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Abstract Like many traditional Chinese herbal medicines, preparations from Radix Dipsaci are at risk of contamination by harmful mycotoxins; however, there have been no reports of actual contamination. In this study, we developed an analytical method to simultaneously detect eight mycotoxins in Radix Dipsaci and estimate the exposure risk for consumers. We have developed an analytical method utilizing ultra-high performance liquid chromatography and tandem mass spectrometry to accurately determine the levels of AFB1, AFB2, AFG1, AFG2, OTA, ZEN, T-2 and ST mycotoxins in 45 batches of Radix Dipsaci sourced from major medicinal herb markets across five regions in China. We also analyzed migration of mycotoxins from the raw herbs into water decoction. Based on these results and data on human consumption of the herbal medicine, we estimated risk of exposure and acceptable exposure limits to mycotoxins in the Radix Dipsaci using the “margin of exposure (MOE)” method. Of the 45 batches of Radix Dipsaci, 48.89% contained at least one of the eight mycotoxins, 24.44% contained one, 17.78% contained two and 6.67% contained three. The most frequent mycotoxins were aflatoxin B1, present in 35.56% of batches (at 0.25–34.84 μg/kg); aflatoxin G1, 15.56% (1.99–44.05 μg/kg); and ochratoxin A, 22.22% (16.11–143.38 μg/kg). These three mycotoxins transferred from the raw herb into water decoction at respective rates of 20.20%, 29.14%, and 24.80%. The 95th percentile values of the MOE risk factors for health effects of AFB1 were below 10,000 at high doses but above 10,000 at low doses of Radix Dipsaci long-term treatment. With the reduction in duration of exposure years, the MOE values of AFB1 and AFG1 gradually reverted to within the acceptable range. The mean, 50th, and 95th percentile values of the MOE risk factors for health effects of OTA exceeded 10,000 regardless of whether consumers received a low or high dose of Radix Dipsaci treatment for durations ranging from 1 to lifetime. Based on this exposure and a typical human diet, we have estimated the respective 20-year exposure limits for Radix Dipsaci to be 5.821 μg/kg, 4.035 μg/kg, and 56.073 μg/kg for the three mycotoxins under consideration. Contamination with multiple mycotoxins is frequently observed in Radix Dipsaci, and the three most prevalent contaminants have been found to leach into water decoctions, thereby posing a potential health hazard for individuals consuming this herbal preparation. This work highlights the need to monitor herbal medicines for mycotoxin contamination in order to protect consumers.

Abstract Background Continuous monocropping obstacles are common in plants, especially medicinal plants, resulting in disease outbreaks and productivity reductions. Foliar disease, mainly caused by Fusarium oxysporum, results in a severe decrease in the yield of Pseudostellaria heterophylla annually. Determining an effective biomethod to alleviate this disease is urgently needed to improve its productivity and quality. Results This study screened thirty-two keystone bacterial genera induced by pathogens in P. heterophylla rhizosphere soil under continuous monocropping conditions. Pseudomonas, Chryseobacterium, and Flavobacterium, referred to as the beneficial microbiota, were significantly attracted by pathogen infection. The P. palleroniana strain B-BH16-1 can directly inhibit the growth and spore formation of seven primary pathogens of P. heterophylla foliar disease by disrupting fusaric acid production via the emission of volatile organic compounds (VOCs). In addition, strain B-BH16-1 enhances the disease resistance of P. heterophylla by obliterating the pathogen and assembling beneficial microbiota. Conclusion Pathogen-induced Pseudomonas reshaped phyllosphere microbial communities via direct antagonism of pathogens and indirect disruption of the pathogen virulence factor biosynthesis to enhance disease suppression and improve yields. These results show that inhibiting pathogen virulence biosynthesis to reshape the plant microbial community using disease-induing probiotics will be an innovative strategy for managing plant disease, especially under continuous monoculture conditions.

BackgroundPseudostellaria heterophylla is used in traditional Chinese medicine, so ensuring an adequate supply of plant material with high levels of bioactive components is important.MethodsUsing an optimized maximum entropy niche model and assays of bioactive components from cultivation samples, this study started from the plant’s natural distribution area and estimated correlations of ecological factors with not only abundance of the plant but also abundance of polysaccharides and heterophyllin B. These correlations were combined with the spatial analysis function in ArcGIS to generate maps of the suitability of different habitats in China for cultivating P. heterophylla under current climate conditions and different models of climate change.ResultsThe following ecological factors emerged as particularly important for habitat suitability: precipitation of driest month and driest quarter, annual precipitation, annual mean temperature, temperature seasonality, and mean temperature of coldest quarter, contributing to a cumulative total of 87%. Under current climate conditions, optimum habitats of P. heterophylla were mainly distributed in the southwestern region (Guizhou) and eastern regions (Anhui, Zhejiang, Fujian, Jiangsu) of China, and only 0.197×106 km2 of these areas were optimum habitat. In future climate change scenarios, the optimal habitat area of P. heterophylla exhibited an increase across different time periods under the SSP5-8.5 climate scenario. By the 2090s, distribution area of high heterophyllin B content under SSP5-8.5 climate scenarios will increase significantly, distribution area of high polysaccharide content had little change under all three climate scenarios (SSP 1-2.6, 2-4.5, 5-8.5). The center of mass of suitable habitat migrates southwestward under scenario SSP 1-2.6 and SSP 2-4.5, while it migrates northward under scenario SSP 5-8.5. Under the three climate scenarios, the center of mass of suitable habitat migrated consistently with that of high polysaccharide content but differed from that of high heterophyllin B content.ConclusionThese findings provide a crucial foundation for cultivating P. heterophylla with superior medicinal properties, developing adaptive management strategies to enhance conservation efforts, and ensuring sustainable utilization in the face of global climate change.

Eucommia ulmoides Oliver is a medicinal plant of significant economic importance. Its cortex has been employed for centuries to alleviate various conditions such as lumbar pain, knee pain, and osteoporosis. Additionally, E. ulmoides possesses substantial industrial value. With the growing demand for this medicinal herb, ensuring its sustainable supply has become imperative. Climate change has caused habitat restrictions or migration of medicinal plants. Therefore, predicting the impact of climate change on the distribution of E. ulmoides is crucial for its conservation and sustainable use. This study evaluated the potential distribution of E. ulmoides across China under various climate change scenarios since the last interglacial period by modeling suitable areas based on 257 distribution records and 19 major environmental factors related to E. ulmoides. The model selection process initially identified the MaxEnt model as the most suitable. The optimized MaxEnt model, with RM = 2.0 and FC = LQHPT settings, generated the most precise predictions. Results indicate that the minimum temperature of the coldest month, annual mean temperature, and annual precipitation significantly affect the distribution of E. ulmoides. Under current environmental conditions, highly suitable areas for E. ulmoides are found in Southwest and Southeast China, with a total suitable habitat area of 23.12 × 104 km2. However, the range of suitable habitat has shifted due to global warming’s negative impact. Under different climate scenarios, suitable areas for E. ulmoides have either increased or decreased, with expansions primarily in high-latitude regions. Future climate scenarios predict shifts in the centroid of suitable E. ulmoides habitat towards Yichang City in Hubei Province. The findings of this study support the development, artificial cultivation, and conservation of E. ulmoides resources.

BackgroundGastrodiae rhizoma (GR) refers to the dried tuber of Gastrodia elata Bl. and has been used for many centuries to treat brain diseases, such as Alzheimer’s disease, major depressive disorder, and cerebral ischemia. However, the processing of GR is complex and varied, resulting in unstable clinical treatment effects. The processing protocols significantly affect the active ingredients and curative effects of GR. We can optimize the processing of GR by identifying quality markers to treat brain diseases.MethodsFresh tubers of G. elata Bl. were processed under eight different protocols, and their resulting contents of potentially bioactive compounds were compared using liquid chromatography mass spectrometry to screen the potential quality markers of GR through stoichiometric analysis. The potential quality markers of GR targeting Alzheimer’s disease, major depressive disorder, and cerebral ischemia were identified by network pharmacology, and the potentially neuroprotective effects of these components were validated through simulated docking to likely protein targets. Finally, a fit degree analysis was carried out using different composition ratios and proportions of the disease component degree value, and the therapeutic effects of different processing methods on Alzheimer’s disease, major depressive disorder, and cerebral ischemia were outlined clearly.ResultsWe identified 32 potential therapeutic components and screened 13 quality markers in GR, of which five quality markers (galactinol, glucosyringic acid, parishins C and E, and S-(4-hydroxybenzyl)-glutathione) showed efficacy against all three brain diseases. Furthermore, steaming and microwave-drying during processing can optimize the components of these quality markers for treating the three diseases.ConclusionProcessing protocols significantly affect the therapeutic components of GR and may also impact its effectiveness in treating brain diseases. Accordingly, optimizing the processing methods of GR to correspond to different therapeutic purposes may improve its efficacy against brain diseases.

Effects of stress on obesity have been thoroughly studied in high-fat diet fed mice, but not in normal diet fed mice, which is important to clarify because even on a normal diet, some individuals will become obese under stress conditions. Here we compared mice that showed substantial weight gain or loss under chronic mild stress while on a normal diet; we compared the two groups in terms of cognitive function, hypothalamic–pituitary–adrenal signaling, neurogenesis and activation of microglia in hippocampus, gene expression and composition of the gut microbiome. Chronic mild stress induced diet-independent obesity in approximately 20% of animals, and it involved inflammatory responses in peripheral and central nervous system as well as hyperactivation of the hypothalamic–pituitary–adrenal signaling and of microglia in the hippocampus, which were associated with cognitive deficits and impaired hippocampal neurogenesis. It significantly increased in relative abundance at the phylum level (Firmicutes), at the family level (Prevotellaceae ucg − 001 and Lachnospiraceae NK4a136), at the genus level (Dubosiella and Turicibacter) for some enteric flora, while reducing the relative abundance at the family level (Lactobacillaceae and Erysipelotrichaceae), at the genus level (Bacteroidota, Alistipes, Alloprevotella, Bifidobacterium and Desulfovibrio) for some enteric flora. These results suggest that stress, independently of diet, can induce obesity and cognitive decline that involve dysfunctional gut microbiota. These insights imply that mitigation of hypothalamic–pituitary–adrenal signaling and microglial activation as well as remodeling of gut microbiota may reverse stress-induced obesity and associated cognitive decline.

Abstract Background The Gastrodia elata Bl. is an orchid, and its growth demands the presence of Armillaria species. The strong competitiveness of Armillaria species has always been a concern of major threat to other soil organisms, thus disrupting the equilibrium of soil biodiversity. Introducing other species to where G. elata was cultivated, could possibly alleviate the problems associated with the disequilibrium of soil microenvironment; however, their impacts on the soil microbial communities and the underlying mechanisms remain unclear. To reveal the changes of microbial groups associated with soil chemical properties responding to different cultivation species, the chemical property measurements coupled with the next-generation pyrosequencing analyses were applied with soil samples collected from fallow land, cultivation of G. elata and Phallus impudicus, respectively. Results The cultivation of G. elata induced significant increases (p

Medicinal herbs have been increasingly used for therapeutic purposes against a diverse range of human diseases worldwide. However, inevitable contaminants, including mycotoxins, in medicinal herbs can cause serious problems for humans despite their health benefits. The increasing consumption of medicinal plants has made their use a public health problem due to the lack of effective surveillance of the use, efficacy, toxicity, and quality of these natural products. Radix Dipsaci is commonly utilized in traditional Chinese medicine and is susceptible to contamination with mycotoxins. Here, we evaluated the mycotoxins, mycobiota and toxigenic fungi in the traditional medicine Radix Dipsaci. A total of 28 out of 63 Radix Dipsaci sample batches (44.4%) were found to contain mycotoxins. Among the positive samples, the contamination levels of AFB1, AFG1, AFG2, and OTA in the positive samples ranged from 0.52 to 32.13 μg/kg, 5.14 to 20.05 μg/kg, 1.52 to 2.33 μg/kg, and 1.81 to 19.43 μg/kg respectively, while the concentrations of ZEN and T-2 were found to range from 2.85 to 6.33 μg/kg and from 2.03 to 2.53 μg/kg, respectively. More than 60% of the contaminated samples were combined with multiple mycotoxins. Fungal diversity and community were altered in the Radix Dipsaci contaminated with various mycotoxins. The abundance of Aspergillus and Fusarium increased in the Radix Dipsaci contaminated with aflatoxins (AFs) and ZEN. A total of 95 strains of potentially toxigenic fungi were isolated from the Radix Dipsaci samples contaminated with mycotoxins, predominantly comprising Aspergillus (73.7%), Fusarium (20.0%), and Penicillium (6.3%). Through morphological identification, molecular identification, mycotoxin synthase gene identification and toxin production verification, we confirmed that AFB1 and AFG1 primarily derive from Aspergillus flavus, OTA primarily derives from Aspergillus westerdijkiae, ZEN primarily derives from Fusarium oxysporum, and T-2 primarily derives from Fusarium graminearum in Radix Dipsaci. These data can facilitate our comprehension of prevalent toxigenic fungal species and contamination levels in Chinese herbal medicine, thereby aiding the establishment of effective strategies for prevention, control, and degradation to mitigate the presence of fungi and mycotoxins in Chinese herbal medicine.

Background: Aflatoxin B1, which can penetrate the blood-brain barrier and kill neural cells, can contaminate traditional herbal medicines, posing a significant risk to human health. The present study examined cellular, cognitive and behavioral consequences of aflatoxin B1 contamination of the anti-osteoporotic medicine Radix Dipsaci. Methods: A mouse model of osteoporosis was created by treating the animals with all-trans-retinoic acid. Then the animals were treated intragastically with water decoctions of Radix Dipsaci that contained detectable aflatoxin B1 or not. The animals were compared in terms of mineral density and mineral salt content of bone, production of pro-inflammatory factors, neurogenesis and microglial activation in hippocampus, as well as behavior and cognitive function. Results: Contamination of Radix Dipsaci with aflatoxin B1 significantly reduced the medicine's content of bioactive saponins. It destroyed the ability of the herbal decoction to improve mineral density and mineral salt content in the bones of diseased mice, and it induced the production of the oxidative stress marker malondialdehyde as well as the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α. Aflatoxin B1 contamination inhibited formation of new neurons and increased the proportion of activated microglia in the hippocampus. These neurological changes were associated with anhedonia, behavioral despair, and deficits in short-term memory and social memory. Conclusion: Contamination of Radix Dipsaci with aflatoxin B1 not only eliminates the herbal decoction's anti-osteoporotic effects, but it also induces neurotoxicity that can lead to cognitive decline and behavioral abnormalities. Such contamination should be avoided through tightly regulated production and quality control of medicinal herbs.

Sweating is one of the most important processing methods of Chinese medicinal herbs. However, the high temperature and humidity environment required for sweating Chinese medicinal herbs makes it very easy for fungi to breed, especially toxigenic fungi. The mycotoxins produced by these fungi will then contaminate the Chinese medicinal herbs. In this study, we explored the changes in mycobiota, toxigenic fungi, and mycotoxins with and without sweating in Radix Dipsaci (RD), a typical representative of traditional Chinese medicine that requires processing through sweating. We also isolated and identified the toxigenic fungi from RD, whether they were subjected to sweating treatment or not, and examined their toxigenic genes and ability. The results showed that the detection rate of mycotoxins (aflatoxins, ochratoxins, zearalenone, and T-2 toxin) in RD with sweating was 36%, which was 2.25-fold higher than that in RD without sweating. We also detected T-2 toxin in the RD with sweating, whereas it was not found in the RD without sweating. The sweating process altered the fungal composition and increased the abundance of Fusarium and Aspergillus in RD. Aspergillus and Fusarium were the most frequently contaminating fungi in the RD. Morphological and molecular identification confirmed the presence of key toxigenic fungal strains in RD samples, including A. flavus, A. westerdijkiae, F. oxysporum and F. graminearum. These four fungi, respectively, carried AflR, PKS, Tri7, and PKS14, which were key genes for the biosynthesis of aflatoxins, ochratoxins, zearalenone, and T-2 toxin. The toxigenic ability of these four fungal strains was verified in different matrices. We also found that A. flavus, A. westerdijkiae, and F. oxysporum were isolated in RD both with sweating and without sweating, but their isolation frequency was significantly higher in the RD with sweating than in the RD without sweating. F. graminearum was not isolated from RD without sweating, but it was isolated from RD with sweating. These findings suggest that the sweating process promotes the expansion of toxigenic fungi and increases the risk of combined mycotoxin contamination in RD.

Houttuynia Cordata (HC) is a widely distributed plant in Asia and is used extensively for both food and medicinal purposes. A preliminary investigation found that HC is often bleached with sodium metabisulfite solution during its field processing, leading to health risks. In this study, the effects of sodium metabisulfite on the quality of HC were comprehensively evaluated using volatile and non-volatile targeted metabolomic methods. The results revealed a positive correlation between the extent of chemical composition changes and the bleaching time. These notable changes mainly occurred at the initial stage of bleaching. Subsequently, an untargeted UPLC/Q-TOF MS method was used to explore the potential chemical bleaching markers in bleached HC. The marker 1-hydroxy-3-oxodecane-1-sulfonic acid was subsequently prepared, isolated, and identified. Market sample verification further validated the accuracy and effectiveness of this marker.

Communication among the brain, gut and microbiota in the gut is known to affect the susceptibility to stress, but the mechanisms involved are unclear. Here we demonstrated that stress resistance in mice was associated with more abundant Lactobacillus and Akkermansia in the gut, but less abundant Bacteroides, Alloprevotella, Helicobacter, Lachnoclostridium, Blautia, Roseburia, Colidextibacter and Lachnospiraceae NK4A136. Stress-sensitive animals showed higher permeability and stronger immune responses in their colon, as well as higher levels of pro-inflammatory cytokines in serum. Their hippocampus also showed more extensive microglial activation, abnormal interactions between microglia and neurons, and lower synaptic plasticity. Transplanting fecal microbiota from stress-sensitive mice into naïve ones perturbed microglia-neuron interactions and impaired synaptic plasticity in the hippocampus, translating to more depression-like behavior after stress exposure. Conversely, transplanting fecal microbiota from stress-resistant mice into naïve ones protected microglia from activation and preserved synaptic plasticity in the hippocampus, leading to less depression-like behavior after stress exposure. These results suggested that gut microbiota may influence resilience to chronic psychological stress by regulating microglia-neuron interactions in the hippocampus.

Studies from rodents to primates and humans indicate that individuals vary in how resilient they are to stress, and understanding the basis of these variations may help improve treatments for depression. Here we explored the potential contribution of the gut microbiome to such variation. Mice were exposed to chronic unpredictable mild stress (CUMS) for 4 weeks then allowed to recover for 3 weeks, after which they were subjected to behavioral tests and categorized as showing low or high stress resilience. The two types of mouse were compared in terms of hippocampal gene expression using RNA sequencing, fecal microbiomes using 16S RNA sequencing, and extent of neurogenesis in the hippocampus using immunostaining of brain sections. Fecal microbiota were transplanted from either type of mouse into previously stress-exposed and stress-naïve animals, and the effects of the transplantation on stress-induced behaviors and neurogenesis in the hippocampus were examined. Finally, we blocked neurogenesis using temozolomide to explore the role of neurogenesis promoted by fecal microbiota transplantation in enhancing resilience to stress. Results showed that highly stress-resilient mice, but not those with low resilience, improved significantly on measures of anhedonia, behavioral despair, and anxiety after 3-week recovery from CUMS. Their feces showed greater abundance of Lactobacillus, Bifidobacterium and Romboutsia than feces from mice with low stress resilience, as well as lower abundance of Staphylococcus, Psychrobacter and Corynebacterium. Similarly, highly stress-resilient mice showed greater neurogenesis in hippocampus than animals with low stress resilience. Transplanting fecal microbiota from mice with high stress resilience into previously CUMS-exposed recipients rescued neurogenesis in hippocampus, facilitating recovery from stress-induced depression and cognitive decline. Blockade of neurogenesis with temozolomide abolished recovery of recipients from CUMS-induced depression and cognitive decline in mice transplanted with fecal microbiota from mice with high stress resilience. In conclusion, our results suggested that remodeling of the gut microbiome after stress may reverse stress-induced impairment of hippocampal neurogenesis and thereby promote recovery from stress-induced depression.

Armillaria members play important roles in the nutrient supply and growth modulation of Gastrodia elata Bl., and they will undergo severe competition with native soil organisms before colonization and become symbiotic with G. elata. Unraveling the response of soil microbial organisms to symbiotic fungi will open up new avenues to illustrate the biological mechanisms driving G. elata’s benefit from Armillaria. For this purpose, Armillaria strains from four main G. elata production areas in China were collected, identified, and co-planted with G. elata in Guizhou Province. The result of the phylogenetic tree indicated that the four Armillaria strains shared the shortest clade with Armillaria gallica. The yields of G. elata were compared to uncover the potential role of these A. gallica strains. Soil microbial DNA was extracted and sequenced using Illumina sequencing of 16S and ITS rRNA gene amplicons to decipher the changes of soil bacterial and fungal communities arising from A. gallica strains. The yield of G. elata symbiosis with the YN strain (A. gallica collected from Yunnan) was four times higher than that of the GZ strain (A. gallica collected from Guizhou) and nearly two times higher than that of the AH and SX strains (A. gallica collected from Shanxi and Anhui). We found that the GZ strain induced changes in the bacterial community, while the YN strain mainly caused changes in the fungal community. Similar patterns were identified in non-metric multidimensional scaling analysis, in which the GZ strain greatly separated from others in bacterial structure, while the YN strain caused significant separation from other strains in fungal structure. This current study revealed the assembly and response of the soil microbial community to A. gallica strains and suggested that exotic strains of A. gallica might be helpful in improving the yield of G. elata by inducing changes in the soil fungal community.

Sweating is one of the most important primary processing methods of Chinese medicinal materials. Dipsacus asper is a typical representative of sweating treatment that is recommended by the Chinese Pharmacopoeia. The color change of the fracture surface of the root is the prominent feature of sweating treatment. However, few studies have focused on the mechanism of color change during sweating treatment. In this study, widely targeted metabolomics and ITS high-throughput sequencing technologies were applied to detect metabolites and microbial structure and diversity in the root of D. asper during sweating treatment. A total of 667 metabolites, including 36 downregulated and 78 upregulated metabolites, were identified in D. asper following sweating treatment. The significantly differential metabolites were divided into 12 classes, including terpenoids and phenolic acids. Moreover, all the differential terpenoids were upregulated and 20 phenolic acids showed a significant change after sweating treatment. In addition, microbial community diversity and richness increased following sweating treatment. The composition of microbial communities revealed that the relative abundances of Ascomycota and Basidiomycota significantly changed after sweating treatment. Correlation analysis revealed that Ascomycota (Fusarium sp., Macrophomina sp., Ilyonectria sp., Memnoniella sp., Penicillium sp., Cyphellophora sp., Neocosmospora sp., unclassified_f_Nectriaceae, and unclassified_o_Saccharomycetales) and Basidiomycota (Armillaria sp.) were associated with the content of terpenoids (6-deoxycatalpol and laciniatoside III) and phenolic acids (3-(4-hydroxyphenyl)-propionic acid, ethyl caffeate, 4-O-glucosyl-4-hydroxybenzoic acid, 2-acetyl-3-hydroxyphenyl−1-O-glucoside, 4-O-glucosyl-3,4-dihydroxybenzyl alcohol, 3-O-feruloylquinic acid, 3,4-O-dicaffeoylquinic acid methyl ester, O-anisic acid, and coniferyl alcohol). We speculate that the Ascomycota and Basidiomycota affect the content of terpenoids and phenolic acids, resulting in color change during sweating treatment in D. asper. This study provides a foundation for analyzing the mechanism involved in the processing of Chinese medicinal materials.

Background: Chronic aflatoxin B1 (AFB1) exposure may increase the risk of multiple neuropsychiatric disorders. Stress is considered one of the main contributors to major depressive disorder. Whether and how chronic AFB1 exposure affects vulnerability to stress is unclear. Methods: Mice were exposed for three weeks to AFB1 (100 µg/kg/d) and/or chronic mild stress (CMS). The vulnerability behaviors in response to stress were assessed in the forced swimming test (FST), sucrose preference test (SPT), and tail suspension test (TST). Microglial pyroptosis was investigated using immunofluorescence, enzyme-linked immunosorbent assays, and western blot assay in the hippocampus of mice. Hippocampal neurogenesis and the effects of AFB1-treated microglia on proliferation and differentiation of neural stem/precursor cells (NSPCs) were assessed via immunofluorescence in the hippocampus of mice. Results: Mice exposed to CMS in the presence of AFB1 exhibited markedly greater vulnerability to stress than mice treated with CMS or AFB1 alone, as indicated by reduced sucrose preference and longer immobility time in the forced swimming test. Chronic aflatoxin B1 exposure resulted in changes in the microglial morphology and increase in TUNEL+ microglia and GSDMD+ microglia in the hippocampal dentate gyrus. When mice were exposed to both CMS and AFB1, pyroptosis-related molecules (such as NLRP3, caspase-1, GSDMD-N, and interleukin-1β) were significantly upregulated in the hippocampus. These molecules were also significantly enhanced by AFB1 in primary microglial cultures. AFB1-treated mice showed decrease in the numbers of BrdU+, BrdU-DCX+, and BrdU-NeuN+ cells in the hippocampal dentate gyrus, as well as the percentages of BrdU+ cells that were NeuN+ in the presence or absence of CMS when compared with vehicle-treated mice. The combination of AFB1 and CMS exacerbated these effects to an even greater extent. The number of DCX+ cells correlated negatively with the percentage of ameboid microglia, TUNEL+ microglia and GSDMD+ microglia in the hippocampal dentate gyrus. AFB1-treated microglia suppressed the proliferation and neuronal differentiation of NSPCs in vitro. Conclusion: Chronic AFB1 exposure induces microglial pyroptosis, promoting an adverse neurogenic microenvironment that impairs hippocampal neurogenesis, which may render mice more vulnerable to stress.

Abstract Background The natural compound asperosaponin VI has shown potential as an antidepressant, but how it works is unclear. Here, we explored its effects on mice exposed to chronic mild stress (CMS) and the underlying molecular pathways. Methods Mice were exposed to CMS for 3 weeks followed by asperosaponin VI (40 mg/kg) or imipramine (20 mg/kg) for another 3 weeks. Depression-like behaviors were assessed in the forced swimming test (FST), sucrose preference test (SPT), tail suspension test (TST). Microglial phenotypes were evaluated using immunofluorescence staining, real-time quantitative PCR and enzyme-linked immunosorbent assays in hippocampus of mice. In some experiments, stressed animals were treated with the PPAR-γ antagonist GW9662 to examine its involvement in the effects of asperosaponin VI. Blockade of PPAR-γ in asperosaponin VI-treated primary microglia in the presence of lipopolysaccharide (LPS) was executed synchronously. The nuclear transfer of PPAR-γ in microglia was detected by immunofluorescence staining in vitro and in vivo. A co-cultured model of neuron and microglia was used for evaluating the regulation of ASA VI on the microglia–neuron crosstalk molecules. Results Asperosaponin VI ameliorated depression-like behaviors of CMS mice based on SPT, TST and FST, and this was associated with a switch of hippocampal microglia from a pro-inflammatory (iNOS+-Iba1+) to neuroprotective (Arg-1+-Iba1+) phenotype. CMS reduced the expression levels of PPAR-γ and phosphorylated PPAR-γ in hippocampus, which asperosaponin VI partially reversed. GW9662 treatment prevented the nuclear transfer of PPAR-γ in asperosaponin VI-treated microglia and inhibited the induction of Arg-1+ microglia. Blockade of PPAR-γ signaling also abolished the ability of asperosaponin VI to suppress pro-inflammatory cytokines while elevating anti-inflammatory cytokines in the hippocampus of CMS mice. The asperosaponin VI also promoted interactions between hippocampal microglia and neurons by enhancing CX3CL1/CX3CR1 and CD200/CD200R, and preserved synaptic function based on PSD95, CamKII β and GluA levels, but not in the presence of GW9662. Blockade of PPAR-γ signaling also abolished the antidepressant effects of asperosaponin VI in the SPT, TST and FST. Conclusion CMS in mice induces a pro-inflammatory microglial phenotype that causes reduced crosstalk between microglia and neuron, inflammation and synaptic dysfunction in the hippocampus, ultimately leading to depression-like behaviors. Asperosaponin VI may ameliorate the effects of CMS by inducing microglia to adopt a PPAR-γ-dependent neuroprotective phenotype.

Borneol is a precious monoterpenoid with two chiral structures, (-)-borneol and (+)-borneol. Bornyl diphosphate synthase is the key enzyme in the borneol biosynthesis pathway. Many (+)-bornyl diphosphate synthases have been reported, but no (-)-bornyl diphosphate synthases have been identified. Blumea balsamifera leaves are rich in borneol, almost all of which is (-)-borneol. In this study, we identified a high-efficiency (-)-bornyl diphosphate synthase (BbTPS3) from B. balsamifera that converts geranyl diphosphate (GPP) to (-)-bornyl diphosphate, which is then converted to (-)-borneol after dephosphorylation in vitro. BbTPS3 exhibited a Km value of 4.93 ± 1.38 μM for GPP, and the corresponding kcat value was 1.49 s−1. Multiple strategies were applied to obtain a high-yielding (-)-borneol producing yeast strain. A codon-optimized BbTPS3 protein was introduced into the GPP high-yield strain MD, and the resulting MD-B1 strain produced 1.24 mg·L-1 (-)-borneol. After truncating the N-terminus of BbTPS3 and adding a Kozak sequence, the (-)-borneol yield was further improved by 4-fold to 4.87 mg·L-1. Moreover, the (-)-borneol yield was improved by expressing the fusion protein module of ERG20F96W-N127W-YRSQI-t14-BbTPS3K2, resulting in a final yield of 12.68 mg·L-1 in shake flasks and 148.59 mg·L-1 in a 5-L bioreactor. This work is the first reported attempt to produce (-)-borneol by microbial fermentation.

Dipsacus asperoides is a perennial herb, the roots of which are abundant in asperosaponin VI, which has important medicinal value. However, the molecular mechanism underlying the biosynthesis of asperosaponin VI in D. asperoides remains unclear. In present study, a comprehensive investigation of asperosaponin VI biosynthesis was conducted at the levels of metabolite and transcript during root development. The content of asperosaponin VI was significantly accumulated in two-leaf stage roots, and the spatial distribution of asperosaponin VI was localized in the xylem. The concentration of asperosaponin VI gradually increased in the root with the development process. Transcriptome analysis revealed 3916 unique differentially expressed genes (DEGs) including 146 transcription factors (TFs) during root development in D. asperoides. In addition, α-linolenic acid metabolism, jasmonic acid (JA) biosynthesis, JA signal transduction, sesquiterpenoid and triterpenoid biosynthesis, and terpenoid backbone biosynthesis were prominently enriched. Furthermore, the concentration of JA gradually increased, and genes involved in α-linolenic acid metabolism, JA biosynthesis, and triterpenoid biosynthesis were up-regulated during root development. Moreover, the concentration of asperosaponin VI was increased following methyl jasmonate (MeJA) treatment by activating the expression of genes in the triterpenoid biosynthesis pathway, including acetyl-CoA acetyltransferase (DaAACT), 3-hydroxy-3-methylglutaryl coenzyme A synthase (DaHMGCS), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (DaHMGCR). We speculate that JA biosynthesis and signaling regulates the expression of triterpenoid biosynthetic genes and facilitate the biosynthesis of asperosaponin VI. The results suggest a regulatory network wherein triterpenoids, JA, and TFs co-modulate the biosynthesis of asperosaponin VI in D. asperoides.

Bletilla ochracea Schltr. (Orchidaceae) is a traditional medicinal plant widely distributed in the south-central part of China. The complete chloroplast genome of B. ochracea was sequenced using the Illumina HiSeq X Ten platform. The chloroplast genome was 160,018 bp in length, which contained two short inverted repeat (IRa and IRb) regions of 26,295 bp and was separated by a large single copy (LSC) region of 88,270 bp and a small single copy (SSC) region of 19,158 bp. The GC content of the whole chloroplast genome was 37.2%. The chloroplast DNA of B. ochracea consisted of 114 distinct genes, including 80 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. The phylogenetic tree showed that B. ochracea was sister to B. formosana. Meanwhile, the monophyletic clade formed by all species of genus Bletilla was closely related to genus Thunia.

Given the ability of akebia saponin D (ASD) to protect various types of stem cells, in the present study, we hypothesized that ASD could promote the proliferation, differentiation, and survival of neural stem/precursor cells (NSPCs), even in a microglia-mediated inflammatory environment, thereby mitigating inflammation-related neuropsychopathology. We established a mouse model of chronic neuroinflammation by exposing animals to low-dose lipopolysaccharide (LPS, 0.25 mg/kg/d) for 14 days. The results showed that chronic exposure to LPS strikingly reduced hippocampal levels of PI3K and pAkt and neurogenesis in mice. In the presen of a microglia-mediated inflammatory niche, the PI3K-Akt signaling in cultured NSPCs was inhibited, promoting their apoptosis and differentiation into astrocytes, while decreasing neurogenesis. Conversely, ASD strongly increased the levels of PI3K and pAkt and stimulated NSPC proliferation, survival and neuronal differentiation in the microglia-mediated inflammatory niche in vitro and in vivo. ASD also restored the synaptic function of hippocampal neurons and ameliorated depressive- and anxiety-like behaviors and cognitive impairment in mice chronically exposed to LPS. The results from network pharmacology analysis showed that the PI3K-AKT pathway is one of the targets of ASD to against major depressive disorder (MDD), anxiety and Alzheimer’s disease (AD). And the results from molecular docking based on computer modeling showed that ASD is bound to the interaction interface of the PI3K and AKT. The PI3K-Akt inhibitor LY294002 blocked the therapeutic effects of ASD in vitro and in vivo. These results suggested that ASD protects NSPCs from the microglia-mediated inflammatory niche, promoting their proliferation, survival and neuronal differentiation, as well as ameliorating depressive- and anxiety-like behaviors and cognitive impairment by activating the PI3K-AKT pathway. Our work suggests the potential of ASD for treating Alzheimer’s disease, depression and other cognitive disorders involving impaired neurogenesis by microglia-mediated inflammation.

Bletilla striata (Thunb.) Rchb.f. is a perennial herb belonging to the Orchidaceae family. Its tubers are used in traditional Chinese medicine to treat gastric ulcers, inflammation, silicosis tuberculosis, and pneumogastric hemorrhage. It has been reported that different soil types can affect the growth of B. striata and the accumulation of secondary metabolites in its tubers, but the biological mechanisms underlying these effects remain unclear. In this study, we compared agronomic traits and the accumulation of secondary metabolites (extractum, polysaccharide, total phenol, militarine) in B. striata grown in sandy loam or sandy clay soil. In addition, we compared physicochemical properties and microbial communities between the two soil types. In pot experiments, we tested how irradiating soil or transplanting microbiota from clay or loam into soil affected B. striata growth and accumulation of secondary metabolites. The results showed that sandy loam and sandy clay soils differed significantly in their physicochemical properties as well as in the structure and composition of their microbial communities. Sandy loam soil had higher pH, SOM, SOC, T-Ca, T-N, T-Mg, T-Mn, T-Zn, A-Ca, A-Mn, and A-Cu than sandy clay soil, but significantly lower T-P, T-K, T-Fe, and A-P content. Sandy loam soil showed 7.32% less bacterial diversity based on the Shannon index, 19.59% less based on the Ace index, and 24.55% less based on the Chao index. The first two components of the PCoA explained 74.43% of the variation in the bacterial community (PC1 = 64.92%, PC2 = 9.51%). Similarly, the first two components of the PCoA explained 58.48% of the variation in the fungal community (PC1 = 43.67%, PC2 = 14.81%). The microbiome associated with sandy clay soil can promote the accumulation of militarine in B. striata tubers, but it inhibits the growth of B. striata. The accumulation of secondary metabolites such as militarine in B. striata was significantly higher in sandy clay than in sandy loam soil. Conversely, B. striata grew better in sandy loam soil. The microbiome associated with sandy loam soil can promote the growth of B. striata, but it reduces the accumulation of militarine in B. striata tubers. Pot experiment results further confirmed that the accumulation of secondary metabolites such as militarine was higher in soil transplanted with loam microbiota than in soil transplanted with clay microbiota. These results may help guide efforts to improve B. striata yield and its accumulation of specific secondary metabolites.

Abstract Background The ternary cropping system of Gastradia elata depends on a symbiotic relationship with the mycorrhizal fungi Armillaria mellea, which decays wood to assimilate nutrition for the growth of G. elata. The composition of microbe flora as key determinants of rhizoshere and mycorrhizoshere soil fertility and health was investigated to understand how G. elata and A. mellea impacted on its composition. The next generation pyrosequencing analysis was applied to assess the shift of structure of microbial community in rhizoshere of G. elata and mycorrhizoshere of A. mellea compared to the control sample under agriculture process. Results The root-associated microbe floras were significantly impacted by rhizocompartments (including rhizoshere and mycorrhizoshere) and agriculture process. Cropping process of G. elata enhanced the richness and diversity of the microbial community in rhizoshere and mycorrhizoshere soil. Furthermore, planting process of G. elata significantly reduced the abundance of phyla Basidiomycota, Firmicutes and Actinobacteria, while increased the abundance of phyla Ascomycota, Chloroflexi, Proteobacteria, Planctomycetes, and Gemmatimonadetes in rhizoshere and mycorrhizoshere. Besides, A. mellea and G. elata significantly enriched several members of saprophytoic and pathogenic fungus (i.e., Exophiala, Leptodontidium, Cosmospora, Cercophora, Metarhizium, Ilyonectria, and Sporothrix), which will enhance the possibility of G. elata disease incidence. At the same time, the ternary cropping system significantly deterred several members of beneficial ectomycorrhizal fungus (i.e., Russula, Sebacina, and Amanita), which will reduce the ability to protect G. elata from diseases. Conclusions In the ternary cropping system of G. elata, A. mellea and G. elata lead to imbalance of microbial community in rhizoshere and mycorrhizoshere soil, suggested that further studies on maintaining the balance of microbial community in A. mellea mycorrhizosphere and G. elata rhizosphere soil under field conditions may provide a promising avenue for high yield and high quality G. elata.

Fusarium wilt (FW) is a primary replant disease that affects Pseudostellaria heterophylla (Taizishen) and is caused by Fusarium oxysporum, which occurs widely in China under the continuous monocropping regime. However, the ternary interactions among the soil microbiota, P. heterophylla, and F. oxysporum remain unknown. We investigated the potential interaction relationship by which the pathogen-mediated P. heterophylla regulates the soil and the tuberous root microbiota via high-throughput sequencing. Plant–pathogen interaction assays were conducted to measure the arrival of F. oxysporum and Pseudomonas poae at the tuberous root via qPCR and subsequent seedling disease incidence. A growth assay was used to determine the effect of the tuberous root crude exudate inoculated with the pathogen on P. poae. We observed that pathogen-mediated P. heterophylla altered the diversity and the composition of the microbial communities in its rhizosphere soil and tuberous root. Beneficial microbe P. poae and pathogen F. oxysporum were significantly enriched in rhizosphere soil and within the tuberous root in the FW group with high severity. Correlation analysis showed that, accompanied with FW incidence, P. poae co-occurred with F. oxysporum. The aqueous extract of P. heterophylla tuberous root infected by F. oxysporum substantially promoted the growth of P. poae isolates (H1-3-A7, H2-3-B7, H4-3-C1, and N3-3-C4). These results indicated that the extracts from the tuberous root of P. heterophylla inoculated with F. oxysporum might attract P. poae and promote its growth. Furthermore, the colonization assay found that the gene copies of sucD in the P. poae and F. oxysporum treatment (up to 6.57 × 1010) group was significantly higher than those in the P. poae treatment group (3.29 × 1010), and a pathogen-induced attraction assay found that the relative copies of sucD of P. poae in the F. oxysporum treatment were significantly higher than in the H2O treatment. These results showed that F. oxysporum promoted the colonization of P. poae on the tuberous root via F. oxysporum mediation. In addition, the colonization assay found that the disease severity index in the P. poae and F. oxysporum treatment group was significantly lower than that in the F. oxysporum treatment group, and a pathogen-induced attraction assay found that the disease severity index in the F. oxysporum treatment group was significantly higher than that in the H2O treatment group. Together, these results suggest that pathogen-mediated P. heterophylla promoted and assembled plant-beneficial microbes against plant disease. Therefore, deciphering the beneficial associations between pathogen-mediated P. heterophylla and microbes can provide novel insights into the implementation and design of disease management strategies.

The morphology of microglial cells is often closely related to their functions. The mechanisms that regulate microglial ramification are not well understood. Here we reveal the biological mechanisms by which astrocytes regulate microglial ramification. Morphological variation in mouse microglial cultures was measured in terms of cell area as well as branch number and length. Effects on microglial ramification were analyzed after microinjecting the toxin L-alpha-aminoadipic acid (L-AAA) in the mouse cortex or hippocampus to ablate astrocytes, and after culturing microglia on their own in an astrocyte-conditioned medium (ACM) or together with astrocytes in coculture. TGF-β expression was determined by Western blotting, immunohistochemistry, and ELISA. The TGF-β signaling pathway was blocked by the TGF-β antibody to assess the role of TGF-β on microglial ramification. The results showed that microglia had more and longer branches and smaller cell bodies in brain areas where astrocytes were abundant. In the mouse cortex and hippocampus, ablation of astrocytes by L-AAA decreased number and length of microglial branches and increased the size of cell bodies. Similar results were obtained with isolated microglia in culture. However, isolated microglia were able to maintain their multibranched structure for a long time when cultured on astrocyte monolayers. Ameboid microglia isolated from P0 to P3 mice showed increased ramification when cultured in ACM or on astrocyte monolayers. Microglia cultured on astrocyte monolayers showed more complex branching structures than those cultured in ACM. Blocking astrocyte-derived TGF-β decreased microglial ramification. Astrocytes induced the formation of protuberances on branches of microglia by forming glial fibers that increased traction. These experiments in mice suggest that astrocytes promote microglial ramification by forming glial fibers to create traction and by secreting soluble factors into the surroundings. For example, astrocyte-secreted TGF-β promotes microglia to generate primitive branches, whose ramification is refined by glial fibers.

Plant cyclic peptides (CPs) are a large group of small molecule metabolites found in a wide variety of plants, including traditional Chinese medicinal plants. However, the majority of plant CPs have not been studied for their biosynthetic mechanisms, including heterophyllin B (HB), which is one of the characteristic chemical components of Pseudostellaria heterophylla. Here, we screened the precursor gene (prePhHB) of HB in P. heterophylla and functionally identified its correctness in vivo and in vitro. First, we developed a new method to screen the precursors of HB from 16 candidate linear peptides. According to transcriptome sequencing data, we cloned the genes that encoded the HB precursor peptides and confirmed that the prePhHB-encoded precursor peptide could enzymatically synthesize HB. Next, we generated the transgenic tobacco that expressed prePhHB, and the results showed that HB was detected in transgenic tobacco. Moreover, we revealed that prePhHB gene expression is positively correlated with HB accumulation in P. heterophylla. Mutations in the prePhHB gene may influence the accumulation of HB in P. heterophylla. These results suggest that HB is ribosomally synthesized and posttranslationally modified peptide (RiPP) derived from the precursor gene prePhHB-encoded precursor peptide, and the core peptide sequence of HB is IFGGLPPP in P. heterophylla. This study developed a new idea for the rapid identification of Caryophyllaceae-type CP precursor peptides via RNA-sequencing data mining.

[This corrects the article DOI: 10.1371/journal.pone.0164235.].

Pseudostellaria heterophylla (Miq.) Pax is a mild tonic herb widely cultivated in the Southern part of China. The tuberous roots of P. heterophylla accumulate high levels of secondary metabolism products of medicinal value such as saponins, flavonoids, and isoquinoline alkaloids. Despite numerous studies on the pharmacological importance and purification of these compounds in P. heterophylla, their biosynthesis is not well understood. In the present study, we used Illumina HiSeq 4000 sequencing platform to sequence the RNA from flowers, leaves, stem, root cortex and xylem tissues of P. heterophylla. We obtained 616,413,316 clean reads that we assembled into 127, 334 unique sequences with an N50 length of 951 bp. Among these unigenes, 53,184 unigenes (41.76%) were annotated in a public database and 39, 795 unigenes were assigned to 356 KEGG pathways; 23,714 unigenes (8.82%) had high homology with the genes from Beta vulgaris. We discovered 32, 095 DEGs in different tissues and performed GO and KEGG enrichment analysis. The most enriched KEGG pathway of secondary metabolism showed up-regulated expression in tuberous roots as compared with the ground parts of P. heterophylla. Moreover, we identified 72 candidate genes involved in triterpenoids saponins biosynthesis in P. heterophylla. The expression profiles of 11 candidate unigenes were analyzed by quantitative real-time PCR (RT-qPCR). Our study established a global transcriptome database of P. heterophylla for gene identification and regulation. We also identified the candidate unigenes involved in triterpenoids saponins biosynthesis. Our results provide an invaluable resource for the secondary metabolites and physiological processes in different tissues of P. heterophylla.