The Liquid Fermentation Process for Mycelia of Poria cocos (Agaricomycetes) by Single-Factor Experimentation and Response Surface Methodology.

Liquid fermentation could yield substantial mycelia mass and valuable secondary metabolites in large-scale production within a short, fermented duration. The liquid fermented process of mycelia of Poria cocos was optimized using a combination of single-factor experimentation and response surface methodology (RSM) to obtain more extract of P. cocos. The optimal conditions were determined as follows: The carbon source concentration at 1%, the nitrogen source concentration at 1%, the inoculum volume at 7% and a culture time of 9 d. Under these conditions, the ethyl acetate extract mass of P. cocos mycelia reached 0.0577 ± 0.0041 mg. There were significant interactions between nitrogen source concentration and cultivation time. The predicted values by the mathematical model based on the response surface analysis showed a close agreement with experimental data.

Identification of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing.

BACKGROUND: Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids. RESULT: In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach. CONCLUSION: The findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.

Guizhi Fuling capsule relieves memory deficits by inhibition of microglial neuroinflammation through blocking JAK2/STAT3 pathway in presenilin1/2 conditional double knockout mice.

Chronic neuroinflammation has been regarded as an important part of the pathological initiation of Alzheimer's disease (AD), which is associated with the regulation of microglial activation. Preventing microglial activation to inhibit neuroinflammation may become a potential target for the treatment of neurodegenerative diseases. Guizhi Fuling capsule (GZFL) has a strong repression on inflammatory responses. Here, the presenilin1/2 conditional double knockout (PS cDKO) mice, a well-established mouse model of AD, were divided into: WT mice (WT), WT mice+GZFL (WT+GZFL), PS cDKO mice (cDKO), and PS cDKO mice+GZFL (cDKO+GZFL). Mice in the WT+GZFL and cDKO+GZFL group were fed standard chow containing 2000 ppm GZFL for 90 days. After 60 days of GZFL treatment, mice were given to behavioral tests for 30 days in order to explore the effects of GZFL on cognitive and motor function. Then, mice were sacrificed for examining the effects of GZFL on inflammation. Furthermore, primary microglia were obtained from neonatal Sprague-Dawley rats and pretreated with or without GZFL (50 µg/ml) for 1 h in the absence or presence of lipopolysaccharide (LPS) (100 ng/ml) stimulation to speculate whether the underlying mechanism of GZFL's anti-inflammatory potential was closely associated with Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Our findings indicated that GZFL has the ability to alleviate memory deficits in PS cDKO mice, which attributes to the improvement of neuroinflammation by inhibiting microglial activation and the levels of pro-inflammatory mediators. In addition, GZFL could inverse the tau hyperphosphorylation and the lessened expression of synaptic proteins in hippocampus of PS cDKO mice. Furthermore, GZFL prevented LPS-induced neuroinflammatory responses in primary microglia by decreasing the levels of pro-inflammatory mediators. It is noteworthy that therapeutic effects of GZFL on memory impairment are depended on the inhibition of neuroinflammatory responses by the blockage of JAK2/STAT3 signaling pathway. Taken together, GZFL may be an effective compound Chinese medicine for the improvement and postponement of neurodegenerative progression in AD.

Inhibition of Osteoclast Differentiation and Promotion of Osteogenic Formation by Wolfiporia extensa Mycelium.

Osteoporosis, Greek for "porous bone," is a bone disease characterized by a decrease in bone strength, microarchitectural changes in the bone tissues, and an increased risk of fracture. An imbalance of bone resorption and bone formation may lead to chronic metabolic diseases such as osteoporosis. Wolfiporia extensa, known as "Bokryung" in Korea, is a fungus belonging to the family Polyporaceae and has been used as a therapeutic food against various diseases. Medicinal mushrooms, mycelium and fungi, possess approximately 130 medicinal functions, including antitumor, immunomodulating, antibacterial, hepatoprotective, and antidiabetic effects, and are therefore used to improve human health. In this study, we used osteoclast and osteoblast cell cultures treated with Wolfiporia extensa mycelium water extract (WEMWE) and investigated the effect of the fungus on bone homeostasis. Subsequently, we assessed its capacity to modulate both osteoblast and osteoclast differentiation by performing osteogenic and anti-osteoclastogenic activity assays. We observed that WEMWE increased BMP-2-stimulated osteogenesis by inducing Smad-Runx2 signal pathway axis. In addition, we found that WEMWE decreased RANKL-induced osteoclastogenesis by blocking c-Fos/NFATc1 via the inhibition of ERK and JNK phosphorylation. Our results show that WEMWE can prevent and treat bone metabolic diseases, including osteoporosis, by a biphasic activity that sustains bone homeostasis. Therefore, we suggest that WEMWE can be used as a preventive and therapeutic drug.

Effect of Poria cocos Mushroom Polysaccharides (PCPs) on the Quality and DNA Methylation of Cryopreserved Shanghai White Pig Spermatozoa.

In this study, we explore the effects of Poria cocos mushroom polysaccharides (PCPs) on the quality and DNA methylation of the cryopreserved spermatozoa of Shanghai white pigs. A total of 24 ejaculates (three ejaculate samples per boar) from eight Shanghai white pigs were manually collected. The pooled semen was diluted with a based extender supplemented with different concentrations of PCPs (0, 300, 600, 900, 1200, and 1500 µg/mL). Once thawed, the quality of the spermatozoa and their antioxidant function were assessed. In the meantime, the effect of spermatozoa DNA methylation was also analyzed. The results show that compared with the control group, 600 µg/mL of PCPs significantly improves the spermatozoa viability (p < 0.05). The motility and plasma membrane integrity of the frozen-thawed spermatozoa are significantly higher after treatment with 600, 900, and 1200 µg/mL of PCPs compared with the control group (p < 0.05). In comparison with the control group, the percentages of acrosome integrity and mitochondrial activity are significantly enhanced after the application of 600 and 900 µg/mL PCPs (p < 0.05). The reactive oxygen species (ROS), the malondialdehyde (MDA) levels, and the glutathione peroxidase (GSH-Px) activity, in comparison with the control group, are significantly decreased in all groups with PCPs (all p < 0.05). The enzymatic activity of superoxide dismutase (SOD) in spermatozoa is significantly higher in the treatment with 600 µg/mL of PCPs than in the other groups (p < 0.05). As compared with the control group, a significant increase in the catalase (CAT) level is found in the groups with PCPs at 300, 600, 900, and 1200 µg/mL (all p < 0.05). In comparison with the control group, the 5-methylcytosine (5-mC) levels are significantly decreased in all groups with PCPs (all p < 0.05). As a result of these findings, a certain amount of PCPs (600-900 µg/mL) added to the cryodiluent can significantly improve the quality of Shanghai white pig spermatozoa and can also reduce the methylation of spermatozoa DNA caused by cryopreservation. This treatment strategy may establish a foundation for the cryopreservation of semen from pigs.

First report on the regulation and function of carbon metabolism during large sclerotia formation in medicinal fungus Wolfiporia cocos.

The medicinal fungus Wolfiporia cocos colonizes and then grows on the wood of Pinus species, and utilizes a variety of Carbohydrate Active Enzymes (CAZymes) to degrades wood for the development of large sclerotia that is mostly built up of beta-glucans. Some differentially expressed CAZymes were revealed by comparisons between the mycelia cultured on potato dextrose agar (PDA) and sclerotia formed on pine logs in previous studies. Here, different profile of expressed CAZymes were revealed by comparisons between the mycelia colonization on pine logs (Myc.) and sclerotia (Scl.b). To further explore the regulation and function of carbon metabolism in the conversion of carbohydrates from Pine species by W. cocos, the transcript profile of core carbon metabolism was firstly analyzed, and it was characterized by the up-regulated expression of genes in the glycolysis pathway (EMP) and pentose phosphate pathway (PPP) in Scl.b, as well as high expression of genes in the tricarboxylic acid cycle (TCA) in both Myc. and Scl.b stages. The conversion between glucose and glycogen and between glucose and ß-glucan was firstly identified as the main carbon flow in the differentiation process of W. cocos sclerotia, with a gradual increase in the content of ß-glucan, trehalose and polysaccharide during this process. Additionally, gene functional analysis revealed that the two key genes (PGM and UGP1) may mediate the formation and development of W. cocos sclerotia possibly by regulating ß-glucan synthesis and hyphal branching. This study has shed light on the regulation and function of carbon metabolism during large W. cocos sclerotium formation and may facilitate its commercial production.

Four polysaccharides isolated from Poria cocos mycelium and fermentation broth supernatant possess different activities on regulating immune response.

Four polysaccharide fractions were isolated and purified from the culture supernatant and mycelium of Poria cocos, and differences in their immunomodulatory activity were investigated. The average molecular weights of EPS-0M, EPS-0.1M, IPS-0M, and IPS-0.1M were 1.77 × 103, 2.01 × 103, 0.03 × 103 and 4.97 × 103 kDa, respectively. They all mainly consisted of 5 monosaccharides, including glucose, mannose, galactose, fucose and rhamnose, but with different molar ratios. At a dose of 50 µg/mL, EPS-0M, EPS-0.1M, and IPS-0.1M significantly increased the production of nitric oxide (NO), as well as the mRNA and protein levels of pro-inflammatory factors including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) in RAW264.7 cells, suggesting that they enhanced macrophage-mediated innate immunity. Moreover, based on the in vitro inflammation model of lipopolysaccharide (LPS)-stimulated RAW264.7 cells, EPS-0M, EPS-0.1M and IPS-0M but not IPS-0.1M could inhibit the LPS-induced excessive inflammatory response, including NO, IL-6, TNF-α, IL-1ß production and gene transcription. Interestingly, IPS-0M showed a relatively poor immunostimulatory effect, but had the strongest inhibitory effect against the LPS-induced RAW264.7 inflammatory response. Furthermore, our results indicate that the nuclear factor-kappa B (NF-κB) pathway is associated with the immunomodulatory effects of the polysaccharide samples on RAW264.7 cells. This study can provide a reference for the more targeted application of different polysaccharide components from Poria cocos for human health.

The Effects of Poria cocos on Rho Signaling-Induced Regulation of Mobility and F-Actin Aggregation in MK-801-Treated B35 and C6 Cells.

Methods: B35 neuronal cells and C6 glial cells were incubated with MK-801 for 7 days followed by MK-801, MK801 in combination with water extracts of P. cocos (PRP for P. cocos cum Radix Pini or WP for White Poria) treatment for an additional 7 days. Analysis of cell mobility, F-actin aggregation, and Rho signaling modulation was performed to clarify the roles of PRP or WP in MK-801-treated B35 and C6 cells. Results: MK-801 decreases B35 cell mobility, whereas the inhibited cell migration ability and F-actin aggregation in MK-801-treated B35 or C6 cells could be reversed by PRP or WP. The CDC42 expression in B35 or C6 cells would be reduced by MK-801 and restored by treating with PRP or WP. The RhoA expression was increased by MK-801 in both B35 and C6 cells but was differentially regulated by PRP or WP. In B35 cells, downregulation of PFN1, N-WASP, PAK1, and ARP2/3 induced by MK-801 can be reversely modulated by PRP or WP. PRP or WP reduced the increase in the p-MLC2 expression in B35 cells treated with MK-801. The reduction in ROCK1, PFN1, p-MLC2, and ARP2/3 expression in C6 cells induced by MK-801 was restored by PRP or WP. Reduced N-WASP and PAK1 expression was differentially regulated by PRP or WP in MK-801-treated C6 cells.

Guizhi Fuling pill attenuates liver fibrosis in vitro and in vivo via inhibiting TGF-ß1/Smad2/3 and activating IFN-γ/Smad7 signaling pathways.

Liver fibrosis resulting from chronic liver injuries (CLI) is a common health problem globally. Guizhi Fuling pill (GZFL), a modern preparation from traditional Chinese medicine, exhibited anti-dysmenorrhea, anti-inflammatory, and immune-regulative effects. However, the effect of GZFL on liver fibrosis remains unknown. In this research, LX-2 cells were stimulated with acetaldehyde for mimicking liver fibrosis progression in vitro. In addition, carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis was established as well. The data revealed GZFL obviously suppressed the proliferation and triggered the apoptosis of acetaldehyde-stimulated LX-2 cells. In addition, GZFL prevented acetaldehyde-induced activation of LX-2 cells via downregulation of TGF-ß1, p-Smad2, p-Smad3, CUGBP1, and upregulation of p-STAT1 and Smad7. Meanwhile, GZFL significantly alleviated CCl4­induced liver fibrosis, as evidenced by the decrease of ALT and AST levels. Moreover, GZFL downregulated the expressions of TGF-ß1, p-Smad2, p-Smad3, and CUGBP1 in CCl4-treated mice. Furthermore, GZFL remarkably elevated the levels of IFN-γ, p-STAT1, and Smad7 in CCl4-treated mice. To sum up, GZFL was able to inhibit liver fibrosis in vitro and in vivo through suppressing TGF-ß1/Smad2/3-CUGBP1 signaling and activating IFN-γ/STAT1/Smad7 signaling. Thus, GZFL might have a potential to act as a therapeutic agent for anti-fibrotic therapy.

Effect of carbohydrate-feeding strategy on the production, physiochemical changes, anti-inflammation activities of polysaccharides of Poria cocos.

The aim of this research was to physiochemically characterize the structure and study the pharmaceutical benefits of the polysaccharide (PS) produced by Poria cocos using two selected carbohydrates (sucrose, and potato dextrose broth) in the in vitro culture system. A direct dosage effect was shown as sucrose- or PDB-based medium on the PS yield of Paragalago cocos. Very low-molecular-weight PS (<1 kDa) were largely synthesized by sucrose and PDB feeding. Sucrose-feeding mycelia of P. cocos results in a direct dosage effect in the fructose component in the PS. Sucrose and PDB feeding increased the glucose content but decreased the galactose content of PS. This study examined the anti-inflammatory activities of PS in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. At 100 µg/mL and 50 µg/mL, PS from 10 g/L PDB- treatment, denoted as PDB 10, pretreatment showed maximal inhibition of TNF-α and IL-6 release, respectively. Mechanically, PDB10 attenuated IκB from degradation in LPS-induced macrophages, and down-regulated LPS-induced phosphorylation of ERK/AKT/p-38. PDB10 showed dose-dependent inhibition of the LPS induced TGFRII signaling pathways.

Analysis of weighted gene co-expression network of triterpenoid-related transcriptome characteristics from different strains of Wolfiporia cocos.

The fungus Wolfiporia cocos has wide-ranging and important medicinal value, and its dried sclerotia are used as a traditional Chinese medicine. Modern studies have shown that triterpenoid, the active ingredient of W. cocos, have a variety of pharmacological effects. The aim of our research was to determine the key genes related to triterpenoid biosynthesis, which may be useful for the genetic modification of cell-engineered bacteria for triterpenoid biosynthesis. In this study, two monospore strains, DZAC-WP-H-29 (high-yielding) and DZAC-WP-L-123 (low-yielding), were selected from the sexually propagated offspring of strain 5.78 of W. cocos, and the mycelia were cultured for 17, 34, and 51 days, respectively. Weighted gene co-expression network analysis (WGCNA) method was used to analyze transcriptional expressions. The results show that eight core genes (ACAT1-b, hgsA, mvd1, SQLE, erg6, TAT, erg26, and erg11) are associated with the triterpenoid synthesis pathway, and Pm20d2 and norA outside the pathway may be important genes that influence the biosynthesis and accumulation of W. cocos triterpenoid. The biosynthesis of W. cocos triterpenoid is closely related to the expression of sterol metabolic pathway genes. The role of these genes in triterpenoid synthesis complements our knowledge on the biosynthesis and accumulation of W. cocos triterpenoid, and also provides a reference for the target gene modification of engineered bacteria for the fermentation production of triterpenoid.

Removal of Aflatoxin B1 by Edible Mushroom-Forming Fungi and Its Mechanism.

Aflatoxins (AFs) are biologically active toxic metabolites, which are produced by certain toxigenic Aspergillus sp. on agricultural crops. In this study, five edible mushroom-forming fungi were analyzed using high-performance liquid chromatography fluorescence detector (HPLC-FLD) for their ability to remove aflatoxin B1 (AFB1), one of the most potent naturally occurring carcinogens known. Bjerkandera adusta and Auricularia auricular-judae showed the most significant AFB1 removal activities (96.3% and 100%, respectively) among five strains after 14-day incubation. The cell lysate from B. adusta exhibited higher AFB1 removal activity (35%) than the cell-free supernatant (13%) after 1-day incubation and the highest removal activity (80%) after 5-day incubation at 40 °C. In addition, AFB1 analyses using whole cells, cell lysates, and cell debris from B. adusta showed that cell debris had the highest AFB1 removal activity at 5th day (95%). Moreover, exopolysaccharides from B. adusta showed an increasing trend (24-48%) similar to whole cells and cell lysates after 5- day incubation. Our results strongly suggest that AFB1 removal activity by whole cells was mainly due to AFB1 binding onto cell debris during early incubation and partly due to binding onto cell lysates along with exopolysaccharides after saturation of AFB1 binding process onto cell wall components.

Functional Analysis of Sterol O-Acyltransferase Involved in the Biosynthetic Pathway of Pachymic Acid in Wolfiporia cocos.

Pachymic acid from Wolfiporia cocos possesses important medicinal values including anti-bacterial, anti-inflammatory, anti-viral, invigorating, anti-rejection, anti-tumor, and antioxidant activities. However, little is known about the biosynthetic pathway from lanostane to pachymic acid. In particular, the associated genes in the biosynthetic pathway have not been characterized, which limits the high-efficiency obtaining and application of pachymic acid. To characterize the synthetic pathway and genes involved in pachymic acid synthesis, in this study, we identified 11 triterpenoids in W. cocos using liquid chromatography tandem mass spectrometry (LC-MS/MS), and inferred the putative biosynthetic pathway from lanostane to pachymic acid based on analyzing the chemical structure of triterpenoids and the transcriptome data. In addition, we identified a key gene in the biosynthetic pathway encoding W. cocos sterol O-acyltransferase (WcSOAT), which catalyzes tumolusic acid to pachymic acid. The results show that silence of WcSOAT gene in W. cocos strain led to reduction of pachymic acid production, whereas overexpression of this gene increased pachymic acid production, indicating that WcSOAT is involved in pachymic acid synthesis in W. cocos and the biosynthesis of W. cocos pachymic acid is closely dependent on the expression of WcSOAT gene. In summary, the biosynthetic pathway of pachymic acid and the associated genes complement our knowledge on the biosynthesis of W. cocos pachymic acid and other triterpenoids, and also provides a reference for target genes modification for exploring high-efficiency obtaining of active components.

Comparative Studies on Polysaccharides, Triterpenoids, and Essential Oil from Fermented Mycelia and Cultivated Sclerotium of a Medicinal and Edible Mushroom, Poria Cocos.

Poria cocos, an important medicinal and edible fungus, is well known in East Asia. The main active components are water-soluble polysaccharides (WPS) and triterpenoids. Due to the growing market demand, long cultivation period, and consumption of pine trunk during cultivation, alternative methods for producing P. cocos or its active components should be investigated. In this study, WPS, triterpenoids, monosaccharide composition, and essential oil in fermented mycelia and cultivated sclerotium were analyzed using UV spectrophotometry, HPLC, pre-column derivatization, and HS-GC/MS, respectively. Our results showed that the WPS and triterpenoids in mycelia are several times higher than those in sclerotium. Among the 62 compounds identified by HS-GC/MS analysis from the essential oil obtained from the fermentation media and a fresh external layer, the two main fragrances in common were linalool and methyl phenylacetate. Our results suggested that it is applicable to produce polysaccharides and triterpenoids by the fermentation of P. cocos, and a strategy to improve triterpenoid production in the fermentation process was proposed.

Occurrence, distribution and estimated intake of mercury and selenium from sclerotia of the medicinal fungus Wolfiporia cocos from China.

The contamination and distribution of mercury and selenium in the Chinese medicinal fungus Wolfiporia cocos was investigated. The sclerotial mercury concentrations ranged from 0.0043 to 0.027 mg kg1 dry biomass (db) in the inner white part and 0.019-0.074 mg kg-1 db in the shell (outer part), while selenium concentrations ranged from < 0.00048 to 0.0040 mg kg-1 db (white) and 0.0034-0.038 mg kg-1 db (shell). Positive correlations were found for mercury, as well as for mercury and selenium but they were not consistent for both morphological parts. Mercury concentrations exceeded selenium in 16 of 17 white part pools (molar quotient 0.53 to > 10) and in 11 of 17 shell pools (quotient 0.37 to 3.2). The estimated maximal exposure to mercury contained in sclerotial products based on 45 g per capita daily intake for a 60 kg individual over one week, was 0.000020 mg kg-1 body mass (bm; white) and 0.000055 mg kg-1 bm (shell) on a daily basis, and 0.00014 mg kg-1 bm (white) and 0.00039 mg kg-1 bm (shell) on a weekly basis. Relative to mercury, the corresponding intake rates of selenium were considered very low, i.e., they averaged on a daily basis at 0.00075 µg kg-1 bm (white) and 0.0097 µg kg-1 bm (shell) with maximum intake at 0.0030 µg kg-1 bm (white) and 0.028 µg kg-1 bm (shell).

Triterpenes from Poria cocos are revealed as potential retinoid X receptor selective agonists based on cell and in silico evidence.

Poria cocos is an edible and medicinal fungus that is widely used in Traditional Chinese Medicines as well as in modern applications. Retinoid X receptor (RXR) occupies a central place in nuclear receptor signaling, and a pharmacological RXR-dependent pathway is involved in myeloid cell function. Here, structural information for 82 triterpenes from P. cocos and 17 known RXR agonists was collected in a compound library and retrieved for a molecular docking study. Three triterpenes, 16α-hydroxytrametenolic acid (HTA), pachymic acid (PA), and polyporenic acid C (PPAC), were identified as novel RXR-specific agonists based on luciferase reporter assays and in silico evidence. Treatment with HTA, PA, and PPAC significantly induced differentiation of the human promyelocytic leukemia cell line HL-60 with EC50 values of 21.0 ± 0.52, 6.7 ± 0.37, and 9.4 ± 0.65 µM, respectively. These effects were partly blocked by the RXR antagonist UVI3003, suggesting that an RXR-dependent pathway may play an important role in their anti-acute promyelocytic leukemia (APL) effects. Taken together, triterpenes from P. cocos are revealed as naturally occurring RXR selective agonists with the potential for anti-cancer activity. These results suggest a novel approach to the treatment or prevention of APL.

Comparative Analysis of the Characteristics of Triterpenoid Transcriptome from Different Strains of Wolfiporia cocos.

The dried sclerotia of Wolfiporia cocos (Schwein.) Ryvarden & Gilb., a traditional Chinese medicine, has triterpenoid as its main active component. Breeding high-yield triterpenoid in W. cocos is an important research topic at present. We screened out two monosporal strains from the same W. cocos 5.78, high-yielding DZAC-Wp-H-29 (H) and low-yielding DZAC-Wp-L-123 (L), and cultured mycelia for 17 days, 34 days, and 51 days, respectively. Transcriptome analysis results showed that triterpenoid synthesis is closely related to gene expression in triterpenoid synthesis pathways (hydroxymethyl glutaryl-CoA reductase (HMGCR), farnesyl diphosphate synthase (FDPS), 4-hydroxybenzoate polyprenyltransferase (COQ2), C-8 sterol isomerase (ERG2), sterol O-acyltransferase (ACAT), tyrosine aminotransferase (TAT), torulene dioxygenase (CAO2), and sterol-4alpha-carboxylate 3-dehydrogenase (erg26)), and is limited by the expression of enzyme M20 combined with domain protein peptide (Pm20d2), aryl-alcohol dehydrogenase (norA), ISWI chromatin-remodeling complex ATPase ISW2, GroES-like protein (adh), cytochrome P450 (ftmP450-1), and unknown proteins unigene0001029 and unigene0011374. In addition, maintaining high triterpenoid accumulation in W. cocos may require a stable membrane structure, so the accumulation ability may be related to the high synthesis ability of sterols. The low accumulation of triterpenoid in W. cocos may be due to the products of key enzymes increasing flow to other pathways.

Microbiome-metabolomics reveals gut microbiota associated with glycine-conjugated metabolites and polyamine metabolism in chronic kidney disease.

Dysbiosis of the gut microbiome and related metabolites in chronic kidney disease (CKD) have been intimately associated with the prevalence of cardiovascular diseases. Unfortunately, thus far, there is a paucity of sufficient knowledge of gut microbiome and related metabolites on CKD progression partly due to the severely limited investigations. Using a 5/6 nephrectomized (NX) rat model, we carried out 16S rRNA sequence and untargeted metabolomic analyses to explore the relationship between colon's microbiota and serum metabolites. Marked decline in microbial diversity and richness was accompanied by significant changes in 291 serum metabolites, which were mediated by altered enzymatic activities and dysregulations of lipids, amino acids, bile acids and polyamines metabolisms. Interestingly, CCr was directly associated with some microbial genera and polyamine metabolism. However, SBP was directly related to certain microbial genera and glycine-conjugated metabolites in CKD rats. Administration of poricoic acid A (PAA) and Poria cocos (PC) ameliorated microbial dysbiosis as well as attenuated hypertension and renal fibrosis. In addition, treatments with PAA and PC lowered serum levels of microbial-derived products including glycine-conjugated compounds and polyamine metabolites. Collectively, the present study confirmed the CKD-associated gut microbial dysbiosis and identified a novel dietary and therapeutic strategy to improve the gut microbial dysbiosis and the associated metabolomic disorders and retarded the progression of kidney disease in the rat model of CKD.

Production of a novel Poria cocos immunomodulatory protein in Pichia pastoris: cloning, expression, purification and activities assays.

In this study, the cDNA of immunomodulatory protein from Poria cocos (PCP) was amplified by reverse transcription polymerase chain reaction and used to transform P. Pastoris cells, resulting in rPCP expression as a secreted protein to a concentration of ~ 38 mg/L following methanol induction in shake flasks. Approximately 1.6 mg of high purity rPCP was obtained from a 100-mL culture by Ni+-affinity chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated rPCP as a homologous dimer glycoprotein formed by different molecular-weight monomers. Peptide-N-glycosidase F-mediated deglycosylation analysis showed the presence of an N-glycosylated rPCP monomer, and bioactivity assays showed that rPCP activity upregulated tumor necrosis factor (TNF)-α and interleukin-1ß transcription and increased TNF-α secretion from mouse macrophage RAW 264.7 cells. Shortly, we demonstrated successful purification of active rPCP from P. pastoris, which promoted further study of its biological activities and medical applications.

Transcriptome analysis of three cultivars of Poria cocos reveals genes related to the biosynthesis of polysaccharides.

Poria cocos (P. cocos) polysaccharides (PCPs) are used to improve immunity and possess antitumor activities. We compared three cultivars of P. cocos (5.78, XJ 28 and JHYH) PCP contents. Then we determined that malZ, galA, SORD, gnl and bglX are key enzymes within the PCP biosynthetic pathway by using HiSeq2500 transcriptome and qRT-PCR validation. Our results provide more detailed information about the PCP biosynthesis pathway at the molecular level in P. cocos and establish the functions for the molecular breeding to produce polysaccharides in general for therapeutic use in Chinese medicinal plants.