Single-cell RNA sequencing data locate ALDH1A2-mediated retinoic acid synthetic pathway to glomerular parietal epithelial cells.

Aldehyde dehydrogenase 1, family member A2, is a retinoic acid-synthesizing enzyme encoded by Aldh1a2 in mice and ALDH1A2 in humans. This enzyme is indispensable for kidney development, but its role in kidney physiology and pathophysiology remains to be fully defined. In this review, we mined single-cell and single-nucleus RNA sequencing databases of mouse and human kidneys and found that glomerular parietal epithelial cells (PECs) express a full set of genes encoding proteins needed for cellular vitamin A uptake, intracellular transport, and metabolism into retinoic acid. In particular, Aldh1a2/ALDH1A2 mRNAs are selectively enriched in mouse and human PECs. Aldh1a2 expression in PECs is greatly increased in a mouse model of anti-glomerular basement membrane glomerulonephritis and moderately induced in a mouse model of ischemia-reperfusion acute kidney injury. Aldh1a2 expression in PECs is substantially repressed in a chronic kidney disease mouse model combining diabetes, hypertension, and partial nephrectomy and is moderately repressed in mouse models of focal segmental glomerulosclerosis and diabetic nephropathy. Single-nucleus RNA sequencing data show that ALDH1A2 mRNA expression in PECs is diminished in patients with chronic kidney disease associated with diabetes, hypertension and polycystic kidney disease. In addition to data mining, we also performed Spearman's rank correlation coefficient analyses and identified gene transcripts correlated with Aldh1a2/ALDH1A2 transcripts in mouse PECs and PEC subtypes, and in human PECs of healthy subjects and patients with AKI or CKD. Furthermore, we conducted Gene Ontology pathway analyses and identified the biological pathways enriched among these Aldh1a2/ALDH1A2-correlated genes. Our data mining and analyses led us to hypothesize that ALDH1A2-mediated retinoic acid synthesis in PECs plays a yet-undefined role in the kidney and that its dysregulation mediates injury. Conditional, PEC-selective Aldh1a2 knockout, RNA silencing and transgenic mouse models will be useful tools to test this hypothesis. Clinical studies on genetics, epigenetics, expression and functions of ALDH1A2 and other genes needed for retinoic acid biosynthesis and signaling are also warranted.

Novel role for epalrestat: protecting against NLRP3 inflammasome-driven NASH by targeting aldose reductase.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a progressive and inflammatory subtype of nonalcoholic fatty liver disease (NAFLD) characterized by hepatocellular injury, inflammation, and fibrosis in various stages. More than 20% of patients with NASH will progress to cirrhosis. Currently, there is a lack of clinically effective drugs for treating NASH, as improving liver histology in NASH is difficult to achieve and maintain through weight loss alone. Hence, the present study aimed to investigate potential therapeutic drugs for NASH. METHODS: BMDMs and THP1 cells were used to construct an inflammasome activation model, and then we evaluated the effect of epalrestat on the NLRP3 inflammasome activation. Western blot, real-time qPCR, flow cytometry, and ELISA were used to evaluate the mechanism of epalrestat on NLRP3 inflammasome activation. Next, MCD-induced NASH models were used to evaluate the therapeutic effects of epalrestat in vivo. In addition, to evaluate the safety of epalrestat in vivo, mice were gavaged with epalrestat daily for 14 days. RESULTS: Epalrestat, a clinically effective and safe drug, inhibits NLRP3 inflammasome activation by acting upstream of caspase-1 and inducing ASC oligomerization. Importantly, epalrestat exerts its inhibitory effect on NLRP3 inflammasome activation by inhibiting the activation of aldose reductase. Further investigation revealed that the administration of epalrestat inhibited NLRP3 inflammasome activation in vivo, alleviating liver inflammation and improving NASH pathology. CONCLUSIONS: Our study indicated that epalrestat, an aldose reductase inhibitor, effectively suppressed NLRP3 inflammasome activation in vivo and in vitro and might be a new therapeutic approach for NASH.

Optimal transplantation strategy using human induced pluripotent stem cell-derived cardiomyocytes for acute myocardial infarction in nonhuman primates.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have the potential to be a therapeutic option for myocardium restoration. However, hiPSC-CMs of varying maturation and transplantation routes exhibit different reactivity and therapeutic effects. We previously demonstrated that the saponin+ compound induces more mature hiPSC-CMs. The safety and efficacy of multi-route transplantation of saponin+ compound-induced hiPSC-CMs in a nonhuman primate with myocardial infarction will be investigated for the first time in this study. Our findings indicate that optimized hiPSC-CMs transplanted via intramyocardial and intravenous routes may affect myocardial functions by homing or mitochondrial transfer to the damaged myocardium to play a direct therapeutic role as well as indirect beneficial roles via anti-apoptotic and pro-angiogenesis mechanisms mediated by different paracrine growth factors. Due to significant mural thrombosis, higher mortality, and unilateral renal shrinkage, intracoronary transplantation of hiPSC-CMs requires closer attention to anticoagulation and caution in clinical use. Collectively, our data strongly indicated that intramyocardial transplantation of hiPSC-CMs is the ideal technique for clinical application; multiple cell transfers are recommended to achieve steady and protracted efficacy because intravenous transplantation's potency fluctuates. Thus, our study offers a rationale for choosing a therapeutic cell therapy and the best transplantation strategy for optimally induced hiPSC-CMs.

Capsaicin functions as a selective degrader of STAT3 to enhance host resistance to viral infection.

Although STAT3 has been reported as a negative regulator of type I interferon (IFN) signaling, the effects of pharmacologically inhibiting STAT3 on innate antiviral immunity are not well known. Capsaicin, approved for the treatment of postherpetic neuralgia and diabetic peripheral nerve pain, is an agonist of transient receptor potential vanilloid subtype 1 (TRPV1), with additional recognized potencies in anticancer, anti-inflammatory, and metabolic diseases. We investigated the effects of capsaicin on viral replication and innate antiviral immune response and discovered that capsaicin dose-dependently inhibited the replication of VSV, EMCV, and H1N1. In VSV-infected mice, pretreatment with capsaicin improved the survival rate and suppressed inflammatory responses accompanied by attenuated VSV replication in the liver, lung, and spleen. The inhibition of viral replication by capsaicin was independent of TRPV1 and occurred mainly at postviral entry steps. We further revealed that capsaicin directly bound to STAT3 protein and selectively promoted its lysosomal degradation. As a result, the negative regulation of STAT3 on the type I IFN response was attenuated, and host resistance to viral infection was enhanced. Our results suggest that capsaicin is a promising small-molecule drug candidate, and offer a feasible pharmacological strategy for strengthening host resistance to viral infection.

Bergapten inhibits NLRP3 inflammasome activation and pyroptosis via promoting mitophagy.

Inhibition of NLRP3 inflammasome activation produces potent therapeutic effects in a wide array of inflammatory diseases. Bergapten (BeG), a furocoumarin phytohormone present in many herbal medicines and fruits, exibits anti-inflammatory activity. In this study we characterized the therapeutic potential of BeG against bacterial infection and inflammation-related disorders, and elucidated the underlying mechanisms. We showed that pre-treatment with BeG (20 µM) effectively inhibited NLRP3 inflammasome activation in both lipopolysaccharides (LPS)-primed J774A.1 cells and bone marrow-derived macrophages (BMDMs), evidenced by attenuated cleaved caspase-1 and mature IL-1ß release, as well as reduced ASC speck formation and subsequent gasdermin D (GSDMD)-mediated pyroptosis. Transcriptome analysis revealed that BeG regulated the expression of genes involved in mitochondrial and reactive oxygen species (ROS) metabolism in BMDMs. Moreover, BeG treatment reversed the diminished mitochondrial activity and ROS production after NLRP3 activation, and elevated the expression of LC3-II and enhanced the co-localization of LC3 with mitochondria. Treatment with 3-methyladenine (3-MA, 5 mM) reversed the inhibitory effects of BeG on IL-1ß, cleaved caspase-1 and LDH release, GSDMD-N formation as well as ROS production. In mouse model of Escherichia coli-induced sepsis and mouse model of Citrobacter rodentium-induced intestinal inflammation, pre-treatment with BeG (50 mg/kg) significantly ameliorated tissue inflammation and injury. In conclusion, BeG inhibits NLRP3 inflammasome activation and pyroptosis by promoting mitophagy and maintaining mitochondrial homeostasis. These results suggest BeG as a promising drug candidate for the treatment of bacterial infection and inflammation-related disorders.

Single cell landscape of parietal epithelial cells in healthy and diseased states.

The biology and diversity of glomerular parietal epithelial cells (PECs) are important for understanding podocyte regeneration and crescent formation. Although protein markers have revealed the morphological heterogeneity of PECs, the molecular characteristics of PEC subpopulations remain largely unknown. Here, we performed a comprehensive analysis of PECs using single-cell RNA sequencing (scRNA-seq) data. Our analysis identified five distinct PEC subpopulations: PEC-A1, PEC-A2, PEC-A3, PEC-A4 and PEC-B. Among these subpopulations, PEC- A1 and PEC-A2 were characterized as podocyte progenitors while PEC-A4 represented tubular progenitors. Further dynamic signaling network analysis indicated that activation of PEC-A4 and the proliferation of PEC-A3 played pivotal roles in crescent formation. Analyses suggested that upstream signals released by podocytes, immune cells, endothelial cells and mesangial cells serve as pathogenic signals and may be promising intervention targets in crescentic glomerulonephritis. Pharmacological blockade of two such pathogenic signaling targets, proteins Mif and Csf1r, reduced hyperplasia of the PECs and crescent formation in anti-glomerular basement membrane glomerulonephritis murine models. Thus, our study demonstrates that scRNA-seq-based analysis provided valuable insights into the pathology and therapeutic strategies for crescentic glomerulonephritis.

Scoparone suppresses mitophagy-mediated NLRP3 inflammasome activation in inflammatory diseases.

Recent evidence shows that targeting NLRP3 inflammasome activation is an important means to treat inflammasome-driven diseases. Scoparone, a natural compound isolated from the Chinese herb Artemisia capillaris Thunb, has anti-inflammatory activity. In this study we investigated the effect of scoparone on NLRP3 inflammasome activation in inflammatory diseases. In LPS-primed, ATP or nigericin-stimulated mouse macrophage J774A.1 cells and bone marrow-derived macrophages (BMDMs), pretreatment with scoparone (50 µM) markedly restrained canonical and noncanonical NLRP3 inflammasome activation, evidenced by suppressed caspase-1 cleavage, GSDMD-mediated pyroptosis, mature IL-1ß secretion and the formation of ASC specks. We then conducted a transcriptome analysis in scoparone-pretreated BMDMs, and found that the differentially expressed genes were significantly enriched in mitochondrial reactive oxygen species (ROS) metabolic process, mitochondrial translation and assembly process, as well as in inflammatory response. We demonstrated in J774A.1 cells and BMDMs that scoparone promoted mitophagy, a well-characterized mechanism to control mitochondrial quality and reduce ROS production and subsequent NLRP3 inflammasome activation. Mitophagy blockade by 3-methyladenine (3-MA, 5 mM) reversed the protective effects of scoparone on mitochondrial damage and inflammation in the murine macrophages. Moreover, administration of scoparone (50 mg/kg) exerted significant preventive effects via inhibition of NLRP3 activation in mouse models of bacterial enteritis and septic shock. Collectively, scoparone displays potent anti-inflammatory effects via blocking NLRP3 inflammasome activation through enhancing mitophagy, highlighting a potential action mechanism in treating inflammasome-related diseases for further clinical investigation.

Can eastern wisdom resolve western epidemics? Traditional Chinese medicine therapies and the opioid crisis.

The widespread use of opioids to treat chronic pain led to a nation-wide crisis in the United States. Tens of thousands of deaths annually occur mainly due to respiratory depression, the most dangerous side effect of opioids. Non-opioid drugs and non-pharmacological treatments without addictive potential are urgently required. Traditional Chinese medicine (TCM) is based on a completely different medical theory than academic Western medicine. The scientific basis of acupuncture and herbal treatments as main TCM practices has been considerably improved during the past two decades, and large meta-analyses with thousands of patients provide evidence for their efficacy. Furthermore, opinion leaders in the United States favor non-pharmacological techniques including TCM for pain management to fight the opioid crisis. We advocate TCM as therapeutic option without addictive potential and without life-threatening side effects (e.g., respiratory depression) to treat chronic pain patients suffering from opioid misuse. The evidence suggests that: (1) opioid misuse cannot be satisfactorily managed with standard medication; (2) opinion leaders in the United States favor to consider non-opioid and non-pharmacological treatment strategies including those from TCM to treat acute and chronic pain conditions; (3) large meta-analyses provide scientific evidence for the clinical activity of acupuncture and herbal TCM remedies in the treatment of chronic pain. Future clinical trials should demonstrate the safety of TCM treatments if combined with Western medical practices to exclude negative interactions between both modalities.

In vitro screening and toxic mechanism exploring of leading components with potential hepatotoxicity of Herba Epimedii extracts.

Herba Epimedii is a famous Chinese edible herb, and due to its potential hepatotoxic effects, the safety associated with this herb has attracted a great deal of attention. In this study, the components of four types of the Herba Epimedii extracts were identified by HPLC-MS/MS. Among these components, 11 components that were present in all four extracts and could be obtained as reference substances were evaluated for their ability of cytotoxicity in HL-7702 and HepG2 cells, resulting in the identification of icarisid I and sagittatoside A as the most relevant with respect to the toxicity of the extracts. The targeted toxicological effects were further investigated using a series of correlated biological indicators to elucidate potentially hepatotoxic mechanisms. The results showed that the extracts and the selected compounds had varying degrees of influence on the leakage of ALT, AST and LDH; the activity of SOD, GSH and MDA; the increase in intercellular ROS; and the decrease in MMP. Among the tested substances, the ethanol extracts exhibited stronger hepatotoxicity, with icarisid I and sagittatoside A correlating with this toxic effect, and the hepatoxic mechanisms of which may be associated with damaged cell structure, increased oxidative stress and induction of apoptosis.

Combining the wisdoms of traditional medicine with cutting-edge science and technology at the forefront of medical sciences.

BACKGROUND: A central topic is to bring traditional medicine to a new horizon by integrating the latest advances in genomic, metabolomic, and system biological approaches, in order to re-examine the wisdom and knowledge of traditional Chinese medicine (TCM) and other traditional medicines. PURPOSE: A new consortium has been formed at a conference of the Harvard Medical School, Boston, on October 29-30, 2018. The main goal was to build a collaborative platform for the scientific investigation of traditional medicine with cutting edge sciences and technologies at the forefront of biomedicine. RESULTS: Traditional medicines are largely experience-based, but the scientific basis is largely non-satisfactory. Therefore, the transformation from experience-based to evidence-based medicine would be an important step forward. The consortium covers three main fields: TCM diagnostics, acupuncture and TCM pharmacology. Diseases occur because of regulatory imbalances of holistic physiological display and genetic information/expression related to systems biology and energy consumption/release (e.g. cold and hot) within body. As organs are interconnected by meridians, affecting the meridians by acupuncture and medicinal herbs restores healthy organ function and body balance. There are two concepts in herbal medicine: The traditional way is based on complex herbal mixtures. The second concept is related to Western pharmacological drug development including the isolation of bioactive phytochemicals, which are subjected to preclinical and clinical investigations. CONCLUSION: Development of collaborative scientific project to integrate the best of both worlds - Western and Eastern medicine into a "One World Integrative Medicine" for the sake of patients worldwide.

Synthesis, characterization, and anticancer activity of ruthenium(II)-ß-carboline complex.

Four [Ru(tpy)(N-N)(L)] type complexes: [Ru(tpy)(bpy)(Nh)](2+) (Ru1, tpy = 2,2';6',2″-terpyridine, bpy = 2'2-bipyridine, Nh = Norharman), [Ru(tpy)(phen)(Nh)](2+) (Ru2, phen = 1,10-phenanthroline), [Ru(tpy)(dpa)(Nh)](2+) (Ru3, dpa = 2,2'-dipyridylamine) and [Ru(tpy)(dip)(Nh)](2+) (Ru4, dip = 4,7-diphenyl-1,10-phenanthroline) were presented as anticancer drugs. In vitro cytotoxicity assays indicated that these complexes showed anticancer activity against various cancer cells. Flow cytometry and signaling pathways analysis demonstrated that these complexes induced apoptosis via the mitochondrial pathway, as evidenced by the loss of mitochondrial membrane potential and the release of cytochrome c. The resulting accumulation of p53 proteins from phosphorylation at serine-15 and serine-392 was correlated with an increase in p21 and caspase activation. Taken together, these findings suggested that Ru1-Ru4 may contribute to the future development of improved chemotherapeutics against human cancers.

A ruthenium(II) ß-carboline complex induced p53-mediated apoptosis in cancer cells.

A ruthenium(II) ß-carboline complex [Ru(tpy)(Nh)3](2+) (tpy = 2,2':6',2″-terpyridine, Nh = Norharman, Ru1) has been synthesized and characterized. This complex induced apoptosis against various cancer cell lines and had high selectivity between tumor cells and normal cells. In vivo examination indicated Ru1 decreased mouse MCF-7 and HepG2 tumor growth. Signaling pathways analysis demonstrated that this complex induced apoptosis via the mitochondrial pathway, as evidenced by the loss of mitochondrial membrane potential (MMP, ΔΨm) and the release of cytochrome c. The resulting accumulation of p53 proteins from phosphorylation at Ser-15 and Ser-392 correlated with an increase in p21 and caspase activation. Taken together, these findings suggest that Ru1 exhibits high and selective cytotoxicity induced p53-mediated apoptosis and may contribute to the future development of improved chemotherapeutics against human cancers.

[Construction of genome BAC library for single Branchiostoma belcheri individual].

As one of the closest living invertebrate relatives of vertebrates, amphioxus (subphylum Cephalochordata) occupies a key position in animal evolution and is becoming the best available proxy and model animal for studying the last common ancestor of all chordates, especially vertebrates. As long-term continuous culturing of amphioxus in laboratory became reliable, for pushing this animal to be a more successful model system, whole-genome sequencing of one or more species derived from this branch will be another urgent issue needed to address. In the present study, we described the construction and characterization of a bacterial artificial chromosome (BAC) library, using a single individual of Chinese amphioxus (Branchiostoma belcheri). High quality genomic DNA extracted from the spermary was partially digested with EcoRand EcoRmethylase. Desirable DNA fragments were isolated by pulsed field gel electrophoresis (PFGE), ligated to linearized and phosphorylased carrier pCC1BAC, and then transformed to Escherichia coli EPI300. The constructed library consists of 44 706 clones with the average insert fragment size around 80 kb as estimated by PFGE. The representation of the library is about 9 equivalents to the amhioxus genome. These results indicate that the BAC library will be useful for functional genomic studies and facilitate the whole-genome sequencing of Chinese amphioxus.

Tracing the origins of Hakka and Chaoshanese by mitochondrial DNA analysis.

Hakka and Chaoshanese are two unique Han populations residing in southern China but with northern Han (NH) cultural traditions and linguistic influences. Although most of historical records indicate that both populations migrated from northern China in the last two thousand years, no consensus on their origins has been reached so far. To shed more light on the origins of Hakka and Chaoshanese, mitochondrial DNAs (mtDNAs) of 170 Hakka from Meizhou and 102 Chaoshanese from Chaoshan area, Guangdong Province, were analyzed. Our results show that some southern Chinese predominant haplogroups, e.g. B, F, and M7, have relatively high frequencies in both populations. Although median network analyses show that Hakka/Chaoshanese share some haplotypes with NH, interpopulation comparison reveals that both populations show closer affinity with southern Han (SH) populations than with NH. In consideration of previous results from nuclear gene (including Y chromosome) research, it is likely that matrilineal landscapes of both Hakka and Chaoshanese have largely been shaped by the local people during their migration southward and/or later colonization in southern China, and factors such as cultural assimilation, patrilocality, and even sex-bias in the immigrants might have played important roles during the process.

[Flavonoids from Diospyros kaki inhibit the adhesion between lymphocyte and dorsal root ganglion].

OBJECTIVE: To investigate the effects of flavonoids from the leaves of Diospyros kaki L (FLDK) on the adhesion between the lymphocyte and the neurone. METHODS: Centrifugal assay for fluorescence-bsaed cell adhesion was used to assay the adhesion between the lymphocyte and the dorsal root ganglion (DRG). RESULTS: The adhesion was significantly suppressed in the presence of FLDK dose-dependently at 5, 25 microg/mL concentration. FLDK was also effective to inhibit the adhesion under the challenge of ICAM-1 by 28.5% and 50.1%, respectively. Furthermore, FLDK enforced the inhibition of anti-NCAM antibody on the lymphocyte adhesion to DRG cells. CONCLUSION: FLDK might contribute to the prevention and treatment of the inflammation injury under neuron insult such as ischemia/reperfusion, neurotrauma and other neurodegenerative disease by inhibiting the adhesion between lymphocytes and neurons.

Adenosine monophosphate-activated protein kinase inhibits cardiac hypertrophy through reactivating peroxisome proliferator-activated receptor-alpha signaling pathway.

The activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) has been shown to inhibit cardiac hypertrophy, however, the mechanism remains unclear. Rat models of cardiac hypertrophy were created with transaortic constriction (TAC) to investigate the mechanistic role of AMPK involved. RT-PCR and Western blot analyses indicated that hypertrophy marker genes ANP and beta-MHC expression were up-regulated in the myocardium of TAC rats. We also observed that the expressions of peroxisome proliferator-activated receptor-alpha (PPARalpha) and its target genes, carnitine palmitoyl transferase-capital I, Ukrainian (CPT-capital I, Ukrainian) and medium-chain acyl-COA dehydrogenases (MCAD), were down-regulated, and the fatty acid oxidation was decreased in TAC rats. Treatment of TAC animals with 5-aminoimidazole 1 carboxamide ribonucleoside (AICAR, 0.5 mg/g body wt), a specific activator of AMPK, inhibited cardiac hypertrophy in TAC and reversed PPARalpha, CPT-I and MCAD expression and fatty acid oxidation. Similar observations were made in hypertrophied cardiomyocytes induced by phenylephrine in vitro. Treatment of hypertrophied cardiomyocytes with Compound C, a specific AMPK inhibitor, showed an effect opposite to that of AICAR. The effect of AICAR on cardiac hypertrophy was blocked after PPARalpha was silenced by transfection of cardiomyocytes with PPARalpha-siRNA. Luciferase activity assay suggested that AICAR elevates PPARalpha transcriptional activity. These results indicate that AMPK plays an important role in the inhibition of cardiac hypertrophy by activating the PPARalpha signaling pathway.

Recombinant interferon-alpha2b poly(lactic-co-glycolic acid) microspheres: pharmacokinetics-pharmacodynamics study in rhesus monkeys following intramuscular administration.

AIM: Investigation into pharmacokinetic-pharmacodynamic properties of interferon- alpha (IFN-alpha)2b-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) in rhesus monkey primates. METHOD: IFN-alpha2b was loaded with biodegradable PLGA with 3 inherent viscosities using a double emulsion and solvent evaporation method. The particle size, surface morphology, and in vitro release profiles were investigated. Two groups of rhesus monkeys (n=3) were injected intramuscularly with either 3 MIU/kg commercial IFN-alpha2b lyophilized powder or IFN-alpha2b-loaded PLGA microspheres (inherent viscosity of 0.89 dL/g). In vitro release was determined by Lowry protein assay. The serum IFN and neopterin levels were determined by the enzyme-linked immunosorbent assay (ELISA) method to evaluate biological activity of the microspheres in rhesus monkeys. RESULTS: The IFN-alpha2b microspheres with 3 inherent viscosities (0.39, 0.89, and 1.13 dL/g) were entirely spherical and had a smooth surface. The average diameter of each type was 45.55, 81.23, and 110.25 microm, respectively. The in vitro release was 30 d. The pharmacokinetic-pharmacodynamic properties between the IFN-alpha2b microspheres and IFN-alpha2b lyophilized powder were significantly different (P<0.05). CONCLUSION: The drug residence time for the IFN-alpha2b of the PLGA microsphere with an inherent viscosity of 0.89 dL/g in plasma significantly increased and had a longer time of biological effects in rhesus monkeys following intramuscular administration.

Role of P-glycoprotein in the intestinal absorption of glabridin, an active flavonoid from the root of Glycyrrhiza glabra.

Glabridin is a major constituent of the root of Glycyrrhiza glabra, which is commonly used in the treatment of cardiovascular and central nervous system diseases. This study aimed to investigate the role of P-glycoprotein (PgP/MDR1) in the intestinal absorption of glabridin. The systemic bioavailability of glabridin was approximately 7.5% in rats, but increased when combined with verapamil. In single-pass perfused rat ileum with mesenteric vein cannulation, the permeability coefficient of glabridin based on drug disappearance in luminal perfusates (P(lumen)) was approximately 7-fold higher than that based on drug appearance in the blood (P(blood)). Glabridin was mainly metabolized by glucuronidation, and the metabolic capacity of intestine microsomes was 1/15 to 1/20 of that in liver microsomes. Polarized transport of glabridin was found in Caco-2 and MDCKII monolayers. Addition of verapamil in both apical (AP) and basolateral (BL) sides abolished the polarized transport of glabridin across Caco-2 cells. Incubation of verapamil significantly altered the intracellular accumulation and efflux of glabridin in Caco-2 cells. The transport of glabridin in the BL-AP direction was significantly higher in MDCKII cells overexpressing PgP/MDR1 than in the control cells. Glabridin inhibited PgP-mediated transport of digoxin with an IC(50) value of 2.56 microM, but stimulated PgP/MDR1 ATPase activity with a K(m) of 25.1 microM. The plasma AUC(0-24h) of glabridin in mdr1a(-/-) mice was 3.8-fold higher than that in wild-type mice. These findings indicate that glabridin is a substrate for PgP and that both PgP/MDR1-mediated efflux and first-pass metabolism contribute to the low oral bioavailability of glabridin.

St. John's wort attenuates irinotecan-induced diarrhea via down-regulation of intestinal pro-inflammatory cytokines and inhibition of intestinal epithelial apoptosis.

Diarrhea is a common dose-limiting toxicity associated with cancer chemotherapy, in particular for drugs such as irinotecan (CPT-11), 5-fluouracil, oxaliplatin, capecitabine and raltitrexed. St. John's wort (Hypericum perforatum, SJW) has anti-inflammatory activity, and our preliminary study in the rat and a pilot study in cancer patients found that treatment of SJW alleviated irinotecan-induced diarrhea. In the present study, we investigated whether SJW modulated various pro-inflammatory cytokines including interleukins (IL-1beta, IL-2, IL-6), interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) and intestinal epithelium apoptosis in rats. The rats were treated with irinotecan at 60 mg/kg for 4 days in combination with oral SJW or SJW-free control vehicle at 400 mg/kg for 8 days. Diarrhea, tissue damage, body weight loss, various cytokines including IL-1beta, IL-2, IL-6, IFN-gamma and TNF-alpha and intestinal epithelial apoptosis were monitored over 11 days. Our studies demonstrated that combined SJW markedly reduced CPT-11-induced diarrhea and intestinal lesions. The production of pro-inflammatory cytokines such as IL-1beta, IFN-gamma and TNF-alpha was significantly up-regulated in intestine. In the mean time, combined SJW significantly suppressed the intestinal epithelial apoptosis induced by CPT-11 over days 5-11. In particular, combination of SJW significantly inhibited the expression of TNF-alpha mRNA in the intestine over days 5-11. In conclusion, inhibition of pro-inflammatory cytokines and intestinal epithelium apoptosis partly explained the protective effect of SJW against the intestinal toxicities induced by irinotecan. Further studies are warranted to explore the potential for STW as an agent in combination with chemotherapeutic drugs to lower their dose-limiting toxicities.

Reversal of resistance to oxazaphosphorines.

The oxazaphosphorines including cyclophosphamide (CPA), ifosfamide (IFO) and trofosfamide are one important group of alkylating agents. However, resistance is the major hindrance for success of oxazaphosphorine chemotherapy. The mechanism of resistance to oxazaphosphorines is not fully identified, but recently some novel insights into these aspects have been generated by using sensitive analytical techniques and powerful pharmacogenetic techniques. Potential mechanisms for oxazaphosphorine resistance include decreased activation by cytochrome P450s (e.g. CYP3A4, CYP2C9 and CYP2B6), increased deactivation of the agents by deactivating enzymes such as aldehyde dehydrogenases (ALDHs), increased cellular thiol level, increased DNA repair capacity, and altered cellular apoptotic response to DNA repair, e.g. deficient apoptosis due to lack of cellular mechanisms to result in cell death following DNA damage. In addition, decreased cellular accumulation of cytotoxic species of oxazaphosphorines may also play a role in the resistance. This review highlights the pharmacology of oxazaphosphorine anticancer drugs and possible agents that reverse the resistance to these agents. Possible agents that can overcome oxazaphosphorine resistance include inducers of CYPs, modulators of GSTs and ALDHs, modulators of DNA repair process, antisense oligonucleotides against genes encoding various enzymes and signalling proteins, and novel gene delivery systems. Most of these agents have been investigated in preclinical studies and promising results have been observed. To date, several types of these agents are being evaluated in Phase III trials in cancer patients. Further studies are needed to identify the molecular targets associated with resistance to oxazaphosphorines.