Comparative analysis of the liver-protective effects of raw and stir-fried semen of Hovenia dulcis in rats via gas chromatography-mass spectrometry-based serum metabolomic profiling and chemometrics.

In this study, we used a serum metabolomics methodology based on GC coupled with MS (GC-MS) to investigate the liver-protective effects of raw and stir-fried semen of Hovenia dulcis in rats models of carbon tetrachloride-induced liver injury. Multivariate statistical analysis, such as principal component analysis and orthogonal partial least squares discriminant analysis, were performed to examine changes in the metabolic state of rats with carbon tetrachloride-induced liver injury, as well as the recovery pattern of rats pretreated with the raw and stir-fried semen of H. dulcis. Liver tissues were subjected to histopathological examination. A total of 47 biomarkers were predicted to contribute to the dynamic pathological processes in the liver injury, such as phenylalanine, glutamic acid, glycine, arachidonic acid and linoleic acid. Further analysis revealed that pathways associated with phenylalanine, tyrosine and tryptophan biosynthesis, and linoleic acid metabolism were altered in the injured liver, and that pretreatment with raw and stir-fried semen of H. dulcis abolished the changes in the aforementioned metabolic pathways.

[Identification of a novel mutation of factor XII gene in a family with coagulation FXII deficiency].

OBJECTIVE: To analyze genetic mutation and molecular pathogenesis in a family affected with inherited coagulation factor XII(FXII) deficiency. METHODS: Activated partial thromboplastin time (APTT), FXII procoagulant activity (FXII:C), FXII antigen (FXII:Ag) and other coagulants were measured. For affected members of the family, exons 1-14 and flanking intronic regions of the FXII gene were amplified with polymerase chain reaction (PCR) and sequenced thereafter. Expression plasmids containing mutant FXII cDNA was constructed and transfected into COS7 cells transiently. Expressions of FXII:Ag and FXII:C were analyzed. RESULTS: The proband has manifested a prolonged APTT of 108.1 s (reference range: 27.0-41.0 s). Her husband has a normal APTT. Other members of the family had slightly increased APTT. The FXII:C and FXII:Ag of the proband have both dropped to about 0.01 (reference range: 0.72-1.13). The FXII:C levels of her husband, son, daughter and grandchild were 0.57, 0.24, 0.14, 0.16, respectively. And the FXII:Ag levels in her husband, son, daughter and grandchild were 0.55, 0.27, 0.15, 0.21, respectively. The proband and her daughter have both carried a heterozygous deletional mutation 6800-6808delAGCTGGGAG (6800-6808del9bp) in exon 9. For the promoter region of the FXII gene, the genotypes of the proband, her son, daughter and grandchild was TT, whilst that of her husband was CT. Expression study has shown that, whilst the mutant FXII protein has accumulated in the cells similar to wild-type protein, its secretion has reduced approximately by half. CONCLUSION: A novel deletional mutation 6800-6808del9bp has been identified in the FXII gene. Although mutant FXII protein can still accumulate in cells, its secretion has become insufficient. The 6800-6808del9bp mutation and 46T/T have both contributed to the pathogenesis of FXII deficiency in the family, but may have not been the sole cause.

Network pharmacology analysis of the pharmacological mechanism of Artemisia lavandulaefolia DC. in rheumatoid arthritis.

OBJECTIVE: The traditional She medicine is a notable type of traditional Chinese medicine, which has been applied for a long history despite the lack of sufficient mechanistic understanding. Our study revealed the possible molecular mechanism of sesquiterpene 5α-Hydroxycostic acid, active ingredient of traditional She medicine Artemisia lavandulaefolia DC., in the treatment of rheumatoid arthritis (RA). METHODS: RA-fibroblast like synoviocytes (RA-FLSs) were treated with 5α-Hydroxycostic acid, Anthemidin, and methotrexate (MTX). CCK-8 and ELISA were used to measure the resultant viability of RA-FLSs and to quantify pro-inflammatory cytokines. Target genes of 5α-Hydroxycostic acid and Anthemidin, RA-related differentially expressed genes, and RA-related genes were retrieved by bioinformatics analyses, results of which were further intersected to identify candidate genes. The protein-protein interaction network was constructed to develop the pharmacophore model. The molecular docking was simulated to determine the core target androgen receptor (AR) for subsequent molecular mechanism investigation in vitro. RESULTS: The 5ɑ-Hydroxycostic acid, Anthemidin, or MTX of different concentrations inhibited the viability of RA-FLSs, and downregulated the levels of proinflammatory cytokines. The pharmacophore model and molecular docking of 10 candidate targets with 5α-Hydroxycostic acid were successfully established. In vitro experiments provided evidence confirming that 5α-Hydroxycostic acid elevated AR expression to inhibit inflammatory responses of RA-FLSs and degradation of extracellular matrix. CONCLUSION: Therefore, this study reveals the active ingredient sesquiterpene 5α-Hydroxycostic acid of traditional She medicine Artemisia lavandulaefolia DC., and illustrates potential molecular mechanism in RA treatment by upregulating AR expression. This study is the first to report the effect of the active ingredient sesquiterpenes in traditional She medicine A.lavandulaefolia DC on RA and elucidate the underlying molecular mechanism associated with up-regulated AR expression. This study provides new insights into the mechanistic understanding of traditional She medicine in the treatment of RA.

New prenylated flavonoid glycosides from the stems of Celastrus orbiculatus with their anti-tumor activities.

Two new prenylated flavonoid glycosides (1-2), together with five known compounds (3-7) were isolated from the EtOAc-soluble extract of the stems of Celastrus orbiculatus. The structures of new compounds were elucidated with spectroscopic and physico-chemical analyses. All isolates were evaluated for in vitro cytotoxic activities against HepG2, MCF-7, A549, and A2780 cancer cells. Among them, compound 1 showed potential antiproliferative activity on A2780 cells with IC50 value of 10.76 µM. In addition, compound 2 exhibited selective cytotoxic activity on A2780 cells with IC50 value of 26.30 µM.

Preoperative Serum Calcitonin Level and Ultrasonographic Characteristics Predict the Risk of Metastatic Medullary Thyroid Carcinoma: Functional Analysis of Calcitonin-Related Genes.

Background: Early cervical lymph node (LN) metastasis is an important cause of poor survival in patients with medullary thyroid cancer (MTC). This study evaluated whether the preoperative serum calcitonin level in combination with ultrasonographic features of MTC can be used to assess the LN status as well as predict the risk of metastasis in patients with MTC. Methods: We retrospectively analyzed the clinical data of 95 patients with MTC, and a nomogram model was constructed and validated. Using integrated database analysis of The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx), we mined pathways wherein CALCA is involved, identified calcitonin-related genes, and analyzed their functions. Results: Correlation analysis revealed a significant association between the infiltrating range, diameter, calcification, blood flow, the preoperative serum calcitonin level, and metastasis. The metastasis risk-prediction model showed great accuracy in determining the risk of metastasis in MTC (area under the curve of the receiver operating characteristic [ROC] curve: 0.979 [95% confidence interval 0.946-1.000]). Decision curve analysis (DCA) showed that the model has excellent clinical utilization potential. Significantly, CALCA, the mRNA for calcitonin, was highly expressed in thyroid cancer tissues and associated with the cytokine-cytokine receptor and neuroactive ligand-receptor interaction pathways as well as the cell-adhesion molecules. ROC curve indicated that the CNTFR, CD27, GDF6, and TSLP genes, which are related to the cytokine-cytokine receptor interaction pathway, could indicate the risk of metastasis in MTC. Conclusions: The preoperative serum calcitonin level, in combination with ultrasonographic features, can be used to predict the risk of metastasis in patients with MTC and constitute a noninvasive accurate method for preoperative diagnosis of MTC.

Plumbagin relieves rheumatoid arthritis through nuclear factor kappa-B (NF-κB) pathway.

This study aimed to explore the effects of plumbagin on rheumatoid arthritis (RA) and its mechanism. The RA cell model was simulated following the treatment of interleukin-1ß (IL-1ß). After the treatment of various concentrations of plumbagin, the impact of plumbagin on the cell viability was examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The collagen-induced arthritis (CIA) model was established using the solution of bovine type II collagen. Hematoxylin-eosin staining was used to observe the changes of ankle joint tissue, while enzyme-linked immunosorbent assay and western blot were applied to detect the level of inflammatory cytokines. Plumbagin inhibited the viability of human fibroblast-like synoviocytes (HFLS) at the concentration of 1 ~ 3.5 µM. The inhibitory effect of 1 µM plumbagin on cell proliferation was similar to that of methotrexate, the drug used as the positive control. Plumbagin downregulated the levels of inflammatory cytokines and matrix metalloproteinases (MMPs) in IL-1ß-treated HFLS, and suppressed the activation of IκB and nuclear factor kappa-B (NF-κB) as well as the entry of p65 into the nucleus. It was also demonstrated in animal experiments that plumbagin inhibited the activation of NF-κB pathway, down-regulated the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and MMPs, and alleviated joint damage in CIA-modeled mice. Collectively speaking, plumbagin might down-regulate the levels of inflammatory cytokines and MMPs through inhibiting the activation of the NF-κB pathway, thereby attenuating RA-induced damage to cells and joints.Abbreviations: CIA: Collagen-induced arthritis; ELISA: Enzyme-linked immuno sorbent assay; HFLS: Human fibroblast-like synoviocytes; IL-6: Interleukin-6; IL-1ß: Interleukin-1ß; NF-κB: nuclear factor kappa-B; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MMPs: Matrix metalloproteinase; OD: Optical density; RA: Rheumatoid arthritis; SDS: Sodium dodecyl sulfate; SD: Standard deviation; TNF-α: Tumor necrosis factor-α; PVDF: Polyvinylidene fluoride.

[Gene analysis in four inherited coagulation Fâ « deficiency pedigree].

OBJECTIVE: To identify the genotype and pathogenesis in four Chinese pedigrees with Factor Ⅻ deficiency. METHODS: Activated partial thromboplastin time (APTT), FⅫ procoagulant activity (FⅫ∶C), FⅫ antigen（FⅫ∶Ag）and other coagulant parameters were detected. The FⅫ deficiency Pedigree members,all exons,boundary introns including the splice junctions of the FⅫ gene were amplified with Polymerase chain reaction (PCR). Expression plasmids were constructed by mutagenesis based on the wild-type and transfected into COS7 cells. FⅫ∶C and FⅫ∶Ag of the expression levels were tested in the supernatant and cell lysate. RESULTS: The four probands presented prolonged APTT with all the values of FⅫ∶C and FⅫ∶Ag were low to 2% and 1%, respectively. There were common 46C/T polymorphism in the promoter regions of FⅫ gene in four pedigrees. Proband A was heterozygous for two mutations, g.5741-5742delCA (His101Gln) and g.7142insertC (Lys346Gln). Proband B was a heterozygous deletion mutation g.6800-6808del9bp. The results of the transfection revealed that FⅫ∶Ag in cell lysates and conditioned media protein FⅫ6800-6808del9bp were 85.6% and 51.9%. The FⅫ∶C in the conditioned media was 56.4%. Proband C was a heterozygous mutation g.8699G>A(Gly542Ser). Proband D was a homozygous mutation 8699G>A, whose parents with consanguineous marriage. CONCLUSION: Four mutations, g.5741-5742delCA, g.7142insertC, g.6800-6808del9bp and g.8699G>A with 46C/T polymorphism in the promoter regions of FⅫ gene, were identified in the four Factor Ⅻ deficiency pedigrees. The two mutations g.5741-5742delCA and g.6800-6808del9bp were first found in China. FⅫ 6800-6808del9bp expressed in vitro suggested that almost normal proteinum synthesis but defect proteinum secretion.