Vitamin D binding protein (VDBP) hijacks twist1 to inhibit vasculogenic mimicry in hepatocellular carcinoma.

Rationale: Vitamin D (VD) has been suggested to have antitumor effects, however, research on the role of its transporter vitamin D-binding protein (VDBP, gene name as GC) in tumors is limited. In this study, we demonstrated the mechanism underlying the inhibition of vasculogenic mimicry (VM) by VDBP in hepatocellular carcinoma (HCC) and proposed an anti-tumor strategy of combining anti-PD-1 therapy with VD. Methods: Three-dimensional cell culture models and mice with hepatocyte-specific GC deletion were utilized to study the correlation between VDBP expression and VM. A patient-derived tumor xenograft (PDX) model was further applied to validate the therapeutic efficacy of VD in combination with an anti-PD-1 drug. Results: The study revealed that VDBP expression is negatively correlated with VM in HCC patients and elevated VDBP expression is associated with a favorable prognosis. The mechanism studies suggested VDBP hindered the binding of Twist1 on the promoter of VE-cadherin by interacting with its helix-loop-helix DNA binding domain, ultimately leading to the inhibition of VM. Furthermore, VD facilitated the translocation of the vitamin D receptor (VDR) into the nucleus where VDR interacts with Yin Yang 1 (YY1), leading to the transcriptional activation of VDBP. We further demonstrated that the combination of VD and anti-PD-1 led to an improvement in the anti-tumor efficacy of an anti-PD-1 drug. Conclusion: Collectively, we identified VDBP as an important prognostic biomarker in HCC patients and uncovered it as a therapeutic target for enhancing the efficacy of immune therapy.

Enzymatic activity of 38 CYP2C9 genotypes on ibuprofen.

BACKGROUND AND OBJECTIVE: Ibuprofen, a common non-steroidal anti-inflammatory drug, is used clinically for pain relief and antipyretic treatment worldwide. However, regular or long-term use of ibuprofen may lead to a series of adverse reactions, including gastrointestinal bleeding, hypertension and kidney injury. Previous studies have shown that CYP2C9 gene polymorphism plays an important role in the elimination of various drugs, which leads to the variation in drug efficacy. This study aimed to evaluate the effect of 38 CYP2C9 genotypes on ibuprofen metabolism. METHODS: Thirty-eight recombinant human CYP2C9 microsomal enzymes were obtained using a frugiperda 21 insect expression system according to a previously described method. Assessment of the catalytic function of these variants was completed via a mature incubation system: 5 pmol CYP2C9*1 and 38 CYP2C9 variants recombinant human microsomes, 5 µL cytochrome B5, ibuprofen (5-1000 µM), and Tris-HCl buffer (pH 7.4). The ibuprofen metabolite contents were determined using HPLC analysis. HPLC analysis included a UV detector, Plus-C18 column, and mobile phase [50% acetonitrile and 50% water (containing 0.05% trifluoroacetic acid)]. The kinetic parameters of the CYP2C9 genotypes were obtained by Michaelis-Menten curve fitting. RESULTS: The intrinsic clearance (CLint) of eight variants was not significantly different from CYP2C9*1; four CYP2C9 variants (CYP2C9*38, *44, *53 and *59) showed significantly higher CLint (increase by 35%-230%) than that of the wild-type; the remaining twenty-six variants exhibited significantly reduced CLint (reduced by 30%-99%) compared to that of the wild-type. CONCLUSION: This is the first systematic evaluation of the catalytic characteristics of 38 CYP2C9 genotypes involved ibuprofen metabolism. Our results provide a corresponding supplement to studies on CYP2C9 gene polymorphisms and kinetic characteristics of different variants. We need to focus on poor metabolizers (PMs) with severely abnormal metabolic functions, because they are more susceptible to drug exposure.

[Formation and development of "Xiang thinking" in traditional Chinese medicine].

"Xiang thinking" is the main thinking mode in traditional Chinese medicine(TCM) with both philosophical and scientific connotations, and has an important influence on the emergence and development of TCM. This study systematically expounded the philosophical connotation and characteristics of "Xiang thinking", and its application in the construction of TCM theory, clinical syndrome differentiation and treatment, the formation of medicinal properties, and interpretation of efficacy of Chinese medicine. "Xiang thinking" in TCM develops and changes continuously with practical application, and its historical evolution can be summarized into three stages, i.e., "Ying Xiang(corresponding to Xiang)" "Fa Xiang(following Xiang)", and "Bian Xiang(differentiating Xiang)". The understanding of Xiang is based on the means and methods of human observation of things and the backgrounds of the philosophy, science, and development. The result of comparison depends on the depth and breadth of Xiang. In the real world, Xiang is showing new construction characteristics with multiple dimensions and levels. Therefore, this study proposed the thinking of "Bian Xiang", which is expected to provide a thinking approach that can realize the transformation from "Xiang thinking" to scientific research for the exploration and innovative research on life origin in the field of contemporary and TCM.

[Metabolites of endophytic fungus Nigrospora sphaerica S5 from Myoporum bontioides].

The endophytic fungus Nigrospora sphaerica S5 derived from the semi-mangrove plant Myoporum bontioides was fermented. Its metabolites were purified by column chromatography. Nine compounds were obtained and identified as terezine P(1), 3-(1-hydroxyethyl)-4-methyl dihydrofuran-2(3H)-one(2), methylhydroheptelidate(3), hydroheptelidic acid(4), 5, 7-dimethoxy-4, 6-dimethylphthalide(5),(3R,4S)-(-)-4-hydroxymellein(6), pestalopyrone(7), indole-3-formaldehyde(8) and p-hydroxybenzaldehyde(9) by spectroscopic techniques. Terezine P(1) was a new alkaloid belonging to the terezine class with a pyrazine ring. Compounds 2-7 were lactones, of which 3 and 4 belonged to sesquiterpenes. Compounds 8 and 9 were indole alkaloids and phenols, respectively. Compounds 3-6 were purified from Nigrospora sp. for the first time. These compounds showed different degrees of antibacterial activity against Staphylococcus aureus, Escherichia coli of O6 serotype and E. coli of O78 serotype.

Integrative bioinformatics analysis to identify the effects of circadian rhythm on Crohn's disease.

Background: Crohn's disease (CD) is a multifactorial inflammatory bowel disease characterized by complex aberrant autoimmune disorders. Currently, the involvement of the circadian rhythm in the pathogenesis of CD is unknown. Methods: Bulk and single-cell RNA-seq data and associated clinical data from patients with CD were downloaded from the Gene Expression Omnibus (GEO). Single-sample gene set enrichment analysis was performed to calculate the enrichment score (ES) of circadian rhythm-related genes. Differential expression analysis was used to identify differentially expressed genes. Functional enrichment analysis was used to explore potential disease mechanisms. CIBERSORT was used to estimate immune cell abundance. Single-cell RNA-seq data were analyzed using the R package "Seurat." Results: The ES of circadian rhythm-related genes was lower in the CD tissue than in the normal tissue. Ubiquitin-specific protease 2 (USP2), a circadian rhythm-related gene, was identified as a potential modulator of CD pathogenesis. USP2 expression was reduced in CD and was associated with disease severity. Moreover, the analysis of bulk RNA-seq and single-cell RNA-seq data showed that monocyte and neutrophil abundance was elevated in CD and was negatively correlated with USP2 expression. It should be noted that USP2 expression in acinar cells was negatively correlated with monocyte and neutrophil abundance. Functional enrichment analysis revealed several canonical pathways to be enriched in CD, including the interleukin-17 signaling pathway, tumor necrosis factor signaling pathway, cytokine-cytokine receptor interaction, toll-like receptor signaling pathway, and nod-like receptor signaling pathway. Conclusion: Aberrant expression of circadian rhythm-related genes is correlated with CD pathogenesis. USP2 might be related to crosstalk among the different cell types in CD. These findings provide insights into future chronotherapy for CD.

Integrative Analysis of the Gut Microbiota and Metabolome in Obese Mice with Electroacupuncture by 16S rRNA Gene Sequencing and HPLC-MS-based Metabolic Profiling.

Acupuncture has been used to treat numerous diseases such as obesity in China for thousands of years. Several mechanisms of acupuncture on obesity have been surveyed based on metabolomics, but the effects of acupuncture on the alterations in the gut flora are still unclear. In this study, an integrated approach based on 16S rRNA gene sequencing combined with high-performance liquid chromatography-mass spectrometry (HPLC-MS) metabolic profiling was conducted to investigate the effects of acupuncture on high-fat-diet-induced obesity through the regulation of the relative abundances of gut microbiota and their relationships with biomarker candidates. A total of 10 significantly altered bacterial genera and 11 metabolites were recognized, which recovered to normal levels after electroacupuncture treatment. The relative abundances of the bacterial families Muribaculaceae,Lachnospiraceae,Desulfovibrionaceae,Helicobacteraceae, Prevotellaceae,Ruminococcaceae,Rikenellaceae,Deferribacteraceae,Bacteroidaceae andTannerellaceaewere remarkedly changed among the three groups. Potential biomarkers, including LysoPC(0:0/16:0) ([Formula: see text]1),PC(0:0/18:0) ([Formula: see text]2),Cholic acid([Formula: see text]3),LysoPC(16:0) ([Formula: see text]4), 3[Formula: see text],6[Formula: see text],7[Formula: see text]-Trihydroxy-5[Formula: see text]-cholanoic acid([Formula: see text]5), 5beta-Cyprinolsulfate([Formula: see text]6),PC(18:0/0:0) ([Formula: see text]7), 1-Nitro-5-hydroxy-6-glutathionyl-5,6-dihydronaphthalene([Formula: see text]8),Glycocholic acid([Formula: see text]9),[Formula: see text]-Arginine([Formula: see text]10) andGulonic acid([Formula: see text]11), were involved in several metabolic pathways, such as the glycerophospholipid metabolism and primary bile acid biosynthesis. Interestingly, there was a strong correlation between the perturbed gut flora in Bilophila and Bifidobacterium and the altered intestinal metabolite of 3[Formula: see text],6[Formula: see text],7[Formula: see text]-Trihydroxy-5[Formula: see text]-cholanoic acid and Cholanoic acid and [Formula: see text]-Arginine. This finding suggested that the effects of electroacupuncture might change the proportions of Bilophila and Bifidobacterium by regulating the constituents of the functional metabolite of 3[Formula: see text],6[Formula: see text],7[Formula: see text]-Trihydroxy-5[Formula: see text]-cholanoic acid and Cholanoic acid and [Formula: see text]-Arginine. These results indicated that the effects of electroacupuncture focused on custom metabolic pathways as well as depend on the changes in the gut microbiota in obesity. These findings suggest that the 16S rRNA gene sequencing and HPLC-MS-based metabolomics approach can be applied to comprehensively assess the effects of traditional Chinese medicines.

[Effect of granules made by new-medicinal parts of Crocus sativus on hyperuricemia in rats and its mechanism].

To explore the effect of the granules made by new-medicinal parts of Crocus sativus(NMPCS) on hyperuricemia(HUA) in rats, the rat model of HUA was established by intramuscular injection of 3% potassium oxonate and intraperitoneal injection of 4% pyrazinamide. The content of serum uric acid was monitored every week for 3 consecutive weeks. After the experiment, the levels of serum uric acid, urine uric acid, serum creatinine, blood urea nitrogen(BUN), and xanthine oxidase(XOD) were determined. The protein and gene expressions of XOD were determined by Western blot method and fluorescence quantitative polymerase chain reaction(qPCR), and the morphological changes in the liver tissue were performed by hematoxylin-eosin(HE) staining. The results showed that as compared with the model group, the levels of serum uric acid in the positive drug group and the low, medium, and high-dose NMPCS groups were lower(P<0.05), the levels of urine uric acid in the high-dose NMPCS group were decreased(P<0.01), and there was no statistical difference in the medium and low-dose NMPCS groups. The levels of BUN in the high and low-dose NMPCS groups were decreased(P<0.05), and the levels of serum creatinine did not change in the administration groups. The positive drug group and the low, medium, and high-dose NMPCS groups significantly reduced the liver damage, with only a few hepatocytes vacuolization and a small number of red blood cells in the central venous area. The nephridial tissue structure was slightly abnormal, with a small number of red blood cell infiltration, and no obvious inflammatory cell infiltration was found in the glomerulus in these groups. No degeneration was found in renal tubular epithelial cells, with mild glomerular and tubular lesions and a small amount of sodium urate deposition and crystallization in the positive drug group and the low, medium, and high-dose NMPCS groups. The relative protein expressions of XOD in the positive drug group and the high dose NMPCS group were decreased(P<0.05), and the relative mRNA expressions of XOD in the positive drug group and the high and low-dose NMPCS groups were decreased as well(P<0.05). The above results show that NMPCS reduces uric acid in rats with HUA by regulating XOD, which provides a certain experimental basis for the development of NMPCS as a new medicine for the treatment of HUA.

Biological Activities of Two Essential Oils from Pogostemon cablin and Eupatorium fortunei and Their Major Components against Fungi Isolated from Panax notoginseng.

Panax notoginseng (Burkill) F.H.Chen (Araliaceae), of which the dry root and rhizome are precious traditional Chinese medicine, suffers severely from diseases during planting. Essential oils (EOs) with antimicrobial activity are a possibility for the development of green pesticides. We extracted EOs from Pogostemon cablin (Blanco) Benth. and Eupatorium fortunei Turcz., respectively and tested their inhibitory rates on fungi isolated from diseased P. notoginseng by the Oxford cup method. The compounds of the EO were identified by GC/MS and the minimum inhibitory concentrations (MICs) of the EOs and their main components were evaluated by the 96-well plate method. We also mixed P. cablin EO, E. fortunei EO and hymexazol in pairs to explore whether their combinations produce stronger antifungal effects than individual components. Finally, we evaluated the effects of the EOs against Fusarium oxysporum in vivo. P. cablin EO and E. fortunei EO exhibited different antifungal activities against fungi, with the inhibitory rates of 21.02 %-100 % and 54.84 %-100 % and MICs of 0.07-0.88 mg/mL and 0.20-1.17 mg/mL, respectively. Pogostone (24.96 %) and thymol (15.64 %) were the major compounds of P. cablin EO and E. fortunei EO, respectively, and they exhibited stronger antifungal activities than EOs, with MICs of 0.008-0.078 mg/mL and 0.12-0.31 mg/mL, respectively. Moreover, hymexazol was mixed with E. fortunei EO, and the inhibitory effect against Cylindrocarpon destructans was enhanced with a synergistic effect. The disease incidence and disease index of EO treatments decreased significantly in vivo. Based on our study, P. cablin EO and E. fortunei EO have great potential to be developed into green fungicides for use in agriculture to control diseases of P. notoginseng.

Pseudorivibacter rhizosphaerae gen. nov., sp. nov., isolated from rhizosphere soil of Camellia sinensis (L.) O. Ktze and emended description of the genus Rivibacter.

A Gram-stain-negative, facultative anaerobic, motile and straight rod-shaped bacterium, designated strain C1-9T, was isolated from rhizosphere soil of Camellia sinensis (L.) O. Ktze collected from a tea garden in Huize, south-western PR China. Cells were oxidase-positive and catalase-negative. Growth occurred at 20-40 °C and pH 6.0-10.0, with an optimal growth at 30 °C and pH 7.0. The respiratory quinone was detected as ubiquinone-8 (Q-8). The major fatty acids were identified as summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and summed feature 8 (C18 : 1ω7c or C18 : 1ω6c). The cellular polar lipids contained phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, three unidentified phospholipids, two unidentified lipids, one unidentified aminophospholipid and one unidentified aminolipid. The polyamine types were detected as 1,8-diaminooctane and 2-hydroxyputrescine. The genomic DNA G+C content was 68.6 mol%. Based on the results of 16S rRNA gene sequence analysis, strain C1-9T (MF687442) showed highest sequence similarity to Rivibacter subsaxonicus DSM 19570T (97.1 %). The phylogenetic tree based on 16S rRNA gene sequences showed that strain C1-9T clustered close to R. subsaxonicus DSM 19570T, Methylibium petroleiphilum CCTCC AB 2014193T and species belonging to the genera Rhizobacter and Piscinibacter. The phylogenomic tree indicated that strain C1-9T formed a clade with R. subsaxonicus. The average nucleotide identity value was 76.0 % between strain C1-9T and R. subsaxonicus DSM 19570T, which is lower than the prokaryotic species delineation threshold of 95.0-96.0 %. The polyphasic taxonomic characteristics indicated that strain C1-9T represents a novel species of a new genus within the order Burkholderiales, for which the name Pseudorivibacter rhizosphaerae gen. nov., sp. nov. (type strain C1-9T = KCTC 62325T=CGMCC 1.13864T) is proposed.

Monitoring Antifungal Agents of Artemisia annua against Fusarium oxysporum and Fusarium solani, Associated with Panax notoginseng Root-Rot Disease.

Root rot of Panax notoginseng has received great attention due to its threat on the plantation and sustainable utilization of P. notoginseng. To suppress the root-rot disease, natural ingredients are of great importance because of their environment friendly properties. In this study, we found that the methanol extract from Artemisia annua leaves has strong antifungal effects on the growth of Fusarium oxysporum and Fusarium solani resulting into root-rot disease. Essential oil (EO) thereof was found to be the most active. GC-MS analysis revealed 58 ingredients and camphor, camphene, ß-caryophyllene, and germacrene D were identified as the major ingredients. Further antifungal assays showed that the main compounds exhibit various degrees of inhibition against all the fungi tested. In addition, synergistic effects between A. annua EO and chemical fungicides were examined. Finally, in vivo experiments were conducted and disclosed that P. notoginseng root rot could be largely inhibited by the petroleum ether extract from A. annua, indicating that A. annua could be a good source for controlling P. notoginseng root-rot.

[Intestinal absorption of puerarin nanocrystalline self-stabilizing pickering emulsion in rats].

To study the intestinal absorption characteristics of drug's nanocrystalline self-stabilizing Pickering emulsion (NSSPE) in situ in rats. Rat single-pass intestinal perfusion model was established, and high performance liquid chromatography (HPLC) was used to detect the concentration of puerarin in rat intestinal perfusion solution, assay the absorption rate constant (Ka) and the intestinal apparent permeability coefficient (Papp) of NSSPE in duodenum, jejunum, ileum, and colon, which were compared with those of raw material, nanocrystal and normal emulsion, respectively. For NSSPE, the Ka and Papp values were in the following order: duodenum>jejunum>ileum (P<0.05)>colon (P<0.01). However, there was no obvious difference between jejunum and ileum. As compared with raw material, nanocrystal and normal emulsion, the Ka and Papp values of NSSPE in duodenum were significantly higher than those of other three preparations (P<0.05); and the Ka and Papp values of NSSPE in jejunum and colon were significantly higher than those of raw material, nanocrystal and normal emulsion (P<0.01); and the Ka and Papp of NSSPE in ileum were also higher than those of raw material and normal emulsion (P<0.05), but had no obvious difference with nanocrystal. The results showed that NSSPE could significantly improve the absorption of puerarin in the intestine of rats.

Characterization of Three Glucosyltransferase Genes in Tartary Buckwheat and Their Expression after Cold Stress.

Anthocyanins confer the red color in the hypocotyl of tartary buckwheat sprouts. Uridine diphosphate (UDP)-glucose:flavonoid 3-O-glycosyltransferase (UFGT) stabilizes anthocyanin by attaching the glucosyl moiety from UDP-glucose to the C3 hydroxyl of anthocyanin. In this study, we characterized three UFGT-like genes, designated FtUFGT1, 2, and 3 from tartary buckwheat. The results revealed that FtUFGT1, FtUFGT2, and FtUFGT3 can convert cyanidin to cyanidin 3-O-glucoside, with specific activities of 20.01 × 10(-3), 8.93 × 10(-3), and 20.24 × 10(-3) IU/mg, respectively. The active-site residues of the C-terminal domains and the N-terminal domains are important for the donor and acceptor recognition of these proteins. The expression of the three FtUFGTs paralleled the tissue-specific anthocyanin accumulation. After cold treatment, the increased content of anthocyanin was accompanied by the up-regulated expression of the three FtUFGTs. Among these three UGFT gene members, FtUFGT3 showed the highest expression level and the highest specific activity, suggesting that FtUFGT3 might be the major gene involved in anthocyanin biosynthesis. These results suggested that the FtUFGT genes, FtUFGT3 in particular, might be important candidates for anthocyanin formation in tartary buckwheat sprouts.

Scutellarin attenuates endothelium-dependent aasodilation impairment induced by hypoxia reoxygenation, through regulating the PKG signaling pathway in rat coronary artery.

Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant. Our previous study has demonstrated that SCU relaxes mouse aortic arteries mainly in an endothelium-depend-ent manner. In the present study, we investigated the vasoprotective effects of SCU against HR-induced endothelial dysfunction (ED) in isolated rat CA and the possible mechanisms involving cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG). The isolated endothelium-intact and endothelium-denuded rat CA rings were treated with HR injury. Evaluation of endothelium-dependent and -independent vasodilation relaxation of the CA rings were performed using wire myography and the protein expressions were assayed by Western blotting. SCU (10-1 000 µmol·L(-1)) could relax the endothelium-intact CA rings but not endothelium-denuded ones. In the intact CA rings, the PKG inhibitor, Rp-8-Br-cGMPS (PKGI-rp, 4 µmol·L(-1)), significantly blocked SCU (10-1 000 µmol·L(-1))-induced relaxation. The NO synthase (NOS) inhibitor, NO-nitro-L-arginine methylester (L-NAME, 100 µmol·L(-1)), did not significantly change the effects of SCU (10-1 000 µmol·L(-1)). HR treatment significantly impaired ACh-induced relaxation, which was reversed by pre-incubation with SCU (500 µmol·L(-1)), while HR treatment did not altered NTG-induced vasodilation. PKGI-rp (4 µmol·L(-1)) blocked the protective effects of SCU in HR-treated CA rings. Additionally, HR treatment reduced phosphorylated vasodilator-stimulated phosphoprotein (p-VASP, phosphorylated product of PKG), which was reversed by SCU pre-incubation, suggesting that SCU activated PKG phosphorylation against HR injury. SCU induces CA vasodilation in an endothelium-dependent manner to and repairs HR-induced impairment via activation of PKG signaling pathway.