Molecular genetic and clinical characteristic analysis of primary signet ring cell carcinoma of urinary bladder identified by a novel OR2L5 mutation.

To get a better understanding of the genetic basis of primary signet ring cell carcinoma (SRCC) of the bladder, which is highly rare and not yet explored. First, by using immunohistochemistry to find histological pathological characteristics. Second, a massively parallel whole-exome sequencing (WES) was performed on a 58-year-old male patient who had painless macroscopic hematuria and was pathologically diagnosed with primary SRCC of the bladder, followed by comparing with genes of ordinary urothelial cancer (UC) from TCGA. Furthermore, a population-based analysis using the SEER database was performed to investigate the prognosis (SRCC vs. UC). We identified 63 copy number variations (CNVs) with gain counts and 181 CNVs with loss counts. Totally 4515 mutations were discovered in C > T with a success rate of greater than 89%. The most frequently mutated pathway was RTK-RAS which has 85 genes involved in carcinogenic signaling. Final screening on predisposing genes is performed after filtering based on ACMG. Moreover, several driver genes, including NBN, KCTD18, SPATA13, ANKRD36, OR2L5, MALRD1, and LSMEM1, were detected. Sanger sequencing of germline DNA revealed the presence of a mutant base A/G of OR2L5 in the sequence, which was discovered for the first time in primary SRCC of the bladder. Furthermore, the immunohistochemical profile showed that primary SRCC of the bladder were positive for CK7, CK20, GATA-3, and expression of CK(AE1/AE2), EMA, and Ki67. In the SEER-based study, the patients with primary SRCC of the bladder got a worse prognosis compared to those with UC with median months overall survival (OS) 14 vs. 41, respectively, P = 0001, even after adjusting the variables in the Cox regression model, the SRCC of the bladder showed worse survival HR = 1.119, 95% CI = (1.081-1.328), P = 0.0001. These results imply that suppression of potential driver mutations may be a viable adjuvant treatment approach for primary SRCC in the bladder in place of standard chemotherapy, a possibility that warrants further clinical investigation.

A Comprehensive Update of Cerebral Organoids between Applications and Challenges.

The basic technology of stem cells has been developed and created organoids, which have established a strong interest in regenerative medicine. Different cell types have been used to generate cerebral organoids, which include interneurons and oligodendrocytes (OLs). OLs are fundamental for brain development. Abundant studies have displayed that brain organoids can recapitulate fundamental and vital features of the human brain, such as cellular regulation and distribution, neuronal networks, electrical activities, and physiological structure. The organoids contain essential ventral brain domains and functional cortical interneurons, which are similar to the developing cortex and medial ganglionic eminence (MGE). So, brain organoids have provided a singular model to study and investigate neurological disorder mechanisms and therapeutics. Furthermore, the blood brain barrier (BBB) organoids modeling contributes to accelerate therapeutic discovery for the treatment of several neuropathologies. In this review, we summarized the advances of the brain organoids applications to investigate neurological disorder mechanisms such as neurodevelopmental and neurodegenerative disorders, mental disorders, brain cancer, and cerebral viral infections. We discussed brain organoids' therapeutic application as a potential therapeutic unique method and highlighted in detail the challenges and hurdles of organoid models.

ARID1A downregulation promotes cell proliferation and migration of colon cancer via VIM activation and CDH1 suppression.

According to our prior findings, ARID1A expression is decreased in colon cancer, which has a poor prognosis. In this study, we investigated the ARID1A-VIM/CDH1 signalling axis's role in colon cancer proliferation and migration. The differentially expressed genes in cells that might be controlled by ARID1A were discovered by a database screening for ARID1A knockout. qPCR was used to analyse ARID1A and EMT markers expression levels in colon cancer. We utilized siRNA RID1A to explore the influence of ARID1A silencing on EMT in CRC cells. The function of ARID1A in the colon was investigated utilizing the wound healing, transwell and CCK-8 WST- assays. The molecular mechanism by which ARID1A regulates VIM and CDH1 was elucidated using chip-qPCR. Numerous genes involved in EMT were dysregulated in the absence of ARID1A. VIM expression increased in cells lacking ARID1A expression and vice versa. Many COAD samples with high ARID1A mRNA expression had low VIM mRNA expression, despite the relevance. CDH1 gene was positively correlated with ARID1A. Moreover, siRNA-ARID1A-transfected cells accelerated cell migration and invasion and increased cell proliferation rate in vitro. Chip-qPCR analysis showed that ARID1A binds to the promoters of both genes and changes their expression in colon cancer. ARID1A inactivation is associated with VIM activation and CDH1 suppression, which might serve as crucial molecules influencing COAD prognosis, accelerate tumour progression, and shorten patients' survival time, and promote metastases of COAD. Thus, depletion of ARID1A can be therapeutically exploited by targeting downstream effects to improve cancer treatment-related outcomes.

Aberrantly hypermethylated ARID1B is a novel biomarker and potential therapeutic target of colon adenocarcinoma.

Background and Objective: Understanding the tumor microenvironment (TME) and immune cell infiltration (ICI) may help guide immunotherapy efforts for colon cancer (COAD). However, whether ARID1B is truly regulated by hypermethylation or linked to immune infiltration remains unknown. The current work focused on the ARID1B gene expression and methylation in COAD, as well as its relation with ICI. Methods and Results: Multiple tools based on TCGA were used to analyze the differences in the expression of the ARID1B gene, DNA methylation, and its association with various clinicopathological features, somatic mutations, copy number variation, and the prognosis of patients with COAD. According to the analysis results, patients with high mRNA, low methylation levels showed better overall survival than patients with low mRNA, high methylation levels. The correlation analysis of immune cell infiltration and immune checkpoint gene expression showed that the infiltration rates of the main ICI subtypes, cancer-associated fibroblast, and myeloid cells were significantly enriched and correlated with ARID1B in COAD. An association between ARID1B expression and immune infiltration in COAD was found by correlating ICI indicators with ARID1B expression in the immune cell composition of the COAD microenvironment. Notably, M2 chemokines were related to ARID1B expression, while M1 chemokines were not. Conclusion: This study provided evidence that ARID1B may have a role in the pathogenesis of COAD. The specific underlying mechanisms that could be responsible for ARID1B's downregulation in COAD will need to be investigated in the future.

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.

Biochemical and clinical predictors in pregnant women with antiphospholipid syndrome and systemic lupus erythematosus: comprehensive update.

BACKGROUND: Autoimmune diseases as antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE) could cause many maternal complications. The most common maternal complications of autoimmune diseases are lupus flare, hypertension, nephritis, preeclampsia (PE), eclampsia, and poor pregnancy outcomes which including preterm delivery and pregnancy loss. Only the lupus anticoagulant in the greatest prospective multicenter study has been associated with adverse pregnancy outcomes of the APS. PURPOSE: This review aims to provide a comprehensive update for predictors in pregnant women with APS/SLE. METHODS: These data have been collected from clinical and pathological studies, systematic reviews, and meta-analysis. RESULTS: In recent years the SLE and APS demonstrated to have different and valuable clinical and biomarker predictors for the pregnancy outcome. Treatment of pregnant women with APS is low molecular weight heparin (LMWH) and aspirin; however, around 75% of this management is considered successful. CONCLUSION: This review summarizes recent research that focuses on biochemical and clinical predictors of adverse pregnancy outcomes (APOs) of pregnant women with SLE and APS. Furthermore, we have collected more evidence that confirms the safety and efficacy of hydroxychloroquine (HCQ) preventing APOs.

Enhancement of gemcitabine efficacy by K73-03 via epigenetically regulation of miR-421/SPINK1 in gemcitabine resistant pancreatic cancer cells.

BACKGROUND: Gemcitabine (GCB) is a first-line chemotherapeutic drug for pancreatic cancer (PCa). However, the resistance begins developing within weeks of chemotherapy. SPINK1 overexpression enhances resistance to chemotherapy. In a recent study, our laboratory established that the oleanolic acid (OA) derivative, K73-03, had a strong inhibitory effect on a SPINK1 overexpressed PCa cells. PURPOSE: In our current study, we studied the enhancement of GCB inhibitory effect by K73-03, a new novel OA derivative, alone or in combination with GCB on the GCB-resistant PCa cells by mitochondrial damage through regulation of the miR-421/SPINK1. METHODS: We detected the binding between miR-421 and SPINK1-3'-UTR in GCB-resistant PCa cells using Luciferase reporter assays. Cells viability, apoptosis, migration, and mitochondrial damage were investigated. RESULTS: The results demonstrated that the combination of K73-03 and GCB suppressed the growth of AsPC-1 and MIA PaCa-2 cells synergistically, with or without GCB resistance. Mechanistic findings showed that a combination of K73-03 and GCB silences SPINK1 epigenetically by miR-421 up-regulating, which leads to mitochondrial damage and inducing apoptosis in GCB-resistant PCa cells. CONCLUSION: We found an interesting finding that the 73-03 in combination with GCB can improve GCB efficacy and decrease PCa resistance, which induced apoptosis and mitochondrial damage through epigenetic inhibition of SPINK1 transcription by miR-421 up-regulation. This was the first study that used OA derivatives on GCB-resistant PCa cells, so this combined strategy warrants further investigation.

PD-1/PDL-1 Inhibitors and Cardiotoxicity; Molecular, Etiological and Management Outlines.

Background: The US Food and Drug Administration (FDA) has approved several immunotherapeutic drugs for cancer since 2010, and many more are still being evaluated in other clinical studies. These inhibitors significantly increase response rates and result in the treatment of patients with advanced cancer. However, cancer immunotherapy leads to essential cardiac toxicity properties that have become distinct from other cancer patients' care and are mostly related to their etiology. Aim of review: As potential implications, the occurrence of cardiovascular adverse events is particularly challenging and needs a comprehensive understanding of overall cancer-related etiology, clinical outcomes with different variable severity, and management. Key scientific concepts of review: In terms of improving the overall survival of patients with cancer, clinicians should be careful in selecting either programmed cell death-1 (PD-1) or its programmed cell death ligand (PDL-1) inhibitors by evaluating their risk and clinical benefit for early intervention and decrease the level of morbidity and mortality of their patients. This review focuses on the effectiveness of PD-1/PL-1 antibodies and associated cardiotoxicity adverse events, including etiological mechanisms, diagnosis, and treatment.

TL1A/TNFR2 Axis Enhances Immunoregulatory Effects of Bone Marrow Derived Mesenchymal Stem Cell by Indian Hedgehog Signaling Pathway.

BACKGROUND AND OBJECTIVES: The immunomodulatory potential of mesenchymal stem cells (MSCs) can be regulated by a variety of molecules, especially cytokines. The inflammatory cytokine, TNF-like ligand 1A (TL1A), has been reported as an inflammation stimulator in-multiple autoimmune diseases. Here, we studied the effects of TL1A/TNF-receptor 2 (TNFR2) pathway on the therapeutic potency of bone marrow-derived MSCs (BMSCs). METHODS AND RESULTS: BMSCs, fibroblast-like synoviocytes (FLSs), and H9 and jurkat human T lymphocytes were used in this study. BMSCs paracrine activities, differentiation, proliferation, and migration were investigated after stimulation with TL1A, and intervened with anti-TNFR2. Additionally, the effects of TL1A on BMSCs therapeutic potency were evaluated by treating RA-FLSs, and H9 and jurkat T cells with TL1A-stimulated BMSCs conditioned medium (CM). Indian hedgehog (IHH) involvement was determined by gene silencing and treatment by recombinant IHH (rIHH). TL1A induced BMSCs stemness-related genes, COX-2, IL-6, IDO, TGF-ß and HGF through TNFR2. Also, TL1A corrected biased differentiation and increased proliferation, and migration through TNFR2. Meanwhile, CM of TL1A-stimulated BMSCs decreased the inflammatory markers of RA-FLSs and T cells. Moreover, TL1A-stimulated BMSCs experienced IHH up-regulation coupled with NF-κB and STAT3 signaling up-regulation, while p53 and oxidative stress were down-regulated. Furthermore, treatment of BMSCs by rIHH increased their anti-inflammatory effects. More importantly, knockdown of IHH decreased the ability of TL1A-stimulated BMSCs to alleviating the inflammation in RA-FLSs and T cells. CONCLUSIONS: This study reports the effects of TL1A/TNFR2 pathway on the biological behaviors and therapeutic potency of BMSCs through IHH. These findings could introduce novel procedures to increase the stemness of MSCs in cellular therapy.

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.

Selaginellin B induces apoptosis and autophagy in pancreatic cancer cells via the JAK2/STAT3 signaling pathway.

Selaginella tamariscina (ST), a well-known traditional medicinal plant, has been used to treat various cancers, including pancreatic cancer. However, the underlying mechanism by which Selaginellin B, a natural pigment isolated and purified from ST, protects against pancreatic cells has yet to be fully elucidated. In the present study, the biological functions of Selaginellin B were investigated using apoptosis, migration and colony formation assays in ASPC-1 and PANC-1 cells. In addition, apoptosis-associated proteins were detected by Western blotting. Our results demonstrated that Selaginellin B induced apoptosis, as evidenced by the increased cleaved caspase-3 level and Bax/Bcl-2 ratio. Moreover, Selaginellin B led to a marked up-regulation of the ratio of LC3-II/LC3-I in ASPC-1 and PANC-1 cells, respectively. Furthermore, reverse pharmacophore screening, molecular docking and molecular dynamics simulation studies revealed that Janus kinase 2 (JAK2) may be a potential target for Selaginellin B. In summary, the results of the present research have demonstrated that Selaginellin B is an effective anticancer agent against PANC-1 and ASPC-1 cells, and the compound holds great promise for the treatment of pancreatic cancer.

miR-421 up-regulation by the oleanolic acid derivative K73-03 regulates epigenetically SPINK1 transcription in pancreatic cancer cells leading to metabolic changes and enhanced apoptosis.

SPINK1 overexpression promotes cancer cell aggressiveness and confers chemo-resistance to multiple drugs in pancreatic cancer. Oleanolic acid (OA) derivatives possess active effects against different cancers. Here we report the effect of K73-03, a new novel OA derivative, against pancreatic cancer through mitochondrial dysfunction via miR-421/SPINK1 regulation. We examined the binding ability of miR-421 with SPINK1-3'UTR Luciferase reporter assays. Moreover, miR-421/SPINK1 expressions in pancreatic cancer, with or without K73-03 treatment, were evaluated. Cells viability, migration, autophagy, mitochondrial function and apoptosis were examined with or without K73-03 treatment. We established that the K73-03 effect on the miR-421 that plays a crucial role in the regulation of SPINK1 in pancreatic cancer. Our findings indicated that K73-03 inhibited the mitochondrial function that led to inducing autophagy and apoptosis through epigenetic SPINK1 down-regulation via miR-421 up-regulation in pancreatic cancer. Furthermore, the inhibition of miR-421 expression in pancreatic cancer cells abolished the efficacy of K73-03 against SPINK1 oncogenic properties. We found an interesting finding that the interaction between miR-421 and SPINK1 is related to mitochondrial function through the effect of K73-03. Further, SPINK1 appear to be the molecular targets of K73-03 especially more than gemcitabine.

Cinnamaldehyde protects against rat intestinal ischemia/reperfusion injuries by synergistic inhibition of NF-κB and p53.

Our preliminary study shows that cinnamaldehyde (CA) could protect against intestinal ischemia/reperfusion (I/R) injuries, in which p53 and NF-κB p65 play a synergistic role. In this study, we conducted in vivo and in vitro experiments to verify this proposal. SD rats were pretreated with CA (10 or 40 mg · kg-1 · d-1, ig) for 3 days, then subjected to 1 h mesenteric ischemia followed by 2 h reperfusion. CA pretreatment dose-dependently ameliorated morphological damage and reduced inflammation evidenced by decreased TNF-α, IL-1ß, and IL-6 levels and MPO activity in I/R-treated intestinal tissues. CA pretreatment also attenuated oxidative stress through restoring SOD, GSH, LDH, and MDA levels in I/R-treated intestinal tissues. Furthermore, CA pretreatment significantly reduced the expression of inflammation/apoptosis-related NF-κB p65, IKKß, IK-α, and NF-κB p50, and downregulated apoptotic protein expression including p53, Bax, caspase-9 and caspase-3, and restoring Bcl-2, in I/R-treated intestinal tissues. We pretreated IEC-6 cells in vitro with CA for 24 h, followed by 4 h hypoxia and 3 h reoxygenation (H/R) incubation. Pretreatment with CA (3.125, 6.25, and 12.5 µmol · L-1) significantly reversed H/R-induced reduction of IEC-6 cell viability. CA pretreatment significantly suppressed oxidative stress, NF-κB activation and apoptosis in H/R-treated IEC-6 cells. Moreover, CA pretreatment significantly reversed mitochondrial dysfunction in H/R-treated IEC-6 cells. CA pretreatment inhibited the nuclear translocation of p53 and NF-κB p65 in H/R-treated IEC-6 cells. Double knockdown or overexpression of p53 and NF-κB p65 caused a synergistic reduction or elevation of p53 compared with knockdown or overexpression of p53 or NF-κB p65 alone. In H/R-treated IEC-6 cells with double knockdown or overexpression of NF-κB p65 and p53, CA pretreatment caused neither further decrease nor increase of NF-κB p65 or p53 expression, suggesting that CA-induced synergistic inhibition on both NF-κB and p53 played a key role in ameliorating intestinal I/R injuries. Finally, we used immunoprecipitation assay to demonstrate an interaction between p53 and NF-κB p65, showing the basis for CA-induced synergistic inhibition. Our results provide valuable information for further studies.

MicroRNA-4316 inhibits gastric cancer proliferation and migration via directly targeting VEGF-A.

BACKGROUND AND AIMS: microRNAs (miRNAs) have been reported to regulate proliferation and migration by down-regulating the expression of target genes. The aims of this study were to investigate whether miR-4316 inhibited proliferation and migration by downregulating vascular endothelial growth factor A (VEGF-A) and its clinical significance in gastric cancer (GC). METHODS: The clinical tissues of the GC patients for miR-4316 and VEGF-A were detected by qRT-PCR. The protein levels of VEGF-A and c-Met were determined by western blotting. Cell Proliferation, migration, and colony forming assays were conducted to show whether miR-4316 affects proliferation by CCK-8, migration by transwell, wound healing and colony formation assays. The bioinformatic methods and luciferase reporter assay were applied to detect the relationship between miRNA and VEGF-A on its targeting 3-untranslated regions (3-UTRs). CCK-8, colony formation, wound healing, and transwell assay were performed to explore the function of miR-4316. RESULTS: The results of qRT-PCR indicated that miR-4316 expression level was significantly downregulated in human GC tissues and GC cell lines compared with their control. miR-4316 inhibited proliferation, migration and colony formation in GC cell lines by reducing VEGF-A. And western blot results indicated that miR-4316 significantly inhibited GC through repressing VEGF-A and c-Met. The investigation of Luciferase assay indicated that VEGF-A is a direct target gene of miR-4316. CONCLUSIONS: miR-4316 suppressed proliferation and migration of GC through the VEGF-A gene. MiR-4316 acts as a tumor suppressor by targeting VEGF-A and this indicated that MiR-4316 might be a potential therapeutic target for GC.

Protection of diabetes-induced kidney injury by phosphocreatine via the regulation of ERK/Nrf2/HO-1 signaling pathway.

Diabetic nephropathy is the most common long-term complication of diabetes mellitus. The Methylglyoxal (MGO) production is mainly by metabolic pathways, such as lipolysis and glycolysis, its increases in the DM enhances oxidative stress and plays a crucial role in the diabetic nephrotic pathogenesis. Phosphocreatine (PCr) can improve lipopolysaccharide, ox-LDL-induced atherosclerosis, and alleviate vascular endothelial cell injury in diabetes. The aim of our present study is to examine the potential role of phosphocreatine (PCr) as a molecule protects against diabetes-induced Kidney Injury in-vitro and in-vivo through ERK/Nrf2/HO-1 signaling pathway. NRK-52E cells treatment with PCr obviously suppressed MGO-induced change of viability, apoptosis, coupled with decreased Bax/Bcl-2ratio, casapse-9 and caspase-3expressions. We determined the generation of reactive oxygen species (ROS) using membrane permeable fluorescent probe DCFH-DA as well as intracellular calcium by flow cytometry. ERK, Nrf2 and HO-1 expressions were determined by Western blot. PCr pretreatment significantly returned the oxidative stress enzymes to normal condition in-vitro and in-vivo. PCr pretreatment significantly reduced apoptosis, calcium and ROS production, induced by MGO, in NRK-52E cells. Moreover, pretreatment with PCr significantly inhibited cleaved caspase-3, cleaved caspase-9 and p-ERK expressions, while increased Nrf-2 and HO-1 expressions. Furthermore, PCr pretreatment significantly decreased p-ERK expression of MGO-induced injury in NRK-52E cells transfected with p-ERK cDNA. In conclusion, the renal protective effect of PCr in-vitro and in-vivo depends on suppressing apoptosis and ROS generation through ERK mediated Nrf-2/HO-1 pathway, suggesting that PCr may be a novel therapeutic candidate for the diabetic nephropathy treatment.

Phosphocreatine Improves Cardiac Dysfunction by Normalizing Mitochondrial Respiratory Function through JAK2/STAT3 Signaling Pathway In Vivo and In Vitro.

Diabetic cardiomyopathy (DCM) is one of the common cardiovascular complications in patients with diabetes. Accumulating evidence has demonstrated that DCM is thoroughly related to mitochondrial energy impairment and increases the generation of reactive oxygen species (ROS). Therefore, an ongoing study is developing strategies to protect cardiac mitochondria from diabetic complications, especially from hyperglycemia. Phosphocreatine (PCr) plays a major metabolic role in cardiac muscular cells including intracellular concentration of ATP which affects the activity of the myocardium. We hypothesized that PCr might improve oxidative phosphorylation and electron transport capacity in mitochondria impaired by hyperglycemia in vivo and in vitro. Also, we aimed to evaluate the protective effect of PCr against DCM through the JAK2/STAT3 signaling pathway. The mitochondrial respiratory capacity from rats and H9C2 cells was measured by high-resolution respirometry (HRR). Expressions of proteins Bax, Bcl-2, caspase 3, caspase 9, cleaved caspase 3, and cleaved caspase 9, as well as JAK2/STAT3 signaling pathways, were determined by western blotting. ROS generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Type 1 diabetes mellitus was induced in Wistar male rats by a single intraperitoneal injection of streptozotocin (STZ) (80 mg/kg body weight). Our results revealed that PCr possessed protective effects against DCM injury by improving the mitochondrial bioenergetics and by positively exerting protective effects against DCM in vivo and in vitro, not only improving diabetes symptom, resulting in changes of cardiac tissue using hematoxylin and eosin (H&E) stain, but also ameliorating biochemical changes. Moreover, PCr increased Bcl-2, caspase 3, and caspase 9 protein expressions and decreased Bax, cleaved caspase 3, and cleaved caspase 9 expressions as well as the JAK2/STAT3 signaling pathway. In conclusion, PCr improves mitochondrial functions and exerts an antiapoptotic effect in vivo and in vitro exposed to oxidative stress by hyperglycemia through the JAK2/STAT3 signaling pathway. Our findings suggest that PCr medication is a possible therapeutic strategy for cardioprotection.

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.