A Metabolomics Study of the Effects of Eleutheroside B on Glucose and Lipid Metabolism in a Zebrafish Diabetes Model.

(1) Background: Diabetes is a common metabolic disease that seriously endangers human health. In the present study, we investigated the therapeutic effects of the active ingredient Eleutheroside B (EB) from the traditional Chinese medicine Eleutheroside on diabetes mellitus in a zebrafish model. Concomitant hepatic injury was also analysed, along with the study of possible molecular mechanisms using metabolomics technology. This work should provide some theoretical references for future experimental studies. (2) Methods: A zebrafish diabetes model was constructed by soaking in a 1.75% glucose solution and feeding a high-fat diet. The intervention drug groups were metformin (100 µg∙mL-1) and EB (50, 100, and 150 µg∙mL-1) via water-soluble exposure for 30 days. Glucose, TG, TC, LDL-C, and HDL-C were evaluated in different treatment groups. GLUT4 protein expression was also evaluated in each group, and liver injury was observed by HE staining. Metabolomics techniques were used to investigate the mechanism by which EB regulates endogenous markers and metabolic pathways during the development of diabetes. (3) Results: All EB treatment groups in diabetic zebrafish showed significantly reduced body mass index (BMI) and improved blood glucose and lipid profiles. EB was found to upregulate GLUT4 protein expression and ameliorate the liver injury caused by diabetes. Metabolomics studies showed that EB causes changes in the metabolic profile of diabetic zebrafish. These were related to the regulation of purine metabolism, cytochrome P450, caffeine metabolism, arginine and proline metabolism, the mTOR signalling pathway, insulin resistance, and glycerophospholipid metabolism. (4) Conclusions: EB has a hypoglycaemic effect in diabetic zebrafish as well as significantly improving disorders of glycolipid metabolism. The mechanism of action of EB may involve regulation of the mTOR signalling pathway, purine metabolism, caffeine metabolism, and glycerophospholipid metabolism.

Ameliorating effects of Acanthopanax koreanum extract and components on nicotine dependence and withdrawal symptoms.

Tobacco smoking is a serious health problem in society. While smoking rates are declining, smoking remains a serious risk to national health. Currently, there are several medications available to aid in smoking cessation. However, these medications have the disadvantages of low success rates in smoking cessation and various side effects. Therefore, natural-based smoking cessation aids are being suggested as a good alternative due to their accessibility and minimal side effects. The roots and stems of Acanthopanax koreanum (AK) Nakai, a plant that is native to Jeju Island, South Korea, have traditionally been used as tonic and sedatives. Moreover, eleutheroside B and chlorogenic acid are the main components of AK stem extract. In the present study, we investigated the effect of 70% ethanol AK extract and its components on ameliorating nicotine dependence and withdrawal symptoms by using behavioural tests in mice. In addition, alterations in the dopaminergic and DRD1-EPAC-ERK-CREB pathways were observed using dopamine ELISA and western blotting using mouse brains. Our findings demonstrate that the AK extract and its components effectively mitigated the effects of nicotine treatment in behavioural tests. Furthermore, it normalized the dopamine concentration and the expression level of nicotine acetylcholine receptor α7. Additionally, it was observed that AK extract and its components led to the normalization of DRD1, ERK and CREB expression levels. These results indicate that AK extract exhibits effects in ameliorating nicotine dependence behaviour and alleviating withdrawal symptoms. Moreover, EB and CGA are considered potential marker components of AK extract.

Safety and efficacy of a feed additive consisting of a tincture derived from the roots of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. (taiga root tincture) for use in dogs, cats and horses (FEFANA asbl).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of a tincture from the roots of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. (taiga root tincture) when used as a sensory additive in feed for dogs, cats and horses. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive is safe for dogs, cats and horses at the maximum proposed use level of 460.7, 489.5 and 140.7 mg/kg complete feed, respectively. The additive was considered safe for consumers when used at the proposed conditions of use in horses for meat production. The additive under assessment should be considered as irritant to skin and eyes, and as a skin and respiratory sensitiser. The use of the taiga root tincture as a flavour in feed for horses was not expected to pose a risk for the environment. Since the root of E. senticosus has flavouring properties and its function in feed would be essentially the same as that in food, no further demonstration of efficacy is considered necessary for the tincture under assessment.

[Eleutheroside B induces apoptosis and autophagy of lung cancer cells by regulating PI3K/Akt/mTOR pathway].

This study investigated the effect of eleutheroside B on apoptosis and autophagy of lung cancer A549 and H460 cells and its molecular mechanism. MTT assay was used to detect the cytotoxicity of eleutheroside B at 5, 10, 15, 20, 25, 30, 35, 40, and 45 mmol·L~(-1) on lung cancer cells. Trypan blue exclusion assay was used to detect the effect of eleutheroside B on the survival rate of lung cancer A549 and H460 cells at different time. Colony formation assay was used to detect the effect of eleutheroside B on the proliferation of lung cancer A549 and H460 cells. AO/EB fluorescence double staining and Hoechst 33342 fluorescence staining were used to detect the effect of eleutheroside B on apoptosis of lung cancer A549 and H460 cells, and Western blot was used to detect apoptosis-related proteins to explore the apoptosis-related molecular mechanism. AO fluorescence staining and Western blot were used to detect the expression of autophagic vesicles and autophagy-related proteins P62 and LC3. The results showed that compared with the control group, eleutheroside B inhibited the growth of lung cancer A549 and H460 cells in a concentration-dependent manner. The optimal effect time of eleutheroside B on lung cancer A549 and H460 cells was 24 h, and the optimal concentrations were 28.64 and 22.16 mmol·L~(-1), respectively. Eleutheroside B could inhibit the colony formation of A549 and H460 cells. Compared with the control group, eleutheroside B could promote the formation of apoptotic bodies and induce cell apoptosis, as well as induce the expression of mitochondrial pathway-related proteins. Under the effect of eleutheroside B, the acidic autophagy vacuole in lung cancer cells increased, LC3Ⅱ expression increased, P62 protein expression decreased, and PI3K, p-Akt, and p-mTOR protein expression decreased in the PI3K/Akt/mTOR pathway. Studies have shown that eleutheroside B can inhibit the growth of lung cancer cells, reduce colony formation, induce apoptosis of lung cancer cells through mitochondrial pathway, and induce autophagy. The mechanism may be related to the PI3K/Akt/mTOR pathway.

Eleutheroside B induces apoptosis and autophagy of lung cancer cells by regulating PI3K/Akt/mTOR pathway / 中国中药杂志

This study investigated the effect of eleutheroside B on apoptosis and autophagy of lung cancer A549 and H460 cells and its molecular mechanism. MTT assay was used to detect the cytotoxicity of eleutheroside B at 5, 10, 15, 20, 25, 30, 35, 40, and 45 mmol·L~(-1) on lung cancer cells. Trypan blue exclusion assay was used to detect the effect of eleutheroside B on the survival rate of lung cancer A549 and H460 cells at different time. Colony formation assay was used to detect the effect of eleutheroside B on the proliferation of lung cancer A549 and H460 cells. AO/EB fluorescence double staining and Hoechst 33342 fluorescence staining were used to detect the effect of eleutheroside B on apoptosis of lung cancer A549 and H460 cells, and Western blot was used to detect apoptosis-related proteins to explore the apoptosis-related molecular mechanism. AO fluorescence staining and Western blot were used to detect the expression of autophagic vesicles and autophagy-related proteins P62 and LC3. The results showed that compared with the control group, eleutheroside B inhibited the growth of lung cancer A549 and H460 cells in a concentration-dependent manner. The optimal effect time of eleutheroside B on lung cancer A549 and H460 cells was 24 h, and the optimal concentrations were 28.64 and 22.16 mmol·L~(-1), respectively. Eleutheroside B could inhibit the colony formation of A549 and H460 cells. Compared with the control group, eleutheroside B could promote the formation of apoptotic bodies and induce cell apoptosis, as well as induce the expression of mitochondrial pathway-related proteins. Under the effect of eleutheroside B, the acidic autophagy vacuole in lung cancer cells increased, LC3Ⅱ expression increased, P62 protein expression decreased, and PI3K, p-Akt, and p-mTOR protein expression decreased in the PI3K/Akt/mTOR pathway. Studies have shown that eleutheroside B can inhibit the growth of lung cancer cells, reduce colony formation, induce apoptosis of lung cancer cells through mitochondrial pathway, and induce autophagy. The mechanism may be related to the PI3K/Akt/mTOR pathway.

Eleutheroside B ameliorated high altitude pulmonary edema by attenuating ferroptosis and necroptosis through Nrf2-antioxidant response signaling.

High altitude pulmonary edema (HAPE) is a potentially fatal condition induced by exposure to high-altitude environment. Eleutheroside B is a naturally active polyphenolic substance that has previously demonstrated anti-inflammatory, antioxidant and antidepressant properties. However, the effects of eleutheroside B on HPAE are unknown. Here, eleutheroside B (50 mg/kg and 100 mg/kg) was applied to HAPE rats. Eleutheroside B alleviated lung edema and decreased levels of tumor necrosis factor-α, interleukin-1ß, vascular endothelial growth factor, and total proteins in the bronchoalveolar lavage fluid. Eleutheroside B reversed the acid-base disturbances by HAPE. In addition, eleutheroside B reversed the oxidative stress. Eleutheroside B pretreatment facilitated the translocation of nuclear factor E2-related factor 2 (Nrf2) into the nucleus, contributing to the inhibition of ferroptosis and necroptosis. ML385 confirmed the role of Nrf2 in ferroptosis and necroptosis. Collectively, the beneficial effects of eleutheroside B against HAPE were associated with the inhibition of ferroptosis and necroptosis through Nrf2-antioxidant response signaling.

Metabolome and transcriptome analysis of eleutheroside B biosynthesis pathway in Eleutherococcus senticosus.

Eleutheroside B (syringin) is a medicinal active ingredient extracted from Eleutherococcus senticosus (Ruper. et Maxim.) Maxim with high clinical application value. However, its synthesis pathway remains unknown. Here, we analyzed the eleutheroside B biosynthesis pathway in E. senticosus. Consequently, metabolomic and transcriptomic analyses identified 461 differentially expressed genes (DEGs) and 425 metabolites. Further, we identified 7 DEGs and 67 metabolites involved in the eleutheroside B biosynthetic pathway in the eleutheroside B high and low plants. The correlation between the gene and metabolites was explored using the pearson correlation coefficient (PCC) analysis. Caffeoyl-CoA O-methyltransferase, caffeic acid-O-methyltransferase, ß-amyrin synthase (ß-AS) genes, NAC5, and HB5 transcription factors were identified as candidate genes and transcription factors related to the eleutheroside B synthesis. Eleutheroside B content was the highest at the young stage of the leaves both in the high and low eleutheroside B plants. Quantitative real-time polymerase chain reaction revealed that phenylalanine ammonia-lyase1, cinnamate 4-hydroxylase, ß-AS, and leucoanthocyanidin reductase gene had higher expression levels at the young stage of the leaves in the low eleutheroside B plants but lower expression levels in the high eleutheroside B plants. In the present study, we complemented the eleutheroside B biosynthetic pathway by analyzing the expression levels of relevant genes and metabolite accumulation patterns.

Tea saponin additive to extract eleutheroside B and E from Eleutherococcus senticosus by ultrasonic mediation and its application in a semi-pilot scale.

The safety of ethanol in operations and its effects on human health are gradually being questioned. Under this premise, we attempted to use the natural surfactant tea saponin, which originates from the processing residues of camellia oil, as the additive of the extraction solvent and to extract eleutheroside B and eleutheroside E in the roots and rhizomes of E. senticosus by ultrasonic mediation. After a single-factor experiment, extraction kinetics at different powers and reaction temperatures, and Box-Behnken design optimization, the optimal conditions obtained were 0.3% tea saponin solution as the extraction solvent, 20 mL/g liquid-solid ratio, 250 W ultrasonic irradiation power (43.4 mW/g ultrasonic power density) and 40 min ultrasonic irradiation time. Under optimal conditions, satisfactory yields of eleutheroside B (1.06 ± 0.04 mg/g) and eleutheroside E (2.65 ± 0.12 mg/g) were obtained with semi pilot scale ultrasonic extraction equipment. The experiments showed that compared with the traditional thermal extraction process, the extraction time is significantly reduced at lower operating temperatures.

Mitochondrial dysfunction is involved in the cellular activity inhibition by eleutheroside B in SMMC-7721 and HeLa cells.

Eleutheroside B, also known as syringin, has been shown to have various pharmacological activities such as anti-inflammatory, anti-irradiation and antidepressant, but there are few studies on its anti-cancer activity. Its anti-tumor effect on SMMC-7721 cells has not been revealed. Moreover, whether it induces autophagy is still unclear. Thus, the present study investigated whether Eleutheroside B induces apoptosis, autophagy and cellular motility in SMMC-7721 cells and HeLa cells, and explored the underlying molecular mechanisms. SMMC-7721 cells and HeLa cells treated with Eleutheroside B and cell viability measured by MTT assay and trypan blue dye exclusion assay. Apoptosis checked by flow cytometry combined, fluorescent staining. Apoptotic signal proteins and autophagy proteins were checked by Western blot. This study showed that Eleutheroside B inhibited the cell proliferation and blocked cell cycle, migration and invasion as well. Moreover, Eleutheroside B induced apoptosis in SMMC-7721 cells and HeLa cells. It upregulated Bax expression, while simultaneously decreasing Bcl-2 expression. Further elucidation of the mechanism revealed that Eleutheroside B induced mitochondrial dysfunction, with mitochondrial membrane potential collapse and cytochrome c release, suggesting that Eleutheroside B induced apoptosis by triggering mitochondrial pathway. Most importantly, Eleutheroside B could induce autophagy in SMMC-7721 cells and HeLa cells. Taken together, these results suggested Eleutheroside B is a potential therapeutic candidate for HCC and Human cervical cancer.

Eleutheroside B-loaded poly (lactic-co-glycolic acid) nanoparticles protect against renal fibrosis via Smad3-dependent mechanism.

Although renal fibrosis is a common complication of chronic kidney disease (CKD), effective options for its treatment are currently limited. In this study, we evaluated the renal protective effect and possible mechanism of eleutheroside B. In order to solve the allergic reactions, side effects, and low oral bioavailability of eleutheroside B, we successfully prepared PLGA (poly [lactic-co-glycolic acid])-eleutheroside B nanoparticles (NPs) with the diameter of about 128 nm. In vitro and in vivo results showed that eleutheroside B could inhibit expression levels of α-smooth muscle actin (α-SMA) and collagen I. Molecular docking results showed that eleutheroside B bound to Smad3 and significantly decreased the expression of phospho-Smad3 (p-Smad3). Silencing Smad3 reversed the fibrotic protective effect of eleutheroside B in HK2 cells. Furthermore, small animal imaging showed that NPs can selectively accumulate in the UUO kidneys of mice, and retention time reached as long as 7 days. In conclusion, our results suggested that eleutheroside B is a potential drug to protect renal fibrosis and PLGA-eleutheroside B NPs could facilitate specific targeted therapy for renal fibrosis.

Combination of Geniposide and Eleutheroside B Exerts Antidepressant-like Effect on Lipopolysaccharide-Induced Depression Mice Model.

OBJECTIVE: To study the antidepressant-like effect and action mechanism of geniposide and eleutheroside B combination treatment on the lipopolysaccharide (LPS)-induced depression mice model. METHODS: Depression mice model was established by lipopolysaccharide (LPS) injection. Totally 48 mice were randomly divided into 6 groups (8 rats per group) according to a random number table, including normal, model, fluoxetine (20 mg/kg), geniposide (100 mg/kg) + eleutheroside B (100 mg/kg), geniposide + eleutheroside B + WAY 100635 (0.03 mg/kg), geniposide + eleutheroside B+ N-methyl-D-aspartic acid receptor (NMDA, 75 mg/kg) groups, respectively. After continuous administration for 10 days, autonomic activity tests after 30 min of administration were performed on the 10th day. On the 11th day, except for the normal group, the mice in the other groups were intraperitoneally injected with LPS (1 mg/kg), and the behavioral tests were performed 4 h later. Enzyme linked immunosorbent assay was used to detect tumor necrosis factor alpha (TNF- α) and interleukin-1 ß (IL-1 ß) levels in mice serum. The mRNA expression of indoleamine 2,3-dioxygenase (IDO) and nuclear transcription factor (NF- κB) were detected by real-time quantitative polymerase chain reaction. Western-blot analysis was used to detect IDO and NF- κB protein expressions in hippocampus tissue. RESULTS: Compared with the normal group, a single administration of LPS increased the immobility time in the forced swimming test (FST) and tail suspension test (TST, P<0.01), without affecting autonomous activity. Compared with the model group, fluoxetine and geniposide + eleutheroside B administration significantly improved the immobility time of depressed mice in the FST and TST, decreased serum IL-1 ß content, inhibited the expression levels of NF- κ B gene and protein in hippocampus tissues (P<0.05 or P<0.01). Compared with the model group, geniposide + eleutheroside B treatment significantly reduced serum TNF-α content and inhibited IDO mRNA and protein expressions in hippocampus (P<0.05 or P<0.01). In addition, NMDA partly prevented the inhibition of IDO mRNA expression by geniposide + eleutheroside B; NMDA and WAY-100635 also partly prevented the reduction of IL-1 ß content induced by geniposide + eleutheroside B treatment (P<0.05 or P<0.01). CONCLUSIONS: The combination of geniposide and eleutheroside B showed a certain antidepression-like effect. Its main mechanism of action may be contributed to inhibiting the activation of NF- κB, decreasing the proinflammatory cytokines such as TNF-α, IL-1 ß, and inhibiting in the neuroinflammatory reaction. Additionally, it also affects tryptophan metabolism, reduces the expression of a key enzyme of tryptophan metabolism, IDO. And this antidepressant-like effect may be mediated by 5-hydroxytryptamine and glutamate systems.

Eleutheroside B, a selective late sodium current inhibitor, suppresses atrial fibrillation induced by sea anemone toxin II in rabbit hearts.

Eleutheroside B (EB) is the main active constituent derived from the Chinese herb Acanthopanax senticosus (AS) that has been reported to possess cardioprotective effects. In this study we investigated the effects of EB on cardiac electrophysiology and its suppression on atrial fibrillation (AF). Whole-cell recording was conducted in isolated rabbit atrial myocytes. The intracellular calcium ([Ca2+]i) concentration was measured using calcium indicator Fura-2/AM fluorescence. Monophasic action potential (MAP) and electrocardiogram (ECG) synchronous recordings were conducted in Langendorff-perfused rabbit hearts using ECG signal sampling and analysis system. We showed that EB dose-dependently inhibited late sodium current (INaL), transient sodium current (INaT), and sea anemone toxin II (ATX II)-increased INaL with IC50 values of 167, 1582, and 181 µM, respectively. On the other hand, EB (800 µM) did not affect L-type calcium current (ICaL), inward rectifier potassium channel current (IK), and action potential duration (APD). Furthermore, EB (300 µM) markedly decreased ATX II-prolonged the APD at 90% repolarization (APD90) and eliminated ATX II-induced early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), and triggered activities (TAs). Moreover, EB (200 µM) significantly suppressed ATX II-induced Na+-dependent [Ca2+]i overload in atrial myocytes. In the Langendorff-perfused rabbit hearts, application of EB (200 µM) or TTX (2 µM) substantially decreased ATX II-induced incidences of atrial fibrillation (AF), ventricular fibrillation (VF), and heart death. These results suggest that augmented INaL alone is sufficient to induce AF, and EB exerts anti-AF actions mainly via blocking INaL, which put forward the basis of pharmacology for new clinical application of EB.

A comprehensive in silico exploration of pharmacological properties, bioactivities and COX-2 inhibitory potential of eleutheroside B from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim.

Eleutherococcus senticosus (Rupr. & Maxim.) Maxim., popularly known as 'Siberian ginseng', is an important medicinal plant. Pharmacologically active compounds of this plant are called eleutherosides and among them, eleutheroside B is the most prevalent. The E. senticosus has been reported to have many medicinal properties however; very few studies are reported to understand the medicinal properties of eleutheroside B. Consequently, in the present study various computational tools have been used to predict the drug-likeness, bioactivities, and pharmacokinetic properties of eleutheroside B. Besides, the inhibitory potential of eleutheroside B has been investigated against cyclooxygenase 2 (COX-2) enzyme. This study suggests that eleutheroside B is a drug-like compound with bioactivity score (-0.08 to 0.38), having satisfactory pharmacokinetic values. Metabolism and toxicities were further studied using FAME3, GLORY, pred-hERG and Endocrine Disruptome tools. No severe toxicities (Ames, hepatotoxicity, cardiotoxicity, skin sensitization) were predicted. Rat acute toxicity, ecotoxicity and cell line cytotoxicity were evaluated based on GUSAR and CLC-pred. The compound has been predicted as non-toxic (class 5), non-hERG inhibitor and less likely to cause adverse drug interactions. Molecular docking against COX-2 enzyme revealed strong hydrogen bonds (SER530, TYR355, LEU352, SER353, VAL349, TYR385, MET522) and hydrophobic interaction (LEU352) with eleutheroside B. The docking score (-6.97 kcal/mol) suggested that this molecule can be utilized as an anti-inflammatory agent as well as a potential anticancer drug in the future. Hence, this is a comprehensive integrated in silico approach to establish the anti-inflammatory mechanism of eleutheroside B in the background of its potential in future drug development.Communicated by Ramaswamy H. Sarma.

Combination of Geniposide and Eleutheroside B Exerts Antidepressant-like Effect on Lipopolysaccharide-Induced Depression Mice Model / 中国结合医学杂志

OBJECTIVE@#To study the antidepressant-like effect and action mechanism of geniposide and eleutheroside B combination treatment on the lipopolysaccharide (LPS)-induced depression mice model.@*METHODS@#Depression mice model was established by lipopolysaccharide (LPS) injection. Totally 48 mice were randomly divided into 6 groups (8 rats per group) according to a random number table, including normal, model, fluoxetine (20 mg/kg), geniposide (100 mg/kg) + eleutheroside B (100 mg/kg), geniposide + eleutheroside B + WAY 100635 (0.03 mg/kg), geniposide + eleutheroside B+ N-methyl-D-aspartic acid receptor (NMDA, 75 mg/kg) groups, respectively. After continuous administration for 10 days, autonomic activity tests after 30 min of administration were performed on the 10th day. On the 11th day, except for the normal group, the mice in the other groups were intraperitoneally injected with LPS (1 mg/kg), and the behavioral tests were performed 4 h later. Enzyme linked immunosorbent assay was used to detect tumor necrosis factor alpha (TNF- α) and interleukin-1 β (IL-1 β) levels in mice serum. The mRNA expression of indoleamine 2,3-dioxygenase (IDO) and nuclear transcription factor (NF- κB) were detected by real-time quantitative polymerase chain reaction. Western-blot analysis was used to detect IDO and NF- κB protein expressions in hippocampus tissue.@*RESULTS@#Compared with the normal group, a single administration of LPS increased the immobility time in the forced swimming test (FST) and tail suspension test (TST, P<0.01), without affecting autonomous activity. Compared with the model group, fluoxetine and geniposide + eleutheroside B administration significantly improved the immobility time of depressed mice in the FST and TST, decreased serum IL-1 β content, inhibited the expression levels of NF- κ B gene and protein in hippocampus tissues (P<0.05 or P<0.01). Compared with the model group, geniposide + eleutheroside B treatment significantly reduced serum TNF-α content and inhibited IDO mRNA and protein expressions in hippocampus (P<0.05 or P<0.01). In addition, NMDA partly prevented the inhibition of IDO mRNA expression by geniposide + eleutheroside B; NMDA and WAY-100635 also partly prevented the reduction of IL-1 ß content induced by geniposide + eleutheroside B treatment (P<0.05 or P<0.01).@*CONCLUSIONS@#The combination of geniposide and eleutheroside B showed a certain antidepression-like effect. Its main mechanism of action may be contributed to inhibiting the activation of NF- κB, decreasing the proinflammatory cytokines such as TNF-α, IL-1 β, and inhibiting in the neuroinflammatory reaction. Additionally, it also affects tryptophan metabolism, reduces the expression of a key enzyme of tryptophan metabolism, IDO. And this antidepressant-like effect may be mediated by 5-hydroxytryptamine and glutamate systems.

Eleutheroside B Protects against Acute Kidney Injury by Activating IGF Pathway.

Acute kidney injury (AKI) is a common, complex, and severe clinical syndrome characterized by rapid decline in renal function, combined with tissue damage. Currently, the prevention and treatment of AKI are focused on symptomatic treatment, rather than treating the underlying causes. Therefore, there is no specific treatment to prevent renal injury except for renal dialysis. In this study, we used cisplatin-induced AKI mouse and human kidney-2 (HK-2) cell models to evaluate the renal protective effect of eleutheroside B, an active compound in traditional Chinese medicines. MTT assay was used to detect the effect of eleutheroside B on proliferation of human HK-2 cells in presence and in absence of cisplatin. Western blot and immunostaining were used to detect the protein level of kidney injury molecule-1 (KIM-1), cleaved caspase-3, receptor-interacting protein kinase (RIPK)-1, and RIPK-3. Real-time PCR was used to detect the mRNA levels of chemokines (like monocyte chemotactic protein 1, MCP-1) and pro-inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor (TNF-α). Flow cytometry assay was used to detect apoptosis of HK-2 cells. In vivo results showed that eleutheroside B reduced the increase in serum creatinine and blood urea nitrogen (BUN) levels in the AKI model. Periodic acid-Schiff staining and Western blot analysis of KIM-1 showed that eleutheroside B alleviated tubular cell injury. Further, eleutheroside B reduced macrophage infiltration and production of inflammatory cytokines, inhibited the activation of nuclear factor (NF)-κB, and inhibited apoptosis and programmed necrosis. The mechanism may be that eleutheroside B can activate the insulin-like growth factor (IGF) pathway and its downstream pathway by downregulating the expression of IGFBP-7, thus promoting cell proliferation. Therefore, our results suggest that eleutheroside B is a potential drug for AKI treatment.

Eleutheroside B increase tight junction proteins and anti-inflammatory cytokines expression in intestinal porcine jejunum epithelial cells (IPEC-J2).

Eleutheroside B (EB) is a phenylpropanoid glycoside with anti-inflammatory properties, neuroprotective abilities, immunomodulatory effects, antinociceptive effects, and regulation of blood glucose. The aim of this study was to investigate the effects of EB on the barrier function in the intestinal porcine epithelial cells J2 (IPEC-J2). The IPEC-J2 cells were inoculated into 96-well plates at a density of 5 × 103 cells per well for 100% confluence. The cells were cultured in the presence of EB at concentrations of 0, 0.05, 0.10, and 0.20 mg/ml for 48 hr. Then, 0.10 mg/ml was selected as the suitable concentration for the estimation of transepithelial electric resistance (TEER) value, alkaline phosphatase activity, proinflammatory cytokines mRNA expression, tight junction mRNA and protein expression. The results of this study indicated that the supplementation of EB in IPEC-J2 cells decreased cellular membrane permeability and mRNA expression of proinflammatory cytokines, including interleukin-6 (IL-6), interferon-γ (INF-γ), and tumour necrosis factor-α (TNF-α). The supplementation of EB in IPEC-J2 cells increased tight junction protein expression and anti-inflammatory cytokines, interleukin 10 (IL-10) and transforming growth factor beta (TGF-ß). In addition, the western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) results indicated that EB significantly (p < 0.05) increased the mRNA and protein expression of intestinal tight junction proteins, Claudin-3, Occludin, and Zonula Occludins protein-1 (ZO-1). Therefore, dietary supplementation of EB may increase intestinal barrier function, tight junction protein expression, anti-inflammatory cytokines, and decrease proinflammatory cytokines synthesis in IPEC-J2 cells.

Inhibition viral RNP and anti-inflammatory activity of coumarins against influenza virus.

Influenza viruses pose a severe threat to human health and a significant increase in antiviral drug-resistant among influenza viruses worldwide has been observed. Therefore, there is an urgent need to develop the new antiviral drugs, specifically from the natural products. In this study, the anti-viral and anti-inflammatory activities of coumarins against influenza A virus in vitro were investigated. One of the derivatives eleutheroside B1 showed a wide spectrum of anti- human influenza virus effect with the IC50 value of 64-125µg/ml in vitro, but it showed no effects against avian influenza virus. The time of addition was done and the results indicated that it had a potent antiviral effect when added at 0-6h, and also the virus yield was reduced by 60%. The influenza virus ribonucleoprotein was inhibited at 200µg/ml, and also the NP mRNA expression was inhibited at 50 and 200µg/ml. The expression level of cytokines and chemokines influenced by eleutheroside B1 was further demonstrated, the IL-6, CXCL-8, CCL-2 expression were all inhibited by the eleuthe roside B1 at concentration 200µg/ml. The findings of study suggest that eleutheroside B1 can be as potential agent to develop for the prevention and treatment of influenza A virus.

Effect of eleutheroside B/E on proliferation and expression of PPARγand TGF-β1 in HBZY-1 cells cultured with high glucose / 中国病理生理杂志

[ ABSTRACT] AIM:To explore the effects and mechanism of eleutheroside ( ETS) B or E on the proliferation of HBZY-1 cells treated with high glucose.METHODS:The HBZY-1 cells were cultured under high glucose condition.The 4th generation of HBZY-1 cells was used for determining the optimal cell density, which was consistent with the growth reg-ulation curve of the cells.The cells were divided into 6 groups:low glucose ( LG) group, high glucose ( HG) group, high glucose plus ETS-B/E ( low dose, medium dose and high dose) groups, and high glucose plus losartan ( LTG) group.Af-ter all cells were treated with the corresponding drugs at 24 h, 48 h and 72 h, the inhibitory rate of the proliferation was measured, and the expression of TGF-β1 and PPARγwas detected by immunocytochemistry and Western blotting.RE-SULTS:The best cell density was 2 000 cells/well, which was complied with the basic rules of the cell growth, and high glucose significantly promoted the HBZY-1 cell proliferation.At each time point, the inhibitory effects of ETS-B/E were significantly different between HG group and LTG group on the proliferation of the HBZY-1 cells ( P<0.05) .The expres-sion of TGF-β1 was significantly inhibited, and the expression of PPARγwas significantly promoted by ETS-B/E ( P<0.05).ETS-E showed stronger effect than ETS-B (P<0.05) in a concentration-and time-dependent manner.CONCLU-SION:ETS-B/E significantly inhibits the proliferation of HBZY-1 cells under high glucose condition by decreasing TGF-β1 expression and promoting PPARγexpression.

Development of sample preparation method for eleutheroside B and E analysis in Acanthopanax senticosus by ionic liquids-ultrasound based extraction and high-performance liquid chromatography detection.

An ionic liquids-based ultrasonic-assisted extraction (ILUAE) method was successfully developed for extracting eleutheroside B and E from Radix Acanthopanax senticosus. Thirteen 1-alkyl-3-methylimidazolium ionic liquids with different cations and anions were investigated and 1-butyl-3-methylimidazolium bromide ([C4mim]Br) solution was selected as the solvent. The conditions for ILUAE, including the ionic liquid concentration, soaking time, ultrasonic power, ultrasonic time, solid-liquid ratio and number of extraction cycles, were optimized. With the proposed method, the energy consumption time was reduced to 30 min, whereas conventional method requires about 4h. The proposed method had good recovery (97.96-103.39%) and reproducibility (RSD, n=5; 3.3% for eleutheroside B, 4.6% for eleutheroside E). ILUAE was an efficient, rapid and simple sample preparation technique that showed high reproducibility and was environmental friendly.

Eleutheroside B or E enhances learning and memory in experimentally aged rats.

Eleutheroside B or E, the main component of Acanthopanax, can relieve fatigue, enhance memory, and improve human cognition. Numerous studies have confirmed that high doses of acetylcholine significantly attenuate clinical symptoms and delay the progression of Alzheimer's disease. The present study replicated a rat model of aging induced by injecting quinolinic acid into the hippocampal CA1 region. These rats were intraperitoneally injected with low, medium and high doses of eleutheroside B or E (50, 100, 200 mg/kg), and rats injected with Huperzine A or PBS were used as controls. At 4 weeks after administration, behavioral tests showed that the escape latencies and errors in searching for the platform in a Morris water maze were dose-dependently reduced in rats treated with medium and high-dose eleutheroside B or E. Hematoxylin-eosin staining showed that the number of surviving hippocampal neurons was greater and pathological injury was milder in three eleutheroside B or E groups compared with model group. Hippocampal homogenates showed enhanced cholinesterase activity, and dose-dependent increases in acetylcholine content and decreases in choline content following eleutheroside B or E treatment, similar to those seen in the Huperzine A group. These findings indicate that eleutheroside B or E improves learning and memory in aged rats. These effects of eleutheroside B or E may be mediated by activation of cholinesterase or enhanced reuse of choline to accelerate the synthesis of acetylcholine in hippocampal neurons.