Phosphocreatine Ameliorates Diabetes And Its Liver Injury In Mice By Inhibiting The Endoplasmic Reticulum Stress-mediated Hepatic Apoptosis In Vitro And In Vivo

Background Hepatocellular cells damaged represents an increasing morbidity and mortality among diabetic patients.Hyperglycemia been identified as the primary cause in the pathogenesis of liver damage in DM patients.Hepatocellular cells damaged associated with Diabetes mellitus(DM)are common complications in Diabetes mellitus patients(DM).Phosphocreatine(PCr)C4H10N3O5P also known as Creatine phosphate;Phosphorylcreatine;N-phosphocreatine;phosphorylated creatine molecule serves as a rapidly mobilizable of high-energy reserve of phosphates in brain and skeletal muscle to recycle adenosine triphosphate ATP,which could be recognize as the energy currency of the cell.It was reported to possess many functions.However,whether PCr possesses protective effects against diabeties associated liver injury is not reported.This study aimed to evaluate whether PCr protect liver cells against liver injury associated with DM and to report the mechanism involved in its action in vivo and in vitro.Materials and Methods(In vitro)Methylglyoxal(MGO),is an active metabolite of glucose,can cause hepatocellular injury with hyperglycemia which has an affinity for the progression of diabetes-associated liver cells damage.Hep G2 cells were cultured and cells were pre-treated with MGO for two hours,then cells were treated with different concentrations of PCr(5m M,10 m M,20 m M)respectively.Cell morphology,cytotoxicity,and apoptosis were studied using MTT assay;flowcytometry Annexin V-FITC cells apoptosis assay;Calcien AM proliferation assay were assessed respectively.Protein expression levels were assessed using western blotting for apoptotic proteins Bax;Bcl-2;and Caspase3;and Caspase9 and CHOP,ATF4,IRS ER stress proteins expression.Animal model(In Vivo)Wistar rats received a single intraperitoneal injection of streptozotocin(80mg/kg body weight)and treated with PCr consequently grouped to 20mg/kg and 50mg/kg body weight.Biochemical assay of liver tissues homogenate was performed to analyze liver metabolites such as MDA,GSH and SOD using spectrophotometry,liver tissues proteins isolated were evaluated for apoptosis and ER stress proteins expression using western blot.Results In general treatment with PCr in Cell line experiments shown that MGO induced Hep G2 cells damage and apoptosis while treated cells with different concentrations of PCr enhanced the cells proliferation,decreased apoptosis and ameliorated cells healing.Western blot results revealed an increase of apoptotic proteins such as Caspase 3,and Caspase 9,Bax,while antiapoptotic protein Bcl2 was decreased in MGO group and reversed by PCr treated cells in time and dose dependent manner.Furthermore,mechanistic study was carried out to elaborated the protective mechanism of PCr on liver cells ER stress,revealed a significant increase in ER stress proteins level in MGO induced liver damage,while this effect reversed by PCr treatment cells in dose and time dependent manner.In vivo,a study regarding diabetes associated liver injury with the treatment of PCr shown a reduced blood sugar level,and PCr possessed protective effects against STZ-induced diabetic associated liver endothelial cells injury in vivo.Furthermore,Diabetes animal model was obtained to verify and check whether the effect of PCr could ameliorate liver tissue injury in vivo,our results revealed a protective effect and quick recover after injury induced as shown in histology staining of liver tissues.Moreover,Biochemical assay was performed on liver tissues such as GSH,MDA,SOD were measured using spectrophotometry,revealing an anti-oxidants effect of PCr may involve in its protective mechanism.Furthermore,the apoptotic proteins level and ER stress level was decreased in PCr treated animals comparing to STZ induced diabetes associated liver injury.Also decreased in Bcl-2/Bax protein ratio,and decrease of Caspase-3 and 9 proteins expression.Conclusion our study revealed that treating with PCr in diabetes associated liver injury models possess protective effect in vivo and in vitro as well as anti-oxidant,anti-apoptotic effect.ER stress amelioration may involve in the mechanism of PCr protective effect via ER stress signaling pathway.Which may suggest a possible therapeutic strategy for diabetes associated liver injury patients.