Multiomics Analysis Reveals Leucine Deprivation Promotes Bile Acid Synthesis by Upregulating Hepatic CYP7A1 and Intestinal Turicibacter sanguinis in Mice.

BACKGROUND: Leucine, a branched-chain amino acid, participates in the regulation of lipid metabolism and the composition of the intestinal microbiota. However, the related mechanism remains unclear. OBJECTIVES: Here, we aimed to reveal the potential mechanisms by which hepatic CYP7A1 (a rate-limiting enzyme for bile acid [BA] synthesis) and gut microbiota coregulate BA synthesis under leucine deprivation. METHODS: To this end, 8-wk-old C57BL/6J mice were fed with either regular diets or leucine-free diets for 1 wk. Then, we investigated whether secondary BAs were synthesized by Turicibacter sanguinis in 7-wk-old C57BL/6J germ-free mice gavaged with T. sanguinis for 2 wk by determining BA concentrations in the plasma, liver, and cecum contents using liquid chromatography-tandem mass spectrometry. RESULTS: The results showed that leucine deprivation resulted in a significant increase in total BA concentration in the plasma and an increase in the liver, but no difference in total BA was observed in the cecum contents before and after leucine deprivation. Furthermore, leucine deprivation significantly altered BA profiles such as taurocholic acid and ω-muricholic acid in the plasma, liver, and cecum contents. CYP7A1 expression was significantly upregulated in the liver under leucine deprivation. Leucine deprivation also regulated the composition of the gut microbiota; specifically, it significantly upregulated the relative abundance of T. sanguinis, thus enhancing the conversion of primary BAs into secondary BAs by intestinal T. sanguinis in mice. CONCLUSIONS: Overall, leucine deprivation regulated BA profiles in enterohepatic circulation by upregulating hepatic CYP7A1 expression and increasing intestinal T. sanguinis abundance. Our findings reveal the contribution of gut microbiota to BA metabolism under dietary leucine deprivation.

Discovery and pharmacological characterization of 1,2,3,4-tetrahydroquinoline derivatives as RORγ inverse agonists against prostate cancer.

The retinoic acid receptor-related orphan receptor γ (RORγ) is regarded as an attractive therapeutic target for the treatment of prostate cancer. Herein, we report the identification, optimization, and evaluation of 1,2,3,4-tetrahydroquinoline derivatives as novel RORγ inverse agonists, starting from high throughput screening using a thermal stability shift assay (TSA). The representative compounds 13e (designated as XY039) and 14a (designated as XY077) effectively inhibited the RORγ transcriptional activity and exhibited excellent selectivity against other nuclear receptor subtypes. The structural basis for their inhibitory potency was elucidated through the crystallographic study of RORγ LBD complex with 13e. Both 13e and 14a demonstrated reasonable antiproliferative activity, potently inhibited colony formation and the expression of AR, AR regulated genes, and other oncogene in AR positive prostate cancer cell lines. Moreover, 13e and 14a effectively suppressed tumor growth in a 22Rv1 xenograft tumor model in mice. This work provides new and valuable lead compounds for further development of drugs against prostate cancer.

Liver receptor homolog-1: structures, related diseases, and drug discovery.

Liver receptor homolog-1 (LRH-1), a member of the nuclear receptor superfamily, is a ligand-regulated transcription factor that plays crucial roles in metabolism, development, and immunity. Despite being classified as an 'orphan' receptor due to the ongoing debate surrounding its endogenous ligands, recent researches have demonstrated that LRH-1 can be modulated by various synthetic ligands. This highlights the potential of LRH-1 as an attractive drug target for the treatment of inflammation, metabolic disorders, and cancer. In this review, we provide an overview of the structural basis, functional activities, associated diseases, and advancements in therapeutic ligand research targeting LRH-1.

Cinnamaldehyde Alleviates Bone Loss by Targeting Oxidative Stress and Mitochondrial Damage via the Nrf2/HO-1 Pathway in BMSCs and Ovariectomized Mice.

Osteoporosis (OP) is typically brought on by disruption of bone homeostasis. Excessive oxidative stress and mitochondrial dysfunction are believed to be the primary mechanisms underlying this disorder. Therefore, in order to restore bone homeostasis effectively, targeted treatment of oxidative stress and mitochondrial dysfunction is necessary. Cinnamaldehyde (CIN), a small molecule that acts as an agonist for the nuclear factor erythroid 2-related factor (Nrf2), has been found to possess antiapoptotic, anti-inflammatory, and antioxidant properties. We found that CIN, while rescuing apoptosis, can also reduce the accumulation of reactive oxygen species (ROS) to improve mitochondrial dysfunction and thus restore the osteogenic differentiation potential of BMSCs disrupted by hydrogen peroxide (H2O2) exposure. The role of CIN was preliminarily considered to be a consequence of Nrf2/HO-1 axis activation. The ovariectomized mice model further demonstrated that CIN treatment ameliorated oxidative stress in vivo, partially reversing OVX-induced bone loss. This improvement was seen in the trabecular microarchitecture and bone biochemical indices. However, when ML385 was concurrently injected with CIN, the positive effects of CIN were largely blocked. In conclusion, this study sheds light on the intrinsic mechanisms by which CIN regulates BMSCs and highlights the potential therapeutic applications of these findings in the treatment of osteoporosis.

Discovery of human pancreatic lipase inhibitors from root of Rhodiola crenulata via integrating bioactivity-guided fractionation, chemical profiling and biochemical assay.

Although herbal medicines (HMs) are widely used in the prevention and treatment of obesity and obesity-associated disorders, the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood. Recently, we assessed the inhibitory potentials of several HMs against human pancreatic lipase (hPL, a key therapeutic target for human obesity), among which the root-extract of Rhodiola crenulata (ERC) showed the most potent anti-hPL activity. In this study, we adopted an integrated strategy, involving bioactivity-guided fractionation techniques, chemical profiling, and biochemical assays, to identify the key anti-hPL constituents in ERC. Nine ERC fractions (retention time = 12.5-35 min), obtained using reverse-phase liquid chromatography, showed strong anti-hPL activity, while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS/MS). Among the identified ERC constituents, 1,2,3,4,6-penta-O-galloyl-ß-d-glucopyranose (PGG) and catechin gallate (CG) showed the most potent anti-hPL activity, with pIC50 values of 7.59 ± 0.03 and 7.68 ± 0.23, respectively. Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner, with inhibition constant (K i) values of 0.012 and 0.082 µM, respectively. Collectively, our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC, as well as to elucidate their anti-hPL mechanisms. These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.

Peri-implantation expression and regulation of ITGB8 in goat uterus.

Ruminants have a superficial implantation pattern. The extended conceptus attaches to the receptive endometrium to form the cotyledonary placenta. During the attachment, a large number of events occur at the maternal-fetal interface. However, the related molecular mechanisms have not been fully understood. Integrin beta8 (ITGB8) is a subunit of integrin beta involved in embryo implantation. In this study, we determined peri-implantation expression and regulation of ITGB8 in goat uterus. The mRNA and protein levels of ITGB8 were both high in goat endometrial luminal epithelium (LE) and superficial glandular epithelium (sGE) during the adhesion period (Days 16-19 of pregnancy). Such expression profile was opposite to that of microRNA-187 (miR-187). Then, we validated that miR-187 targeted the 3' untranslated region (UTR) of ITGB8 in primary goat endometrial epithelial cells (EECs). In EECs, inhibition of miR-187 resulted in not only up-regulated ITGB8 level but also reduced cell proliferation and focal adhesion kinase (FAK) activity. Moreover, ITGB8 and miR-187 were regulated by interferon tau (IFNT). Altogether, in goat, the miR-187/ITGB8 axis may be involved in conceptus attachment and is downstream of IFNT. Our results will help us better understand the mechanisms of ruminant implantation and may provide a useful tool to improve the reproduction ratio for ruminants.

Discovery of human pancreatic lipase inhibitors from root of Rhodiola crenulata via integrating bioactivity-guided fractionation,chemical profiling and biochemical assay / 药物分析学报

Although herbal medicines(HMs)are widely used in the prevention and treatment of obesity and obesity-associated disorders,the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood.Recently,we assessed the inhibitory potentials of several HMs against human pancreatic lipase(hPL,a key therapeutic target for human obesity),among which the root-extract of Rhodiola crenulata(ERC)showed the most potent anti-hPL activity.In this study,we adopted an integrated strategy,involving bioactivity-guided fractionation techniques,chemical profiling,and biochemical assays,to identify the key anti-hPL constituents in ERC.Nine ERC fractions(retention time=12.5-35 min),obtained using reverse-phase liquid chromatography,showed strong anti-hPL activity,while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS/MS).Among the identified ERC constituents,1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose(PGG)and catechin gallate(CG)showed the most potent anti-hPL activity,with pIC50 values of 7.59±0.03 and 7.68±0.23,respectively.Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner,with inhibition constant(Ki)values of 0.012 and 0.082 μM,respectively.Collectively,our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC,as well as to elucidate their anti-hPL mechanisms.These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.

Flavonoids in Ampelopsis grossedentata as covalent inhibitors of SARS-CoV-2 3CLpro: Inhibition potentials, covalent binding sites and inhibitory mechanisms.

Coronavirus 3C-like protease (3CLpro) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CLpro inhibitory activity, but the key constituents with SARS-CoV-2-3CLpro inhibitory effect and their mechanisms were unrevealed. Herein, a practical strategy via integrating bioactivity-guided fractionation and purification, mass spectrometry-based peptide profiling and time-dependent biochemical assay, was applied to identify the crucial constituents in AGE and to uncover their inhibitory mechanisms. The results demonstrated that the flavonoid-rich fractions (10-17.5 min) displayed strong SARS-CoV-2-3CLpro inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CLpro inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CLpro in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CLpro at Cys300 and Cys44, while dihydromyricetin and isodihydromyricetin covalently bound at Cys300. Covalent docking coupling with molecular dynamics simulations showed the detailed interactions between the orthoquinone form of myricetin and two covalent binding sites (surrounding Cys300 and Cys44) of SARS-CoV-2 3CLpro. Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CLpro, while the newly identified SARS-CoV-2 3CLpro inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.

Discovery and characterization of flavonoids in vine tea as catechol-O-methyltransferase inhibitors.

Vine tea has been used as a traditionally functional herbal tea in China for centuries, which exhibits paramount potential for chronic metabolic diseases. Herein, the inhibitory potential of vine tea toward human catechol-O-methyltransferase (hCOMT) was investigated. A practical bioactivity-guided fractionation combined with chemical profiling strategy was developed to identify the naturally occurring hCOMT inhibitors. Five flavonoids in vine tea displayed moderate to strong inhibition on hCOMT with IC50 values ranging from 0.96 µM to 42.47 µM, in which myricetin was the critically potent constituent against hCOMT. Inhibition kinetics assays and molecular docking simulations showed that myricetin could bind to the active site of COMT and inhibited COMT-catalyzed 3-BTD methylation in a mixed manner. Collectively, our findings not only suggested that the strong hCOMT inhibition of vine tea has guiding significance in the drug exposure of catechol drugs, but also identified a promising lead compound for developing more efficacious hCOMT inhibitors.

Comparison of 18 F-FDG PET/CT and 68 Ga-DOTATATE PET/CT in the Targeted Imaging of Culprit Tumors Causing Osteomalacia.

OBJECTIVE: To assess and compare the performance of fluorine-18-labeled fluorodeoxyglucose positron emission tomography (18 F-FDG-PET/ CT) and gallium-68-labeled tetraazacyclododecanetetraacetic acid-DPhe1-Tyr3-octreotate (68 Ga- DOTATATE) PET/CT in the targeted imaging of culprit tumors causing osteomalacia. METHODS: This was a clinical retrospective analysis. We analyzed 13 patients (five men, eight women; mean age, 49 years; range, 19-55 years) with suspicion of tumor-induced osteomalacia (TIO) between March 2017 and October 2019. All patients underwent two functional imaging methods to locate the culprittumors. Studies were performed on a PET/CT scanner. The injection doses of 18 F- FDG and 68 Ga-DOTATATE were 0.5mCi/kg and approximately 5.0mCi, respectively. In the two scans, the whole body was captured from head to toe 45 to 60 min after intravenous tracer injection. 68 Ga-DOTATATE PET/CT and 18 F-FDG PET/CT imaging results locate culprit tumors according to the following criteria: (i) abnormal foci uptake concentration was observed locally, and the uptake level was higher than the background level of the right lobe of the liver; (ii) combined CT showed or did not have obvious abnormal density changes; and (iii) non-specific ingestion lesions due to fracture, arthritis, necrosis of femoral head are excluded. Compared with the results of pathological examination and clinical follow-up, the sensitivity, specificity and accuracy of 68 Ga-DOTATATE PET/CT imaging and 18 F-FDG PET/CT imaging for TIO were analyzed. RESULTS: All patients had symptoms of osteomalacia and hypophosphatemia. The lag time (symptoms to PET diagnosis) ranged from 2 to 12 years. There were eight cases of TIO patients and five cases of non-TIO patients confirmed by surgery, pathology and follow-up. Among the eight TIO patients, there were six cases (75.0%) of PMTs, one case (12.5%) of giant cell tumor, one case (12.5%) of hemangiopericutoma. Most (n = 6, 75.0%) of the confirmed tumors in our patient population were in the lower extremities, followed by craniofacial regions (n = 1, 12.5%), and torso (n = 1, 12.5%), respectively. Among the five non-TIO patients, there were two cases of Fanconi syndrome, one case of rickets, and two cases of sporadic osteomalacia hypophosphorus. The culprit tumors could be located either in the bone (n = 5, 62.5%) or the soft tissue (n = 3, 37.5%). 18 F-FDG PET/CT was able to localize the tumor in six (6/13, 46.1%) patients. 68 Ga-DOTATATE PET/CT detected tumor in 8 (83.3%) of 13 patients. The sensitivity of 68 Ga-DOTATATE PET/CT imaging and 18 F-FDG PET/CT imaging in the evaluation of TIO in our patient population were 100% (8/8) vs 75% (6/8). The specificity of the two different methods was 80% (4/5). The overall accuracy was 92.3% (12/13) vs 76.9% (10/13). CONCLUSIONS: 68 Ga-DOTATATE PET/CT is very effective in assessing hypophosphatemia patients with TIO typical symptoms compared with 18 F-FDG. Therefore, in clinically suspected cases of hypophosphatemic osteomalacia, 68 Ga-DOTATATE PET/CT should be preferred as an imaging modality investigation to avoid delay in the treatment of this disease.

Connective tissue growth factor gene expression in goat endometrium during estrous cycle and early pregnancy.

Embryo implantation is crucial for a successful pregnancy. Although many essential molecular modulators and pathways have been identified, the precise mechanisms of the process in goat remain largely unknown. CCN2 is a connective tissue growth factor participating in many biological processes; however, its presence or function in goat uterus has not yet been reported. In this study, we determined the expression and regulation of CCN2 in goat uterus. CCN2 was not detected by in situ hybridization at ED0 (Day 0 of the estrous cycle), but at ED6 (metestrus), ED12 (dioestrus), and ED16 (proestrus), with high signals in luminal epithelium, superficial glands, and caruncula matrix. During early pregnancy, CCN2 was also detected in these locations on D0 and D6 (pre-receptive uterus). The signals significantly increased on D16 and D19 (receptive uterus), and remained at high levels on D25 and D30. Similarly, the RT-qPCR assays showed that the mRNA level of CCN2 was relatively low on D0 and D6, increased on D16, peaked on D19, and kept high thereafter. Moreover, CCN2 was up-regulated not only in ovariectomized ewes subcutaneously injected with 17ß-estradiol and progesterone (separately or together), but also in cultured goat uterine epithelial cells treated with the two hormones or interferon tau (IFNτ). In conclusion, CCN2 expression may be induced by 17ß-estradiol, progesterone, and IFNτ in the luminal epithelium of goat receptive uterus, suggesting that CCN2 is involved in goat embryo adhesion during early pregnancy.

Design, Synthesis and Biological Evaluation of Novel 4-Substituted Coumarin Derivatives as Antitumor Agents.

Herein, fifteen new compounds containing coumarin, 1,2,3-triazole and benzoyl- substituted arylamine moieties were designed, synthesized and tested in vitro for their anticancer activity. The results showed that all tested compounds had moderate antiproliferative activity against MDA-MB-231, a human breast cancer cell line, under both normoxic and hypoxic conditions. Furthermore, the 4-substituted coumarin linked with benzoyl 3,4-dimethoxyaniline through 1,2,3-triazole (compound 5e) displayed the most prominent antiproliferative activities with an IC50 value of 0.03 µM, about 5000 times stronger than 4-hydroxycoumarin (IC50 > 100 µM) and 20 times stronger than doxorubicin (IC50 = 0.60 µM). Meanwhile, almost all compounds revealed general enhancement of proliferation-inhibiting activity under hypoxia, contrasted with normoxia. A docking analysis showed that compound 5e had potential to inhibit carbonic anhydrase IX (CA IX).