Design, Synthesis, and In Vivo Evaluation of Isosteviol Derivatives as New SIRT3 Activators with Highly Potent Cardioprotective Effects.

Cardiovascular diseases (CVDs) persist as the predominant cause of mortality, urging the exploration of innovative pharmaceuticals. Mitochondrial dysfunction stands as a pivotal contributor to CVDs development. Sirtuin 3 (SIRT3), a prominent mitochondrial deacetylase known for its crucial role in protecting mitochondria against damage and dysfunction, has emerged as a promising therapeutic target for CVDs treatment. Utilizing isosteviol, a natural ent-beyerene diterpenoid, 24 derivatives were synthesized and evaluated in vivo using a zebrafish model, establishing a deduced structure-activity relationship. Among these, derivative 5v exhibited significant efficacy in doxorubicin-induced cardiomyopathy in zebrafish and murine models. Subsequent investigations revealed that 5v selectively elevated SIRT3 expression, leading to the upregulation of SOD2 and OPA1 expression, effectively preventing mitochondrial dysfunction, mitigating oxidative stress, and preserving cardiomyocyte viability. As a novel structural class of SIRT3 activators with robust therapeutic effects, 5v emerges as a promising candidate for further drug development.

Oridonin enhances cytotoxic activity of natural killer cells against lung cancer.

BACKGROUND: Oridonin is a Chinese herbal medicine exhibiting anti-tumor properties; however, its immune modulation capacity has yet to be elucidated. Our objective in this study was to determine whether oridonin enhances the anti-tumor activity of natural killer (NK) cells against lung cancer cells. METHODS: LDH-releasing assays were used to investigate the effects of oridonin on NK-92MI cell activity against lung cancer cells. Flow cytometry and real-time PCR were used to examine the effects of oridonin on degranulation markers, cytotoxic factors, activating receptors on NK-92MI cells, and ligands in lung cancer cells. Western blot analysis provided insight into the mechanisms underlying the observed effects. RESULTS: Oridonin enhanced the cytotoxic effects of NK-92MI cells against A549 lung cancer cells. This effect involved upregulating the expression of the degranulation marker CD107a and IFN-γ as well as activating receptors on NK cells and their ligand MICA/B. Oridonin also inhibited STAT3 phosphorylation in A549 cells and NK-92MI cells. A lung cancer mouse model confirmed the anti-tumor effects of oridonin and NK-92MI cells, wherein both treatments alone suppressed tumor growth. Oridonin was also shown to have a synergistic effect on the anti-tumor activity of NK-92MI cells. CONCLUSIONS: The ability of oridonin to enhance the cytotoxic effects of NK cells indicates its potential as a novel therapeutic agent for the treatment of lung cancer.

Oridonin suppresses gastric cancer SGC-7901 cell proliferation by targeting the TNF-alpha/androgen receptor/TGF-beta signalling pathway axis.

Statistics provided by GLOBOCAN list gastric cancer as the sixth most common, with a mortality ranking of third highest for the year 2020. In China, a herb called Rabdosia rubescens (Hemsl.) H.Hara, has been used by local residents for the treatment of digestive tract cancer for hundreds of years. Oridonin, the main ingredient of the herb, has a curative effect for gastric cancer, but the mechanism has not been previously clarified. This study mainly aimed to investigate the role of TNF-alpha/Androgen receptor/TGF-beta signalling pathway axis in mediating the proliferation inhibition of oridonin on gastric cancer SGC-7901 cells. MTT assay, cell morphology observation assay and fluorescence assay were adopted to study the efficacy of oridonin on cell proliferation. The network pharmacology was used to predict the pathway axis regulated by oridonin. Western blot assay was adopted to verify the TNF-α/Androgen receptor/TGF-ß signalling pathway axis regulation on gastric cancer by oridonin. The results showed Oridonin could inhibit the proliferation of gastric cancer cells, change cell morphology and cause cell nuclear fragmentation. A total of 11signaling pathways were annotated by the network pharmacology, among them, Tumour necrosis factor alpha (TNF-α) signalling pathway, androgen receptor (AR) signalling pathway and transforming growth factor (TGF-ß) signalling pathway account for the largest proportion. Oridonin can regulate the protein expression of the three signalling pathways, which is consistent with the results predicted by network pharmacology. These findings indicated that oridonin can inhibit the proliferation of gastric cancer SGC-7901 cells by regulating the TNF-α /AR /TGF-ß signalling pathway axis.

Discovery of New D-Ring Modified Isosteviol Derivatives as Potent Cardioprotective Agents against Oxidative Stress-Trigged Damage.

Cardiovascular diseases (CVDs) are a major global health concern, and oxidative stress is known to play a central role in their pathogenesis. The identification of new agents capable of inhibiting oxidative stress presents a promising strategy for preventing and treating CVDs. Natural products and their derivatives offer a valuable source for drug discovery, and isosteviol, a readily available natural product, is known to exhibit cardioprotective effects. In this study, 22 new D-ring modified isosteviol derivatives were synthesized and evaluated for their cardioprotective effect in vivo using the zebrafish cardiomyopathy model. The findings revealed that derivative 4e exhibited the most potent cardioprotective effect, surpassing its parent compound isosteviol and the positive drug levosimendan. At 1 µM, derivative 4e significantly protected the cardiomyocytes from injury, while at 10 µM it effectively maintained normal heart phenotypes, preventing cardiac dysfunction in zebrafish. Further investigation demonstrated that 4e protected cardiomyocytes from oxidative stress-induced damage by inhibiting reactive oxygen species overaccumulation, activating superoxide dismutase 2 expression, and enhancing the endogenous antioxidant defense system. These results suggest that isosteviol derivatives, particularly 4e, have the potential to serve as a novel class of cardioprotective agents for the prevention and treatment of CVDs.

Oridonin inhibits Hela cell proliferation via downregulation of glutathione metabolism: a new insight from metabolomics.

OBJECTIVES: This study aims to elucidate Oridonin' s inhibitory mechanism to cervical cancer using metabolomics methods and pharmacological assays. METHODS: Network pharmacology and KEGG pathway analysis are used to identify overlapped targets and involved metabolic pathways. UPLC-MS/MS metabolomics analysis is used to determine altered metabolites after Oridonin treatment. Other bioassays are also employed to uncover the changes in critical molecules that are highly related to altered metabolites. KEY FINDINGS: Seventy-five overlapped targets are identified between Oridonin and cervical cancer. Twenty-one metabolites involved in tricarboxylic acid cycle glutathione metabolism, branched-chain amino acid metabolism and so on changes significantly after Oridonin treatment. Oridonin treatment significantly reduces the content of cysteine and inhibit the catalytic activity of glutamine-cysteine ligase subunit, a rate-limiting enzyme for the synthesis of glutathione. As a result, the content of glutathione is also reduced. The antioxidant enzyme glutathione peroxidase 4 which uses glutathione as a cofactor, is inactivated, resulting in a burst release of reactive oxygen species. The ATP content is also significantly reduced in Hela cells after Oridonin treatment. CONCLUSIONS: This study finds that Oridonin treatment induces Hela cell apoptosis possibly via inhibition of the glutathione metabolism.

Research Progress and Future Development Potential of Oridonin in Pharmacological Activities.

In recent years, attention has increasingly focused on herbal medicines and their bioactive components attributed to their multi-target pharmacological activity and low side effects. Oridonin is a natural diterpenoid extracted from the traditional Chinese herb and is one of the main active components of Rabdosia rubescens. Modern pharmacological studies have shown that oridonin has anti-tumor, anti-bacterial, anti-inflammatory, anti-oxidant, cardiovascular protective, immunomodulatory, and other effects. Based on the published literature in recent years, we outline the pharmacological activities of oridonin, aiming to provide a theoretical basis for the design and development of new oridonin-based drugs, as well as to facilitate the process of oridonin for clinical use.

Oridonin Protects against Myocardial Ischemia-Reperfusion Injury by Inhibiting GSDMD-Mediated Pyroptosis.

Pyroptosis serves a crucial function in various types of ischemia and reperfusion injuries. Oridonin, a tetracycline diterpene derived from Rabdosia rubescens, can significantly inhibit the aggregation of NLRP3-mediated inflammasome. This experiment is aimed at investigating the effect of oridonin on pyroptosis in mice cardiomyocytes. Based on the models of myocardial ischemia/reperfusion (I/R) and hypoxia/reoxygenation (H/R), Evans Blue/TTC double staining, TUNEL staining, and Western blotting were applied to determine the effects of oridonin on myocardial damage, cellular activity and signaling pathways involved in pyroptosis. During I/R and H/R treatments, the extent of gasdermin D-N domains was upregulated in cardiomyocytes. Apart from that, oridonin improved cell survival in vitro and decreased the myocardial infarct size in vivo by also downregulating the activation of pyroptosis. Finally, the expression levels of ASC, NLRP3 and p-p65 were markedly upregulated in cardiomyocytes after H/R treatment, whereas oridonin suppressed the expression of these proteins. The present experiment revealed that myocardial I/R injury and pyroptosis can be alleviated and inhibited by oridonin pretreatment via NF-κB/NLRP3 signaling pathway, both in vivo and in vitro. Therefore, oridonin may serve as a potentially novel agent for the clinical treatment of myocardial ischemia-reperfusion injuries.

Synthesis and in vivo evaluation of new steviol derivatives that protect against cardiomyopathy by inhibiting ferroptosis.

Cardiovascular diseases (CVDs) remain the leading cause of death globally. Inhibiting ferroptosis and thus preventing cardiac cell death is a promising and effective strategy for cardiomyopathy prevention and therapy. Steviol, an ent-kaurene diterpenoid, possesses broad-spectrum bioactivity. In the present study, with the aim to discover new agents for CVDs treatment, 30 derivatives of steviol, including 22 new ones, were synthesized, and evaluated their protective activity in vivo using the doxorubicin (DOX) induced zebrafish cardiomyopathy model. Our results firstly demonstrated that steviol has promising cardioprotective activity and further modification of steviol can greatly improve the activity. Among the new derivatives, 16d and 16e show the most potent activity. Both 16d (1 µM) and 16e (0.1 µM) effectively maintain the normal heart shape and prevent the cardiac dysfunction impaired by DOX in zebrafish. Their therapeutic efficacy is much superior to the parent natural product, steviol, and positive drug, levosimendan. Further study demonstrated that 16d and 16e inhibit DOX-induced ferroptosis and thus protect cardiomyopathy, by suppressing the glutathione depletion, iron accumulation, and lipid peroxidation, decreasing reactive oxygen species overaccumulation, and restoring the mitochondrial membrane potential. Consequently, due to their unique structure and significant cardioprotective activity with ferroptosis inhibition, new steviol derivatives 16d and 16e merit further research for the development of new cardioprotective drug candidates.

Oridonin suppresses particulate-induced NLRP3-independent IL-1α release to prevent crystallopathy in the lung.

The human body is exposed to various particulates of industrial, environmental, or endogenous origin. Invading or intrinsic particulates can induce inflammation by aberrantly activating the immune system, thereby causing crystallopathies. When immune cells such as macrophages phagocytose the particulates, their phagolysosomal membranes undergo mechanical damage, eventually leading to pyroptotic cell death accompanied by the release of inflammatory cytokines, including interleukin (IL)-1α and IL-1ß. The nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is responsible for particulate-induced IL-1ß release and is therefore regarded as a potential therapeutic target for inflammation-mediated crystallopathies. However, IL-1α is released after particulate stimulation in an NLRP3 inflammasome-independent manner and plays a critical role in disease development. Therefore, drugs that exert potent anti-inflammatory effects by comprehensively suppressing particulate-induced responses, including IL-1ß release and IL-1α release, should be developed. Here, we found that oridonin, a diterpenoid isolated from Isodon japonicus HARA, strongly suppressed particulate-induced cell death, accompanied by the release of IL-1α and IL-1ß in mouse and human macrophages. Oridonin reduced particulate-induced phagolysosomal membrane damage in macrophages without affecting phagocytosis of particulates. Furthermore, oridonin treatment markedly suppressed the symptoms of silica particle-induced pneumonia, which was attributed to the release of IL-1α independently of NLRP3. Thus, oridonin is a potential lead compound for developing effective therapeutics for crystallopathies attributed to NLRP3-dependent as well as NLRP3-independent inflammation.

Glaucocalyxin A induces apoptosis of non-small cell lung carcinoma cells by inhibiting the PI3K/Akt/GSK3ß pathway.

Lung cancer is one of the fastest growing malignancies in morbidity and mortality, and current therapies are in general not sufficiently effective for this deadly disease. This study characterizes the anticancer effects of glaucocalyxin A (GLA) and explores the underlying mechanisms using human non-small cell lung carcinoma (NSCLC) cells. First, our data showed that GLA suppressed the viability of cancer cells, whereas no effect was observed in the normal bronchial epithelial cell BEAS-2B cells. Second, GLA inhibited colony formation, induced apoptosis of cancer cells. Third, GLA downregulated the expression of B-cell lymphoma-2 (Bcl-2) protein; upregulated the expression of Bcl2-associated X protein (Bax), and strengthened cleavage of caspase 3 and polyadenyl diphosphate ribose polymerase (PARP). Fourth, GLA also diminished mitochondrial membrane potential and inhibited phosphatidylinositol 3-kinase (PI3K)/Akt/ glycogen synthase kinase-3ß (GSK3ß) pathway. In addition, injection of GLA (20 mg/kg) every 2 days significantly inhibited A549 xenograft tumour growth, accompanied by increased apoptosis and decreased proliferation. Together, our study provides evidence that the anticancer effect of GLA in NSCLC is mediated by inducing apoptosis through inhibiting PI3K/Akt/GSK3ß pathway and suggests that GLA may be used as a promising natural medicine for NSCLC therapy.

Potential of diterpenes as antidiabetic agents: Evidence from clinical and pre-clinical studies.

Diterpenes are a diverse group of structurally complex natural products with a wide spectrum of biological activities, including antidiabetic potential. In the last 25 years, numerous diterpenes have been investigated for antidiabetic activity, with some of them reaching the stage of clinical trials. However, these studies have not been comprehensively reviewed in any previous publication. Herein, we critically discussed the literature on the potential of diterpenes as antidiabetic agents, published from 1995 to September, 2021. In the period under review, 427 diterpenes were reported to have varying degrees of antidiabetic activity. Steviol glycosides, stevioside (1) and rebaudioside A (2), were the most investigated diterpenes with promising antidiabetic property using in vitro and in vivo models, as well as human subjects. All the tested pimaranes consistently showed good activity in preclinical evaluations against diabetes. Inhibitions of α-glucosidase and protein tyrosine phosphatase 1B (PTP 1B) activities and peroxisome proliferator-activated receptors gamma (PPAR-γ) agonistic property, were the most frequently used models for studying the antidiabetic activity of diterpenes. The molecular mechanisms of action of the diterpenes include increased GLUT4 translocation, and activation of phosphoinositide 3-kinase (PI3K) and AMP-activated protein kinase (AMPK)-dependent signaling pathways. This review revealed that diterpenes hold promising antidiabetic potential while stevioside (1) and rebaudioside A (2) are the only diterpenes that were advanced to the clinical trial stage of the drug discovery pipeline. Diterpenes belonging to the abietane, labdane, pimarane and kaurane classes have shown promising activity in in vitro and in vivo models of diabetes and should be further investigated.

Glaucocalyxin A Attenuates IL-1ß-Induced Inflammatory Response and Cartilage Degradation in Osteoarthritis Chondrocytes via Inhibiting the Activation of NF-κB Signaling Pathway.

Glaucocalyxin A (GLA) is a bioactive natural compound with anti-inflammatory activity. Herein, the role of GLA in osteoarthritis (OA) was evaluated. Our results demonstrated that the IL-1ß-induced inducible nitric oxide synthase (iNOS) and cyclooygenase-2 (COX-2) expression, two enzymes resulting in the release of nitric oxide (NO) and PGE2, were also prevented by GLA in chondrocytes. Moreover, GLA suppressed inflammatory cytokines production in chondrocytes. In addition, the elevated expressions of MMPs and ADAMTSs and the degradation of aggrecan and collagen II were reversed by GLA in chondrocytes. Furthermore, GLA decreased p-p65 level and suppressed the nuclear p65 accumulation in the nucleus of chondrocytes. Collectively, we concluded that GLA attenuated inflammatory response in chondrocytes via NF-κB pathway. These findings suggested that GLA might become an effective agent for OA treatment.

Anti-Cancer Properties of Stevia rebaudiana; More than a Sweetener.

Stevia rebaudiana Bertoni is a perennial shrub from Paraguay that is nowadays widely cultivated, since it is increasingly being utilized as a sugar substitute in various foodstuffs due to its sweetness and minimal caloric content. These properties of the plant's derivatives have spurred research on their biological activities revealing a multitude of benefits to human health, including antidiabetic, anticariogenic, antioxidant, hypotensive, antihypertensive, antimicrobial, anti-inflammatory and antitumor actions. To our knowledge, no recent reviews have surveyed and reported published work solely on the latter. Consequently, our main objective was to present a concise, literature-based review of the biological actions of stevia derivatives in various tumor types, as studied in in vitro and in vivo models of the disease. With global cancer estimates suggesting a 47% increase in cancer cases by 2040 compared to 2020, the data reviewed in this article should provide a better insight into Stevia rebaudiana and its products as a means of cancer prevention and therapy within the context of a healthy diet.

Isosteviol Sodium Ameliorates Dextran Sodium Sulfate-Induced Chronic Colitis through the Regulation of Metabolic Profiling, Macrophage Polarization, and NF-κB Pathway.

Inflammatory bowel diseases (IBDs) constitute a group of chronic intestinal conditions prominently featuring deranged metabolism. Effective pharmacological treatments for IBDs are lacking. Isosteviol sodium (STV-Na) exhibits anti-inflammatory activity and may offer therapeutic benefits in chronic colitis. However, the associated mechanism remains unclear. This study is aimed at exploring the therapeutic effects of STV-Na against chronic colitis in terms of metabolic reprogramming and macrophage polarization. Results show that STV-Na attenuated weight loss and colonic pathological damage and restored the hematological and biochemical parameters in chronic colitis mice models. STV-Na also restored intestinal permeability by increasing the goblet cell numbers, which was accompanied by lowered plasma lipopolysaccharide and diamine oxidase levels. Metabolomic analysis highlighted 102 candidate biomarkers and 5 vital pathways that may be crucial in the potential pharmacological mechanism of STV-Na in regulating intestinal inflammation and oxidative stress. These pathways were glycerophospholipid metabolism, phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, the pentose phosphate pathway, and phosphonate and phosphinate metabolism. Furthermore, STV-Na significantly decreased M1 macrophage polarization in the spleen and colon. The mRNA and protein levels of IL-1ß, TNF-α, and NF-κB/p65 in colonic tissue from the colitis mice were decreased after the STV-Na treatment. Overall, STV-Na could alleviate chronic colitis by suppressing oxidative stress and inflammation levels, reprogramming the metabolic profile, inhibiting macrophage polarization, and suppressing the NF-κB/p65 signaling pathway. STV-Na remains a promising candidate drug for treating IBDs.

Mechanism of neuroprotective effect of stevioside on cerebral ischemia-reperfusion injury via PPAR-γ activation.

OBJECTIVE: The aim of this work was to explore the possible protective effects and its mechanism of stevioside on cerebral ischemia reperfusion (CIR) induced neuron damages. METHODS: Middle cerebral artery occlusion/reperfusion (MCAO/R) rat models were constructed. The rats were treated with stevioside treatment, PPAR-γ antagonist GW9662, PPAR-γ activator pioglitazone or PI3K/AKT inhibitor LY294002 before neurological deficits were assessed using modified Neurological Severity Scale (mNSS) scores. The infarct size, brain injury, apoptotic cells, inflammatory cytokines in neurons extracted from MCAO/R rats were determined by TTC staining, H&E staining, TUNEL staining, qRT-PCR and Western blot, respectively. RESULTS: Stevioside attenuates MCAO/R-induced neuronal apoptosis and inflammation by regulating PPAR-γ expression. Besides, PPAR-γ activates PI3K/AKT signaling pathway. Moreover, PPAR-γ antagonist GW9662 or PI3K/AKT inhibitor LY294002 abrogated the anti-apoptosis and anti-inflammatory effects of stevioside on MCAO/R rats. CONCLUSION: Stevioside alleviates MCAO/R-induced neuronal apoptosis and inflammation by upregulating PPAR-γ to activate PI3K/AKT signaling pathway.

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.

Antinociceptive Activity of Compounds from the Aqueous Extract of Melampodium divaricatum.

A decoction prepared from the aerial parts of Melampodium divaricatum showed antinociceptive and antihyperalgesic responses when tested in the formalin model in mice. From the CH2 Cl2 fraction of the decoction, two non-previously reported secondary metabolites, 3-O-ß-D-glucopyranosyl-16α-hydroxy-ent-kaurane (1) and melampodiamide (2) [(2'R*,4'Z)-2'-hydroxy-N-[(2S*,3S*,4R*)-1,3,4-trihydroxyoctadec-2-yl]tetracos-4-enamide] were separated and characterized by spectroscopic, spectrometric, and computational techniques. The flavonoids isoquercitrin and hyperoside, which possessed noted antinociceptive properties, were obtained from the active AcOEt fraction of the decoction. The chemical composition of the essential oil of the plant was also analyzed by gas chromatography-mass spectrometry. The major constituents were (E)-caryophyllene, germacrene D, ß-elemene, δ-elemene, γ-patchoulene, and 7-epi-α-selinene. Headspace solid-phase microextraction analysis detected (E)-caryophyllene as the main volatile compound of the plant.

Renoprotective Effect of Oridonin in a Mouse Model of Acute Kidney Injury via Suppression of Macrophage Involved Inflammation.

Ischemia-reperfusion injury (IRI) is the major cause of acute kidney injury (AKI). The previous studies demonstrated that Oridonin can protect kidney against IRI-induced AKI, but the underlying molecular mechanism is unclear. In this study, it showed that Oridonin significantly improved kidney damage, and inhibited the expression of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and MCP-1, as well as macrophage marker F4/80 in kidney and the secretion of inflammatory cytokins in serum of AKI mice in vivo. In addition, Oridonin also effectively reduced the expression and secretion of lipopolysaccharide (LPS)-induced inflammatory factors in macrophage cell line RAW264.7 in vitro. Notably, Oridonin strongly downregulated Mincle and AKT/nuclear factor-kappaB (NF-κB) signaling both in vivo and in vitro, and the results of cellular recovery experiments of overexpression of Mincle in macrophage suggested that Oridonin suppressed inflammatory response of macrophage through inhibiting Mincle, which may be the underlying mechanism of Oridonin improving injury in kidney of AKI mice. In summary, the above results indicated that Oridonin can protect kidney from IRI-induced inflammation and injury by inhibiting the expression of Mincle in macrophage.

Glaucocalyxin A suppresses osteoclastogenesis induced by RANKL and osteoporosis induced by ovariectomy by inhibiting the NF-κB and Akt pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Glaucocalyxin A (GLA), the most abundant active component of the aboveground sections of Rabdosia japonica (Burm. f.) Hara var. glaucocalyx (Maxim.) Hara, possesses various pharmacological activities, such as antioxidant, antithrombosis, anticoagulation, antibacterial, antitumor, anti-inflammatory activities. According to previous studies, inflammation is closely associated with osteoclast differentiation and activity. Although GLA has demonstrated effective anti-inflammatory properties, its effects on osteoclast differentiation remain unclear. AIM OF THE STUDY: To examine the possible inhibitory effects of GLA and its molecular mechanisms in osteogenesis induced by RANKL as well as ovariectomy (OVX)-induced osteoporosis (OP) in mice. MATERIALS AND METHODS: Tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining, and a bone resorption pit assay were applied for identifying the effects of GLA on the differentiation of osteoclasts and the function of bone resorption. The mRNA expression of the genes related to osteoclast differentiation was measured by quantitative PCR. Protein expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-fos and phosphorylation of inhibitor of nuclear factor kappa B (IκBα), protein kinase B (AKT), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 in RANKL-induced osteoclasts was determined using western blotting. The effect of GLA on OP was studied using a mouse model of OVX. RESULTS: At nontoxic concentrations ≤0.5 µM in vitro, GLA suppressed the formation of osteoclasts induced by RANKL with the decreased number and area size of TRAP-positive multinuclear osteoclasts, and the resorption of bone function by reducing F-actin ring number and bone resorption pit areas. It also reduced the expression of the genes specific for osteoclasts, which included genes encoding NFATc1, cathepsin K, c-fos, TRAP, vacuolar-type ATPase d2, and dendritic cell-specific transmembrane protein. Moreover, GLA repressed NF-κB and Akt pathway activation induced by RANKL. Micro-CT analysis of femur samples indicated decreased bone loss and greater trabecular bone density after GLA treatment, which showed that GLA played a protective role by inhibiting bone loss in OVX-induced OP mice in vivo. CONCLUSIONS: Our study is the first to show that GLA has significant therapeutic potential in OP, which is the disease of osteoclast increase caused by estrogen deficiency.

Synthesis and in vivo screening of isosteviol derivatives as new cardioprotective agents.

Isosteviol, an ent-beyerane diterpenoid, has been repeatedly reported to possess potent cardioprotective activity. With the aim of discovering new cardioprotective derivatives from isosteviol, 47 compounds, including 40 new ones, were synthesized and evaluated in vivo using the easy-handling and efficient zebrafish model. The structure-activity relationship of this type of compounds was thus discussed. Of these compounds, new derivative 15d exhibited the most pronounced efficacy in vivo. Our results indicated that 15d could effectively prevent the doxorubicin-induced morphological distortions and cardiac dysfunction in zebrafish. Its cardioprotective activity is much better than that of isosteviol, and Levosimendan in zebrafish model. The molecular mechanism underlying in H9c2 cells indicated that 15d protected cardiomyocyte death and damage through inhibiting the reactive oxygen species overproduction, restoring the mitochondrial membrane potential and maintaining morphology of mitochondrial. Thus, 15d merits further development as a potential cardioprotective clinical trial candidate. The present study is a successful example to combine synthesis, structure-activity relationship study and in vivo screening to effectively discover new cardioprotective agents from isosteviol.