Therapeutic Potential of Peucedanum japonicum Thunb. and Its Active Components in a Delayed Corneal Wound Healing Model Following Blue Light Irradiation-Induced Oxidative Stress.

Blue light is reported to be harmful to eyes by inducing reactive oxygen species (ROS). Herein, the roles of Peucedanum japonicum Thunb. leaf extract (PJE) in corneal wound healing under blue light irradiation are investigated. Blue-light-irradiated human corneal epithelial cells (HCECs) show increased intracellular ROS levels and delayed wound healing without a change in survival, and these effects are reversed by PJE treatment. In acute toxicity tests, a single oral administration of PJE (5000 mg/kg) does not induce any signs of clinical toxicity or body weight changes for 15 days post-administration. Rats with OD (oculus dexter, right eye) corneal wounds are divided into seven treatment groups: NL (nonwounded OS (oculus sinister, left eye)), NR (wounded OD), BL (wounded OD + blue light (BL)), and PJE (BL + 25, 50, 100, 200 mg/kg). Blue-light-induced delayed wound healing is dose-dependently recovered by orally administering PJE once daily starting 5 days before wound generation. The reduced tear volume in both eyes in the BL group is also restored by PJE. Forty-eight hours after wound generation, the numbers of inflammatory and apoptotic cells and the expression levels of interleukin-6 (IL-6) largely increase in the BL group, but these values return to almost normal after PJE treatment. The key components of PJE, identified by high-performance liquid chromatography (HPLC) fractionation, are CA, neochlorogenic acid (NCA), and cryptochlorogenic acid (CCA). Each CA isomer effectively reverses the delayed wound healing and excessive ROS production, and their mixture synergistically enhances these effects. The expression of messenger RNAs (mRNAs) related to ROS, such as SOD1, CAT, GPX1, GSTM1, GSTP1, HO-1, and TRXR1, is significantly upregulated by PJE, its components, and the component mixture. Therefore, PJE protects against blue-light-induced delayed corneal wound healing via its antioxidative, anti-inflammatory, and antiapoptotic effects mechanistically related to ROS production.

De Novo Assembly and Species-Specific Marker Development as a Useful Tool for the Identification of Scutellaria L. Species.

Scutellaria L. (family Lamiaceae) includes approximately 470 species found in most parts of the world and is commonly known as skullcaps. Scutellaria L. is a medicinal herb used as a folk remedy in Korea and East Asia, but it is difficult to identify and classify various subspecies by morphological methods. Since Scutellaria L. has not been studied genetically, to expand the knowledge of species in the genus Scutellaria L., de novo whole-genome assembly was performed in Scutellaria indica var. tsusimensis (H. Hara) Ohwi using the Illumina sequencing platform. We aimed to develop a molecular method that could be used to classify S.indica var. tsusimensis (H. Hara) Ohwi, S. indica L. and three other Scutellaria L. species. The assembly results for S.indica var. tsusimensis (H. Hara) Ohwi revealed a genome size of 318,741,328 bp and a scaffold N50 of 78,430. The assembly contained 92.08% of the conserved BUSCO core gene set and was estimated to cover 94.65% of the genome. The obtained genes were compared with previously registered Scutellaria nucleotide sequences and similar regions using the NCBI BLAST service, and a total of 279 similar nucleotide sequences were detected. By selecting the 279 similar nucleotide sequences and nine chloroplast DNA barcode genes, primers were prepared so that the size of the PCR product was 100 to 1000 bp. As a result, a species-specific primer set capable of distinguishing five species of Scutellaria L. was developed.

Peucedanum japonicum Thunberg and Its Active Components Mitigate Oxidative Stress, Inflammation and Apoptosis after Urban Particulate Matter-Induced Ocular Surface Damage.

We previously demonstrated that urban particulate matter (UPM) exposure decreases the migration activity and survival of human corneal epithelial cells (HCECs). Herein, we investigated the potential to improve the corneal wound-healing ability of Peucedanum japonicum Thunb. leaf extract (PJE) and its active components on UPM-induced ocular surface damage in vitro and in vivo. PJE effectively assisted wound healing without altering HCEC survival and enhanced catalase (CAT), heme oxygenase 1 (HO1) and glutathione peroxidase 1 (GPX1) antioxidant gene expression. A corneal wound was uniformly induced on the right eye in all experimental animals and divided into eight groups such as two control groups (wounded right eye group-NR and non-wounded left eye group-NL), UPM treated group and PJEs (25, 50, 100, 200, 400 mg/kg) treated groups. Corneal abrasion model rats exposed to UPM showed delayed wound healing compared to unexposed rats, but wound healing was dose-dependently enhanced by PJE oral administration. Seventy-two hours after wound generation, inflammatory cells, apoptotic cells and interleukin-6 (IL-6) expression were increased substantially after UPM exposure, but PJE treatment significantly reduced the wound to an almost normal level while enhancing re-epithelialization without changing corneal thickness. Next, we tried to identify the key molecules for enhancing wound healing through fractionation. The major compounds in the fraction, confirmed by high-performance liquid chromatography (HPLC), were chlorogenic acid (CA), neochlorogenic acid (NCA) and cryptochlorogenic acid (CCA). Each type of CA isomers showed slightly different half maximal effective (EC50) and maximal effective (ECmax) concentrations, and their mixtures synergistically enhanced HCEC migration. Thus, corneal abrasion wound recovery after UPM exposure improved after PJE treatment, and the active PJE components were identified, providing an important basis to develop therapeutics for ocular surface damage using PJE.

SR-5, the specific ratio of Korean multi-herbal formula: An evaluation of antiulcerogenic effects on experimentally induced gastric ulcers in mice.

PURPOSE: Previously, we demonstrated that the specific ratio of Korean multi-herbal formula (SR-5) exhibits hepatoprotective properties against ethanol-induced hepatic damage in rats. Chronic and excessive alcohol consumption is a major etiological factor involved in gastric disease and ulcer development induced by the inflammatory response and oxidative stress. METHODS: The present study evaluated the gastroprotective effects of SR-5 (100, 150, and 200 mg/kg) against hydrochloride acid/ethanol (HCl/EtOH)-induced and indomethacin/hydrochloride acid (INDO/HCl)-induced gastritis in a mouse model and the mechanisms involved. RESULTS: All the tested doses of SR-5 significantly inhibited gastric lesions in the HCl/EtOH-induced ulcer model mice. Similarly, all the tested doses of SR-5 significantly inhibited gastric lesions in the INDO/HCl-induced ulcer model mice. Furthermore, mice pretreated with SR-5 had significantly increased gastric levels of enzymatic and nonenzymatic antioxidants, namely, catalase (CAT) and glutathione (GSH), with concomitant reductions in malondialdehyde (MDA) and reactive oxygen species (ROS) levels compared with those in the HCl/EtOH or INDO/HCl group. SR-5 suppressed the expression of nuclear factor-kappa B (NF-κB)/p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) to their normal values. CONCLUSION: These findings are the first to demonstrate the powerful protective effect of SR-5 against gastric injury development and provide hope for clinical application.

Combined antihypertensive effect of unripe Rubus coreanus Miq. and Dendropanax morbiferus H. Lév. Extracts in 1 kidney-1 clip hypertensive rats and spontaneously hypertensive rats.

BACKGROUND: We previously showed that enzymatically hydrolyzed Dendropanax morbiferus H. Lév. leaf (Hy-DP) and unripe Rubus coreanus Miq. (5-uRCK) extracts exhibit potent vasodilator effects on isolated aortic rings from rats partly through endothelium-dependent and endothelium-independent mechanisms. These two extracts have different mechanisms of action; however, their combined effect on antihypertensive activity has not been explored. METHODS: The present study aims to investigate the effect of a chronic optimized mixture (HDR-2, composed of Hy-DP and 5-uRCK in a 2:1 mass ratio) on vascular tension and blood pressure in two different hypertensive rat models. RESULTS: The results showed that HDR-2 concentration-dependently relaxed endothelium-intact and endothelium-denuded aortic rings precontracted with phenylephrine. Antihypertensive effects were assessed in vivo on a 1 kidney-1 clip (1 K-1C) rat model of hypertension and spontaneously hypertensive rats (SHRs). Acute HDR-2 treatment significantly decreased systolic blood pressure (SBP) 3 h posttreatment in both models. Chronic HDR-2 administration also significantly decreased SBP in the hypertensive rat models. Moreover, HDR-2 increased eNOS protein expression and phosphorylation levels in the aorta. CONCLUSION: Chronic HDR-2 administration may effectively improve vascular function by decreasing plasma angiotensin-converting enzyme (ACE) activity and AngII levels. HDR-2 significantly improved acetylcholine (ACh)-induced aortic endothelium-dependent relaxation and affected sodium nitroprusside (SNP)-induced endothelium-independent relaxation in SHRs.

The adipokine Retnla deficiency increases responsiveness to cardiac repair through adiponectin-rich bone marrow cells.

Resistin-like alpha (Retnla) is a member of the resistin family and known to modulate fibrosis and inflammation. Here, we investigated the role of Retnla in the cardiac injury model. Myocardial infarction (MI) was induced in wild type (WT), Retnla knockout (KO), and Retnla transgenic (TG) mice. Cardiac function was assessed by echocardiography and was significantly preserved in the KO mice, while worsened in the TG group. Angiogenesis was substantially increased in the KO mice, and cardiomyocyte apoptosis was markedly suppressed in the KO mice. By Retnla treatment, the expression of p21 and the ratio of Bax to Bcl2 were increased in cardiomyocytes, while decreased in cardiac fibroblasts. Interestingly, the numbers of cardiac macrophages and unsorted bone marrow cells (UBCs) were higher in the KO mice than in the WT mice. Besides, phosphorylated histone H3(+) cells were more frequent in bone marrow of KO mice. Moreover, adiponectin in UBCs was notably higher in the KO mice compared with WT mice. In an adoptive transfer study, UBCs were isolated from KO mice to transplant to the WT infarcted heart. Cardiac function was better in the KO-UBCs transplanted group in the WT-UBCs transplanted group. Taken together, proliferative and adiponectin-rich bone marrow niche was associated with substantial cardiac recovery by suppression of cardiac apoptosis and proliferation of cardiac fibroblast.

Gemigliptin suppresses salivary dysfunction in streptozotocin-induced diabetic rats.

Patients with diabetes commonly experience hyposalivation, which induces discomfort in eating, swallowing, dryness, smell, and speaking, as well as increases the incidence of periodontal disease. Dipeptidyl peptidase-4 (DPP4) inhibitors are frequently used as antidiabetic drugs that lower glucose levels by utilizing similar mechanisms; however, additional protective functions of each gliptin have been discovered. In this study, the protective roles of gemigliptin, a DPP4 inhibitor, against salivary dysfunction under diabetic conditions were investigated. Streptozotocin-induced diabetic rats received gemigliptin 10 mg/kg or 100 mg/kg via oral gavage for 3 weeks. The weights of salivary gland tissues, saliva secretion, and antioxidant capacity in salivary glands were reduced after diabetes induction, but were significantly preserved following gemigliptin treatment. In salivary gland analysis, expression of apoptotic proteins, as well as amylase and aquaporin-5 (AQP5) protein expression, were increased following gemigliptin treatment. Furthermore, the number of TUNEL-positive cells decreased after gemigliptin treatment. Therefore, gemigliptin has protective roles against salivary dysfunction observed in diabetes, mediated via antioxidant, anti-apoptotic, and salivary secretion mechanisms. These results may help in selecting a suitable drug for patients with diabetes experiencing salivary dysfunction.

Long-Term Exposure to Urban Particulate Matter on the Ocular Surface and the Incidence of Deleterious Changes in the Cornea, Conjunctiva and Retina in Rats.

We investigated the time-dependent deleterious ocular changes induced by urban particulate matter (UPM) in vitro and in vivo. UPM treatment decreased human corneal epithelial cell migration and survival. Fluorescein scores were consistently increased by UPM application for 16 weeks. One week of rest at 2 or 4 weeks led to a recovery trend, whereas two weeks of rest at 8 weeks induced no change. UPM treatment decreased the tear film break-up time at 2 weeks, which was thereafter maintained until 16 weeks. No changes were found after periods of rest. UPM-treated eyes exhibited greater corneal epithelium thickness than normal eyes at 2 weeks, which recovered to normal at 4 and 8 weeks and was significantly decreased at 16 weeks. Apoptotic cell number in the epithelium was increased at 2 weeks, which remained constant except at 8 weeks. IL-6 expression in the cornea of the right eye continually increased for 16 weeks, and significant recovery was only observed at 8 weeks after 2 weeks of rest. Ocular pressure was significantly increased in the right eye at 12 and 16 weeks. Topical UPM application to the eye induced deleterious changes to various closely related parts of the eye.

Endothelium-dependent and endothelium-independent vasorelaxant effects of unripe Rubus coreanus Miq. and Dendropanax morbiferus H. Lév. extracts on rat aortic rings.

BACKGROUND: Many clinical trials on antihypertensive drugs have confirmed the usefulness of these drugs in regulating blood pressure effectively. However, all the drugs usually require long-term use; thus, economic burdens as well as some adverse effects, including headache, diarrhea, skin rash, edema, fever, and liver and kidney dysfunction, accompany their use. Therefore, we attempted to identify natural medications for treating hypertension. We investigated the antihypertensive effects of Dendropanax morbiferus H. Lév. extract (DP), enzymatically hydrolyzed DP extract (Hy-DP) and 5% unripe Rubus coreanus Miq. ethanol extract (5-uRCK). METHODS: Extracts of the unripe R. coreanus were made using 20 volumes of 5% ethanol at 100 °C for 4 h. The dried leaves of D. morbiferus were subjected to enzymatic hydrolysis by protease, trypsin, bromelain and papain to increase L-arginine and GABA levels. Vasorelaxant effects of these extracts were evaluated on rat aorta precontracted with phenylephrine. In addition, hippocampal neurons, RAW 264.7 macrophages and human umbilical vein endothelial cells (HUVECs) were used to exam nitric oxide (NO) production and NO synthase (NOS) gene expression. RESULTS: DP, Hy-DP and 5-uRCK dose-dependently relaxed isolated rat aortic rings contracted with phenylephrine; however, Hy-DP was more effective than DP. L-NAME and ODQ differentially inhibited Hy-DP- and 5-uRCK-induced relaxation; both L-NAME and ODQ completely blocked 5-uRCK-mediated relaxation. Endothelium-denuded aortic ring relaxation was induced much less by 5-uRCK than by Hy-DP. Therefore, 5-uRCK and Hy-DP induced vascular relaxation by endothelium-dependent and partially endothelium-dependent mechanisms, respectively. Hy-DP and 5-uRCK induced eNOS gene expression and NO production in endothelial cells but did not change iNOS/nNOS expression or NO production in macrophages or neuronal cells. Both Hy-DP and 5-uRCK effectively induced vascular relaxation via similar but slightly different mechanisms. The best effective combination was investigated after mixing Hy-DP and 5-uRCK at different ratios. The 2:1 Hy-DP:5-uRCK mixture inhibited ACE, cGMP- and cAMP-dependent phosphodiesterase activity and vascular relaxation better than the other mixtures. CONCLUSION: In conclusion, Hy-DP and 5-uRCK exert antihypertensive effects through different endothelium-dependent or endothelium-independent mechanisms. These findings may greatly help elucidate the mechanisms of clinical efficacy of Hy-DP:5-uRCK mixtures used for blood pressure regulation.

Unripe Rubus coreanus Miquel Extract Containing Ellagic Acid Regulates AMPK, SREBP-2, HMGCR, and INSIG-1 Signaling and Cholesterol Metabolism In Vitro and In Vivo.

Our previous study demonstrated that a 5% ethanol extract of unripe Rubus coreanus (5-uRCK) has hypo-cholesterolemic and anti-obesity activity. However, the molecular mechanisms of its effects are poorly characterized. We hypothesized that 5-uRCK and one of its major bioactive compounds, ellagic acid, decrease cellular and plasma cholesterol levels. Thus, we investigated the hypocholesterolemic activity and mechanism of 5-uRCK in both hepatocytes and a high-cholesterol diet (HCD)-induced rat model. Cholesterol in the liver and serum was significantly reduced by 5-uRCK and ellagic acid. The hepatic activities of HMG-CoA and CETP were reduced, and the hepatic activity of LCAT was increased by both 5-uRCK extract and ellagic acid, which also caused histological improvements. The MDA content in the aorta and serum was significantly decreased after oral administration of 5-uRCK or ellagic acid. Further immunoblotting analysis showed that AMPK phosphorylation in the liver was induced by 5-uRCK and ellagic acid, which activated AMPK, inhibiting the activity of HMGCR by inhibitory phosphorylation. In contrast, 5-uRCK and ellagic acid suppressed the nuclear translocation and activation of SREBP-2, which is a key transcription factor in cholesterol biosynthesis. In conclusion, our results suggest that 5-uRCK and its bioactive compound, ellagic acid, are useful alternative therapeutic agents to regulate blood cholesterol.

Ethyl Pyruvate Prevents Renal Damage Induced by Methylglyoxal-Derived Advanced Glycation End Products.

The renal accumulation of advanced glycation end products (AGEs) is a causative factor of various renal diseases, including chronic kidney disease and diabetic nephropathy. AGE inhibitors, such as aminoguanidine and pyridoxamine, have the therapeutic activities for reversing the increase in renal AGE burden. This study evaluated the inhibitory effects of ethyl pyruvate (EP) on methylglyoxal- (MGO-) modified AGE cross-links with proteins in vitro. We also determined the potential activity of EP in reducing the renal AGE burden in exogenously MGO-injected rats. EP inhibited MGO-modified AGE-bovine serum albumin (BSA) cross-links to collagen (IC50 = 0.19 ± 0.03 mM) in a dose-dependent manner, and its activity was stronger than aminoguanidine (IC50 = 35.97 ± 0.85 mM). In addition, EP directly trapped MGO (IC50 = 4.41 ± 0.08 mM) in vitro. In exogenous MGO-injected rats, EP suppressed AGE burden and MGO-induced oxidative injury in renal tissues. These activities of EP on the MGO-mediated AGEs cross-links with protein in vitro and in vivo showed its pharmacological potential for inhibiting AGE-induced renal diseases.

Aucuba japonica Extract and Aucubin Prevent Desiccating Stress-Induced Corneal Epithelial Cell Injury and Improve Tear Secretion in a Mouse Model of Dry Eye Disease.

Dry eye disease is affected by a broad range of causes such as age, lifestyle, environment, medication and autoimmune diseases. These causes induce tear instability that activates immune cells and promotes expression of inflammatory molecules. In this study, we investigated the therapeutic effects of an ethanolic extract of Aucuba japonica (AJE) and its bioactive compound, aucubin, on dry eye disease. The human corneal cells were exposed to desiccation stress induced by exposing cells to air, so that viability was decreased. On the other hand, pre-treatment of AJE and aucubin restored cell survival rate depending on the dose under the dry condition. This result was confirmed again by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The mRNA expression of inflammatory molecules was reduced by the pretreatment of AJE and aucubin under the dry state. The therapeutic effects of AJE and aucubin were examined in the animal model for dry eye induced by unilateral excision of the exorbital lacrimal gland. Declined tear volumes and corneal irregularity in the dry eye group were fully recovered by the administration of AJE and aucubin. The apoptotic cells on the cornea were also decreased by AJE and aucubin. Therefore, this study suggests that administration of AJE can be a novel therapeutic for dry eye disease and that the pharmacological activities of AJE may be in part due to its bioactive compound, aucubin.

A novel system-level approach using RNA-sequencing data identifies miR-30-5p and miR-142a-5p as key regulators of apoptosis in myocardial infarction.

This study identified microRNAs involved in myocardial infarction (MI) through a novel system-level approach using RNA sequencing data in an MI mouse model. This approach involved the extraction of DEGs and DEmiRs from RNA-seq data in sham and MI samples and the subsequent selection of two miRNAs: miR-30-5p (family) and miR-142a-5p, which were downregulated and upregulated in MI, respectively. Gene Set Enrichment Analysis (GSEA) using the predicted targets of the two miRNAs suggested that apoptosis is an essential gene ontology (GO)-associated term. In vitro functional assays using neonatal rat ventricular myocytes (NRVMs) demonstrated that miR-30-5p is anti-apoptotic and miR-142a-5p is pro-apoptotic. Luciferase assays showed that the apoptotic genes, Picalm and Skil, and the anti-apoptotic genes, Ghr and Kitl, are direct targets of miR-30-5p and miR-142a-5p, respectively. siRNA studies verified the results of the luciferase assays for target validation. The results of the system-level high throughput approach identified a pair of functionally antagonistic miRNAs and their targets in MI. This study provides an in-depth analysis of the role of miRNAs in the pathogenesis of MI which could lead to the development of therapeutic tools. The system-level approach could be used to identify miRNAs involved in variety of other diseases.

Intramyocardial Injection of Stem Cells in Pig Myocardial Infarction Model: The First Trial in Korea.

Although cell therapy is emerged for cardiac repair, its efficacy is modest by intracoronary infusion. Therefore, we established the intramyocardial delivery technique using a left ventricular (LV) mapping system (NOGA® XP) using 18 pigs. After adipose tissue-derived mesenchymal stem cells (ATSCs) were delivered intramyocardially to porcine infarcted heart, LV ejection fraction (EF) was increased, and LV chamber size was decreased. We proved the therapeutic effect of intramyocardial injection of ATSC through a LV mapping system in the porcine model for the first time in Korea. The adoption of this technique may accelerate the translation into a clinical application in the near future.

The optimization of cell therapy by combinational application with apicidin-treated mesenchymal stem cells after myocardial infarction.

Although mesenchymal stem cells (MSC) have been shown to be safe in preclinical studies of cardiovascular disease, multiple meta-analyses have debated whether functional improvement is significant or not. The cardiac differentiation from MSC is achievable using cardiogenic factors, however, the high cost and long culture period may limit the applications. Here, we developed a novel method to optimize the therapeutic outcome for myocardial infarction (MI). Treatment of MSC with apicidin, a histone deacetylase inhibitor, dramatically increased the expressions of cardiac markers such as GATA4, Nkx2.5, and cardiac troponin I (cTnI). In AC/MSC, stemness-related genes and yes-associated protein (YAP), a potent oncogene that drives cell proliferation, were significantly suppressed. Furthermore apicidin treatment or YAP knockdown downregulated miR-130a expression followed by induction of cardiac markers in MSC. In the comparison study, we found that both cardiac gene induction and angiogenesis were most prominent in the mixture of non-treated MSC and AC/MSC (Mix). Using mouse MI model, we show that application of Mix was strongly associated with cardiac differentiation of injected MSC and improved cardiac performance. Our results suggest that suppression of YAP/miR-130a shifts MSC cell fate toward cardiac lineage and identify apicidin as a potential pharmacological target for therapeutic development.

Tauroursodeoxycholic acid (TUDCA) attenuates pressure overload-induced cardiac remodeling by reducing endoplasmic reticulum stress.

Pressure overload in the heart induces pathological hypertrophy and is associated with cardiac dysfunction. Apoptosis and fibrosis signaling initiated by the endoplasmic reticulum stress (ERS) is known to contribute to these maladaptive effects. The aim of this study was to investigate whether reduction of ERS by a known chemical chaperone, tauroursodeoxycholic acid (TUDCA) can attenuate pressure overload-induced cardiac remodeling in a mouse model of transverse aortic constriction (TAC). Oral administration of TUDCA at a dose of 300 mg/kg body weight (BW) in the TUDCA-TAC group reduced ERS markers (GRP78, p-PERK, and p-eIf2α), compared to the Vehicle (Veh)-TAC group. TUDCA administration, for 4 weeks after TAC significantly reduced cardiac hypertrophy as shown by the reduced heart weight (HW) to BW ratio, and expression of hypertrophic marker genes (ANF, BNP, and α-SKA). Masson's trichrome staining showed that myocardial fibrosis and collagen deposition were also significantly reduced in the TUDCA-TAC group. We also found that TUDCA significantly decreased expression of TGF-ß signaling proteins and collagen isoforms. TUDCA administration also reduced cardiac apoptosis and the related proteins in the TUDCA-TAC group. Microarray analysis followed by gene ontology (GO) and pathway analysis demonstrated that extracellular matrix genes responsible for hypertrophy and fibrosis, and mitochondrial genes responsible for apoptosis and fatty acid metabolism were significantly altered in the Veh-TAC group, but the alterations were normalized in the TUDCA-TAC group, suggesting potential of TUDCA in treatment of heart diseases related to pressure-overload.

ENOblock, a unique small molecule inhibitor of the non-glycolytic functions of enolase, alleviates the symptoms of type 2 diabetes.

Type 2 diabetes mellitus (T2DM) significantly impacts on human health and patient numbers are predicted to rise. Discovering novel drugs and targets for treating T2DM is a research priority. In this study, we investigated targeting of the glycolysis enzyme, enolase, using the small molecule ENOblock, which binds enolase and modulates its non-glycolytic 'moonlighting' functions. In insulin-responsive cells ENOblock induced enolase nuclear translocation, where this enzyme acts as a transcriptional repressor. In a mammalian model of T2DM, ENOblock treatment reduced hyperglycemia and hyperlipidemia. Liver and kidney tissue of ENOblock-treated mice showed down-regulation of known enolase target genes and reduced enolase enzyme activity. Indicators of secondary diabetic complications, such as tissue apoptosis, inflammatory markers and fibrosis were inhibited by ENOblock treatment. Compared to the well-characterized anti-diabetes drug, rosiglitazone, ENOblock produced greater beneficial effects on lipid homeostasis, fibrosis, inflammatory markers, nephrotoxicity and cardiac hypertrophy. ENOblock treatment was associated with the down-regulation of phosphoenolpyruvate carboxykinase and sterol regulatory element-binding protein-1, which are known to produce anti-diabetic effects. In summary, these findings indicate that ENOblock has potential for therapeutic development to treat T2DM. Previously considered as a 'boring' housekeeping gene, these results also implicate enolase as a novel drug target for T2DM.

Natural product derivative BIO promotes recovery after myocardial infarction via unique modulation of the cardiac microenvironment.

The cardiac microenvironment includes cardiomyocytes, fibroblasts and macrophages, which regulate remodeling after myocardial infarction (MI). Targeting this microenvironment is a novel therapeutic approach for MI. We found that the natural compound derivative, BIO ((2'Z,3'E)-6-Bromoindirubin-3'-oxime) modulated the cardiac microenvironment to exert a therapeutic effect on MI. Using a series of co-culture studies, BIO induced proliferation in cardiomyocytes and inhibited proliferation in cardiac fibroblasts. BIO produced multiple anti-fibrotic effects in cardiac fibroblasts. In macrophages, BIO inhibited the expression of pro-inflammatory factors. Significantly, BIO modulated the molecular crosstalk between cardiac fibroblasts and differentiating macrophages to induce polarization to the anti-inflammatory M2 phenotype. In the optically transparent zebrafish-based heart failure model, BIO induced cardiomyocyte proliferation and completely recovered survival rate. BIO is a known glycogen synthase kinase-3ß inhibitor, but these effects could not be recapitulated using the classical inhibitor, lithium chloride; indicating novel therapeutic effects of BIO. We identified the mechanism of BIO as differential modulation of p27 protein expression and potent induction of anti-inflammatory interleukin-10. In a rat MI model, BIO reduced fibrosis and improved cardiac performance. Histological analysis revealed modulation of the cardiac microenvironment by BIO, with increased presence of anti-inflammatory M2 macrophages. Our results demonstrate that BIO produces unique effects in the cardiac microenvironment to promote recovery post-MI.

5-Azacytidine modulates interferon regulatory factor 1 in macrophages to exert a cardioprotective effect.

Macrophages are actively involved in inflammatory responses during the progression of cardiac injury, including myocardial infarction (MI). A previous study showed that 5-azacytidine (5AZ), a DNA methylation inhibitor, can ameliorate cardiac injury by shifting macrophages toward an anti-inflammatory phenotype via iNOS inhibition. Here, we show that the beneficial effect of 5AZ is associated with sumoylation of interferon regulatory factor-1 (IRF1) in macrophages. IRF1 is a critical transcription factor for iNOS induction and is antagonized by IRF2. In the stimulated macrophages, IRF1 accumulated in the nucleus without degradation by 5AZ treatment. In animal study, 5AZ administration resulted in significant improvements in cardiac function and fibrosis. IRF1-expressing macrophages were more abundant in the 5AZ-treated MI group than in the PBS-treated MI group. Because sumoylated IRF1 is known to mimic IRF2, we examined the IRF1 sumoylation. Sumoylated IRF1 was resistant to degradation and significantly increased in the 5AZ-treated MI group. Collectively, 5AZ had a protective effect after MI by potentiation of IRF1 sumoylation and is suggested as a novel therapeutic intervention for cardiac repair.

Involvement of miR-34c in high glucose-insulted mesenchymal stem cells leads to inefficient therapeutic effect on myocardial infarction.

High glucose-insulted bone marrow-derived mesenchymal stem cells (BMCs) showed impaired angiogenesis along with downregulation of stem cell factor (SCF). This study was designed to determine the involvement of microRNAs (miR), which are actively involved in the physiological function of stem cells. We observed that miR-34c was significantly induced by high glucose treatment and blunted tube formation of BMCs. Stem cell factor (SCF) was confirmed as a target of miR-34c by 3'-UTR promoter analysis and Western blot. SCF knockdown by siRNA induced Krüppel-like factor 4 (KLF4) and resulted in the blockade of angiogenesis of BMCs. Sequentially, KLF4 overexpression completely blocked tube formation through inducing PAI-1 (plasminogen activator inhibitor-1). To study the action of miR-34c in terms of the therapeutic potential of BMCs, myocardial infarction (MI) was induced by ligation of the coronary artery in nude mice, BMCs transfected with miR-control or miR-34c were injected into the infarcted myocardium 7 days later, and histological studies were performed 2 weeks later. Cardiac fibrosis was 18.24±4.7% in the miR-34c-BMC group and 10.01±0.2% in the miR-control-BMC group (p<0.05). Cardiac function and vessel density were decreased in the miR-34c-BMC group compared with the miR-con-BMC group. Particularly, miR-34c-BMCs failed to incorporate into vessels. Our results show that the angiogenic activity of BMCs is finely regulated by the miR-34c-SCF-KLF4 axis, which is a potent translational target for optimizing the therapeutic activity of autologous BMCs for cardiac repair.