The sweating process promotes toxigenic fungi expansion and increases the risk of combined contamination of mycotoxins in Radix Dipsaci.

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.

Chronic exposure to aflatoxin B1 increases hippocampal microglial pyroptosis and vulnerability to stress in mice.

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-1ß) 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.

Gastrodin programs an Arg-1+ microglial phenotype in hippocampus to ameliorate depression- and anxiety-like behaviors via the Nrf2 pathway in mice.

BACKGROUND: Regulating the microglial phenotype is an attractive strategy for treating diseases of the central nervous system such as depression and anxiety. Gastrodin can quickly cross the blood-brain barrier and mitigate microglia-mediated inflammation, which widely used to treat a variety of central nervous system diseases associated with microglial dysfunction. However, the molecular mechanism by which gastrodin regulates the functional phenotype of microglia remains unclear. PURPOSE: Since the transcription factor "nuclear factor erythroid 2-related factor 2″ (Nrf2) is associated with the anti-inflammatory effects of gastrodin, we hypothesized that gastrodin induces Nrf2 expression in microglia and thereby programs an anti-inflammatory phenotype. STUDY DESIGN: Male C57BL/6 mice, treated or not with gastrodin, were given lipopolysaccharide (LPS) at 0.25 mg/kg/d for 10 days to induce chronic neuroinflammation. The effects of gastrodin on microglial phenotypes, neuroinflammation and depression- and anxiety-like behaviors were evaluated. In another experiment, animals were treated with Nrf2 inhibitor ML385 throughout the 13-day gastrodin intervention period. METHODS: The effects of gastrodin on depression- and anxiety-like behaviors were evaluated through the sucrose preference test, forced swimming test, open field test and elevated plus-maze test; as well as its effects on morphology and molecular and functional phenotypes of hippocampal microglia through immunohistochemistry, real-time PCR and enzyme-linked immunosorbent assays. RESULTS: Chronic exposure to LPS caused hippocampal microglia to secrete inflammatory cytokines, their somata to enlarge, and their dendrites to lose branches. These changes were associated with depression- and anxiety-like behaviors. Gastrodin blocked these LPS-induced alterations and promoted an Arg-1+ microglial phenotype that protected neurons from injury. The effects of gastrodin were associated with Nrf2 activation, whereas blockade of Nrf2 antagonized gastrodin. CONCLUSION: These results suggest that gastrodin acts via Nrf2 to promote an Arg-1+ microglial phenotype, which buffers the harmful effects of LPS-induced neuroinflammation. Gastrodin may be a promising drug against central nervous system diseases that involve microglial dysfunction.

Akebia saponin D acts via the PPAR-gamma pathway to reprogramme a pro-neurogenic microglia that can restore hippocampal neurogenesis in mice exposed to chronic mild stress.

BACKGROUND: Using drugs to modulate microglial function may be an effective way to treat disorders, such as depression, that involve impaired neurogenesis. Akebia saponin D (ASD) can cross the blood-brain barrier and exert anti-inflammatory and neuroprotective effects, so we wondered whether it might influence adult hippocampal neurogenesis to treat depression. METHODS: We exposed C57BL/6 mice to chronic mild stress (CMS) as a model of depression and then gave them ASD intraperitoneally once daily for 3 weeks. We investigated the effects of ASD on microglial phenotype, hippocampal neurogenesis, and animal behavior. The potential role of the peroxisome proliferator-activated receptor-gamma (PPAR-γ) or BDNF-TrkB pathway in the pro-neurogenesis and anti-depressant of ASD was identified using there inhibitors GW9662 and K252a, respectively. The neurogenic effects of ASD-treated microglia were evaluated using conditioned culture methods. RESULTS: We found that CMS upregulated pro-inflammatory factors and inhibited hippocampal neurogenesis in dentate gyrus of mice, while inducing depressive-like behaviors. Dramatically, ASD (40 mg/kg) treatment reprogrammed an arginase (Arg)-1+ microglial phenotype in dentate gyrus, which increased brain-derived neurotrophic factor (BDNF) expression and restored the hippocampal neurogenesis, and partially ameliorated the depressive-like behaviors of the CMS-exposed mice. K252a or neurogenesis inhibitor blocked the pro-neurogenic, anti-depressant effects of ASD. Furthermore, ASD activated PPAR-γ in dentate gyrus of CMS mice as well as in primary microglial cultures treated with lipopolysaccharide. Blocking the PPAR-γ using GW9962 suppressed the ASD-reprogrammed Arg-1+ microglia and BDNF expression in dentate gyrus of CMS mice. Such blockade abolished the promoted effects of ASD-treated microglia on NSPC proliferation, survival, and neurogenesis. The pro-neurogenic and anti-depressant effects of ASD were blocked by GW9962. CONCLUSION: These results suggested that ASD acts via the PPAR-γ pathway to induce a pro-neurogenic microglia in dentate gyrus of CMS mice that can increase BDNF expression and promote NSPC proliferation, survival, and neurogenesis.

Spatial distribution and sources of heavy metals in the sediment and soils of the Yancheng coastal ecosystem and associated ecological risks.

Studies of heavy metal pollution are essential for the protection of coastal environments. In this study, positive matrix factorization (PMF) and a GeoDetector model were used to evaluate the sources of heavy metal contamination and associated ecological risks along the Yancheng Coastal Wetland. The distribution of heavy metals was shown to be greatly affected by clay content, except for Cr in shoal. Components from 6.5 to 9φ have the strongest ability to absorb heavy metals, where the effects of Cd and Zn sequestration in the wetlands were most apparent. The abilities of various wetland environments to sequester heavy metals were shown to be Spartina alterniflora wetland > woodland > Phragmites australis wetland > aquaculture pond > shoal > paddy > meadow > dry land. The sources of the heavy metals included parent soil material (59%), agriculture (15%), and industrial pollutants (26%). According to the single-factor pollution index, there was no evidence of pollution except Cr and Pb. In general, the heavy metal pollution was insignificant. The order of pollution loading index was shoal > paddy field > dry land > Spartina Alterniflora wetland > aquaculture ponds > woodland > meadow > Phragmites australis wetland. The ecological harm of heavy metal exposure was slight except for Cd and Hg, where vehicle emissions appeared to be the main cause of heavy metal pollution.

Akebia saponin D protects hippocampal neurogenesis from microglia-mediated inflammation and ameliorates depressive-like behaviors and cognitive impairment in mice through the PI3K-Akt pathway.

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.

Gasdermin D-mediated microglial pyroptosis exacerbates neurotoxicity of aflatoxins B1 and M1 in mouse primary microglia and neuronal cultures.

Aflatoxin B1 (AFB1) disrupts the blood-brain barrier by poisoning the vascular endothelial cells and astrocytes that maintain it. It is important to examine whether aflatoxin B1 or its metabolite, aflatoxin M1 (AFM1), affect microglia, which as the "immune cells" in the brain may amplify their damaging effects. Here we evaluated the toxicity of AFB1 and AFM1 against primary microglia and found that both aflatoxins decreased the viability of primary microglia and increased the leakage of lactate dehydrogenase, gamma-H2AX expression, nuclear lysis, necrosis and apoptosis in a dose-dependent manner. The potential contribution of microglia to the toxic effects of aflatoxins was assessed in transwell co-culture experiments involving microglia, neurons, astrocytes, oligodendrocytes or neural stem/precursor cells. And we found that the toxic effects of both aflatoxins on various types of nervous system cells were greater in the presence of microglia than in their absence. We also found that both aflatoxins induced gasdermin D-mediated microglial pyroptosis and inflammatory cytokine expression by activating the NLRP3 inflammasome. Blockade of gasdermin D activity in AFB1- or AFM1-treated primary microglia using dimethyl fumarate (DMF) reduced the release of IL-1ß, IL-18 and nitric oxide, as well as the neurotoxicity of microglia-conditioned medium to neurons, astrocytes, oligodendrocytes and neural stem/precursor cells. These data suggested that the toxicity of AFB1 and AFM1 on various cells of the central nervous system is due, remarkably, the gasdermin D-mediated microglial pyroptosis exacerbates their neurotoxicity.

Asperosaponin VI ameliorates the CMS-induced depressive-like behaviors by inducing a neuroprotective microglial phenotype in hippocampus via PPAR-γ pathway.

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 ß 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.

[Correlation analysis between continuous cropping obstacle of Gastrodia elata and Ilyonectria fungi and relieving strategy].

The continuous cropping obstacle of Gastrodia elata is outstanding, but its mechanism is still unclear. In this study, microbial changes in soils after G. elata planting were investigated to explore the mechanism correlated with continuous cropping obstacle. The changes of species and abundance of fungi and bacteria in soils planted with G. elata after 1, 2, and 3 years were compared. The pathogenic fungi that might cause continuous cropping diseases of G. elata were isolated. Finally, the prevention and control measures of soil-borne fungal diseases of G. elata were investigated with the rotation planting pattern of "G. elata-Phallus impudicus". The results showed that G. elata planting resulted in the decrease in bacterial and fungal community stability and the increase in harmful fungus species and abundance in soils. This change was most obvious in the second year after G. elata planting, and the soil microbial community structure could not return to the normal level even if it was left idle for another two years. After G. elata planting in soils, the most significant change was observed in Ilyonectria cyclaminicola. The richness of the Ilyonectria fungus in soils was significantly positively correlated with the incidence of G. elata diseases. When I. cyclaminicola was inoculated in the sterile soil, the rot rate of G. elata was also significantly increased. After planting one crop of G. elata and one to three crops of P. impudicus, the fungus community structure in soils gradually recovered, and the abundance of I. cyclaminicola decreased year by year. Furthermore, the disease rate of G. elata decreased. The results showed that the cultivation of G. elata made the Ilyonectria fungi the dominant flora in soils, and I. cyclaminicola served as the main pathogen of continuous cropping diseases of G. elata, which could be reduced by rotation planting with P. impudicus.

Mapping the Plasticity of Morphology, Molecular Properties and Function in Mouse Primary Microglia.

Microglia exert diverse functions by responding in diverse ways to different stimuli, yet little is known about the plasticity of various phenotypes that microglia display. We used interferon (IFN)-γ, interleukin (IL)-4 and IL-10 to induce different phenotypes in mouse primary microglia. RNA sequencing was used to identify genes differentially expressed in response to stimulation, and the different stimulated populations were compared in terms of morphology, proliferative capacity, phagocytic ability and neurotoxicity. IFN-γ induced an "immunodefensive" phenotype characterizing both induction of filopodia and upregulation of inducible nitric oxide synthase (iNOS) and tumor necrosis factor α. Microglia with this phenotype mediated an acute inflammatory response accompanied by excellent proliferative capacity and neurotoxicity, and remained susceptible to remodeling for up to 48 h after initial stimulation. IL-4 induced an enduring "neuroimmunoregulatory" phenotype involving induction of lamellipodium and persistent upregulation of arginase (Arg)-1 and YM-1 expression. Microglia with this phenotype remained susceptible to remodeling for up to 24 h after initial stimulation. IL-10 induced an "immunosuppressive" phenotype involving induction of ameba-like morphology and upregulation of transforming growth factor ß and IL-10 as well as inhibition of inflammation. This phenotype was accompanied by inhibition of self-proliferation, while its morphology, molecular properties and function were the least susceptible to remodeling. IFN-γ, IL-4, or IL-10 appear to induce substantially different phenotypes in microglia. The immunodefensive microglia induced by IFN-γ showed remarkable plasticity, which may help repair CNS inflammation damage under pathological condition. Chronic activation with IL-10 decreases microglial plasticity, which may help protect the brain form the immune response. Our research justifies and guides further studies into the molecular pathways that operate in each phenotype to help multitasking microglia regulate homeostasis in the brain.