Ginsenoside F2 Restrains Hepatic Steatosis and Inflammation by Altering the Binding Affinity of Liver X Receptor Coregulators.

Background: Ginsenoside F2 (GF2), the protopanaxadiol-type constituent in Panax ginseng, has been reported to attenuate metabolic dysfunction-associated steatotic liver disease (MASLD). However, the mechanism of action is not fully understood. Here, this study investigates the molecular mechanism by which GF2 regulates MASLD progression through liver X receptor (LXR). Methods: To demonstrate the effect of GF2 on LXR activity, computational modeling of protein-ligand binding, Time-resolved fluorescence resonance energy transfer (TR-FRET) assay for LXR cofactor recruitment, and luciferase reporter assay were performed. LXR agonist T0901317 was used for LXR activation in hepatocytes and macrophages. MASLD was induced by high-fat diet (HFD) feeding with or without GF2 administration in WT and LXRα-/- mice. Results: Computational modeling showed that GF2 had a high affinity with LXRα. LXRE-luciferase reporter assay with amino acid substitution at the predicted ligand binding site revealed that the S264 residue of LXRα was the crucial interaction site of GF2. TR-FRET assay demonstrated that GF2 suppressed LXRα activity by favoring the binding of corepressors to LXRα while inhibiting the accessibility of coactivators. In vitro, GF2 treatments reduced T0901317-induced fat accumulation and pro-inflammatory cytokine expression in hepatocytes and macrophages, respectively. Consistently, GF2 administration ameliorated hepatic steatohepatitis and improved glucose or insulin tolerance in WT but not in LXRα-/- mice. Conclusion: GF2 alters the binding affinities of LXRα coregulators, thereby interrupting hepatic steatosis and inflammation in macrophages. Therefore, we propose that GF2 might be a potential therapeutic agent for the intervention in patients with MASLD.

Ginsenoside F1-Mediated Telomere Preservation Delays Cellular Senescence.

Telomeres play pivotal roles in processes closely related to somatic senescence and aging, making them a compelling target for interventions aimed at combating aging and age-related pathologies. Ginsenoside, a natural compound, has emerged as a potential remedy for promoting healthy aging, yet how it protects telomeres remains incompletely understood. Here, we show that treatment of F1 can effectively restore the level of TRF2, thereby preserving telomere integrity. This restoration leads to inhibition of the DNA damage response and improvements in mitochondrial function and, ultimately, delays in cellular senescence. Conversely, depletion of TRF2 causes mitochondrial dysfunction, accompanied by increased oxidative stress, autophagy inhibition, insufficient energy metabolism, and the onset of cellular senescence. These observations underscore the critical role of TRF2 in maintaining telomere integrity and direct association with the initiation of cellular senescence. We conduct a further analysis, suggesting F1 could bind in proximity to the TRF2 heterodimer interface, potentially enhancing dimerization stability. These findings suggest that F1 may be a promising natural remedy for anti-aging, and restoring TRF2 could potentially prevent telomere-dependent diseases commonly associated with the aging process.

Novel Split Intein-Mediated Enzymatic Channeling Accelerates the Multimeric Bioconversion Pathway of Ginsenoside.

Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.

Ginsenoside Rh2 mitigates doxorubicin-induced cardiotoxicity by inhibiting apoptotic and inflammatory damage and weakening pathological remodelling in breast cancer-bearing mice.

OBJECTIVES: There are presently a few viable ways to reduce cardiotoxicity of doxorubicin (Dox). The combination of chemotherapy agents with natural compounds delivers greater efficacy and reduces adverse effects in recent researches for cancer treatment. Here, we examined the potential effect of ginsenoside Rh2 on a Dox-based regimen in chemotherapy treatment. MATERIALS AND METHODS: Human breast tumour (MDA-MB-231) xenograft nude mice, human cardiac ventricle fibroblasts, and human umbilical vein endothelial cells (HUVEC) were employed in the present study. Histology, immunohistochemistry, immunofluorescence, western blot, antibody array, and RNA-sequencing analyses were utilized to assess the protective effect of Rh2 on cardiotoxicity induced by Dox and the underlying mechanisms. RESULTS: Rh2-reduced cardiotoxicity by inhibiting the cardiac histopathological changes, apoptosis and necrosis, and consequent inflammation. Pathological remodelling was attenuated by reducing fibroblast to myofibroblast transition (FMT) and endothelial-mesenchymal transition (EndMT) in hearts. RNA-sequencing analysis showed that Dox treatment predominantly targets cell cycle and attachment of microtubules and boosted tumour necrosis, chemokine and interferon-gamma production, response to cytokine and chemokine, and T cell activation, whereas Rh2 regulated these effects. Intriguingly, Rh2 also attenuated fibrosis via promoting senescence in myofibroblasts and reversing established myofibroblast differentiation in EndMT. CONCLUSIONS: Rh2 regulates multiple pathways in the Dox-provoked heart, proposing a potential candidate for cancer supplement and therapy-associated cardiotoxicity.

Ginsenoside F1 Protects the Brain against Amyloid Beta-Induced Toxicity by Regulating IDE and NEP.

Ginsenoside F1, the metabolite of Rg1, is one of the most important constituents of Panax ginseng. Although the effects of ginsenosides on amyloid beta (Aß) aggregation in the brain are known, the role of ginsenoside F1 remains unclear. Here, we investigated the protective effect of ginsenoside F1 against Aß aggregation in vivo and in vitro. Treatment with 2.5 µM ginsenoside F1 reduced Aß-induced cytotoxicity by decreasing Aß aggregation in mouse neuroblastoma neuro-2a (N2a) and human neuroblastoma SH-SY5Y neuronal cell lines. Western blotting, real-time PCR, and siRNA analysis revealed an increased level of insulin-degrading enzyme (IDE) and neprilysin (NEP). Furthermore, liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis confirmed that ginsenoside F1 could pass the blood-brain barrier within 2 h after administration. Immunostaining results indicate that ginsenoside F1 reduces Aß plaques in the hippocampus of APPswe/PSEN1dE9 (APP/PS1) double-transgenic Alzheimer's disease (AD) mice. Consistently, increased levels of IDE and NEP protein and mRNA were observed after the 8-week administration of 10 mg/kg/d ginsenoside F1. These data indicate that ginsenoside F1 is a promising therapeutic candidate for AD.

High fat diet-induced brain damaging effects through autophagy-mediated senescence, inflammation and apoptosis mitigated by ginsenoside F1-enhanced mixture.

BACKGROUND: Herbal medicines are popular approaches to capably prevent and treat obesity and its related diseases. Excessive exposure to dietary lipids causes oxidative stress and inflammation, which possibly induces cellular senescence and contribute the damaging effects in brain. The potential roles of selective enhanced ginsenoside in regulating high fat diet (HFD)-induced brain damage remain unknown. METHODS: The protection function of Ginsenoside F1-enhanced mixture (SGB121) was evaluated by in vivo and in vitro experiments. Human primary astrocytes and SH-SY5Y cells were treated with palmitic acid conjugated Bovine Serum Albumin, and the effects of SGB121 were determined by MTT and lipid uptake assays. For in vivo tests, C57BL/6J mice were fed with high fat diet for 3 months with or without SGB121 administration. Thereafter, immunohistochemistry, western blot, PCR and ELISA assays were conducted with brain tissues. RESULTS AND CONCLUSION: SGB121 selectively suppressed HFD-induced oxidative stress and cellular senescence in brain, and reduced subsequent inflammation responses manifested by abrogated secretion of IL-6, IL-1ß and TNFα via NF-κB signaling pathway. Interestingly, SGB121 protects against HFD-induced damage by improving mitophagy and endoplasmic reticulum-stress associated autophagy flux and inhibiting apoptosis. In addition, SGB121 regulates lipid uptake and accumulation by FATP4 and PPARα. SGB121 significantly abates excessively phosphorylated tau protein in the cortex and GFAP activation in corpus callosum. Together, our results suggest that SGB121 is able to favor the resistance of brain to HFD-induced damage, therefore provide explicit evidence of the potential to be a functional food.

CRISPRi-Guided Metabolic Flux Engineering for Enhanced Protopanaxadiol Production in Saccharomyces cerevisiae.

Protopanaxadiol (PPD), an aglycon found in several dammarene-type ginsenosides, has high potency as a pharmaceutical. Nevertheless, application of these ginsenosides has been limited because of the high production cost due to the rare content of PPD in Panax ginseng and a long cultivation time (4-6 years). For the biological mass production of the PPD, de novo biosynthetic pathways for PPD were introduced in Saccharomyces cerevisiae and the metabolic flux toward the target molecule was restructured to avoid competition for carbon sources between native metabolic pathways and de novo biosynthetic pathways producing PPD in S. cerevisiae. Here, we report a CRISPRi (clustered regularly interspaced short palindromic repeats interference)-based customized metabolic flux system which downregulates the lanosterol (a competing metabolite of dammarenediol-II (DD-II)) synthase in S. cerevisiae. With the CRISPRi-mediated suppression of lanosterol synthase and diversion of lanosterol to DD-II and PPD in S. cerevisiae, we increased PPD production 14.4-fold in shake-flask fermentation and 5.7-fold in a long-term batch-fed fermentation.

Novel enzymatic elimination method for the chromatographic purification of ginsenoside Rb3 in an isomeric mixture.

BACKGROUND: The separation of isomeric compounds from a mixture is a recurring problem in chemistry and phytochemistry research. The purification of pharmacologically active ginsenoside Rb3 from ginseng extracts is limited by the co-existence of its isomer Rb2. The aim of the present study was to develop an enzymatic elimination-combined purification method to obtain pure Rb3 from a mixture of isomers. METHODS: To isolate Rb3 from the isomeric mixture, a simple enzymatic selective elimination method was used. A ginsenoside-transforming glycoside hydrolase (Bgp2) was employed to selectively hydrolyze Rb2 into ginsenoside Rd. Ginsenoside Rb3 was then efficiently separated from the mixture using a traditional chromatographic method. RESULTS: Chromatographic purification of Rb3 was achieved using this novel enzymatic elimination-combined method, with 58.6-times higher yield and 13.1% less time than those of the traditional chromatographic method, with a lower minimum column length for purification. The novelty of this study was the use of a recombinant glycosidase for the selective elimination of the isomer. The isolated ginsenoside Rb3 can be used in further pharmaceutical studies. CONCLUSIONS: Herein, we demonstrated a novel enzymatic elimination-combined purification method for the chromatographic purification of ginsenoside Rb3. This method can also be applied to purify other isomeric glycoconjugates in mixtures.

Identification of novel paraben-binding peptides using phage display.

Parabens are alkyl esters of 4-hydroxybenzoic acid, which is derived from a family of synthetic esters of p-hydroxybenzoic acid. Among all the kinds of paraben, two parabens (methyl paraben, MP; and n-propyl paraben, PP) are the most generally used as preservatives in personal care products, such as cosmetics, pharmaceuticals, and food also, and are often presented together. However, a number of studies have reported that the toxicity of parabens affects the water environment, and human as well. This study utilized M13 phage display technology to provide easy, efficient, and relatively inexpensive methods to identify peptides that bind to MP and PP, respectively, to remove in wastewater. At first, biopanning was performed, to sort MP and PP specific binding phages, and three cases of experiment, including negative control (NC), which could sort unspecific binding phage, were conducted at the same time. Phage binding affinity tests were substituted by concentration reduction using antibody conjugated magnetic beads, and paraben concentration was measured by HPLC. Analysis showed that the MP concentration reduction of 38% was the highest in M4 phage, while the PP concentration reduction of 44% was the highest in P3 phage. We successfully screened two peptides specific to MP and PP, namely, MP4 and PP3, respectively; the results showed that the MP concentration reduction in MP4 was the highest at 44%, and the PP concentration reduction in PP3 was the highest at 39%, and their specificity was measured by the capture rate between target and control. In conclusion, the phage display technique shows applicability to the removal of parabens in water; furthermore, it also shows the possibility of the detection or removal of other chemicals.

Removal of Trans-2-nonenal Using Hen Egg White Lysosomal-Related Enzymes.

2-Nonenal is a long-chain aliphatic aldehyde containing nine carbons and an unsaturated bond. 2-Nonenal is the primary cause of odor associated with aging, with an unpleasant greasy and grassy odor. Lysosome, mitochondria, and peroxisome are significant organelles in eukaryotic cells that contain various hydrolases that degrade biomolecules. Proteins in mitochondria and peroxisome also contain aldehyde dehydrogenase. We performed trans-2-nonenal treatment using lysosomal-related enzymes extracted from hen egg white (HEW). As trans-2-nonenal is more structurally stable than cis-2-nonenal, it was selected as the target aldehyde. HEW contains various biologically active proteins and materials such as albumin, ovotransferrin, lysosome, peroxisome, and mitochondria. Here, complementary experiments were conducted to evaluate the role of lysosomal-related enzymes in the treatment of trans-2-nonenal. The activity of lysosomal-related enzymes was confirmed via antimicrobial test against E. coli. HPLC analysis was used to determine the reduction of trans-2-nonenal. The trans-2-nonenal treatment depended on the reaction time and enzyme concentration. Materials considered as an intermediate from trans-2-nonenal treatment were detected by GC/MS spectrometer. Under acidic conditions (pH 6), lysosomal-related enzymes were the most efficient in the treatment of trans-2-nonenal. Furthermore, based on differential pH testing, we found the conditions under which all the 50 ppm trans-2-nonenal was removed. Therefore, our results suggest that the lysosomal-related enzymes reduced trans-2-nonenal, suggesting clinical application as anti-aging deodorants.

Characterization of a Novel Ginsenoside MT1 Produced by an Enzymatic Transrhamnosylation of Protopanaxatriol-Type Ginsenosides Re.

BACKGROUND: Ginsenosides, triterpene saponins of Panax species, are considered the main active ingredients responsible for various pharmacological activities. Herein, a new protopanaxatriol-type ginsenoside called "ginsenoside MT1" is described; it was accidentally found among the enzymatic conversion products of ginsenoside Re. METHOD: We analyzed the conversion mechanism and found that recombinant ß-glucosidase (MT619) transglycosylated the outer rhamnopyranoside of Re at the C-6 position to glucopyranoside at C-20. The production of MT1 by trans-rhamnosylation was optimized and pure MT1 was obtained through various chromatographic processes. RESULTS: The structure of MT1 was elucidated based on spectral data: (20S)-3ß,6α,12ß,20-tetrahydroxydammarene-20-O-[α-L-rhamnopyranosyl(1→2)-ß-D-glucopyranoside]. This dammarane-type triterpene saponin was confirmed as a novel compound. CONCLUSION: Based on the functions of ginsenosides with similar structures, we believe that this ginsenoside MT1 may have great potential in the development of nutraceutical, pharmaceutical or cosmeceutical products.

Doxorubicin-induced normal breast epithelial cellular aging and its related breast cancer growth through mitochondrial autophagy and oxidative stress mitigated by ginsenoside Rh2.

Clinical dose of doxorubicin (100 nM) induced cellular senescence and various secretory phenotypes in breast cancer and normal epithelial cells. Herein, we reported the detailed mechanism underlying ginsenoside Rh2-mediated NF-κB inhibition, and mitophagy promotion were evaluated by antibody array assay, western blotting analysis, and immunocytostaining. Ginsenoside Rh2 suppressed the protein levels of TRAF6, p62, phosphorylated IKK, and IκB, which consequently inactivated NF-κB activity. Rh2-mediated secretory phenotype was delineated by the suppressed IL-8 secretion. Senescent epithelial cells showed increased level of reactive oxygen species (ROS), which was significantly abrogated by Rh2, with upregulation on SIRT 3 and SIRT 5 and subsequent increase in SOD1 and SOD2. Rh2 remarkably favored mitophagy by the increased expressions of PINK1 and Parkin and decreased level of PGC-1α. A decreased secretion of IL-8 challenged by mitophagy inhibitor Mdivi-1 with an NF-κB luciferase system was confirmed. Importantly, secretory senescent epithelial cells promoted the breast cancer (MCF-7) proliferation while decreased the survival of normal epithelial cells demonstrated by co-culture system, which was remarkably alleviated by ginsenoside Rh2 treatment. These data included ginsenoside Rh2 regulated ROS and mitochondrial autophagy, which were in large part attributed to secretory phenotype of senescent breast epithelial cells induced by doxorubicin. These findings also suggested that ginsenoside Rh2 is a potential treatment candidate for the attenuation of aging related disease.

D­galactose induces astrocytic aging and contributes to astrocytoma progression and chemoresistance via cellular senescence.

The administration of D­galactose triggers brain aging by poorly understood mechanisms. It is generally recognized that D­galactose induces oxidative stress or affects protein modifications via receptors for advanced glycated end products in a variety of species. In the present study, we aimed to investigate the involvement of astrocytes in D­galactose­induced brain aging in vitro. We found that D­galactose treatment significantly suppressed cell viability and induced cellular senescence. In addition, as of the accumulation of senescent cells, we proposed that the senescence­associated secretory phenotype (SASP) can stimulate age­related pathologies and chemoresistance in brain. Consistently, senescent astrocytic CRT cells induced by D­galactose exhibited increases in the levels of IL­6 and IL­8 via NF­κB activation, which are major SASP components and inflammatory cytokines. Conditioned medium prepared from senescent astrocytic CRT cells significantly promoted the viability of brain tumor cells (U373­MG and N2a). Importantly, conditioned medium greatly suppressed the cytotoxicity of U373­MG cells induced by temozolomide, and reduced the protein expression levels of neuron marker neuron­specific class III ß­tubulin, but markedly increased the levels of c­Myc in N2a cells. Thus, our findings demonstrated that D­galactose treatment might mimic brain aging, and that D­galactose could contribute to brain inflammation and tumor progression through inducing the accumulation of senescent­secretory astrocytes.

Minor ginsenoside F1 improves memory in APP/PS1 mice.

Ginseng has been shown to produce a cognitive improvement effect. The key molecular components in ginseng that produce pharmacological effects are ginsenosides. Previous studies reported a memory improvement effect of a few major ginsenosides. However, the identity of specific minor ginsenosides mediating such function remains unknown. Here, we report that a minor ginsenoside F1 improves memory function in APPswe/PSEN1dE9 (APP/PS1) double-transgenic Alzheimer's disease (AD) model mice. After 8-wk oral administration of F1 jelly, we observed that spatial working memory, but not context-dependent fear memory, was restored in AD mice. To search for a possible underlying molecular and cellular mechanism, we investigated the effect of F1 on Aß plaque. We observed F1 administration reduced the Aß plaque area and density in the cortex, but not in the hippocampus of AD mice. Next, we tested for the effect of F1 on the expression level of key molecules involved in learning and memory. Results from Western blot assay revealed that an abnormally reduced level of a phosphorylated form of CREB in the hippocampus of AD mice was restored to a normal level by F1 administration. Moreover, in the same animals, BDNF level was augmented in the cortex. Our results, therefore, suggest that minor ginsenoside F1 constitutes a promising target to develop therapeutic agents for AD.

High-density immobilization of a ginsenoside-transforming ß-glucosidase for enhanced food-grade production of minor ginsenosides.

Use of recombinant glycosidases is a promising approach for the production of minor ginsenosides, e.g., Compound K (CK) and F1, which have potential applications in the food industry. However, application of these recombinant enzymes for food-grade preparation of minor ginsenosides are limited by the lack of suitable expression hosts and low productivity. In this study, Corynebacterium glutamicum ATCC13032, a GRAS strain that has been used extensively for the industrial-grade production of additives for foodstuffs, was employed to express a novel ß-glucosidase (MT619) from Microbacterium testaceum ATCC 15829 with high ginsenoside-transforming activity. A cellulose-binding module was additionally fused to the N-terminus of MT619 for immobilization on cellulose, which is an abundant and safe material. Via one-step immobilization, the fusion protein in cell lysates was efficiently immobilized on regenerated amorphous cellulose at a high density (maximum 984 mg/g cellulose), increasing the enzyme concentration by 286-fold. The concentrated and immobilized enzyme showed strong conversion activities against protopanaxadiol- and protopanaxatriol-type ginsenosides for the production of CK and F1. Using gram-scale ginseng extracts as substrates, the immobilized enzyme produced 7.59 g/L CK and 9.42 g/L F1 in 24 h. To the best of our knowledge, these are the highest reported product concentrations of CK and F1, and this is the first time that a recombinant enzyme has been immobilized on cellulose for the preparation of minor ginsenosides. This safe, convenient, and efficient production method could also be effectively exploited in the preparation of food-processing recombinant enzymes in the pharmaceutical, functional food, and cosmetics industries.

Cytochrome P450 Monooxygenase-Mediated Metabolic Utilization of Benzo[a]Pyrene by Aspergillus Species.

Soil-dwelling fungal species possess the versatile metabolic capability to degrade complex organic compounds that are toxic to humans, yet the mechanisms they employ remain largely unknown. Benzo[a]pyrene (BaP) is a pervasive carcinogenic contaminant, posing a significant concern for human health. Here, we report that several Aspergillus species are capable of degrading BaP. Exposing Aspergillus nidulans cells to BaP results in transcriptomic and metabolic changes associated with cellular growth and energy generation, implying that the fungus utilizes BaP as a growth substrate. Importantly, we identify and characterize the conserved bapA gene encoding a cytochrome P450 monooxygenase that is necessary for the metabolic utilization of BaP in Aspergillus We further demonstrate that the fungal NF-κB-type velvet regulators VeA and VelB are required for proper expression of bapA in response to nutrient limitation and BaP degradation in A. nidulans Our study illuminates fundamental knowledge of fungal BaP metabolism and provides novel insights into enhancing bioremediation potential.IMPORTANCE We are increasingly exposed to environmental pollutants, including the carcinogen benzo[a]pyrene (BaP), which has prompted extensive research into human metabolism of toxicants. However, little is known about metabolic mechanisms employed by fungi that are able to use some toxic pollutants as the substrates for growth, leaving innocuous by-products. This study systemically demonstrates that a common soil-dwelling fungus is able to use benzo[a]pyrene as food, which results in expression and metabolic changes associated with growth and energy generation. Importantly, this study reveals key components of the metabolic utilization of BaP, notably a cytochrome P450 monooxygenase and the fungal NF-κB-type transcriptional regulators. Our study advances fundamental knowledge of fungal BaP metabolism and provides novel insight into designing and implementing enhanced bioremediation strategies.

Triterpenoid-biosynthetic UDP-glycosyltransferases from plants.

Triterpenoid saponins are naturally occurring structurally diverse glycosides of triterpenes that are widely distributed among plant species. Great interest has been expressed by pharmaceutical and agriculture industries for the glycosylation of triterpenes. Such modifications alter their taste and bio-absorbability, affect their intra-/extracellular transport and storage in plants, and induce novel biological activities in the human body. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze glycosylation using UDP sugar donors. These enzymes belong to a multigene family and recognize diverse natural products, including triterpenes, as the acceptor molecules. For this review, we collected and analyzed all of the UGT sequences found in Arabidopsis thaliana as well as 31 other species of triterpene-producing plants. To identify potential UGTs with novel functions in triterpene glycosylation, we screened and classified those candidates based on similarity with UGTs from Panax ginseng, Glycine max, Medicago truncatula, Saponaria vaccaria, and Barbarea vulgaris that are known to function in glycosylate triterpenes. We highlight recent findings on UGT inducibility by methyl jasmonate, tissue-specific expression, and subcellular localization, while also describing their catalytic activity in terms of regioselectivity for potential key UGTs dedicated to triterpene glycosylation in plants. Discovering these new UGTs expands our capacity to manipulate the biological and physicochemical properties of such valuable molecules.

Ginsenoside Rh2 Ameliorates Doxorubicin-Induced Senescence Bystander Effect in Breast Carcinoma Cell MDA-MB-231 and Normal Epithelial Cell MCF-10A.

The anthracycline antibiotic doxorubicin is commonly used antineoplastic drug in breast cancer treatment. Like most chemotherapy, doxorubicin does not selectively target tumorigenic cells with high proliferation rate and often causes serve side effects. In the present study, we demonstrated the cellular senescence and senescence associated secretory phenotype (SASP) of both breast tumor cell MDA-MB-231 and normal epithelial cell MCF-10A induced by clinical dose of doxorubicin (100 nM). Senescence was confirmed by flattened morphology, increased level of beta galactose, accumulating contents of lysosome and mitochondrial, and elevated expression of p16 and p21 proteins. Similarly, SASP was identified by highly secreted proteins IL-6, IL-8, GRO, GM-CSF, MCP-1, and MMP1 by antibody array assay. Reciprocal experiments, determined by cell proliferation and apoptosis assays and cell migration and cell invasion, indicated that SASP of MDA-MB-231 cell induces growth arrest of MCF-10A, whereas SASP of MCF-10A significantly stimulates the proliferation of MDA-MB-231. Interestingly, SASP from both cells powerfully promotes the cell migration and cell invasion of MDA-MB-231 cells. Treatment with the natural product ginsenoside Rh2 does not prevent cellular senescence or exert senolytic. However, SASP from senescent cells treated with Rh2 greatly attenuated the above-mentioned bystander effect. Altogether, Rh2 is a potential candidate to ameliorate this unwanted chemotherapy-induced senescence bystander effect.

Systematic Dissection of the Evolutionarily Conserved WetA Developmental Regulator across a Genus of Filamentous Fungi.

Asexual sporulation is fundamental to the ecology and lifestyle of filamentous fungi and can facilitate both plant and human infection. In Aspergillus, the production of asexual spores is primarily governed by the BrlA→AbaA→WetA regulatory cascade. The final step in this cascade is controlled by the WetA protein and governs not only the morphological differentiation of spores but also the production and deposition of diverse metabolites into spores. While WetA is conserved across the genus Aspergillus, the structure and degree of conservation of the wetA gene regulatory network (GRN) remain largely unknown. We carried out comparative transcriptome analyses of comparisons between wetA null mutant and wild-type asexual spores in three representative species spanning the diversity of the genus Aspergillus: A. nidulans, A. flavus, and A. fumigatus We discovered that WetA regulates asexual sporulation in all three species via a negative-feedback loop that represses BrlA, the cascade's first step. Furthermore, data from chromatin immunoprecipitation sequencing (ChIP-seq) experiments in A. nidulans asexual spores suggest that WetA is a DNA-binding protein that interacts with a novel regulatory motif. Several global regulators known to bridge spore production and the production of secondary metabolites show species-specific regulatory patterns in our data. These results suggest that the BrlA→AbaA→WetA cascade's regulatory role in cellular and chemical asexual spore development is functionally conserved but that the wetA-associated GRN has diverged during Aspergillus evolution.IMPORTANCE The formation of resilient spores is a key factor contributing to the survival and fitness of many microorganisms, including fungi. In the fungal genus Aspergillus, spore formation is controlled by a complex gene regulatory network that also impacts a variety of other processes, including secondary metabolism. To gain mechanistic insights into how fungal spore formation is controlled across Aspergillus, we dissected the gene regulatory network downstream of a major regulator of spore maturation (WetA) in three species that span the diversity of the genus: the genetic model A. nidulans, the human pathogen A. fumigatus, and the aflatoxin producer A. flavus Our data show that WetA regulates asexual sporulation in all three species via a negative-feedback loop and likely binds a novel regulatory element that we term the WetA response element (WRE). These results shed light on how gene regulatory networks in microorganisms control important biological processes and evolve across diverse species.

Possible role of ginsenoside Rb1 in skin wound healing via regulating senescent skin dermal fibroblast.

Cellular senescence suppresses cancer by inducing irreversible cell growth arrest. Nevertheless, senescent cells is proposed as causal link with aging and aging-related pathologies. The physiological beneficial functions of senescent cells are still of paucity. Here we show that senescent human dermal fibroblast accelerates keratinocytes scratch wound healing and stimulates differentiation of fibroblast. Using oxidative stress (100 µM H2O2 exposure for 1 h) induction, we successfully triggered fibroblast senescence and developed senescence associated secretory phenotype (SASP). The induction of SASP was regulated by p38MAPK/MSK2/NF-κB pathway. Interestingly, inhibition of p38MAPK activation only partially suppressed SASP. However, SASP was significantly inhibited by SB747651A, a specific MSK inhibitor. Additionally, we demonstrate that SASP stimulates migration of keratinocytes and myofibroblast transition of fibroblast, through fold-increased secretion of growth factors, platelet-derived growth factor AA (PDGF-AA) and AB (PDGF-AB), transforming growth factor beta 1 (TGF-ß1) and beta 2 (TGF-ß2), vascular endothelial growth factor A (VEGF-A) and D (VEGF-D), vascular endothelial growth factor receptor 2 (VEGFR2) and 3 (VEGFR3). Importantly, we also confirmed ginsenoside Rb1 promoted SASP-mediated healing process via p38MAPK/MSK2/NF-κB pathway. The results pointed to senescent fibroblast as a potential mechanism of wound healing control in human skin. Further, it provided a candidate targeted for wound therapy.