Gene silencing dynamics are modulated by transiently active regulatory elements.

Transcriptional enhancers have been extensively characterized, but cis-regulatory elements involved in acute gene repression have received less attention. Transcription factor GATA1 promotes erythroid differentiation by activating and repressing distinct gene sets. Here, we study the mechanism by which GATA1 silences the proliferative gene Kit during murine erythroid cell maturation and define stages from initial loss of activation to heterochromatinization. We find that GATA1 inactivates a potent upstream enhancer but concomitantly creates a discrete intronic regulatory region marked by H3K27ac, short noncoding RNAs, and de novo chromatin looping. This enhancer-like element forms transiently and serves to delay Kit silencing. The element is ultimately erased via the FOG1/NuRD deacetylase complex, as revealed by the study of a disease-associated GATA1 variant. Hence, regulatory sites can be self-limiting by dynamic co-factor usage. Genome-wide analyses across cell types and species uncover transiently active elements at numerous genes during repression, suggesting that modulation of silencing kinetics is widespread.

Interspecies regulatory landscapes and elements revealed by novel joint systematic integration of human and mouse blood cell epigenomes.

Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state Regulatory Potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbored distinctive transcription factor binding motifs that were similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we showed that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.

Up-regulation of BTN3A1 on CD14+ cells promotes Vγ9Vδ2 T cell activation in psoriasis.

Vγ9Vδ2 T cells play an important role in the development and progression of psoriasis vulgaris (PV), but how they promote skin inflammation and the molecular mechanisms underlying Vγ9Vδ2 T cell dysfunction are poorly understood. Here, we show that circulating Vγ9Vδ2 T cells are decreased and exhibit enhanced proliferation and increased production of IFN-γ and TNF-α in PV patients. Monocytes from PV patients express higher levels of the phosphoantigen sensor butyrophilin 3A1 (BTN3A1) than monocytes from healthy controls. Blockade of BTN3A1 suppresses Vγ9Vδ2 T cell activation and abolishes the difference in Vγ9Vδ2 T cell activation between PV patients and healthy controls. The CD14+ cells in PV skin lesions highly express BTN3A1 and juxtapose to Vδ2 T cells. In addition, IFN-γ induces the up-regulation of BTN3A1 on monocytes. Collectively, our results demonstrate a crucial role of BTN3A1 on monocytes in regulating Vγ9Vδ2 T cell activation and highlight BTN3A1 as a potential therapeutic target for psoriasis.

FeP-Based Nanotheranostic Platform for Enhanced Phototherapy/Ferroptosis/Chemodynamic Therapy.

Ferroptosis is an iron-dependent and lipid peroxides (LPO)-overloaded programmed damage cell death, induced by glutathione (GSH) depletion and glutathione peroxide 4 (GPX4) inactivation. However, the inadequacy of endogenous iron and reactive oxygen species (ROS) restricts the efficacy of ferroptosis. To overcome this obstacle, a near-infrared photo-responsive FeP@PEG NPs is fabricated. Exogenous iron pool can enhance the effect of ferroptosis via the depletion of GSH and further regulate GPX4 inactivation. Generation of ·OH derived from the Fenton reaction is proved by increased accumulation of lipid peroxides. The heat generated by photothermal therapy and ROS generated by photodynamic therapy can enhance cell apoptosis under near-infrared (NIR-808 nm) irradiation, as evidenced by mitochondrial dysfunction and further accumulation of lipid peroxide content. FeP@PEG NPs can significantly inhibit the growth of several types of cancer cells in vitro and in vivo, which is validated by theoretical and experimental results. Meanwhile, FeP@PEG NPs show excellent T2-weighted magnetic resonance imaging (MRI) property. In summary, the FeP-based nanotheranostic platform for enhanced phototherapy/ferroptosis/chemodynamic therapy provides a reliable opportunity for clinical cancer theranostics.

Unlinking an lncRNA from Its Associated cis Element.

Long non-coding (lnc) RNAs can regulate gene expression and protein functions. However, the proportion of lncRNAs with biological activities among the thousands expressed in mammalian cells is controversial. We studied Lockd (lncRNA downstream of Cdkn1b), a 434-nt polyadenylated lncRNA originating 4 kb 3' to the Cdkn1b gene. Deletion of the 25-kb Lockd locus reduced Cdkn1b transcription by approximately 70% in an erythroid cell line. In contrast, homozygous insertion of a polyadenylation cassette 80 bp downstream of the Lockd transcription start site reduced the entire lncRNA transcript level by >90% with no effect on Cdkn1b transcription. The Lockd promoter contains a DNase-hypersensitive site, binds numerous transcription factors, and physically associates with the Cdkn1b promoter in chromosomal conformation capture studies. Therefore, the Lockd gene positively regulates Cdkn1b transcription through an enhancer-like cis element, whereas the lncRNA itself is dispensable, which may be the case for other lncRNAs.

Direct modulation bandwidth enhancement of uncooled DFB laser operating over a wide temperature range based on groove-in-trench waveguide structure.

Uncooled direct modulation DFB laser offers high speed transmission rate over a wide temperature range with high reliability and low cost, making it a cost-effective light source choice for 5G fronthaul and data center applications. However, a significant 3dB bandwidth decrease can be observed in high temperature for conventional DFB lasers. We present an uncooled DFB laser operating up to 85°C with extended direct modulation bandwidth and high reliability based on a novel groove-in-trench ridge waveguide structure, where two narrow grooves penetrating the active layer are etched symmetrically in the two conventional trenches by deep wet etching, respectively. By optimizing the distance between the groove and the mesa stripe, we obtain a 3dB bandwidth of 15.3 GHz at 85°C, which is a 3.7 GHz improvement compared with the conventional ridge waveguide DFB laser. Transmissions of 25 Gb/s NRZ signal at 25°C and 85°C with clear eye openings have been demonstrated. It also achieves 25 Gb/s transmission over 10 km optical fiber with a low power penalty of 0.5 dB for a bit error rate of 10-12 at 85°C. In addition, the result of 2000-hour aging test shows that the proposed groove-in-trench structure DFB lasers have the same excellent reliability as the conventional ones.

Experiment demonstration of high speed 1.3 µm grating assisted surface-emitting DFB lasers.

Surface emitting lasers are attractive light sources for silicon integrated photonic circuits. High speed direct operation is of great importance for these lasers in high capacity and low cost on-chip communication system. Here, we demonstrate a 1.3 µm surface emitting ridge-waveguide distributed feedback (DFB) laser with second order grating and λ/4 phase shift grating, which can achieve a 24 Gb/s operation over a wide temperature. The fabricated lasers can achieve low threshold current as 6.8 mA, and 12.5 mA at 20, and 70°C, respectively. Stable single mode operation has been observed with high side mode suppression ratio (SMSR) > 40 dB at all temperatures (20-70 °C). Meanwhile, the surface emitting optical power can reach 1.7 mW at high temperature as 70 °C. 3 dB bandwidth of small signal response is 21 GHz and 12 GHz at 20 °C and 70 °C respectively. The far-field divergence angle of surface emitting beam is 13.4°×20.2° of 10 µm length second order grating coupler. The proposed laser may have great advantages of single mode, high speed modulation and good temperature tolerance. In addition, compared with conventional DFB lasers, the surface emitting DFB laser has no additional manufacturing process, which is simple to fabricate and easy to integrate with silicon platform.

Activation of Autophagy Induces Monocrotaline-Induced Pulmonary Arterial Hypertension by FOXM1-Mediated FAK Phosphorylation.

PURPOSE: It has been shown that activation of autophagy promotes the development of pulmonary arterial hypertension (PAH). Meanwhile, forkhead box M1 (FOXM1) has been found to induce autophagy in several types of cancer. However, it is still unclear whether FOXM1 mediates autophagy activation in PAH, and detailed mechanisms responsible for these processes are indefinite. METHOD: PAH was induced by a single intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-smooth muscle actin (α-SMA) staining, and Ki67 staining were performed to evaluate the development of PAH. The protein levels of FOXM1, phospho-focal adhesion kinase (p-FAK), FAK, and LC3B were determined by immunoblotting or immunohistochemistry. RESULTS: FOXM1 protein level and FAK activity were significantly increased in MCT-induced PAH rats, this was accompanied with the activation of autophagy. Pharmacological inhibition of FOXM1 or FAK suppressed MCT-induced autophagy activation, decreased RVSP, RVHI and %MT in MCT-induced PAH rats, and inhibited the proliferation of pulmonary arterial smooth muscle cells and pulmonary vessel muscularization in MCT-induced PAH rats. CONCLUSION: FOXM1 promotes the development of PAH by inducing FAK phosphorylation and subsequent activation of autophagy in MCT-treated rats.

Factors affecting physicians using mobile health applications: an empirical study.

BACKGROUND: Mobile health applications (mHealth apps) have created innovative service channels for patients with chronic diseases. These innovative service channels require physicians to actively use mHealth apps. However, few studies investigate physicians' participation in mHealth apps. OBJECTIVE: This study aims to empirically explore factors affecting physicians' usage behaviors of mHealth apps. Based on the extended Unified Theory of Acceptance and Use of Technology (UTAUT2) and mHealth apps features, we propose a research model including altruism, cognitive trust, and online ratings. METHODS: We collected data from physicians who have used mHealth apps and conducted a factor analysis to verify the convergence and discriminative effects. We used a hierarchical regression method to test the path coefficients and statistical significance of our research model. In addition, we adopted bootstrapping approach and further analyzed the mediating effects of behavioral intention between all antecedent variables and physicians' usage behavior. Finally, we conducted three robustness analyses to test the validity of results and tested the constructs to verify the common method bias. RESULTS: Our results support the effects of performance expectancy, effort expectancy, social influence, and altruism on the behavioral intentions of physicians using mHealth apps. Moreover, facilitating conditions and habits positively affect physicians using mHealth apps through the mediating effort of behavioral intention. Physicians' cognitive trust and online rating have significant effects on their usage behaviors through the mediating efforts of behavioral intention. CONCLUSIONS: This study contributes to the existing literature on UTAUT2 extension of physicians' acceptance of mHealth apps by adding altruism, cognitive trust, and online ratings. The results of this study provide a novel perspective in understanding the factors affecting physicians' usage behaviors on mHealth apps in China and provide such apps' managers with an insight into the promotion of physicians' active acceptance and usage behaviors.

Identification of novel Aß-LilrB2 inhibitors as potential therapeutic agents for Alzheimer's disease.

LilrB2 is an Aß receptor with high affinity, which not only contributes to memory deficits but also mediates the loss of synaptic plasticity. Thus, Aß-LilrB2 interaction inhibitors (ALIs) might be a potential therapeutic strategy for Alzheimer's disease. In this study, an ELISA-based interaction assay was established as a novel approach to identify ALIs and was used to screen 110 compounds from a compound library. Among the 110 compounds, four compounds presented IC50 values lower than the positive control flusipirilene. The two phenyl-1,3,5-triazine derivatives (compound 103 and 104) displayed inhibitory activities with the IC50 of 0.23 µM and 0.05 µM respectively. The neuroprotection activities of the hit compounds were evaluated in SH-SY5Y cell line. Compound 104 presented good safety and neuroprotective effects against Aß. Further study of its effect on the downstream pathway of Aß indicated that compound 104 was able to reverse the Aß induced cofilin dephosphorylation, tau hyperphosphorylation and neurite outgrowth inhibition. The docking study showed that fluspirilene and compound 104 were favorably positioned into the Ben 3 and 4 binding pockets via their aromatic ring, which was similar to that reported for Aß. Based on these facts, compound 104 can be identified as a potential ALI which might be of therapeutic importance for AD treatment.

Regulation of gene expression by miR-144/451 during mouse erythropoiesis.

The microRNA (miRNA) locus miR-144/451 is abundantly expressed in erythrocyte precursors, facilitating their terminal maturation and protecting against oxidant stress. However, the full repertoire of erythroid miR-144/451 target messenger RNAs (mRNAs) and associated cellular pathways is unknown. In general, the numbers of mRNAs predicted to be targeted by an miRNA vary greatly from hundreds to thousands, and are dependent on experimental approaches. To comprehensively and accurately identify erythroid miR-144/451 target mRNAs, we compared gene knockout and wild-type fetal liver erythroblasts by RNA sequencing, quantitative proteomics, and RNA immunoprecipitation of Argonaute (Ago), a component of the RNA-induced silencing complex that binds miRNAs complexed to their target mRNAs. Argonaute bound ∼1400 erythroblast mRNAs in a miR-144/451-dependent manner, accounting for one-third of all Ago-bound mRNAs. However, only ∼100 mRNAs were stabilized after miR-144/451 loss. Thus, miR-144 and miR-451 deregulate <10% of mRNAs that they bind, a characteristic that likely applies generally to other miRNAs. Using stringent selection criteria, we identified 53 novel miR-144/451 target mRNAs. One of these, Cox10, facilitates the assembly of mitochondrial electron transport complex IV. Loss of miR-144/451 caused increased Cox10 mRNA and protein, accumulation of complex IV, and increased mitochondrial membrane potential with no change in mitochondrial mass. Thus, miR-144/451 represses mitochondrial respiration during erythropoiesis by inhibiting the production of Cox10.

Role of ultrasound in the diagnosis of primary and recurrent dermatofibrosarcoma protuberans.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a rare, low- to intermediate-grade sarcoma, which represents a diagnostic imaging challenge. This study aimed to analyze the clinical and ultrasound features of primary and recurrent DFSP to improve the diagnosis. METHODS: Clinical, imaging, and pathological data from a total of 58 patients (23 patients with primary DFSP and 35 patients with recurrent DFSP) were retrospectively reviewed. RESULTS: There was no statistically significant difference in age, sex, tumor size, or echogenicity between the two groups. Most of the primary DFSP lesions involved the overlying dermis and hypodermis, while most of the recurrent DFSP lesions were fixated to more deeply seated structures at the original surgical incision. Red nodules on the skin were found more frequently in the primary group. There were statistically significant differences in the type of lesion and ultrasound tumor morphology (p < 0.050). The lesions in the primary group showed more tentacle-like projections or a "claw" sign, while the lesions in the recurrent group were more commonly oval, lobulated, and irregularly shaped. Hypervascularity was common in both groups. CONCLUSIONS: For primary DFSP, a slow-growing, red nodule on the skin involving the overlying dermis and hypodermis, more frequently a hypoechoic mass with tentacle-like projections or a "claw" sign, was observed. For recurrent DFSP, palpable subcutaneous nodules or subcutaneous masses at the original surgical incision and oval, lobulated, and irregularly shaped lesions were more commonly observed. This may be useful for improving diagnostic accuracy.

Mvda is required for zebrafish early development.

BACKGROUND: The MVD gene mutations are identified in porokeratosis, which is considered a skin-specific autoinflammatory keratinization disease. However, the biological function of MVD gene remains largely unknown. Therefore, we analyzed the function of mvda gene, orthologous to the human MVD gene, in developing zebrafish. METHODS: Morpholino antisense oligonucleotide technique was used to generate mvda loss-of-function phenotypes. Knockdown of mvda was confirmed by RT-PCR and Sanger sequencing. Scanning and transmission electron microscopy were performed to analyze the morphology of the epidermis. Angiogenesis study was presented using the Tg(fli1a:EGFP)y1 transgenic strain. In addition, acridine orange staining was used to examine the apoptotic cells in vivo. RESULTS: As expected, the mvda morphants showed abnormal morphology of the epidermis. Moreover, we observed ectopic sprouts in trunk angiogenesis and impaired formation of the caudal vein plexus in the mvda-deficient zebrafish. Besides, increased apoptosis was found throughout the tail, heart, and eyes in mvda zebrafish morphants. CONCLUSIONS: These findings indicated the essential role of mvda in the early development of zebrafish. This was the first in vivo knockdown study of the zebrafish mvda gene, which might offer insight into the biological function of the human MVD gene.

Influence of Online Social Support on the Public's Belief in Overcoming COVID-19.

The sudden outbreak of coronavirus disease 2019 (COVID-19) in early 2020 has dramatically changed people's lives. Some countries have taken mass home quarantine to control the virus. However, the side effects of quarantine have rarely been interrogated by current COVID-19 research. This study thus investigates the effects of online social support on the public's beliefs in overcoming COVID-19 by embracing their cognition and emotion during the epidemic. First, by crawling and content analysis of the messages posted on "Baidu COVID-19 bar", this study identified 5 types of online social support given or received by the public during COVID-19. On this basis, a model explaining the public's beliefs was developed from the perspectives of online social support, cognition and emotion. 334 valid online questionnaires were collected to examine the proposed model and hypotheses. The results show that cognition has a direct effect on the belief, while emotion affects the belief via a full mediating effect of cognition. Tangible support and esteem support can directly affect the public's beliefs, and educational level significantly moderates these effects. In addition, the public's cognition is influenced by informational support, however, emotion is not influenced by social support but by other factors (e.g., information disclosure, material supplies and frustration caused by the epidemic). These research results provide a deep insight into how to reduce the negative effects of quarantine, consolidate the theoretical basis of the public's beliefs, and have important practical implications for individuals and the government in dealing with such emergencies.

Chrm3 protects against acinar cell necrosis by stabilizing caspase-8 expression in severe acute pancreatitis mice model.

Acinar cells in acute pancreatitis (AP) die through apoptosis and necrosis, the impacts of which are quite different. Early clinical interference strategies on preventing the progress of AP to severe acute pancreatitis (SAP) are the elimination of inflammation response and inhibition of necrosis. Muscarinic acetylcholine receptor M3 was encoded by Chrm3 gene. It is one of the best-characterized receptors of pancreatic ß cells and regulates insulin secretion, but its function in AP remains unclear. In this study, we explored the function of Chrm3 gene in the regulation of cell death in l-arginine-induced SAP animal models. We found that Chrm3 was upregulated in pancreatitis, and we further confirmed the localization of Chrm3 resided in both pancreatic islets and acinar cell membranes. The reduction of Chrm3 decreased the pathological lesion of SAP and reduced amylase activities in serum. Consistently, Chrm3 can suppress acinar cells necrosis markedly, but has no effect on regulating apoptosis after l-arginine treatment. It was shown that Chrm3 attenuated acinar cells necrosis at least in part by stabilizing caspase-8. Thus, this study indicates that Chrm3 is critical participants in SAP, and regulation of Chrm3 expression might be a useful therapeutic strategy for preventing pathologic necrosis.

Polarization and direction-controlled asymmetric multifunctional metadevice for focusing, vortex and Bessel beam generation.

Integrating multiple independent functionalities into one single photonic device has been an important part in optoelectronic system. In this paper, we here propose a kind of asymmetric multifunctional metadevice operating at 1550 nm (in optical communication band), which can manipulate the light with four different functions, depending on the polarization and illumination direction of incident light. As a proof of our concept, we design this metadevice composed of the upper metasurface layer, middle grating layer and lower metasurface layer. For x-polarized incident light, the metadevice under forward illumination works as transmissive focusing lens and vortex beam generator of y-polarized light, while under backward illumination it acts as a reflective vortex beam generator. In contrast, for y-polarized incident light, the metadevice under forward illumination behaves as a reflective Bessel beam generator, while a combination of transmissive vortex beam generator and focusing lens of x-polarized light under backward illumination. Our findings may motivate the realization of high-performance multifunctional metadevices and extend the application in complex integrated optical system.

A cellular model for Wilson's disease using patient-derived induced pluripotent stem cells revealed aberrant ß-catenin pathway during osteogenesis.

Wilson's disease (WD) is a rare autosomal recessive disorder of copper metabolism caused by an ATP7B gene mutation. Except for hepatic, neurological symptoms, lower bone mineral density is another most frequent clinical features of WD, but the underlying mechanisms have not been fully understood. This article aims to use induced pluripotent stem cells (iPSCs) to establish cellular osteoblasts model related to WD to identify abnormal osteogenesis and signaling pathways. In this study, we successfully produced functional osteoblasts from normal and WD iPSCs through embryoid bodies (EBs) formation method, and then we found WD osteoblasts may have a lower osteogenesis activity than normal controls by detection of osteogenic marker genes and mineralization ability. Further, through gene expression profiling, detection of ß-catenin in total protein and nuclear protein, and the nuclear localization of ß-catenin, we identified and validated that low osteogenic activity in WD may be due to abnormal ß-catenin pathway. Interestingly, we found SKL2001, a small molecule can reverse decreased osteogenesis of WD. In summery, our results suggested that the low bone density of WD may caused by abnormal ß-catenin signaling pathway, and these may provided a new target for the treatment of WD.

C-X-C Chemokine Receptor 4 in Diffuse Large B Cell Lymphoma: Achievements and Challenges.

Diffuse large B cell lymphoma (DLBCL), an aggressive cancer of the B cells, is the most common subtype of non-Hodgkin lymphoma (NHL) worldwide. In China, the cases of DLBCL increase yearly. C-X-C chemokine receptor 4 (CXCR4) has been implicated in the migration and trafficking of malignant B cells in several hematological malignancies, and only a few reports have been published on the role of CXCR4 in the metastasis of DLBCL. This review summarizes the relevant perspectives on the functional mechanism, prognostic significance, and therapeutic applications of the CXCL12/CXCR4 axis in DLBCL, in particular DLBCL with bone marrow involvement.

Icariin Promotes the Osteogenic Action of BMP2 by Activating the cAMP Signaling Pathway.

Icariin (ICA) is the main active flavonoid glucoside from herbs of the genus Epimedium; in traditional Chinese medicine, these herbs have long been prescribed for the treatment of bone fractures and osteoporosis. Several studies have shown that treatment with ICA can increase osteogenic differentiation and reduce bone loss in vivo and in vitro. However, the definite signaling pathway of this osteogenic effect remains unclear. In this study, we selected bone morphogenetic protein 2 (BMP2)-induced osteoblastic differentiation of multipotent mesenchymal progenitor C2C12 cells as a model of osteoblast differentiation. We investigated the effects of ICA on C2C12 cells osteogenic differentiation and the underlying molecular mechanisms. We found that ICA could enhance BMP2-mediated osteoblastic differentiation of C2C12 cells in a dose-dependent manner. Treatment with ICA activated the cAMP/PKA/CREB signaling axis in a time-dependent manner. Blocking cAMP signaling using the PKA selective inhibitor H89 significantly inhibited the stimulatory effect of ICA on osteogenesis. Therefore, the osteoinductive potential and the low cost of ICA indicate that it is a promising alternative treatment or promoter for enhancing the therapeutic effects of BMP2.

Shenxian-Shengmai Oral Liquid Reduces Myocardial Oxidative Stress and Protects Myocardium from Ischemia-Reperfusion Injury.

BACKGROUND/AIMS: Shenxian-shengmai (SXSM) oral liquid, a Chinese patent compound medicine, has been used to treat sinus bradyarrhythmias induced by mild sick sinus syndrome in clinical practice. Myocardial ischemia, in particular in serious or right coronary-related heart diseases, can cause bradyarrhythmias and cardiac dysfunction. Moreover, reperfusion of ischemic myocardium is associated with additional myocardial damage known as myocardial ischemia-reperfusion (I/R) injury. This study was designed to evaluate the effects of SXSM on bradyarrhythmias and cardiac dysfunction induced by myocardial I/R injury, and to explore the underlying mechanisms. METHODS: Administration of SXSM to adult male Sprague Dawley (SD) rats was achieved orally by gavage and control rats were given equivalent deionized water every day for 14 days. After the last administration, the heart was connected with the Langendorff perfusion apparatus and both groups were subjected to ischemia for 20 min followed by reperfusion for 40 min to induce myocardial I/R injury. Heart rate (HR), left ventricular developed pressure (LVDP), the maximal increase rate of left ventricular pressure (+dp/dtmax) and the maximal decrease rate of left ventricular pressure (-dp/dtmax) were recorded by a physiological signal acquisition system. The heart treated with ischemic preconditioning (IPC) for 3 times at a range of 5 min/time before ischemia served as a positive control group. The hearts without I/R injury served as control group. After reperfusion, superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-Px) activities in the myocardium were determined by appropriate assay kits. Myocardial SOD1 and glutamate cysteine ligase catalytic subunit (GCLC) expression were assessed by western blot analysis. For the in vitro study, SXSM serum was prepared according to the serum pharmacological method and neonatal rat cardiomyocytes were isolated from the heart of new born SD rats. Neonatal rat cardiomyocytes were pretreated with SXSM serum and subjected to H2O2 or anoxia/ reoxygenation (A/R) treatment to induce oxidative damage. Cell viability was evaluated using a Cell Counting Kit-8 (CCK8) assay. Levels of reactive oxygen species (ROS), SOD, GSH and GSH-Px in cardiomyocytes were determined by appropriate assay kits. SOD1 and GCLC expression were assessed by western blot analysis. Buthionine-[S, R]-sulfoximine (BSO), a GCLC inhibitor, and SOD1 siRNA were also used for identifying the cardiac protective targets of SXSM. RESULTS: SXSM and ischemic preconditioning (IPC) significantly increased heart rate during myocardial reperfusion and protected cardiac function against myocardial I/R injury, including an increase in left ventricular diastolic pressure (LVDP), the maximal increase rate of left ventricular pressure (+dp/dtmax) and the maximal decrease rate of left ventricular pressure (-dp/dtmax). We also found that SXSM and IPC improved the expansion of myocardial interstitium, the structural abnormality and morphological changes of cardiomyocytes induced by I/R injury. Meanwhile, SXSM protected cardiomyocytes against the oxidative damage induced by H2O2 and A/R injury through reducing intracellular ROS levels. Moreover, SXSM increased SOD activity through enhancing SOD1 expression and increased GSH content through promoting GCLC expression as well as GSH-Px activity. BSO and SOD1 siRNA counteracted anti-arrhythmic and cardiac protective effect of SXSM, suggesting that the therapeutic targets of SXSM might be SOD1 and GCLC. CONCLUSION: SXSM is effective in protecting the myocardium from I/R injury, with myocardial SOD1 and GCLC being the potential therapeutic targets.