Although the central nervous system (CNS) consists of highly heterogeneous populations of neurones and glial cells, clustered into diverse anatomical regions with specific functions, there are some conditions, including alertness, awareness and attention that require simultaneous, coordinated and spatially homogeneous activity within a large area of the brain. During such events, the brain, representing only about two percent of body mass, but consuming one fifth of body glucose at rest, needs additional energy to be produced. How simultaneous energy procurement in a relatively extended area of the brain takes place is poorly understood. This mechanism is likely to be impaired in neurodegeneration, for example in Alzheimer’s disease, the hallmark of which is brain hypometabolism. Astrocytes, the main neural cell type producing and storing glycogen, a form of energy in the brain, also hold the key to metabolic and homeostatic support in the central nervous system and are impaired in neurodegeneration, contributing to the slow decline of excitation-energy coupling in the brain. Many mechanisms are affected, including cell-to-cell signalling. An important question is how changes in cellular signalling, a process taking place in a rather short time domain, contribute to the neurodegeneration that develops over decades. In this review we focus initially on the slow dynamics of Alzheimer’s disease, and on the activity of locus coeruleus, a brainstem nucleus involved in arousal. Subsequently, we overview much faster processes of vesicle traffic and cytosolic calcium dynamics, both of which shape the signalling landscape of astrocyte-neurone communication in health and neurodegeneration. Locus coeruleus (LC) is a brainstem nucleus located in the pons in the lateral floor of the fourth ventricle, which contains neurones with neuromelanin granules. These give LC colouring and an alternative name of nucleus pigmentosus pontis or “heavily pigmented nucleus of the pons” . This coLC nucleus is abundantly vascularised , indicatThe mechanisms that facilitate or prevent neurodegeneration are poorly understood, however they may be related to the global coordinating function of LC that starts in prenatal period when the nucleus is already formed. It has been noted that LC efferents are associated with the development of various brain areas, especially the neocortex . In ratsDeveloping brain is growing in mass, which is associated with a problem for cell-to-cell signalling. Distances between cells become far greater than those reachable by diffusion–based signalling . To bypa2+) changes revealed robust NA-mediated signalling predominantly through α1-ARs in neocortical astrocytes but not in neurones i [2+]i [2+]i slows down peptidergic vesicles and endolysosomes [2+]i was remarkable: the movement of vesicles was almost halted, with only a jitter remaining . At least some of the peptidergic vesicles carry ATP and a similar attenuation of their mobility was observed when astrocytes were stimulated [In astrocytes vesicle traffic is maintained by an elaborated system regulated by fluctuations in [Ca2+]i ,71. The 2+]i ,71,72. G]i [2+]i , whereasysosomes . Similarysosomes  and in tysosomes . In thesysosomes . The effimulated .n-ethylmaleimide-sensitive fusion factor attachment protein receptor (SNARE) complex formation i but also other cytosolic second messengers such as cAMP . In astrocytes, binding of InsP3 to InsP3Rs located on the endoplasmic reticulum (ER) [2+]i through the release of Ca2+ from these organelles. Activation of ryanodine receptors on the ER may also increase cytosolic Ca2+ levels through Ca2+-induced release of Ca2+ from the ER i was increased several fold in astroglial cultures treated, for several hours, with oligomeric β-amyloid1–42 [1–40 [25–35 [2+ elevations, sometimes Ca2+ oscillations [2+ elevations more readily). It has been suggested that β-amyloid can generally increase astroglial Ca2+ excitability [2+ release and store-operated Ca2+ entry, SOCE [When astrocytes are exposed to β-amyloid, complex effects on intracellular Caobserved ,147 whicobserved . Restingloid1–42 ,150. Thi42 [1–40  or β-amy0 [25–35  were empllations ,156,157.llations ,152. Thetability ,147 by iry, SOCE ,159.2+ signalling has been reported in several AD animal models. In APP/PS1 mice [swe) and mutant presenilin 1 (PS1ΔE9) an increase in the resting Ca2+ concentrations and Ca2+ hyperactivity with abnormal long-projecting intercellular Ca2+ waves were detected in astrocytes surrounding senile plaques. Similar high-frequency intercellular Ca2+ waves were detected in astrocytes at the pre-plaque stages in APPSwe mice model [2+ excitability of astrocytes from AD model mice may be an elevated ATP release from reactive astrocytes that leads to over-activation of P2Y1 purinergic receptors [An increased astroglial CaPS1 mice  harbourice model . The undeceptors .2+ signalling could be associated with the mutated presenilin 1 as observed in primary astroglial cultures from neonatal 3xTg-AD mice [2+ oscillations compared to the control cells [2+ channel expression [2+ transients and SOCE in primary cultured astroglial cells from Tg5469 AD mice [2+ levels were elevated two-fold compared to the controls also in astrocytes obtained from a Down syndrome mouse model (Trisomy 16 mice) that shares several key features with AD [2+ transport ATPase (SERCA) inhibitor cyclopiazonic acid, which unmasks leakage of Ca2+ from the ER, large intracellular Ca2+ elevations were observed, reflecting higher ER Ca2+ content compared to controls. A positive correlation between the amplitude of cyclopiazonic acid-induced intracellular Ca2+ elevations and the resting Ca2+ levels was observed [Abnormal Ca-AD mice ,159. In ol cells . The SOCol cells . The abopression . On the  AD mice . Resting with AD . When thobserved .2+-dependent enzymes, intracellular Ca2+ channels and SOCE. Upregulation of metabotropic glutamate receptor mGluR5 has been observed in vitro upon 24–72 h exposure of cultured astrocytes to oligomeric β-amyloid (100 nM to 20 μM) [swe/PS1ΔE9 mice senile plaques [1–42  ,151,163 o 20 μM) , in cort plaques , in post plaques  and late–100 nM; ) increasmutation . Anothereceptors ,171.2+ and cAMP. Increased Ca2+ and cAMP excitability that occurs at short time-scale regulates astrocyte cellular metabolism, morphology and vesicle traffic. The latter controls the surface signalling properties of astrocytes and their capacity to provide sufficient amounts of energy via augmented aerobic glycolysis. These processes may be altered during decades in neurodegeneration, such as taking place in AD. If LC neurones, which are vulnerable due to their relatively high metabolic rate, are reduced in numbers, e.g., during neurodegeneration, NA-mediated synchronous brain “reset” is impaired. On the other hand, even if the LC neurones are intact, alterations in vesicle traffic of astrocytes, affecting signalling landscape of these cells, together with impaired astroglial Ca2+ and possibly cAMP systems may also play an additional role in facilitating the long-term course of neurodegeneration. Since the excitation-energy coupling occurs mainly in the astrocytes, more emphasis is needed to understand how astrocytes interact with the noradrenergic signals at the level of both Ca2+ and cAMP signalling, and how they cope with the demand of coherent spatio-temporal signalling in the relatively large brain areas taking place in a long-time domain.There are conditions where relatively large areas of the CNS need to be synchronously activated. This involves the stimulation of LC neurones and NA release, which primarily excites astrocytes, since the density of adrenergic receptors is almost an order of magnitude higher in astroglia vs. neurones ,32. Thus


Existing guidelines do not offer a quick, efficient alternative to the patient’s recollection of relevant clinical features during anamnesis and physical examination for chronic urticaria (CU). This study aimed to identify specific items reflecting the main characteristics of CU that should be included in a comprehensive medical history for patients with CU. We also aimed to clarify possible eliciting factors for CU to support accurate diagnosis of the disease.A panel of postgraduate dermatologists conducted a literature search for relevant studies on CU using Medline, the Cochrane database, and PubMed.We identified82 articles from which we drew a collection of items to inform development of an easy-to-use checklist and collection of items that should be included in a correct medical history. The final version of the checklist included42 items across two areas: essential clues for anamnesis and diagnosis of CU; and typical symptoms/parameters or characteristics according to subtype, etiology, and laboratory findings. Items included time of disease onset; duration, shape, size, color, and distribution of wheals; associated angioedema; atopy; and triggering factors.Our guide provides an easy-to-use tool to support clinicians to focus, orient themselves, and save time in medical consultations for CU, allowing better diagnosis and management of this disease.The online version of this article (doi:10.1186/s40413-017-0165-0) contains supplementary material, which is available to authorized users. Urticaria is characterized by the development of wheals (hives), angioedema, or both . Three cThe prevalence of CU in the general population ranges from 0.5–5%, with the incidence of CU in Western countries estimated at 1.4% per year . An ArgeUrticaria is classified into two major types according to duration: acute urticaria (<6 weeks) and CU (≥6 weeks) . For cliThere is a subgroup of CSU patients (40%) who has autoantibodies against the FcεRI, the high affinity receptor for IgE, that can stimulate and activate mast cells. The presence of these autoantibodies may be clinically important in a group of severely affected, treatment-resistant patients, where immunomodulatory treatments may be valuable. Patients with autoantibodies have no distinctive diagnostic clinical features. However, they do tend to have more severe urticaria .In contrast, CINDU has known triggering stimuli, which can be classified as symptomatic dermographism, caused by cold, delayed pressure, sunlight, heat, vibration, cholinergic, contact, or water . There iWe included urticarial vasculitis (UV) in this study. UV is not a form of CU, however is necessary to exclude it in patients with CU. In UV, the lesions are painful, burning, and tender, with plaques lasting for more than 24 h (sometimes up to 72 h). The wheals are associated with residual purpura or hyperpigmentation, and can have a central dark red or brown macule signifying underlying purpura and vasculitis. Other characteristics of UV are swelling, residual bruising, and edema from focused pressure; in such cases, skin biopsy would confirm or reject the diagnosis –13. A stThis study aimed to determine a collection of items covering the main characteristics of CU that should be included in a comprehensive medical history to support thorough anamnesis and physical examination. We also evaluated possible eliciting factors of CU to aid accurate diagnosis of the disease and obtain comprehensive information on each patient that will then allow to select the best approach for treatment.It is important to emphasize that this collection of items should not limit physical examination findings, which can vary and include features of other forms of urticaria; however, it provides a way to orient physicians in thinking about CU.For the purpose of this study, a panel of postgraduate dermatologists conducted a literature search. Inclusion criteria were: studies on the approach for patients with urticaria, anamnesis, physical examination, clinical features, characterization of lesions, and factors triggering CU; studies in English and Spanish; and articles published before 2016. Databases searched included Medline, the Cochrane database, and PubMed. Studies about the prevalence and treatment of CU were excluded.The criterion used to identify relevant items was specific patterns or features of lesions described in the selected studies that correlated with the forms of CU. For example, localized hives appearing after a temperature change (cold or heat urticaria), and those induced by pressure .These items were included in the first draft and subsequently, a medical history was performed. A new revision and agreement between authors was done until the final version of the checklist was designed.Essential features for anamnesis and diagnosis of CU.Typical symptoms/parameters or characteristics according to CU subtype, etiology, and laboratory findings.In total, 82 articles were selected and used to develop a 42-item checklist for taking a comprehensive medical history for CU. The guide and checklist are presented in Additional files Very rapid (less than 2 h): CINDU: dermographism, 6–7 min; cold urticaria, 2–5 min; heat urticaria, a few minutes to hours ; aquagenMore than 2 h: Pressure urticaria, 4–8 h .Time of disease onsetLess than 24 h: CINDU (<2 h), which includes dermographism (<30 min), aquagenic urticaria (<60 min) , solar uMore than 24 h: Pressure urticaria(24 h) ; UV; yellowish to red brown, urticaria pigmentosa (mastocytosis) ; erythemDistribution: CINDU from pressure, solar and dermographic stimuli share a localized distribution . PressurShape, size, color, and distribution of whealsOnset symptomsDermographic urticaria can sometimes begin with erythema and hives . Cold urAssociated angioedemaThrough inherited deficiency of C1 esterase inhibitor, maybe localized or generalized; triggered by physical trauma, stress, drugs, or food; gastrointestinal compromise (angioedema) . TypicalAssociated subjective symptoms of lesionsItching, pain, and burning in pressure urticaria and UV. Burning in UV  and coldFamily and personal history regarding urticaria and atopySome reports mention a marked prevalence of atopy in CSU and CINDU, with the most common cases being allergic asthma, rhinitis, and contact allergy , 30.Physical stimuli in CINDU: Cold (cold urticaria); heat (heat urticaria); sunlight (solar urticaria); water (aquagenic urticaria); pressure (pressure urticaria), machines that transmit vibration such as mixers or pruners (vibratory urticaria); rubbing (dermographic urticaria); physical exercise (cholinergic urticaria); and stress (CSU). Surgical implantations and events during surgery: implants, intrauterine contraceptive devices , surgicaStreptococcus spp., Staphylococcus spp., Yersinia, Helicobacter pylori, Treponema pallidum); viruses; parasites, [Infections: The association between infections and urticaria can be considered more direct in cases where urticaria disappears after treatment of infection. The role of infections in chronic urticaria as a causative agent in CU is still controversial however in a systematic review has been reported as an etiologic in approximately 6% of cases . Identifnworms]) ; and funnworms]) .Contact: latex, nickel, animals, steam cooking, perfumes, and pollens (rare cases reported) , 9.Drugs: NSAIDs can trigger CSU and angioedema , 37.Provoking factors for whealsDiurnal and nocturnal variationPruritus of urticaria in children and adults displays extensive temporal variability. It is worse nocturnally in chronic urticaria, often disturbing sleep, and is bothersome to a lesser extent upon awakening . Recent Psychosomatic and psychiatric diseasesMainly anxiety and depression . PatientGastric/intestinal problemsH. pylori. Although H. pylori eradication has no discernible effects on CSU beyond that of standard CSU therapy, H. pylori eradication should only be initiated in accordance with currently accepted indications in its treatment guidelines [Patients with symptoms of dyspepsia should undergo to a routine test to screen for idelines .Relationship to the menstrual cycleMenstrual Cycle is an aggravating factor in CSU. A rare form of urticaria is related to the menstrual cycle, appearing 7–10 days before menstruation , 46. TheSmoking habits Agents such as tobacco may aggravate CU .Type of workParticularly vulnerable populations include healthcare workers, hairdressers, and food handlers . Other vPrevious diagnostic procedures/resultsRoutine screening for malignancies in the diagnosis of underlying causes for urticaria is not suggested; however, if underlying causes are suspected, further research is necessary . A prevaChecklist items included:Physicians must promptly detect problems to successfully weigh the importance of findings in clinical settings and react appropriately. A thorough history and physical examination to identify all possible eliciting factors and causes of CU are important for diagnosis .International guidelines for the diagnosis and management of CU are available, with most recommending diagnostic procedures when a doctor has a clinical suspicion of CU. These existing guidelines provide example recommendations to differentiate between CSU, CINDU, and other differential diagnoses, and to measure disease activity and impact. However, they do not offer a quick or efficient alternative to a patient’s recollection of relevant clinical features during anamnesis and physical examination. Most existing guidelines are general, and not particularly useful for clinical practice and informing oriented history-taking in the first doctor-patient meeting.Physicians need an easy-to-use tool that helps them to quickly and easily diagnose this pathology. Therefore, this checklist has general and specific parameters ordered according to the sequence of a comprehensive clinical history. In addition, the checklist is able to be used by physicians independent of specialty  without causing confusion as to the various differential diagnoses. The checklist will also contribute to the proper management of patients with CU from the start of clinical occurrence, achieving better control of the disease and reducing its social and economic impact. Delay in diagnosis statistically significantly increases severity, correspondingly increasing healthcare costs  and wages lost because of absences from work .We developed an easy-to-use tool to support the early correct diagnosis and management of CU and facilitate healthcare providers/physicians’ diagnostic workup, clinical approach and allow to select the best approach for treatment in patients with CU.To our knowledge, this is the first checklist available for diagnostic workup and management of patients with CU since 2010. In 2014, a clinical history of CU was published that provided a model for questions directed to patients suspected of this pathology , 14, 54.In the last 30 years, attention has been focused on new ways to understand and manage urticaria. This includes the recent addition of novel drugs to the therapeutic arsenal, updating of clinical practice guidelines, and publication of pathophysiologic insights. Three decades ago, it would not have been possible to develop easy-to-use clinical guidance such as that developed here.There are some limitations to our checklist. First, in CU, etiology is a major challenge for physicians because the frequency and relevance of infectious diseases vary among patient groups and geographical regions. We recommend further research to develop specific tools adapted to the context of each country. In addition, our study does not provide a “real-world” evaluation of the checklist or its impact. The present study did not intend to validate the role of our checklist in the medical history; consequently, face validity and reliability need to be established in a future study. Future studies should also evaluate the usefulness of this checklist. The initial version of the checklist was in Spanish, and it is also necessary to explore the usefulness of the checklist in other languages. A final limitation is that the checklist may be considered too long for some physicians. However, the length of the checklist is justified by relevant parameters that cannot be omitted from a clinical history for a patient with CU.A strength of our study was that we performed an extended literature search to identify semiological and interrogatory characteristics that permitted our checklist to include all items necessary to collect comprehensive information from patients with CU. In a previous study, we found that knowledge of guideline recommendations among physicians who treat patients with urticaria in Ecuador was low . The diaUrticaria consultations can be frustrating for both patients and physicians, because lesions may persist, factors that elicit urticaria are confusing, and there is a risk of misdiagnosis. The easy-to-use checklist developed in the present study should be adapted to the context of each country to reflect the most common CU etiology. Finally, this checklist will help focus and orient physicians, and save time in medical consultations, allowing physicians to perform better diagnosis and management of this disease.Additional file 1:Chronic urticaria medical history (DOCX 2047 kb)Additional file 2:Checklist for a complete chronic urticaria medical history (DOCX 1489 kb)


Despite the considerable progress in understanding the molecular bases of acute myeloid leukemia (AML), new tools to link disease biology to the unpredictable patient clinical course are still needed. Herein, high-throughput metabolomics, combined with the other “-omics” disciplines, holds promise in identifying disease-specific and clinically relevant features.In this study, we took advantage of nuclear magnetic resonance (NMR) to trace AML-associated metabolic trajectory employing two complementary strategies. On the one hand, we performed a prospective observational clinical trial to identify metabolic changes associated with blast clearance during the first two cycles of intensive chemotherapy in nine adult patients. On the other hand, to reduce the intrinsic variability associated with human samples and AML genetic heterogeneity, we analyzed the metabolic changes in the plasma of immunocompromised mice upon engraftment of primary human AML blasts.Combining the two longitudinal approaches, we narrowed our screen to seven common metabolites, for which we observed a mirror-like trajectory in mice and humans, tracing AML progression and remission, respectively. We interpreted this set of metabolites as a dynamic fingerprint of AML evolution.Overall, these NMR-based metabolomic data, to be consolidated in larger cohorts and integrated in more comprehensive system biology approaches, hold promise for providing valuable and non-redundant information on the systemic effects of leukemia.The online version of this article (doi:10.1186/s13045-016-0346-2) contains supplementary material, which is available to authorized users. Re-programming energy metabolism is a hallmark of cancer . Acute mH) , all patients had achieved complete remission.To study the metabolic changes associated with AML in patients, we enrolled nine newly diagnosed patients in a prospective trial  Fig. . At the H) according to the presence or absence of mutations in IDH genes , highlighting 30 metabolites significantly contributing to the model . PB samples were collected over time from the same animals before and after AML infusion for a total of nine time points (tpM)  and in the exponential phase of disease (tp5M and tp6M). Possibly due to the lower number of variables present in the mouse model, OPLS-DA  mice were infused with 2 × 10pM) Fig. . AML bected Fig. . We thus-DA Fig.  pinpoint-DA Fig. . The incCombining the two longitudinal approaches to trace AML in patients and mice, we narrowed our screen to seven metabolites representative of AML tumor burden: leucine, valine, alanine, glutamine, citrate, acetoacetate, and lipids CH2CO. We interpreted this set of metabolites as a dynamic fingerprint of AML evolution that could be followed in a mirror-like trajectory in both studies Fig. .Fig. 3HeCollectively, our results highlight how NMR-based metabolomics might provide valuable and non-redundant information on the systemic effects of leukemia, to be consolidated in larger cohorts, and integrated in more comprehensive system biology approaches. Although the limited number of cases investigated in this study does not allow a meaningful comparison to previous reports , 7, the


PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton’s tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*), which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton’s tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to evaluate the combination of idelalisib and ONO/GS-4059 (NCT02457598).Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton’s tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton’s tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of  B-cell receptor (BCR) signaling is a key driver of pathogenesis in many types of lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) . The BCRThe class I PI3K family, which includes the catalytic p110 α, β, γ and δ isoforms, are often mutationally or constitutively activated in a variety of cancers . PI3Kδ e®  and Imbruvica® (ibrutinib) [Clinical trials have recently demonstrated significant efficacy with inhibitors that disrupt BCR signaling, including Zydeligrutinib) , 8. Idelrutinib) . Ibrutinrutinib) . Despite2 and 95% air at 37°C. Compounds used in this study include: idelalisib , GS-649443 (Gilead Sciences) [TMD8 cells were obtained from the Tokyo Medical and Dental University, and OCI-LY10 cells were obtained from University Health Network. Both cell lines were cultured in RPMI-1640 medium supplemented with 20% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin . Idelalisib and ibrutinib resistant TMD8 were cultured in the presence of idelalisib (1 μM) or ibrutinib (10–20 nM), respectively, and grown in a humidified atmosphere of 5% COciences) , BYL719,ciences) –16, ibruIDELA-R). Ibrutinib-resistant TMD8 cells were generated by continuous passaging in the presence of ibrutinib for 12 weeks then dose-escalating to 10 or 20 nM until stable resistance to ibrutinib was established (TMD8BTKi-R). Parallel cultures were grown in the presence of 0.1% DMSO as passage-matched, drug-sensitive control lines (TMD8IDELA-S and TMD8BTKi-S). Sensitive and resistant TMD8 cells were clonally isolated through two rounds of single cell limiting dilution.Idelalisib-resistant TMD8 cells were generated by continuous passage in the presence of 1 μM idelalisib for 8 weeks until stable resistance to idelalisib was established . Data was plotted as percent of vehicle (DMSO) control using GraphPad Prism . For apoptosis assays, cells were seeded at 0.2 X 106/mL in a 12 well dish. Apoptosis was measured after 48 hours using FITC Annexin V Apoptosis Detection Kit II or PE Annexin V Apoptosis Detection Kit I  following manufacturer's protocol. Labeled cells were measured by flow cytometry using BD LSRII and analyzed using FACSDiva™ (BD Biosciences).Cells were seeded at 20,000 cells/well in 96-well plates. Test compounds were added in quadruplicate using the HP D300 Digital Dispenser (Hewlett Packard Inc.). Inhibition of cell viability was assessed after 96 hours using CellTiter Glomax values), or idelalisib (1μM), MK2206 (1μM), GSK2334470 (1μM) for 2 hours or 24 hours. Cells were harvested in lysis buffer  containing: Protease Inhibitor Cocktail (Roche Diagnostics Corp), and phosphatase inhibitor sets 1 and 2 (EMD Millipore). Following 30 minutes on ice, cell lysates were cleared by centrifugation at 12,500 rpm for 10 minutes at 4°C. Lysates were analyzed by Simple Western using Peggy Sue™  or by Novex SDS-PAGE gels  blotted to Immobilon-F membrane . A standard curve using recombinant proteins was generated to measure PI3K isoform concentration by Simple Western; data was processed using Compass software (ProteinSimple). Western blots were quantified using LI-COR Imager and LI-COR Odyssey Image Studio Software version 5.2 . The following antibodies were purchased from Cell Signaling Technology : p-AKT (S473) , p-AKT (T308) #2965 rabbit monoclonal, AKT , p-ERK (T202/Y204) , ERK , p-S6RP (S235/236) , S6 , p-GSK3β (S9) ,p-STAT3 (Y705) , p-SFK (Y416) , p-IκBα (S32) , IκBα , p-SYK (Y525/526) , p-BTK (Y223) , BTK , PTEN , A20/TNFAIP3 , and actin . The c-MYC antibody  was purchased from Abcam, Inc. . GSK3β  and the actin used in western blots  were purchased from EMDMillipore. PI3Kγ  was purchased from Jena Bioscience . The p-SYK (Y352) antibody  was purchased from Sigma-Aldrich . Secondary IRDye-conjugated anti-mouse and anti-rabbit antibodies were purchased from LI-COR Biosciences.Cells were grown overnight in RPMI-1640 medium supplemented with 10% FBS and treated the following morning with idelalisib (420nM), ONO/GS-4059 (320nM), or in combination  using Vivo Manager® software . Mice were administered twice daily either vehicle, GS-649443 at 1 and 5 mg/kg, or ONO/GS-4059 at 5 and 10 mg/kg, alone or in combination, by oral gavage at a dosing volume of 5 ml/kg. All test compounds were formulated into 5% (v/v) N-Methyl-2-pyrrolidone (NMP) / 55% (v/v) Polyethylene Glycol 300 (PEG) 300 / 40% (v/v) Water / 1% (w/v) Vitamin E D-α-Tocopherol Polyethylene Glycol 1000 Succinate (TPGS). The tumor volume was calculated, taking length to be the longest diameter across the tumor and width to be the corresponding perpendicular diameter, using the following formula: (length×width2)/2. The tumor growth inhibition (TGI) was calculated based on tumor volume (TV) measured at the beginning and the end of compound or vehicle treatment:These studies were approved by the Institutional Animal Care and Use Committee at Omeros Corp. in Seattle, Washington, which complies with all provisions of the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals (NIH Publication No. 15–8013) and the United States Department of Agriculture Animal Welfare Regulations . Animal care and research procedures were performed in dedicated rooms within their animal facility using their program of husbandry under the direction of a veterinarian board certified by the American College of Laboratory Animal Medicine. The animal facility provides a barrier-type environment free of all rodent pathogens using individually ventilated microisolator cages for mice; irradiated corncob bedding, ad libitum irradiated food , ad libitum ultrafiltered and UV irradiated water, and species-appropriate nesting material as enrichment. Room temperatures were maintained at 70–72 degrees F and 30–70% relative humidity with a 12:12 light: dark photoperiod. Male CB17-SCID mice were treated with 1.44 Gy whole body irradiation using a Immunohistochemistry was performed on the Ventana Discovery Ultra  autostainer using the manufacturer’s instructions and reagents. The slides were immunolabeled for p-S6 (S235/236)  and c-MYC , and counterstained with hematoxylin.Characterization of mutations and gene expression were identified by whole exome sequencing  and RNA-Seq , respectively. DNA sequencing reads were aligned to human reference genome by BWA  and sing50 was determined using a sigmoidal dose-response (variable slope) curve generated from quadruplicate samples. Statistical significance was performed by student’s t-test for cell viability and two-tailed paired t-test for apoptosis experiments (GraphPad). The mixed effect Analysis of Variance (ANOVA) model for repeated measure was used to determine the treatment effect on tumor growth. The model fitted on tumor volume included factors of treatment, time, and their interaction. Baseline tumor volume was also included as covariate. The covariance among repeated measurements was assumed with ante-dependence structure. The 8 mono- and combination-treatment groups were compared to vehicle control with Dunnett’s multiple comparison adjustment. Each of the 4 combination-treatment groups was also compared to the 2-corresponding mono-dose groups. A multivariate t method was applied for multiple comparison adjustment. A logarithm transformation was applied on tumor volume to meet model assumptions. The analysis was performed using SAS® v9.2 . A one-way ANOVA method was used to compare treatment groups to vehicle control in pharmacodynamic tumor samples. Log transformation was applied when needed. No multiple comparison adjustments were applied.Cell viability EC50 for the PI3Kδ-selective inhibitor idelalisib and the pan PI3K inhibitor GDC-0941, was 24 nM and 46 nM, respectively  as compared to p110α, p110β, and p110γ  , ONO/GS-4059 EC50 shifted from 7 nM (single agent) to 2 and 1.5 nM. In OCI-LY10, the addition of ONO/GS-4059 (25 nM) maximally shifted the EC50 of idelalisib from 122 to 13 nM  . Both TMectively . When idto 13 nM . In TMD8to 13 nM .The effect of the combination of these compounds was characterized on downstream signaling pathways implicated in malignant B-cell survival and proliferation. Idelalisib elicited a stronger inhibition of p-AKT (S473) and p-ERK (T202/Y204)  than ONO/GS-4059 at 2 hours  . As expein vitro, the efficacy of PI3Kδ and BTK inhibition in combination was assessed in a TMD8 xenograft model. Due to the short half-life of idelalisib in mice, the PI3Kδ tool compound GS-649443 was used in vivo. GS-649443 is more potent but displays similar selectivity to idelalisib across the class I PI3K family . The combination of GS-649443 and ONO/GS-4059 at the lowest and highest dose groups led to a 110% and 120% inhibition of tumor growth, respectively. All combinations of GS-649443 and ONO/GS-4059 strongly inhibited tumor growth, leading to tumor regression (P < 0.0001). No decreases in body weight were observed in any of the treatment groups (data not shown). Pharmacodynamic results in the individual TMD8 mouse tumors showed a 35% decrease in p-BTK with ONO/GS-4059  led to the establishment of a cell line with acquired resistance (TMD8IDELA-R), while a passage-matched, vehicle control line (TMD8IDELA-S) remained sensitive to idelalisib as assessed by cell viability   . Acquire in the TMD8IDELA-R line showed higher constitutive p-AKT levels (16-fold), with little to no upregulation of p-BTK and c-MYC , were identified by WES as a mechanism of acquired resistance. However, protein profiling of the PI3K pathway revealed PTEN loss in the TMD8IDELA-R which was not predicted by WES or RNAseq  suggesting that the upregulation of PI3Kγ may not be contributing to the observed resistance , while the pan-PI3K inhibitor BKM120 showed some decrease in inhibitory activity (EC50 0.76 vs 1.2 uM for DMSO vs. resistant line) as it has selectivity for PI3K α>δ>β>γ [IDELA-R, as shown by upregulation of p-AKT S473 and T308, p-S6RP S235/236, and p-GSK3β . As single agents, no induction of apoptosis was observed with idelalisib or GSK2334470 while a small increase was seen with MK-2206 (33%) compared to vehicle control (24%) in TMD8IDELA-R (AKT and phosphoinositide-dependent kinase-1 (PDK1)-targeted inhibitors were used to determine if the upregulation of downstream PI3K pathway signaling was a driver of idelalisib resistance. The AKT inhibitor MK-2206 and the PDK1 inhibitor GSK2334470 had little to no single or combination agent activity on the TMD88IDELA-R . ApoptosIDELA-R line. As shown previously, TMD8IDELA-R displayed higher constitutive activation of the PI3K pathway as demonstrated by higher levels of p-AKT S473, T308 and p-S6RP S235/236 . These lines showed complete resistance to ibrutinib and ONO/GS-4059 as compared to a passage-matched, vehicle control line , which was confirmed by Sanger sequencing. WES analysis also revealed a novel mutation in TNFα-induced protein 3  in clonal isolates from the 10 nM treated clones only . Protein expression profiling in these lines showed a loss of A20 and an increase in p-IκBα in the TMD8A20-Q143*, signifying NF-κB pathway activation, while total and p-BTK Y223 remained unchanged , none displayed a shift in sensitivity to the combination of PI3Kδ and PDK1/AKT inhibition (data not shown). The same lack of ability to overcome innate resistance in vitro with idelalisib and ONO/GS-4059 was also observed in this cell line screen.In our resistance model, bypass of the PI3Kδ requirement for PI3K pathway hyperactivation is likely due to the observed loss of PTEN. Central to this pathway is the activation of PDK1and subsequent phosphorylation and activation of AKT, which was highly upregulated in our model (16-fold). Cell survival was shown to be dependent on PDK1/AKT, as demonstrated by both cell viability and signal transduction alterations with PI3Kδ and PDK1/AKT inhibition. We also investigated whether a combination that is effective in restoring sensitivity in this resistance setting  could overcome innate resistance. Of 12 DLBCL lines insensitive to idelalisib  and PLCγ2, have been previously associated with resistance to the BTK inhibitor ibrutinib in CLL and MCL –43. Charerations . A20, a erations . AlthougUpregulation of SRC family kinase (SFK) and SYK phosphorylation, in conjunction with the observed BTK-C481F mutation, suggests upregulation of an alternate survival pathway that confers cross resistance to both idelalisib and ONO/GS-4059. Targeting of the SYK pathway has been implicated recently in several reports as a mechanism to overcome drug resistance in CLL and DLBCL so it is not surprising that this may serve as a bypass mechanism of resistance , 47. An in vitro data demonstrate that ONO/GS-4059 is an effective BTK inhibitor and has demonstrated efficacy in the clinic [in vivo. The evaluation of combination therapy provides an appealing mechanism to lower or limit the toxicity of each drug in a dose sparing manner. Clinical trials are currently underway to test this hypothesis with idelalisib and ONO/GS-4059 .Clinically, there is limited experience with CLL patients in the context of disease progression while on ibrutinib or idelalisib therapy. A recent study showed that overall response rate for use of ibrutinib after idelalisib discontinuation or use of idelalisib after ibrutinib discontinuation was 77% and 50% respectively, suggesting non-overlapping mechanisms of resistance . Use of e clinic . AdditioS1 FigCell viability was assessed with idelalisib and ONO/GS-4059 at fixed concentrations  or with ONO/GS-4059 and idelalisib at fixed concentrations (156 or 625 nM); mean ± SEM, n = 4.(EPS)Click here for additional data file.S2 FigCell viability with idelalisib and GS-649443 was assessed by CellTiterGlo after 96 hours, mean ± SEM, n = 4.(EPS)Click here for additional data file.S3 FigClonal isolates of idelalisib resistant TMD8 and a DMSO passage-matched clone were treated with idelalisib; cell viability was assessed by CellTiterGlo assay, mean ± SEM, n = 4.(EPS)Click here for additional data file.S4 Fig2-fold of TMD8IDELA-S versus TMD8IDELA-R clones, mean ± SEM.Boxplots generated from RNAseq data, y-axis is log(EPS)Click here for additional data file.S5 FigIDELA-S and TMD8IDELA-R were assessed by RNAseq analysis. (B) Protein lysates were generated from TMD8IDELA-S and TMD8IDELA-R cells and analyzed by Simple Western. (C) Cells were treated with the PI3Kδ/γ inhibitor IPI-145 and viability was assessed after 96 hours by CellTiterGlo assay, mean ± SEM, n = 4.(A) PIK3CG expression levels of TMD8(EPS)Click here for additional data file.S6 FigIDELA-S and TMD8IDELA-R cells, and analyzed by western blot  or Simple Western .Protein lysates were generated for TMD8(EPS)Click here for additional data file.S7 FigBTK-C481F lines, and protein expression of p-SFK Y416, p-SYK Y352, total SYK and actin was analyzed by Simple Western.Protein lysates were generated for TMD8 (DMSO control) and TMD8(EPS)Click here for additional data file.S1 TableCell viability with ibrutinib, ONO/GS-4059, idelalisib and GS-649443 was assessed by 96 hour CellTiterGlo assay.(PPTX)Click here for additional data file.


The potential roles of GORK (depolarization-activated guard cell outward-rectifying potassium) channels and RBOHD (respiratory burst oxidase homologue D) in plant adaptive responses to hypoxia were investigated in the context of tissue specificity  in roots of Arabidopsis. The expression of GORK and RBOHD was down-regulated by 2- to 3-fold within 1 h and 24 h of hypoxia treatment in Arabidopsis wild-type (WT) roots. Interestingly, a loss of the functional GORK channel resulted in a waterlogging-tolerant phenotype, while rbohD knockout was sensitive to waterlogging. To understand their functions under hypoxia stress, we studied K+, Ca2+, and reactive oxygen species (ROS) distribution in various root cell types. gork1-1 plants had better K+ retention ability in both the elongation and mature zone compared with the WT and rbohD under hypoxia. Hypoxia induced a Ca2+ increase in each cell type after 72 h, and the increase was much less pronounced in rbohD than in the WT. In most tissues except the elongation zone in rbohD, the H2O2 concentration had decreased after 1 h of hypoxia, but then increased significantly after 24 h of hypoxia in each zone and tissue, further suggesting that RBOHD may shape hypoxia-specific Ca2+ signatures via the modulation of apoplastic H2O2 production. Taken together, our data suggest that plants lacking functional GORK channels are more capable of retaining K+ for their better performance under hypoxia, and that RBOHD is crucial in hypoxia-induced Ca2+ signalling for stress sensing and acclimation mechanism.Regulation of root cell K The increasing frequency of flooding in southeast Asia, east Africa, and southern India . Waterlo+ participates in many defence-related processes under hypoxic stress, such as detoxifying reactive oxygen species (ROS), preventing cytosolic acidification, and possibly formation of aerenchyma  channels  and superoxide (O2·−) are both produced by a number of intracellular and extracellular sources H oxidases partially inhibited ABA-induced stomatal closure H, decreased synthesis of ATP , and gen sources . One of  sources . RBOHs cf anoxia . Also, agnalling . Also, b+ homeostasis and therefore possibly acclimation to hypoxia. Moreover, the tissue-specific context of these hypoxia responses in roots needs to be investigated. In this work, we have studied profiles of K+, Ca2+, superoxide, and H2O2 distribution in specific tissues (epidermis and stele) and zones (elongation and mature) in Arabidopsis roots in the WT, and gork1-1 and rbohD mutants. We report that the loss of a functional GORK channel in Arabidopsis leads to better K+ retention and results in a more hypoxia-tolerant phenotype. We also show that NADPH oxidase activity affects Ca2+ distribution and functions in Ca2+ signalling under hypoxia stress.The roles and interaction between GORK channels and NADPH oxidase in hypoxic responses of roots requires elucidation since both can impact cellular signalling and KArabidopsis thaliana WT Columbia-0 (Col-0) and two loss-of-function mutants gork1-1 (SALK_082258) and rbohD (SALK_021661) (both in Col-0 background) were obtained from the Arabidopsis Biological Resource Centre (http://www.Arabidopsis.org/abrc/). Seeds were surface-sterilized by 20% commercial bleach [1% (v/v) NaClO] for 10 min, washed with sterilized distilled water five times, and then stratified in fresh distilled water at 4 °C for 2 d. Seeds were then sown in Petri dishes containing 1% (w/v) phytogel, half-strength Murashige and Skoog (MS) medium, and 0.5% (w/v) sucrose at pH 5.7, sealed with 3M micro-pore tape , and then placed in a growth chamber and positioned vertically to allow root growth along the surface of the medium. The conditions in the growth chamber were 16 h/8 h light/dark cycles, with 100 µmol m−2 s−1 photon flux density during the light period, and temperature at 22 °C. Unless specified, all chemicals were of analytical grade from Sigma-Aldrich .2   agar which was dissolved by boiling while being stirred, and the solution was cooled down to room temperature, and then bubbled with high purity N2.The whole seedlings were submerged in agar solution in a transparent container and were exposed to 16 h/8 h light/dark cycles, enabling plant photosynthetic activity; this approach was taken following earlier work on Arabidopsis , the hypoxic solution contained 5 mM NaCl, 5 mM Ca2+-MES, pH 6.1, with 0.2% (w/v) agar. For Ca2+ measurement, again using a dye (see below), the hypoxic solution was 10 mM KCl, 5 mM Na+-MES, pH 6.1, with 0.2% (w/v) agar. For ROS measurement, the hypoxic solution was 10 mM KCl, 5 mM Ca2+-MES, pH 6.1, with 0.2% (w/v) agar. Seedlings used for control were exposed to air under the same light and temperature regime.Hypoxic treatment was imposed by submerging the 10-day-old Arabidopsis seedlings in 0.2% (w/v) agar solution pre-bubbled with high purity Ngork1-1, rbohD, and WT seeds were sown in 0.2 litre pots filled with peat moss, perlite, vermiculite, and coarse sand , and grown for 3 weeks at 21 °C using a 12/12 h light [photosynthetically active radiation (PAR) 100 µmol m−2 s−1]/dark regime. Waterlogging treatment was implemented by immersing pots of 3-week-old plants at water level 0.5 cm above the soil surface and leaving them for another 3 weeks. The above-ground biomass was determined as fresh weight per plant. Leaf chlorophyll content was measured with a SPAD meter . Measurements were taken for at least six biological replicates for each treatment in a single experiment.In the phenotyping experiments, stratified GORK; and forward 5'-TGCGGGTGCCCATTTAAC-3' and reverse 5'-CTTTCACAAACCACCAGTAGC-3' for RBOHD. Amplification of the RNA polymerase II subunit (RPB2) (forward primer 5'-TTCCCCGTTCCGATAACT-3' and reverse primer 5'-ATGCTCTGCCGTCCACC-3') gene was used as an internal control. The PCR program was two steps: one cycle of 95 °C, 10 min; 40 cycles of 95 °C, 15 s; 60 °C, 15 s; 72 °C, 15 s. The amplification of the target genes was monitored every cycle by SYBR-green fluorescence. Three biological and three technical replicates were performed for each treatment.Total RNA was extracted from roots of 10-day-old Arabidopsis WT (Col-0) with TRIZOL reagent  according to 2+ and K+ concentration, respectively, in Arabidopsis root cells. The optimal concentrations of Calcium Green-5N and APG-2 dye were established in our previous work  or Ca2+  measuring buffers, respectively. After 1, 24, and 72 h of hypoxic treatment, Arabidopsis seedlings were incubated in the dye-containing measuring buffers for 3 h in the dark at room temperature. They were then washed in distilled water for 3 min to remove the residual dyes before measuring the fluorescence intensity. For each treatment, at least nine biological replicates  were measured; for each of them, between 20 and 30 cells  were averaged.The Calcium Green-5N, AM  and Asante Potassium Green-2  dyes were employed to measure the Ca+ concentration in Arabidopsis root cells was also determined using the fluorescent dye APG-2. For imaging analysis, several lines were drawn across the tissue region of interest in the elongation and mature zones with Leica Application Suite X software . For each treatment, at least nine biological replicates  were measured; for each of them ~60 cells  were averaged. Continuous fluorescence was quantified in arbitrary units by LAS AF software based on intensity  was used to measure fluorescent signals from roots, as described in detail in + flux was measured using the MIFE  technique. The specific details relating to the MIFE theory and electrode fabrication and calibration are available in our previous publications  with 0.2% agar pre-bubbled with N2 in a sealed plastic container for 3 d under a photon flux density of 100 µmol m−2 s−1 and at 22 °C. Prior to the MIFE measurement, one Arabidopsis root was cut into 2 cm long segment from the tip and then immobilized horizontally in a 10 ml Perspex measuring chamber (10.5 × 0.8 × 2.0 cm). For treatment, 9 ml of BSM solution with 0.2% agar pre-bubbled with N2 were added to the chamber at 21 °C. For the control, roots were exposed only to BSM solution. Net K+ flux was measured by microelectrodes in the elongation zone (0.4 mm from the root tip) and mature zone (5 mm from the root tip) 2 h after the treatment. The net ion flux between the two positions was recorded by MIFE CHART software and calculated by the MIFEFLUX program. For each treatment and two different zones, at least 12 replicates were measured for each treatment.Net K2O2-sensitive fluorescent probe cell-permeant 2',7'-dichlorodihydrofluorescein diacetate  and superoxide fluorescent dye dihydroethidium (DHE) AM ester  were employed to measure H2O2 and superoxide concentration in Arabidopsis root cells, respectively . After 1, 24, and 72 h of hypoxic treatment, Arabidopsis seedlings were incubated in the dye-containing measuring buffers for 30 min in the dark. The stained seedlings were washed in distilled water for 3 min to remove residual dyes before measuring fluorescence intensity in elongation epidermal, elongation stelar, mature epidermal, and mature stelar root cells. The fluorescence images of superoxide and H2O2 were collected with excitation at 488 nm and 514 nm for DCF and DHE, respectively, and emission at 517–527 nm for DCF and 590–630 nm for DHE. Then, fluorescent images were analysed with Image J software  based on integrated density similar to ion concentration measurements. For each treatment and ROS species, at least nine biological replicates  were measured; for each of them, between 20 and 30 cells  were averaged. Relative total cell superoxide and H2O2 concentration data shown in The HP<0.05. The statistical significance of phenotypes in the control and waterlogging treatment was tested by paired samples t-test. The significance levels are *P<0.05, **P<0.01, and ***P<0.001.Statistical analysis was performed using IBM SPSS Statistics 21 . All data in the figures are means ±SE. The significant differences were compared using Duncan’s multiple range test. Different lower case letters represent significant difference between genotypes and/or treatments at GORK and RBOHD in roots of 10-day-old WT Arabidopsis after 1, 24, and 72 h of hypoxic treatments  reduced the shoot fresh weight in the WT and rbohD, but gork1-1 plants showed a waterlogging-tolerant phenotype  after 24 h of hypoxia, but in the mature zone it was decreased after 72 h of hypoxia compared with 24 h  in the WT; likewise, in rbohD, the K+ concentration was 1.6- to 2.2-fold higher than that in the WT . Interestingly, with the clear trend in stress-induced accumulation of Ca2+ in each zone and tissue after 72 h of hypoxia, the increase was much less pronounced in rbohD than in the WT. In epidermal cells, there were only 1.4- and 1.5-fold increases in rbohD between 72 h of hypoxia and control in the elongation and mature zone, respectively. At the same time, in the WT, these increases were 2.7- and 4.8-fold, respectively. In stelar cells, rbohD showed 4.0- and 3.3-fold increases and there were 4.6- and 3.7-fold increases in the WT  can be activated when there is insufficient V-ATPase, arguing that this switch is beneficial to roots under anoxic treatment  and oxalate oxidases  signal transduction pathway in Arabidopsis via an NADPH oxidase mechanism , leading to more apoplastic Ca2+ entering the cytosol  is key for the propagation of the ROS wave in plants (2+ waves and ROS signalling. Based on the present result, we suggest that RBOHD may interfere with Ca2+ signalling under hypoxic stress.The production of ROS by RBOHs is identified to be synergistically activated by the binding of Caied here . Comparie latter , which m+ accumulation compared with the WT in different zones and tissues under hypoxic stress. By preventing hypoxia stress-induced K+ leak, the gork1-1 mutant showed higher waterlogging tolerance compared with the WT. Reduced NADPH oxidase activity resulted in the hypoxia-sensitive phenotype in the rbohD mutant, with a less pronounced increase of Ca2+ concentration and higher superoxide accumulation in the elongation zone compared with the WT. We suggest that RBOHD is a crucial component interfering with stress-induced Ca2+ signalling.This study highlighted the essential roles of GORK channels and NADPH oxidase as components of plant adaptive response mechanisms to hypoxia in Arabidopsis roots. A loss-of-function mutant of the GORK channel exhibits higher KJXB onlineSupplementary data are available at Figure S1. Effect of hypoxic stress on Ca2+ distribution in Arabidopsis roots.Figure S2. Effect of hypoxic stress on superoxide distribution in Arabidopsis roots.Figure S3. Effect of hypoxic stress on H2O2 distribution in Arabidopsis roots.supplementary_figures_S1_S3Click here for additional data file.


The aim of this study is to systematically evaluate the clinical efficacy and safety of the traditional Chinese medicine prescription Jade Screen combined with desloratadine in the treatment of chronic urticaria. Two researchers independently conducted literature searches. The extracted data were analyzed using Rev Man 5.2.3 software. The established retrieval time range of the various databases was up to 15 March, 2017.P < 0.00001), while its recurrence rate (P < 0.00001) and symptom score (P = 0.006) were both significantly lower than the latter. The rate of adverse reaction in the combination group was lower than that when orally taking desloratadine alone (P = 0.74), and the serum level of total IgE in the combination group was lower than that when orally taking desloratadine alone (P = 0.82); however, the results of the rate of adverse reaction and the serum level of total IgE were insignificant. Sixteen randomized controlled trials were included in this study. The results of the meta-analysis showed that the total effective rate of using Jade Screen and desloratadine in combination to treat chronic urticaria was higher than that with desloratadine alone ( Using Jade Screen and desloratadine together to treat chronic urticaria gains a better clinical effect than using desloratadine alone.  Urticaria is a common disease in dermatology, characterized by transient, localized, edematous wheals of various sizes on skin mucous membranes and accompanied by severe itching. If wheals occur every day or almost every day for more than six weeks, such condition is called chronic urticaria . Approxi⟪General Treatise on Causes and Manifestations of All Diseases⟫, ⟪Invaluable Prescriptions for Ready Reference⟫, and ⟪Complete Works of Diagnosis and Treatment for Surgical Diseases and other works⟫. Most modern doctors believe that rheumatism attacks the skin but decreased resistance and transportation dysfunction of the spleen and stomach are the keys to the disease [ Astragalus membranaceus [Astragalus membranaceus (Fisch.) Bunge]; Rhizoma Atractylodis Macrocephalae (bighead atractylodes rhizome); and Radix Saposhnikoviae [Saposhnikovia divaricata (Turcz.) Schischk]. Astragalus membranaceus can nourish qi and strengthen the exterior to enhance the grain of the skin and the texture of the subcutaneous flesh and prevent it from evil invasion; Rhizoma Atractylodis Macrocephalae strengthens the middle warmer and invigorates the spleen to recover vital qi, and Radix Saposhnikoviae dispels wind and disperses evil to prevent external invasion and help cure various diseases. The compatibility of the three medicines has the functions of nourishing qi, strengthening the exterior, and arresting sweat. With sweet flavor and warm nature, Astragalus membranaceus tonifies qi and strengthens the exterior, while Rhizoma Atractylodis Macrocephalae invigorates the spleen and eliminates dampness. The combination of Astragalus membranaceus and Rhizoma Atractylodis Macrocephalae can greatly tonify lung and stomach qi, make the spleen and stomach healthy and vigorous, replenish the fleshy exterior, and prevent evil invasion, while Radix Saposhnikoviae relieves the exterior syndrome by dispersing wind-evil.Urticaria is called “hidden rash” or “red and white wandering wind” in traditional Chinese medicine. As the “Treatise on Needling” in the Sù Wèn (“Plain Questions”) states, when shaoyin qi is excessive, skin numbness and hidden rash will occur, which is also recorded in  disease . It can  disease . Jade ScAmong published clinical studies, some reports argue that Jade Screen combined with desloratadine can achieve a good therapeutic effect on chronic urticaria. To comprehensively assess the clinical efficacy and safety of this classical ancient prescription, we conducted this systematic review.⟪Lishizhen Medicine and Materia Medica Research⟫, ⟪China Journal of Experimental Traditional Medical Formulae⟫, ⟪Journal of Clinical Dermatology⟫, ⟪the Chinese Journal of Dermatovenereology⟫, ⟪Liaoning Journal of Traditional Chinese Medicine⟫, and relevant conference papers and proceedings after 2010 to improve the recall ratio.Records from the following databases in Chinese or English have been retrieved up to 15 March, 2017: Cochrane Library, PubMed, Embase, SinoMed, CNKI, VIP, and WanFang Data. The retrieval strategy was based on the combination of subject terms and free words. Chinese search terms were “Jade Screen”, “dilvleitading”, “xunmazhen”, etc.; English search terms were “Yu Ping Feng”, “Jade Screen”, “traditional Chinese medicine”, “Urticaria”, “Urticarias”, “desloratadine”, “Clarinex” and so on. Manual retrieval was conducted in Jade Screen combined with desloratadine treatment of chronic urticaria in randomized controlled trials was included, including literature limited to the Chinese or English languages and regardless of whether the studies were blinded.⟪Chinese Medical Association clinical treatment guidelines. Skin disease and STD Volume⟫, ⟪Practical Dermatology⟫, ⟪Clinical dermatology⟫,” ⟪Dermatology⟫ , Among the 16 studies included , there wP = 0.37, I2 = 8%). The fixed effects model revealed that the recurrence rate of Jade Screen combined with desloratadine for the treatment of chronic urticaria was significantly lower than that of the oral use of desloratadine alone , This method , 29, 31 P = 0.93, I2 = 0%). Meta-analysis suggested that the rate of adverse reaction of Jade Screen combined with desloratadine for the treatment of chronic urticaria was lower than that of the oral use of desloratadine alone but failed to show a statistical significance , Seven studies adopted this method , 28–31, P = 0.04, I2 = 64%). The random effects model indicated that the symptom score of Jade Screen combined with desloratadine for the treatment of chronic urticarial was significantly lower than that of the oral use of desloratadine alone , This method , 27, 31 P = 0.00001, I2 = 99%). The random effects model meta-analysis did not show a significant change between Jade Screen combined with the desloratadine group and the oral use of desloratadine alone , This method , 30 was x-coordinate and standard error logOR value as the y-coordinate. The analysis result of the funnel plot showed no mean symmetric distribution on the two sides of the plot, suggesting some publication bias possibly due to the limited number of the published papers with negative results, For the 16 research studies included, a funnel plot of overall response rate was drawn with the OR value of each paper as the Chronic urticaria is a clinically common skin disease that is hard to heal and often reoccurs even after it is suppressed; thus, it is worrisome to patients. The cause of this disease might be associated with food, drug, infection, inhalant, physical, genetic, endocrine, and mental factors . HoweverIgE is primarily generated from the plasma cells in the mucous membranes of the respiratory tract  and the digestive tract. It plays a crucial role in mediating the occurrence and development of allergic diseases . Modern In this research, we made a systematic evaluation on the functions of Jade Screen combined with desloratadine in treating chronic urticaria. Through a meta-analysis of the published studies, this research increased the sample size, enhanced the research credibility, and provided a reliable theoretical basis for clinical use.First, the results of total effective rate and recurrence rate suggested that the total effective rate of the combination of traditional Chinese and Western medicine in treating chronic urticaria was higher than the single use of desloratadine, while the recurrence rate was lower than the single use of desloratadine.Second, the results of occurrence rate of adverse reaction revealed that the combination of traditional Chinese and Western medicine and the single use of desloratadine showed no difference in the occurrence rate of adverse reactions. The adverse reactions of both treatments were mainly characterized by xerostomia, nausea, headache, dizziness, drowsiness, and, to a lesser extent, lack of strength.Third, the results of the symptom score and total serum IgE showed high heterogeneity. The primary reason could be the small number of articles reviewed and their poor quality. Second, the published research literature about the curative effect of chronic urticaria in the clinic, the criteria for clinical symptom assessment, and intervention measures performance still cause much confusion, and no unified cure-judging criteria have been adopted, thus increasing the heterogeneity between the results of different research and having a high risk of selective bias.The results of this study have satisfied our goal of this meta-analysis. The result has proven the effective therapeutic effect, fewer adverse reactions, and good safety of the traditional Chinese prescription Jade Screen in treating chronic urticaria. Thus, it can be used as an option for the treatment of chronic urticaria.This research assesses the curative effect of Jade Screen in treating chronic urticaria according to the Methods for Cochrane Systematic Review for the first time. With limited cases included and low quality, the research fails to make a completely reliable positive conclusion. The limitations of the research included are as follows: (1) the research sites of all the articles were located in China, which is very likely to cause potential location bias; (2) all patients included were Chinese, and all the articles were written in Chinese or English, possibly resulting in potential selection bias and English language bias. (3) The articles included varied in dose, which may lead to potential drug dose bias. The advantages and deficiencies of the research were as follows. Deficiencies, (4) the methodological quality of the research included was low. All the research included mention “randomization”, but only a few expound on how to generate a random sequence, and most research failed to describe how to realize allocation concealment. (5) The articles included have no unified diagnosis and efficacy determination criteria, increasing the heterogeneity between the results of different research and a high risk of selective bias. On the one hand, there is absence of the latest guidelines , 39 prepWith the development of examining approaches and the emphasis on patients' quality of life in recent years, many articles have included laboratory indices and quality of life into the clinical efficacy system of chronic urticaria . For exa(1) The total effective rate of the combined use of Jade Screen and desloratadine was greater than that of desloratadine alone.(2) The recurrence rate of the combined use of Jade Screen and desloratadine was greater than that of desloratadine alone.(3) There was no difference in the incidence of adverse reactions between the two treatments.(4) The present research results have showed that, in the treatment of chronic urticaria, the combination of traditional Chinese medicine and Western medicine is significantly better than using Western medicine alone. The therapeutic method provides a new thought for the treatment of chronic urticaria. However, due to the low quality of the currently selected articles, it is a necessity to carry out randomized controlled trials with strict designs in the future.


It also significantly decreased the apoptotic index. In vitro, ESMS (400 μg/mL) inhibited mitochondrial-dependent myocardial apoptosis by modulating the expression of caspase-3 and the Bcl-2/Bax ratio, and improved mitochondrial function through increasing mitochondrial membrane potential and cellular ATP content. ESMS restored intracellular Ca2+ and downregulated the expression of Calcineurin A (CnA), thus inhibiting phosphorylation of dynamin-related protein 1 (Drp1) at Ser616 and increasing phosphorylation of Drp1 at Ser637 to prevent cardiomyocyte mitochondrial fission. Above-mentioned results demonstrated ESMS suppressed mitochondrial-mediated apoptosis in oxygen glucose deprivation (OGD) injured H9c2 cardiomyocytes. These findings suggested that ESMS attenuated MI-induced HF by regulating Ca2+ homeostasis and suppressing mitochondrial mediated apoptosis through the modulation of Ca2+-calcineurin-mediated Drp1 signaling pathways. Our results provide insight into the mechanism and clinical applications of SMS and suggest a potential therapeutic strategy for HF.Sheng-Mai-San (SMS) is a well-known traditional Chinese medicine (TCM) complex prescription used to treat heart failure (HF) and angina in clinic. However, its potential therapeutic mechanisms remain unclear. The present study evaluated the cardioprotection of extract of SMS (ESMS) on myocardial ischemia (MI)-induced HF, and explored the underlying molecular mechanisms. The results demonstrated that ESMS (728.0 mg/kg) significantly attenuated MI injury-induced HF by improving cardiac function and pathological changes, decreasing lactate dehydrogenase (LDH), creatine kinase (CK) activities, and brain natriuretic peptide (BNP) levels; increasing ATPase activity; and reducing intracellular Ca Heart failure (HF) is a complex clinical syndrome and is recognized as one of the most burdensome diseases in the world ,2. AccorOver the past centuries, traditional Chinese medicine (TCM) has been used to prevent or treat imbalances in the body. Scientific evidence supports the safety and efficacy of TCM for the treatment of specific ailments. Treatments consisting of multiple drugs directed against multiple pathways and targets have stronger therapeutic efficacy for complex multifactorial diseases ,8. The ePanax ginseng C. A. Mey, Ophiopogon japonicus (Thunb.) Ker-Gawl., and Schisandra chinensis (Turcz.) Baill. (1:3:1.5). SMS is used for the treatment of palpitations, lassitude, and shortness of breath, which are symptoms of effort angina in clinical practice. Both clinical and basic studies show that SMS can be used for the prevention and treatment of cardio-cerebral ischemic diseases, it has shown better efficacy and fewer side effects than standard medical treatments [Sheng-Mai-San (SMS) is a well-known TCM complex prescription composed of eatments ,12. Preveatments ,14,15. Ieatments ,17. Meaneatments ,18,19. HCardiac mitochondria are responsible for generating energy in the form of ATP through oxidative phosphorylation. Mitochondria are critical for cardiac function, structural and functional abnormalities of mitochondria contribute to several common cardiovascular diseases including HF ,21. Mito2+) homeostasis is important for cellular homeostasis [2+, the second messenger in the heart, relates the muscle contraction and relaxation in the cardiac cycle to mitochondrial energy production [2+ within the cardiomyocytes. Meanwhile, Ca2+ homeostasis is disrupted, leading to alterations of mitochondrial structure and function [2+ levels modulated the activation of calcineurin [2+ homeostasis and suppressing mitochondrial fission might be beneficial for the treatment of MI-induced cardiomyocyte injury.Evidence suggests that calcium ion  in vivo on heart structural and functional impairment caused by permanent coronary artery ligation (CAL)-induced MI following HF, and in the H9c2 cardiomyocyte cell line subjected to oxygen glucose deprivation (OGD) injury in vitro. Then, we investigated the therapeutic effect of ESMS and explored its underlying mechanisms of ESMS. We found that ESMS attenuated MI injury-induced HF by modulated CaEchocardiography was performed to determine the effects of ESMS on cardiac function. Representative images of M-mode echocardiograms are shown in The levels of lactate dehydrogenase (LDH), creatine kinase (CK), and brain natriuretic peptide (BNP) in serum were evaluated to determine the effects of ESMS on myocardial damage. As shown in The pathological changes in the myocardium were examined by hematoxylin-eosin (H&E) staining of the heart. As shown in +-K+-ATPase, Ca2+-Mg2+-ATPase, and Ca2+-ATPase activities were significantly decreased in the model group compared with those in the sham group, whereas the ESMS (728.0 mg/kg) significantly increased ATPase activity. Meanwhile, since elevation of intracellular Ca2+ levels could result in cardiac hypertrophic responses, we assessed whether the Ca2+ levels were affected. As shown in 2+ concentration was significantly increased in the model group, whereas the ESMS (728.0 mg/kg) significantly reduced the intracellular Ca2+ levels.As shown in Myocardial apoptosis is an essential contributor to cardiac dysfunction after MI injury. The anti-apoptotic effects of ESMS were detected using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. As shown in The deleterious effects of different durations of OGD were investigated in H9c2 cells. The results showed that the viability of H9c2 cells decreased significantly by approximately 50% after 12 h of OGD . Assessm2+ concentration, whereas ESMS significant reduced the intracellular Ca2+ levels. During OGD, Na+-K+-ATPase, Ca2+-Mg2+-ATPase and Ca2+-ATPase activities were significantly decreased compared with control group, while ESMS significantly increased ATPase activity. Previous studies showed that intracellular Ca2+ levels modulate the activity of calcineurin; therefore, we observed whether the expression of CnA was affected. Western blot analysis showed that CnA expression was increased in H9c2 cells subjected to OGD, whereas the ESMS (400 μg/mL) significantly inhibited increases in CnA expression.As shown in Mitochondrial membrane potential was assessed using the JC-1 probe. As shown in Bcl-2 and Bax play important roles in the regulation of cell apoptosis. As shown in ® Deep Red FM . Representative images in Mitochondria show a flexible reticular formation in normal cells, whereas cells bearing predominantly fragmented or spherical mitochondria are considered to have undergone mitochondrial fission ,34. To i2+ homeostasis, ventricular remodeling, altered expression or function of proteins, and defects in many organelles and cellular pathways [HF is an important and growing public health concern, and a leading cause of morbidity and mortality in industrialized countries worldwide . MI has pathways . Therefo2+-calcineurin-mediated Drp1 signaling pathways, regulated Ca2+ homeostasis, and inhibited mitochondrial-mediated apoptosis, thereby preventing further deterioration. These findings provide the first evidence of the protective mechanism of ESMS against myocardial injury during the progression from MI to HF.The formula prescription SMS has been widely used for the treatment of cardiovascular diseases, with demonstrated clinical efficacy in the treatment of angina an HF in China ,14,15. PEchocardiography is clinically utilized to evaluate cardiac structure and function ,37. CompThe activities of certain cardiac marker enzymes reflect the pathological process of myocardial diseases. Myocardial enzymes such as LDH and CK are the most common biomarkers for MI and HF ,40. In aThe loss of cardiomyocytes caused by apoptosis plays a leading role in various heart diseases, and, even worse, it unavoidably results in HF, as shown by accumulating evidence . After i2+ cycling plays a crucial role in myocyte contracting and relaxing. A series of specialized regulatory proteins precisely controls the intracellular Ca2+ homeostasis, including transcription factors, ion channels and Ca2+ binding proteins [2+ handling in HF may be beneficial. The current understanding of Ca2+ homeostasis during HF is largely focused on the end-stage of the disease, whereas little is known about changes in Ca2+ homeostasis during disease development. Existing data indicate that Ca2+ cycling is initially increased at early stages [2+ concentration increases, resulting in acidosis; the decrease of Na+-K+-ATPase and Ca2+-ATPase activities result in impaired Ca2+ reuptake into the sarcoplasmic reticulum (SR), further increasing intracellular Ca2+ concentration [+-K+-ATPase, Ca2+-Mg2+-ATPase, and Ca2+-ATPase activities and reduced intracellular Ca2+ concentration, thereby normalizing of intracellular Ca2+ levels.Based on the results that ESMS protected against cardiomyocyte apoptosis induced by OGD, we investigated the potential mechanisms underlying the effect of ESMS against MI injury. In cardiomyocytes, Caproteins ,46. Moreproteins . Studiesy stages ,48. Durintration . The resMitochondrial dynamics include fission and fusion processes that are controlled by critical regulatory proteins. Aberrant or increased fission leads to increased mitochondrial fragmentation and mitochondrial death or mitophagy . HoweverMitochondria provides at least 90% energy for cardiomyocytes, and serves as an important mediator in cardiomyocyte apoptotic signaling pathway . In the 2+ leads to alterations of mitochondrial structure and function. Previous studies showed that cytoplasmic Ca2+ levels increase leading to activation of calcineurin, and sustained calcineurin activation is sufficient to damage cardiac structure and function [MI-disrupted Cafunction . In addifunction . Activatfunction ,31,33.2+ overload [2+-calcineurin signaling pathways [Ophiopogon japonicas reduces intracellular Ca2+ overload and suppressed ER stress and stress-mediated apoptosis in cardiomyocytes [As previously described, ESMS consists of bioactive compounds, some of which may protect against myocardial injury. Modern pharmacological studies illustrated that ginsenosides can be effective in improving ischemic myocardium metabolism, scavenging the free radicals, protecting the myocardial ultrastructure, and lessening the Caoverload . Ginsenooverload ,55. Ginspathways ,57. Ginspathways ,59. In amyocytes . Other imyocytes ,61. It hPanax ginseng C.A. MEY., water extract of Ophiopogon japonicus (Thunb.) KER-GAWL and Schisandra chinensis (Turcz.) BAILL at a ratio of 1:3:1.5. ESMS was provided from Tasly Pharmaceutical Co., Ltd. , and its quality was confirmed by HPLC analysis . All procedures and assessments were approved by the Animal Ethics Committee of the School of Chinese Materia Medica, China Pharmaceutical University . The experiments were carried out in accordance with the National Institutes of Health Guide for the care and use of laboratory animals . Mice were allowed a 1-week acclimatization period prior to entry into any experimental protocol. Prior to experiments, all animals were randomized into experimental groups and measured blindly.The model was produced as previously described . The micThe mice were randomized into six groups: sham, myocardial ischemia model, Bisoprolol, and three ESMS groups treated with different doses. According to the protocol, the treatment groups were orally administered ESMS  or Bisoprolol (8.3 mg/kg). The sham and model groups were treated with the same volume of double distilled water. The treatment was maintained for three weeks (once a day).After treatment, mice underwent an echocardiographic by using a Vevo 2100 Micro-imaging System  with a 30-MHz probe. The mice were anesthetized using 2.5% isoflurane. Each mouse was transferred to dorsal recumbency and then was placed on a heated imaging platform after fully anesthetized. The following parameters were calculated: LVEF, LVFS, IVS; d, LVPW; d, LVID; d, LV Vol; d, LV Mass, and RWT. All measurements were averaged for three consecutive cardiac cycles by an experienced technician who was the study groupings.At the end of the experiment, the heart tissue was removed, fixed with 4% paraformaldehyde, embedded in paraffin, sliced into sections of 5 μm thickness, stained with hematoxylin–eosin and Masson’s trichrome, and mounted. The histopathological changes were observed with a light microscope .At the end of the experiment, blood samples were collected. Serum samples were obtained by centrifugation of the blood samples at 15 °C for 10 min at 3500 rpm. LDH and CK activities were determined using respective kits , and BNP was measured using an ELISA kits , according to the manufacturer’s instructions.+-K+-ATPase, Ca2+-Mg2+-ATPase, Ca2+-ATPase activities, and Ca2+ levels were determined using the respective kits , following the manufacturer’s instructions. Heart tissue samples were weighed, homogenized in lysis buffer, and centrifuged at 4 °C for 10 min at 16,000× g. Caspase-3 activity was determined using a caspase-3 activity assay kit , following the manufacturer’s instructions.At the end of the experiment, the hearts were removed and gently washed in 0.9% sodium chloride. LV samples were taken, weighed and homogenized in lysis buffer and centrifuged at 4 °C for 10 min at 3500 rpm. The NaThe myocardium was harvested and fixed in 4% paraformaldehyde solution at three weeks after treatment. TUNEL staining was performed using the In Situ Cell Death Detection Kit, POD  following the manufacturer’s instructions. For each slice, five fields were randomly obtained under a light microscope (Leica). The region of peri-infarcted was analyzed, and the extent of cell apoptosis was expressed as the ratio of TUNEL positive nuclei.2 and 95% air at 37 °C. The cells were grown to 80–90% confluence prior to use in experiments.The H9c2 cardiomyocyte cell line was obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences . H9c2 cells were maintained in DMEM , and exposure to a humidified atmosphere of 5% CO2, 5% CO2, and 1% O2 at 37 °C. There were six different groups: control, OGD exposure (model), various concentrations of ESMS , and TMZ (10 μM) as a positive control for 12 h during OGD treatment. ESMS was dissolved in none-glucose DMEM, and TMZ was dissolved in phosphate buffer saline (PBS). Mitochondrial division inhibitor (Mdivi-1) was dissolved in dimethyl sulfoxide , which was diluted with none-glucose DMEM to ensure the final concentration of DMSO was 1:10,000.The H9c2 cell cultures were subjected to OGD for 12 h by incubation in a deoxygenated glucose-free balanced salt solution. The OGD injury was produced by incubation with none-glucose DMEM and exposure to a hypoxic environment of 94% NCell viability was determined through 3--2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded at 10,000 cells/well in 96-well plates. After different treatments, cells were incubated with MTT at a final concentration of 0.5 mg/mL for another 4 h. Then, the medium was discarded, 150 μL of DMSO was added with shaking for 10 min to dissolve the formazan crystals. The absorbance was read measured using a microplate reader  at a detection wavelength of 570 nm, with a reference wavelength of 650 nm, and cell viability was expressed as a percentage of the untreated control values. After different treatments, the culture supernatants were collected. The release of LDH was tested to further measure the extent of cell injury. The activity of LDH was determined using a LDH activity assay kit, following the manufacturer’s instructions.After each treatment, H9c2 cells were collected, washed with cold PBS, resuspended in Annexin V-FITC binding buffer. Then, the cells were incubated with Annexin V-FITC/PI for 5 min in the dark. Cellular fluorescence was analyzed by a flow cytometric assay using MACSQuant Analyzer 10 . At least 10,000 cells were detected in all experiments, and the data were analyzed with FCS Express V3 .+-K+-ATPase, Ca2+-Mg2+-ATPase, and Ca2+-ATPase activities were determined using the respective kit following the manufacturer’s instructions. For assessment of caspase activity, cardiomyocytes were lysed in ice-cold lysis buffer and centrifuged at 4 °C for 10 min at 16,000× g. Caspase-3 activity was determined using a caspase-3 activity assay kit, following the manufacturer’s instructions.H9c2 cardiomyocytes were lysed in ice-cold lysis buffer and centrifuged at 4 °C for 10 min at 3500 rpm. The NaH9c2 cells were seeded at a density of 10,000 cells/dish in 35-mm confocal dishes . After each treatment, the cells were washed three times with PBS and then incubated with Fluo-3 AM (10 μM) for 30 min at 37 °C. Then, the solution was discarded, extracellular unloaded free Fluo-3/AM was completely washed off with PBS, and images were scanned with a confocal laser scanning microscope . Fluorescence intensity was measured. (Fluo-3/AM: excitation/emission = 488/525 nm).H9c2 cells were seeded at a density of 10,000 cells/dish in 35-mm confocal dishes (Glass Bottom Dish). After each treatment, the cells were incubated with JC-1 staining solution (10 μg/mL) for 25 min at 37 °C. The cells were washed three times with PBS and immediately analyzed with a confocal laser scanning microscope. Green fluorescence reflects JC-1 monomers, which enters into the cytosol following mitochondrial membrane depolarization. Red fluorescence is ejected from JC-1 aggregation in the inner membrane of mitochondria. Data were expressed as the ratio of red to green fluorescence intensity, which indicates the level of depolarization of the mitochondrial membrane .® Deep Red FM: excitation/emission = 644/665 nm; Alexa Fluor 488 conjugated Donkey Anti-Goat IgG (H+L) antibody: excitation/emission = 493/519 nm; DAPI: excitation/emission = 340/488 nm).H9c2 cells were seeded at a density of 10,000 cells/dish in 35-mm confocal dishes , and treated with ESMS and Mdivi-1 (20 μM) with the OGD injury. After each treatment, the cells were washed with PBS. Then they were incubated at 37 °C with Mito Tracker® Deep Red FM (400 nM) for 30 min. The cells were followed up with fixing, permeabilizing, and incubation with anti-Drp1 primary antibody. The cells were subsequently incubated with an Alexa Fluor 488 conjugated Donkey Anti-Goat IgG (H+L) antibody and DAPI. The structures of mitochondria were viewed with a confocal laser scanning microscope. . Equal amounts of proteins (35 μg) were separated by 10% SDS-PAGE, and subsequently transferred to polyvinyl difluoride (PVDF) membranes  by electrophoresis. The membranes were blocked with 3% BSA and stained with primary antibodies against Bax (1:1000), Bcl-2 (1:1000) and caspase-3 (1:1000), all from CST ; CnA ; Drp1 ; p-Drp1(Ser637) ; p-Drp1(Ser616) ; and β-actin  for 16 h at 4 °C. The primary antibodies were followed by incubation with the HRP-conjugated secondary antibodies . The visualization of protein signal was detected using enhanced chemiluminescence . The results were quantified using Image Lab™ software .p < 0.05 were considered statistically significant. In addition, all statistical analyses were performed using GraphPad Prism 6.01 .All data were presented as the means ± SD. One-way analysis of variance (ANOVA) was performed to analyze the data, followed by Dunnett’s post-hoc test when the data involved three or more groups. Values of 2+-calcineurin-mediated Drp1 signaling pathways, thereby inhibiting mitochondrial mediated apoptosis. These findings provided insight into the function and mechanism of ESMS in the treatment MI-induced HF.Our findings indicated for the first time that ESMS can attenuate MI-induced HF by improving cardiac function, repairing cardiac structural damage, and reducing histopathological damage. ESMS attenuated MI injury-induced HF by modulating Ca