Characterization and Applications of Permeabilized Hepatocytes in Drug Discovery.

Hepatocytes are one of the most physiologically relevant in vitro liver systems for human translation of clearance and drug-drug interactions (DDI). However, the cell membranes of hepatocytes can limit the entry of certain compounds into the cells for metabolism and DDI. Passive permeability through hepatocytes can be different in vitro and in vivo, which complicates the human translation. Permeabilized hepatocytes offer a useful tool to probe mechanistic understanding of permeability-limited metabolism and DDI. Incubation with saponin of 0.01% at 0.5 million cells/mL and 0.05% at 5 million cells/mL for 5 min at 37°C completely permeabilized the plasma membrane of hepatocytes, while leaving the membranes of subcellular organelles intact. Permeabilized hepatocytes maintained similar enzymatic activity as intact unpermeabilized hepatocytes and can be stored at -80°C for at least 7 months. This approach reduces costs by preserving leftover hepatocytes. The relatively low levels of saponin in permeabilized hepatocytes had no significant impact on the enzymatic activity. As the cytosolic contents leak out from permeabilized hepatocytes, cofactors need to be added to enable metabolic reactions. Cytosolic enzymes will no longer be present if the media are removed after cells are permeabilized. Hence permeabilized hepatocytes with and without media removal may potentially enable reaction phenotyping of cytosolic enzymes. Although permeabilized hepatocytes work similarly as human liver microsomes and S9 fractions experimentally requiring addition of cofactors, they behave more like hepatocytes maintaining enzymatic activities for over 4 h. Permeabilized hepatocytes are a great addition to the drug metabolism toolbox to provide mechanistic insights.

[Total saponins of Panax japonicus alleviates CCl4-induced acute liver injury in rats by regulating the PI3K/AktNF-κB signaling pathway].

OBJECTIVE: To investigate the protective effect of total saponins of Panax japonicus (TSPJ) against CCl4-induced acute liver injury (ALI) in rats and explore the underlying pharmacological mechanisms. METHODS: Male SD rat models of CCl4-induced ALI were given intraperitoneal injections of distilled water, 100 mg/kg biphenyl bisabololol, or 50, 100, and 200 mg/kg TSPJ during modeling (n=8). Liver functions (AST, ALT, TBil and ALP) of the rats were assessed and liver pathologies were observed with HE staining. Immunohistochemistry was used to detect the expressions of PI3K/Akt/NF-κB signaling pathway molecules in liver tissue; ELISA was used to determine the levels of T-SOD, GSH-Px, and MDA. Western blotting was performed to detect the expression levels of PI3K-Akt and SIRT6-NF-κB pathways in the liver tissue. RESULTS: Network pharmacological analysis indicated that the key pathways including PI3K/Akt mediated the therapeutic effect of TSPJ on ALI. In the rat models of ALI, treatments with biphenyl bisabololol and TSPJ significantly ameliorated CCl4-induced increase of serum levels AST, ALT, ALP, TBil and MDA and decrease of T-SOD and GSH-Px levels (all P < 0.01). The rat models of ALI showed significantly increased expression of p-NF-κB (P < 0.01), decreased expressions of PI3K, p-Akt and SIRT6 proteins, and elevated expression levels of p-NF-κB, TNF-α and IL-6 proteins in the liver, which were all significantly improved in the treatment groups (P < 0.05 or 0.01). CONCLUSION: TSPJ can effectively alleviate CCl4-induced ALI in rats by suppressing inflammatory responses and oxidative stress in the liver via regulating the PI3K/Akt and SIRT6/NF-κB pathways.

Application of fenugreek in ruminant feed: implications for methane emissions and productivity.

Background: Human demand for meat and dairy products will increase as a result of economic development and population growth, and the farming of ruminants, such as cattle and sheep, will also increase. Methane (CH4) emission from the enteric fermentation of ruminant livestock is a major source of greenhouse gas emissions and a significant contributor to global warming. Meanwhile, growth performance is often limited and animals are more vulnerable to diseases in high-density, intensive farming, greatly reducing livestock productivity, so developing ways to reduce CH4 emissions and improve ruminant productivity has become a research hotspot. Studies have reported that fenugreek (Trigonella foenum-graecum L.) as feed additives have the potential to reduce ruminant methane and improve the productivity. However, systematic reviews of such studies are lacking. Methodology: In this review, databases of Google Scholar, Web of Science, PubMed, Scopus and Science Direct were used for the literature search. The initial keywords search was fenugreek or Trigonella foenum-graecum L. For more focused search, we added terms such as methane, rumen fermentation, growth, milk production and antioxidants. All were done for ruminants. The literature that conforms to the theme of this article is selected, summarized, and finally completed this article. Results: By regulating the rumen microbiome (suppressing protozoans, methanogenic bacteria, and fungi), fenugreek can lower CH4 emissions according to many in vitro anaerobic fermentation experiments. Fenugreek secondary metabolites (saponins and tannins) are responsible for this impact, but it is still unclear exactly how they work. Therefore, more long-term in vivo experiments are needed to verify its efficacy. Fenugreek is also rich in alkaloids, amino acids, flavonoids, saponins and phenolic acids. These compounds have been shown to have beneficial effects on ruminant growth, lactation, and total antioxidant capacity. Therefore, fenugreek has a great opportunity to develop into a new green feed additive. Conclusions: This review provides a summary of the effect of fenugreek and its bioactive compounds on rumen fermentation, CH4 emissions and production performance by ruminants. In addition, based on the available data, the possible biochemical pathway of fenugreek to reduce CH4 emissions in ruminants was described. Overall, the livestock feed industry has the opportunity to develop natural, environmentally-friendly feed additives based on fenugreek.

Analysis of gut microbiota metabolites of platycodin D and activity verification.

As the main saponin component of Platycodon grandiflorum A.DC, Platycodin D has been reported to have an anti-obesity effect. Due to poor oral absorption, the intestinal microflora usually transforms saponins into potential bioactive substances. In this study, we profiled the metabolic changes of platycodin D by incubating it with intestinal microflora extracted from mice feces subjected to either a standard control diet or a high-fat diet. A UPLC-LTQ-Orbitrap-MS method was used for rapid analysis of the metabolic profile of platycodin D. A total of 10 compounds were identified 9 of which were assessed to be metabolized by intestinal microflora. Dehydroxylation and deglycosylation were the major metabolic process of platycodin D. The metabolic profile of platycodin D biotransformed by intestinal microflora was elucidated based on the metabolite information. Platycodin D and its metabolites had anti-inflammatory effects in LPS-stimulated RAW 264.7 cells. Only platycodin D could alleviate lipid accumulation in FFA-treated HepG2 cells.

Immunofluorescence analysis of human eosinophils.

A prominent inflammatory cell type in allergic diseases is the eosinophil, a granulated white blood cell that releases pro-inflammatory cytokines. Eosinophil-derived cytokines, including interleukin-9 (IL-9) and interleukin-13 (IL-13), can skew the immune response towards an allergic phenotype. Unfortunately, it is challenging to immunolabel and collect quantifiable images of eosinophils given their innate autofluorescence and ability to nonspecifically bind to antibodies. Hence, it is important to optimize permeabilization, blocking, and imaging conditions for eosinophils. Here, we show enhanced protocols to ensure that measured immunofluorescence represents specific immunolabelling. To test this, eosinophils were purified from human blood, adhered to glass coverslips, stimulated with or without platelet-activating factor (PAF), fixed with paraformaldehyde, and then permeabilized with Triton X-100 or saponin. Cells were then blocked with goat serum or human serum and incubated with antibodies labelling cytokines (IL-9 and IL-13) and secretory organelles (CD63 for crystalloid granules and transferrin receptor [TfnRc] for recycling endosomes). Carefully selected isotype controls were used throughout, and cells were imaged using Deltavision super-resolution microscopy. Intensities of fluorescent probes were quantified using Volocity software. Our findings show that permeabilization with saponin, blockage with human serum, and using concentrations of antibodies up to 10 µg/ml allowed us to detect marked differences in fluorescence intensities between isotypes and test antibodies. With the achievement of sufficient qualitative and quantitative measures of increased test probe intensity compared to respective isotypes, these results indicate that our protocol allows for optimal immunolabelling of eosinophils. Using this protocol, future studies may provide further insights into trafficking mechanisms within this important inflammatory cell type.

Exploration of the immuno-adjuvant effect and mechanism of Anemoside B4 through network pharmacology and experiment verification.

BACKGROUND: Extensive investigation has been undertaken about the utilization of saponin adjuvants in vaccines intended for veterinary and human applications. AB4 is the main constituent of the traditional Chinese medicine, Pulsatilla chinensis (Bunge) Regel, and has immunomodulatory activity. However, there is a paucity of reports on AB4 as a potential adjuvant. PURPOSE: The objective of this work was to clarify the adjuvant role of AB4 and the molecular mechanisms that underlie its immunomodulatory actions. STUDY DESIGN AND METHODS: The immunomodulatory effects of AB4 were investigated using network pharmacological analyses. These effects were validated by evaluating the developmental status of the immune organs and by using the following techniques: ELISA for the quantification of serum-specific antibodies to determine immune-related cytokine levels; the MTS method for the assessment of proliferative activity of splenic lymphocytes; flow cytometry to analyze lymphocyte and dendritic cell activation status; and western blotting for mechanistic analysis at the protein level. RESULTS: The network pharmacological analysis predicted a total of 52 targets and 12 pathways for AB4 to exert immunomodulatory effects. In a mouse model with immunity to OVA, the introduction of AB4 resulted in the enhancement of immunological organ growth and maturation, elevation of blood antibodies targeting OVA, and amplification of the production of cytokines associated with Th1 and Th2 immune responses. Additionally, the administration of AB4 resulted in a notable augmentation of lymphocyte proliferation and an elevation in the CD4+/CD8+ T lymphocyte ratios. Furthermore, the administration of AB4 enhanced the maturation process of DCs in the draining LNs and increased the production of co-stimulatory factors and MHC II molecules. AB4 induces the upregulation of TLR4 and IKK proteins, as well as the phosphorylation of NF-κB p65 protein within the TLR4/NF-κB signaling cascade, while concurrently suppressing the expression of IκBα protein. CONCLUSION: The specific immunoadjuvant effects of AB4 have been demonstrated to modulate the growth and maturation of immune organs and enhance the secretion and cellular activity of pertinent immune molecules. The utilization of network pharmacology, combined within and in vivo vitro assays, clarified the adjuvant function of AB4, which potentially involves the regulation of the TLR4/NF-κB signaling pathway.

Astragaloside IV mitigates hypoxia-induced cardiac hypertrophy through calpain-1-mediated mTOR activation.

BACKGROUND: Astragaloside IV (AsIV), a key functioning element of Astragalus membranaceus, has been recognized for its potential cardiovascular protective properties. However, there is a need to elucidate the impacts of AsIV on myocardial hypertrophy under hypoxia conditions and its root mechanisms. PURPOSE: This study scrutinized the influence of AsIV on cardiac injury under hypoxia, with particular emphasis on the role of calpain-1 (CAPN1) in mediating mTOR pathways. METHODS: Hypoxia-triggered cardiac hypertrophy was examined in vivo with CAPN1 knockout and wild-type C57BL/6 mice and in vitro with H9C2 cells. The impacts of AsIV, 3-methyladenine, and CAPN1 inhibition on hypertrophy, autophagy, apoptosis, [Ca2+]i, and CAPN1 and mTOR levels in cardiac tissues and H9C2 cells were investigated. RESULTS: Both AsIV treatment and CAPN1 knockout mitigated hypoxia-induced cardiac hypertrophy, autophagy, and apoptosis in mice and H9C2 cells. Moreover, AsIV, 3-methyladenine, and CAPN1 inhibition augmented p-mTOR level but reduced [Ca2+]i and CAPN1 level. Additionally, lentivirus-mediated CAPN1 overexpression in H9C2 cells exacerbated myocardial hypertrophy, apoptosis, and p-mTOR inhibition under hypoxia. Specifically, AsIV treatment reversed the impacts of increased CAPN1 expression on cardiac injury and the inhibition of p-mTOR. CONCLUSION: These findings suggest that AsIV may alleviate cardiac hypertrophy under hypoxia by attenuating apoptosis and autophagy through CAPN1-mediated mTOR activation.

Adventitious root culture of Lessertia frutescens for the production of triterpenoid saponins and polysaccharides.

Lessertia frutescens is a perennial shrub of commercial importance in South Africa, but the scarcity of plant resources has limited current product production. In this study, to provide an alternative approach for obtaining L. frutescens material, adventitious roots (ARs) were induced from sterilized seedlings and cultured in a suspension culture system. During this process, selection tests were conducted to find a suitable auxin and its concentration for AR induction and a suitable basal medium for AR growth and metabolite accumulation; a kinetic study was then performed to constructure kinetic models. The results showed that compared to other auxins and concentrations, indole-3-butyric acid at 3 mg/L was suitable for increasing the number and length of ARs during AR induction. In AR suspension culture, Schenk and Hildebrandt (SH) was better than other basal media, and the maximum AR fresh (86.9 g/L) or dry weight (5.5 g/L), total triterpenoid saponin (92.6 mg/g DW), and polysaccharide (114.7 mg/g DW) contents were determined in the 1.5×SH medium. In addition, AR biomass and metabolite contents reached the maximum on day 42. The kinetic models for AR growth and triterpenoid and polysaccharide production were constructed, providing the basis for further optimization of culture conditions and large-scale culture.

A new insight into material basis of rhizoma Paridis saponins in alleviating pain.

ETHNOPHARMACOLOGICAL RELEVANCE: Paris polyphylla, as a traditional Chinese herbal medicine, was often used to relieve inflammation and pain. Rhizoma Paridis saponins (RPS) as the main active components of Paris polyphylla have excellent analgesic effects. AIM OF THE STUDY: Determine the analgesic material basis of RPS. MATERIALS AND METHODS: LC-MS/MS was used to analyze RPS, plasma after intravenous injection of RPS, and oral administration of RPS. H22 plantar pain model was established to explore the analgesic material basis of RPS. Moreover, correlation analysis, network pharmacology, RT-PCR and molecular docking were applied in this research. RESULTS: RPS had dose-dependently analgesic effects in acetic acid- and formalin-induced pain models. LC-MS/MS detection indicated that diosgenin as the metabolite of RPS mainly distributed in brain tissues. The addition of antibiotics increased the anti-tumor effect of RPS, but reduced its analgesic effect. Network pharmacology, RT-PCR and molecular docking showed that diosgenin exerted its analgesic effect through SRC and Rap1 signaling pathway. CONCLUSION: Diosgenin exhibited analgesic effects, while saponins had good anti-tumor effects in RPS. This discovery provided a better indication for the later application of RPS in anti-tumor and analgesic settings.

Hepatoprotective efficacy and interventional mechanism of the panaxadiol saponin component in high-fat diet-induced NAFLD mice.

Dietary administration is a promising strategy for intervention in non-alcoholic fatty liver disease (NAFLD). Our research team has identified a biologically active component, the panaxadiol saponin component (PDS-C) isolated from total saponins of panax ginseng, which has various pharmacological and therapeutic functions. However, the efficacy and mechanism of PDS-C in NAFLD were unclear. This study aimed to elucidate the hepatoprotective effects and underlying action mechanism of PDS-C in NAFLD. Mice were fed a high-fat diet (HFD) for 8 weeks to induce NAFLD and treated with PDS-C and metformin as the positive control for 12 weeks. PDS-C significantly alleviated liver function, hepatic steatosis and blood lipid levels, reduced oxidative stress and inflammation in NAFLD mice. In vitro, PDS-C has been shown to reduce lipotoxicity and ROS levels while enhancing the antioxidant and anti-inflammatory capabilities in HepG2 cells induced by palmitic acid. PDS-C induced AMPK phosphorylation, leading to upregulation of the Nrf2/HO1 pathway expression and downregulation of the NFκB protein level. Furthermore, our observations indicate that PDS-C supplementation improves insulin resistance and glucose homeostasis in NAFLD mice, although its efficacy is not as pronounced as metformin. In conclusion, these results demonstrate the hepatoprotective efficacy of PDS-C in NAFLD and provide potential opportunities for developing functional products containing PDS-C.

Total Astragalus saponins can reverse type 2 diabetes mellitus-related intestinal dysbiosis and hepatic insulin resistance in vivo.

Objective: Intestinal flora homeostasis in rats with type 2 diabetes mellitus (T2DM) was evaluated to explore the effects of total Astragalus saponins (TAS) on hepatic insulin resistance (IR). Methods: Six-week-old male Sprague-Dawley rats were fed high-fat and high-sugar diet for 4 weeks and intraperitoneally injected with streptozotocin to induce T2DM, and they were then randomly divided into control, model, metformin, and TAS groups. Stool, serum, colon, and liver samples were collected after 8 weeks of drug administration for relevant analyses. Results: TAS reduced fasting blood glucose, 2-hour postprandial blood glucose, area under the curve of oral glucose tolerance test, glycated serum protein, homeostasis model assessment of insulin resistance, total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels in T2DM rats but increased insulin, C-peptide, and high-density lipoprotein cholesterol levels. Moreover, TAS improved the morphology and structure of liver and colon tissues and improved the composition of the intestinal microbiome and bacterial community structure at different taxonomic levels. In addition, TAS increased the protein expression of hepatic IRS-1, PI3K, PDK1, and p-AKT and decreased the protein expression of p-GSK-3ß. Meanwhile, TAS increased the mRNA expression of liver PDK1, PI3K, and GS and decreased the mRNA expression of GSK-3ß. Conclusion: TAS can ameliorate T2DM-related abnormal glucose and blood lipid metabolism, intestinal dysbiosis, and IR.

Astragaloside IV reduces lung injury in lethal sepsis via promoting treg cells expansion and inhibiting inflammatory responses.

Sepsis is a systemic inflammatory response syndrome caused by an infection progressing to sepsis-associated organ failure (such as lung injury). Our previous review revealed that Astragaloside IV (ASI-IV), one of the primary bioactive ingredients in Astragalus membranaceus (Fisch) Bge (Huang-Qi), had been shown to exert anti-inflammatory and immunomodulatory effects. Nevertheless, it is still unclear whether ASI-IV could attenuate septic lung injury via activating regulatory T-cells (Tregs). This study was designed to evaluate the therapeutic potential of ASI-IV on sepsis-induced lung injury and to further explore its underlying mechanism. In the murine models of cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) induced sepsis, ASI-IV can markedly improve the survival rate and reduce inflammatory lung injury, protect mice against exacerbated inflammatory responses by decreasing myeloid cell infiltration and down-regulating IL-6 and TNF-α in lung tissue. Meanwhile, Treg cell-related gene expression, including Foxp3 and IL-10, significantly increased after ASI-IV treatment. Furthermore, ASI-IV notably promoted the differentiation of naïve CD4+ T cells into T regulatory cells without obviously affecting Th1 and Th17 differentiation. Our results indicated that ASI-IV could attenuate septic lung injury by promoting Treg cell expansion and inhibiting inflammatory responses. It represents a promising agent for the treatment of sepsis.

Hemolysis by Saponin Is Accelerated at Hypertonic Conditions.

Saponins are a large group of organic amphiphilic substances (surfactants) mainly extracted from herbs with biological activity, considered as one of the main ingredients in numerous remedies used in traditional medicine since ancient times. Anti-inflammatory, antifungal, antibacterial, antiviral, antiparasitic, antitumor, antioxidant and many other properties have been confirmed for some. There is increasing interest in the elucidation of the mechanisms behind the effects of saponins on different cell types at the molecular level. In this regard, erythrocytes are a very welcome model, having very simple structures with no organelles. They react to changing external conditions and substances by changing shape or volume, with damage to their membrane ultimately leading to hemolysis. Hemolysis can be followed spectrophotometrically and provides valuable information about the type and extent of membrane damage. We investigated hemolysis of erythrocytes induced by various saponin concentrations in hypotonic, isotonic and hypertonic media using measurements of real time and end-point hemolysis. The osmotic pressure was adjusted by different concentrations of NaCl, manitol or a NaCl/manitol mixture. Unexpectedly, at a fixed saponin concentration, hemolysis was accelerated at hypertonic conditions, but was much faster in NaCl compared to mannitol solutions at the same osmotic pressure. These findings confirm the colloid-osmotic mechanism behind saponin hemolysis with pore formation with increasing size in the membrane.

Achyranthes bidentate extracts protect the IL-1ß-induced osteoarthritis of SW1353 chondrocytes.

Osteoarthritis, the most common joint disease worldwide, is a degenerative disease characterized by cartilage degeneration and inflammation. The active ingredients in the traditional Chinese medicinal plant Achyranthes bidentate can be used to treat waist, leg, and joint pain caused by rheumatism arthralgia. In this study, we identified the optimal microwave extraction protocol for saponins from A. bidentate, evaluated their protective effects against IL-1ß-induced inflammation in SW1353 human chondrocytes, and explored their protective pathway. The microwave-extraction parameters required to obtain the maximum yield of A. bidentate saponins using 80% ethanol were identified using response surface methodology. The parameters were solid-liquid ratio, 1:10; extraction time, 20 min; power, 721 W; temperature, 65 °C. The actual yield of saponins extracted was to be 194.01 µg/mg extract. The SW1353 cells were pretreated with A. bidentate extract (ABE) at a concentration of 50 or 100 µg/mL for 3 h, after which an inflammatory response was stimulated using IL-1ß. The ABE significantly reduced the expression of proinflammatory factors IL-6, TNF-α, COX-2, iNOS, PGE2, and NO, and inhibited NF-κB activity, effectively attenuating the inflammatory response. ABE also inhibited MMP13 and ADAMTS-5 expression, reducing IL-1ß-induced degradation of the extrachondral matrix. This confirmed that ABE effectively inhibits NF-κB activity and reduces IL-1ß-induced inflammation, extracellular matrix degradation, and expression of apoptotic proteins Bax and caspase-3. Therefore, ABE has potential as a new botanical drug for preventing osteoarthritis.

Asperosaponin VI protects alcohol-induced hepatic steatosis and injury via regulating lipid metabolism and ER stress.

BACKGROUND: Asperosaponin VI (AVI) is a natural triterpenoid saponin isolated from Dipsacus asper Wall with documented anti-inflammatory and bone protective effects. Our previous work reported that AVI protects the liver of septic mice from acute inflammatory damage. In this paper, we further explored the protective effect and the potential mechanisms of AVI in alcoholic fatty liver disease (AFLD). METHODS: The Lieber-Decarli model was constructed to evaluate the effect of AVI on AFLD in C57BL/6 J mice. Additional in vitro work was performed to investigate HepG2 cells exposed to alcohol, then analyzed the degree of liver injury by detecting the ALT and AST levels both in the liver and serum. H&E staining and Sirius red staining were used to evaluate the histopathology variations in the liver. Further, observe lipid droplets in the cytoplasm by Oil Red O staining. We detected the expression of inflammatory cytokines with qualitative PCR; ROS, MDA, SOD, and GSH-px levels were analyzed to observe oxidative stress. Finally, exploring the activation of AMPK signaling pathway by real-time PCR and Western blotting. RESULTS: Histological examination of liver tissue combined with serum ALT and AST levels showed a significant protective effect of AVI against alcoholic liver injury in AFLD mice. Compared with the model group, AVI evidently improved antioxidant capacity, reduced inflammatory response and lipid accumulation both in vitro and in vivo. For mechanically, it was found that AVI up-regulated phosphorylation level of AMP-activated protein kinase (AMPK) and inhibited the endoplasmic reticulum stress (ER) pathway in AFLD. CONCLUSION: AVI protects mice from alcohol-induced hepatic steatosis and liver injury through activating AMPK signaling and repress ER stress, suggesting that it might be a potential therapeutic agent for AFLD.

Tomato root-associated Sphingobium harbors genes for catabolizing toxic steroidal glycoalkaloids.

IMPORTANCE: Saponins are a group of plant specialized metabolites with various bioactive properties, both for human health and soil microorganisms. Our previous works demonstrated that Sphingobium is enriched in both soils treated with a steroid-type saponin, such as tomatine, and in the tomato rhizosphere. Despite the importance of saponins in plant-microbe interactions in the rhizosphere, the genes involved in the catabolism of saponins and their aglycones (sapogenins) remain largely unknown. Here we identified several enzymes that catalyzed the degradation of steroid-type saponins in a Sphingobium isolate from tomato roots, RC1. A comparative genomic analysis of Sphingobium revealed the limited distribution of genes for saponin degradation in our saponin-degrading isolates and several other isolates, suggesting the possible involvement of the saponin degradation pathway in the root colonization of Sphingobium spp. The genes that participate in the catabolism of sapogenins could be applied to the development of new industrially valuable sapogenin molecules.

Multi-omics analysis reveals the mechanism of action of ophiopogonin D against pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with limited therapeutic strategies. Therefore, there is an urgent need to search for safe and effective drugs to treat this condition. Ophiopogonin D (OP-D), a steroidal saponin compound extracted from ophiopogon, possesses various pharmacological properties, including anti-inflammatory, antioxidant, and antitumor effects. However, the potential pharmacological effect of OP-D on pulmonary fibrosis remains unknown. PURPOSE: The aim of this study was to investigate whether OP-D can improve pulmonary fibrosis and to explore its mechanism of action. METHODS: The effect of OP-D on pulmonary fibrosis was investigated in vitro and in vivo using a mouse model of IPF induced by bleomycin and an in vitro model of human embryonic lung fibroblasts induced by transforming growth factor-ß1 (TGF-ß1). The mechanism of action of OP-D was determined using multi-omics techniques and bioinformatics. RESULTS: OP-D attenuated epithelial-mesenchymal transition and excessive deposition of extracellular matrix in the lungs, promoted the apoptosis of lung fibroblasts, and blocked the differentiation of lung fibroblasts into myofibroblasts. The multi-omics techniques and bioinformatics analysis revealed that OP-D blocked the AKT/GSK3ß pathway, and the combination of a PI3K/AKT inhibitor and OP-D was effective in alleviating pulmonary fibrosis. CONCLUSION: This study demonstrated for the first time that OP-D can reduce lung inflammation and fibrosis. OP-D is thus a potential new drug for the prevention and treatment of pulmonary fibrosis.

Saikosaponin D attenuates inflammatory response and cell apoptosis of lipopolysaccharide-induced lung epithelial cells.

BACKGROUND: Acute lung injury (ALI) is a prevalent complication of sepsis with high mortality rate. Saikosaponin D (SSD) is a triterpenoid saponin that has been reported to alleviate sepsis-triggered renal injury in mice. Nonetheless, the therapeutic effect of SSD on sepsis-evoked ALI is unclarified. METHODS: Lipopolysaccharide (LPS) from Escherichia coli 055:B5 was utilized to stimulate lung epithelial cell line MLE-12. A mouse model of sepsis was established. CCK-8 assay was employed for determining cytotoxicity. ELISA was utilized for determining proinflammatory cytokine production. Flow cytometry and western blotting were implemented for evaluating cell apoptosis. Hematoxylin-eosin staining was conducted for histologic analysis of murine lung tissues. RESULTS: SSD alleviated LPS-triggered inflammation and cell apoptosis of MLE-12 cells. SSD treatment ameliorated the pathological damages, inflammatory response, and cell apoptosis in the lungs of septic mice. CONCLUSION: SSD protects against sepsis-triggered ALI by inhibiting inflammation and cell apoptosis in MLE-12 cells and septic mouse mice.

Evolutionary roles of polyploidization-derived structural variations in the phenotypic diversification of Panax species.

Genomic structural variations (SVs) are widespread in plant and animal genomes and play important roles in phenotypic novelty and species adaptation. Frequent whole genome duplications followed by (re)diploidizations have resulted in high diversity of genome architecture among extant species. In this study, we identified abundant genomic SVs in the Panax genus that are hypothesized to have occurred through during the repeated polyploidizations/(re)diploidizations. Our genome-wide comparisons demonstrated that although these polyploidization-derived SVs have evolved at distinct evolutionary stages, a large number of SV-intersecting genes showed enrichment in functionally important pathways related to secondary metabolites, photosynthesis and basic cellular activities. In line with these observations, our metabolic analyses of these Panax species revealed high diversity of primary and secondary metabolites both at the tissue and interspecific levels. In particular, genomic SVs identified at ginsenoside biosynthesis genes, including copy number variation and large fragment deletion, appear to have played important roles in the evolution and diversification of ginsenosides. A further herbivore deterrence experiment demonstrated that, as major triterpenoidal saponins found exclusively in Panax, ginsenosides provide protection against insect herbivores. Our study provides new insights on how polyploidization-derived SVs have contributed to phenotypic novelty and plant adaptation.

The NAC Transcription Factor PgNAC41-2 Gene Involved in the Regulation of Ginsenoside Biosynthesis in Panax ginseng.

Ginseng (Panax ginseng C.A. Meyer) is a perennial herb of the Araliaceae family, a traditional and valuable Chinese herb in China. The main active component of ginseng is ginsenoside. The NAC transcription factors belong to a large family of plant-specific transcription factors, which are involved in growth and development, stress response and secondary metabolism. In this study, we mapped the NAC gene family on 24 pairs of ginseng chromosomes and found numerous gene replications in the genome. The NAC gene PgNAC41-2, found to be highly related to ginsenoside synthesis, was specifically screened. The phylogeny and expression pattern of the PgNAC41-2 gene were analyzed, along with the derived protein sequence, and a structure model was generated. Furthermore, the PgNAC41-2 gene was cloned and overexpressed by a Rhizobium rhizogenes mediated method, using ginseng petioles as receptor material. The saponin content of the transformed material was analyzed to verify the function of the NAC transcription factor in ginseng. Our results indicate that the PgNAC41-2 gene positively regulates the biosynthesis of saponins.