Isosteviol sodium attenuates high fat/high cholesterol-induced myocardial dysfunction by regulating the Sirt1/AMPK pathway.

A fat-rich diet triggers obesity, and promotes cardiomyocyte injury. Till now, no prior investigations suggested a beneficial role of Isosteviol Sodium (STVNa) in cardiac activity in high fat diet (HFD)-exposed obese rats. However, there is evidence that STVNa accelerates healing of multiple tissue injuries. Herein, we explored the underlying mechanism behind the STVNa-based protection against HFD-induced myocardial dysfunction (MCD) in a rat model of myocardial injury. We employed dosages of 1, 10, and 20 mg/kg STVNa to treat MCD in rats fed with a HFD. Based on our results, STVNa repressed MCD (as indicated by ecocardiographic analysis), myocardium function, pathological structure, and myocardial enzymes. Mechanistically, the STVNa-mediated protection against HFD-induced MCD involved inhibition of inflammation and oxidative stress. Furthermore, using Western blot analysis, we revealed that the critical members of the Sirt1/AMPK network were markedly activated in the STVNa-treated group, relative to HFD-fed controls. Collectively, these evidences suggested that the STVNa offered strong protection against HFD-induced MCD. Moreover, this effect was mediated by the activation of the Sirt1/AMPK network, which, in turn, promoted lipid metabolism.

The protective effect of isosteviol sodium on cardiac function and myocardial remodelling in transverse aortic constriction rat.

Pathological hypertrophy contributes to heart failure and there is not quite effective treatment to invert this process. Isosteviol has been shown to protect the heart against ischaemia-reperfusion injury and isoproterenol-induced cardiac hypertrophy, but its effect on pressure overload-induced cardiac hypertrophy is still unknown. Pressure overload induced by transverse aortic constriction (TAC) causes cardiac hypertrophy in rats to mimic the pathological condition in human. This study examined the effects of isosteviol sodium (STVNa) on cardiac hypertrophy by the TAC model and cellular assays in vitro. Cardiac function test, electrocardiogram analysis and histological analysis were conducted. The effects of STVNa on calcium transient of the adult rat ventricular cells and the proliferation of neonatal rat cardiac fibroblasts were also studied in vitro. Cardiac hypertrophy was observed after 3-week TAC while the extensive cardiac dysfunction and electronic remodelling were observed after 9-week TAC. Both STVNa and sildenafil (positive drug) treatment reversed the two process, but STVNa appeared to be more superior in some aspects and did not change calcium transient considerably. STVNa also reversed TAC-induced cardiac fibrosis in vivo and TGF-ß1-induced fibroblast proliferation in vitro. Moreover, STVNa, but not sildenafil, reversed impairment of the autonomic nervous system induced by 9-week TAC.

Protective effect of isosteviol sodium against LPS-induced multiple organ injury by regulating of glycerophospholipid metabolism and reducing macrophage-driven inflammation.

Sepsis is a severe inflammatory disorder that can lead to multiple organ injury. Isosteviol sodium (STV-Na) is a terpenoid derived from stevioside that exerts anti-inflammatory, antioxidant and antiapoptotic activities. However, the influence of STV-Na on sepsis remains unknown. Here, we assessed the potential effects of STV-Na on sepsis and multiple organ injury induced by lipopolysaccharide (LPS). We found that STV-Na increased the survival rate of mice treat with LPS, significantly improved the functions of the heart, lung, liver, and kidney, reduced the production of inflammatory cytokines and decreased macrophage infiltration. Moreover, Multiorgan metabolomics analysis demonstrated that glutathione metabolism, purine metabolism, glycerophospholipid metabolism and pantothenate and CoA biosynthesis, were significantly altered by STV-Na. This study provides novel insights into the metabolite changes of multiple organ injury in septic mice, which may help characterize the underlying mechanism and provide an improved understanding of the therapeutic effects of STV-Na on sepsis.

Isosteviol sodium protects the cardiomyocyte response associated with the SIRT1/PGC-1α pathway.

Cardiomyocyte dysfunction is attributed to excess oxidative damage, but the molecular pathways involved in this process have not been completely elucidated. Evidence indicates that isosteviol sodium (STVNa) has cardioprotective effects. We therefore aimed to identify the effect of STVNa on cardiomyocytes, as well as the potential mechanisms involved in this process. We established two myocardial hypertrophy models by treating H9c2 cells with high glucose (HG) and isoprenaline (ISO). Our results showed that STVNa reduced H9c2 mitochondrial damage by attenuating oxidative damage and altering the morphology of mitochondria. The results also indicated that STVNa had a positive effect on HG- and ISO-induced damages via mitochondrial biogenesis. The protective effects of STVNa on cardiomyocytes were associated with the regulation of the SIRT1/PGC-1α signalling pathway. Importantly, the effects of STVNa involved different methods of regulation in the two models, which was confirmed by experiments using an inhibitor and activator of SIRT1. Together, the results provide the basis for using STVNa as a therapy for the prevention of cardiomyocyte dysfunctions.

Isosteviol Sodium Attenuates High Fat/High Cholesterol-Induced Kidney Dysfunction by Inhibiting Inflammation, Oxidative Stress and Apoptosis.

The sodium salt of isosteviol (STVNa) is a beyerane diterpene synthesized through acid hydrolysis of stevioside. STVNa improves multiple types of tissue injuries. However, it is not known how isosteviol sodium affects high-fat and high cholesterol diet (HFD)-induced kidney. Therefore, in this study we examined the potential molecular mechanism underlying STVNa mediated protective effect against high fat/high cholesterol-induced kidney dysfunction in HFD-induced kidney injury. Sprague-Dawley (SD) rats were allocated into six groups: the normal group, HFD group and HFD treated with three doses of STVNa, fenofibrate treatment group. The results indicated that HFD induced kidney injury evident by a 60% increase in serum creatinine (CRE) leves. In addition, there was a significant accumulation of triglycerides (approx. 60%), fatty acids (approx. 50%) and total cholesterol (approx. 2.5 fold) in the kidneys. STVNa inhibited HFD-induced kidney injury evident by reducing the increased levels of serum CRE. Specifically, STVNa attenuated HFD-induced kidney injury by inhibiting inflammation, oxidative stress, and apoptosis. These findings indicate that STVNa has a therapeutic potential for HFD-induced kidney dysfunction. The mechanisms of this pharmacological effect are through the inhibition of inflammation, oxidative stress and apoptosis.

STVNa Attenuates Isoproterenol-Induced Cardiac Hypertrophy Response through the HDAC4 and Prdx2/ROS/Trx1 Pathways.

Recent data show that cardiac hypertrophy contributes substantially to the overall heart failure burden. Mitochondrial dysfunction is a common feature of cardiac hypertrophy. Recent studies have reported that isosteviol inhibits myocardial ischemia-reperfusion injury in guinea pigs and H9c2 cells. This work investigated the protective mechanisms of isosteviol sodium (STVNa) against isoproterenol (Iso)-induced cardiac hypertrophy. We found that STVNa significantly inhibited H9c2 cell and rat primary cardiomyocyte cell surface, restored mitochondrial membrane potential (MMP) and morphological integrity, and decreased the expression of mitochondrial function-related proteins Fis1 and Drp1. Furthermore, STVNa decreased reactive oxygen species (ROS) levels and upregulated the expression of antioxidant factors, Thioredoxin 1 (Trx1) and Peroxiredoxin 2 (Prdx2). Moreover, STVNa restored the activity of histone deacetylase 4 (HDAC4) in the nucleus. Together, our data show that STVNa confers protection against Iso-induced myocardial hypertrophy primarily through the Prdx2/ROS/Trx1 signaling pathway. Thus, STVNA is a potentially effective treatment for cardiac hypertrophy in humans.

Isosteviol Sodium Inhibits Astrogliosis after Cerebral Ischemia/Reperfusion Injury in Rats.

Previous reports have indicated that isosteviol sodium (STVNa) has neuroprotective effects against acute focal cerebral ischemia in rats; however, the exact underlying mechanisms and ideal treatment paradigm are not known. To find a reasonable method for STVNa administration and to determine its possible therapeutic mechanisms, we characterized the protective effects of single-dose and multiple-dose STVNa in cerebral ischemic/reperfusion (I/R) injury in rats. Single and multiple treatments with 10 mg/kg STVNa were administered intraperitoneally after injury to investigate its neuroprotective effects. Neurobehavioral deficits and infarct volume were assessed 7 d after ischemia. Both STVNa treatments reduced infarct volumes, improved neurological behaviors, preserved cellular morphology, enhanced neuronal survival, and suppressed cell apoptosis. Multiple treatments performed better than single treatment. Reactive astrogliosis was apparent at 7 d after injury and was significantly inhibited by multiple STVNa treatments but not single treatment. These results indicate that STVNa exerts neuroprotection by different mechanisms in the acute and delayed phases of I/R. Specifically, STVNa neuroprotection in the delayed phase of injury was found to be accompanied with the inhibition of astrogliosis.

The investigation of protective effects of isosteviol sodium on cerebral ischemia by metabolomics approach using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Cerebral ischemia remains a major cause of mortality and a long-term disability with limited therapies. Isosteviol sodium (STV-Na) was proved to exert significant protective effects on cerebral ischemia, but the protective mechanism was not understood. In this study, the protective effects of STV-Na on cerebral ischemia were investigated by the metabolomics approach based on ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry technique. The models of ischemic rats were established and the brain tissues were employed for metabolomics analyses. The principal component analysis showed that the model group was clearly separated from the sham group, while both STV-Na and edaravone groups were located between the sham and the model groups, which indicated that STV-Na as well as edaravone had protective effects on cerebral ischemia. Eighteen differential metabolites which had significant differences between the sham and the model groups were screened and identified. After the administration of STV-Na, all 18 differential metabolites were regulated to the levels between the sham and the model groups, and 12 of them presented significant differences between the model and STV-Na groups. The pathway analysis indicated that the protective effects of STV-Na on cerebral ischemia might be associated with the regulation of several metabolic pathways, i.e. glycerophospholipid metabolism, arachidonic acid metabolism and linoleic acid metabolism.

Development of a liquid formulation of poorly water-soluble isosteviol sodium using the co-solvent technology.

An intravenously injectable liquid formulation of the poorly water-soluble isosteviol sodium (ISVNa) that has a great clinical potential for cardiovascular diseases was developed using the co-solvent technology. The pH and composition of the co-solvent were optimized to obtain a stable liquid formulation (termed as STVNa) based on saline at pH 10.0 containing 25% (v/v) of ethanol and 20% (v/v) of propylene glycol. STVNa was physicochemically stable upon storage for more than 3 months under various conditions. In vitro studies showed that STVNa did not induce hemolytic effects up to 9.1% (v/v) after 3 h of incubation and it was cytocompatible up to 50 µg/mL in H2C9 cells. Furthermore, STVNa showed acceptable safety and pharmacokinetic parameters comparable with those of ISVNa in saline (dissolved at 60 °C) upon i.v. injection in Wistar rats. Overall, the results demonstrated that STVNa is a promising formulation of ISVNa for clinical translation.

Isosteviol Sodium (STVNA) Reduces Pro-Inflammatory Cytokine IL-6 and GM-CSF in an In Vitro Murine Stroke Model of the Blood-Brain Barrier (BBB).

Early treatment with glucocorticoids could help reduce both cytotoxic and vasogenic edema, leading to improved clinical outcome after stroke. In our previous study, isosteviol sodium (STVNA) demonstrated neuroprotective effects in an in vitro stroke model, which utilizes oxygen-glucose deprivation (OGD). Herein, we tested the hypothesis that STVNA can activate glucocorticoid receptor (GR) transcriptional activity in brain microvascular endothelial cells (BMECs) as previously published for T cells. STVNA exhibited no effects on transcriptional activation of the glucocorticoid receptor, contrary to previous reports in Jurkat cells. However, similar to dexamethasone, STVNA inhibited inflammatory marker IL-6 as well as granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion. Based on these results, STVNA proves to be beneficial as a possible prevention and treatment modality for brain ischemia-reperfusion injury-induced blood-brain barrier (BBB) dysfunction.

Isosteviol Sodium Exerts Anti-Colitic Effects on BALB/c Mice with Dextran Sodium Sulfate-Induced Colitis Through Metabolic Reprogramming and Immune Response Modulation.

PURPOSE: Inflammatory bowel diseases (IBDs) are global health problems that are associated with immune regulation, but clinical IBDs treatment is currently inadequate. Effective preventive or therapeutic methods for immune disorders rely on controlling the function of immune cells. Isosteviol sodium (STV-Na) has antioxidant activity, but the therapeutic effect of STV-Na against IBD remain undocumented. Herein, we investigated the therapeutic effect of STV-Na in mice models with IBDs. METHODS: Mice received 3.5% DSS for 7 days to establish IBD models. Intraperitoneal STV-Na was given 2 days before DSS and lasted for 9 days. Commercially available drugs used in treating IBDs (5-aminosalicylic acid, dexamethasone, and infliximab) were used as positive controls. Samples were collected 7 days after colitis induction. Histopathological score, biochemical parameters, molecular biology methods, and metabolomics were used for evaluating the therapeutic effect of STV-Na. RESULTS: Our data revealed that STV-Na could significantly alleviate colon inflammation in mice with colitis. Specifically, STV-Na treatment improved body weight loss, increased colon length, decreased histology scores, and restored the hematological parameters of mice with colitis. The untargeted metabolomics analysis revealed that metabolic profiles were restored by STV-Na treatment. Furthermore, STV-Na therapy suppressed the number of CD68 macrophages and F4/80 cell infiltration. And STV-Na suppressed M1 and M2 macrophage numbers along with the mRNA expressions of proinflammatory cytokines. Moreover, STV-Na administration increased the number of regulatory T (Treg) cells while decreasing Th1/Th2/Th17 cell counts in the spleen. Additionally, STV-Na treatment restored intestinal barrier disruption in DSS-triggered colitis tissues by ameliorating the TJ proteins, increasing goblet cell proportions, and mucin protein production, and decreasing the permeability to FITC-dextran, which was accompanied by decreased plasma LPS and DAO contents. CONCLUSION: These results indicate that STV-Na can ameliorate colitis by modulating immune responses along with metabolic reprogramming, and could therefore be a promising therapeutic strategy for IBDs.

Therapeutic evaluation and metabolic reprograming of isosteviol sodium in a rat model of ischemic cardiomyopathy.

Ischemia heart disease, one of the lethal cardiovascular diseases, irreversibly impairs cardiac function and is recognized as the primary risk factor for mortality in industrialized countries. The myocardial ischemia treatment still faces a considerable degree of increasing unmet needs. Isosteviol sodium (STVNa) and its derivatives have been proven to effectively alleviate metabolic diseases, hypertension, and heart hypertrophy. Little is known about how STVNa confers the cardioprotective effect during acute myocardial ischemia (AMI). In the present study, a rat model of acute ST-segment-elevation myocardial ischemia by left anterior descending (LAD) ligation was established. Compared to the AMI model group, STVNa administration (4 mg/kg, twice a day) well preserved left ventricle function by ejection fraction (45.10 ± 10.39 vs. 73.64 ± 13.15, p = 0.0013) and fractional shortening (22.94 ± 6.28 vs. 44.00 ± 11.05, p = 0.0017). Further analysis shows that high-dose STVNa (4 mg/kg) significantly improved the hemodynamics in AMI rats, with LVSP (88.25 ± 12.78 vs 99.75 ± 5.10, p = 0.018), max dP/dt (2978.45 ± 832.46 vs 4048.56 ± 827.23, p = 0.096), LVEDP (19.88 ± 2.00 vs 22.26 ± 3.21, p = 0.04) and left ventricular relaxation time constant (Tau) (0.030 ± 0.006 vs 0.021 ± 0.004, p = 0.021). Mechanically, STVNa administration retained the myocardial levels of phosphorylated AMPK, and CPT1b. Moreover, STVNa significantly increased the total energy expenditure, and reduced fatty acid accumulation through mitochondrial oxidative phosphorylation, which was supported by the indirect calorimetry and cellular energy analysis. Taken together, these findings suggest that STVNa is a potential cardioprotection agent for ischemic cardiomyopathy, likely through improving energy homeostasis, left ventricular hemodynamics, and heart function.

Neuroprotective Effects of Isosteviol Sodium in Murine Brain Capillary Cerebellar Endothelial Cells (cerebEND) After Hypoxia.

Ischemic stroke is one of the leading causes of death worldwide. It damages neurons and other supporting cellular elements in the brain. However, the impairment is not only confined to the region of assault but the surrounding area as well. Besides, it also brings about damage to the blood-brain barrier (BBB) which in turn leads to microvascular failure and edema. Hence, this necessitates an on-going, continuous search for intervention strategies and effective treatment. Of late, the natural sweetener stevioside proved to exhibit neuroprotective effects and therapeutic benefits against cerebral ischemia-induced injury. Its injectable formulation, isosteviol sodium (STVNA) also demonstrated favorable results. Nonetheless, its effects on the BBB have not yet been investigated to date. As such, this present study was designed to assess the effects of STVNA in our in vitro stroke model of the BBB.The integrity and permeability of the BBB are governed and maintained by tight junction proteins (TJPs) such as claudin-5 and occludin. Our data show increased claudin-5 and occludin expression in oxygen and glucose (OGD)-deprived murine brain capillary cerebellar endothelial cells (cerebEND) after STVNa treatment. Likewise, the upregulation of the transmembrane protein integrin-αv was also observed. Finally, cell volume was reduced with the simultaneous administration of STVNA and OGD in cerebEND cells. In neuropathologies such as stroke, the failure of cell volume control is a major feature leading to loss of cells in the penumbra as well as adverse outcomes. Our initial findings, therefore, point to the neuroprotective effects of STVNA at the BBB in vitro, which warrant further investigation for a possible future clinical intervention.

Lipidomics study of the protective effects of isosteviol sodium on stroke rats using ultra high-performance supercritical fluid chromatography coupling with ion-trap and time-of-flight tandem mass spectrometry.

Isosteviol sodium (STV-Na) was reported to possess significant protective effects on ischemic stroke in recent years. However, the protective mechanism of STV-Na against stroke was still unclear. In this work, an untargeted lipidomics approach based on the ultra high-performance supercritical fluid chromatography coupling with ion-trap and time-of-flight tandem mass spectrometry (UHSFC-IT-TOF/MS) was employed to investigate the lipid profiles of stroke rats with STV-Na treatment for the first time. The possible mechanism of STV-Na was further elucidated. The UHSFC-IT-TOF/MS-based method achieved a fast separation of various lipids within 9 min with a qualified repeatability. Multivariate statistical analysis was used to show differences in lipid profiles induced by stroke and STV-Na treatment. The results showed a clear separation of the model group and the sham group, with the STV-Na group as well as EDA group located much closer to the sham group than the model group, which was consistent with the results of physiological and pathological assays, indicating the protective effects of STV-Na. Fifteen differential lipids that presented significant differences between the sham group and the model group were screened and identified. With the treatment of STV-Na, 15 differential lipids in stroke rats showed a tendency to the normal levels. Among them, 6 lipids were significantly reversed to the normal levels by STV-Na. The results of pathway analysis suggested the protective effects of STV-Na might be related to the regulation of several metabolic pathways including glycerophospholipid metabolism, arachidonic acid metabolism and sphingolipid metabolism. This work demonstrated that the UHSFC-IT-TOF/MS-based lipidomics profiling method was a useful tool to investigate the protective effects of STV-Na against stroke.