Protective effect of esculentoside A against myocardial infarction via targeting C-X-C motif chemokine receptor 2.

Myocardial infarction (MI) is the primary cause of cardiac mortality. Esculentoside A (EsA), a triterpenoid saponin, has anti-inflammatory and antioxidant activities. However, its effect on MI remains unknown. In this study, the protective effect and mechanisms of EsA against MI were investigated. EsA significantly alleviated hypoxia-induced HL-1 cell injury, including increasing cell viability, inhibiting reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) and lactate dehydrogenase (LDH) leakage. In mouse MI model by left coronary artery (LAD) ligating, EsA obviously restored serum levels of creatine kinase isoenzymes (CK-MB), cardiac troponin I (cTnI), superoxide dismutase (SOD) and malondialdehyde (MDA). In addition, the cardioprotective effect of EsA was further confirmed by infarct size, electrocardiogram and echocardiography. Mechanistically, the targeted binding relationship between EsA and C-X-C motif chemokine receptor 2 (CXCR2) was predicted by molecular docking and dynamics, and validated by small molecule pull-down and surface plasmon resonance tests. EsA inhibited CXCR2 level both in vitro and in vivo, correspondingly alleviated oxidative stress by suppressing NOX1 and NOX2 and relieved inflammation through inhibiting p65 and p-p65. It demonstrated that EsA could play a cardioprotective role by targeting CXCR2. However, the effect of EsA against MI was abolished in combination with CXCR2 overexpression both in vitro and in vivo. This study revealed that EsA showed excellent cardioprotective activities by targeting CXCR2 to alleviate oxidative stress and inflammation in MI. EsA may function as a novel CXCR2 inhibitor and a potent candidate for the prevention and intervention of MI in the future.

Comparative proteomic analysis of cerebral cortex revealed neuroprotective mechanism of esculentoside A on Alzheimer's disease.

Esculentoside A (EsA), isolated from phytolacca esculenta, is a saponin showing neuroprotective effect in the mouse models of Alzheimer's disease (AD). To investigate its action target and underlying mechanism, this study used the proteomics technique of isobaric tags for relative and absolute quantification (iTRAQ) to analyze the differentially expressed proteins (DEPs) in the cerebral cortex of EsA-treated and untreated triple-transgenic 3 × Tg-AD model mice. Proteomic comparison revealed 250, 436, and 903 DEPs in three group pairs, i.e. AD/Wild-type (WT), AD+5 mg/kg EsA/AD, AD+10 mg/kg EsA/AD, respectively. Among them 28 DEPs were commonly shared by three group pairs, and 25 of them showed reversed expression levels in the diseased group under the treatment of both doses of EsA. Bioinformatics analysis revealed that these DEPs were mainly linked to metabolism, synapses, apoptosis, learning and memory. EsA treatment restored the expression of these proteins, including amyloid precursor protein (APP), cathepsin B (Cstb), 4-aminobutyrate aminotransferase (Abat), 3-phosphoinositide-dependent protein kinase-1 (PDK1), carnitine palmitoyltransferase1 (Cpt1) and synaptotagmin 17 (Syt17), thereby ameliorated the spatial learning and memory of AD mice. Collectively, this study reveals for the first time the profound effect of EsA on the cerebral cortex of AD mice, which might be a potential therapeutic agent for the treatment of AD.

Esculentoside A ameliorates BSCB destruction in SCI rat by attenuating the TLR4 pathway in vascular endothelial cells.

BACKGROUND AND AIMS: Overexpressed MMP-9 in vascular endothelial cells is involved in blood spinal cord barrier (BSCB) dysfunction in spinal cord injury (SCI). Esculentoside A (EsA) has anti-inflammatory and cell protective effects. This study aimed to evaluate its effects on neuromotor function in SCI rats, as well as the potential mechanisms. METHODS: The therapeutic effect of EsA in SCI rats was investigated using Basso-Beattie-Bresnahan (BBB) scores, a grid walk test and histological analyses. To assess the protective role of EsA in the BSCB and in oxygen glucose deprivation/reoxygenation (OGD/R)-induced hBMECs, the BSCB function, tight junctions (TJ) protein (ZO-1 and claudin-5) expression, structure of the BSCB and Matrix metalloproteinase-9 (MMP-9) expression were observed via Evans blue (EB) detection, immunofluorescence analyses and western blotting. Molecular docking simulations and additional experiments were performed to explore the potential mechanisms by which EsA maintains the function of the BSCB in vivo and in vitro. RESULTS: EsA treatment improved BBB scores, reduced cavity formation and the loss of neuronal cells, demonstrating an improvement in motor function in SCI rats. In vivo experiments showed that EsA decreased the infiltration of blood cells and inflammatory mediators (IL-1ß, IL-6 and TNF-α) and protected the structure of TJs in the rat spinal cord and in OGD/R-induced hBMECs. EsA inhibited the activation of Toll-like receptor 4 (TLR4) signalling, which may be related to the protective effect of EsA against MMP-9-induced BSCB damage. CONCLUSIONS: EsA downregulated MMP-9 expression in vascular endothelial cells, protected BSCB function in SCI rats and attenuated TLR4 signalling and thus provide new options for the treatment of SCI.

Esculentoside A ameliorates DNCB-induced atopic dermatitis by suppressing the ROS-NLRP3 axis via activating the Nrf2 pathway.

Atopic dermatitis (AD) is a chronic inflammatory skin condition with a high prevalence. Inflammation and oxidative stress are strongly associated with AD progression. Esculentoside A (EsA) inhibits inflammation and oxidative stress in various diseases. However, whether EsA mitigates AD by suppressing inflammation and oxidative stress remains unknown. A mouse model of AD was constructed by the induction of 1-chloro-2,4-dinitrochlorobenzene (DNCB). The mechanism of EsA and its effects on AD symptoms, pathology, inflammation and oxidative stress were investigated through histopathological staining, enzyme-linked immunosorbent assay, blood cells analysis, colorimetric measurement and western blot analysis. EsA improved the clinical symptoms and increased clinical skin scores in AD mice. Skin thickening of the epidermis and dermal tissues and the mast cell numbers in AD mice were reduced with the EsA treatment. EsA decreased the relative mRNA level of thymic stromal lymphopoietin, interleukin (IL)-4, IL-5 and IL-13; the serum concentrations of immunoglobulin E (IgE) and IL-6; and the numbers of white blood cells (WBC) and WBC subtypes, including basophil, lymphocytes, eosinophil, neutrophil and monocytes in DNCB-induced mice. DNCB caused higher levels of oxidative stress, which was reversed with the administration of EsA. Mechanically, EsA upregulated the expression of Nrf2 but downregulated the level of NLRP3 inflammasome in AD mice. The inhibitor of Nrf2 significantly recovered the EsA-induced changes in the NLRP3 inflammasome proteins in DNCB-treated mice. Therefore, EsA improved the clinical and pathological symptoms, inflammation and oxidative stress experienced by DNCB-induced mice and was involved in the inactivation of NLRP3 inflammasome by activating Nrf2.

The protective effects of Esculentoside A through AMPK in the triple transgenic mouse model of Alzheimer's disease.

BACKGROUND: Neurofibrillary tangles comprising hyperphosphorylated tau are vital factors associated with the pathogenesis of Alzheimer's disease (AD). The elimination or reduction of hyperphosphorylated and abnormally aggregated tau is a valuable measure in AD therapy. Esculentoside A (EsA), isolated from Phytolacca esculenta, exhibits pharmacotherapeutic efficacy in mice with amyloid beta-induced AD. However, whether EsA affects tau pathology and its specific mechanism of action in AD mice remains unclear. PURPOSE: To investigate the roles and mechanisms of EsA in cognitive decline and tau pathology in a triple transgenic AD (3 × Tg-AD) mouse model. METHODS: EsA (5 and 10 mg/kg) was administered via intraperitoneal injection to 8-month-old AD mice for eight consecutive weeks. Y-maze and novel object recognition tasks were used to evaluate the cognitive abilities of mice. Potential signaling pathways and targets in EsA-treated AD mice were assessed using quantitative proteomic analysis. The NFT levels and hippocampal synapse numbers were investigated using Gallyas-Braak silver staining and transmission electron microscopy, respectively. Western blotting and immunofluorescence assays were used to measure the expression of tau-associated proteins. RESULTS: EsA administration attenuated memory and recognition deficits and synaptic damage in AD mice. Isobaric tags for relative and absolute quantitation proteomic analysis of the mouse hippocampus revealed that EsA modulated the expression of some critical proteins, including brain-specific angiogenesis inhibitor 3, galectin-1, and Ras-related protein 24, whose biological roles are relevant to synaptic function and autophagy. Further research revealed that EsA upregulated AKT/GSK3ß activity, in turn, inhibited tau hyperphosphorylation and promoted autophagy to clear abnormally phosphorylated tau. In hippocampus-derived primary neurons, inhibiting AMP-activated protein kinase (AMPK) activity through dorsomorphin could eliminate the effect of EsA, as revealed by increased tau hyperphosphorylation, downregulated activity AKT/GSK3ß, and blocked autophagy. CONCLUSIONS: To our knowledge, this study is the first to demonstrate that EsA attenuates cognitive decline by targeting the pathways of both tau hyperphosphorylation and autophagic clearance in an AMPK-dependent manner and it shows a high reference value in AD pharmacotherapy research.

Esculentoside A could attenuate apoptosis and inflammation in TNBS-induced ulcerative colitis via inhibiting the nuclear translocation of NF-κB.

Background: Esculentoside A (EsA) has had a remarkable curative effect on a variety of experimental acute and chronic inflammatory and autoimmune diseases. However, the role of EsA in the pathological process of ulcerative colitis (UC) is still unknown. Methods: Rat colonic smooth muscle cells (SMCs) were identified by immunofluorescence. The effect of EsA and/or lipopolysaccharide (LPS) on the viability, proliferation, and apoptosis of SMCs was explored via 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU) staining, flow cytometry, and TdT-mediated dUTP nick end labeling (TUNEL) staining, respectively. The changes of apoptosis-related proteins were performed via western blotting. The expression and nuclear translocation of NF-κB were detected via western blotting, immunohistochemistry (IHC), and immunofluorescence staining, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of IL-6 and TNF-α. Results: The EsA treatment greatly up-regulated the viability of LPS-suppressed SMCs. The LPS-induced cell apoptosis was significantly reversed by EsA treatment, which was achieved via down-regulating Bax and cleaved caspase-3 expression and up-regulating Bcl-2 expression. In addition, LPS-induced IL-6, TNF-α expression and NF-κB activation were also largely decreased when treated with EsA. In vivo, the TNBS-induced colon injury including crypt destruction and crypt deformation, disorder, epithelial cell remains or complete destruction, and inflammatory cell infiltration was recovered by EsA treatment. The secretion of IL-6 and TNF-α in the serum of the model group was also down-regulated by EsA treatment. The expression of Bax, cleaved caspase-3, and Bcl-2 showed similar trends as those observed in the in vitro experiments. Conclusions: Our data provides supportive evidence that EsA can relieve the symptoms of UC and be used as a drug candidate for the treatment of UC.

Esculentoside A alleviates cognitive deficits and amyloid pathology through peroxisome proliferator-activated receptor γ-dependent mechanism in an Alzheimer's disease model.

BACKGROUND: Alzheimer's disease (AD) is characterized clinically by cognitive deficits and pathologically by amyloid-ß (Aß) deposition and tau aggregation, as well as the brain atrophy. Esculentoside A (EsA), a neuroprotective saponin, is isolated from Phytolacca esculenta and shows potent health-promoting effects in a variety of experimental models. However, there are minimal reports on the effects of EsA on triple transgenic AD mice. PURPOSE: The current research aimed at investigating the protective effects and underlying mechanisms of EsA on the mitigation of cognitive deficits and pathology in triple transgenic AD mice. METHODS: Triple transgenic AD mice (3 × Tg-AD) of 8 months old received intraperitoneal treatment of 5 or 10 mg/kg EsA for 8 consecutive weeks. Morris water maze test and open field test were made to evaluate the cognitive function and degree of anxiety of the mice. Liquid chromatography with tandem mass spectrometry analysis was performed to characterize and to quantify EsA in the blood and brain of mice. Immunofluorescence assay and Western blot were adopted to measure the levels of peroxisome proliferator-activated receptor gamma (PPARγ) and key proteins in Aß pathology, ER stress- and apoptosis-associated pathways. The combination of EsA with PPARγ were theoretically calculated by molecular docking programs and experimentally confirmed by the bio-layer interferometry technology. RESULTS: Supplemental EsA could improve the cognitive deficits of 3 × Tg-AD mice. EsA penetrated the brain-blood barrier to exert a strong effect on AD mice, evidenced as decreasing Aß generation, reducing the degrees of oxidative and ER stress, and mitigating neuronal apoptosis through the increase of PPARγ expression. In the culture of primary neurons, addition of PPARγ inhibitor GW9662 eliminated the effects of EsA on AD pathologies. Direct combination of EsA with PPARγ were demonstrated by molecular docking programs and bio-layer interferometry technology. CONCLUSIONS: For the first time, these outcomes revealed that EsA could penetrate the brain-blood barrier to exert a strong effect on ameliorating cognitive deficits in 3 × Tg-AD mice and exert neuroprotective effects toward AD pathology via PPARγ-dependent mechanism.

Study on pharmacodynamics and pharmacokinetics of Phytolacca acinosa Roxb.And its mechanism / 中国药理学通报

Aim To explore the diuretic effect, diuretic mechanism and pharmacokinetics of Phytolacca acinosa Roxb., and clarify its "quantity-time-effect" relationship.Methods Firstly, qualified rats were modeled by water load model, given different doses of Phytolacca acinosa Roxb.aqueous extract, then the diuretic effect was investigated.Secondly, Western blot was used to detect the protein expression of aquaporins AQP2, AQP4 and the angiotensin II receptors ATGR1, ATGR2, and renin in the RAAS system in kidney tissues.Thirdly, the established LC-MS/MS biological analysis method was used to detect the esculentoside A(EsA)content in the plasma, calculate the pharmacokinetic parameters and analyze the correlation between the blood concentration and the drug effect.Results The water load model was successfully established.Compared with the model group, hydrochlorothiazide had a significant diuretic effect(P<0.01).Low, medium and high dose groups of Phytolacca acinosa Roxb.all had obvious diuretic effects(P<0.01), EsA also had a significant diuretic effect(P<0.05).Phytolacca acinosa Roxb.aqueous extract and EsA significantly down-regulated the expression of AQP2, AQP4, ATGR1 and renin protein.The pharmacokinetic results showed that the Cmax and AUC0-t of EsA in the plasma of rats in the low, medium, and high dose groups of aqueous extract increased with the increase of the dose.Conclusions Phytolacca acinosa Roxb.had a diuretic effect, which is related to inhibiting the expression of aquaporins AQP2 and AQP4 and inhibiting the expression of angiotensin II type 1 receptor and renin, thereby inhibiting the reabsorption of renal tubules and collecting ducts.

A potential antiviral activity of Esculentoside A against binding interactions of SARS-COV-2 spike protein and angiotensin converting enzyme 2 (ACE2).

The recent emergence of the novel coronavirus (SARS-CoV-2) has resulted in a devastating pandemic with global concern. However, to date, there are no regimens to prevent and treat SARS-CoV-2 virus. There is an urgent need to identify novel leads with anti-viral properties that impede viral pathogenesis in the host system. Esculentoside A (EsA), a saponin isolated from the root of Phytolacca esculenta, is known to exhibit diverse pharmacological properties, especially anti-inflammatory activity. To our knowledge, SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) to enter host cells. This is mediated through the proteins of SARS-CoV-2, especially the spike glycoprotein receptor binding domain. Thus, our primary goal is to prevent virus replication and binding to the host, which allows us to explore the efficiency of EsA on key surface drug target proteins using the computational biology paradigm approach. Here, the anti-coronavirus activity of EsA in vitro and its potential mode of inhibitory action on the S-protein of SARS-CoV-2 were investigated. We found that EsA inhibited the HCoV-OC43 coronavirus during the attachment and penetration stage. Molecular docking results showed that EsA had a strong binding affinity with the spike glycoprotein from SARS-CoV-2. The results of the molecular dynamics simulation revealed that EsA had higher stable binding with the spike protein. These results demonstrated that Esculentoside A can act as a spike protein blocker to inhibit SARS-CoV-2. Considering the poor bioavailability and low toxicity of EsA, it is suitable as novel lead for the inhibitor against binding interactions of SARS-CoV-2 of S-protein and ACE2.

Integrating non-targeted metabolomics and toxicology networks to study the mechanism of Esculentoside A-induced hepatotoxicity in rats.

Esculentoside A (EsA) is a kind of triterpenoid saponins from the root tuber of Phytolacca acinosa Roxb. It has extensive medicinal activity, such as antibacterial, anti-inflammatory, immune regulation, and cell proliferation inhibition. However, some researches suggested that EsA can cause hepatotoxicity, whose mechanism is not precise. To ensure the safety and reliability in the clinical use of Phytolacca acinosa Roxb., it is necessary to establish a rapid and accurate method to evaluate the toxicity, analyze and verify the toxicity mechanism of EsA. Therefore, this research explored the mechanism of hepatotoxicity induced by EsA in rats and analyzed endogenous metabolites' changes in rat plasma by combining network toxicology with non-targeted metabolomics. We obtained 58 critical targets of EsA induced hepatotoxicity in rats based on the strategy of network toxicology, including albumin, mitogen-activated protein kinase 1, Caspase-3, etc. Many important pathways were obtained by Kyoto Encyclopedia of Genes and Genomes enrichment analysis, such as HIF-1 signaling pathway, TNF signaling pathway, IL-17 signaling pathway, and other concerning pathways. Sixteen biomarkers, including 5-hydroxykynurenamine, N-acetylserotonin, palmitic acid, etc., were screened from rat plasma using Ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS), mainly involve Glycerophospholipid metabolism, Tryptophan metabolism, and other metabolic pathways. Further analysis showed that EsA may induce liver injury by activating oxidative stress and energy metabolism disorders, triggering inflammation and apoptosis.

Esculentoside A rescues granulosa cell apoptosis and folliculogenesis in mice with premature ovarian failure.

Follicular atresia is one of the main processes for the loss of granulosa cells and oocytes from the mammalian ovary and any impairment to premature ovarian failure. Large numbers of studies have demonstrated that granulosa cell apoptosis causes follicular atresia, yet the rescue of these cells remains elusive. We aimed to use Esculentoside A (3-O-b-D-glucopyranosyl-1, 4-b-D-xylopyranosyl) phytolaccagenin, a saponin extracted from Phytolacca esculenta roots, as a potential rescue agent for the apoptosis of granulosa cells. Our results revealed the rescue of normal body and ovary weights, normal ovarian histo-architecture of ovaries, and hormones levels with regular estrus cycle. Consistently, the expression of proliferating and anti-apoptotic markers, i.e. KI67 and BCL-2 in granulosa cells, was enhanced. Meanwhile, the expressions of pro-apoptotic markers, which were BAX and CASPASEs (CASPASE-9 and CASPASE-3), were prominently reduced in Esculentoside A-induced premature ovarian failure mice. Additionally, PPARγ, a potential therapeutic target, has also rescued its expression by treating the premature ovarian failure mice with Esculentoside A. Our results advocated that Esculentoside A could restore folliculogenesis in premature ovarian failure mice. Furthermore, it has the potential to be investigated as a therapeutic agent for premature ovarian failure.

Esculentoside A protects against osteoarthritis by ameliorating inflammation and repressing osteoclastogenesis.

Osteoarthritis is a relatively common disorder of articular deterioration related to cartilage damage, subchondral bone remodelling, inflammation and metabolism. Agents that can inhibit cartilage degradation and osteoclastogenesis are required for the prevention and treatment of osteoarthritis. Esculentoside A, the highest concentration triterpene saponin isolated from the root of Phytolacca esculenta, has commonly been used for the treatment of chronic bronchitis. However, the role esculentoside A plays in ameliorating osteoarthritis has not been reported. We found that esculentoside A suppresses the expression of IL-1ß-induced inflammatory and metabolic factors (IL-6, IL-8, TNF-α, MMP2, MMP3 and MMP13). In addition, esculentoside A restrains osteoclast formation by inhibiting the marker gene expression of NFATc1 and c-Fos. Our results indicate that esculentoside A markedly suppresses IL-1ß-induced NF-κB and MAPK signalling pathway activation in chondrocytes, and inhibits RANKL-induced osteoclast precursor generation. Finally, treatment with esculentoside A inhibits the progressive cartilage degeneration and osteoclastogenesis in osteoarthritis mouse models. In summary, these results demonstrate that esculentoside A could be a latent therapeutic reagent for the treatment of osteoarthritis.

Analysis of Mechanism of Phytolaccae Radix and Its Processed Products on Doxorubicin-induced Nephropathy in Rats / 中国实验方剂学杂志

Objective: To compare the protective effect of Phytolaccae Radix and its processed products on nephropathy induced by doxorubicin (DOX) in rats, and explore its mechanism. Method: A rat model of nephropathy was established by a single tail intravenous injection of DOX hydrochloride. Content of esculentoside A (EsA) in Phytolaccae Radix and its processed products was determined by HPLC-ELSD. Contents of serum total protein (TP), albumin (Alb), urea nitrogen (BUN), serum creatinine (SCr), total cholesterol (TC) and urine protein (UP) were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression of transforming growth factor-β (TGF-β) in renal tissue of rats was examined by real-time quantitative polymerase chain reaction (Real-time PCR) and immunohistochemistry. Result: A single intravenous injection of DOX could induce a severe nephrotic syndrome associated with decreased serum TP, Alb and elevated serum BUN, SCr, TC, and a high urinary excretion of protein (Pβ in renal tissue of model group rats was significantly higher than that of blank group (PPPConclusion: Phytolaccae Radix and its processed products can improve the symptoms of DOX nephropathy model rats in different degrees, among which the vinegar prepared products have the strongest effect, and this effect may be related to the reduction of TGF-β expression in renal tissue.

Esculentoside A suppresses breast cancer stem cell growth through stemness attenuation and apoptosis induction by blocking IL-6/STAT3 signaling pathway.

Breast cancer stem cells (CSCs) survive in inflammatory microenvironment, their survival are regulated by inflammatory cytokines and signaling pathways. Esculentoside A (EsA), a triterpene saponin derived from the root of Phytolacca esculenta, possesses antiinflammation effects; but whether it has anticancer activity is unknown. The purpose of this study is to test the inhibitory effect of EsA on the growth of breast CSCs and to elucidate its probable mechanisms of action. The proliferation inhibitory effect of EsA on breast CSCs in vitro were determined by cytotoxicity, mammosphere formation inhibition, apoptotic cell detection assays, and in vivo tumor growth inhibition experiment. The possible molecular mechanisms elucidating the inhibitory effect of EsA on breast CSC growth were examined with western blotting. EsA caused proliferation and mammosphere formation inhibition of breast CSCs; induced breast CSCs apoptotic death; suppressed the growth of tumors generated from breast CSCs significantly; the expressions of stemness proteins including ALDH1A1, Sox2, and Oct4 were downregulated; proapoptotic proteins, Bax and cleaved caspase-3 were upregulated, whereas the antiapoptotic protein Bcl-2 was reduced; IL-6/STAT3 pathway proteins including IL-6, phosphorylated STAT3 (Tyr705), and STAT3 (Ser727) were downregulated significantly in EsA-treated breast CSCs and tumor tissues. EsA inhibited breast CSC growth in vitro and in vivo through stemness attenuation and apoptosis induction by blocking IL-6/STAT3 signaling pathway; it might serve as a novel candidate agent for human breast cancer treatment and/or prevention.

Esculentoside A specifically binds to ribosomal protein S3a and impairs LPS-induced signaling in macrophages.

Esculentoside A (EsA), a saponin isolated from Phytolacca esculenta, is reported as a potent suppressor of pro-inflammatory functions of macrophages. However, little is known about the target proteins of EsA for its anti-inflammatory activity. In the present study, to identify the intracellular target for EsA, affinity resins bearing immobilized EsA were used to capture binding proteins of EsA from RAW264.7 cell lysates. Mass spectrography and Western blot analysis of captured proteins indicated that ribosomal protein S3a preferentially bound to EsA affinity resin. Competition experiment further demonstrated that free EsA can disturb the specific interaction between recombinant RPS3a and affinity resin. Surface Plasmon Resonance analysis confirmed that EsA directly bound to RPS3a. Lentivirus-mediated RNAi RPS3a resulted in suppression of TNF-α and IL-6 production and impediment of signal transduction in LPS-stimulated RAW264.7 cells, indicating that RPS3a is required for LPS-triggered signaling during induction of pro-inflammatory cytokines. In addition, EsA inhibited the expression of inflammatory factors more strongly in the case of RPS3a interference. These results suggest that EsA exerts its anti-inflammatory activity by targeting RPS3a and impairing its signaling function. These new findings not only extended our understanding on the intracellular mechanisms of EsA, but also indicated RPS3a as an essential component for LPS-mediated pro-inflammatory signaling, thus implying RPS3a as a novel therapeutic target for anti-inflammatory therapy.

Esculentoside A exerts anti-inflammatory activity in microglial cells.

Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. This study was designed to evaluate the pharmacological effects of EsA on lipopolysaccharide (LPS)-stimulated BV2 microglia and primary microglia cells. Our results indicated that EsA pretreatment significantly decreased LPS-induced production of Nitric Oxide (NO) and Prostaglandin E2 (PGE2) and impeded LPS-mediated upregulation of pro-inflammatory mediators' expression such as nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-a (TNF-α) in both BV2 microglia and primary microglia cells. Moreover, EsA markedly suppressed nuclear factor-κB p65 (NF-κB p65) translocation by blocking IκB-α phosphorylation and degradation in LPS-treated BV2 cells. EsA also decreased phosphorylation level of mitogen-activated protein kinases (MAPKs) and inhibited NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome mediated caspase-1 activation in LPS-stimulated BV2 cells. Additionally, EsA decreased ß-amyloid1-42 (Aß1-42)-induced production of TNF-α, IL-1ß and IL-6 in primary microglia. Thus, EsA might be a promising therapeutic agent for alleviating neuroinflammatory diseases.

Esculentoside A inhibits LPS-induced BV2 microglia activation through activating PPAR-γ.

Neuroinflammation has been recognized as a factor in the pathogenesis of neurodegenerative diseases. Esculentoside A (EsA), a saponin isolated from Phytolacca esculenta, has been reported to have anti-inflammatory activity. However, little research has been reported on the anti-neuroinflammatory effects of EsA. EsA concentration-dependently suppressed LPS-induced TNF-α, IL-1ß, and PGE2 production. LPS-induced NF-κB activation was inhibited by treatment of EsA. Furthermore, EsA concentration-dependently up-regulated the expression of PPAR-γ. In addition, GW9662, a specific PPAR-γ inhibitor, reversed the anti-inflammatory effects of EsA. In conclusion, these results indicate that EsA exerts anti-inflammatory effects by activating PPAR-γ.

Esculentoside A inhibits LPS-induced acute kidney injury by activating PPAR-γ.

Acute kidney injury (AKI) is a major clinical problem associated with high morbidity and mortality. Esculentoside A (EsA), a kind of saponin isolated from the root of the Chinese herb Phytolaca esculenta, has been reported to have anti-inflammatory effect. In this study, we aimed to investigate the protective effects of EsA on LPS-induced AKI in mice. The protective effects of EsA was evaluated by detecting kidney histological change, blood urea nitrogen (BUN) and creatinine levels, and inflammatory cytokines production. The results showed that EsA significantly attenuated LPS-induced kidney histological change, as well as BUN and creatinine levels. EsA also inhibited LPS-induced TNF-α, IL-1ß, and IL-6 production. LPS-induced NF-κB activation was significantly suppressed by treatment of EsA. In addition, EsA up-regulated the expression of PPAR-γ in a dose-dependent manner. In conclusion, EsA protected mice effectively from LPS-induced AKI by PPAR-γ, which subsequently inhibited LPS-induced inflammatory response.

Esculentoside A ameliorates cecal ligation and puncture-induced acute kidney injury in rats.

Esculentoside A (EsA), a saponin isolated from Phytolacca esculenta, can attenuate acute liver and lung injury. However, whether EsA has a protective effect against sepsis-induced acute kidney injury (AKI) has not been reported. In this study, EsA (2.5, 5, or 10 mg/kg) was given to rats with sepsis induced by cecal ligation and puncture (CLP). We found that EsA improved the survival of septic rats in a dose-dependent manner. In addition, EsA lowered the kidney tubular damage score and decreased blood urea nitrogen and creatinine. Moreover, EsA inhibited excessive generation of pro-inflammatory tumor necrosis factor-α, IL-1ß, and IL-6 in the serum and downregulated cyclooxygenase-2 and inducible nitric oxide synthase in the renal tissues of septic rats. EsA also suppressed the production of malonaldehyde and the activity of myeloperoxidase in the septic kidney and enhanced the activity of superoxide dismutase and glutathione. The anti-inflammatory and antioxidative effects of a high dose of EsA were comparable to those of dexamethasone. Mechanically, EsA inhibited CLP-induced increases in high-mobility group box 1, Toll-like receptor-4, and myeloid differentiation primary response 88 and nuclear accumulation of nuclear factor kappa B p65 in renal tissues. In vitro, lipopolysaccharide-induced alteration of AKI-related factors in HK-2 cells, which had been evaluated in vivo, was inhibited after EsA administration. Taken together, our study suggests that EsA effectively protects rats against septic AKI caused by CLP.

Esculentoside A suppresses lipopolysaccharide-induced pro-inflammatory molecule production partially by casein kinase 2.

ETHNOPHARMACOLOGICAL RELEVANCE: Esculentoside A (EsA) is a saponin isolated from the root of Phytolacca esculenta, an herb which has long been used in Traditional Chinese Medicine for various inflammatory diseases. EsA has been reported to have potent anti-inflammatory properties both in vitro and in vivo. AIM OF THE STUDY: The present study focused on the molecular mechanism of EsA for its anti-inflammatory effects in RAW264.7 cells stimulated with lipopolysaccharide (LPS). METHODS AND RESULTS: Enzyme Linked Immunosorbent Assay (ELISA) showed EsA dose dependently inhibited the production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and nitric oxide in RAW264.7 cells. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay further confirmed the suppression of LPS-induced TNF-α, IL-6 and iNOS gene expression by EsA on a transcriptional level. Moreover, EsA treatment markedly suppressed LPS-stimulated IκB phosphorylation and degradation as well as LPS-stimulated luciferase reporter construct driven by κB response elements in RAW264.7 cells. In addition, EsA significantly reduced LPS-induced stimulation of p38 and JNK, but not ERK1/2, phosphorylation. Furthermore, we used a computational method called "reverse docking" to search the possible binding proteins of EsA from the potential drug target database (PDTD), and focused on CK2 as the primary binding protein of EsA. Afterward, we further tested EsA directly interacts with recombinant CK2 using SPR assay. In CK2 kinase activity assay, EsA inhibited recombinant CK2 holoenzyme activity obviously in a dose-dependent manner. In addition, TBB (4, 5, 6, 7-tetrabromo-2-benzotriazole, a pharmacological inhibitor of CK2) blocked IL-6 release in a dose-dependent manner, whereas co-treatment of cells with EsA and TBB did not have an additive effect. CONCLUSIONS: Taken together, these results indicate that EsA blocks the LPS-induced pro-inflammatory molecules expression, at least in part, by impediment of LPS-triggered activation of NF-κB and p38/JNK MAPK pathways in macrophages. Furthermore, we discovered for the first time EsA as a ligand for CK2, which was involved in the inhibition of EsA to the expression of inflammatory cytokines. These findings extended our understanding on the cellular and molecular mechanisms responsible for the anti-inflammatory activity of EsA.