Liver X Receptor Agonists Inhibit the Phospholipid Regulatory Gene CTP: Phosphoethanolamine Cytidylyltransferase-Pcyt2

Metabolic pulse-chase experiments demonstrated that 25-hydroxycholesterol (25-OH), the endogenous activator of the liver X receptor (LXR), significantly reduced the biosynthesis of phosphatidylethanolamine via CDP-ethanolamine (Kennedy) pathway at the step catalyzed by CTP: phosphoethanolamine cytidylyltransferase (Pcyt2). In the mouse embryonic fibroblasts C3H10T1/2, the LXR synthetic agonist TO901317 lowered Pcyt2 promoter-luciferase activity in a concentration-dependent manner. Furthermore, 25-OH and TO901317 reduced mouse Pcyt2 mRNA and protein levels by 35–60%. The inhibitory effects of oxysterols and TO901317 on the Pcyt2 promoter function, mRNA and protein expression were conserved in the human breast cancer cells MCF-7. These studies identify the Pcyt2 gene as a novel target whereby LXR agonists may indirectly modulate inflammatory responses and atherosclerosis.

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Different Induction Effects of Praziquantel Racemate and Enantiomers on the Enzyme CYP3A4 Mediated by Pregnane X Receptor and Its Variants

Current Drug Metabolism ◽

10.2174/1389200221999210104204057 ◽

2021 ◽

Vol 21 ◽

Author(s):

Ran Meng ◽

Xueli Zhang ◽

Haina Wang ◽

Danlu Zhang ◽

Xin Zhao

Keyword(s):

Hepg2 Cells ◽

Pregnane X Receptor ◽

Luciferase Reporter ◽

Dependent Manner ◽

Luciferase Reporter Gene ◽

Concentration Dependent Manner ◽

Protein Levels ◽

Mrna And Protein Expression ◽

Polymerase Chain ◽

Expression Plasmids

Background: Praziquantel (PZQ), which possesses an asymmetric center, is classified as a pyrazinoisoquinoline and has been the mainstay in the treatment of schistosomiasis since 1980. PZQ undergoes a pronounced first-pass metabolism in the liver through the CYP450 system which could be mediated by nuclear receptors. Objective: The purpose of this study was to investigate the possible different induction effects of CYP3A4 by PZQ racemate and enantiomers via the pregnane X receptor (PXR) and the effect of PXR polymorphism on the induction potency of PZQs. Methods: The dual-luciferase reporter gene systems constructed in HepG2 cells were used to measure the abilities of PZQs to induce CYP3A4 expression mediated by PXR. The mRNA and protein levels of CYP3A4 were evaluated by polymerase chain reaction (PCR) and western blotting, respectively. Results: In HepG2 cells transfected with PXRwt, PXR158, PXR163, PXR370 or PXR403 expression plasmids, PZQ racemate and its enantiomers up-regulated the luciferase activity in a concentration-dependent manner, while reaching saturation after transfected with PXR379 expression plasmids. The mRNA and protein expression of CYP3A4 was effectively activated in PXR-transfected HepG2 cells. The induction ability of CYP3A4 mediated by PXR activation by PZQ racemate and its enantiomers were statistically different between the same PXR group and different PXR groups. Conclusion: The enantioselective induction effects of PZQs on CYP3A4 were related to the enantioselective activations of PXR by PZQs and were influenced by the PXR gene polymorphism. These findings provide a basis for further understanding the enantiomeric metabolism and the variable efficacy of PZQs.

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Mn Inhibits GSH Synthesis via Downregulation of Neuronal EAAC1 and Astrocytic xCT to Cause Oxidative Damage in the Striatum of Mice

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/4235695 ◽

2018 ◽

Vol 2018 ◽

pp. 1-15 ◽

Cited By ~ 7

Author(s):

Xinxin Yang ◽

Haibo Yang ◽

Fengdi Wu ◽

Zhipeng Qi ◽

Jiashuo Li ◽

...

Keyword(s):

Oxidative Stress ◽

Oxidative Damage ◽

Dependent Manner ◽

Protein Levels ◽

Key Factor ◽

Protein Sulfhydryl ◽

Mrna And Protein Expression ◽

The Brain

Excessive manganese (Mn) can accumulate in the striatum of the brain following overexposure. Oxidative stress is a well-recognized mechanism in Mn-induced neurotoxicity. It has been proven that glutathione (GSH) depletion is a key factor in oxidative damage during Mn exposure. However, no study has focused on the dysfunction of GSH synthesis-induced oxidative stress in the brain during Mn exposure. The objective of the present study was to explore the mechanism of Mn disruption of GSH synthesis via EAAC1 and xCT in vitro and in vivo. Primary neurons and astrocytes were cultured and treated with different doses of Mn to observe the state of cells and levels of GSH and reactive oxygen species (ROS) and measure mRNA and protein expression of EAAC1 and xCT. Mice were randomly divided into seven groups, which received saline, 12.5, 25, and 50 mg/kg MnCl2, 500 mg/kg AAH (EAAC1 inhibitor) + 50 mg/kg MnCl2, 75 mg/kg SSZ (xCT inhibitor) + 50 mg/kg MnCl2, and 100 mg/kg NAC (GSH rescuer) + 50 mg/kg MnCl2 once daily for two weeks. Then, levels of EAAC1, xCT, ROS, GSH, malondialdehyde (MDA), protein sulfhydryl, carbonyl, 8-hydroxy-2-deoxyguanosine (8-OHdG), and morphological and ultrastructural features in the striatum of mice were measured. Mn reduced protein levels, mRNA expression, and immunofluorescence intensity of EAAC1 and xCT. Mn also decreased the level of GSH, sulfhydryl, and increased ROS, MDA, 8-OHdG, and carbonyl in a dose-dependent manner. Injury-related pathological and ultrastructure changes in the striatum of mice were significantly present. In conclusion, excessive exposure to Mn disrupts GSH synthesis through inhibition of EAAC1 and xCT to trigger oxidative damage in the striatum.

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Abstract 492: Arachidonic Acid Induces Activation of Platelet PF4 and Par-1 mRNA, Which Is Attenuated by Aspirin

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a492 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Liang Hu ◽

Michael A Nardi ◽

Michael Merolla ◽

Yajaira Suarez ◽

Jeffrey Berger

Keyword(s):

Arachidonic Acid ◽

Platelet Activation ◽

Mrna Levels ◽

Dependent Manner ◽

Thiazole Orange ◽

Platelet Activity ◽

Protein Levels ◽

Reticulated Platelets ◽

Mrna And Protein Expression ◽

Dose Dependent

Arachidonic acid (AA) is converted to thromboxane A2 via the cyclooxygenase pathway; however its exact mechanism of platelet activation is uncertain. Inhibition of this pathway via aspirin highlights the importance of this pathway in decreasing thrombotic events. In the present study, we investigate the effect of AA on platelet activity indicators (leukocyte- and monocyte-platelet aggregation [LPA, MPA] and reticulated platelets [RP]), as well as the expression (mRNA and protein) of platelet markers PF4 and Par-1, previously well established platelet transcripts with quantitative determinations. To this end, whole blood was incubated with AA (150mM) for 30 min at room temperature in the absence or presence of aspirin (1mM) prior to addition of antibodies for platelet activity indicators, and isolating platelets for mRNA and protein expression. LPA and MPA were significantly increased after AA stimulation in a dose dependent manner, and were inhibited by aspirin treatment. AA significantly increased PF4 and Par-1 protein level as determined by flow cytometry and western blot assays. Pretreatment with aspirin also attenuated this increase in protein levels. Surprisingly, AA stimulation significantly increased thiazole orange staining (a measure of nucleic acids), another marker of increased platelet activity. Importantly, these results suggest that AA-mediated platelet activation produced an overall increase in platelet total RNA content. To confirm these findings, we analyzed the mRNA expression of PF4 and Par-1 by quantitative real time PCR from platelets treated with AA. Interestingly, AA significantly up-regulated the platelet mRNA transcripts of PF4 and Par-1 by 40% to 60%, and pretreatment with aspirin completely attenuated this effect supporting the specificity of the AA effect on platelet RNA. Altogether, these data suggest that platelet mRNA is affected by AA stimulation, which is attenuated by pretreatment with aspirin. However, the mechanisms responsible for the increased mRNA levels and expression of PF4 and Par-1 (processing of pre-RNA to mRNA) require further investigation. Importantly, our findings provide novel insight regarding platelet activation and a better understanding of mediators in the processes of thrombosis and hemostasis.

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Modulatory effect of antibiotics on cytokine production by human monocytes in vitro.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.6.1366 ◽

1996 ◽

Vol 40 (6) ◽

pp. 1366-1370 ◽

Cited By ~ 132

Author(s):

K Morikawa ◽

H Watabe ◽

M Araake ◽

S Morikawa

Keyword(s):

Antimicrobial Agents ◽

Inflammatory Responses ◽

Interleukin 1 ◽

Immunomodulatory Activity ◽

Lymphocyte Function ◽

Dependent Manner ◽

Human Monocytes ◽

Concentration Dependent Manner ◽

Cytokine Synthesis

Some antimicrobial agents have been reported to modify the host immune and inflammatory responses both in vivo and in vitro. Fosfomycin (FOM) and clarithromycin (CAM) have immunomodulatory activity on human lymphocyte function. In the present study, we examined the effects of FOM and CAM on cytokine synthesis by lipopolysaccharide (LPS)-stimulated human monocytes in comparison with that of dexamethasone in vitro. The three drugs demonstrated positive or negative effects on the synthesis of various cytokines by LPS-primed monocytes. They suppressed the synthesis of tumor necrosis factor alpha, interleukin 1 alpha (IL-1 alpha), IL-1 beta, the IL-1 receptor antagonist, and granulocyte-macrophage colony-stimulating factor in a concentration-dependent manner at concentrations between 1.6 and 40 micrograms/ml. On the contrary, the drugs showed different actions on the synthesis of IL-6 and IL-10. Namely, FOM enhanced both IL-6 and IL-10 synthesis, CAM enhanced only IL-10 synthesis, but dexamethasone deeply suppressed the synthesis of both cytokines. These data indicate that antibacterial agents may modify acute-phase inflammatory responses through their effects on cytokine synthesis by monocytes.

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The Antinociceptive, Antioxidant and Anti-Inflammatory Effects of 5-Fluoro-2-Oxindole during Inflammatory Pain

Antioxidants ◽

10.3390/antiox9121249 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1249

Author(s):

Alejandro Redondo ◽

Gabriela Riego ◽

Olga Pol

Keyword(s):

Inflammatory Pain ◽

Inflammatory Responses ◽

Protoporphyrin Ix ◽

Mitogen Activated Protein Kinase ◽

Oxidative Stress Marker ◽

Heme Oxygenase 1 ◽

Peripheral Inflammation ◽

Dependent Manner ◽

Protein Levels ◽

Antinociceptive Effects

Recent studies demonstrate that 5-fluoro-2-oxindole inhibits neuropathic pain but the antinociceptive actions of this drug and its effects on the plasticity, oxidative and inflammatory changes induced by peripheral inflammation as well as on the effects and expression of µ-opioid receptors (MOR) have not been evaluated. In C57BL/6 male mice with inflammatory pain provoked by the subplantar administration of complete Freund’s adjuvant (CFA), we evaluated: (1) the antinociceptive actions of 5-fluoro-2-oxindole and its reversion with the HO-1 inhibitor, tin protoporphyrin IX (SnPP); (2) the effects of 5-fluoro-2-oxindole in the protein levels of mitogen-activated protein kinase (MAPK), Nrf2, NADPH quinone oxidoreductase1 (NQO1), heme oxygenase 1 (HO-1), oxidative stress marker (4-hydroxy-2-nonenal; 4-HNE), inducible nitric oxide synthase (NOS2), microglial markers (CD11b/c and IBA-1), and MOR in the spinal cord and/or paw of animals with inflammatory pain; (3) the antinociceptive effects of morphine in 5-fluoro-2-oxindole pre-treated animals. Treatment with 5 and 10 mg/kg of 5-fluoro-2-oxindole inhibited the allodynia and hyperalgesia induced by CFA in a different, time-dependent manner. These effects were reversed by SnPP. Treatment with 5-fluoro-2-oxindole increased the expression of NQO1, HO-1 and MOR and inhibited the CFA-induced upregulation of phosphorylated MAPK, 4-HNE, NOS2, CD11b/c and IBA-1 in spinal cords and/or paws. The local effects of morphine were improved with 5-fluoro-2-oxindole. This work reveals that 5-fluoro-2-oxindole inhibits the plasticity, oxidative and inflammatory responses provoked by peripheral inflammation and potentiates the antinociceptive effects of morphine. Thus, treatment with 5-fluoro-2-oxindole alone and/or combined with morphine are two remarkable new procedures for chronic inflammatory pain management.

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Signal regulatory protein α1 is involved in the inhibitory effect of glucocorticoid receptor on the proliferation of murine macrophage RAW264.7 cell and mouse peritoneal macrophage

Journal of Molecular Endocrinology ◽

10.1677/jme-08-0021 ◽

2008 ◽

Vol 41 (5) ◽

pp. 393-403 ◽

Cited By ~ 3

Author(s):

Xiaohui Wang ◽

Yidong Li ◽

Xiaoyan Zhu ◽

Yan Wang ◽

Fei Diao ◽

...

Keyword(s):

Rna Interference ◽

Peritoneal Macrophage ◽

Inflammatory Responses ◽

Regulatory Protein ◽

Mouse Peritoneal Macrophage ◽

Raw264.7 Cells ◽

Dependent Manner ◽

Proliferation Inhibition ◽

Murine Macrophage ◽

Concentration Dependent Manner

Glucocorticoid (GC) effectively suppresses immune and inflammatory responses and inhibits the growth of several types of cells, but the role of GC and its receptor on macrophage proliferation is unclear. In our previous work, we found RAW-GR(−) cells (murine macrophage RAW264.7 cells stably transfected with GR-siRNA expression vector by RNA interference) grew faster by about twofold. In this study, we further explored the role and mechanisms of GC/GR on the proliferation of macrophage. We found that the growth of RAW264.7 cells was inhibited by dexamethasone (Dex) in a concentration-dependent manner. The mRNA and protein levels of signal regulatory protein α1 (SIRPA) were induced by GC/GR in RAW264.7 cells and SIRPA expression was decreased remarkably in RAW-GR(−) cells. Overexpression of SIRPA negatively regulated the proliferation of RAW-GR(−) cells, and inhibition of SIRPA expression by a small from RNA interference attenuated Dex-induced proliferation inhibition in RAW264.7 cells. The proliferation inhibition of GC/GR was also found in mouse peritoneal macrophage, which was associated with the increase in SIRPA induced by GC/GR as well. In addition, elevation of the expression of CDK2, cyclinD1, and cyclinB1, but not phosphorylated ERK1/2 and p38, was found in RAW-GR(−) cells. In conclusion, we provided the novel evidences that GC/GR inhibited the growth of RAW264.7 cells and mouse peritoneal macrophage, and the antiproliferative effect of GC/GR on these cells was at least in part a result from GC/GR-induced SIRPA expression. Up-regulation of CDK2, cyclinD1, and cyclinB1 was also related to the increased proliferation of RAW-GR(−) cells.

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Decreased levels of sRAGE in follicular fluid from patients with PCOS

Reproduction ◽

10.1530/rep-16-0359 ◽

2017 ◽

Vol 153 (3) ◽

pp. 285-292 ◽

Cited By ~ 6

Author(s):

BiJun Wang ◽

Jing Li ◽

QingLing Yang ◽

FuLi Zhang ◽

MengMeng Hao ◽

...

Keyword(s):

Regression Analysis ◽

Granulosa Cells ◽

Follicular Fluid ◽

Cultured Cells ◽

Dependent Manner ◽

Vascular Endothelial ◽

Protein Levels ◽

Pcos Group ◽

Mrna And Protein Expression ◽

Endothelial Growth Factor

This study aimed to explore the association between soluble receptor for advanced glycation end products (sRAGE) levels in follicular fluid and the number of oocytes retrieved and to evaluate the effect of sRAGE on vascular endothelial growth factor (VEGF) in granulosa cells in patients with polycystic ovarian syndrome (PCOS). Two sets of experiments were performed in this study. In part one, sRAGE and VEGF protein levels in follicular fluid samples from 39 patients with PCOS and 35 non-PCOS patients were measured by ELISA. In part two, ovarian granulosa cells were isolated from an additional 10 patients with PCOS and cultured. VEGF and SP1 mRNA and protein levels, as well as pAKT levels, were detected by real-time PCR and Western blotting after cultured cells were treated with different concentrations of sRAGE. Compared with the non-PCOS patients, patients with PCOS had lower sRAGE levels in follicular fluid. Multi-adjusted regression analysis showed that high sRAGE levels in follicular fluid predicted a lower Gn dose, more oocytes retrieved, and a better IVF outcome in the non-PCOS group. Logistic regression analysis showed that higher sRAGE levels predicted favorably IVF outcomes in the non-PCOS group. Multi-adjusted regression analysis also showed that high sRAGE levels in follicular fluid predicted a lower Gn dose in the PCOS group. Treating granulosa cells isolated from patients with PCOS with recombinant sRAGE decreased VEGF and SP1 mRNA and protein expression and pAKT levels in a dose-dependent manner.

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Pyridoxine Stimulates Filaggrin Production in Human Epidermal Keratinocytes

10.21203/rs.3.rs-295371/v1 ◽

2021 ◽

Author(s):

Miyuki Fujishiro ◽

Shoichi Yahagi ◽

Shota Takemi ◽

Takafumi Sakai ◽

ichiro sakata

Keyword(s):

Pyridoxal Phosphate ◽

Seborrheic Dermatitis ◽

Epidermal Differentiation ◽

Disulfonic Acid ◽

Epidermal Keratinocytes ◽

Marker Genes ◽

Dependent Manner ◽

Human Epidermal Keratinocytes ◽

Concentration Dependent Manner ◽

Protein Levels

Abstract Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical studies have revealed that PN deficiency causes skin problems such as seborrheic dermatitis and stomatitis. However, the detailed effects of PN and its mechanism of action in epidermal function are poorly understood. In this study, we examined the effects of PN on epidermal function in normal human epidermal keratinocytes and found that PN specifically causes an increase in the expression of profilaggrin mRNA, among marker genes of terminal epidermal differentiation. In addition, PN treatment caused an increase in the production of filaggrin protein in a concentration-dependent manner. Treatment with P2x purinoceptor antagonists, namely, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate and TNP-ATP hydrate, induced an increase in the filaggrin protein levels. Moreover, we showed that elevated filaggrin production induced upon PN treatment was suppressed by ATP (known as P2x purinoceptor agonist). This study is the first to report that PN causes an increase in filaggrin transcription and production, and these results suggest that PN-induced filaggrin production may be a useful target as a daily care component in atopic dermatitis, wherein filaggrin levels are specifically reduced.

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Abstract 15558: Characterization of Sarcolemmal K Atp Channels in Human Ipsc-derived Cardiomyocytes

Circulation ◽

10.1161/circ.142.suppl_3.15558 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Robert Geiger ◽

Naheed Fatima ◽

Michael Klein ◽

Robert E Goldstein ◽

Mark C HAIGNEY ◽

...

Keyword(s):

Katp Channel ◽

Field Potential ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Protein Levels ◽

Typical Composition ◽

Human Ipsc ◽

Action Potential Shortening

Background: The ATP-sensitive potassium channel (KATP) plays a key role in protecting heart muscle during metabolic challenges such as ischemia. KATP activation causes action potential shortening that reduces calcium entry and contraction thus reducing calcium overload induced damage and preserving energy reserves. Cardiomyocytes derived from human inducible pluripotent stem cell (hiPSC) have emerged as a model to study cardiac function, however there are few studies that have focused on KATP. Methods: In the present study, cardiomyocytes were either generated from hiPSC using heparin- based chemically defined media or purchased from Cellular Dynamics (iCells2). Expression of the pore-forming (Kir6.2) and regulatory (SUR1 & SUR2) subunits of the KATP channel during differentiation were assessed using western blot. KATP function was assessed by measuring the field potential duration (FPD) and spontaneous beat rate in a confluent monolayer using the Axion Maestro multielectrode array system. Cells were probed using the KATP activators P1075 and diazoxide, specific for SUR2 and SUR1, respectively. Results: We found that the pore-forming subunit of the sarcolemmal KATP channel (Kir6.2) was expressed in iPSC and maintained throughout the course of differentiation. Consistent with the typical composition of sarcolemmal KATP, we observe a significant increase of SUR2 but little SUR1 protein following Wnt inhibition. Functionally, the FPD is markedly reduced by P1075 in a concentration-dependent manner, with 24% reduction at 100 nM and 92 % reduction at 100 μM. Moreover, glibenclamide 10μM reduces FPD shortening confirming a role for KATP. Finally, we observe little change in FPD when cells are exposed to diazoxide (100 μM) consistent with reduced SUR1 protein levels. Conclusion: These results indicate that cardiomyocytes derived from human iPSC express the KATP channel composed of primarily the SUR2 isoform and suggest that iPSC derived cardiomyocytes would be an effective model for studying the role of KATP during metabolic challenges.

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Enhancement of Cellular Antioxidant-Defence Preserves Diastolic Dysfunction via Regulation of Both Diastolic Zn2+and Ca2+and Prevention of RyR2-Leak in Hyperglycemic Cardiomyocytes

Oxidative Medicine and Cellular Longevity ◽

10.1155/2014/290381 ◽

2014 ◽

Vol 2014 ◽

pp. 1-15 ◽

Cited By ~ 20

Author(s):

Erkan Tuncay ◽

Esma N. Okatan ◽

Aysegul Toy ◽

Belma Turan

Keyword(s):

Diastolic Dysfunction ◽

Ryanodine Receptors ◽

Diabetic Rats ◽

Oxidative Stress Marker ◽

Macromolecular Complex ◽

Resting Cells ◽

Dependent Manner ◽

Antioxidant Defence ◽

Concentration Dependent Manner ◽

Protein Levels

We examined whether cellular antioxidant-defence enhancement preserves diastolic dysfunction via regulation of both diastolic intracellular free Zn2+and Ca2+levels (Zn2+iandCa2+i) levelsN-acetyl cysteine (NAC) treatment (4 weeks) of diabetic rats preserved altered cellular redox state and also prevented diabetes-induced tissue damage and diastolic dysfunction with marked normalizations in the restingZn2+iandCa2+i. The kinetic parameters of transient changes in Zn2+and Ca2+under electrical stimulation and the spatiotemporal properties of Zn2+and Ca2+sparks in resting cells are found to be normal in the treated diabetic group. Biochemical analysis demonstrated that the NAC treatment also antagonized hyperphosphorylation of cardiac ryanodine receptors (RyR2) and significantly restored depleted protein levels of both RyR2 and calstabin2. Incubation of cardiomyocytes with 10 µM ZnCl2exerted hyperphosphorylation in RyR2 as well as higher phosphorphorylations in both PKA and CaMKII in a concentration-dependent manner, similar to hyperglycemia. Our present data also showed that a subcellular oxidative stress marker, NF-κB, can be activated if the cells are exposed directly to Zn2+. We thus for the first time report that an enhancement of antioxidant defence in diabetics via directly targeting heart seems to prevent diastolic dysfunction due to modulation of RyR2 macromolecular-complex thereby leading to normalizedCa2+iandZn2+iin cardiomyocytes.

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