Bone-muscle crosstalk following exercise plus Ursolic acid by myomiR-133a/Cx43-Runx2 axis in aged type 2 diabetes rat models.

Natural bioactive compound, Ursolic acid (UA), plus different types of exercise may exert the action on glycemic control, leading to clinical benefits in the prevention and treatment of aging/diabetes-associated complications. So, this study examined the effects of eight weeks combination of 250 mg of UA per day per kilogram of body weight of rat as well as resistance/endurance training on miR-133a expression across serum, bone marrow, skeletal muscle, and Connexin 43 (Cx43)-Runt-related transcription factor 2 (Runx2) signaling axis in high-fat diet and low-dose streptozotocin-induced T2D (here, HFD/STZ-induced T2D). The study was conducted on 56 male Wistar rats (427 ± 44 g, 21 months old), having HFD/STZ-induced T2D randomly assigned into 7 groups of 8 including (1) sedentary non-diabetic old rats (C); (2) sedentary type 2 diabetes animal model (D); (3) sedentary type 2 diabetes animal model + UA (DU); (4) endurance-trained type 2 diabetes animal model (DE); (5) resistance-trained type 2 diabetes animal model (DR); (6) endurance-trained type 2 diabetes animal model + UA (DEU); and (7) resistance-trained type 2 diabetes animal model + UA (DRU). Resistance training included a model of eight weeks of ladder resistance training at 60-80% maximal voluntary carrying capacity (MVCC) for five days/week. Treadmill endurance exercise protocol included eight weeks of repetitive bouts of low-/high-intensity training with 30%-40% and 60%-75% maximal running speed for five days/week, respectively. UA Supplementary groups were treated with 500 mg of UA per kg of high-fat diet per day. The results revealed significant supplement and exercise interaction effects for the BM miR-133a (p = 0.001), the bone marrow Runx2 (p = 0.002), but not the serum miR-133a (p = 0.517), the skeletal muscle miR-133a (p = 0.097) and the Cx43 (p = 0.632). In conclusion, only eight weeks of resistance-type exercise could affect miR-133a profile in muscles and osteoblast differentiation biomarker RUNX2 in aged T2D model of rats. 250 mg of UA per kilogram of body weight rat per day was administered orally, less than the sufficient dose for biological and physiological impacts on osteoblast differentiation biomarkers in aged T2D model of rats following eight weeks.

Ursolic Acid Enhances Myelin Repair in Adult Mice Brains and Stimulates Exhausted Oligodendrocyte Progenitors to Remyelinate.

In multiple sclerosis patients, long-term inflammation makes the oligodendrocyte progenitor cells (OPCs) exhausted; therefore, a new therapy that makes them responsive to insults to participate in remyelination is highly in demand. Here, we investigated the effect of ursolic acid (UA) on myelin repair after mid-term and long-term demyelination periods induced by 6 or 12 weeks of cuprizone treatment followed by 2 weeks of recovery with or without UA. Immunohistochemistry studies and myelin genes expression assessment were used to evaluate the myelination status of mouse corpora callosa and the cellular mechanisms of myelin repair. Results showed that UA significantly promoted recovery from myelin loss after discontinuing 6 or 12 weeks of cuprizone feeding, as measured by luxol fast blue (LFB), fluoroMyelin (FM), anti-myelin basic protein (MBP) staining, and oligodendrocyte progenitor cell counts. It led to reduced inflammation and gliosis as evaluated by glial fibrillary acidic protein (GFAP), Iba1, or other marker gene transcripts. Following long-term demyelination, gliosis and TNF-α were observed as potential players in lesion pathology, which were restored by UA. An increased IL-10 may contribute to UA anti-inflammatory effect and making responsive the exhausted OPCs. UA increased the number of new oligodendrocyte lineage cells and myelination. Our findings indicated that UA can enhance myelin repair after cuprizone challenge through the prevention of gliosis and increasing the newly generated myelin.

Myelin Protection by Ursolic Acid in Cuprizone-Induced Demyelination in Mice.

Neuronal survival in multiple sclerosis (MS) and other demyelinating diseases depends on the preservation of myelin and remyelination of axons. Myelin protection is the main purpose to decrease myelin damage in the central nervous system (CNS). Ursolic acid (UA) as a natural product in apple is suggested to protect neural cells. This study is the first to demonstrate an effect for UA on CNS myelin loss induced by cuprizone toxin. In the current study, we hypothesized that daily treatment with UA in drinking water (1 mg/mL) prevents myelin damage by 6 weeks administration of CPZ in mice pellet which lead to corpus callosum axonal demyelination. We assessed the myelin content and the number of myelinating cells in corpus callosum by FluoroMyelin and luxol fast blue staining as well as by immunostaining against MBP and Olig2. Our finding indicated that UA could decrease the extent of demyelination area and enhanced myelin stain intensity within CC and protected oligodendrocyte lineage cells against cuprizone toxin. We could conclude that myelinated structures could be protected by UA in corpus callosum, which provide favorable evidence for the possibility of application of UA in demyelinating diseases and traumatic injuries.

Mounting evidence validates Ursolic Acid directly activates SIRT1: A powerful STAC which mimic endogenous activator of SIRT1.

Ursolic Acid (UA), a pentacyclic triterpenoid compound, plays a vital role in aging process. However, the role of UA in the regulation of aging and longevity is still controversial as we have previously demonstrated that UA increases SIRT1 protein level in aged-mice. Here, we reveal that UA directly activates SIRT1 in silico, in vitro and in vivo. We have identified that UA binds to outer surface of SIRT1 and leads to tight binding of substrates to enzyme in comparison with Resveratrol (RSV) and control. Furthermore, our results indicate that UA drives the structure of SIRT1 toward a closed state (an active form of enzyme). Interestingly, our experimental findings are in agreement with the molecular dynamic results. Based on our data, UA increases the affinity of enzyme for both substrates with decreasing Km value, while enhances the Vmax of enzyme. Additionally, we have determined that UA heightened SIRT1 catalytic efficiency by 2 folds compared with RSV. Thereby, to identify the endogenous activator of SIRT1, UA was administrated to aged-mice and then the tissues were isolated. According to our results, it can be concluded that UA increases SIRT1 activity and mimics Lamin A and AROS behavior in the living cells.

Ursolic Acid Mediates Hepatic Protection through Enhancing of anti-aging Biomarkers.

BACKGROUND: Age-associated loss of liver function has been recognized for decades. But, the mechanism driving liver regeneration and its decline with age remains elusive. OBJECTIVE: Hence, to support of our previous studies about anti-aging effects of Ursolic Acid (UA), a compound which is extensively present in apple peels. The aim of this study is to address whether UA might alter sensors of the cell metabolic state such as SIRT1, SIRT6, PGC-1ß and Klotho proteins. METHODS: To evaluate the effect of UA on hepatic indicated proteins, mice were administrated with UA twice daily for 7 days. The involvements of these proteins in the UA-mediated effect harmony hepatic protection were investigated by immunofluorescence microscopy technique. RESULTS: Our findings clearly illustrated that UA enhanced SIRT1 (~ 5 ± 0.2 folds) and SIRT6 (~ 8 ± 0.5 folds) proteins levels in hepatic, p<0.001. In addition, the data showed that UA increased PGC-1ß (~ 7 ± 0.4 folds) protein overexpression, p<0.001. Moreover, we showed that UA upregulated Klotho (~ 3.5 ± 0.2 folds) protein in order to improve hepatic performance, p<0.01. CONCLUSION: Our results suggest that UA through increasing of SIRT1 up-regulation ameliorate reverse cholesterol transport, fatty acid use and oxidative stress defense. In addition, it seems that UA by enhancing of SIRT6 expression promotes cholesterol homeostasis through repressing SREBP1 and SREBP2. Reciprocally, UA might be involved in VLDL synthesis and exportation through PGC-1ß up-regulation. Finally, UA might be as key regulators of mineral homeostasis and bile acid/cholesterol metabolism, by inducing Klotho overexpression.

Ursolic acid regulates aging process through enhancing of metabolic sensor proteins level.

We previously reported that Ursolic Acid (UA) ameliorates skeletal muscle performance through satellite cells proliferation and cellular energy status. In studying the potential role of the hypothalamus in aging, we developed a strategy to pursue UA effects on the hypothalamus anti-aging proteins such as; SIRT1, SIRT6, PGC-1ß and α-Klotho. In this study, we used a model of aging animals (C57BL/6). UA dissolved in Corn oil (20mg/ml) and then administrated (200mg/Kg i.p injection) to mice, twice daily for 7days. After treatment times, the mice perfused and the hypothalamus isolated for preparing of tissue to Immunofluorescence microscopy. The data illustrated that UA significantly increased SIRT1 (∼3.5±0.3 folds) and SIRT-6 (∼1.5±0.2 folds) proteins overexpression (P<0.001). In addition, our results showed that UA enhanced α-Klotho (∼3.3±0.3) and PGC-1ß (∼2.6±0.2 folds) proteins levels (P<0. 01). In this study, data were analyzed using SPSS 16 (ANOVA test). To the best of our knowledge, it seems that UA through enhancing of anti-aging biomarkers (SIRT1 and SIRT6) and PGC-1ß in hypothalamus regulates aging-process and attenuates mitochondrial-related diseases. In regard to the key role of α-Klotho in aging, our data indicate that UA may be on the horizon to forestall diseases of aging.

Short-term ursolic acid promotes skeletal muscle rejuvenation through enhancing of SIRT1 expression and satellite cells proliferation.

Ursolic acid (UA) is a triterpenoid compound, which exerts its influences on the skeletal muscles. However, the mechanisms underlying these effects are still unclear. In this study, muscle satellite cells were isolated and purified by high-throughput pre-plating method (∼>60%) from 10 days old mice skeletal muscles. Evaluation of paired-box 7 (Pax7) expressions then confirmed the purification. Treatment of the cells with UA showed that UA up-regulated SIRT1 (∼35 folds) and overexpressed PGC-1α (∼175 folds) gene significantly. Moreover, the number of muscle satellite cells, which accompanied by initiation of neomyogenesis in the animal skeletal muscles, was increased (∼3.4 times). We also evaluated UA-mediated changes in the cellular energy status in the skeletal muscles. The results revealed that in the UA-treated mice, ATP and ADP contents in the various skeletal muscle tissue types, including: Gastrocnemius (Gas), Tibialis Anterior (Tib) and Gluteus Maximus (Glu) have been significantly decreased (P≤0.001); 2.2, 3.2, 2 times for ATP, and 9.6, 35.7, 11.6 times for ADP, respectively; however to compensate this process mitochondrial biogenesis occurred (12.33%±1.5 times). Furthermore, a rise in ATP/ADP ratio was observed 2.5, 4.5, 2.05 times for Gas, Tib and Glu muscles, respectively (P≤0.001). Alternatively, UA enhanced the expression of myoglobin (∼2 folds) in concert with remodeling of glycolytic muscle fibers to mainly fast IIA (∼30%) and slow-twitch (∼4%) types as well. Finally, our study indicated that UA indirectly mimicked beneficial effects of short-term calorie restriction and exercise (fast-oxidative) by directing the skeletal muscle composition toward oxidative metabolism.

Ursolic acid ameliorates aging-metabolic phenotype through promoting of skeletal muscle rejuvenation.

Ursolic acid (UA) is a lipophilic compound, which highly found in apple peels. UA has some certain features, of the most important is its anabolic effects on skeletal muscles, which in turn plays a prominent role in the aging process, encouraged us to evaluate skeletal muscle rejuvenation. This study seeks to address the two following questions: primarily, we wonder to know if UA increases anti-aging biomarkers (SIRT1 and PGC-1α) in the isolated satellite cells, to pave the way for satellite cells proliferation. The results revealed that UA elevated the expression of SIRT1 (∼ 35 folds) and PGC-1α (∼ 175 folds) genes. The other question that needs to be asked, however, is to understand whether it is possible to generalize the in vitro findings to in vivo. For this, a study was designed to investigate the effects of UA on the cellular energy status in the animal models (C57BL/6 mice). We found that UA decreased cellular energy charges such as ATP (∼ 3 times) and ADP (∼ 18 times). With respect to the role of UA in energy expenditure and as an anti-aging biomarker, one might wonder to elucidate skeletal muscle rejuvenation as well as satellite cells proliferation and neomyogenesis. The results illustrated that UA boosted neomyogenesis through enhancing the number of satellite cells. In addition, rejuvenation effects of UA on the skeletal muscle promptly encouraged us to reexamine the performance of skeletal muscles. The results indicated that UA through increasing myoglobin expression (∼ 2 folds) accompanied with transforming of glycolytic to fast oxidative status chiefly and slow-twitch muscle fibers. To the best of our knowledge, it seems that UA might be considered as a potential candidate for treatment of pathological conditions associated with muscular atrophy and dysfunction, including skeletal muscle atrophy, amyotrophic lateral sclerosis (ALS), sarcopenia and metabolic diseases of the muscles.

Luciferin-Regenerating Enzyme Mediates Firefly Luciferase Activation Through Direct Effects of D-Cysteine on Luciferase Structure and Activity.

Luciferin-regenerating enzyme (LRE) contributes to in vitro recycling of D-luciferin. In this study, reinvestigation of the luciferase-based LRE assay is reported. Here, using quick change site-directed mutagenesis seven T-LRE (Lampyris turkestanicusLRE) mutants were constructed and the most functional mutant of T-LRE (T(69)R) was selected for this research and the effects of D- and L-cysteine on T(69)R T-LRE-luciferase-coupled assay are examined. Our results demonstrate that bioluminescent signal of T(69)R T-LRE-luciferase-coupled assay increases and then reach equilibrium state in the presence of 5 mm D-cysteine. In addition, results reveal that 5 mm D- and L-cysteine in the absence of T(69)R T-LRE cause a significant increase in bioluminescence intensity of luciferase over a long time as well as decrease in decay rate. Based on activity measurements, far-UV CD analysis, ANS fluorescence and DLS (Dynamic light scattering) results, D-cysteine increases the activity of luciferase due to weak redox potential, antiaggregatory effects, induction of changes in conformational structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE on luciferase bioluminescent intensity, the majority of increase in luciferase light output and time-course originate from the direct effects of D-cysteine on structure and activity of firefly luciferase.

Directed improvement of luciferin regenerating enzyme binding properties: implication of some conserved residues in luciferin-binding domain.

Luciferin regenerating enzyme (LRE) contributes to in vitro recycling of d-luciferin to produce persistent and longer light emission by luciferase. Luciferin binding domains I and II among LREs regarded as potential candidates for luciferin-binding sites. In this study, for the first time, amino acids T69, G75 and K77 located at luciferin binding domain I of LRE from L. turkestanicus (T-LRE) substituted by using site-directed mutagenesis. Single mutant T(69)R increased luciferase light output more than two-fold over a longer time in comparison with a wild-type and other mutants of T-LRE. Nevertheless, double mutant (K(77)E/T(69)R) increased the amount of bioluminescent signal more than two-fold over a short time. In addition, G(75)E, K(77)E and G(75)E/T(69) R mutants did not improve luciferin-luciferase in vitro bioluminescence. Based on our results, addition of K(77)E/G(75)E and K(77)E/G(75)E/T(69)R mutants caused intermediate changes in bioluminescence from in vitro luciferin-luciferase reaction. These findings indicated that the amino acids in question are possible to be located within T-LRE active site. It may also be suggested that substituted Arg69 (Arg218) plays an important role in luciferin binding and the existence of Gly75 as well as Lys77 is essential for T-LRE which has already evolved to have different functions in nature.

Red blood cell ATP/ADP & nitric oxide: The best vasodilators in diabetic patients.

BACKGROUND: Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose) levels that result from defects in insulin secretion, or action, or both. Inspired by previous report the release of ATP from RBCs, which may participate in vessel dilation by stimulating NO production in the endothelium through purinergic receptor signaling and so, the aim of this study is to clearly determined relationship between RBC ATP/ADP ratio with nitric oxide. METHODS: The ATP/ADP ratio of erythrocytes among four groups of normal individuals (young & middle age), athletes' subjects and diabetic patients were compared and the relationship between ATP/ADP ratio and NO level of plasma was determined with AVOVA test and bioluminescence method. RESULTS: ATP/ADP level in four groups normal (young & middle age), athletes, diabetes] are measured and analyzed with ANOVA test that show a significant difference between groups (P-value < 0.001). A significant positive correlation was found between RBC ATP/ADP content (r = 0.705; P < 0.001). Plasma NO content is also analyzed with ANOVA test which shows a significant difference between groups. CONCLUSION: In this study, a positive relationship between RBC ATP/ADP ratio and NO was found. Based on the obtained result, higher RBC ATP/ADP content may control the ratio of plasma NO in different individuals, also this results show that ATP can activate endothelial cells in NO production and is a main factor in releasing of NO from endothelial cells.