Improving insulin resistance by sulforaphane via activating the Bacteroides and Lactobacillus SCFAs-GPR-GLP1 signal axis.

Background: Insulin resistance (IR) is closely associated with non-alcoholic fatty liver disease (NAFLD), and the gut microbiome contributes to the development of NAFLD. Sulforaphane (SFN) is a phytochemical in cruciferous vegetables that could improve lipid metabolism disorder. However, whether SFN can alleviate IR in NAFLD by regulating the intestinal flora remains unclear. Methods: SFN was administered to high fat diet (HFD)-fed Wistar rats for 10 weeks. Gut microbiota was analysed by 16S rRNA sequencing and the short chain fatty acids (SCFAs) by gas chromatography. The expression of tight junction protein and the numbers of Lactobacillus, Bacteroides and Bifidobacterium were determined by qPCR. The expression of G-protein-coupled receptor 41/43 (GPR41/43) was determined by western blot. A randomized controlled trial (RCT) was conducted in NAFLD patients with broccoli seed tablets (rich in SFN, 42 mg d-1) as intervention for 12 weeks. Thirty-six volunteers with abnormal glucose before the broccoli seed tablet treatment were selected in the intervention group to analyze their blood glucose, insulin, homeostasis model assessment-insulin resistance index (HOMA-IRI), homeostasis model assessment-insulin sensitivity index (HOMA-ISI) and glucagon-like peptide (GLP-1). Results: SFN reduced blood glucose and HOMA-IRI while increasing insulin sensitivity in HFD rats. SFN reduced glycogen synthase kinase 3 (GSK-3), phosphoenolpyruvate carboxykinase (PEPCK) activity, and phosphorylation of serine residues of IRS-2 induced by HFD. SFN reshaped the gut microbiota composition of HFD-induced rats and, especially, increased the content of Bacteroidaceae, Lactobacillaceae and Bifidobacteriaceae, which are related to the improvement from SFN of the blood glucose and HOMA-IRI. The increased numbers of Bacteroides and Lactobacillus were the targets of SFN to enhance the expression of tight junction proteins ZO-1 and occludin, thereby lowering lipopolysaccharide content to reduce inflammation, ultimately alleviating IR. Bacteroides and Lactobacillus produced SCFAs, which activated GPR41/43 to secrete GLP1. Moreover, it was also confirmed in RCT that SFN intervention increased the level of GLP1 in NAFLD patients, which was positively correlated with the reduction of blood glucose and HOMA-IR. Conclusions: SFN alleviated IR in NAFLD via the Bacteroides and Lactobacillus SCFAs-GPR41/43-GLP1 axis and protected the intestinal mucosal barrier to decrease inflammation.

Photophobia and Visual Triggers in Vestibular Migraine.

INTRODUCTION: Vestibular migraine (VM) is a prevalent vestibular disorder characterized by episodic vertigo. However, the relationship between photophobia and visual triggers in VM remains unexplored. We investigated the correlation of photophobia during the VM attack with interictal photosensitivity and visually triggering dizziness in patients with VM. METHODS: We enrolled patients diagnosed with VM, with or without photophobia, across seven specialized vertigo and headache clinics in China. Healthy individuals were also included as a control group. Using a cross-sectional survey design, we collected data related to light intensity and dizziness frequency triggered by flicker, glare, and eyestrain using the Headache Triggers Sensitivity and Avoidance Questionnaire. RESULTS: A total of 366 patients were recruited. The photosensitivity and frequency of dizziness induced by flicker, glare, and eyestrain observed in patients with VM and photophobia were significantly elevated compared with those in patients without photophobia and control participants (P < 0.001). A significant positive correlation was observed between photosensitivity levels and dizziness frequency triggered by flicker, glare, and eyestrain in patients with VM and photophobia (P < 0.001). CONCLUSIONS: This study unequivocally established a positive association of ictal photophobia with interictal photosensitivity and visually triggering dizziness, strongly advocating the need for further research on exposure-based therapies for managing VM. CLINICAL TRIALS REGISTRATION: ClinicalTrial.gov Identifier, NCT04939922, retrospectively registered, 14th June 2021.

Identification and Functional Assessment of Eight CYP3A4 Allelic Variants *39-*46 Detected in the Chinese Han Population.

Cytochrome P450 3A4 (CYP3A4), a key enzyme, is pivotal in metabolizing approximately half of the drugs used clinically. The genetic polymorphism of the CYP3A4 gene significantly influences individual variations in drug metabolism, potentially leading to severe adverse drug reactions (ADRs). In this study, we conducted a genetic analysis on CYP3A4 gene in 1163 Chinese Han individuals to identify the genetic variations that might affect their drug metabolism capabilities. For this purpose, a multiplex polymerase chain reaction (PCR) amplicon sequencing technique was developed, enabling us to perform the genotyping of CYP3A4 gene efficiently and economically on a large scale. As a result, a total of 14 CYP3A4 allelic variants were identified, comprising six previously reported alleles and eight new nonsynonymous variants that were nominated as new allelic variants *39-*46 by the PharmVar Association. Further, functional assessments of these novel CYP3A4 variants were undertaken by coexpressing them with cytochromes P450 oxidoreductase (CYPOR) in Saccharomyces cerevisiae microsomes. Immunoblot analysis indicated that with the exception of CYP3A4.40 and CYP3A4.45, the protein expression levels of most new variants were similar to that of the wild-type CYP3A4.1 in yeast cells. To evaluate their catalytic activities, midazolam was used as a probe drug. The results showed that variant CYP3A4.45 had almost no catalytic activity, whereas the other variants exhibited significantly reduced drug metabolism abilities. This suggests that the majority of the CYP3A4 variants identified in the Chinese population possess markedly altered capacities for drug metabolism. SIGNIFICANCE STATEMENT: In this study, we established a multiplex polymerase chain reaction (PCR) amplicon sequencing method and detected the maximum number of new CYP3A4 variants in a single ethnic population. Additionally, we performed the functional characterizations of these eight novel CYP3A4 allele variants in vitro. This study not only contributes to the understanding of CYP3A4 genetic polymorphism in the Chinese Han population but also holds substantial reference value for their potential clinical applications in personalized medicine.

Characterization of 15 CYP2J2 variants identified in the Chinese Han population on the metabolism of ebastine and terfenadine in vitro.

Genetic polymorphism of the cytochrome P450 (CYP) gene can significantly influence the metabolism of endogenous and xenobiotic compounds. However, few studies have focused on the polymorphism of CYP2J2 and its impact on drug catalytic activity, especially in the Chinese Han population. In this study, we sequenced the promoter and exon regions of CYP2J2 in 1,163 unrelated healthy Chinese Han individuals using the multiplex PCR amplicon sequencing method. Then, the catalytic activities of the detected CYP2J2 variants were evaluated after recombinant expression in S. cerevisiae microsomes. As a result, CYP2J2*7, CYP2J2*8, 13 variations in the promoter region and 15 CYP2J2 nonsynonymous variants were detected, of which V15A, G24R, V68A, L166F and A391T were novel missense variations. Immunoblotting results showed that 11 of 15 CYP2J2 variants exhibited lower protein expression than wild-type CYP2J2.1. In vitro functional analysis results revealed that the amino acid changes of 14 variants could significantly influence the drug metabolic activity of CYP2J2 toward ebastine or terfenadine. Specifically, 4 variants with relatively higher allele frequencies, CYP2J2.8, 173_173del, K267fs and R446W, exhibited extremely low protein expression and defective catalytic activities for both substrates. Our results indicated that a high genetic polymorphism of CYP2J2 could be detected in the Chinese Han population, and most genetic variations in CYP2J2 could influence the expression and catalytic activity of CYP2J2. Our data significantly enrich the knowledge of genetic polymorphisms in CYP2J2 and provide new theoretical information for corresponding individualized medication in Chinese and other Asian populations.

Mitochondria Transplantation from Stem Cells for Mitigating Sarcopenia.

Sarcopenia is defined as the age-related loss of muscle mass and function that can lead to prolonged hospital stays and decreased independence. It is a significant health and financial burden for individuals, families, and society as a whole. The accumulation of damaged mitochondria in skeletal muscle contributes to the degeneration of muscles with age. Currently, the treatment of sarcopenia is limited to improving nutrition and physical activity. Studying effective methods to alleviate and treat sarcopenia to improve the quality of life and lifespan of older people is a growing area of interest in geriatric medicine. Therapies targeting mitochondria and restoring mitochondrial function are promising treatment strategies. This article provides an overview of stem cell transplantation for sarcopenia, including the mitochondrial delivery pathway and the protective role of stem cells. It also highlights recent advances in preclinical and clinical research on sarcopenia and presents a new treatment method involving stem cell-derived mitochondrial transplantation, outlining its advantages and challenges.

Identification and in vitro functional assessment of 10 CYP2C9 variants found in Chinese Han subjects.

Cytochrome P450 2C9 (CYP2C9) participates in about 15% of clinical drug metabolism, and its polymorphism is associated with individual drug metabolism differences, which may lead to the adverse drug reactions (ADRs). In this study, 1163 Chinese Han individuals were recruited to investigate their distribution pattern of CYP2C9 gene and find out the variants that may affect their drug metabolic activities. We successfully developed a multiplex PCR amplicon sequencing method and used it for the genetic screening of CYP2C9 in a large scale. Besides the wild type CYP2C9*1, totally 26 allelic variants of CYP2C9 were detected, which included 16 previously reported alleles and 10 new non-synonymous variants that had not been listed on the PharmVar website. The characteristics of these newly detected CYP2C9 variants were then evaluated after co-expressing them with CYPOR in S. cerevisiae microsomes. Immunoblot analysis revealed that except for Pro163Ser, Glu326Lys, Gly431Arg and Ile488Phe, most of newly detected variants showed comparable protein expression levels to wild type in yeast cells. Two typical CYP2C9 probe drugs, losartan and glimepiride, were then used for the evaluation of metabolic activities of variants. As a result, 3 variants Thr301Met, Glu326Lys, and Gly431Arg almost lost their catalytic activities and most of other variants exhibited significantly elevated activities for drug metabolism. Our data not only enriches the knowledge of naturally occurring CYP2C9 variants in the Chinese Han population, but also provides the fundamental evidence for its potential clinical usage for personalized medicine in the clinic.

Increased iron deposition in nucleus accumbens associated with disease progression and chronicity in migraine.

BACKGROUND: Migraine is one of the world's most prevalent and disabling diseases. Despite huge advances in neuroimaging research, more valuable neuroimaging markers are still urgently needed to provide important insights into the brain mechanisms that underlie migraine symptoms. We therefore aim to investigate the regional iron deposition in subcortical nuclei of migraineurs as compared to controls and its association with migraine-related pathophysiological assessments. METHODS: A total of 200 migraineurs (56 chronic migraine [CM], 144 episodic migraine [EM]) and 41 matched controls were recruited. All subjects underwent MRI and clinical variables including frequency/duration of migraine, intensity of migraine, 6-item Headache Impact Test (HIT-6), Migraine Disability Assessment (MIDAS), and Pittsburgh Sleep Quality Index (PSQI) were recorded. Quantitative susceptibility mapping was employed to quantify the regional iron content in subcortical regions. Associations between clinical variables and regional iron deposition were studied as well. RESULTS: Increased iron deposition in the putamen, caudate, and nucleus accumbens (NAC) was observed in migraineurs more than controls. Meanwhile, patients with CM had a significantly higher volume of iron deposits compared to EM in multiple subcortical nuclei, especially in NAC. Volume of iron in NAC can be used to distinguish patients with CM from EM with a sensitivity of 85.45% and specificity of 71.53%. As the most valuable neuroimaging markers in all of the subcortical nuclei, higher iron deposition in NAC was significantly associated with disease progression, and higher HIT-6, MIDAS, and PSQI. CONCLUSIONS: These findings provide evidence that iron deposition in NAC may be a biomarker for migraine chronicity and migraine-related dysfunctions, thus may help to understand the underlying vascular and neural mechanisms of migraine. TRIAL REGISTRATION: ClinicalTrials.gov, number NCT04939922.

Assessments of CYP­inhibition­based drug-drug interaction between vonoprazan and poziotinib in vitro and in vivo.

CONTEXT: Poziotinib and vonoprazan are two drugs mainly metabolized by CYP3A4. However, the drug-drug interaction between them is unknown. OBJECTIVE: To study the interaction mechanism and pharmacokinetics of poziotinib on vonoprazan. MATERIALS AND METHODS: In vitro experiments were performed with rat liver microsomes (RLMs) and the contents of vonoprazan and its metabolite were then determined with UPLC-MS/MS after incubation of RLMs with vonoprazan and gradient concentrations of poziotinib. For the in vivo experiment, rats in the poziotinib treated group were given 5 mg/kg poziotinib by gavage once daily for 7 days, and the control group was only given 0.5% CMC-Na. On Day 8, tail venous blood was collected at different time points after the gavage administration of 10 mg/kg vonoprazan, and used for the quantification of vonoprazan and its metabolite. DAS and SPSS software were used for the pharmacokinetic and statistical analyses. RESULTS: In vitro experimental data indicated that poziotinib inhibited the metabolism of vonoprazan (IC50 = 10.6 µM) in a mixed model of noncompetitive and uncompetitive inhibition. The inhibitory constant Ki was 0.574 µM and the binding constant αKi was 2.77 µM. In vivo experiments revealed that the AUC(0-T) (15.05 vs. 90.95 µg/mL·h) and AUC(0-∞) (15.05 vs. 91.99 µg/mL·h) of vonoprazan increased significantly with poziotinib pretreatment. The MRT(0-∞) of vonoprazan increased from 2.29 to 5.51 h, while the CLz/F value decreased from 162.67 to 25.84 L/kg·h after pretreatment with poziotinib. CONCLUSIONS: Poziotinib could significantly inhibit the metabolism of vonoprazan and more care may be taken when co-administered in the clinic.

Identification and drug metabolic characterization of four new CYP2C9 variants CYP2C9*72-*75 in the Chinese Han population.

Cytochrome 2C9 (CYP2C9), one of the most important drug metabolic enzymes in the human hepatic P450 superfamily, is required for the metabolism of 15% of clinical drugs. Similar to other CYP2C family members, CYP2C9 gene has a high genetic polymorphism which can cause significant racial and inter-individual differences in drug metabolic activity. To better understand the genetic distribution pattern of CYP2C9 in the Chinese Han population, 931 individuals were recruited and used for the genotyping in this study. As a result, seven synonymous and 14 non-synonymous variations were identified, of which 4 missense variants were designated as new alleles CYP2C9*72, *73, *74 and *75, resulting in the amino acid substitutions of A149V, R150C, Q214H and N418T, respectively. When expressed in insect cell microsomes, all four variants exhibited comparable protein expression levels to that of the wild-type CYP2C9 enzyme. However, drug metabolic activity analysis revealed that these variants exhibited significantly decreased catalytic activities toward three CYP2C9 specific probe drugs, as compared with that of the wild-type enzyme. These data indicate that the amino acid substitution in newly designated variants can cause reduced function of the enzyme and its clinical significance still needs further investigation in the future.

Evaluation of commonly used cardiovascular drugs in inhibiting vonoprazan metabolism in vitro and in vivo.

As a novel acid-suppressing drug, vonoprazan shows the potential to replace traditional proton-pump inhibitors. With its widespread use, some adverse effects that require further study have emerged due to drug-drug interactions. Our study is the first experiment that evaluated the drug-drug interactions of eleven common cardiovascular drugs that inhibit vonoprazan metabolism in vitro and in vivo. Rat liver microsome incubation and molecular simulation docking were applied to explore the inhibition mechanism. Amlodipine and nifedipine showed inhibitory effects on vonoprazan metabolism in both rat and human liver microsomes in the first evaluation part in vitro. The inhibition mechanism analysis results demonstrated that amlodipine and nifedipine might inhibit the metabolism of vonoprazan by a mixed type of competitive and non-competitive inhibition. However, the pharmacokinetic data of the vonoprazan prototype revealed that amlodipine affected vonoprazan in vivo while nifedipine did not. Thus, more attention should be paid when amlodipine is prescribed with vonoprazan. Furthermore, the changes in its carboxylic acid metabolites MI hinted at a complex situation. Molecular simulation suggested the CYP2B6 enzyme may contribute more to this than CYP3A4, and further inhibitory experiments preliminarily verified this speculation. In conclusion, the use of vonoprazan with cardiovascular drugs, especially amlodipine, should receive particular attention in clinical prescriptions.

Effects of Simvastatin on the Metabolism of Vonoprazan in Rats Both in vitro and in vivo.

Purpose: To study the potential drug-drug interactions between simvastatin and vonoprazan and to provide the scientific basis for rational use of them in clinical practice. Methods: An incubation system was established with rat liver microsomes, and the main metabolite of vonoprazan M-I was detected by UPLC-MS/MS. The IC50 value of simvastatin was then calculated and its inhibitory mechanism against vonoprazan was also analyzed. Twelve SD rats were randomly divided into 2 groups, then they were given simvastatin or saline for 2 weeks continuously. On the day of the experiment, both groups were intragastrically administered with vonoprazan once, followed by the collection of blood at different time points. Then the plasma concentration of vonoprazan and M-I in rats were detected by UPLC-MS/MS. Results: In vitro experiments revealed that simvastatin could inhibit the metabolism of vonoprazan, and its inhibition type belonged to the mixed non-competitive and competitive inhibition model. In vivo experiments in rats demonstrated that the area under concentration time curve (AUC) of vonoprazan was decreased but the clearance (CLz/F) of it was increased in the simvastatin administrated group, as compared to those of the control group. However, M-I in simvastatin treated group exhibited the higher AUC and lower CLz/F values compared to those in the control group. These data indicated that multiple doses of simvastatin administration could reduce the plasma concentration of vonoprazan and accelerate its metabolic rate in rats. Conclusion: Simvastatin could inhibit the metabolism of vonoprazan in vitro but multiple doses of simvastatin exhibited the opposite effect In vivo. Altogether, our data indicated that an interaction existed between simvastatin and vonoprazan and additional cares might be taken when they were co-administrated in clinic.

Cyanidin-3-o-glucoside (C3G) inhibits vascular leakage regulated by microglial activation in early diabetic retinopathy and neovascularization in advanced diabetic retinopathy.

Cyanidin-3-O-glucoside (C3G) is a kind of anthocyanin which shows strong anti-inflammation, anti-tumor and anti-oxidant properties. This paper was designed to explore the potential effects of C3G on diabetic retinopathy (DR). C57BL/6 mice were administrated with streptozotocin (STZ) or vehicle control for the establishment of diabetic models. To simulate hyperglycemia and hypoxia, D-glucose (30 mM) and CoCl2 (200 µm/l) were utilized to treat HRECs, respectively. The migration, invasion, inflammation and tube formation abilities of cells were evaluated with the adoption of wound healing, transwell, ELISA and tube formation assays, respectively. Besides, immunofluorescence staining was utilized to detect proliferation and retinal vessels. Evans blue permeation assay were performed to evaluate the vascular leakage in DR mice. Moreover, western blot and qPCR were used to quantify the mRNA and protein expressions of ionized calcium-binding adapter molecule (Iba)-1 and tight junction proteins. Results showed that C3G alleviated the inflammation, microglial activation and angiogenesis in DR mice. Moreover, the proliferation and inflammation of BV2 cells induced by high glucose (HG) were suppressed by C3G. Evans blue permeation assay demonstrated the potency of C3G in attenuating vascular leakage. In addition, C3G suppressed the migration, invasion and angiogenesis of human retinal endothelial cells (HRECs) DR model in vitro.By confirming the role of C3G in inhibiting vascular leakage regulated by microglia activation in early DR and angiogenesis in advanced DR, this study pointed out the potential of C3G as a therapeutic drug for DR management.