum.

BACKGROUND: Colonoscopy is a classic diagnostic method with possible complications including abdominal pain and diarrhoea. In this study, gut microbiota dynamics and related metabolic products during and after colonoscopy were explored to accelerate gut microbiome balance through probiotics. METHODS: The gut microbiota and fecal short-chain fatty acids (SCFAs) were analyzed in four healthy subjects before and after colonoscopy, along with seven individuals supplemented with Clostridium butyricum. We employed 16S rRNA sequencing and GC-MS to investigate these changes. We also conducted bioinformatic analysis to explore the buk gene, encoding butyrate kinase, across C. butyricum strains from the human gut. RESULTS: The gut microbiota and fecal short-chain fatty acids (SCFAs) of four healthy subjects were recovered on the 7th day after colonoscopy. We found that Clostridium and other bacteria might have efficient butyric acid production through bioinformatic analysis of the buk and assessment of the transcriptional level of the buk. Supplementation of seven healthy subjects with Clostridium butyricum after colonoscopy resulted in a quicker recovery and stabilization of gut microbiota and fecal SCFAs on the third day. CONCLUSION: We suggest that supplementation of Clostridium butyricum after colonoscopy should be considered in future routine clinical practice.

Weizmannia coagulans BCF-01: a novel gastrogenic probiotic for Helicobacter pylori infection control.

The widespread prevalence of Helicobacter pylori infection, particularly in China, contributes to the development of gastrointestinal diseases. Antibiotics have limitations, including adverse reactions and increased antibiotic resistance. Therefore, identification of novel gastrogenic probiotics capable of surviving the acidic gastric environment and effectively combating H. pylori infection has potential in restoring gastric microbiota homeostasis. Five novel strains of human gastrogenic Weizmannia coagulans (BCF-01-05) were isolated from healthy gastric mucosa and characterized using 16S rDNA identification. Acid resistance, H. pylori inhibition, and adherence to gastric epithelial cells were evaluated in in-vitro experiments and the molecular mechanism explored in in-vivo experiments. Among the gastric-derived W. coagulans strains, BCF-01 exhibited the strongest adhesion and H. pylori inhibition, warranting further in-vivo safety evaluation. Through 16S rRNA sequencing of a mouse model, BCF-01 was determined to significantly restore H. pylori-associated gastric dysbiosis and increase the abundance of potential probiotic bacteria. Furthermore, BCF-01 enhanced mucosal tight junction protein expression and inhibited the TLR4-NFκB-pyroptosis signaling pathway in macrophages, as demonstrated by qRT-PCR and western blotting.These findings highlight the potential of BCF-01 in the prevention and control of H. pylori infection. Specifically, treatment with BCF-01 effectively restored gastric microecology and improved H. pylori-mediated mucosal barrier destruction while reducing inflammation through inhibition of the TLR4-NFκB-pyroptosis signaling pathway in macrophages. BCF-01 is a promising alternative to traditional triple therapy for H. pylori infections, offering minimal side effects with high suitability for high-risk individuals.

Soluble CD80 oral delivery by recombinant Lactococcus suppresses tumor growth by enhancing antitumor immunity.

CD80 is an important co-stimulatory molecule that participates in the immune response. Soluble CD80 can induce T cell activation and overcome PDL1-mediated immune suppression. In this study, we aimed to construct recombinant Lactococcus lactis for oral delivery of the soluble CD80 (hsCD80) protein or the fusion protein containing the cholera toxin B subunit (CTB) and hsCD80 (CTB-hsCD80) under the control of the nisin-inducible expression system. The recombinant L. lactis expressed and secreted hsCD80 or CTB-hsCD80 fusion proteins after induction by nisin in vitro and in the enteric cavity. Additionally, the CTB-hsCD80 fusion protein showed uptake by intestinal epithelial cells, was cleaved by the furin protease, and was released as free hsCD80 protein into the blood circulation. Orally administered hsCD80 and CTB-hsCD80 containing L. lactis increased the proportion of activated T cells in the spleen and intestinal epithelium, inhibited tumor growth, and prolonged the survival of tumor-bearing mice. The hsCD80-containing L. lactis showed greater therapeutic effects on primary colonic adenoma in APCmin/- mice and completely suppressed tumor growth. Further, recombinant CTB-hsCD80 in L. lactis was more efficient than hsCD80-containing bacteria in inhibiting the growth of xenografted colon cancer and melanoma cells. hsCD80 engineered probiotics may serve as a promising new approach for antitumor immunotherapy, especially for colorectal cancer.

Bacteroides dorei BDX-01 alleviates DSS-induced experimental colitis in mice by regulating intestinal bile salt hydrolase activity and the FXR-NLRP3 signaling pathway.

Background: The relationships among intestinal dysbiosis, bile acid (BA) metabolism disorders, and ulcerative colitis pathogenesis are now recognized. However, how specific strains regulate BA metabolism to alleviate colitis is still unclear. This study investigated the effects of Bacteroides dorei on the development of acute colitis and elucidated the underlying mechanisms. Methods: The safety of BDX-01 was evaluated in vitro and in vivo. 2.5% dextran sulfate sodium (DSS) induced colitis in C57BL/6 mice, Caco-2, and J774A.1 cells were used to evaluate the anti-inflammatory effect of BDX-01. qPCR and Western blotting were used to detect the expression of inflammatory pathways. Microbiota composition was analyzed by 16S rRNA gene sequencing. Enzyme activity analysis and targeted metabolomics were used to analyze fecal bile salt hydrolase (BSH) and BA levels. Antibiotic-induced pseudo-germ-free mice were used to investigate the role of gut microbiota in the alleviation of colitis by BDX-01. Results: We confirmed the safety of novel strain Bacteroides dorei BDX-01 in vitro and in vivo. Oral BDX-01 administration significantly ameliorated the symptoms and pathological damage of DSS-induced acute colitis. Moreoever, 16S rRNA sequencing and enzyme activity analysis showed that BDX-01 treatment increased intestinal BSH activity and the abundance of bacteria harboring this enzyme. Targeted metabolomics revealed that BDX-01 significantly increased intestinal BA excretion and deconjugation. Certain BAs act as FXR agonists. The ß-muricholic acid (ßMCA): taurine ß-muricholic acid (T-ßMCA) and cholic acid (CA): taurocholic acid (TCA) ratios and the deoxycholic acid (DCA) level decreased markedly in the colitis models but increased substantially in BDX-01-treated mice. The colonic farnesoid X receptor (FXR) and fibroblast growth factor 15 (FGF15) were upregulated in mice treated with BDX-01. BDX-01 downregulated the expression of colonic proinflammatory cytokines pyrin domain-containing 3 (NLRP3), ASC, cleaved caspase-1, and IL-1ß. Antibiotic treatment didn't abolish the protective effect of BDX-01 on colitis. In vitro studies showed TßMCA abolished the effects of BDX-01 on FXR activation and inhibition of the NLRP3 inflammasome activation. Conclusion: BDX-01 improved DSS-induced acute colitis by regulating intestinal BSH activity and the FXR-NLRP3 signaling pathway. Our findings indicate that BDX-01 is a promising probiotic to improve the management of ulcerative colitis.

Tim3scFv-Transforming Lactobacillus Inhibits Transplanted Tumor of Renal-Cell Carcinoma.

INTRODUCTION: T-cell immunoglobulin-3 (Tim-3) antibody drugs can treat malignant renal tumors but are expensive. To overcome this limitation, Lactococcus lactis host bacteria were used to express Tim-3 single-chain antibodies. METHODS: The pLAN-CTB-Tim3scFv plasmid was constructed using molecular cloning technology and transformed into Lactococcus lactis. Expression and immune activity of proteins in the transformed bacteria were analyzed using Western blotting and enzyme-linked immunosorbent assay in vitro. A mouse subcutaneously transplanted tumor model of renal adenocarcinoma was constructed. The promoting effect of transformed bacteria on mouse spleen lymphocyte activation and their inhibitory effect on transplanted tumors were analyzed. RESULTS: Transformed L. lactis NZ-CTB-Tim3scFv and NZ-Tim3scFv were successfully constructed. CTB-Tim3scFv secreted by NZ-CTB-Tim3scFv showed immunological activity. Compared with the NZ-Tim3scFv and NZ-Vector groups, the subgroups of splenic lymphocytes in the NZ-CTB-Tim3scFv group had a higher proportion of CD3+CD4+, CD3+CD8a+, and CD3+CD69+ cells. Ki67 and CD31 expression in the NZ-CTB-Tim3scFv group was significantly reduced. Tumor volume in the NZ-CTB-Tim3scFv group increased the least. DISCUSSION/CONCLUSION: Secretion of CTB-Tim3scFv promoted the proliferation and activation of spleen lymphocytes and inhibited growth, cell proliferation, and angiogenesis of tumors. The proposed method is low cost and convenient with potential to become a new immunotherapy approach for renal-cell carcinoma.

FDCSP Is an Immune-Associated Prognostic Biomarker in HPV-Positive Head and Neck Squamous Carcinoma.

Background: Head and neck squamous carcinoma (HNSC) poses a major threat to human life. The role of human papillomavirus (HPV) infection in the initiation and progression of HNSC is becoming more widely accepted. HPV-positive (HPV+) HNSC has shown unique responses to cancer therapies, which may be due to differences in immune cell infiltration. It is critical to determine how the immune responses to HPV in HNSC are regulated. Methods: Transcriptome data of HNSC from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database were analyzed. Then, the CIBERSORT algorithm was used to calculate immune cell infiltration in HNSC. FDCSP expression level was detected by qPCR in the HNSC tissues collected from the Nanfang Hospital. Results: Follicular dendritic cell secreted protein (FDCSP) was highly expressed in HPV+ HNSC, and higher expression of FDSCP was associated with a favorable prognosis. In HPV+ HNSC samples, FDCSP significantly increased the proportion of T follicular helper cells (TFHs). FDCSP expression was also found to be associated with TP53 mutation status in HPV+ HNSC. The function of FDCSP was intimately connected to chemokine pathways, particularly with the C-X-C motif chemokine ligand 13 (CXCL13). We verified that the high expression of FDCSP in HPV+ HNSC and higher FDCSP is closely related to prognosis in HNSC samples we collected by qPCR. Conclusions: Collectively, these findings may provide fresh evidence that FDCSP is a potential chemokine-associated prognostic biomarker in HPV+ HNSC.

Construction and Characterization of n6-Methyladenosine-Related lncRNA Prognostic Signature and Immune Cell Infiltration in Kidney Renal Clear Cell Carcinoma.

Background: Kidney renal clear cell carcinoma (KIRC) lacks effective prognostic biomarkers and the role and mechanism of N6-methyladenosine (m6A) modification of long noncoding RNAs (lncRNAs) in KIRC remain unclear. Methods: We extracted standard mRNA-sequencing and clinical data from the TCGA database. The prognostic risk model was obtained by Lasso regression and Cox regression. We randomly divided the samples into training and test sets, each taking half of the cases. Based on Lasso regression and Cox regression for training set, the prognostic risk signature was constructed; risk scores were calculated with the R package "glmnet." Based on the median value of the prognostic risk score, risk scores were calculated for each patient and we divided all KIRC samples into high-risk and low-risk groups. Then, high- and low-risk subtypes were established and their prognosis, clinical features, and immune infiltration microenvironment were evaluated in test set and the entire sampled data set. The reliability of the prognostic model was confirmed by receiver operating characteristic curve analysis. Results: We found 28 prognostic m6A-related lncRNAs and established a m6A-related lncRNAs prognostic signature. Risk score=AC015813.1∗(0.0086)+EMX2OS∗(-0.0101)+LINC00173∗(0.0309)+PWAR5∗(-0.0146)+SNHG1∗(0.0043). The signature showed a better predictive ability than other clinical indicators, including tumor node metastasis classification (TNM), histological, and pathological stages. In the high-risk group, M0 macrophages, CD8+ T cells, and regulatory T cells had significantly higher scores. Contrarily, in the low-risk group, activated dendritic cells, M1 macrophages, mast resting cells, and monocytes had significantly higher scores. In the high-risk group, LSECtin was overexpressed. In the low-risk group, PD-L1 was overexpressed. Moreover, high-risk patients may benefit more from AZ628. Conclusions: In conclusion, prognosis prediction of patients with KIRC and new insights for immunotherapy are provided by the m6A-related lncRNA prognostic signature.

Improving obesity and lipid metabolism using conjugated linoleic acid.

BACKGROUND: Conjugated linoleic acid (CLA) can prevent fatty acid accumulation induced by a high-fructose diet and improve lipid metabolism disorders in patients. OBJECTIVES: We aimed to investigate the effect of CLA on obesity and lipid metabolism and its possible mechanism. METHODS: Eight-month-old male BKS.Cg-Dock7m +/+ Leprdb /JNju (db/db) mice (n = 12) were fed a CLA mix composed of equivalent c9, t11-CLA and t10, c12-CLA for 1 month. The effect of CLA on body weight, water and food intake, and triglyceride (TG) and total cholesterol (TC) levels was investigated. PPARα, PPARγ and CD36 expression was determined by quantitative PCR and western blotting. Additionally, the expression of these three genes was studied in HepG2 cells treated with CLA and linoleic acid. RESULTS: CLA treatment notably reduced the dietary and water intake of db/db mice, effectively reduced body weight, and decreased serum TG and TC levels (p < 0.05). Increased expression of PPARα (p < 0.05) and decreased expression of CD36 (p < 0.001) were observed in the liver of mice that were fed CLA. CLA increased PPARα expression (p < 0.001) and decreased PPARγ (p < 0.001) and CD36 expression (p < 0.01) in HepG2 cells. CONCLUSIONS: Our results showed that CLA can improve lipid metabolism in obese mice through upregulation of PPARα expression and downregulation of CD36 expression.

Bioinformatics Analyses Indicate That Cathepsin G (CTSG) is a Potential Immune-Related Biomarker in Oral Squamous Cell Carcinoma (OSCC).

PURPOSE: Plenty of studies showed that the immune system was associated with cancer initiation and progression. This study aimed to explore the prognostic biomarkers from immune-related genes (IRGs) in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) and IRGs and transcription factors (TFs) were extracted. Then, the co-expression network between IRGs and TFs was constructed using the "WGCNA" package in R software. Furthermore, a gene expression signature according to IRGs was constructed to predict OSCC prognosis and its accuracy was validated by survival analysis. Subsequently, correlation analyses between risk-score and immune cells level and clinical parameters were performed. Finally, immune-related biomarkers were selected and further investigated using gain-of-function assays in vitro. RESULTS: A total of 32 normal cases and 317 OSCC cases were selected in our study. Differentially-expressed analysis indicated that there were 381 differentially-expressed IRGs and 62 TFs in OSCC. Among them, 25 TFs and 21 IRGs were enrolled in the co-expression network. Furthermore, we found that gene expression signature on the basis of 10 IRGs could predict the prognosis accurately and a high-risk score based on gene expression signature meant a high T classification, terminal clinical stage, and low immune cells level in OSCC. Finally, cathepsin G (CTSG) was identified as a potential immune-related biomarker and therapeutic target in OSCC. CONCLUSION: In conclusion, IRGs were directly involved in the development and progression of OSCC. Furthermore, CTSG was identified as a potential independent biomarker and might be an immunotherapeutic target in OSCC treatment.

Bioinformatic identification of key pathways, hub genes, and microbiota for therapeutic intervention in Helicobacter pylori infection.

The pathogenic mechanisms of Helicobacter pylori infection remain to be defined, and potential interventional microbiota are just beginning to be identified. In this study, gene-set enrichment analysis (GSEA) was used to integrate three H. pylori infection microarray data sets from the gene expression omnibus database and identified ten hallmark gene sets and 35 Kyoto encyclopedia of genes and genomes (KEGG) pathways that differed between healthy and Helicobacter pylori-infected individuals. Weighted gene co-expression network analysis (WGCNA) performed on two of the data sets identified three key gene coexpression modules. These modules contained 54 enriched KEGG pathways, 25 of which overlapped with the GSEA analysis, suggesting potentially important roles in H. pylori-infection. We selected 116 hub genes from the three key modules for in vitro validation at the transcriptional level using H. pylori Sydney Strain 1 and verified the upregulation of 80. WGCNA of the microbiomes based on 20 mucosal samples and a sequence read archive data set revealed four microbiota modules correlated with H. pylori infection. The negatively correlated modules contained 11 microbiome families. These findings provide new insight into the pathogenesis of H. pylori infection and systematically identify 25 key pathways, 80 upregulated hub genes, and 11 families of candidate interventional microbiota for further research.

NF-κB-mediated lncRNA AC007271.3 promotes carcinogenesis of oral squamous cell carcinoma by regulating miR-125b-2-3p/Slug.

Oral squamous cell carcinoma (OSCC) is the most common oral cancer. The molecular mechanisms of this disease are not fully understood. Our previous studies confirmed that dysregulated function of long non-coding RNA (lncRNA) AC007271.3 was associated with a poor prognosis and overexpression of AC007271.3 promoted cell proliferation, migration, invasion, and inhibited cell apoptosis in vitro, and promoted tumor growth in vivo. However, the underlying mechanisms of AC007271.3 dysregulation remained obscure. In this study, our investigation showed that AC007271.3 functioned as competing endogenous RNA by binding to miR-125b-2-3p and by destabilizing primary miR-125b-2, resulted in the upregulating expression of Slug, which is a direct target of miR-125b-2-3p. Slug also inhibited the expression of E-cadherin but N-cadherin, vimentin, and ß-catenin had no obvious change. The expression of AC007271.3 was promoted by the canonical nuclear factor-κB (NF-κB) pathway. Taken together, these results suggested that the classical NF-κB pathway-activated AC007271.3 regulates EMT by miR-125b-2-3p/Slug/E-cadherin axis to promote the development of OSCC, implicating it as a novel potential target for therapeutic intervention in this disease.

Novel Lactobacillus reuteri HI120 Affects Lipid Metabolism in C57BL/6 Obese Mice.

Intestinal probiotics are a primary focus area of current medical research. Probiotics such as bifidobacteria and lactobacilli can positively impact obesity and other metabolic diseases by directly or indirectly affecting lipid metabolism. However, the precise mechanisms of these effects remain unclear. In our previous work, the novel strain Lactobacillus reuteri HI120 was isolated and identified. HI120 expresses high levels of linoleic isomerase, resulting in the production of large amounts of conjugated linoleic acid (CLA) when mixed with linoleic acid (LA). As HI120 can efficiently transform LA into CLA, the effect of HI120 on the lipid metabolism in C57BL/6 obese mice was studied and the underlying molecular mechanism was explored in vitro. The results revealed no significant change in the diet, body weight, and serum triglyceride levels in mice. However, serum cholesterol levels were significantly decreased. The underlying mechanism may involve a CLA-mediated reduction in the gene expression levels of NPC1L1, SREBP-2, and HMG-CR, resulting in reduced cholesterol synthesis and absorption. Thus, HI120 can be developed as a potential probiotic formulation. After oral administration, LA from certain food sources can be converted into CLA in the human intestine to contribute to the prevention and treatment of obesity and hyperlipidemia.

M6A-related bioinformatics analysis reveals that HNRNPC facilitates progression of OSCC via EMT.

Increasing evidence suggests that N6-methyladenosine(m6A) has a vital role in cancer progression. Therefore, we aimed to explore the prognostic relevance of m6A-related genes in oral squamous cell carcinoma (OSCC). First, Expression profiles were downloaded from The Cancer Genome Atlas (TCGA) and m6A-related genes were extracted afterwards. Then, cluster analysis and principal component analysis (PCA) were used to analyze m6A-related genes. And differentially-expressed analysis was performed in R software. Furthermore, a risk model was constructed, and crucial m6A genes were selected to explore its biological effects in OSCC cells. Total of 13 m6A-related genes were extracted and 8 differentially-expressed genes were identified. Subsequently, m6A-based clustering showed 2 subtypes with different clinical outcome. In addition, a risk model was successfully established. Of 13 m6A-related genes, only heterogeneous nuclear ribonucleoprotein C (HNRNPC) might be an independent biomarker and mean unfavorable overall survival in OSCC by univariate and multivariate cox regression analysis. Functional studies revealed that overexpression of HNRNPC promoted carcinogenesis of OSCC via epithelial- mesenchymal transition (EMT). In total, a risk model of m6A-related genes in OSCC was established. Subsequently, HNRNPC was proved to promote OSCC carcinogenesis and be an independent biomarker prognostic biomarker of OSCC, suggesting that it might be a new biomarker and therapeutic target of OSCC.

Fibroblast Activation Protein (FAP) Overexpression Induces Epithelial-Mesenchymal Transition (EMT) in Oral Squamous Cell Carcinoma by Down-Regulating Dipeptidyl Peptidase 9 (DPP9).

PURPOSE: Fibroblast activation protein (FAP) acts as a tumor promoter via epithelial-mesenchymal transition (EMT) in human oral squamous cell carcinoma (OSCC). The present study was designed to investigate the FAP targeting proteins and explore the precise mechanism by which FAP promotes EMT in OSCC. PATIENTS AND METHODS: Proteins interacting with FAP were found and filtered by immunoprecipitation-mass spectrometry (IP-MS). Both DPP9 protein and mRNA were examined in 90 paired OSCC samples and matched normal tissue. DPP9 knockdown was conducted to determine its function in OSCC in vitro and in vivo. RESULTS: Dipeptidyl peptidase 9 (DPP9) was identified as interacting with FAP intracellularly by IP-MS. The levels of both DPP9 protein and mRNA were down-regulated in OSCC tissue. Lower DPP9 expression was correlated with unfavorable survival rates of OSCC patients. DPP9 knockdown accelerates the proliferation of OSCC cells in vitro and in vivo. Overexpression of FAP leads to a reduction in DPP9 expression. Likewise, DPP9 overexpression reverses the proliferation, migration, invasion and EMT induced by FAP during OSCC. CONCLUSION: Our study finds that FAP promotes EMT of OSCC by down-regulating DPP9 in a non-enzymatic manner. FAP-DPP9 pathway could be a potential therapeutic target of OSCC.

LncRNA AC007271.3 promotes cell proliferation, invasion, migration and inhibits cell apoptosis of OSCC via the Wnt/ß-catenin signaling pathway.

AIMS: Long noncoding RNA (lncRNA) AC007271.3 has been identified to be dysregulated in oral squamous cell carcinoma (OSCC) in our previous study. However, the precise role of AC007271.3 in OSCC remains unclear. In this study, we investigated the potential functions and the underlying mechanisms of AC007271.3 in OSCC. MATERIALS AND METHODS: The expression levels of AC007271.3 in OSCC tissues and cell lines were examined using RT-qPCR. The relationship between AC007271.3 level and clinicopathological characteristics was analyzed, and its association with patient prognosis was assessed by the Kaplan-Meier method. The biological function of AC007271.3 and its role in the development of OSCC through Wnt/ß-catenin signaling pathway were studied. KEY FINDINGS: We identified that AC007271.3 was up-regulated and positively correlated with advanced clinical stage, lymph node metastasis, poor histological differentiation and unfavorable prognosis. We explored the expression, function, and molecular mechanism of AC007271.3 in OSCC cells. Overexpression of AC007271.3 remarkably promoted cell proliferation in vitro and in vivo, induced cell migration, invasion and inhibited apoptosis in vitro, while knockdown of AC007271.3 attenuated cell proliferation, migration, invasion and induced apoptosis. Mechanistically, AC007271.3 overexpression substantially increased the expression of ß-catenin and the downstream target molecules CyclinD1, c-myc and Bcl-2, while silencing of AC007271.3 has the opposite effect. Rescued experiments showed that the ability to promote cell proliferation, migration, invasion and inhibiting apoptosis could be reversed when treated with the Wnt/ß-catenin pathway inhibitor. SIGNIFICANCE: Our data indicated that AC007271.3 could promote cell proliferation, invasion and inhibit cell apoptosis of OSCC via the Wnt/ß-catenin signaling pathway, which might provide a novel therapeutic approach for OSCC.

[Conjugated linoleic acid improves glucose and lipid metabolism in diabetic mice].

OBJECTIVE: To analyze the effect of conjugated linoleic acid (CLA) on glucose and lipid metabolism in obese diabetic (db/db) mice. METHODS: db/db mice were randomized for treatment with saline or CLA mixture administered intragastrically. The changes in body weight, dietary intake, water intake, oral glucose tolerance, triglyceride and total cholesterol were recorded after the treatments. HE staining and oil red O staining were used to assess liver pathologies and fatty acid content. The expression levels of PPARα, PPARγ, CD36, CHREBP and SREBP-1c were detected using real-time PCR and Western blotting. HepG2 cells were treated with CLA and linoleic acid and the expressions of PPARα, ACC, P-ACC, and CD36 were detected; the level of acetyl-CoA in the cell supernatant was detected using ELISA. RESULTS: CLA treatment obviously reduced the dietary and water intake of db/db mice, effectively reduced the body weight and decreased serum triglyceride and cholesterol levels (P < 0.05). CLA significantly reduced fasting blood glucose, increased glucose tolerance, reduced the accumulation of lipid droplets in the liver and improved lipid metabolism in db/db mice. The mice showed significantly increased expression of PPARα (P < 0.05) and lowered CD36 expression (P < 0.001) in the liver after CLA treatment. Cellular experiments showed that CLA significantly up-regulated PPARα (P < 0.001) and P-ACC and decreased the expression of CD36 (P < 0.01). ELISA showed that acetyl-CoA was significantly up-regulated in the cells after CLA treatment (P < 0.01). CONCLUSIONS: The mixture of two conjugated linoleic acid isomers can reduce fasting blood glucose, increase glucose tolerance and improve glycolipid metabolism in db/db mice by enhancing the expression of PPARα, increasing P-ACC and inhibiting CD36 expression.

[Intragastric administration of interferon-α-transformed Bifidobacterium promotes lymphocyte proliferation and maturation in mice].

OBJECTIVE: To investigate the effects of intragastric administration of human interferon-α (hIFN-α)-transformed Bifidobacterium on immune functions of mice. METHODS: The E.coli-Bifidobacterium shuttle expression vector containing hIFN-α gene was constructed and transformed into Bifidobacterium. The hIFN-α-transformed Bifidobacterium suspension (1010 /ml) was prepared after induction with 0.2% L-arabinose for hIFN-α expression and administered intragastrically in male Balb/C mice at the dose of 0.1 ml every other day for 2 weeks, with the mice receiving empty vector-transformed Bifidobacteria as the negative control and those having an equal volume of saline as the blank control. The percentages of mononuclear cell subsets in the thymus, spleen and blood were detected in the mice by flow cytometry, and the serum levels of IL-4, IL-12, IFN-γ and TNF-α were assayed using mouse cytokine FlowCytomix Kit. RESULTS: The percentages of CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the thymus, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the spleen, and CD3⁺CD8⁺ cells in the blood all increased significantly in IFN group as compared with those in the negative and blank control groups (P<0.01 or 0.05). The serum level of IFN-γ also increased significantly (P<0.05) while IL-4 level remained unchanged in IFN group compared with those in the two groups. CONCLUSION: Intragastric administration of hIFN-α-transformed Bifidobacterium promotes lymphocyte proliferation and maturation and increases the serum levels of Th1 cytokines in mice.

[Biochemical and pathological analysis of mice with type 2 diabetes mellitus induced by high-fat diet and low-dose streptozotocin injections].

OBJECTIVE: To analyze the biochemical and pathological changes in mice with type 2 diabetes mellitus (T2DM) induced by high-fat diet combined with low-dose streptozotocin (STZ) injections. METHODS: C57BL/6J mice were divided randomly into normal control group (NC group), high-fat diet group (HC group) and high-fat diet plus STZ group (HC+STZ group). The mice were fed on normal chow or a high-fat diet for 1 month before two introperitoneal injections of STZ (40 mg/kg) or citrate buffer with an interval of 24 h as appropriate. Fasting blood glucose (FBG) was detected every week for 4 weeks, and oral glucose tolerance test (OGTT) was performed one month after the injections, after which the biochemical profiles, islet and liver were evaluated by immunohistochemical and pathological analysis. RESULTS: In HC+STZ group, FBG was above the cutoff value (13.89 mmol/L) in 75% of the mice at 1 week after STZ injections and in all the mice at two weeks except for the death of 1 mouse, with a success rate of modeling of 91.3%. FBG in HC group, though slightly higher than that in NC group, remained normal (6.8 mmol/L). The body weight in HC+STZ and HC groups was significantly higher than that in NC group after feeding but without obvious increases after the injections (P<0.01). Blood glucose in HC+STZ group at 0.5 to 2 h after OGTT and the area under curve (AUC) were higher than those in NC and HC groups (P<0.01); the AUC in HC group was a also higher than that in NC group (P<0.05). Plasma creatinine was significantly higher in HC+STZ group than in NC (P<0.01) and HC (P<0.05) groups. Insulin secretion by the islets decreased obviously in HC+STZ and HC group. The mice in HC+STZ group showed atrophy, fibrosis, and vacuolization in the islets with mild fatty liver but no visible renal pathologies. CONCLUSION: High-fat diet and low-dose STZ injections can induce T2DM in mice with very similar biochemical and pathological changes to human T2DM and with such complications as fatty liver.

Relationship between p53 gene codon-72 polymorphisms and hypertrophic scar formation following caesarean section.

The aim of the present study was to determine the relationship between p53 gene codon-72 polymorphisms and hypertrophic scar formation following caesarean section (CS). Blood samples from 260 female patients were collected one week following a CS for the detection of p53 gene polymorphisms using a molecular beacon-coupled quantitative polymerase chain reaction technique. Patients had follow-ups for 12-18 months to observe the scar formation. From these observations, the relationship between the p53 codon-72 polymorphisms and hypertrophic scar formation occurrence was investigated. Among the patients with the CCC/CCC genotype, nine patients had hypertrophic scars and 46 patients showed normal healing, which is a ratio of 0.19. However, the follow-up investigations indicated that the presence of a homozygous or heterozygous C-to-G alteration at the codon-72 site in gene p53 resulted in 13 patients with hypertrophic scars and 192 patients with normal healing, which is a ratio of 0.07. Therefore, these results indicate that patients with the CCC/CCC genotype had a higher risk of developing hypertrophic scars compared with that for patients with the CCC/CGC or CGC/CGC genotypes.

Sphingosine-1-phosphate inhibits ceramide-induced apoptosis during murine preimplantation embryonic development.

Sphingolipids are a complex family of naturally occurring molecules enriched with lipid rafts that contribute to their unique biochemical properties. Sphingolipid metabolites, including ceramide (Cer) and sphingosine-1-phosphate (S1P), are bioactive signaling molecules that regulate cell movement, differentiation, survival, and apoptosis, but their effects on preimplantation development of murine embryos are not well-characterized. In this study, murine zygotes were collected, cultured in vitro, and treated with 50 µM C2-Cer plus various concentrations of S1P. The blastocyst formation rate was decreased in the C2-Cer-treated group, compared with that in the control group and the group treated with 50 µM C2-Cer plus 25, 50, or 100 nM S1P (P < 0.05), respectively. The total cell number of the blastocysts from various treatment groups was similar at 110 hours post-hCG treatment, but that from the group treated with 50 µM C2-Cer was significantly decreased at 120 hours post-hCG treatment, compared with the control group and the group treated with 50 µM C2-Cer plus 50 nM S1P. However, the apoptotic cell number of blastocysts from the group treated with 50 µM C2-Cer was significantly increased at 110 and 120 hours post-hCG treatment, compared with the control group and the group treated with 50 µM C2-Cer plus 50 nM S1P. Moreover, expression of p53 in the group treated with 50 µM C2-Cer was higher than that in the control group and the group treated with 50 µM C2-Cer plus 50 nM S1P (P < 0.05). In conclusion, Cer decreases the blastocyst formation rate and induces embryonic cell apoptosis, but S1P partly inhibits the effects of Cer during preimplantation development of murine embryos.