Although the human genome encodes ∼ 20,000 protein-coding genes, only a very small fraction of these have been explored as potential targets for therapeutic development. The challenge of identifying and validating new protein targets has contributed to the significant reduction in the productivity of the pharmaceutical industry in the recent decade, highlighting the continued need to find new therapeutic targets.The traditional methods to discover new targets are expensive, low throughput and time consuming, usually taking years to validate or invalidate a target. To address these limitations, as a proof of concept, we explored the hydrodynamic tail vein (HTV) injection as a gene delivery method for direct in vivo phenotypic screening of novel secreted factor targets for Type II diabetes therapeutics.High levels and sustained expression of target proteins were observed in diabetic mouse models tested, allowing us to identify multiple novel hormones that may regulate glucose metabolism.These results suggest that HTV is a low-cost, high-throughput method for direct in vivo phenotypic drug screening in metabolic disorders and could be applicable to many other disease areas as well. This method if combined with other approaches such as human genetic studies could provide a significant value to future drug discovery.
Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of both community- and hospital-associated infections. The antibiotic resistance and virulence characteristics of MRSA are largely regulated by two-component signal transduction systems (TCS) including the graRS TCS. To make a relatively comprehensive insight into graRS TCS in MRSA, the bioinformatics analysis of dataset GSE26016 (a S. aureus HG001 WT strain vs. the Δ graRS mutant) from Gene Expression Omnibus (GEO) database was performed, and a total of 563 differentially expressed genes (DEGs) were identified. GO analysis revealed that the DEGs were mainly enriched in the “ de novo ” IMP biosynthetic process, lysine biosynthetic process via diaminopimelate, and pathogenesis; and they were mainly enriched in purine metabolism, lysine biosynthesis, and monobactam biosynthesis in KEGG analysis. WGCNA suggested that the turquoise module was related to the blue module, and the genes in these two modules were associated with S. aureus virulence and infection. To investigate the role of graRS in bacterial virulence, a graRS knockout mutant (Δ graRS ) was constructed using MRSA USA500 2,395 strain as a parent strain. Compared to the wild-type strain, the USA500Δ graRS showed reduced staphyloxanthin production, retarded coagulation, weaker hemolysis on blood agar plates, and a decreased biofilm formation. These altered phenotypes were restored by the complementation of a plasmid-expressed graRS . Meanwhile, an expression of the virulence-associated genes ( coa , hla , hlb , agrA , and mgrA ) was downregulated in the Δ graRS mutant. Consistently, the A549 epithelial cells invasion of the Δ graRS mutant was 4-fold lower than that of the USA500 wild-type strain. Moreover, on the Galleria mellonella infection model, the survival rate at day 5 post infection in the USA500Δ graRS group (55%) was obviously higher than that in the USA500 group (20%), indicating graRS knockout leads to a decreased virulence in vivo . In addition, the deletion of the graRS in the MRSA USA500 strain resulted in its increased susceptibilities to ampicillin, oxacillin, vancomycin, and gentamicin. Our work suggests that the graRS TCS plays an important role in regulating S. aureus virulence in vitro and in vivo and modulate bacterial resistance to various antibiotics.
Deep brain stimulation (DBS) is a neuromodulation method that modulates neuronal activity. A trend in the treatment of Alzheimer's disease (AD) is targeting key points of neural circuits with DBS. Here, we explored the effects of DBS targeted to the entorhinal cortex (EC) on neurons in the hippocampal CA1 in a mouse model of preclinical AD. Specifically, we recorded field potential signals from CA1 in preclinical AD mice after DBS of the EC (1 h/day for 21 days of 100 μA, 90 μs, 10 Hz, biphasic square wave pulse) with in-vivo electrophysiology and evaluated corresponding changes in behavior with the open field task and Morris water maze (MWM) task. We also assessed changes in pathological markers and neurogenesis in the hippocampus with immunohistological staining. DBS of the EC increased theta and gamma power and modulated theta in the high gamma band (50–100 Hz) in preclinical AD mice. After DBS of the EC, these mice performed better in the MWM task and exhibited reduced deposition of beta-amyloid and neuronal changes including significant increases in proliferating neurons and immature neurons. This is the first study to target the EC with DBS and analyze resulting neural oscillations in the hippocampal CA1 in a model of preclinical AD. The findings support the use of DBS as a potential treatment for AD.
Background: In vivo fluorescence imaging in the second near-infrared (NIR-II, 1000-1700 nm) window using organic fluorophores has great advantages, but generally suffers from a relatively low fluorescence quantum yield (mostly less than 2%). In this study, organic nanoparticles (L1013 NPs) with a high fluorescence quantum yield (9.9%) were systhesized for in vivo imaging. Methods: A molecule (BTPPA) with donor-acceptor-donor structure and aggregation-induced emission enabling moieties was prepared. BTPPA molecules were then encapsulated into nanoparticles (L1013 NPs) using a nanoprecipitation method. The L1013 NPs were intravenously injected into the mice (including normal, stroke and tumor models) for vascular and tumor imaging. Results: L1013 NPs excited at 808 nm exhibit NIR-II emission with a peak at 1013 nm and an emission tail extending to 1400 nm. They have a quantum yield of 9.9% and also show excellent photo/colloidal stabilities and negligible in vitro and in vivo toxicity. We use L1013 NPs for noninvasive real-time visualization of mouse hindlimb and cerebral vessels (including stroke pathology) under a very low power density (4.6-40 mW cm‒2) and short exposure time (40-100 ms). Moreover, L1013 NPs are able to localize tumor pathology, with a tumor-to-normal tissue ratio of 11.7±1.3, which is unusually high for NIR-II fluorescent imaging through passive targeting strategy. Conclusion: L1013 NPs demonstrate the potential for a range of clinical applications, especially for tumor surgery.
GPR81 is an orphan G protein-coupled receptor (GPCR) that has a high degree of homology to the nicotinic acid receptor GPR109A. GPR81 expression is highly enriched and specific in adipocytes. However, the function and signaling properties of GPR81 are unknown because of the lack of natural or synthetic ligands. Using chimeric G proteins that convert Gi-coupled receptors to Gq-mediated inositol phosphate (IP) accumulation, we show that GPR81 can constitutively increase IP accumulation in HEK293 cells and suggest that GPR81 couples to the Gi signaling pathway. We also constructed a chimeric receptor that expresses the extracellular domains of cysteinyl leukotriene 2 receptor (CysLT2R) and the intracellular domains of GPR81. We show that the CysLT2R ligand, leukotriene D4 (LTD4), is able to activate this chimeric receptor through activation of the Gi pathway. In addition, LTD4 is able to inhibit lipolysis in adipocytes expressing this chimeric receptor. These results suggest that GPR81 couples to the Gi signaling pathway and that activation of the receptor may regulate adipocyte function and metabolism. Hence, targeting GPR81 may lead to the development of a novel and effective therapy for dyslipidemia and a better side effect profile than nicotinic acid. GPR81 is an orphan G protein-coupled receptor (GPCR) that has a high degree of homology to the nicotinic acid receptor GPR109A. GPR81 expression is highly enriched and specific in adipocytes. However, the function and signaling properties of GPR81 are unknown because of the lack of natural or synthetic ligands. Using chimeric G proteins that convert Gi-coupled receptors to Gq-mediated inositol phosphate (IP) accumulation, we show that GPR81 can constitutively increase IP accumulation in HEK293 cells and suggest that GPR81 couples to the Gi signaling pathway. We also constructed a chimeric receptor that expresses the extracellular domains of cysteinyl leukotriene 2 receptor (CysLT2R) and the intracellular domains of GPR81. We show that the CysLT2R ligand, leukotriene D4 (LTD4), is able to activate this chimeric receptor through activation of the Gi pathway. In addition, LTD4 is able to inhibit lipolysis in adipocytes expressing this chimeric receptor. These results suggest that GPR81 couples to the Gi signaling pathway and that activation of the receptor may regulate adipocyte function and metabolism. Hence, targeting GPR81 may lead to the development of a novel and effective therapy for dyslipidemia and a better side effect profile than nicotinic acid. cysteinyl leukotriene 2 receptor G protein-coupled receptor inositol phosphate leukotriene D4 pertussis toxin Nicotinic acid has been used for the treatment of dyslipidemia for >50 years. The drug improves multiple cardiovascular risk factors, including increase of HDL and reduction of VLDL, LDL, lipoprotein [a], and triglycerides (TGs), that overall result in a reduction in mortality (1.Carlson L.A. Nicotinic acid: the broad-spectrum lipid drug. A 50th anniversary review.J. Intern. Med. 2005; 258: 94-114Crossref PubMed Scopus (503) Google Scholar). The receptor for nicotinic acid, termed GPR109A (HM74A in humans and PUMA-G in mice), identified in recent years couples to G proteins of the Gi family and is expressed mainly in adipocytes and immune cells (2.Wise A. Foord S.M. Fraser N.J. Barnes A.A. Elshourbagy N. Eilert M. Ignar D.M. Murdock P.R. Steplewski K. Green A. et al.Molecular identification of high and low affinity receptors for nicotinic acid.J. Biol. Chem. 2003; 278: 9869-9874Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 3.Tunaru S. Kero J. Schaub A. Wufka C. Blaukat A. Pfeffer K. Offermanns S. PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect.Nat. Med. 2003; 9: 352-355Crossref PubMed Scopus (657) Google Scholar, 4.Soga T. Kamohara M. Takasaki J. Matsumoto S. Saito T. Ohishi T. Hiyama H. Matsuo A. Matsushime H. Furuichi K. Molecular identification of nicotinic acid receptor.Biochem. Biophys. Res. Commun. 2003; 303: 364-369Crossref PubMed Scopus (285) Google Scholar). Although the precise mechanism of action for nicotinic acid is unknown, GPR109A-mediated inhibition of lipolysis in adipocytes, which results in a reduction in plasma FFA levels, is postulated to play an important role in the improvement of plasma lipid parameters (5.Pike N.B. Wise A. Identification of a nicotinic acid receptor: is this the molecular target for the oldest lipid-lowering drug?.Curr. Opin. Investig. Drugs. 2004; 5: 271-275PubMed Google Scholar, 6.Offermanns S. The nicotinic acid receptor GPR109A (HM74A or PUMA-G) as a new therapeutic target.Trends Pharmacol. Sci. 2006; 27: 384-390Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Changes in plasma FFA levels may also lead to changes in the peripheral tissue responsiveness to insulin as well. There is an observed correlation between increased plasma FFA levels and type 2 diabetes as well as an increased risk of type 2 diabetes in individuals with high plasma FFA levels. Furthermore, increased plasma FFA levels can lead directly to the inhibition of insulin-dependent glucose disposal, increased hepatic gluconeogenesis, and impaired pancreatic islet functions (7.Wang M. Fotsch C. Small-molecule compounds that modulate lipolysis in adipose tissue: targeting strategies and molecular classes.Chem. Biol. 2006; 13: 1019-1027Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Indeed, it has been observed that short-term treatment with nicotinic acid and its analogs improves insulin action (6.Offermanns S. The nicotinic acid receptor GPR109A (HM74A or PUMA-G) as a new therapeutic target.Trends Pharmacol. Sci. 2006; 27: 384-390Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), while sustained reduction in plasma FFAs by treatment with the nicotinic acid analog acipimox improves the clinical course of type 2 diabetes (8.Worm D. Henriksen J.E. Vaag A. Thye-Ronn P. Melander A. Beck-Nielsen H. Pronounced blood glucose-lowering effect of the antilipolytic drug acipimox in noninsulin-dependent diabetes mellitus patients during a 3-day intensified treatment period.J. Clin. Endocrinol. Metab. 1994; 78: 717-721Crossref PubMed Scopus (0) Google Scholar). Therefore, targeting fat tissue and regulating adipocyte lipolysis may be a general strategy to regulate lipid and glucose homeostasis. Despite its great efficacy in improving plasma lipid parameters, nicotinic acid treatment results in a number of undesirable side effects, including flushing in the face and upper body and gastrointestinal upset. Recently, it was reported that in rodents the activation of GPR109A in the epidermal Langerhans cells via the release of prostaglandins mediates the flushing side effect of nicotinic acid (9.Pike N.B. Flushing out the role of GPR109A (HM74A) in the clinical efficacy of nicotinic acid.J. Clin. Invest. 2005; 115: 3400-3403Crossref PubMed Scopus (73) Google Scholar, 10.Benyo Z. Gille A. Kero J. Csiky M. Suchankova M.C. Nusing R.M. Moers A. Pfeffer K. Offermanns S. GPR109A (PUMA-G/HM74A) mediates nicotinic acid-induced flushing.J. Clin. Invest. 2005; 115: 3634-3640Crossref PubMed Scopus (286) Google Scholar). A number of different approaches have been taken to alleviate the side effects, including the use of an extended-release formulation (1.Carlson L.A. Nicotinic acid: the broad-spectrum lipid drug. A 50th anniversary review.J. Intern. Med. 2005; 258: 94-114Crossref PubMed Scopus (503) Google Scholar), receptor-selective agonists/modulators that only activate the antilipolytic pathway while avoiding the activation of the immune-related flush-inducing pathway (11.Richman J.G. Kanemitsu-Parks M. Gaidarov I. Cameron J.S. Griffin P. Zheng H. Guerra N.C. Cham L. Maciejewski-Lenoir D. Behan D.P. et al.Nicotinic acid receptor agonists differentially activate downstream effectors.J. Biol. Chem. 2007; 282: 18028-18036Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), and combination treatment of nicotinic acid with prostaglandin D2 receptor 1 antagonists (12.Cheng K. Wu T.J. Wu K.K. Sturino C. Metters K. Gottesdiener K. Wright S.D. Wang Z. O'Neill G. Lai E. et al.Antagonism of the prostaglandin D2 receptor 1 suppresses nicotinic acid-induced vasodilation in mice and humans.Proc. Natl. Acad. Sci. USA. 2006; 103: 6682-6687Crossref PubMed Scopus (264) Google Scholar). Another alternative strategy is to investigate other adipocyte Gi-coupled G protein-coupled receptors (GPCRs) that are either not expressed in immune cells or whose activation does not induce flushing. GPR81 is an orphan GPCR that belongs to the same subfamily of receptors as GPR109A and GPR109B (13.Lee D.K. Nguyen T. Lynch K.R. Cheng R. Vanti W.B. Arkhitko O. Lewis T. Evans J.F. George S.R. O'Dowd B.F. Discovery and mapping of ten novel G protein-coupled receptor genes.Gene. 2001; 275: 83-91Crossref PubMed Scopus (165) Google Scholar). GPR81 shares ∼51% sequence identity with GPR109A and is colocalized in humans on the same chromosome, 12q24.31, as GPR109A and GPR109B (6.Offermanns S. The nicotinic acid receptor GPR109A (HM74A or PUMA-G) as a new therapeutic target.Trends Pharmacol. Sci. 2006; 27: 384-390Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). GPR81 expression was found to be highest in adipose tissue among a number of human tissues tested (2.Wise A. Foord S.M. Fraser N.J. Barnes A.A. Elshourbagy N. Eilert M. Ignar D.M. Murdock P.R. Steplewski K. Green A. et al.Molecular identification of high and low affinity receptors for nicotinic acid.J. Biol. Chem. 2003; 278: 9869-9874Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Because no ligands have been described for GPR81, the signaling pathway for receptor activation as well as the functions of the receptor are largely unknown. Here, using chimeric G proteins as well as chimeric cysteinyl leukotriene 2 receptor (CysLT2R) and GPR81 receptors, we demonstrate that GPR81 couples to Gi pathways for receptor activation. Furthermore, we show that activation of our chimeric receptor by leukotriene D4 (LTD4) results in the inhibition of lipolysis in adipocytes. These results suggest that activation of adipocyte-specific GPR81 has the potential to promote a similar positive clinical outcome as nicotinic acid treatment, but without the skin-mediated flushing side effect. GPR81 expression in tissues and differentiated adipocytes was analyzed using quantitative PCR. Mouse RNA was from Ambion FirstChoice Total RNA: Mouse Normal Tissue Survey Panel (catalog No. AM7800). Mouse 3T3-L1 preadipocytes were cultured and differentiated into adipocytes as described previously (14.Lee G. Elwood F. McNally J. Weiszmann J. Lindstrom M. Amaral K. Nakamura M. Miao S. Cao P. Learned R.M. et al.T0070907, a selective ligand for peroxisome proliferator-activated receptor gamma, functions as an antagonist of biochemical and cellular activities.J. Biol. Chem. 2002; 277: 19649-19657Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The primers used for GPR81 were 5′-TCTTCCTGCCCCTGACAATC-3′, 5′-CCGTCTCAGGCTCCAAACA-3′, and 5′-6FAM-TCTTGTTCTGCTCGGTCAACG-BHQ1-3′; the primers for CysLT2R/GPR81 were 5′-CAACCATCCATCTCCGTATCAG-3′, 5′-TTGTGCAGTTCCTGCTGTTGT-3′, and 5′-6FAM-AATGGAACCAAATGGCACCTTCAGCAAT-BHQ1-3′; the primers for GPR109A were from ABI (catalog No. Mm02620500 s1); and the primers for GAPDH were from ABI (catalog No. 4352932E). Quantitative PCR was performed on Stratagene Mx3000P quantitative PCR machines with the Stratagene Brilliant QRT-PCR Master Mix Kit, 1-Step (catalog No. 600551) using 100 ng RNA/well and normalized with mouse GAPDH (ABI). HEK293 cells were obtained from the American Type Culture Collection. Yttrium silicate scintillation proximity assay beads were obtained from Amersham. Tritiated inositol (50 to 80 Ci/mmol) was obtained from Amersham. Chimeric G proteins, Gα15, Gα16, aequorin, and GPR81, were cloned by PCR using the published sequence data (15.Coward P. Chan S.D. Wada H.G. Humphries G.M. Conklin B.R. Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors.Anal. Biochem. 1999; 270: 242-248Crossref PubMed Scopus (205) Google Scholar) and inserted into the plasmid pcDNA3.1 or pZeo (Gα16). HEK293 cells were dispensed onto a poly-d-lysine tissue culture-treated 96-well plate at a density of 25,000 cells/well. The next day, the cells (∼80–90% confluent) were transfected with 100 ng of GPR81 and 20 ng of G protein (5:1 ratio) or control vector containing the aequorin gene using Lipofectamine2000 according to the manufacturer's instructions. Six hours after transfection, the medium was replaced with inositol-free DMEM/10% dialyzed FCS supplemented with 1 μCi/ml tritiated inositol. After incubation overnight, the medium was replaced with HBSS/0.01% BSA containing 10 mM LiCl and incubated at 37°C for 1 h. The medium was aspirated, and the cells were fixed with ice-cold 20 mM formic acid. After incubation at 4°C for 5 h, the lysates were added to yttrium silicate scintillation proximity assay beads, allowed to settle overnight, and read on a Beckman TopCount scintillation counter. The extracellular, transmembrane, and intracellular domains for CysLT2R and GPR81 were determined using the TMHMM2.0 software package (Invitrogen, Vector NTI). The chimeric receptor construct consisted of the N terminus, all three extracellular loops, and all seven transmembrane domains from CysLT2R and all three intracellular loops and the C terminus from GPR81. The CysLT2R/GPR81 chimera was synthesized by Blue Heron Biotechnology (Bothell, WA) using GeneMaker technology according to the sequence specified. The chimeric receptor was then amplified using PCR and oligonucleotide primers 5′-CACCATGGAGAGAAAATTTATGTCCTTGC-3′ and 5′-AGCTTCTAGATCAGTGCCACTCAACAATGTGGGGA-3′ and subcloned into pEF6/V5-His-TOPO vector (Invitrogen). CHO cells were transfected with CysLT2R/GPR81 DNA, Aeq DNA, and Gqi9 DNA using Lipofectamine2000 (Invitrogen). Vector plasmid, GPR81, and CysLTR2 DNA were used as controls. A total of 200 ng/ml pertussis toxin (PTX; Calbiochem) was added as indicated to the medium to treat the cells for 18 h. One day after transfection, the cells were resuspended in 20 ml of HBSS buffer containing 2.3 μM coelenterazine F. The cells were labeled for 2 h in the dark with periodic agitation. Aequorin luminescence resulting from intracellular calcium mobilization upon the addition of LTD4 was measured using the microplate luminometer (EG&G Bertholt, Gaithersburg, MD). CysLT2R/GPR81 chimeric receptor was cloned into the KpnI and FseI sites of aP2-pMCS5 transgenic vector (16.Ross S.R. Graves R.A. Greenstein A. Platt K.A. Shyu H.L. Mellovitz B. Spiegelman B.M. A fat-specific enhancer is the primary determinant of gene expression for adipocyte P2 in vivo.Proc. Natl. Acad. Sci. USA. 1990; 87: 9590-9594Crossref PubMed Scopus (184) Google Scholar). The SphI-to-AscI fragment containing the aP2 promoter and the CysLT2R/GPR81 chimeric receptor excised from the vector was gel-purified and injected into the pronuclear embryos of FVB mice. Injected embryos were transferred into the oviducts of pseudopregnant CD1 female mice. Transgenic founder mice were identified by SV40pA sequence-specific PCR genotyping using primers FWD (5′-GATGAGTTTGGACAAACCACA-3′) and REV (5′-CCGGATCATAATCAGCCATAC-3′). One founder, which gave the highest level of chimeric receptor expression, was chosen for all experiments described in this report. All animal experiments were approved by the Institutional Animal Care and Use Committee of Amgen. Adipocytes were released from epididymal fat pads of male CysLT2R/GPR81 transgenic mice or wild-type mice by collagenase (Sigma catalog No. C2674) digestion similar to previously described methods (3.Tunaru S. Kero J. Schaub A. Wufka C. Blaukat A. Pfeffer K. Offermanns S. PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect.Nat. Med. 2003; 9: 352-355Crossref PubMed Scopus (657) Google Scholar, 17.Rodbell M. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis.J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google Scholar). Cells were then washed four times with KRB buffer (Sigma catalog No. K4002) with 3% fatty acid-free BSA (Sigma) and 1 U/ml adenosine deaminase (Biocatalytics, Inc.; ADA-101). The primary adipocytes harvested from 15 mice were plated onto one 24-well plate and then incubated at 37°C with mild shaking in the presence or absence of LTD4. Nicotinic acid was added as a positive control. Aliquots were collected from the centers of the wells hourly for glycerol assay using Free Glycerol Reagent (Sigma catalog No. F6428). An earlier report suggests that GPR81 expression is fairly restricted to adipocytes in humans (2.Wise A. Foord S.M. Fraser N.J. Barnes A.A. Elshourbagy N. Eilert M. Ignar D.M. Murdock P.R. Steplewski K. Green A. et al.Molecular identification of high and low affinity receptors for nicotinic acid.J. Biol. Chem. 2003; 278: 9869-9874Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). To understand the potential physiological role of GPR81 and to confirm this restricted expression pattern, we analyzed GPR81 expression in a mouse tissue panel using quantitative PCR-based analysis. Our results show that, similar to the report for human tissue samples (2.Wise A. Foord S.M. Fraser N.J. Barnes A.A. Elshourbagy N. Eilert M. Ignar D.M. Murdock P.R. Steplewski K. Green A. et al.Molecular identification of high and low affinity receptors for nicotinic acid.J. Biol. Chem. 2003; 278: 9869-9874Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar), GPR81 was expressed mainly in the white adipose tissues and that relatively low levels were detected in other tissues (Fig. 1A). The expression analysis also revealed that GPR81 expression was markedly induced during the 3T3-L1 preadipocyte differentiation process in vitro (Fig. 1A). Because GPR81 and GPR109A share significant sequence homology, GPR109A expression was also analyzed from the same tissue panel as a comparison. Again, similar to the report for human tissues (2.Wise A. Foord S.M. Fraser N.J. Barnes A.A. Elshourbagy N. Eilert M. Ignar D.M. Murdock P.R. Steplewski K. Green A. et al.Molecular identification of high and low affinity receptors for nicotinic acid.J. Biol. Chem. 2003; 278: 9869-9874Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar), GPR109A expression was largely restricted to adipose tissue and spleen (Fig. 1B). GPR109A expression was also markedly induced during the 3T3-L1 preadipocyte differentiation process in vitro, although induction appeared earlier than for GPR81 (Fig. 1B).Fig. 1.Distribution of GPR81 transcripts in mouse tissues. Quantitative PCR analyses of mouse tissue samples with mouse GPR81-specific (A) and mouse GPR109A-specific (B) primers. Samples taken at different times during the mouse 3T3-L1 adipocyte differentiation process were also included. Each bar represents the mean value of triplicate data determinations from a single repeated experiment, and results were normalized with mouse GAPDH. Error bars indicate ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Chimeric G proteins are useful reagents for converting signals generated by Gi-coupled receptors to a Gq-mediated signaling pathway (15.Coward P. Chan S.D. Wada H.G. Humphries G.M. Conklin B.R. Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors.Anal. Biochem. 1999; 270: 242-248Crossref PubMed Scopus (205) Google Scholar, 18.Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha.Nature. 1993; 363: 274-276Crossref PubMed Scopus (606) Google Scholar). This allows for the measurement of Gi-coupled receptor activity in an inositol phosphate (IP) accumulation assay. The IP assay is generally more robust than traditional adenylate cyclase assays (19.Brandish P.E. Hill L.A. Zheng W. Scolnick E.M. Scintillation proximity assay of inositol phosphates in cell extracts: high-throughput measurement of G-protein-coupled receptor activation.Anal. Biochem. 2003; 313: 311-318Crossref PubMed Scopus (79) Google Scholar). The development of chimeric G proteins by Conklin and colleagues (18.Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha.Nature. 1993; 363: 274-276Crossref PubMed Scopus (606) Google Scholar) has had a significant impact upon GPCR research and on orphan GPCR research in particular. Shown in Fig. 2 are the results of transfecting GPR81 into HEK293 cells with or without chimeric G proteins and monitoring of IP accumulation in the presence of 10 mM LiCl to inhibit inositol monophosphatase. In the absence of chimeric G proteins, GPR81 showed no IP accumulation. However, in the presence of Gqi5, Gqi9, myristolyated Gqi5, and Gα15, robust constitutive activity was observed compared with the vector control. Gqi9 showed the highest level of activity, which was ∼4.2-fold greater than the basal level of vector/Gqi9. Myristolyated Gqi5 showed the next highest level of constitutive activity (3.6-fold), although this was not significantly different from that of Gqi9. Gqo5 and Gqo3 also demonstrated constitutive activity to a lesser degree. The basal level of Gqo3 was approximately twice that of the other chimeric G proteins. This is perhaps attributable to the ability of Gqo3 to couple unidentified endogenous Gi-coupled receptors present in HEK293 cells. Interestingly, the human homolog of Gα15, Gα16, showed no activity (20.Offermanns S. Simon M.I. G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C.J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 21.Lee J.W. Joshi S. Chan J.S. Wong Y.H. Differential coupling of mu-, delta-, and kappa-opioid receptors to G alpha16-mediated stimulation of phospholipase C.J. Neurochem. 1998; 70: 2203-2211Crossref PubMed Scopus (87) Google Scholar). These results strongly suggest that GPR81 couples to the Gi signaling pathway in HEK293 cells. They are also consistent with data showing that the nicotinic acid receptor, which is closely related to the GPR81, is also coupled to the Gi signaling pathway, based upon niacin's ability to inhibit forskolin-induced cAMP accumulation (6.Offermanns S. The nicotinic acid receptor GPR109A (HM74A or PUMA-G) as a new therapeutic target.Trends Pharmacol. Sci. 2006; 27: 384-390Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Other Gi-coupled receptors have been shown to couple to chimeric G proteins with a comparable level of activity as GPR81 (22.Xiao, S-H., J. D. Reagan, P. H. Lee, A. Fu, R. Schwandner, X. Zhao, J. Knop, H. Beckmann, and S. W. Young. 2008. High throughput screening for orphan and liganded GPCRS. Comb. Chem. High Throughput Screen. In press.Google Scholar).Fig. 2.Constitutive activity of GPR81 in an inositol phosphate (IP) accumulation assay. HEK293 cells were transiently transfected with GPR81 alone (no G) or in combination with various chimeric G proteins, Gα15, or Gα16. Control vector containing aequorin cDNA served as a negative control. Twenty-four hours after transfection, the cells were incubated with HBSS containing 10 mM LiCl to initiate IP accumulation and measure constitutive activity. A: Data are representative of three experiments. Results are presented as means of quadruplicate determinations ± SD. B: Data from three experiments are averaged and expressed as fold over basal.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because GPR81 is an orphan receptor, it is difficult to study the receptor signaling and function without access to natural or synthetic agonists. To provide more direct evidence that GPR81 couples to the Gi pathway and to study the potential effects of the receptor activation on adipocyte functions, we generated a chimeric receptor between cysteinyl leukotriene receptor, CysLT2R (23.Capra V. Thompson M.D. Sala A. Cole D.E. Folco G. Rovati G.E. Cysteinyl-leukotrienes and their receptors in asthma and other inflammatory diseases: critical update and emerging trends.Med. Res. Rev. 2007; 27: 469-527Crossref PubMed Scopus (151) Google Scholar, 24.Brink C. Dahlen S.E. Drazen J. Evans J.F. Hay D.W. Nicosia S. Serhan C.N. Shimizu T. Yokomizo T. International Union of Pharmacology XXXVII. Nomenclature for leukotriene and lipoxin receptors.Pharmacol. Rev. 2003; 55: 195-227Crossref PubMed Scopus (261) Google Scholar), and GPR81. The chimeric receptor was designed to contain the three intracellular loops and the C terminus of GPR81, with the expectation that the hybrid receptor signaling would resemble that of native GPR81. The chimeric receptor also contained extracellular and transmembrane domains from CysLT2R to allow the response of the chimeric receptor to CysLT2R ligand, LTD4 (Fig. 3A). This strategy has worked well for several other GPCRs (25.Liu T. DeCostanzo A.J. Liu X. Wang H. Hallagan S. Moon R.T. Malbon C.C. G protein signaling from activated rat frizzled-1 to the beta-catenin-Lef-Tcf pathway.Science. 2001; 292: 1718-1722Crossref PubMed Scopus (221) Google Scholar, 26.DeCostanzo A.J. Huang X.P. Wang H.Y. Malbon C.C. The Frizzled-1/(beta(2))-adrenergic receptor chimera: pharmacological properties of a unique G protein-linked receptor.Naunyn Schmiedebergs Arch. Pharmacol. 2002; 365: 341-348Crossref PubMed Scopus (21) Google Scholar, 27.Li H. Malbon C.C. Wang H.Y. Gene profiling of Frizzled-1 and Frizzled-2 signaling: expression of G-protein-coupled receptor chimeras in mouse F9 teratocarcinoma embryonal cells.Mol. Pharmacol. 2004; 65: 45-55Crossref PubMed Scopus (20) Google Scholar, 28.Yin D. Gavi S. Wang H.Y. Malbon C.C. Probing receptor structure/function with chimeric G-protein-coupled receptors.Mol. Pharmacol. 2004; 65: 1323-1332Crossref PubMed Scopus (27) Google Scholar, 29.Yin D. Shumay E. Wang H.Y. Malbon C.C. Yeast Ste2 receptors as tools for study of mammalian protein kinases and adaptors involved in receptor trafficking.J. Mol. Signal. 2006; 1: 2Crossref PubMed Scopus (2) Google Scholar, 30.Gupte J. Cutler G. Chen J.L. Tian H. Elucidation of signaling properties of vasopressin receptor-related receptor 1 by using the chimeric receptor approach.Proc. Natl. Acad. Sci. USA. 2004; 101: 1508-1513Crossref PubMed Scopus (49) Google Scholar).Fig. 3.Construction and signaling of cysteinyl leukotriene 2 receptor (CysLT2R)/GPR81 chimeric receptor. A: Amino acid sequence alignment of CysLT2R, GPR81, and chimeric CysLT2R/GPR81 receptors generated by the Vector NTI program (Invitrogen). B, C: CHO cells were transiently transfected with vectors containing various receptors, chimeric G protein-Gqi9, and aequorin. Calcium flux was converted and measured as luminescent signals from aequorin. RLU, relative light units; PTX, pertussis toxin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3.Construction and signaling of cysteinyl leukotriene 2 receptor (CysLT2R)/GPR81 chimeric receptor. A: Amino acid sequence alignment of CysLT2R, GPR81, and chimeric CysLT2R/GPR81 receptors generated by the Vector NTI program (Invitrogen). B, C: CHO cells were transiently transfected with vectors containing various receptors, chimeric G protein-Gqi9, and aequorin. Calcium flux was converted and measured as luminescent signals from aequorin. RLU, relative light units; PTX, pertussis toxin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next studied the signaling pathways that are activated by the CysLT2R/GPR81 chimeric receptor in response to LTD4 treatment. No activation was observed through the Gs and Gq pathways (data not shown). However, when CysLT2R/GPR81 was cotransfected with Gqi9 into CHO cells, intracellular calcium mobilization occurred in response to LTD4 treatment, and this signaling was PTX-sensitive (Fig. 3B). The wild-type GPR81 receptor did not respond to LTD4 treatment in this assay format (Fig. 3B). As a control for these experiments, the native CysLT2R, when transfected into CHO cells, responded to LTD4 treatment as described previously, and this response was PTX-insensitive, as the native CysLT2R coupled to the Gq signaling pathway (Fig. 3C) (23.Capra V. Thompson M.D. Sala A. Cole D.E. Folco G. Rovati G.E. Cysteinyl-leukotrienes and their receptors in asthma and other inflammatory diseases: critical update and emerging trends.Med. Res. Rev. 2007; 27: 469-527Crossref PubMed Scopus (151) Google Scholar, 24.Brink C. Dahlen S.E. Drazen J. Evans J.F. Hay D.W. Nicosia S. Serhan C.N. Shimizu T. Yokomizo T. International Union of Pharmacology XXXVII. Nomenclature for leukotriene and lipoxin receptors.Pharmacol. Rev. 2003; 55: 195-227Crossref PubMed Scopus (261) Google Scholar). These results demonstrate that the chimeric receptor signals through Gi pathways. Because the intracellular loops and the C-terminal domain of the chimeric receptor are derived from GPR81, these results provided additional evidence that GPR81 is able to signal through Gi proteins for receptor activation. To study the potential effects of GPR81 activation on adipocyte function, transgenic mice overexpressing CysLT2R/GPR81 under of the control of a fat-specific promoter, aP2, were generated. Transgenic animals appeared normal with no apparent abnormalities. Furthermore, there was no difference in body weight or plasma free fatty acid levels in transgenic versus wild-type littermates (data not shown). The relative expression levels of endogenous GPR81 and CysLT2R/GPR81 chimeric receptors in the wild-type and transgenic animals were examined. As shown in Fig. 4A, chimeric receptor expression was detected only in the transgenic animals and not in the wild-type littermates. The effect of transgene on endogenous GPR81 expression was also examined, and a small increase was observed (Fig. 4B). To examine whether the chimeric receptor could affect lipolysis in adipocytes, glycerol release was measured after LTD4 treatment in primary adipocytes isolated from either wild-type or CysLT2R/GPR81 transgenic mice. Nicotinic acid was used as a positive control in these experiments. As shown in Fig. 4C, D, nicotinic acid treatment resulted in the inhibition of lipolysis in both wild-type and CysLT2R/GPR81-expressing adipocytes to a similar extent in a time-dependent manner. In contrast, LTD4 treatment only inhibited lipolysis in adipocytes isolated from CysLT2R/GPR81 transgenic animals but not in cells from wild-type animals. The extent of antilipolytic effects was similar between LTD4 and nicotinic acid treatments (Fig. 4C, D). These results suggest that the activation of native GPR81 receptor in adipocytes may also lead to the inhibition of lipolysis.Fig. 4.Leukotriene D4 (LTD4) inhibits lipolysis in isolated adipocytes expressing CysLT2R/GPR81 chimeric receptor. A, B: Expression levels of CysLT2R/GPR81 chimeric receptor (A) and endogenous GPR81 (B) in the fat of wild-type (WT) or transgenic mice were determined by quantitative PCR analysis and normalized to mouse GAPDH. Results are averages of triplicate data points from three different animals from each group. C, D: Isolated adipocytes from wild-type (C) or CysLT2R/GPR81 transgenic (D) mice were treated with buffer control, LTD4, or nicotinic acid. Glycerol release was measured hourly in the medium using Free Glycerol Reagent. Data are representative of three experiments. Results are presented as means of duplicate determinations. Error bars indicate ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4.Leukotriene D4 (LTD4) inhibits lipolysis in isolated adipocytes expressing CysLT2R/GPR81 chimeric receptor. A, B: Expression levels of CysLT2R/GPR81 chimeric receptor (A) and endogenous GPR81 (B) in the fat of wild-type (WT) or transgenic mice were determined by quantitative PCR analysis and normalized to mouse GAPDH. Results are averages of triplicate data points from three different animals from each group. C, D: Isolated adipocytes from wild-type (C) or CysLT2R/GPR81 transgenic (D) mice were treated with buffer control, LTD4, or nicotinic acid. Glycerol release was measured hourly in the medium using Free Glycerol Reagent. Data are representative of three experiments. Results are presented as means of duplicate determinations. Error bars indicate ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4.Leukotriene D4 (LTD4) inhibits lipolysis in isolated adipocytes expressing CysLT2R/GPR81 chimeric receptor. A, B: Expression levels of CysLT2R/GPR81 chimeric receptor (A) and endogenous GPR81 (B) in the fat of wild-type (WT) or transgenic mice were determined by quantitative PCR analysis and normalized to mouse GAPDH. Results are averages of triplicate data points from three different animals from each group. C, D: Isolated adipocytes from wild-type (C) or CysLT2R/GPR81 transgenic (D) mice were treated with buffer control, LTD4, or nicotinic acid. Glycerol release was measured hourly in the medium using Free Glycerol Reagent. Data are representative of three experiments. Results are presented as means of duplicate determinations. Error bars indicate ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Obesity is commonly associated with insulin resistance, hyperinsulinemia, and dyslipidemia, which are all important cardiovascular risk factors. The identification of novel, safe, and effective therapies for cardiovascular diseases has been the subject of intense pharmaceutical/biotech research efforts. Nicotinic acid still remains the most effective therapy for increased HDL while decreasing other cardiovascular risk factors, including VLDL, LDL, lipoprotein [a], and triglycerides. However, the therapeutic value of nicotinic acid is limited by its major side effect of cutaneous flushing, which is mediated by the expression of GPR109A in immune cells (6.Offermanns S. The nicotinic acid receptor GPR109A (HM74A or PUMA-G) as a new therapeutic target.Trends Pharmacol. Sci. 2006; 27: 384-390Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Clearly, there is much room for other novel effective therapies with more manageable side effect profiles. In this report, we examined the potential signaling pathways and functions of adipocyte-expressed orphan GPR81. Results from constitutive activities observed in the IP accumulation assay using chimeric G proteins that convert Gi signaling to Gq signaling, as well as Gi pathway activation from a chimeric receptor between CysLT2R and GPR81, argue that GPR81 couples to the Gi family of proteins. These findings, together with the well-known effects of antilipolytic activities of Gi activation, suggest that GPR81 may regulate this process in adipocytes as well. Indeed, the activation of adipocytes isolated from transgenic animals expressing the CysLT2R/GPR81 chimeric receptor by LTD4 results in the inhibition of lipolysis in vitro. Although our understanding of the lipid-lowering profile of nicotinic acid is far from complete, it has been proposed that the mechanism of action involves the inhibition of adipocyte lipolysis via activation of the Gi pathway. Given that GPR81 likely couples to the Gi signaling pathway and that its expression is more restricted to adipocytes than GPR109A, we speculate that the activation of GPR81 has potential utility in treating dyslipidemia without the flushing side effect observed with nicotinic acid treatment. Identification of GPR81 ligands could provide better understanding of its functions in adipocyte biology and its potential utility in treating metabolic diseases. The authors thank David Shuford, Vincent Javinal, Jonitha Gardner, Mark Chhoa, Maria Cuellar, James McCabe, the Amgen Transgenic Animal Facility, and the Amgen SF Lab Animal Resources for their technical support.
Abstract Heart failure (HF) and cardiac arrhythmias share overlapping pathological mechanisms that act cooperatively to accelerate disease pathogenesis. Cardiac fibrosis is associated with both pathological conditions. Our previous work identified a link between phytosterol accumulation and cardiac injury in a mouse model of phytosterolemia, a rare disorder characterized by elevated circulating phytosterols and increased cardiovascular disease risk. Here, we uncover a previously unknown pathological link between phytosterols and cardiac arrhythmias in the same animal model. Phytosterolemia resulted in inflammatory pathway induction, premature ventricular contractions (PVC) and ventricular tachycardia (VT). Blockade of phytosterol absorption either by therapeutic inhibition or by genetic inactivation of NPC1L1 prevented the induction of inflammation and arrhythmogenesis. Inhibition of phytosterol absorption reduced inflammation and cardiac fibrosis, improved cardiac function, reduced the incidence of arrhythmias and increased survival in a mouse model of phytosterolemia. Collectively, this work identified a pathological mechanism whereby elevated phytosterols result in inflammation and cardiac fibrosis leading to impaired cardiac function, arrhythmias and sudden death. These comorbidities provide insight into the underlying pathophysiological mechanism for phytosterolemia-associated risk of sudden cardiac death.
Objective: To estimate the predictive value of heart rate (HR)-blood pressure (BP) products of multiplication for compensated shock in children. Methods: The study population consisted of 99 children with shock who had lactate measured before receiving vasopressor agents in Department of Critical Care Medicine of Children's Hospital, Capital Institute of Pediatrics from October 2015 to March 2021. The clinical data including the HR, BP, HR to BP ratio, HR-BP product and lactate at admission and after the correction of shock, as well as the 28-day mortality were collected. According to the outcome at the 28th day, the patients were divided into survival group and non-survival group. Comparisons between groups were performed with unpaired Student t test, or Mann-Whitney U test, or chi-square test. Pearson correlation analysis was used to analyze the correlations between lactate and HR, BP, HR to BP ratio and HR-BP product, respectively. Receiver operating characteristic (ROC) curve was analyzed to evaluate the predictive values of HR, BP, HR to BP ratio and HR-BP product for lactate greater than 2 mmol/L. Results: In these 99 children, 49 were males, and the median age was 3.8 (0.7-6.0) years. The most common type of shock was septic shock (61 cases, 62%), followed by cardiogenic shock (12 cases, 12%), hemorrhagic shock (12 cases, 12%), Kawasaki disease shock syndrome (8 cases, 8%) and anaphylactic shock (6 cases, 6%). Sixty-six patients (67%) survived, and 33 patients (33%) died. ROC curve showed that the area under curves (AUC) of lactate (optimal cutoff value 3.15 mmol/L, sensitivity 96.0%, specificity 54.4%, P<0.01) and HR to systolic blood pressure ratio (HR/SBP) (optimal cutoff value 2.0 times/(min·mmHg), sensitivity 62.5%, specificity 69.0%, P = 0.03) for predicting adverse outcome were 0.769 and 0.649, respectively. There were significant correlations between lactate and HR to diastolic blood pressure (DBP) ratio, HR to mean blood pressure (MBP) ratio, SBP, HR/SBP, MBP, DBP and HR (r= 0.476, 0.452, -0.444, 0.425,-0.410, -0.364, 0.177, all P<0.01), while no significant correlation was found between lactate and the products of HR and BP(all P>0.05). HR/SBP performed better than the other six parameters for predicting lactate>2 mmol/L, with the AUC of 0.872 and the optimal cutoff value of 1.4 bpm/mmHg (sensitivity 92.1%, specificity 70.9%, P<0.01). When MBP was greater than or equal to 65 mmHg, MBP × HR, DBP × HR, SBP × HR, HR, HR/SBP, HR/MBP and HR/DBP were significantly correlated with lactate (r= 0.706, 0.705, 0.669, 0.626, 0.555, 0.502, 0.446, all P<0.01). And MBP × HR performed better for predicting lactate>2 mmol/L than the other six parameters, with the AUC of 0.974 and the optimal cutoff value of 9446 bpm × mmHg (sensitivity 100.0%, specificity 90.9%, P<0.01). Conclusions: The product of HR and BP, especially the MBP × HR, shows higher predictive values for abnormally elevated lactate in children with compensated shock than the HR/SBP does. It is worth recommending for early identification of compensated shock in children.目的： 探讨心率血压乘积识别儿童代偿期休克的价值。 方法： 回顾性分析2015年10月至2021年3月就诊于首都儿科研究所附属儿童医院重症监护室（PICU）99例诊断休克的患儿入PICU时（未应用血管活性药物状态）及休克纠正后的心率、血压、心率血压比值、心率血压乘积、乳酸及28 d转归等临床资料。根据28 d转归分为生存组和死亡组，组间比较应用独立样本t检验、Mann-Whitney U检验、χ²检验。应用Pearson相关分析分析心率、血压、心率血压比值、心率血压乘积与乳酸的相关性，受试者工作特征（ROC）曲线分析心率、血压、心率血压比值、心率血压乘积对乳酸>2 mmol/L的预测价值。 结果： 99例患儿中男49例、女50例，年龄3.8（0.7，6.0）岁，其中脓毒性休克61例（62%），心源性休克12例（12%），失血性休克12例（12%），川崎病休克综合征8例（8%），过敏性休克6例（6%）。随访28 d生存66例（67%），死亡33例（33%）。ROC曲线显示乳酸、心率/收缩压均对28 d死亡有预测价值（均P<0.05），曲线下面积（AUC）分别为0.769、0.649，最佳临界值分别为3.15 mmol/L、2.0 次/（min·mmHg）（1 mmHg=0.133 kPa），灵敏度分别为96.0%、62.5%，特异度分别为54.4%、69.0%。Pearson相关分析显示乳酸与心率/舒张压、心率/平均压、心率/收缩压、心率均正相关（r=0.476、0.452、0.425、0.177，均P<0.01），与收缩压、平均压、舒张压均负相关（r=-0.444、-0.410、-0.364，均P<0.01），与心率血压乘积无相关性（P>0.05）；ROC曲线显示心率/收缩压预测乳酸>2 mmol/L的AUC最大为0.872［最佳临界值1.4 次/（min·mmHg），灵敏度92.1%，特异度70.9%，P<0.01］。当平均压≥65 mmHg时，乳酸与心率平均压乘积、心率舒张压乘积、心率收缩压乘积、心率、心率/收缩压、心率/平均压、心率/舒张压均正相关（r=0.706、0.705、0.669、0.626、0.555、0.502、0.446，均P<0.01）；ROC曲线显示心率平均压乘积预测乳酸>2 mmol/L的AUC最大为0.974［最佳临界值9 446（次· mmHg/min），灵敏度100.0%，特异度90.9%，P<0.01］。 结论： 心率血压乘积尤其是心率平均压乘积对儿童代偿期休克的动脉血乳酸异常升高有较高的预测价值，优于心率/收缩压，值得推荐用于儿童代偿期休克的早期识别。.
Stroke is one of the leading causes of death worldwide that also result in long-term disability.Endogenous neural stem/progenitor cells (NSPCs) within subventricular (SVZ) and dentate gyrus (DG) zone, stimulated by cerebral infarction, can promote neural function recovery.However, the proliferation of eNSPCs triggered by ischemia is not enough to induce neural repair, which may contribute to the permanent disability in stroke patients.In this study, our results showed that following the treatment with artesunate (ART, 150 mg/kg), the functional recovery was significantly improved, the infarct volume was notably reduced, and the expression of Nestin, a proliferation marker of NSPCs in the infarcted cortex, was also increased.Additionally, the proliferative activity of NSPCs with or without oxygen-glucose deprivation/reperfusion was significantly promoted by ART treatment, and the therapeutic concentration was 0.8 μmol/L (without OGD/R) or 0.4 μmol/L (with OGD/R) in the in vitro model.Furthermore, the effects of ART can be abolished by the treatment of PI3K inhibitor wortmannin.The expression levels of related molecules in PI3K/Akt/FOXO-3a/p27 kip1 signaling pathway (p-AKT, p-FOXO-3a, p27 kip1 ) were examined using western blotting.The results suggested ART could inhibit the transcriptional function of FOXO-3a by inducing its phosphorylation, subsequently downregulating p27 kip1 and enhancing neural stem cell proliferation in the infarcted cortex via PI3K/AKT signaling, further alleviating ischemia-reperfusion injury after ischemic stroke.
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