Modulating intestinal barrier function by sphingosine-1-phosphate receptor 1 specific agonist SEW2871 attenuated ANIT-induced cholestatic hepatitis via the gut-liver axis.

Bile acid (BA) homeostasis throughout the enterohepatic circulation system is a guarantee of liver physiological functions. BA circulation disorders is one of the characteristic clinical manifestations of cholestasis, and have a closely relationship with intestinal barrier function, especially ileum. Here, our in vivo and in vitro studies showed that intestinal tight junctions (TJs) were disrupted by α-naphthylisothiocyanate (ANIT), which also down-regulated the protein expression of sphingosine-1-phosphate receptor 1 (S1PR1) in the top of villus of mice ileum. Activating S1PR1 by specific agonist SEW2871 could improve TJs via inhibiting ERK1/2/LKB1/AMPK signaling pathway in the ileum of ANIT-treated mice and ANIT-cultured Caco-2 cells. SEW2871 not only regained ileum TJs by activating S1PR1 in the epithelial cells of ileum mucosa, but also recovered ileum barrier function, which was further verified by the recovered BA homeostasis in mice ileum (content and tissue) by using of high-performance liquid chromatographytandem mass spectrometry (LC-MS/MS). Subsequently, the improved intestinal injury and inflammation further strengthened that SEW2871 modulated intestinal barrier function in ANIT-treated mice. Finally, our data revealed that along with the down-regulated levels of serum lipopolysaccharides (LPS), SEW2871 improved liver function and relieved hepatitis via blocking TLR4/MyD88/NF-kB signaling pathway in ANIT-treated mice. In conclusion, these results demonstrated that activating intestinal S1PR1 by SEW2871 could modulate intestinal barrier function, leading to the improvement of cholestatic hepatitis in ANIT-treated mice via the "gut-liver" axis.

Influence of Zhuanggu Guanjie Pill on Seven Cytochrome P450 Enzymes Based on Probe Cocktail and Pharmacokinetics Approaches.

BACKGROUND: The use of herbal medicines has tremendously increased over the past few decades. Case reports and controlled clinical investigations of herbal-drug interactions have been reported. Since Cytochrome P450 (CYP) enzymes play an important role in drug interactions. The evaluation of the influence of herbal medicines on the activities of CYPs is beneficial to promote scientific and rational clinical use of herbal medicines. OBJECTIVE: Herein, we aimed to develop and validate a method to simultaneously quantify seven CYP cocktail probe drugs consisting of phenacetin (PNC), bupropion (BPP), losartan potassium (LK), omeprazole (OMP), dextromethorphan (DM), chlorzoxazone (CZZ) and midazolam (MDZ) and their respective metabolites in a single acquisition run and use this method to evaluate the influence of Zhuanggu Guanjie Pill (ZGGJP) on seven CYPs. METHODS: A cost-effective and simple UHPLC-(±)ESI-MS/MS method for simultaneous determination of seven probe drugs and metabolites in rat plasma was developed and validated. Male and female rats were randomly divided into three groups and treated with 1.2 g/kg/d ZGGJP, 5 g/kg/d ZGGJP and 0.5% CMC-Na for 14 consecutive days. After 24 h of the last administration, all rats were administrated orally with probe drugs. The influence of ZGGJP on the CYPs was carried out by comparing the metabolic ratio (Cmax, AUC0-t) of metabolites/probe drugs in rats. RESULTS: The calibration curves were linear, with correlation coefficient > 0.99 for seven probe drugs and their corresponding metabolites. Intra- and inter-day precisions were not greater than 15% RSD and the accuracies were within ±15% of nominal concentrations. The ZGGJP showed significant inductive effect on CYP1A2, CYP2B6, CYP2C9 and CYP3A in male and female rats. CONCLUSION: ZGGJP had inductive effects on CYP1A2, CYP2B6, CYP2C9 and CYP3A in male and female rats.

Comparison of bile acids profiles in the enterohepatic circulation system of mice and rats.

Bile acids (BAs) were selected as biomarkers for the diagnosis and prevention of multiple liver diseases, and they were also considered as an important signal transductor via "liver-gut" axis. As important factors for maintaining the normal function and tissue morphology, BA homeostasis throughout the enterohepatic circulation system was guaranteed by BA synthases and transporters, nuclear receptors (NRs) and gut microbiota, all of which presented significant species differences. Thus, we simultaneously quantify BA profiles in the enterohepatic circulation of SD rats and C57BL/6 mice to reveal the species differences of BA homeostasis between these two main rodents of preclinical studies. Our results showed that BA profiles of mice plasma, bile and liver were most dissimilar from these of rats. Meanwhile, BAs profiles also presented obvious species differences in the intestine of mice and rats, especially small intestine. Unlike rats, taurine-conjugated bile acids (T-BAs) were predominant in mice small intestine content and tissue, in which taurocholic acid (TCA) was the most prominent BAs. BAs dynamic analysis showed that compared with rats, mice showed stranger taurine and glycine de-conjugations in lager intestine. However, both the ratios of unconjugated bile acids (Un-BAs) to conjugated BAs, and secondary BAs to primary BAs in mice small content and tissue were all much lower than these in rats. Furthermore, ileum BAs profiles also showed significantly separation trend between rats and mice, whether content or tissue. Our data revealed that the patterns of BAs homeostasis in mice enterohepatic circulation system were significantly different from these in rats (especially in intestine), suggesting that more cautious should be paid to the selection of BAs as biomarkers for disease diagnosis or/and drug induced toxicity, and the certain role and mechanism of individual BA in the pathological process of BA-related diseases via "liver-gut" axis should be verified by using of multiple species.

Study on the effects of Zhuanggu Guanjie Pill, a modern Chinese medicine formula, on the activities and mRNA expression of seven CYP isozymes in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhuanggu Guanjie Pill (ZGGJP), a modern Chinese medicine formula, is composed of 12 herbs and has been used to treat osteoporosis in China for almost 30 years. However, no in vivo study of the influences of ZGGJP on the cytochrome P450 (CYP) activities have been reported. AIM OF THE STUDY: The aim of this study was to evaluate the effects of ZGGJP on the activities and the mRNA expression of CYP enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A) and their corresponding nuclear receptor levels in rats. MATERIALS AND METHODS: After 7 days oral treatment of ZGGJP at low- and high-dose, cocktail solution was given to rats. Blood samples were collected at series of time points. The plasma concentrations of probe drugs and their corresponding metabolites were determined by UPLC-MS/MS. The influence of ZGGJP on the activities of seven CYPs were evaluated the metabolic ratios (Cmax and AUC0-t) for metabolites/probe drugs. In addition, the effects of ZGGJP on the mRNA expression of CYPs and their corresponding nuclear receptors in rat liver were evaluated by real-time PCR. RESULTS: ZGGJP showed significant inductive effects on CYP1A2 and CYP2B6 of both male and female rats. The influence of ZGGJP on CYP2C9 and CYP3A showed gender difference. ZGGJP could induce the activities of CYP2C9 and CYP3A in female rats, but have no influence on the activities in male rats. ZGGJP had no effects on CYP2D6, CYP2C19 and CYP2E1. The mRNA expression results of CYPs were in accordance with the pharmacokinetic results. The mRNA expression levels of constitutive androstane receptor (CAR) and vitamin D receptor (VDR) were increased significantly in female rats at high dosage, but no significant changes were observed in male rats. CONCLUSION: ZGGJP had inductive effects on CYP1A2 and CYP2B6 in both male and female rats. The results showed that ZGGJP could induce the activities of CYP2C9 and CYP3A in female rats, but had no effect in male rats. This may suggest that the influence of ZGGJP on CYP2C9 and CYP3A exhibit gender difference. The inductive effects of ZGGJP on the activities of CYPs, exhibiting gender difference, may be regulated by CAR and VDR. Therefore, co-administration of ZGGJP with other drugs, especially using CYP2C9 and CYP3A substrates in females, may need dose adjustment to avoid herb-drug interaction.

LncRNA BBOX1-AS1 upregulates HOXC6 expression through miR-361-3p and HuR to drive cervical cancer progression.

OBJECTIVES: Over the past years, growing attention has been paid to deciphering the pivotal role of long non-coding RNAs (lncRNAs) in regulating the occurrence and development of human malignancies, cervical cancer (CC) included. Nonetheless, the regulatory role of lncRNA BBOX1 antisense RNA 1 (BBOX1-AS1) has not been explored as yet. MATERIAL AND METHODS: The expression of BBOX1-AS1 was detected by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8), colony formation, TUNEL, Western blot, transwell and immunofluorescence assays testified the critical role of BBOX1-AS1 in CC. The relationship between RNAs (BBOX1-AS1, miR-361-3p, HOXC6 and HuR) was analysed by luciferase reporter, RNA Immunoprecipitation (RIP) and RNA pull-down assays. RESULTS: BBOX1 antisense RNA 1 antisense RNA 1 was revealed to be highly expressed in CC. Decreased expression of BBOX1-AS1 had suppressive effects on CC cell growth and migration. Molecular mechanism assays verified that BBOX1-AS1 had negative interaction with miR-361-3p in CC. Additionally, homeobox C6 (HOXC6) was validated to be a downstream target of miR-361-3p in CC. Furthermore, ELAV-like RNA-binding protein 1, also known as HuR, was uncovered to be capable of regulating the mRNA stability of HOXC6 in CC. More importantly, rescue assays delineated that knockdown of HuR after overexpressing miR-361-3p could reverse BBOX1-AS1 upregulation-mediated effect on CC progression. Similarly, the function induced by BBOX1-AS1 upregulation on CC progression could be countervailed by HOXC6 depletion. CONCLUSIONS: BBOX1 antisense RNA 1 facilitates CC progression by upregulating HOXC6 expression via miR-361-3p and HuR.

FENDRR suppresses cervical cancer proliferation and invasion by targeting miR-15a/b-5p and regulating TUBA1A expression.

BACKGROUND: Previous literature has revealed long non-coding RNAs (lncRNAs) are crucial regulators for cell functions and gene expression. LncRNA fetal-lethal non-coding developmental regulatory RNA (FENDRR) was reported as a biological suppressor in several types of human cancers, yet relevant mechanisms and biological effects of FENDRR with regards to cervical cancer (CC) are not explored until now. METHODS: In this study, quantitative real-time polymerase chain reaction (qRT-PCR) analysis detected gene expression in tissues and cells. Gain- or loss-of-function experiments revealed the biological effects of FENDRR and miR-15a/b-5p on CC cell functions. Bioinformatics tools were used to predict the relevant genes. Mechanism experiments including RNA immunoprecipitation (RIP) assay, pull down assay and luciferase reporter assay depicted the binding situation and coexistence of indicated genes. RESULTS: FENDRR was downregulated in CC tissues and cells, which suppressed CC progression. MiR-15a-5p and miR-15b-5p shared binding sites with FENDRR and had interaction with FENDRR. Tubulin alpha1A (TUBA1A) was downregulated in CC tissues and positively modulated by FENDRR. TUBA1A was the target of miR-15a/b-5p. TUBA1A silencing rescued the effect of FENDRR overexpression on CC cell growth and migration. CONCLUSION: FENDRR inhibits CC progression through upregulating TUBA1A in a miR-15a/b-5p-dependent manner.

LINC00958 facilitates cervical cancer cell proliferation and metastasis by sponging miR-625-5p to upregulate LRRC8E expression.

Accepted as a malignant tumor worldwide, cervical cancer (CC) has attracted much attention for its high incidence and mortality rates. Previous studies have elucidated the critical regulatory function that long noncoding RNAs (lncRNAs) exert on the tumorigenesis and progression of diverse tumors. Although multiple investigations have depicted that LINC00958 has a great impact on the complex biological process of many cancers, knowledge concerning the regulatory role of LINC00958 in CC remains limited and needs to be further explored. In our study, LINC00958 expression was evidently overexpressed in CC tissues and cells. Besides this, LINC00958 negatively regulated miR-625-5p expression and was verified to bind with miR-625-5p in CC. Subsequently, it was testified by a series of experiments that LINC00958 promotes CC cell proliferation and metastasis by sponging miR-625-5p. Furthermore, the leucine-rich repeat containing the eight family member E (LRRC8E) could bind with miR-625-5p, and its expression was negatively modulated by miR-625-5p, whereas positively regulated by LINC00958 in CC. Final rescue assays verified the effects of LINC0095/LRRC8E interaction and miR-625-5p/LRRC8E interaction on CC cell proliferation and metastasis. Collectively, LINC00958 facilitates CC cell proliferation and metastasis via the miR-625-5p/LRRC8E axis.

Quantitative DNA methylation analysis of paired box gene 1 and LIM homeobox transcription factor 1 α genes in cervical cancer.

DNA methylation is associated with tumorigenesis and may act as a potential biomarker for detecting cervical cancer. The aim of the present study was to explore the methylation status of the paired box gene 1 (PAX1) and the LIM homeobox transcription factor 1 α (LMX1A) gene in a spectrum of cervical lesions in an Eastern Chinese population. This single-center study involved 121 patients who were divided into normal cervix (NC; n=28), low-grade squamous intraepithelial lesion (LSIL; n=32), high-grade squamous intraepithelial lesion (HSIL; n=34) and cervical squamous cell carcinoma (CSCC; n=27) groups, according to biopsy results. Following extraction and modification of the DNA, quantitative assessment of the PAX1 and LMX1A genes in exfoliated cells was performed using pyrosequencing analysis. Receiver operating characteristic (ROC) curves were generated to calculate the sensitivity and specificity of each parameter and cut-off values of the percentage of methylation reference (PMR) for differentiation diagnosis. Analysis of variance was used to identify differences among groups. The PMR of the two genes was significantly higher in the HSIL and CSCC groups compared with that in the NC and LSIL groups (P<0.001). ROC curve analysis demonstrated that the sensitivity, specificity and accuracy for detection of CSCC were 0.790, 0.837 and 0.809, respectively, using PAX1; and 0.633, 0.357 and 0.893, respectively, using LMX1A. These results indicated that quantitative PAX1 methylation demonstrates potential for cervical cancer screening, while further investigation is required to determine the potential of LMX1A methylation.

The sphingosine kinase 2 inhibitor ABC294640 inhibits cervical carcinoma cell growth.

ABC294640 is a specific sphingosine kinase 2 (SphK2) inhibitor. The anti-cervical carcinoma activity by ABC294640 was tested in this study. ABC294640 inhibited in vitro growth of the established (C33A and HeLa lines) and primary human cervical carcinoma cells. The SphK2 inhibitor also induced G1-S arrest and apoptosis in cervical carcinoma cells. It was yet non-cytotoxic to SphK2-low human cervical epithelial cells. ABC294640 inhibited SphK activation, causing sphingosine-1-phosphate depletion, signal transducer and activator of transcription 3 in-activation and ceramide production. Bcl-2 is a key resistance factor of ABC294640. Pharmacological Bcl-2 inhibition or Bcl-2 shRNA potentiated ABC294640-induced C33A cell growth inhibition and apoptosis. On the other hand, exogenous over-expression of Bcl-2 attenuated ABC294640's cytotoxicity against C33A cells. In vivo, ABC294640 administration inhibited C33A xenograft tumor growth in mice. Co-administration of the Bcl-2 inhibitor GDC-0199 further potentiated ABC294640's anti-tumor activity. Together, we suggest that ABC294640 might have translational value for the treatment of human cervical carcinoma.