Mechanistic Insights from the Crystal Structure and Computational Analysis of the Radical SAM Deaminase DesII.

Radical S-adenosyl-L-methionine (SAM) enzymes couple the reductive cleavage of SAM to radical-mediated transformations that have proven to be quite broad in scope. DesII is one such enzyme from the biosynthetic pathway of TDP-desosamine where it catalyzes a radical-mediated deamination. Previous studies have suggested that this reaction proceeds via direct elimination of ammonia from an α-hydroxyalkyl radical or its conjugate base (i.e., a ketyl radical) rather than 1,2-migration of the amino group to form a carbinolamine radical intermediate. However, without a crystal structure, the active site features responsible for this chemistry have remained largely unknown. The crystallographic studies described herein help to fill this gap by providing a structural description of the DesII active site. Computational analyses based on the solved crystal structure are consistent with direct elimination and indicate that an active site glutamate residue likely serves as a general base to promote deprotonation of the α-hydroxyalkyl radical intermediate and elimination of the ammonia group.

Modular Access to Chiral Amines via Imine Reductase-Based Photoenzymatic Catalysis.

The development of catalysts serves as the cornerstone of innovation in synthesis, as exemplified by the recent discovery of photoenzymes. However, the repertoire of naturally occurring enzymes repurposed by direct light excitation to catalyze new-to-nature photobiotransformations is currently limited to flavoproteins and keto-reductases. Herein, we shed light on imine reductases (IREDs) that catalyze the remote C(sp3)-C(sp3) bond formation, providing a previously elusive radical hydroalkylation of enamides for accessing chiral amines (45 examples with up to 99% enantiomeric excess). Beyond their natural function in catalyzing two-electron reductive amination reactions, upon direct visible-light excitation or in synergy with a synthetic photoredox catalyst, IREDs are repurposed to tune the non-natural photoinduced single-electron radical processes. By conducting wet mechanistic experiments and computational simulations, we unravel how engineered IREDs direct radical intermediates toward the productive and enantioselective pathway. This work represents a promising paradigm for harnessing nature's catalysts for new-to-nature asymmetric transformations that remain challenging through traditional chemocatalytic methods.

Molecular Insights into Converting Hydroxide Adenosyltransferase into Halogenase.

Halogenation plays a unique role in the design of agrochemicals. Enzymatic halogenation reactions have attracted great attention due to their excellent specificity and mild reaction conditions. S-adenosyl-l-methionine (SAM)-dependent halogenases mediate the nucleophilic attack of halide ions (X-) to SAM to produce 5'-XDA. However, only 11 SAM-dependent fluorinases and 3 chlorinases have been reported, highlighting the desire for additional halogenases. SAM-dependent hydroxide adenosyltransferase (HATase) has a similar reaction mechanism as halogenases but uses water as a substrate instead of halide ions. Here, we explored a HATase from the thermophile Thermotoga maritima MSB8 and transformed it into a halogenase. We identified a key dyad W8L/V71T for the halogenation reaction. We also obtained the best performing mutants for each halogenation reaction: M1, M2 and M4 for Cl-, Br- and I-, respectively. The M4 mutant retained the thermostability of HATase in the iodination reaction at 80 °C, which surpasses the natural halogenase SalL. QM/MM revealed that these mutants bind halide ions with more suitable angles for nucleophilic attack of C5' of SAM, thus conferring halogenation capabilities. Our work achieved the halide ion specificity of halogenases and generated thermostable halogenases for the first time, which provides new opportunities to expand the halogenase repertoire from hydroxylase.

Substrate Conformational Switch Enables the Stereoselective Dimerization in P450 NascB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations.

P450 NascB catalyzes the coupling of cyclo-(l-tryptophan-l-proline) (1) to generate (-)-naseseazine C (2) through intramolecular C-N bond formation and intermolecular C-C coupling. A thorough understanding of its catalytic mechanism is crucial for the engineering or design of P450-catalyzed C-N dimerization reactions. By employing MD simulations, QM/MM calculations, and enhanced sampling, we assessed various mechanisms from recent works. Our study demonstrates that the most favorable pathway entails the transfer of a hydrogen atom from N7-H to Cpd I. Subsequently, there is a conformational change in the substrate radical, shifting it from the Re-face to the Si-face of N7 in Substrate 1. The Si-face conformation of Substrate 1 is stabilized by the protein environment and the π-π stacking interaction between the indole ring and heme porphyrin. The subsequent intermolecular C3-C6' bond formation between Substrate 1 radical and Substrate 2 occurs via a radical attack mechanism. The conformational switch of the Substrate 1 radical not only lowers the barrier of the intermolecular C3-C6' bond formation but also yields the correct stereoselectivity observed in experiments. In addition, we evaluated the reactivity of the ferric-superoxide species, showing it is not reactive enough to initiate the hydrogen atom abstraction from the indole NH group of the substrate. Our simulation provides a comprehensive mechanistic insight into how the P450 enzyme precisely controls both the intramolecular C-N cyclization and intermolecular C-C coupling. The current findings align with the available experimental data, emphasizing the pivotal role of substrate dynamics in governing P450 catalysis.

Photoenzymatic Enantioselective Intermolecular Radical Hydroamination.

Since the discovery of Hofmann-Löffler-Freytag reaction more than 130 years ago, nitrogen-centered radicals have been widely studied in both structures and reactivities1-2. Nevertheless, catalytic enantioselective intermolecular radical hydroamination remains a challenge due to the existence of side reactions, short lifetime of nitrogen-centered radicals, and lack of understanding of the fundamental catalytic steps. In chemistry, nitrogen-centered radicals are produced with radical initiators, photocatalysts, or electrocatalysts. On the other hand, the generation and reaction of nitrogen-centered radicals are unknown in nature. Here we report a pure biocatalytic system by successfully repurposing an ene-reductase through directed evolution for the photoenzymatic production of nitrogen-centered radicals and enantioselective intermolecular radical hydroaminations. These reactions progress efficiently at room temperature under visible light without any external photocatalysts and exhibit excellent enantioselectivities. Detailed mechanistic study reveals that the enantioselectivity originates from the radical-addition step while the reactivity originates from the ultrafast photoinduced electron transfer (ET) from reduced flavin mononucleotide (FMNH-) to nitrogen-containing substrates.

Radical S-Adenosyl Methionine Enzyme BlsE Catalyzes a Radical-Mediated 1,2-Diol Dehydration during the Biosynthesis of Blasticidin S.

The biosynthesis of blasticidin S has drawn attention due to the participation of the radical S-adenosyl methionine (SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated, redox-neutral decarboxylase is unusual because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4'-keto-3'-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogues, as well as computational models based on X-ray crystal structures of the BlsE·SAM (2.09 Å) and BlsE·SAM·CGA (2.62 Å) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4' α-hydroxyalkyl radical as opposed to 1,2-migration of the C3'-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE are discussed.

Naringin attenuates high glucose-induced injuries and inflammation by modulating the leptin-JAK2/STAT3 pathway in H9c2 cardiac cells.

INTRODUCTION: Our previous study showed that naringin (NRG) protects cardiomyocytes against high glucose (HG)-induced injuries by inhibiting p38 mitogen-activated protein kinase (MAPK). Leptin induces hypertrophy in rat cardiomyocytes via p38/MAPK activation. The present study aimed to test the hypothesis that leptin-Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3), which are responsible for leptin's functions, are involved in HG-induced injuries and cardioprotective effects of NRG in cardiomyocytes. MATERIAL AND METHODS: H9c2 cells were exposed to HG for 24 h to establish a cardiomyocyte injury model. Cells were pretreated with NRG and other drugs before exposure to HG. Protein expression was measured by western blot analysis. Cell viability was detected by Cell Counting Kit-8 assay. Apoptotic cells were assessed by Hoechst 33258 staining assay. Intracellular reactive oxygen species levels were determined by dichlorofluorescein diacetate staining. Mitochondrial membrane potential was evaluated using JC-1. An enzyme-linked immunosorbent assay was performed to determine the inflammatory cytokines. RESULTS: NRG significantly attenuated HG-induced increases in leptin and Ob-R expression. Pretreatment with either a leptin antagonist (LA) or NRG markedly ameliorated HG-induced elevation of phosphorylated (p)-JAK2 and p-STAT3, respectively. Pretreatment with NRG, LA, Ob-R antagonist, or AG490 clearly alleviated HG-induced injuries and inflammation. CONCLUSIONS: This study provides new evidence of the NRG protective effects of H9c2 cells against HG-induced injuries possibly via modulation of the leptin-JAK2/STAT3 pathway.

Spin-Regulated Electron Transfer and Exchange-Enhanced Reactivity in Fe4 S4 -Mediated Redox Reaction of the Dph2 Enzyme During the Biosynthesis of Diphthamide.

The [4Fe-4S]-dependent radical S-adenosylmethionine (SAM) proteins is one of large families of redox enzymes that are able to carry a panoply of challenging transformations. Despite the extensive studies of structure-function relationships of radical SAM (RS) enzymes, the electronic state-dependent reactivity of the [4Fe-4S] cluster in these enzymes remains elusive. Using combined MD simulations and QM/MM calculations, we deciphered the electronic state-dependent reactivity of the [4Fe-4S] cluster in Dph2, a key enzyme involved in the biosynthesis of diphthamide. Our calculations show that the reductive cleavage of the S-C(γ) bond is highly dependent on the electronic structure of [4Fe-4S]. Interestingly, the six electronic states can be classified into a low-energy and a high-energy groups, which are correlated with the net spin of Fe4 atom ligated to SAM. Due to the driving force of Fe4-C(γ) bonding, the net spin on the Fe4 moiety dictate the shift of the opposite spin electron from the Fe1-Fe2-Fe3 block to SAM. Such spin-regulated electron transfer results in the exchange-enhanced reactivity in the lower-energy group compared with those in the higher-energy group. This reactivity principle provides fundamental mechanistic insights into reactivities of [4Fe-4S] cluster in RS enzymes.

[Corrigendum] Exogenous hydrogen sulfide exerts proliferation, anti-apoptosis, migration effects and accelerates cell cycle progression in multiple myeloma cells via activating the Akt pathway.

Following the publication of this article, an interested reader drew to the authors' attention that, in Fig. 4 on p. 1913, the t-Akt panel in Fig. 4A looked unexpectedly similar to the ß-actin panel in Fig. 4C. The authors were able to refer back to their original data, and realized that the Figure had been compiled incorrectly; essentially, the data for the t-Akt panel had been duplicated, and the data for the ß-actin panel in Fig. 4C had not been included in the Figure as intended. The revised version of Fig. 4, showing the correct data for the ß-actin panel in Fig. 4C, is shown opposite. This error did not have a significant impact on the results or the conclusions reported in this study. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this Corrigendum, and all of the authors agree to the publication of this Corrigendum. The authors sincerely apologize for this mistake, and regret any inconvenience this mistake has caused. [the original article was published in Oncology Reports 36: 1909-1916, 2016; DOI: 10.3892/or.2016.5014].

Hydrochar from corn stalk used as bio-asphalt modifier: High-temperature performance improvement.

Biomass utilization, even for conversion products like hydrochar or biochar, has an increasing demand because improper disposal can cause intensive pollution. In this study, hydrochar obtained by hydrothermal treatment of corn stalk was added to virgin asphalt as a novel modifier by manual stirring and high-speed shearing. This hydrochar-modified asphalt (HCMA) showed a better high-temperature performance compared to unmodified asphalt, and the optimized dosage was 6 wt% with Rutting Index reaching 76 °C, and its penetration and softening point reaching 31.70 (0.1 mm) and 54.70 °C, respectively. The macroscopic representation of modified asphalt was conducted by microscopic characterization methods such as Fourier Transform Infrared Spectroscopy (FTIR) and Gel Permeation Chromatography (GPC). It was demonstrated that the performance was improved by the good blending state between hydrochar and asphalt. The application of hydrochar in modifying asphalt can reduce pollution and enhance its high-temperature performance, which has a potentially extensive application prospect in pavement engineering in subtropical and tropical climate.

Photoenzymatic enantioselective intermolecular radical hydroalkylation.

Enzymes are increasingly explored for use in asymmetric synthesis1-3, but their applications are generally limited by the reactions available to naturally occurring enzymes. Recently, interest in photocatalysis4 has spurred the discovery of novel reactivity from known enzymes5. However, so far photoinduced enzymatic catalysis6 has not been used for the cross-coupling of two molecules. For example, the intermolecular coupling of alkenes with α-halo carbonyl compounds through a visible-light-induced radical hydroalkylation, which could provide access to important γ-chiral carbonyl compounds, has not yet been achieved by enzymes. The major challenges are the inherent poor photoreactivity of enzymes and the difficulty in achieving stereochemical control of the remote prochiral radical intermediate7. Here we report a visible-light-induced intermolecular radical hydroalkylation of terminal alkenes that does not occur naturally, catalysed by an 'ene' reductase using readily available α-halo carbonyl compounds as reactants. This method provides an efficient approach to the synthesis of various carbonyl compounds bearing a γ-stereocentre with excellent yields and enantioselectivities (up to 99 per cent yield with 99 per cent enantiomeric excess), which otherwise are difficult to access using chemocatalysis. Mechanistic studies suggest that the formation of the complex of the substrates (α-halo carbonyl compounds) and the 'ene' reductase triggers the enantioselective photoinduced radical reaction. Our work further expands the reactivity repertoire of biocatalytic, synthetically useful asymmetric transformations by the merger of photocatalysis and enzyme catalysis.

Exogenous hydrogen sulfide promotes hepatocellular carcinoma cell growth by activating the STAT3-COX-2 signaling pathway.

The effects of hydrogen sulfide (H2S) on cancer are controversial. Our group previously demonstrated that exogenous H2S promotes the development of cancer via amplifying the activation of the nuclear factor-κB signaling pathway in hepatocellular carcinoma (HCC) cells (PLC/PRF/5). The present study aimed to further investigate the hypothesis that exogenous H2S promotes PLC/PRF/5 cell proliferation and migration, and inhibits apoptosis by activating the signal transducer and activator of transcription 3 (STAT3)-cyclooxygenase-2 (COX-2) signaling pathway. PLC/PRF/5 cells were treated with 500 µmol/l NaHS (a donor of H2S) for 24 h. The expression levels of phosphorylated (p)-STAT3, STAT3, cleaved caspase-3 and COX-2 were measured by western blot assay. Cell viability was detected by Cell Counting kit-8 assay. Apoptotic cells were observed by Hoechst 33258 staining. The expression of STAT3 and COX-2 messenger RNA (mRNA) was detected by semiquantitative reverse transcription-polymerase chain reaction. The production of vascular endothelial growth factor (VEGF) was evaluated by ELISA. The results indicated that treatment of PLC/PRF/5 cells with 500 µmol/l NaHS for 24 h markedly increased the expression levels of p-STAT3 and STAT3 mRNA, leading to COX-2 and COX-2 mRNA overexpression, VEGF induction, decreased cleaved caspase-3 production, increased cell viability and migration, and decreased number of apoptotic cells. However, co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and 30 µmol/l AG490 (an inhibitor of STAT3) or 20 µmol/l NS-398 (an inhibitor of COX-2) for 24 h significantly reverted the effects induced by NaHS. Furthermore, co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and 30 µmol/l AG490 markedly decreased the NaHS-induced increase in the expression level of COX-2. By contrast, co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and 20 µmol/l NS-398 inhibited the NaHS-induced increase in the expression level of p-STAT3. In conclusion, the findings of the present study provide evidence that the STAT3-COX-2 signaling pathway is involved in NaHS-induced cell proliferation, migration, angiogenesis and anti-apoptosis in PLC/PRF/5 cells, and suggest that the positive feedback between STAT3 and COX-2 may serve a crucial role in hepatocellular carcinoma carcinogenesis.

[Corrigendum] Heat shock protein 90/Akt pathway participates in the cardioprotective effect of exogenous hydrogen sulfide against high glucose­induced injury to H9c2 cells.

Subsequently to the publication of this article, the authors have realized that the address affiliation for the corresponding author, Chengheng Hu, and the authors Longyun Peng and Xinxue Liao appeared incorrectly. These authors' affiliation information should have appeared as follows (the corrected address affiliation is featured in bold): XIAO KE1,2*, JINGFU CHEN3*, LONGYUN PENG4, WEI ZHANG5, YIYING YANG5, XINXUE LIAO4, LIQIU MO6, RUIXIAN GUO7, JIANQIANG FENG6, CHENGHENG HU4 and RUQIONG NIE2 1Department of Cardiology, Shenzhen Sun Yat­sen Cardiovascular Hospital, Shenzhen; 2Department of Cardiology, Sun Yat­sen Memorial Hospital, Sun Yat­sen University, Guangzhou, Guangdong; 3Department of Cardiovascular Medicine and Dongguan Cardiovascular Institute, The Third People's Hospital of Dongguan City, Dongguan; 4Department of Cardiology and Key Laboratory on Assisted Circulation, Ministry of Health, The First Affiliated Hospital, Sun Yat­sen University; 5Department of Cardiovasology and Cardiac Care Unit (CCU), Huangpu Division of The First Affiliated Hospital, Sun Yat­sen University; 6Department of Anesthesiology, Huangpu Division of The First Affiliated Hospital, Sun Yat­sen University; 7Department of Physiology, Zhongshan School of Medicine, Sun Yat­sen University, Guangzhou, Guangdong, P.R. China *Contributed equally In addition, the address for correspondence in the correspondence box should have appeared as follows: Correspondence to: Professor Chengheng Hu, Department of Cardiology and Key Laboratory on Assisted Circulation, Ministry of Health, The First Affiliated Hospital, Sun Yat­sen University, Guangdong, 58 Zhongshan 2rd Road, Guangzhou 510080, P.R. China E­mail: huchengheng138@163.com The authors regret this error in the affiliations, and apologize for any inconvenience caused. [the original article was published in the International Journal of Molecular Medicine 39: 1001­1010, 2017; DOI: 10.3892/ijmm.2017.2891].

Ang-(1-7) protects HUVECs from high glucose-induced injury and inflammation via inhibition of the JAK2/STAT3 pathway.

Angiotensin (Ang)­1­7, which is catalyzed by angiotensin­converting enzyme 2 (ACE2) from angiotensin­II (Ang­II), exerts multiple biological and pharmacological effects, including cardioprotective effects and endothelial protection. The Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway has been demonstrated to be involved in diabetes­associated cardiovascular complications. The present study hypothesized that Ang­(1­7) protects against high glucose (HG)­induced endothelial cell injury and inflammation by inhibiting the JAK2/STAT3 pathway in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 40 mmol/l glucose (HG) for 24 h to establish a model of HG­induced endothelial cell injury and inflammation. Protein expression levels of p­JAK2, t­JAK2, p­STAT3, t­STAT3, NOX­4, eNOS and cleaved caspase­3 were tested by western blotting. CCK­8 assay was performed to assess cell viability of HUVECs. Apoptotic cell death was analyzed by Hoechst 33258 staining. Mitochondrial membrane potential (MMP) was obtained using JC­1. Superoxide dismutase (SOD) activity was tested by SOD assay kit. Interleukin (IL)­1ß, IL­10, IL­12 and TNF­α levels in culture media were tested by ELISA. The findings demonstrated that exposure of HUVECs to HG for 24 h induced injury and inflammation. This injury and inflammation were significantly ameliorated by pre­treatment of cells with either Ang­(1­7) or AG490, an inhibitor of the JAK2/STAT3 pathway, prior to exposure of the cells to HG. Exposure of the cells to HG also increased the phosphorylation of JAK2/STAT3 (p­JAK2 and p­STAT3). Increased activation of the JAK2/STAT3 pathway was attenuated by pre­treatment with Ang­(1­7). To the best of our knowledge, the findings from the present study provided the first evidence that Ang­(1­7) protects against HG­induced injury and inflammation by inhibiting activation of the JAK2/STAT3 pathway in HUVECs.

Exogenous hydrogen sulfide protects human umbilical vein endothelial cells against high glucose­induced injury by inhibiting the necroptosis pathway.

Hyperglycemia is a key factor in the development of diabetic complications, including the processes of atherosclerosis. Receptor­interacting protein 3 (RIP3), a mediator of necroptosis, is implicated in atherosclerosis development. Additionally, hydrogen sulfide (H2S) protects the vascular endothelium against hyperglycemia­induced injury and attenuates atherosclerosis. On the basis of these findings, the present study aimed to confirm the hypothesis that necroptosis mediates high glucose (HG)­induced injury in human umbilical vein endothelial cells (HUVECs), and that the inhibition of necroptosis contributes to the protective effect of exogenous H2S against this injury. The results revealed that exposure of HUVECs to 40 mM HG markedly enhanced the expression level of RIP3, along with multiple injuries, including a decrease in cell viability, an increase in the number of apoptotic cells, an increase in the expression level of cleaved caspase­3, generation of reactive oxygen species (ROS), as well as dissipation of the mitochondrial membrane potential (MMP). Treatment of the cells with sodium hydrogen sulfide (NaHS; a donor of H2S) prior to exposure to HG significantly attenuated the increased RIP3 expression and the aforementioned injuries by HG. Notably, treatment of cells with necrostatin­1 (Nec­1), an inhibitor of necroptosis, prior to exposure to HG ameliorated the HG­induced injuries, leading to a decrease in ROS generation and a loss of MMP. However, pre­treatment of the cells with Nec­1 enhanced the HG­induced increase in the expression levels of cleaved caspases­3 and ­9. By contrast, pre­treatment with Z­VAD­FMK, a pan ­caspase inhibitor, promoted the increased expression of RIP3 by HG. Taken together, the findings of the present study have demonstrated, to the best of our knowledge for the first time, that exogenous H2S protects HUVECs against HG­induced injury through inhibiting necroptosis. The present study has also provided novel evidence that there is a negative interaction between necroptosis and apoptosis in the HG­treated HUVECs.

[Angiotensin-(1-7) protects cardiac myocytes against high glucose-induced injury by inhibiting ClC-3 chloride channels].

OBJECTIVE: To explore whether angiotensin-(1-7) [Ang-(1-7)] protects cardiac myocytes against high glucose (HG)-induced injury by inhibiting ClC-3 chloride channels. METHOD: H9c2 cardiac cells were exposed to 35 mmol/L glucose for 24 h to establish a cell injury model. The cells were treated with Ang-(1-7) or the inhibitor of chloride channel (NPPB) in the presence of HG for 24 h to observe the changes in HG-induced cell injury. Cell counter kit 8 (CCK-8) assay was used to test the cell viability, and the morphological changes of the apoptotic cells were detected using Hoechst 33258 staining and fluorescent microscopy. The intracellular level of reactive oxygen species (ROS) was examined by DCFH-DA staining, SOD activity in the culture medium was measured using commercial kits, and the mitochondrial membrane potential (MMP) of the cells was tested with rodamine 123 staining. The expression level of cardiac ClC-3 chloride channels was detected with Western blotting. RESULTS: Exposure of H9c2 cardiac cells to 35 mmol/L glucose for 24 h markedly enhanced the expressions of cardiac ClC-3 channel protein (P<0.01). Co-treatment of the cells with 1 µmol/L Ang-(1-7) and HG for 24 h significantly attenuated HG- induced upregulation of ClC-3 channel protein expression (P<0.01). Co-treatment of the cells exposed to HG with 1 µmol/L Ang-(1-7) or 100 µmol/L NPPB for 24 h obviously ameliorated HG-induced injuries as shown by increased cell viability, enhanced SOD activity, decreased number of apoptotic cells, and reduced intracellular ROS generation and loss of MMP (P<0.01). CONCLUSION: ClC-3 channels are involved in HG-induced injury in cardiac cells. Ang-(1-7) protects cardiac cells against HG-induced injury by inhibiting ClC-3 channels.

A novel damage mechanism: Contribution of the interaction between necroptosis and ROS to high glucose-induced injury and inflammation in H9c2 cardiac cells.

Recently, a novel mechanism known as 'programmed necrosis' or necroptosis has been shown to be another important mechanism of cell death in the heart. In this study, we investigated the role of necroptosis in high glucose (HG)-induced injury and inflammation, as well as the underlying mechanisms. In particular, we focused on the interaction between necroptosis and reactive oxygen species (ROS) in H9c2 cardiac cells. Our results demonstrated that the exposure of H9c2 cardiac cells to 35 mM glucose (HG) markedly enhanced the expression level of receptor-interacting protein 3 (RIP3), a kinase which promotes necroptosis. Importantly, co-treatment of the cells with 100 µM necrostatin-1 (a specific inhibitor of necroptosis) and HG for 24 h attenuated not only the increased expression level of RIP3, but also the HG-induced injury and inflammation, as evidenced by an increase in cell viability, a decrease in ROS generation, the attenuation of the dissipation of mitochondrial membrane potential and a decrese in the secretion levels of inflammatory cytokines, i.e., interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. Furthermore, treatment of the cells with 1 mM N-acetyl­L­cysteine (a scavenger of ROS) for 60 min prior to exposure to HG significantly reduced the HG-induced increase in the RIP3 expression level, as well as the injury and inflammatory response described above. Taken together, the findings of this study clearly demonstrate a novel damage mechanism involving the positive interaction between necroptosis and ROS attributing to HG-induced injury and inflammation in H9c2 cardiac cells.

Exogenous hydrogen sulfide eliminates spatial memory retrieval impairment and hippocampal CA1 LTD enhancement caused by acute stress via promoting glutamate uptake.

Acute stress impairs the hippocampus-dependent spatial memory retrieval, and its synaptic mechanisms are associated with hippocampal CA1 long-term depression (LTD) enhancement in the adult rats. Endogenous hydrogen sulfide (H2S) is recognized as a novel gasotransmitter and has the neural protective roles. However, very little attention has been paid to understanding the effects of H2S on spatial memory retrieval impairment. We observed the protective effects of NaHS (a donor of H2S) against spatial memory retrieval impairment caused by acute stress and its synaptic mechanisms. Our results showed that NaHS abolished spatial memory retrieval impairment and hippocampal CA1 LTD enhancement caused by acute stress, but not by glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylic (tPDC), indicating that the activation of glutamate transporters is necessary for exogenous H2S to exert its roles. Moreover, NaHS restored the decreased glutamate uptake in the hippocampal CA1 synaptosomal fraction caused by acute stress. Dithiothreitol (DTT, a disulfide reducing agent) abolished a decrease in the glutamate uptake caused by acute stress, and NaHS eradicated the decreased glutamate uptake caused by 5,5'-dithio-bis(2-nitrobenzoic)acid (DTNB, a thiol oxidizing agent), collectively, revealing that exogenous H2S increases glutamate uptake by reducing disulfide bonds of the glutamate transporters. Additionally, NaHS inhibited the increased expression level of phosphorylated c-Jun-N-terminal kinase (JNK) in the hippocampal CA1 region caused by acute stress. The JNK inhibitor SP600125 eliminated spatial memory retrieval impairment, hippocampal CA1 LTD enhancement and the decreased glutamate uptake caused by acute stress, indicating that exogenous H2S exerts these roles by inhibiting the activation of JNK signaling pathway.

The Opening of ATP-Sensitive K+ Channels Protects H9c2 Cardiac Cells Against the High Glucose-Induced Injury and Inflammation by Inhibiting the ROS-TLR4-Necroptosis Pathway.

BACKGROUND/AIMS: Hyperglycemia activates multiple signaling molecules, including reactive oxygen species (ROS), toll-like receptor 4 (TLR4), receptor-interacting protein 3 (RIP3, a kinase promoting necroptosis), which mediate hyperglycemia-induced cardiac injury. This study explored whether inhibition of ROS-TLR4-necroptosis pathway contributed to the protection of ATP-sensitive K+ (KATP) channel opening against high glucose-induced cardiac injury and inflammation. METHODS: H9c2 cardiac cells were treated with 35 mM glucose (HG) to establish a model of HG-induced insults. The expression of RIP3 and TLR4 were tested by western blot. Generation of ROS, cell viability, mitochondrial membrane potential (MMP) and secretion of inflammatory cytokines were measured as injury indexes. RESULTS: HG increased the expression of TLR4 and RIP3. Necrostatin-1 (Nec-1, an inhibitor of necroptosis) or TAK-242 (an inhibitor of TLR4) co-treatment attenuated HG-induced up-regulation of RIP3. Diazoxide (DZ, a mitochondrial KATP channel opener) or pinacidil (Pin, a non-selective KATP channel opener) or N-acetyl-L-cysteine (NAC, a ROS scavenger) pre-treatment blocked the up-regulation of TLR4 and RIP3. Furthermore, pre-treatment with DZ or Pin or NAC, or co-treatment with TAK-242 or Nec-1 attenuated HG-induced a decrease in cell viability, and increases in ROS generation, MMP loss and inflammatory cytokines secretion. However, 5-hydroxy decanoic acid (5-HD, a mitochondrial KATP channel blocker) or glibenclamide (Gli, a non-selective KATP channel blocker) pre-treatment did not aggravate HG-induced injury and inflammation. CONCLUSION: KATP channel opening protects H9c2 cells against HG-induced injury and inflammation by inhibiting ROS-TLR4-necroptosis pathway.

Heat shock protein 90/Akt pathway participates in the cardioprotective effect of exogenous hydrogen sulfide against high glucose-induced injury to H9c2 cells.

It has been reported that exogenous hydrogen sulfide (H2S) protects against high glucose (HG)-induced cardiac injury and has a modulatory effect on heat shock protein (HSP) and Akt, which play a cardioprotective role. In this study, we examined whether the HSP90/Akt pathway contributes to the protective effects of exogenous H2S against HG-induced injury to H9c2 cardiac cells. Our results revealed that the exposure of H9c2 cardiac cells to 35 mM glucose (HG) for 1 to 24 h decreased the expression of HSP90 and markedly reduced the expression level of phosphorylated (p)-Akt in a time-dependent manner. Co-exposure of the cells to HG and geldanamycin (GA; an inhibitor of HSP90) aggravated the inhibition of the p-Akt expression level by HG. Of note, treatment of the cells with 400 µM NaHS (a donor of H2S) for 30 min prior to exposure to HG significantly attenuated the HG-induced decrease in the expression levels of both HSP90 and p-Akt, along with inhibition of HG-induced cell injury, as indicated by the increase in cell viability and superoxide dismutase (SOD) activity, and by a decrease in the number of apoptotic cells, reactive oxygen species (ROS) generation, as well as by the decreased dissipation of mitochondrial membrance potential (MMP). Importantly, treatment of the cells with GA or LY294002 (an inhibitor of Akt) prior to exposure to NaHS and HG considerably blocked the cardioprotective effects of NaHS against the HG-induced injury mentioned above. On the whole, the findings of this study demonstrate that the inhibition of the HSP90/Akt pathway may be an important mechanism responsible for HG-induced cardiomyocyte injury. We also provide novel evidence that exogenous H2S protects H9c2 cells against HG-induced injury by activating the HSP90/Akt pathway.