A Novel TrxR1 Inhibitor Regulates NK and CD8+ T Cell Infiltration and Cytotoxicity, Enhancing the Efficacy of Anti-PD-1 Immunotherapy against Hepatocarcinoma.

Hepatocellular carcinoma (HCC) has the third highest cancer-related mortality rate globally. The immunosuppressive microenvironment of HCC limits effective treatment options. HCC cells and associated microenvironmental factors suppress NK and T cell infiltration and cytotoxic activities. The abnormal number or function of NK and T cells leads to a lack of immune surveillance. Recently, immunotherapy targeting PD-1 and PD-L1 has been shown to activate functionally exhausted cytotoxic immune cells in some solid tumors. However, the response rate and therapeutic efficacy against solid tumors with little lymphocyte infiltration are limited, especially for HCC. Therefore, new targets and therapeutics that induce tumor cell apoptosis and overcome the problem of depletion of immune cells, thereby inhibiting the immune escape of HCC cells, are urgently required. Butaselen (2-bis[2-(1,2-benzisothiazol-2(2H)-ketone)]butane), an organic molecule containing selenium, is a new type of thioredoxin reductase inhibitor. In this study, we found that butaselen promoted NK and T cell activity and infiltration in the tumor microenvironment in HCC-bearing mice by enhancing the expression of CXCR3, NKG2D, and their respective ligands. When used alone, it can significantly inhibit tumor growth and exert a synergistic effect in combination with PD-1 blockade. We suggested the role of the thioredoxin reductase system in the regulation of the tumor immunosuppressive microenvironment and developed a new effective therapeutic molecule for HCC, revealing the mechanism of butaselen in inhibiting tumor cell immune escape.

Mechanistic target of rapamycin in regulating macrophage function in inflammatory cardiovascular diseases.

The mechanistic target of rapamycin (mTOR) is evolutionarily conserved from yeast to humans and is one of the most fundamental pathways of living organisms. Since its discovery three decades ago, mTOR has been recognized as the center of nutrient sensing and growth, homeostasis, metabolism, life span, and aging. The role of dysregulated mTOR in common diseases, especially cancer, has been extensively studied and reported. Emerging evidence supports that mTOR critically regulates innate immune responses that govern the pathogenesis of various cardiovascular diseases. This review discusses the regulatory role of mTOR in macrophage functions in acute inflammation triggered by ischemia and in atherosclerotic cardiovascular disease (ASCVD) and heart failure with preserved ejection fraction (HFpEF), in which chronic inflammation plays critical roles. Specifically, we discuss the role of mTOR in trained immunity, immune senescence, and clonal hematopoiesis. In addition, this review includes a discussion on the architecture of mTOR, the function of its regulatory complexes, and the dual-arm signals required for mTOR activation to reflect the current knowledge state. We emphasize future research directions necessary to understand better the powerful pathway to take advantage of the mTOR inhibitors for innovative applications in patients with cardiovascular diseases associated with aging and inflammation.

Uptake of Aß by OATPs might be a new pathophysiological mechanism of Alzheimer disease.

BACKGROUND: The accumulation of neurotoxic amyloid-beta (Aß) in the brain is a characteristic of Alzheimer's disease (AD), at the same time, it is possible alterations of liver function could affect brain Aß levels through changes in blood Aß concentration. Over the last decade, a number of reports have shown that P-glycoprotein (encoded by ABC1B1) actively mediates the efflux transport of Aß peptides. However, the mechanism by which Aß peptides enter the cells is not clear. In the preliminary study, we found that the protein expression of organic anion transporting Polypeptide 1a4 (OATP1B1) in the liver tissue of mice with AD was significantly higher than that in the normal mice. In contrast, the protein expression of Oatp1a4 in the brain significantly decreased in mice with AD. OATP1B1, an important drug transporter might be related to the pathophysiology of AD. RESULTS: In this study, we established an OATP1B1-GFP-HEK293T cell model to confirm the OATP1B1 mediated transport of Aß1-42. Compared to the control group of GFP-HEK293Tcells, the uptake of Aß1-42 protein in the OATP1B1-GFP-HEK293T group increased significantly with the increase in concentration of Aß1-42, and also increased significantly with an increase in the duration of incubation. Similar results were observed in the flow cytometry experiment, and the uptake of Aß1-42in HEK293T-OATP1B1 cells was almost twice that in the control group. These results indicate that OATPs may act as an important "carrier" for the transport of Aß1-42 from the blood to the tissues, including liver and brain. CONCLUSIONS: This is a novel and interesting finding and OATP1B1 can be investigated as a new treatment target for AD.

MFN2 silencing promotes neural differentiation of embryonic stem cells via the Akt signaling pathway.

Mitofusin 2 (MFN2) is a regulatory protein participating in mitochondria dynamics, cell proliferation, death, differentiation, and so on. This study aims at revealing the functional role of MFN2 in the pluripotency maintenance and primitive differetiation of embryonic stem cell (ESCs). A dox inducible silencing and routine overexpressing approach was used to downregulate and upregulate MFN2 expression, respectively. We have compared the morphology, cell proliferation, and expression level of pluripotent genes in various groups. We also used directed differentiation methods to test the differentiation capacity of various groups. The Akt signaling pathway was explored by the western blot assay. MFN2 upregulation in ESCs exhibited a typical cell morphology and similar cell proliferation, but decreased pluripotent gene markers. In addition, MFN2 overexpression inhibited ESCs differentiation into the mesendoderm, while MFN2 silencing ESCs exhibited a normal cell morphology, slower cell proliferation and elevated pluripotency markers. For differentiation, MFN2 silencing ESCs exhibited enhanced three germs' differentiation ability. Moreover, the protein levels of phosphorylated Akt308 and Akt473 decreased in MFN2 silenced ESCs, and recovered in the neural differentiation process. When treated with the Akt inhibitor, the neural differentiation capacity of the MFN2 silenced ESCs can reverse to a normal level. Taken together, the data indicated that the appropriate level of MFN2 expression is essential for pluripotency and differentiation capacity in ESCs. The increased neural differentiation ability by MFN2 silencing is strongly related to the Akt signaling pathway.

Identification of cytochrome P450 isoenzymes involved in the metabolism of 23-hydroxybetulinic acid in human liver microsomes.

Context: 23-Hydroxybetulinic acid (23-HBA), a major active constituent of Pulsatilla chinensis (Bunge) Regel (Ranunculaceae), exhibits potential antitumor activity. Its metabolism, however, has not yet been studied.Objective: This study focuses on the metabolism of 23-HBA in vitro by human liver microsomes.Materials and methods: The metabolic kinetics of 23-HBA (0.5-100 µM) and the effects of selective CYP450 (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) inhibitors on metabolism of 23-HBA were evaluated in human liver microsomes incubation system and then determined by LC-MS method. The Michaelis-Menten parameters Km and Vmax were initially estimated by analysing Lineweaver-Burk plot. The clearance (CLint) was also calculated.Results: The Vmax, Km, and CLint of 23-HBA were 256.41 ± 11.20 pmol/min/mg, 11.10 ± 1.07 µM, and 23.10 ± 1.32 µL/min/mg, respectively. The metabolism of 23-HBA was significantly inhibited by furafylline (0.05 µM, p < 0.01) and ketoconazole (0.02 µM, p < 0.05). Ticlopidine (1.3 µM, p < 0.05) could inhibit the metabolism of 23-HBA, while the other inhibitors (sulfaphenazole and quinidine) showed nonsignificant inhibition on the metabolism of 23-HBA.Discussion and conclusions: This is the first investigation of the metabolism of 23-HBA in human liver microsomes. The in vitro study indicates that CYP1A2 and CYP3A4 are mainly involved in the metabolism of 23-HBA. Special attention should be given to the pharmacokinetic and clinical outcomes when 23-HBA was co-administrated with other compounds mainly undergoing CYP1A2/CYP3A4-mediated metabolism. Further studies are needed to evaluate the significance of this interaction and strengthen the understanding of traditional Chinese medicine.

Pharmacokinetic analysis of plasma bicyclol by liquid chromatography-tandem mass spectrometry.

Bicyclol is a synthetic drug widely used to treat chronic hepatitis B. This study aimed to develop a selective, sensitive and high-throughput liquid chromatography-tandem mass spectrometric method for the detection of bicyclol in human plasma. Bicyclol was detected using a multiple reaction monitoring mode, with ammonium adduct ions (m/z 408.2) as the precursor ion and the [M-CH3 ]+ ion (m/z 373.1) subjected to demethylation as the product ion. Chromatographic separation was achieved using a Zobax Eclipse XDB-C18 column with a gradient elution and a mobile phase of 2 mm ammonium formate and acetonitrile. Bicyclol was extracted from plasma matrix by precipitation. A linear detection response was obtained for bicyclol ranging from 0.500 to 240 ng/mL, and the lower limit of quantification was 0.500 ng/mL. The intra- and inter-day precisions were all ≤7.4%, and the accuracies were within ±6.0%. The extraction recovery was >95.9%, and the matrix effects were between 96.0% and 108%. Bicyclol was found to be unstable in human plasma at room temperature, but the degradation was minimized by conducting sample collection and preparation in an ice bath. The validated method was successfully applied to investigate the pharmacokinetics of bicyclol tablets in six healthy Chinese volunteers.

Transport of salvianolic acid B via the human organic anion transporter 1B1 in the liver.

Salvianolic acid B (SAB) has a high concentration in the liver, but the mechanism of its distribution in the liver is unclear. The aim of this study was to investigate the mechanisms of hepatic uptake of SAB. In this study, we first explored the uptake features of SAB in HepG2 cells and the effect of rifampicin on uptake. Then, we explored the effects of SAB on the uptake of pitavastatin in HepG2 cells. Finally, we established an HEK239T-OATP1B1 cell model to confirm whether OATP1B1 mediated the transport of SAB. Results showed that the uptake kinetic parameters Vmax and Km for SAB in HepG2 cells were 21.28 ± 2.06 pmol mg-1 per protein min-1 and 28.47 ± 7.36 µM, respectively. Rifampicin inhibited the uptake of SAB in HepG2 cells (IC50 was 5.85 ± 1.70 µM), and SAB affected the uptake of pitavastatin in HepG2 cells (IC50 was 27.67 ± 1.90 µM). The uptake kinetic parameters Vmax and Km for SAB in HEK239T-OATP1B1 were 60.03 ± 6.16 pmol mg-1 per protein min-1 and 87.24 ± 15.28 µM, respectively, whereas in EGFP-HEK293 cells were 14.04 ± 2.53 pmol mg-1 per protein min-1 and 56.53 ± 15.50 µM. The SAB had no effect of on the expression of OATP1B1 in HEK239T-OATP1B1 cells. In conclusion, this study demonstrated that OATP1B1 contributes to the transport and accumulation of SAB in the liver.

Mulberroside A suppresses PXR-mediated transactivation and gene expression of P-gp in LS174T cells.

Mulberroside A (Mul A) is the main bioactive constituents of Sangbaipi, which is officially listed in the Chinese Pharmacopoeia. The pregnane X receptor (PXR) has been recognized as the critical mediator of human P-glycoprotein (P-gp) gene transactivation. In this study, the effect of Mul A on PXR-mediated transactivation and gene expression of P-gp was investigated. It was found that Mul A significantly suppressed PXR-mediated P-gp luciferase activity induced by rifampicin (Rif). Furthermore, Rif induced an elevation of P-gp expression and transport activity, which was apparently suppressed by Mul A. However, Mul A did not suppress the P-gp luciferase activity, P-gp expression, and function in the absence of Rif. These findings suggest that Mul A suppresses PXR-mediated transactivation and P-gp expression induced by Rif. This should be taken into consideration to predict any potential herb-drug interactions when Mul A or Sangbaipi are co-administered with Rif or other PXR agonist drugs.

UGT1A1 Gene Polymorphism Predicts Irinotecan-Induced Severe Neutropenia and Diarrhea in Chinese Cancer Patients.

BACKGROUND: Irinotecan was widely used in colon cancer and lung cancer, etc., and adverse reactions occur some times. The primary aim of this research is to investigate the association between UGT1A1 gene polymorphisms and irinotecan-related adverse effect in Chinese Han population with a novel kind of gene chip technology. METHODS: UGT1A1*6/*28 gene polymorphisms were detected by PCR and gene chip as well as sequencing. The correlation between UGT1A1 gene polymorphisms and severe delayed diarrhea or neutropenia and effect on response rate and progression-free survival were analyzed. RESULTS: A total of 106 patients receiving irinotecan-based regimens and with detected UGT1A1 gene polymorphisms were enrolled in this research. According to our results, no significant differences of severe diarrhea were found in patients with UGT1A1*6 genotypes (p = 0.608). However, the incidence of severe diarrhea in patients with TA7/7 genotype (66.7%, 4/6) was significantly higher than that in patients with TA6/7 (31.5%, 6/19) or TA6/6 (1.28%, 1/78) genotypes (p < 0.001). The incidence of severe hematologic toxicity in patients with AA (100%, 2/2) was significantly higher than that in patients with GA (33.3%, 7/21) or GG genotype (7.23%, 6/83) (p = 0.011). CONCLUSIONS: In terms of irinotecan-based regimens in cancers, UGT1A1*6 plays a more vital role in hematologic toxicity (p = 0.011) whereas UGT1A1*28 get more involved in diarrhea (p < 0.001).

[Regulation of P-glycoprotein gene expression by PKC/NF-κB-PXR signaling pathway].

P-glycoprotein (P-gp), an ATP binding cassette protein, plays a major role in efflux transport of drugs and xenobiotics due to its abundant expression on several barriers. This study aimed to investigate the potential role of PKC/NF-κB-PXR signaling pathway in modulation of P-gp gene expression in human colon adenocarcinoma LS174T. The effect of PMA on MDR1 luciferase activity was investigated by PXR-MDR1 dual luciferase reporter gene assay. Real-time qPCR assay and Western blot analysis were used to study the gene expression of P-gp and NF-κB, respectively. Compared to the vehicle-treated group, PMA statistically decreased P-gp luciferase activity, mRNA expression and protein expression. Moreover, PMA treatment yielded a significant and dose-dependent increase in RelA/p65 translocation to nucleus. Meanwhile, a remarkable increase of the pho-IκBα status was observed in LS174T cells after treatment with PMA (1-100 nmol·L(-1)). In addition, knockdown of PKCα, NF-κB or PXR can significantly attenuate PMA-induced P-gp suppression. These results suggested that PKC/NF-κB-PXR signaling pathway might play crucial roles in modulation of P-gp gene expression.

Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5.

[OATP1B1 in drug-drug interactions between traditional Chinese medicine Danshensu and rosuvastatin].

The study was designed to explore the drug-drug interactions mechanisms mediated by OATP1B1 between traditional Chinese medicine Danshensu and rosuvastatin. First, the changes of rosuvastatin pharmacokinetics were investigated in presence of Danshensu in rats. Then, the primary rat hepatocytes model was established to explore the effects of Danshensu on the uptake of rosuvastatin by hepatocytes. Finally, HEK293T cells with overexpression of OATP1B1*a and OATP1B1*5 were established using a lentiviral delivery system to explore the effects of Danshensu on the uptake of rosuvastatin. Rosuvastatin pharmacokinetic parameters of C(max0, AUCO(0-t), AUC(0-∞) were increased about 123%, 194% and 195%, by Danshensu in rats, while the CL z/F value was decreased by 60%. Uptake of rosuvastatin in the primary rat hepatocytes was decreased by 3.13%, 41.15% and 74.62%, respectively in the presence of 20, 40 and 80 µmol x L(-1) Danshensu. The IC50 parameters was (53.04 ± 2.43) µmol x L(-1). The inhibitory effect of Danshensu on OATP1B1 mediated transport of rosuvastatin was related to the OATP1B1 gene type. In OATP1B1*5-HEK293T mutant cells, transport of rosuvastatin were reduced by (39.11 ± 4.94)% and (63.61 ± 3.94)%, respectively, by Danshensu at 1 and 10 µmol x L(-1). While transport of rosuvastatin was reduced by (8.22 ± 2.40)% and (11.56 ± 3.04)% and in OATP1B1*1a cells, respectively. Danshensu significantly altered the pharmacokinetics of rosuvastatin in rats, which was related to competitive inhibition of transport by OATPJBI. Danshensu exhibited a significant activity in the inhibition of rosuvastatin transport by OATP1B1*5-HEK293T, but not by OATP1B1*1a, suggesting a dependence on OATP1B1 sequence.

Overview of the roles of Sox2 in stem cell and development.

Sox2 is well known for its functions in embryonic stem (ES) cell pluripotency, maintenance, and self-renewal, and it is an essential factor in generating inducible pluripotent stem (iPS) cells. It also plays an important role in development and adult tissue homeostasis of different tissues, especially the central nervous system. Increasing evidence has shown that aging is a stemness-related process in which Sox2 is also implicated as a key player, especially in the neural system. These distinct roles that Sox2 plays involve delicate regulatory networks consisting of other master transcription factors, microRNAs and signaling pathways. Additionally, the expression level of Sox2 can also be modulated transcriptionally, translationally or post-translationally. Here we will mainly review the roles of Sox2 in stem cell related development, homeostasis maintenance, aging processes, and the underlying molecular mechanisms involved.

Effect of Ursolic Acid on Breast Cancer Resistance Protein-mediated Transport of Rosuvastatin In Vivo and Vitro.

OBJECTIVE: To evaluate whether ursolic acid can inhibit breast cancer resistance protein (BCRP)-mediated transport of rosuvastatin in vivo and in vitro. METHODS: Firstly, we explored the pharmacokinetics of 5-fluorouracil (5-FU, a substrate of BCRP) in rats in the presence or absence of ursolic acid. Secondly, we studied the pharmacokinetics of rosuvastatin in rats in the presence or absence of ursolic acid or Ko143 (inhibitor of BCRP). Finially, the concentration-dependent transport of rosuvastatin and the inhibitory effects of ursolic acid and Ko143 were examined in Madin-Darby Canine Kidney (MDCK) 2-BCRP421CC (wild type) cells and MDCK2-BCRP421AA (mutant type) cells. RESULTS: As a result, significant changes in pharmacokinetics parameters of 5-FU were observed in rats following pretreatment with ursolic acid. Both ursolic acid and Ko143 could significantly affect the pharmacokinetics of rosuvastatin. The rosuvastatin transport in the BCRP overexpressing system was increased in a concentration-dependent manner. However, there was no statistical difference in BCRP-mediated transport of rosuvastatin betweent the wild type cells and mutant cells. The same as Ko143, ursolic acid inhibited BCRP-mediated transport of rosuvastatin in vitro. CONCLUSION: Ursolic acid appears to be a potent modulator of BCRP that affects the pharmacokinetic of rosuvastatin in vivo and inhibits the transport of rosuvastatin in vitro.

Lysophosphatidic acid accelerates lung fibrosis by inducing differentiation of mesenchymal stem cells into myofibroblasts.

Lung fibrosis is characterized by vascular leakage and myofibroblast recruitment, and both phenomena are mediated by lysophosphatidic acid (LPA) via its type-1 receptor (LPA1). Following lung damage, the accumulated myofibroblasts activate and secrete excessive extracellular matrix (ECM), and form fibrotic foci. Studies have shown that bone marrow-derived cells are an important source of myofibroblasts in the fibrotic organ. However, the type of cells in the bone marrow contributing predominantly to the myofibroblasts and the involvement of LPA-LPA1 signalling in this is yet unclear. Using a bleomycin-induced mouse lung-fibrosis model with an enhanced green fluorescent protein (EGFP) transgenic mouse bone marrow replacement, we first demonstrated that bone marrow derived-mesenchymal stem cells (BMSCs) migrated markedly to the bleomycin-injured lung. The migrated BMSC contributed significantly to α-smooth muscle actin (α-SMA)-positive myofibroblasts. By transplantation of GFP-labelled human BMSC (hBMSC) or EGFP transgenic mouse BMSC (mBMSC), we further showed that BMSC might be involved in lung fibrosis in severe combined immune deficiency (SCID)/Beige mice induced by bleomycin. In addition, using quantitative-RT-PCR, western blot, Sircol collagen assay and migration assay, we determined the underlying mechanism was LPA-induced BMSC differentiation into myofibroblast and the secretion of ECM via LPA1. By employing a novel LPA1 antagonist, Antalpa1, we then showed that Antalpa1 could attenuate lung fibrosis by inhibiting both BMSC differentiation into myofibroblast and the secretion of ECM. Collectively, the above findings not only further validate LPA1 as a drug target in the treatment of pulmonary fibrosis but also elucidate a novel pathway in which BMSCs contribute to the pathologic process.

High-fat diet induces early-onset diabetes in heterozygous Pax6 mutant mice.

BACKGROUND: Type 2 diabetes is caused by interactions between genetic and environmental factors. Our previous studies reported that paired box 6 mutation heterozygosity (Pax6(m/+)) led to defective proinsulin processing and subsequent abnormal glucose metabolism in mice at 6 months of age. However, high-fat diet exposure could be an important incentive for diabetes development. In this study, we aimed to develop a novel diabetic model imitating human type 2 diabetes by exposing Pax6(m/+) mice to high-fat diet and to explore the underlying mechanism of diabetes in this model. METHODS: Over 300 Pax6(m/+) and wild-type male weanling mice were randomly divided into two groups and were fed an high-fat diet or chow diet for 6-10 weeks. Blood glucose and glucose tolerance levels were monitored during this period. Body weights, visceral adipose weights, blood lipid profiles and insulin sensitivity (determined with an insulin tolerance test) were used to evaluate obesity and insulin resistance. Proinsulin processing and insulin secretion levels were used to evaluate pancreatic ß cell function. RESULTS: After 6 weeks of high-fat diet exposure, only the Pax6(m/+) mice showed dramatic postloading hyperglycaemia. These mice exhibited significant high-fat diet-induced visceral obesity and insulin resistance and displayed defective prohormone convertase 1/3 production, an increased proinsulin:total insulin ratio and impaired early-phase insulin secretion, because of the Pax6 mutation. Hyperglycaemia worsened progressively over time with the high-fat diet, and most Pax6(m/+) mice on high-fat diet developed diabetes or impaired glucose tolerance after 10 weeks. Furthermore, high-fat diet withdrawal partly improved blood glucose levels in the diabetic mice. CONCLUSIONS: By combining the Pax6(m/+) genetic background with an high-fat diet environment, we developed a novel diabetic model to mimic human type 2 diabetes. This model is characterized by impaired insulin secretion, caused by the Pax6 mutation, and high-fat diet-induced insulin resistance and therefore provides an ideal tool for research on type 2 diabetes pathogenesis and therapies.

Sox2 protects neural stem cells from apoptosis via up-regulating survivin expression.

The transcription factor Sox2 [SRY (sex-determining region Y)-box 2] is essential for the regulation of self-renewal and homoeostasis of NSCs (neural stem cells) during brain development. However, the downstream targets of Sox2 and its underlying molecular mechanism are largely unknown. In the present study, we found that Sox2 directly up-regulates the expression of survivin, which inhibits the mitochondria-dependent apoptotic pathway in NSCs. Although overexpression of Sox2 elevates survivin expression, knockdown of Sox2 results in a decrease in survivin expression, thereby initiating the mitochondria-dependent apoptosis related to caspase 9 activation. Furthermore, cell apoptosis owing to knockdown of Sox2 can be rescued by ectopically expressing survivin in NSCs as well as in the mouse brain, as demonstrated by an in utero-injection approach. In short, we have found a novel Sox2/survivin pathway that regulates NSC survival and homoeostasis, thus revealing a new mechanism of brain development, neurological degeneration and such aging-related disorders.

Effects of Clarithromycin and Ketoconazole on FK506 Metabolism in Different CYP3A4 Genotype Recombinant Metabolic Enzyme Systems.

OBJECTIVE: This study aimed to investigate the effects of clarithromycin and ketoconazole on the pharmacokinetic properties of tacrolimus in different CYP3A4 genotype recombinant metabolic enzyme systems, so as to understand the drug interactions and their mechanisms further. METHOD: The experiment was divided into three groups: a blank control group, CYP3A4*1 group and CYP3A4*18 recombinant enzyme group. Each group was added with tacrolimus (FK506) of a series of concentrations. Then 1 umol/L clarithromycin or ketoconazole was added to the recombinant enzyme group and incubated in the NADPH system for 30 minutes to examine the effects of clarithromycin and ketoconazole on the metabolizing enzymes' activity of different genotypes. The remaining concentration of FK506 in the reaction system was determined using UPLC-MS/MS, and the enzyme kinetic parameters were calculated using the software. RESULTS: The metabolism of CYP3A4*18 to FK506 was greater than that of CyP3А4*1B. Compared with the CYP3A4*1 group, the metabolic rate and clearance of FK506 in the CYP3A4*18 group significantly increased, with Km decreasing. Clarithromycin and ketoconazole inhibit the metabolism of FK506 by affecting the enzyme activity of CYP3A4*1B and CYP3A4*18B. After adding clarithromycin or ketoconazole, the metabolic rate of FK506 significantly decreased in CYP3A4*1 and CYP3A4*18, with Km increasing, Vmax and Clint decreasing. CONCLUSION: Compared with CYP3A4*1, CYP3A4*18 has a greater metabolism of FK506, clarithromycin and ketoconazole can inhibit both the enzymatic activities of CYP3A4*1 and CYP3A4*18, consequently affecting the metabolism of FK506 and the inhibitory on CYP3A4*1 is stronger.