Uncovering the Therapeutic Potential of Phosphocreatine in Diabetic Retinopathy: Mitigating Mitochondrial Dysfunction and Apoptosis via JAK2/STAT3 Signaling Pathway.

Diabetic retinopathy (DR) stands as a prevalent complication of diabetes mellitus, causing damage to the delicate retinal capillaries and potentially leading to visual impairment. While the exact underlying cause of DR remains elusive, compelling research suggests that mitochondrial energy deficiency and the excessive generation of reactive oxygen species (ROS) play pivotal roles in its pathogenesis. Recognizing that controlling hyperglycemia alone fails to reverse the defects in retinal mitochondria induced by diabetes, current strategies seek to restore mitochondrial function as a means of safeguarding against DR. To address this pressing issue, a comprehensive study was undertaken to explore the potential of phosphocreatine (PCr) in bolstering mitochondrial bioenergetics and providing protection against DR via modulation of the JAK2/STAT3 signaling pathway. Employing rat mitochondria and RGC-5 cells, the investigation meticulously assessed the impact of PCr on ROS production, mitochondrial membrane potential, as well as the expression of crucial apoptotic and JAK2/STAT3 signaling pathway proteins, utilizing cutting-edge techniques such as high-resolution respirometry and western blotting. The remarkable outcomes revealed that PCr exerts a profound protective influence against DR by enhancing mitochondrial function and alleviating diabetes-associated symptoms and biochemical markers. Notably, PCr administration resulted in an upregulation of antiapoptotic proteins, concomitant with a downregulation of proapoptotic proteins and the JAK2/STAT3 signaling pathway. These significant findings firmly establish PCr as a potential therapeutic avenue for combating diabetic retinopathy. By augmenting mitochondrial function and exerting antiapoptotic effects via the JAK2/STAT3 signaling pathway, PCr demonstrates promising efficacy both in vivo and in vitro, particularly in counteracting the oxidative stress engendered by hyperglycemia. In summary, our study sheds light on the potential of PCr as an innovative therapeutic strategy for diabetic retinopathy. By bolstering mitochondrial function and exerting protective effects via the modulation of the JAK2/STAT3 signaling pathway, PCr holds immense promise in ameliorating the impact of DR in the face of oxidative stress induced by hyperglycemia.

Protective effects of phosphocreatine on human vascular endothelial cells against hydrogen peroxide-induced apoptosis and in the hyperlipidemic rat model.

Phosphocreatine (PCr) has been shown to have a cardio-protective effect during cardiopulmonary resuscitation (CPR). However, little is known about its impact on atherosclerosis. In this study, we first evaluated the pharmacological effects of PCr on antioxidative defenses and mitochondrial protection against hydrogen peroxide (H2O2) induced human umbilical vascular endothelial cells (HUVECs) damage. Then we investigated the hypolipidemic and antioxidative effects of PCr on hyperlipidemic rat model. Via in vitro studies, H2O2 significantly reduced cell viability and increased apoptosis rate of HUVECs, while pretreatment with PCr abolished its apoptotic effect. PCr could reduce the generation of ROS induced by H2O2. Moreover, PCr could increase the activity of SOD and the content of NO, as well as decrease the activity of LDH and the content of MDA. PCr could also antagonize H2O2-induced up-regulation of Bax, cleaved-caspase3, cleaved-caspase9, and H2O2-induced down-regulation of Bcl-2 and p-Akt/Akt ratio. In addition, PCr reduced U937 cells' adhesion to H2O2-stimulated HUVECs. Via in vivo study, PCr could decrease MDA, TC, TG and LDL-C levels in hyperlipidemic rats. Finally, different-concentration PCr could increase the leaching of TC, HDL, and TG from fresh human atherosclerotic plaques. In conclusion, PCr could suppress H2O2-induced apoptosis in HUVECs and reduce hyperlipidemia through inhibiting ROS generation and modulating dysfunctional mitochondrial system, which might be an effective new therapeutic strategy to further prevent atherosclerosis.

Trimetazidine in heart failure with preserved ejection fraction: a randomized controlled cross-over trial.

AIMS: Impaired myocardial energy homeostasis plays an import role in the pathophysiology of heart failure with preserved ejection fraction (HFpEF). Left ventricular relaxation has a high energy demand, and left ventricular diastolic dysfunction has been related to impaired energy homeostasis. This study investigated whether trimetazidine, a fatty acid oxidation inhibitor, could improve myocardial energy homeostasis and consequently improve exercise haemodynamics in patients with HFpEF. METHODS AND RESULTS: The DoPING-HFpEF trial was a phase II single-centre, double-blind, placebo-controlled, randomized cross-over trial. Patients were randomized to trimetazidine treatment or placebo for 3 months and switched after a 2-week wash-out period. The primary endpoint was change in pulmonary capillary wedge pressure, measured with right heart catheterization at multiple stages of bicycling exercise. Secondary endpoint was change in myocardial phosphocreatine/adenosine triphosphate, an index of the myocardial energy status, measured with phosphorus-31 magnetic resonance spectroscopy. The study included 25 patients (10/15 males/females; mean (standard deviation) age, 66 (10) years; body mass index, 29.8 (4.5) kg/m2 ); with the diagnosis of HFpEF confirmed with (exercise) right heart catheterization either before or during the trial. There was no effect of trimetazidine on the primary outcome pulmonary capillary wedge pressure at multiple levels of exercise (mean change 0 [95% confidence interval, 95% CI -2, 2] mmHg over multiple levels of exercise, P = 0.60). Myocardial phosphocreatine/adenosine triphosphate in the trimetazidine arm was similar to placebo (1.08 [0.76, 1.76] vs. 1.30 [0.95, 1.86], P = 0.08). There was no change by trimetazidine compared with placebo in the exploratory parameters: 6-min walking distance (mean change of -6 [95% CI -18, 7] m vs. -5 [95% CI -22, 22] m, respectively, P = 0.93), N-terminal pro-B-type natriuretic peptide (5 (-156, 166) ng/L vs. -13 (-172, 147) ng/L, P = 0.70), overall quality-of-life (KCCQ and EQ-5D-5L, P = 0.78 and P = 0.51, respectively), parameters for diastolic function measured with echocardiography and cardiac magnetic resonance, or metabolic parameters. CONCLUSIONS: Trimetazidine did not improve myocardial energy homeostasis and did not improve exercise haemodynamics in patients with HFpEF.

Phosphocreatine addition to extender enhances the quality and antioxidant capacity of cryopreserved boar sperm.

Boar sperm are less resistant to drastic changes in the external environment during cryopreservation, mainly because their plasma membranes are rich in unsaturated fatty acids but lack cholesterol and are thus susceptible to lipid peroxidation caused by the attack of reactive oxygen species. This study evaluated the effect of adding phosphocreatine to cryopreservation extenders on boar sperm quality and antioxidant capacity. Different concentrations (0, 5.0, 7.5, 10.0 and 12.5 mmol/L) of phosphocreatine were added to the cryopreservation extender. After thawing, sperm were analysed for morphological parameters, kinetic parameters, acrosome integrity, membrane integrity, mitochondrial activity, DNA integrity and antioxidant enzyme activity. The results showed that 10.0 mmol/L phosphocreatine samples enhanced the boar sperm motility, viability, average path velocity, straight-line velocity, curvilinear velocity and beat cross frequency after cryopreservation and reduced the malformation rate compared to the control group (p < .05). The acrosome integrity, membrane integrity, mitochondrial activity and DNA integrity of boar sperm were higher than those of the control group after adding 10.0 mmol/L phosphocreatine to the cryopreservation extender (p < .05). Extenders containing 10.0 mmol/L phosphocreatine maintained high total antioxidant capacity; elevated the activities of catalase, glutathione peroxidase and superoxide dismutase; reduced malondialdehyde and H2 O2 content (p < .05). Therefore, adding phosphocreatine to the extender is potentially beneficial for boar sperm cryopreservation at an optimal 10.0 mmol/L concentration.

Protection of pancreatic ß-cell by phosphocreatine through mitochondrial improvement via the regulation of dual AKT/IRS-1/GSK-3ß and STAT3/Cyp-D signaling pathways.

Diabetes mellitus (DM) is a metabolic syndrome, caused by insufficient insulin secretion or insulin resistance (IR). DM enhances oxidative stress and induces mitochondrial function in different kinds of cell types, including pancreatic ß-cells. Our previous study has showed phosphocreatine (PCr) can advance the mitochondrial function through enhancing the oxidative phosphorylation and electron transport ability in mitochondria damaged by methylglyoxal (MG). Our aim was to explore the potential role of PCr as a molecule to protect mitochondria from diabetes-induced pancreatic ß-cell injury with insulin secretion deficiency or IR through dual AKT/IRS-1/GSK-3ß and STAT3/Cyclophilin D (Cyp-D) signaling pathways. MG-induced INS-1 cell viability, apoptosis, mitochondrial division and fusion, the morphology, and function of mitochondria were suppressed. Flow cytometry was used to detect the production of intracellular reactive oxygen species (ROS) and the changes of intracellular calcium, and the respiratory function was measured by oxygraph-2k. The expressions of AKT, IRS-1, GSK-3ß, STAT3, and Cyp-D were detected using Western blot. The result showed that the oxidative stress-related kinases were significantly restored to the normal level after the pretreatment with PCr. Moreover, PCr pretreatment significantly inhibited cell apoptosis, decreased intracellular calcium, and ROS production, and inhibited mitochondrial division and fusion, and increased ATP synthesis damaged by MG in INS-1 cells. In addition, pretreatment with PCr suppressed Cytochrome C, p-STAT3, and Cyp-D expressions, while increased p-AKT, p-IRS-1, p-GSK-3ß, caspase-3, and caspase-9 expressions. In conclusion, PCr has protective effect on INS-1 cells in vitro and in vivo, relying on AKT mediated STAT3/ Cyp-D pathway to inhibit oxidative stress and restore mitochondrial function, signifying that PCr might become an emerging candidate for the cure of diabetic pancreatic cancer ß-cell damage.

The effects of phosphocreatine disodium salts plus blueberry extract supplementation on muscular strength, power, and endurance.

BACKGROUND: Numerous studies have demonstrated the efficacy of creatine supplementation for improvements in exercise performance. Few studies, however, have examined the effects of phosphocreatine supplementation on exercise performance. Furthermore, while polyphenols have antioxidant and anti-inflammatory properties, little is known regarding the influence of polyphenol supplementation on muscular strength, power, and endurance. Thus, the purpose of the present study was to compare the effects of 28 days of supplementation with phosphocreatine disodium salts plus blueberry extract (PCDSB), creatine monohydrate (CM), and placebo on measures of muscular strength, power, and endurance. METHODS: Thirty-three men were randomly assigned to consume either PCDSB, CM, or placebo for 28 days. Peak torque (PT), average power (AP), and percent decline for peak torque (PT%) and average power (AP%) were assessed from a fatigue test consisting of 50 maximal, unilateral, isokinetic leg extensions at 180°·s- 1 before and after the 28 days of supplementation. Individual responses were assessed to examine the proportion of subjects that exceeded a minimal important difference (MID). RESULTS: The results demonstrated significant (p < 0.05) improvements in PT for the PCDSB and CM groups from pre- (99.90 ± 22.47 N·m and 99.95 ± 22.50 N·m, respectively) to post-supplementation (119.22 ± 29.87 N·m and 111.97 ± 24.50 N·m, respectively), but no significant (p = 0.112) change for the placebo group. The PCDSB and CM groups also exhibited significant improvements in AP from pre- (140.18 ± 32.08 W and 143.42 ± 33.84 W, respectively) to post-supplementation (170.12 ± 42.68 W and 159.78 ± 31.20 W, respectively), but no significant (p = 0.279) change for the placebo group. A significantly (p < 0.05) greater proportion of subjects in the PCDSB group exceeded the MID for PT compared to the placebo group, but there were no significant (p > 0.05) differences in the proportion of subjects exceeding the MID between the CM and placebo groups or between the CM and PCDSB groups. CONCLUSIONS: These findings indicated that for the group mean responses, 28 days of supplementation with both PCDSB and CM resulted in increases in PT and AP. The PCDSB, however, may have an advantage over CM when compared to the placebo group for the proportion of individuals that respond favorably to supplementation with meaningful increases in muscular strength.

Phosphocreatine attenuates doxorubicin-induced cardiotoxicity by inhibiting oxidative stress and activating TAK1 to promote myocardial survival in vivo and in vitro.

Myocardial apoptosis and necroptosis are the major etiological factor during doxorubicin (DOX) induced cardiotoxicity, and one of the important reasons that limit the drug's clinical application. Up to date, its mechanism has not been fully elucidated. The protective role of phosphocreatine (PCr) in heart surgery and medical cardiology has been observed in numerous clinical trials. This study aimed to evaluate cardioprotective actions of PCr against DOX-induced cardiotoxicity and investigate the underlying mechanism involving in transforming growth factor ß-activated kinase 1 (TAK1) mediated myocardial survive signaling pathway. Male Sprague-Dawleyrats were intraperitoneally (ip) injected with normal saline (NS) or DOX (2 mg/kg) alone or DOX with PCr (200 mg/kg) used as animal model. The data showed that DOX significantly impaired cardiac function and structure, induced oxidative stress, myocardial apoptosis and necroptosis, and dramatically down-regulated the expression level of TAK1, while the intervention of PCr obviously attenuated cardiac dysfunction, oxidative stress, myocardial apoptosis and necroptosis, especially alleviated the decrease of TAK1 expression. In vitro analysis, after H9c2 cells were pretreated with or without PCr (0.5 mM) or N-Acetyl-L-cysteine (NAC, 0.5 mM) or 5Z-7-oxozeaenol (5z-7-Ox, 1 µM) for 1 h, subsequently treated with DOX (1 µM) for 24 h. The results revealed that inhibition of TAK1 further deteriorated apoptotic and necroptotic cell death induced by DOX in H9c2 cells, but didn't affect oxidative stress. While the pretreatment of PCr or NAC enhanced antioxidant activity to reduce oxidative stress, significantly alleviated apoptotic and necroptotic cell death induced by DOX in H9c2 cells. Consistent with the results in vivo, PCr or NAC significantly inhibited the decrease of TAK1 expression induced by DOX. In conclusion, oxidative stress induced by DOX inhibits the expression of TAK1, and leads to myocardial apoptotic and necroptotic death, while the intervention of PCr increases antioxidant activity to alleviate oxidative stress, which in turn activates TAK1 signaling pathway to promote myocardial survival, and finally attenuate DOX-induced cardiotoxicity.

Nourin-Associated miRNAs: Novel Inflammatory Monitoring Markers for Cyclocreatine Phosphate Therapy in Heart Failure.

BACKGROUND: Cyclocreatine phosphate (CCrP) is a potent bioenergetic cardioprotective compound known to preserve high levels of cellular adenosine triphosphate during ischemia. Using the standard Isoproterenol (ISO) rat model of heart failure (HF), we recently demonstrated that the administration of CCrP prevented the development of HF by markedly reducing cardiac remodeling (fibrosis and collagen deposition) and maintaining normal ejection fraction and heart weight, as well as physical activity. The novel inflammatory mediator, Nourin is a 3-KDa formyl peptide rapidly released by ischemic myocardium and is associated with post-ischemic cardiac inflammation. We reported that the Nourin-associated miR-137 (marker of cell damage) and miR-106b-5p (marker of inflammation) are significantly upregulated in unstable angina patients and patients with acute myocardial infarction, but not in healthy subjects. OBJECTIVES: To test the hypothesis that Nourin-associated miR-137 and miR-106b-5p are upregulated in ISO-induced "HF rats" and that the administration of CCrP prevents myocardial injury (MI) and reduces Nourin gene expression in "non-HF rats". METHODS: 25 male Wistar rats (180-220 g) were used: ISO/saline (n = 6), ISO/CCrP (0.8 g/kg/day) (n = 5), control/saline (n = 5), and control/CCrP (0.8 g/kg/day) (n = 4). In a limited study, CCrP at a lower dose of 0.4 g/kg/day (n = 3) and a higher dose of 1.2 g/kg/day (n = 2) were also tested. The Rats were injected SC with ISO for two consecutive days at doses of 85 and 170 mg/kg/day, respectively, then allowed to survive for an additional two weeks. CCrP and saline were injected IP (1 mL) 24 h and 1 h before first ISO administration, then daily for two weeks. Serum CK-MB (U/L) was measured 24 h after the second ISO injection to confirm myocardial injury. After 14 days, gene expression levels of miR-137 and miR-106b-5p were measured in serum samples using quantitative real-time PCR (qPCR). RESULTS: While high levels of CK-MB were detected after 24 h in the ISO/saline rats indicative of MI, the ISO/CCrP rats showed normal CK-MB levels, supporting prevention of MI by CCrP. After 14 days, gene expression profiles showed significant upregulation of miR-137 and miR-106b-5p by 8.6-fold and 8.7-fold increase, respectively, in the ISO/saline rats, "HF rats," compared to the control/saline group. On the contrary, CCrP treatment at 0.8 g/kg/day markedly reduced gene expression of miR-137 by 75% and of miR-106b-5p by 44% in the ISO/CCrP rats, "non-HF rats," compared to the ISO/Saline rats, "HF rats." Additionally, healthy rats treated with CCrP for 14 days showed no toxicity in heart, liver, and renal function. CONCLUSIONS: Results suggest a role of Nourin-associated miR-137 and miR-106b-5p in the pathogenesis of HF and that CCrP treatment prevented ischemic injury in "non-HF rats" and significantly reduced Nourin gene expression levels in a dose-response manner. The Nourin gene-based mRNAs may, therefore, potentially be used as monitoring markers of drug therapy response in HF, and CCrP-as a novel preventive therapy of HF due to ischemia.

Phosphocreatine Promotes Osteoblastic Activities in H2O2-Induced MC3T3-E1 Cells by Regulating SIRT1/FOXO1/PGC-1α Signaling Pathway.

BACKGROUND: Osteoporosis, characterized by bone loss, usually occurs with the increased bone resorption and decreased bone formation. H2O2-induced MC3T3-E1 cells are commonly used for the study of osteoblastic activities, which play a crucial role in bone formation. OBJECTIVE: This study aimed to investigate the effects of Phosphocreatine (PCr) on the osteoblastic activities in H2O2-induced MC3T3-E1 cells and elaborate on the possible molecular mechanism. METHODS: The Osteoprotegerin (OPG)/Receptor Activator of NF-κB Ligand (RANKL) ratio and osteogenic markers were detected to investigate the effects of PCr on osteoblastic activities, and the osteoblastic apoptosis was detected using Hochest staining. Moreover, oxidative stress, Adenosine Triphosphate (ATP) generation and the expression of Sirtuin 1 (SIRT1), Forkhead Box O 1 (FOXO1) and Peroxisome Proliferator-Activated Receptor Γ Coactivator-1α (PGC-1α) were also examined to uncover the possible molecular mechanism in H2O2-induced MC3T3-E1 cells. RESULT: The results showed that PCr promoted the osteoblastic differentiation by increasing the expression levels of osteogenic markers of Alkaline Phosphatase (ALP) and Runt-related transcription factor 2 (Runx2), as well as increased the OPG/RANKL ratio and suppressed the osteoblastic apoptosis in H2O2-induced MC3T3-E1 cells. Moreover, treatment with PCr suppressed reactive oxygen species (ROS) over-generation and promoted the ATP production as well as increased the PGC-1α, FOXO1 and SIRT1 protein expression levels in H2O2-induced MC3T3-E1 cells. CONCLUSION: PCr treatment could promote osteoblastic activities via suppressing oxidative stress and increasing the ATP generation in H2O2-induced MC3T3-E1 cells. In addition, the positive effects of PCr on osteoblasts might be regulated by SIRT1/FOXO1/ PGC-1α signaling pathway.

The neuroprotective effects of phosphocreatine on Amyloid Beta 25-35-induced differentiated neuronal cell death through inhibition of AKT /GSK-3ß /Tau/APP /CDK5 pathways in vivo and vitro.

Alzheimer (AD) is a degenerative disease that can lead memory loss and behavioral dysfunction. Aß protein and phosphorylation of Tau protein are related to the onset of AD. However, at present, its treatment and drugs are limited. The purpose of our study is to evaluate whether phosphocreatine (PCr) could protect neuronal injury induced by Aß protein in vivo and in vitro through AKT/GSK-3ß/Tau/APP/CDK5 pathways. Differentiated PC-12 cells were cultured with Aß25-35 for 24 h, while the mice were injected with D-Galactose for eight weeks, both of them were pretreated with PCr for 2 h. The results showed PCr could obviously induce cells and hippocampus apoptosis using DAPI and TUNEL. PCr decreased the levels of intercellular reactive oxygen species (ROS) and malondialdehyde (MDA), and increased the activities of superoxide dismutase (SOD). Besides, the apoptosis pathway was detected using Western blot, showing that PCr could significantly reduce caspase-3, caspase-9, Bcl-2/Bax expression in vivo and in vitro. At the same time, PCr could decreased Ca2+ and apoptosis by Flow Cytometry in PC-12 cells. We observed that the morphological alteration of hippocampus injury was mitigated with the pretreatment of PCr. Furthermore, PCr pretreatment could decrease Aß25-35-induced PC-12 cells apoptosis with APP cDNA transfection, which up-regulated AKT/GSK-3ß/CDK5 pathways and induced Tau phosphorylation. In summary, PCr could reduce Aß25-35 toxicity to protect neuronal cells via AKT/GSK-3ß/CDK5 pathways.

The myocardial protective effect of monosodium phosphate cardioplegia in cardiopulmonary bypass in infants with an atrial septal defect.

This study aimed to investigate the myocardial protective effect of liquid sodium phosphocreatine cardiac arrest in extracorporeal circulation surgery treating infants with atrial septal defects.Eighty-four infants with atrial septal defects who required extracorporeal circulation surgery treatment at our hospital from January 2016 to June 2018 were divided into an observation group and a control group through a digitally randomized method, with 42 cases in each group. The control group adopted the conventional modified St Thomas II high potassium cold liquid crystal cardiac arrest, while the observation group adopted the liquid sodium phosphocreatine cardiac arrest.The myocardial enzyme indexes of the 2 groups 3, 6, 12, and 24 hours postoperatively were higher than before establishing the cardiopulmonary bypass and the enzyme indexes of the control group at the same time were higher than that of the observation group; adenosine triphosphate, adenosine diphosphate, and other energy levels and the postoperative recovery rate energy levels of the observation group were higher than those in the control group, the difference was statistically significant (P < .05).Liquid sodium phosphocreatine cardiac arrest used in extracorporeal circulation surgery treating infants with atrial septal defects can reduce myocardial ischemia-reperfusion injury, maintain energy supply during ischemia, strengthen the St Thomas II effect, and aid postoperative cardiac function recovery of high potassium cold liquid crystal cardiac arrest used in infants with atrial septal defects and treated with extracorporeal circulation surgery.

Phosphocreatine attenuates Gynura segetum-induced hepatocyte apoptosis via a SIRT3-SOD2-mitochondrial reactive oxygen species pathway.

Purpose: To investigate the mitochondria-related mechanism of Gynura segetum (GS)-induced apoptosis and the protective effect of phosphocreatine (PCr), a mitochondrial respiration regulator. Methods: First, the mechanism was explored in human hepatocyte cell line. The mitochondrial oxidative stress was determined by fluorescence assay. The level of sirtuin 3 (SIRT3), acetylated superoxide dismutase 2 (Ac-SOD2), SOD2, and apoptosis were detected by Western blotting. Mito-TEMPO and cell lines of viral vector-mediated overexpression of SIRT3 and SIRT3H248Y were used to further verify the mechanism of GS-induced apoptosis. GS-induced liver injury mice models were built by GS through intragastric administration and interfered by PCr through intraperitoneal injection. A total of 30 C57BL/6J mice were assigned to 5 groups and treated with either saline, PCr (100 mg/kg), GS (30 g/kg), or PCr (50 or 100 mg/kg)+GS (30 g/kg). Liver hematoxylin and eosin (HE) staining, immunohistochemical analysis, and blood biochemical evaluation were performed. Results: GS induced hepatocyte apoptosis and elevated levels of mitochondrial ROS in L-02 cells. The expression of SIRT3 was decreased. Downregulation of SIRT3 was associated with increased levels of Ac-SOD2, which is the inactivated enzymatic form of SOD2. Conversely, when overexpressing SIRT3 in GS-treated cells, SOD2 activity was restored, and mitochondrial ROS levels and hepatocyte apoptosis declined. Upon administration of PCr to GS-treated cells, they exhibited a significant upregulation of SIRT3 and were protected against apoptosis. In animal experiments, serum ALT level and mitochondrial ROS of the mice treated with GS and 50 mg/kg PCr were significantly attenuated compared with only GS treated. The changes in SIRT3 expression were also consistent with the in vitro results. In addition, immunohistochemical analysis of the mouse liver showed that Ac-SOD2 was decreased in the PCr and GS co-treated group compared with GS treated group. Conclusion: GS caused liver injury by dysregulating mitochondrial ROS generation via a SIRT3-SOD2 pathway. PCr is a potential agent to treat GS-induced liver injury by mitochondrial protection.

Phosphocreatine attenuates endoplasmic reticulum stress-mediated hepatocellular apoptosis ameliorates insulin resistance in diabetes model.

Diabetes mellitus (DM) associated liver damage is a major health burden. Hepatocellular-damage in DM characterized with elevated endoplasmic reticulum stress (ER) and may enhanced insulin-resistance. Phosphocreatine (PCr) a rapidly high-energy-reserve molecule of phosphates naturally occurs in liver, brain and skeletal muscle. This study aimed to investigate the protective effect of PCr on the liver-injury-associated with DM and to report the mechanism involved. Wistar rat's diabetes model was induced using streptozotocin (STZ), and the animals were treated with 20 mg/kg, or 50 mg/kg PCr injection. Blood glucose level, and body wt were recorded. Liver tissues homogenate were analyzed for liver damage markers alanine transaminase (ALT), aspartate transaminase (AST). Liver tissues proteins further evaluated for apoptosis, endoplasmic reticulum stress (ER), and insulin resistance biomarkers using western blotting. Our results revealed that PCr reduced blood glucose level, improved body wt, ameliorates liver function enzymes. Furthermore, PCr upregulates anti-apoptotic Bcl2 proteins expression, and down-regulates significantly pro-apoptotic casp3 and Bax proteins expression in vivo and invitro. Moreover, ER stress CHOP, GRP78 and ATF4 biomarkers level were significantly attenuated in PCr treated animals comparing to STZ diabetes associated liver-damage model with significant improving in insulin-resistance Akt and IRS-1. Our results revealed that treating with PCr in diabetes-associated liver injury models decreased blood glucose level and possess protective effect in-vitro and in-vivo, which could be suggested as potential therapeutic strategy for diabetes associated liver injury patients.

Neuroprotective effect of phosphocreatine on oxidative stress and mitochondrial dysfunction induced apoptosis in vitro and in vivo: Involvement of dual PI3K/Akt and Nrf2/HO-1 pathways.

Methylglyoxal (MGO), an active metabolite of glucose, is observed in high levels in the tissues and blood of diabetic patients. Phosphocreatine (PCr), a high-energy phosphate compound, exhibits a range of pharmacological actions but little is well known of its neuroprotective action. The aim of the present study was to investigate the neuroprotective effects and the possible mechanisms of PCr. Diabetes is closely associated with neurodegenerative diseases, leading not only to the peripheral nervous system (PNS) and but also to central nervous system (CNS) damage. Therefore, we established two rat models of diabetes in vivo induced by MGO and streptozocin (STZ) respectively, while utilized differentiated PC-12 cells in vitro. Treatment of PC-12 cells with PCr markedly attenuated MGO-induced change of viability, apoptosis, accompanied by decreased levels of caspase-3, casapse-9 and Bcl-2/Bax protein ratio. Determination of cellular respiratory function was performed with intact PC-12 cells and homogenized hippocampal neuron tissue of rat. Reactive oxygen species (ROS) generation was assessed by membrane permeable fluorescent probe DCFH-DA. The expressions of Akt, Nrf2 and HO-1 were examined by Western blot. PCr pretreatment significantly reduced oxidative stress-induced high LDH, MDA level, and ROS production of PC-12 cells. PCr pretreatment also significantly decreased mitochondrial dysfunction in vitro and in vivo. In addition, PCr pretreatment increased the expression of p-Akt, Nrf2 and HO-1, and reduced the apoptosis. Moreover, the expression of Cleaved caspase3 was partially increased and the p-Akt, Nrf2 and HO-1 was partially reduced by a PI3K inhibitor (LY294002). While, compared with LY294002 groups, pre-treatment with PCr at the concentrations of 20 mM significantly reduced the expression of Cleaved caspase3 and increased the expression of p-Akt, Nrf2 and HO-1. Molecular docking assay showed that PCr possessed powerful affinity towards to Akt with lower binding energy. In conclusion, the neuroprotective effects of PCr in vitro and in vivo rely on normalizing mitochondrial function and reducing oxidative stress via Akt mediated Nrf2/HO-1 pathway, suggesting that PCr may be a novel therapeutic candidate for the treatment of diabetes-associated neurodegenerative diseases.

Efficacy of phosphocreatine pre-administration on XIAP and Smac in ischemic penumbra of rats with focal cerebral ischemia reperfusion injury.

PURPOSE: To observe the efficacy of phosphocreatine pre-administration (PCr-PA) on X-linked inhibitor of apoptosis protein (XIAP), the second mitochondia-derived activator of caspase (Smac) and apoptosis in the ischemic penumbra of rats with focal cerebral ischemia-reperfusion injury (CIRI). METHODS: A total of 60 healthy male Sprague Dawley (SD) rats were randomly divided into three groups (n=20): group A (the sham operation group), group B <intraperitoneally injected with 20 mg/kg (10 mg/ml) of saline before preparing the ischemia-reperfusion (IR) model>, and group C <intraperitoneally injected with 20 mg/kg (10 mg/ml) of PCr immediately before preparing the IR model>. After 24 h for reperfusion, the neurological function was evaluated and the tissue was sampled to detect expression of XIAP, Smac and caspase-3 positive cells in the ischemic penumbra so as to observe the apoptosis. RESULTS: Compared with group B, neurological deficit scores, numbers of apoptotic cells, expression of Smac,caspase-9 and the numbers of Caspase-3 positive cells were decreased while expression of XIAP were increased in the ischemic penumbra of group C. CONCLUSIONS: Phosphocreatine pre-administration may elicit neuroprotective effects in the brain by increasing expression of X-linked inhibitor of apoptosis protein, reducing expression of second mitochondia-derived activator of caspase, and inhibiting the apoptosis in the ischemic penumbra.

Efficacy of phosphocreatine pre-administration on XIAP and Smac in ischemic penumbra of rats with focal cerebral ischemia reperfusion injury

Abstract Purpose: To observe the efficacy of phosphocreatine pre-administration (PCr-PA) on X-linked inhibitor of apoptosis protein (XIAP), the second mitochondia-derived activator of caspase (Smac) and apoptosis in the ischemic penumbra of rats with focal cerebral ischemia-reperfusion injury (CIRI). Methods: A total of 60 healthy male Sprague Dawley (SD) rats were randomly divided into three groups (n=20): group A (the sham operation group), group B <intraperitoneally injected with 20 mg/kg (10 mg/ml) of saline before preparing the ischemia-reperfusion (IR) model>, and group C <intraperitoneally injected with 20 mg/kg (10 mg/ml) of PCr immediately before preparing the IR model>. After 24 h for reperfusion, the neurological function was evaluated and the tissue was sampled to detect expression of XIAP, Smac and caspase-3 positive cells in the ischemic penumbra so as to observe the apoptosis. Results: Compared with group B, neurological deficit scores, numbers of apoptotic cells, expression of Smac,caspase-9 and the numbers of Caspase-3 positive cells were decreased while expression of XIAP were increased in the ischemic penumbra of group C. Conclusions: Phosphocreatine pre-administration may elicit neuroprotective effects in the brain by increasing expression of X-linked inhibitor of apoptosis protein, reducing expression of second mitochondia-derived activator of caspase, and inhibiting the apoptosis in the ischemic penumbra.

Creatine phosphate disodium salt protects against Dox-induced cardiotoxicity by increasing calumenin.

Inhibiting endoplasmic reticulum stress (ERS)-induced apoptosis may be a new therapeutic target in cardiovascular diseases. Creatine phosphate disodium salt (CP) has been reported to have cardiovascular protective effect, but its effects on ERS are unknown. The aim of this study was to identify the mechanism by which CP exerts its cardioprotection in doxorubicin (Dox)-induced cardiomyocytes injury. In our study, neonatal rats cardiomyocytes (NRC) was randomly divided into control group, model group, and treatment group. The cell viability and apoptosis were detected. grp78, grp94, and calumenin of the each group were monitored. To investigate the role of calumenin, Dox-induced ERS was compared in control and down-regulated calumenin cardiomyocytes. Our results showed that CP decreased Dox-induced apoptosis and relieved ERS. We found calumenin increased in Dox-induced apoptosis with CP. ERS effector C/EBP homologous protein was down-regulated by CP and it was influenced by calumenin. CP could protect NRC by inhibiting ERS, this mechanisms may be associated with its increasing of calumenin.

A Novel Method to Synthesize Phosphocreatine and Phosphocreatine Prodrugs.

BACKGROUND: Adenosine triphosphate (ATP) is the energy currency of the body; it takes part in various and indispensable metabolic processes for the maintenance of cell homeostasis, degrading to its hydrolysis product, adenosine diphosphate (ADP). Efficient ways to restore ATP are therefore necessary in the cells. When the cell lacks energy due to ischemic conditions or high ATP demand, phosphocreatine gives its phosphate group to ADP that converts to ATP, in a reaction catalyzed by the enzyme creatine kinase. For this reason, phosphocreatine is utilized as a pharmacological treatment in human diseases that involve a failure of the cellular energy, most notably in coronary artery disease. OBJECTIVE: Commercially available phosphocreatine is currently synthesized using different methods, each of one characterized by a rather low yield of the final product, probably due to the low reactivity of the guanylating reagent. The aim of this work is to overcome the drawbacks of the synthetic methods currently employed, devising a novel synthetic route to obtain phosphocreatine and phosphocreatine prodrugs in higher yields and purity. METHOD: To obtain a higher yield of the final product and a lower number of sub-products, this method utilizes a new guanylating agent characterized by high reactivity, endowed with a protecting group t-Boc on one of the two nitrogen atoms of the guanidinic function and a protected phosphate on the other one; that compound is then conjugated with an opportune secondary amine. The obtained product is cleaved first with acidic conditions to obtain the phosphocreatine prodrug (phosphocreatine ethyl ester) and then with an enzymatic method to obtain the phosphocreatine. RESULT: Have been obtained in good yield and purity as demonstrated by HPLC and mass spectrometry analysis. CONCLUSION: This novel synthetic route permits to obtain the phosphocreatine molecule in higher yield and purity compared to the methods currently employed with a combination of chemical and enzymatic methods.

Phosphocreatine protects endothelial cells from Methylglyoxal induced oxidative stress and apoptosis via the regulation of PI3K/Akt/eNOS and NF-κB pathway.

Methylglyoxal (MGO), an active metabolite of glucose, can cause cellular injury which has an affinity for the progression of diabetes-associated atherosclerosis. Phosphocreatine (PCr) is a well-known high-energy phosphate compound. However, its protective effects and mechanism in the formation of a diabetes-associated atherosclerosis have not been clarified. In the present study, we investigated whether PCr could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs) and explored the possible mechanisms. Cells were pre-treated with PCr and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were assessed by light microscopy, MTT assay, and Annexin V-FITC respectively. Apoptotic-related proteins were evaluated by Western blotting. Reactive oxygen species (ROS) generation, intracellular calcium and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Our results showed that PCr dose-dependently prevented MGO associated HUVEC cytotoxicity and suppressed MGO activated ROS generation as well as apoptotic biochemical changes such as lactate dehydrogenase, malondialdehyde leakage, loss of MMP, decreased Bcl-2/Bax protein ratio, levels of caspase-3 and 9. In addition, the antiapoptotic effect of PCr enhanced p-Akt/Akt protein ratio, NO synthase (eNOS) activation, NO production and cGMP levels and also was partially suppressed by a PI3K inhibitor (LY294002). Furthermore, PCr also inhibited MGO-induced transcriptional activity of Nuclear factor kappa B (NFκB). In conclusion, our data described that PCr exerts an antiapoptotic effect in HUVECs exposed to oxidative stress by MGO through the mitochondrial pathway and the modulation of PI3K/Akt/eNOS and NF-κB signaling pathway. Thus, it might be a candidate therapeutic agent for diabetic-associated cardiovascular diseases.

Phosphocreatine attenuates angiotensin II-induced cardiac fibrosis in rat cardiomyocytes through modulation of MAPK and NF-κB pathway.

OBJECTIVE: Transforming growth factor ß (TGF-ß) plays crucial roles in Ang II-induced cardiac fibrosis (CF). Phosphocreatine (PCr), one of the important players involved in cellular energy metabolism, is widely used in the treatment of clinical heart failure. However, whether it participates in CF is still unclear. This study aimed to identify the mechanisms involved in PCr and CF. MATERIALS AND METHODS: Rat cardiomyocytes (H9C2) were induced by Ang II followed by treatment of PCr. ERK1 siRNA, ERK2 siRNA and NF-κB siRNA were applied to identify the molecular mechanism. Then CF-related proteins were analyzed by western blot and real-time PCR to confirm the influence of the mechanisms involved in PCr. RESULTS: PCr did protect cardiomyocytes from Ang II-induced fibrosis. Meanwhile, PCr suppressed Ang II-induced up-regulation of TGF-ß. By detecting TGFß-mediated or MAPK pathway associated proteins, PCr inhibited MAPK and NF-κB pathway, thus suppressed Ang II-induced cardiac fibrosis, which was further confirmed by siRNA transfection. CONCLUSIONS: Our study determined that PCr protected cardiomyocytes from Ang II-induced CF through inhibition of MAPK and NF-κB pathway.