Saikosaponins regulate bile acid excretion in mice liver and ileum by activating farnesoid X receptor and bile acid transporter.

Radix Bupleuri exerts effective hepatoprotective and cholagogic effects through its Saikosaponins (SSs) component. Therefore, we attempted to determine the mechanism of saikosaponins used to promote bile excretion by studying their effects on intrahepatic bile flow, focusing on the synthesis, transport, excretion, and metabolism of bile acids. C57BL/6N mice were continuously gavaged with saikosaponin a (SSa), saikosaponin b2 (SSb2 ), or saikosaponin D (SSd) (200 mg/kg) for 14 days. Liver and serum biochemical indices were determined using Enzyme-linked immunosorbent assay (ELISA) kits. In addition, an ultra-performance liquid chromatography-mass spectrometer (UPLC-MS) was used to measure the levels of the 16 bile acids in the liver, gallbladder, and cecal contents. Furthermore, SSs pharmacokinetics and docking between SSs and farnesoid X receptor (FXR)-related proteins were analyzed to investigate the underlying molecular mechanisms. Administration of SSs and Radix Bupleuri alcohol extract (ESS) did not cause significant changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), or alkaline phosphatase (ALP) levels. Saikosaponin-regulated changes in bile acid (BA) levels in the liver, gallbladder, and cecum were closely related to genes involved in BA synthesis, transport, and excretion in the liver. Pharmacokinetic studies indicated that SSs were characterized by rapid elimination (t1/2 as 0.68-2.47 h), absorption (Tmax as 0.47-0.78 h), and double peaks in the drug-time curves of SSa and SSb2 . A molecular docking study revealed that SSa, SSb2 , and SSd docked well with the 16 protein FXR molecules and target genes (<-5.2 kcal/mol). Collectively, saikosaponins may maintain BA homeostasis in mice by regulating FXR-related genes and transporters in the liver and intestine.

Bupleurum chinense exerts a mild antipyretic effect on LPS-induced pyrexia rats involving inhibition of peripheral TNF-α production.

ETHNOPHARMACOLOGICAL RELEVANCE: Bupleuri Radix, the dried roots of Bupleurum chinense DC. (BC) or Bupleurum scorzonerifolium Willd., is one of the most frequently used traditional Chinese medicines. As the species in Xiao-Chai-Hu decoction, BC has been used as an antipyretic medicine with a long history. However, its antipyretic characteristics and underlying mechanism(s) remain unclear. AIM OF THE STUDY: To elucidate the antipyretic characteristics and mechanism(s) of BC used in its traditional way. METHODS: The water extract of BC (BCE) was prepared according to the traditional decocting mode. Murine fever and endotoxemia models were induced by intravenous injection of lipopolysaccharide (LPS). In vitro complement activation assay and the levels of TNF-α, IL-6, IL-1ß, and C5a were determined by ELISA. RESULTS: BCE exerted a confirmed but mild antipyretic effect on LPS-induced fever of rat. In vitro, it significantly lowered LPS-elevated TNF-α in the supernatant of rat complete blood cells and THP-1 cells, but failed to decrease IL-6 and IL-1ß. In murine endotoxemia models, BCE markedly decreased serum TNF-α, but had no impact on IL-6 and IL-1ß. BCE also restricted complement activation in vitro and in vivo. Nevertheless, the mixture of saikosaponin A and D could not suppress supernatant TNF-α of monocytes and serum TNF-α of endotoxemia mice. CONCLUSIONS: The present study dissects the peripheral mechanism for the antipyretic effect of BC used in the traditional way. Our findings indicate that BCE directly suppresses monocyte-produced TNF-α, thus decreasing circulating TNF-α, which may be responsible for its mild but confirmed antipyretic action.

Saikosaponins ameliorate hyperlipidemia in rats by enhancing hepatic lipid and cholesterol metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Hyperlipidemia is the systemic manifestation of abnormal lipid metabolism, characterized by elevated circulating levels of cholesterol and triglyceride and a high risk of cardiovascular events. Radix Bupleuri (RB) is a traditional Chinese herbal product used to treat liver diseases. Our previous study demonstrated that Saikosaponins (SSs), the most potent bioactive ingredients in RB, ameliorate hepatic steatosis. However, whether SSs have anti-hyperlipidemia effects and plausible underlying mechanisms remain elusive. AIM OF THE STUDY: To comprehensively evaluate the lipid-lowering potential of SSs against hyperlipidemia in rats. MATERIALS AND METHODS: RNA sequencing and untargeted metabolomics approaches were applied to analyze the changes in the liver transcriptome and serum lipid profile in long-term high-fat diet feeding-induced hyperlipidemia rats in response to SSs or positive drug simvastatin (SIM) intervention. RESULTS: Our data revealed that SSs significantly alleviated HFD-induced hypertriglyceridemia and hypercholesterolemia. Combined with the analysis of gene ontology enrichment analysis and gene set enrichment analysis, we found that SSs remarkably repaired the unbalanced blood lipid metabolic spectrum in a dose-dependent manner by increasing the hepatic uptake of circulating fatty acids and facilitating mitochondrial respiration in fatty acid oxidation, comparable to SIM group. In addition, SSs markedly modulated cholesterol clearance by promoting intracellular cholesterol efflux, HDL remodeling, LDL particle clearance, and bile acid synthesis. SSs also efficiently protected the liver from lipid overload-related oxidative stress and lipid peroxidation, as well as substantially exaggerated inflammatory response. CONCLUSION: Our research not only unraveled the intricate mechanisms underlying the lipid-lowering functions of SSs but also provided novel perspectives on developing an SSs-based therapeutic strategy for the treatment of hyperlipidemia. CLASSIFICATION: Metabolism.

Drought Stress Stimulates the Terpenoid Backbone and Triterpenoid Biosynthesis Pathway to Promote the Synthesis of Saikosaponin in Bupleurum chinense DC. Roots.

Bupleurum chinense is an important medicinal plant in China; however, little is known regarding how this plant transcribes and synthesizes saikosaponins under drought stress. Herein, we investigated how drought stress stimulates the transcriptional changes of B. chinense to synthesize saikosaponins. Short-term drought stress induced the accumulation of saikosaponins, especially from the first re-watering stage (RD_1 stage) to the second re-watering stage (RD_2 stage). Saikosaponin-a and saikosaponin-d increased by 84.60% and 75.13%, respectively, from the RD_1 stage to the RD_2 stage. Drought stress also stimulated a rapid increase in the levels of the hormones abscisic acid, salicylic acid, and jasmonic acid. We screened 49 Unigenes regarding the terpenoid backbone and triterpenoid biosynthesis, of which 33 differential genes were significantly up-regulated during drought stress. Moreover, one P450 and two UGTs are possibly involved in the synthesis of saikosaponins, while some transcription factors may be involved in regulating the expression of key enzyme genes. Our study provides a reference for the cultivation of B. chinense and a practical means to ensure the quality (safety and effectiveness) of B. chinense for medicinal use, as well as insights into the modernization of the China Agriculture Research System.

Therapeutic effects of total saikosaponins from Radix bupleuri against Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory loss and cognitive dysfunction in the elderly, with amyloid-beta (Aß) deposition and hyperphosphorylation of tau protein as the main pathological feature. Nuclear factor 2 (Nrf2) is a transcription factor that primarily exists in the cytosol of hippocampal neurons, and it is considered as an important regulator of autophagy, oxidative stress, and inflammation. Total saikosaponins (TS) is the main bioactive component of Radix bupleuri (Chaihu). In this study, it was found that TS could ameliorate cognitive dysfunction in APP/PS1 transgenic mice and reduce Aß generation and senile plaque deposition via activating Nrf2 and downregulating the expression of ß-secretase 1 (BACE1). In addition, TS can enhance autophagy by promoting the expression of Beclin-1 and LC3-II, increasing the degradation of p62 and NDP52 and the clearance of phosphorylated tau (p-tau), and reducing the expression of p-tau. It can also downregulate the expression of nuclear factor-κB (NF-κB) to inhibit the activation of glial cells and reduce the release of inflammatory factors. In vitro experiments using PC12 cells induced by Aß, TS could significantly inhibit the aggregation of Aß and reduce cytotoxicity. It was found that Nrf2 knock-out weakened the inhibitory effect of TS on BACE1 and NF-κB transcription in PC12 cells. Moreover, the inhibitory effect of TS on BACE1 transcription was achieved by promoting the binding of Nrf2 and the promoter of BACE1 ARE1. Results showed that TS downregulated the expression of BACE1 and NF-κB through Nrf2, thereby reducing the generation of Aß and inhibiting neuroinflammation. Furthermore, TS can ameliorate synaptic loss and alleviate oxidative stress. In gut microbiota analysis, dysbiosis was demonstrated in APP/PS1 transgenic mice, indicating a potential link between gut microbiota and AD. Furthermore, TS treatment reverses the gut microbiota disorder in APP/PS1 mice, suggesting a therapeutic strategy by remodeling the gut microbe. Collectively, these data shows that TS may serve as a potential approach for AD treatment. Further investigation is needed to clarify the detailed mechanisms underlying TS regulating gut microbiota and oxidative stress.

Quantitative Analysis and Differential Evaluation of Radix Bupleuri Cultivated in Different Regions Based on HPLC-MS and GC-MS Combined with Multivariate Statistical Analysis.

The quality of Radix Bupleuri is greatly affected by its growing environment. In this study, Radix Bupleuri samples that were harvested from seven different regions across northwest China were examined by high-performance liquid chromatography (HPLC) and gas chromatography (GC) coupled with mass spectrometry (MS) to reveal significant differences in quality contributed by the cultivation region. An HPLC-MS method was firstly established and used in the multiple reaction monitoring mode for the quantitative analysis of five saikosaponins in Radix Bupleuri so as to evaluate the difference in the absolute content of saikosaponins attributable to the cultivation region. The effect on the components of Radix Bupleuri was further investigated based on the profiles of the representative saponins and volatile compounds, which were extracted from the Radix Bupleuri samples and analyzed by HPLC-MS and GC-MS. Multivariate statistical analysis was employed to differentiate the Radix Bupleuri samples cultivated in different regions and to discover the differential compositions. The developed quantitative method was validated to be accurate, stable, sensitive, and repeatable for the determination of five saikosaponins. Further statistical tests revealed that the collected Radix Bupleuri samples were distinctly different from each other in terms of both saponins and volatile compounds, based on the provinces where they were grown. In addition, twenty-eight saponins and fifty-eight volatile compounds were identified as the differentially accumulated compositions that contributed to the discrimination of the Radix Bupleuri samples. The Radix Bupleuri samples grown in Shouyang county showed the highest content of saikosaponins. All of the results indicated that the cultivation region significantly affected the accumulation and diversity of the main chemical components of Radix Bupleuri. The findings of this research provide insights into the effect of the cultivation region on the quality of Radix Bupleuri and the differentiation of Radix Bupleuri cultivated in different regions based on the use of HPLC-MS and GC-MS combined with multivariate statistical analysis.

Saikosaponin D attenuates metabolic associated fatty liver disease by coordinately tuning PPARα and INSIG/SREBP1c pathway.

BACKGROUND: Metabolic associated fatty liver disease (MAFLD) is a progressive chronic liver disease, yet there is still a lack of effective pharmacological therapies at present. Saikosaponin D (SSd) has been reported to exhibit hepatoprotective and anti-steatosis activities in our previous research. PURPOSE: The current study aims to further investigate the underlying mechanisms of SSd on MAFLD from the perspectives of the crosstalk between fatty acid (FA) biosynthesis and catabolism to provide strong support for further clinical management of MAFLD. METHODS: A MAFLD mouse model induced by a high-fat diet and glucose-fructose water (HFSW) was used for in vivo study. HepG2 cells, primary mouse hepatocytes and adipocytes were further employed for in vitro studies. RESULTS: SSd improved intracellular lipid accumulation both in the liver and adipose tissues in HFSW-fed mice. Mechanistically, SSd may serve as a potent PPARα agonist, and the activation of PPARα by SSd in both hepatocytes and adipocytes not only promoted FA oxidation but also concurrently induced INSIG1/2 expression, which subsequently inhibited SREBP1c maturation and ultimately FA synthesis. Moreover, the regulative effect of SSd on lipid metabolism was abolished by the PPARα inhibitor, GW6471. CONCLUSION: This study demonstrated that SSd improved lipid homeostasis by coordinately regulating PPARα activation-mediated both inhibition of SREBP1c-dependent FA biosynthesis and induction of FA degradation, and thus shed novel light on the discovery of SSd-based therapeutic strategies for MAFLD.

Suppressing effect of a saikosaponin-enriched extract of Bupleurum falcatum on alcohol and chocolate self-administration in rats.

Recent studies demonstrated that saikosaponin (SS) A and other SSs extracted from Bupleurum falcatum L. (Apiaceae) roots abolished different behaviours motivated by drugs of abuse and palatable foods in rats. The present study was designed to investigate the effect of an SS-enriched extract fraction of B. falcatum roots on operant, oral self-administration of alcohol and chocolate in rats. To this end, female Sardinian alcohol-preferring and Wistar rats were trained to lever-respond for alcohol (15% v/v) and chocolate (5% w/v powdered Nesquik in water), respectively. Acute treatment with B. falcatum extract (0, 0.75, 1.5, and 3 mg/kg, i.p.) reduced, in a dose-related manner, both alcohol and chocolate self-administration. These data confirm the notion that B. falcatum extracts may be a valuable source of pharmacological agents with anti-addictive and anorectic potential. The use of experimental procedures with predictive validity for the human disease adds strength to the translational potential of these results.

Saikosaponin D: review on the antitumour effects, toxicity and pharmacokinetics.

CONTEXT: Bupleuri Radix, the dried root of Bupleurum chinense DC and Bupleurum scorzonerifolium Willd (Apiaceae), is an important medicinal herb widely used to treat cancers for hundreds of years in Asian countries. As the most antitumour component but also the main toxic component in Bupleuri Radix, saikosaponin D (SSD) has attracted extensive attention. However, no summary studies have been reported on the antitumour effects, toxicity and pharmacokinetics of this potential natural anticancer substance. OBJECTIVE: To analyse and summarise the existing findings regarding to the antitumour effects, toxicity and pharmacokinetics of SSD. MATERIALS AND METHODS: We collected relevant information published before April 2021 by conducting a search of literature available in various online databases including PubMed, Science Direct, CNKI, Wanfang database and the Chinese Biological Medicine Database. Bupleurum, Bupleuri Radix, saikosaponin, saikosaponin D, tumour, toxicity, and pharmacokinetics were used as the keywords. RESULTS: The antitumour effects of SSD were multi-targeted and can be realised through various mechanisms, including inhibition of proliferation, invasion, metastasis and angiogenesis, as well as induction of cell apoptosis, autophagy, and differentiation. The toxicological effects of SSD mainly included hepatotoxicity, neurotoxicity, haemolysis and cardiotoxicity. Pharmacokinetic studies demonstrated that SSD had the potential to alter the pharmacokinetics of some drugs for its influence on CYPs and P-gp, and the oral bioavailability and actual pharmacodynamic substances in vivo of SSD are still controversial. CONCLUSIONS: SSD is a potentially effective and relatively safe natural antitumour substance, but more research is needed, especially in vivo antitumour effects and pharmacokinetics of the compound.

Protective effect of dietary supplementation of Bupleurum falcatum L saikosaponins on ammonia exposure-induced ileum injury in broilers.

Ammonia (NH3) at a high concentration has been recognized as a highly poisonous pollutant affecting both air and water quality. NH3, as a stimulus, exerts negative impact on broiler growth and production, but the molecular mechanisms are not clear yet. This study was designed to evaluate the effects of dietary supplementation of Bupleurum falcatum L saikosaponins (SP) on the growth and ileum health status in broilers exposed to NH3. Day-old Arbor Acers broilers (n = 480) were randomly allocated into 1 of 4 treatments. The main factors were dietary SP supplementation (0 or 80 mg/kg of diet) and NH3 challenge (with or without 70 ± 5 ppm NH3). The data of growth, intestinal morphology, and mRNA expression related to ileal function were collected from broilers exposed to NH3 for 7 d. Results showed that NH3 remarkably suppressed growth performance and intestinal development as well as induced biological injuries in the ileum of broilers, resulting from oxidative stress, mucous barrier damage, and immune dysfunction as well as upregulated apoptosis. These negative effects of NH3 were alleviated by the SP supplement. In conclusion, dietary supplementation of SP may be helpful in alleviating the detrimental effects of NH3 on the ileum development in broilers.

Computational assessment of saikosaponins as adjuvant treatment for COVID-19: molecular docking, dynamics, and network pharmacology analysis.

Saikosaponins are major biologically active triterpenoids, usually as glucosides, isolated from Traditional Chinese Medicines (TCM) such as Bupleurum spp., Heteromorpha spp., and Scrophularia scorodonia with their antiviral and immunomodulatory potential. This investigation presents molecular docking, molecular dynamics simulation, and free energy calculation studies of saikosaponins as adjuvant therapy in the treatment for COVID19. Molecular docking studies for 23 saikosaponins on the crystal structures of the extracellular domains of human lnterleukin-6 receptor (IL6), human Janus Kinase-3 (JAK3), and dehydrogenase domain of Cylindrospermum stagnale NADPH-oxidase 5 (NOX5) were performed, and selected protein-ligand complexes were subjected to 100 ns molecular dynamics simulations. The molecular dynamics trajectories were subjected to free energy calculation by the MM-GBSA method. Molecular docking and molecular dynamics simulation studies revealed that IL6 in complex with Saikosaponin_U and Saikosaponin_V, JAK3 in complex with Saikosaponin_B4 and Saikosaponin_I, and NOX5 in complex with Saikosaponin_BK1 and Saikosaponin_C have good docking and molecular dynamics profiles. However, the Janus Kinase-3 is the best interacting partner for the saikosaponin compounds. The network pharmacology analysis suggests saikosaponins interact with the proteins CAT Gene CAT (Catalase) and Checkpoint kinase 1 (CHEK1); both of these enzymes play a major role in cell homeostasis and DNA damage during infection, suggesting a possible improvement in immune response toward COVID-19.

An in-silico evaluation of different Saikosaponins for their potency against SARS-CoV-2 using NSP15 and fusion spike glycoprotein as targets.

The Public Health Emergency of International Concern declared the widespread outbreak of SARS-CoV-2 as a global pandemic emergency, which has resulted in 1,773,086 confirmed cases including 111,652 human deaths, as on 13 April 2020, as reported to World Health Organization. As of now, there are no vaccines or antiviral drugs declared to be officially useful against the infection. Saikosaponin is a group of oleanane derivatives reported in Chinese medicinal plants and are described for their anti-viral, anti-tumor, anti-inflammatory, anticonvulsant, antinephritis and hepatoprotective activities. They have also been known to have anti-coronaviral property by interfering the early stage of viral replication including absorption and penetration of the virus. Thus, the present study was undertaken to screen and evaluate the potency of different Saikosaponins against different sets of SARS-CoV-2 binding protein via computational molecular docking simulations. Docking was carried out on a Glide module of Schrodinger Maestro 2018-1 MM Share Version on NSP15 (PDB ID: 6W01) and Prefusion 2019-nCoV spike glycoprotein (PDB ID: 6VSB) from SARS-CoV-2. From the binding energy and interaction studies, the Saikosaponins U and V showed the best affinity towards both the proteins suggesting them to be future research molecule as they mark the desire interaction with NSP15, which is responsible for replication of RNA and also with 2019-nCoV spike glycoprotein which manage the connection with ACE2. [Formula: see text] Communicated by Ramaswamy H. Sarma.

Drought stress induces biosynthesis of flavonoids in leaves and saikosaponins in roots of Bupleurum chinense DC.

Drought stress affects vegetative and reproductive growth processes and synthesis of secondary metabolites in plants. We assessed relevant indicators of vegetative and reproductive growth in Bupleurum chinense DC. during drought stress. Samples were collected on days 4, 8, 12, 20, and 24 of a drought treatment according to drought stress severity in order to elucidate potential effects on synthesis of flavonoids in leaves and saikosaponins in roots of B. chinense. The results showed that B. chinense can adapt to drought stress mainly by increasing concentrations of osmoregulatory substances (soluble protein and proline) and increasing activity of protective enzymes (superoxide dismutase and catalase), as observed on days 12 and 20 of the treatment. Secondary metabolite concentrations in B. chinense roots and leaves showed significant differences-drought stress increased saikosaponin concentrations in roots by 9.85% and 6.41% during vegetative and reproductive growth, respectively, on day 20, and saikosaponin concentrations in roots were higher during vegetative growth than during reproductive growth. In leaves, large amounts of antioxidants were consumed owing to drought stress, which decreased leaf rutin concentrations by 38.79% and 30.11% during vegetative and reproductive growth, respectively, as observed on day 20; overall, leaf rutin concentrations were lower during vegetative growth than during reproductive growth. Changes in soil water content are known to affect synthesis of secondary metabolites in medicinal plants by altering gene transcription, and affected genes may synergistically respond to soil water changes and alter concentrations of flavonoid in leaves and of saikosaponin in roots. The gene F3H down-regulates flavonoid production in leaves. Squalene epoxidase and ß-amyrin synthase genes may be key genes regulating saikosaponin accumulation, and changes in their expression corresponded to accumulation of saikosaponins. Our results provide insights in B. chinense adaptation to drought stress through physiological changes and regulation of secondary metabolite production in different plant tissues.

The biotransformation of Bupleuri Radix by human gut microbiota.

1. Bupleuri Radix (BR) is a herbal medicine traditionally used orally in oriental countries, which inevitably comes into contact with the intestinal microbiota. However, whether gut microbiota contribute to the biotransformation of BR, and/or the formation of pharmacologically active compounds remains unknown.2. In this study, the main saikosaponins (SAPs) of Bupleurum (including saikosaponin a, b1, b2, c, d, f, h) and BR extract (BRE) were individually incubated with human fecal suspensions (HFS), and metabolic time courses of SAPs and their metabolites by human gut bacteria were systematically characterized.3. Deglycosylation and dehydration were the main metabolic pathways identified for SAPs including newly investigated saikosaponin f (SSf) and saikosaponin h (SSh); dehydration had not been reported previously. A total of 19 dehydrated and deglycosylated metabolites of SAPs were detected and characterized, and 10 of them were newly identified. Moreover, SAPs of BRE were found to be deglycosylated to prosaikogenins. In addition, 13 metabolic pathways related to human gut microbiota were identified for phytochemicals of BRE except for SAPs. Gut microbiota may play a significant role in the biotransformation of BR in humans.

Effects of drought-re-watering-drought on the photosynthesis physiology and secondary metabolite production of Bupleurum chinense DC.

KEY MESSAGE: Drastic changes in soil water content can activate the short-term high expression of key enzyme-encoding genes involved in secondary metabolite synthesis thereby increasing the content of secondary metabolites. Bupleurum chinense DC. is a traditional medicinal herb that is famous for its abundant saikosaponins. In the current study, the effects of drought-re-watering-drought on the photosynthesis physiology and biosynthesis of saikosaponins were investigated in 1-year-old B. chinense. The results showed that alterations in soil moisture altered the photosynthesis physiological process of B. chinense. The dry weight and fresh weight of the roots, photosynthesis capacity, chlorophyll fluorescence parameters, and SOD, POD and CAT activities were significantly reduced, and the contents of SP, soluble sugars, PRO and MDA increased. There were strong correlations between different physiological stress indices. All indices promoted and restricted each other, responded to soil moisture changes synergistically, maintained plant homeostasis and guaranteed normal biological activities. It was found that RW and RD_1 were the key stages of the water-control experiment affecting the expression of saikosaponin-related genes. At these two stages, the expression of multiple genes was affected by changes in soil moisture, with their expression levels reaching several-fold higher than those at the previous stage. We noticed that the expression of saikosaponin synthesis genes (which were rapidly upregulated at the RW and RD_1 stages) did not coincide with the rapid accumulation of saikosaponins (at the RD-2 stage), which were found to correspond to each other at the later stages of the water-control experiment. This finding indicates that there is a time lag between gene expression and the final product synthesis. Rapid changes in the external environment (RW to RD_1) have a short-term promoting effect on gene expression. This study reveals that short-term stress regulation may be an effective way to improve the quality of medicinal materials.

Modification of GSK3ß/ß-catenin signaling on saikosaponins-d-induced inhibition of neural progenitor cell proliferation and adult neurogenesis.

Saikosaponins-d (SSd) is a major bioactive compound isolated from Radix Bupleuri, an herb widely used in traditional Chinese medicine. Emerging studies demonstrate that SSd adversely affects adult neurogenesis and impairs learning and memory. However, the molecular mechanisms remain to be determined. The current study investigated the potential regulatory of GSK3ß/ß-catenin signaling on SSd-induced neurotoxicity. We demonstrated that SSd exposure inhibited the cell viability and proliferation of primary neuronal stem/progenitor cells (NPCs) from hippocampus in a concentration-dependent manner. Significantly, SSd exposure induced activation of GSK3ß and reduced the cellular ß-catenin in NPCs. Treatment with SB216763, a specific inhibitor for GSK3ß activation, we showed that inactivation GSK3ß improved the ß-catenin signaling by inhibiting degradation complex comprising Axin and APC and attenuated SSd-induced toxicity in NPCs. In addition, administration of SB216763 ameliorated SSd-induced inhibition of NPCs proliferation and enhanced radial glial cells in the hippocampus of mice. Moreover, inactivation GSK3ß promoted dendritic arborization and the survival of newborn neurons together with alleviated the impairment of SSd-induced cognitive function in mice. Overall, these data demonstrated that the significant inhibitory effects of SSd on NPCs proliferation and adult neurogenesis via GSK3ß/ß-catenin signaling pathway. Our results contribute to a better understanding of the molecular mechanisms of SSd-induced neurotoxicity.

Saikosaponin d causes apoptotic death of cultured neocortical neurons by increasing membrane permeability and elevating intracellular Ca2+ concentration.

Saikosaponins (SSs) are a class of naturally occurring oleanane-type triterpenoid saponins found in Radix bupleuri that has been widely used in traditional Chinese medicine. As the main active principals of Radix bupleuri, SSs have been shown to suppress mouse motor activity, impair learning and memory, and decrease hippocampal neurogenesis. In the present study, we investigated the effect of five SSs (SSa, SSb1, SSb2, SSc, and SSd) on neuronal viability and the underlying mechanisms in cultured murine neocortical neurons. We demonstrate that SSa, SSb1 and SSd produce concentration-dependent apoptotic neuronal death and induce robust increase in intracellular Ca2+ concentration ([Ca2+]i) at low micromolar concentrations with a rank order of SSd > SSa > SSb1, whereas SSb2 and SSc have no detectable effect on both neuronal survival and [Ca2+]i. Mechanistically, SSd-induced elevation in [Ca2+]i is the primary result of enhanced extracellular Ca2+ influx, which likely triggers Ca2+-induced Ca2+ release through ryanodine receptor activation, but not SERCA inhibition. SSd-induced Ca2+ entry occurs through a non-selective mechanism since blockers of major neuronal Ca2+ entry pathways, including L-type Ca2+ channel, NMDA receptor, AMPA receptor, Na+-Ca2+ exchanger, and TRPV1, all failed to attenuate the Ca2+ response to SSd. Further studies demonstrate that SSd increases calcein efflux and induces an inward current in neocortical neurons. Together, these data demonstrate that SSd elevates [Ca2+]i due to its ability to increase membrane permeability, likely by forming pores in the surface of membrane, which leads to massive Ca2+ influx and apoptotic neuronal death in neocortical neurons.

Advances in Bioculture Technology of Bupleuri Radix and Biosynthesis Pathways of Saikosaponins / 中国实验方剂学杂志

Synthetic biology is an emerging discipline that analyzes the biosynthesis pathways of active constituents in traditional Chinese medicine and explores genes involved in biosynthesis. Bupleuri Radix is one of the most commonly used Chinese medicinal materials with remarkable medicinal value, its index component is saikosaponins, which has significant anti-inflammatory, anti-viral and anti-tumor activities. However, the current wild resources of Bupleuri Radix have been destroyed, and there were some problems in the process of artificial cultivation. The application of biological culture technology and synthetic biology can expand the sources of saikosaponins and protect resources of Bupleuri Radix. The culture conditions of different plants can be followed without a fixed pattern, and the biosynthetic pathways of different medicinal active ingredients are also inconsistent. At present, there is no review report on the culture technology of Bupleuri Radix and the research on the biosynthesis pathway of saikosaponins. This paper introduces the research progress of biological culture techniques, such as callus culture, adventitious root culture, hairy root culture and suspension cell culture used in synthetic biology, and the biosynthesis pathway of saikosaponins and its key enzyme functional genes. It is suggested to optimize the biological culture technology of Bupleuri Radix by referring to the tissue culture technology of other traditional root medicinal materials, so as to provide a reference for the in-depth study on the biosynthesis pathway and metabolic regulation of saikosaponins.

Saikosaponins: a review of pharmacological effects.

Over the past decades, a number of phytochemicals have been reported to possess potent pharmacological effects. Saikosaponins represent a group of oleanane derivatives, usually as glucosides, which are commonly found in medicinal plants Bupleurum spp., which have been used as traditional Chinese medicine for more than 1,000 years in China. Emerging evidence suggests that saikosaponins have many pharmacological effects, including sedation, anticonvulsant, antipyretic, antiviral, immunity, anti-inflammation, antitumor properties, protecting liver and kidney and so on. The present review provides a comprehensive summary and analysis of the pharmacological properties of saikosaponins, supporting the potential uses of saikosaponins as a medicinal agent.

Anti-angiogenic effects of Qingdu granule on breast cancer through inhibiting NFAT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingdu granule (QDG), a traditional Chinese herbal prescription, had anti-tumor effect on breast cancer. However the underlying mechanism of QDG was unclear. THE AIM OF THIS STUDY: The present study aimed to investigate whether QDG could inhibit angiogenesis of breast cancer via acting on nuclear factor of activated T-cells (NFAT) signaling pathway. This was implicated in human umbilical vein endothelial cells (HUVECs) in vitro and breast cancer xenograft model in vivo. MATERIALS AND METHODS: The VEGF165 (15.58 ng/mL) induced human umbilical vein endothelial cells (HUVECs) were treated with serum samples containing tamoxifen (TAM), tacrolimus (FK506), or QDG with three dosages. The migration and canalization capacities of HUVECs were evaluated by transwell migration and tube formation assay. In 72 h-cultured HUVECs, The gene expression, protein amount, and nuclear translocation of NFATc3 were measured. The anti-tumor and anti-angiogenic effects of QDG in vivo were investigated in breast cancer xenograft model. The serum VEGF levels, microvessel density, and protein expressions (immunohistochemistry and western blot) of VEGF, VEGFR2 and NFATc3 were detected. RESULTS: The results showed that, QDG significantly inhibited HUVEC migration and tube formation. It downregulated NFATc3 gene expression, decreased NFATc3 protein amount, and reduced the ratio of NFATc3 nuclear translocation in HUVECs. In breast cancer xenograft model, QDG treatment significantly suppressed tumor growth, inhibited VEGF release, and decreased microvessel density. QDG reduced protein expressions of VEGF, VEGFR2 and NFATc3. CONCLUSION: The results suggested that QDG showed anti-angiogenic effects of breast cancer both in vitro and in vivo. The mechanism might be partially associated with inhibiting NFAT signaling pathway.