Ganoderma lucidum is a mushroom widely used for its edible and medicinal properties. Primary bioactive constituents of G. lucidum are ganoderic triterpenoids (GTs), which exhibit important pharmacological activity. Abscisic acid (ABA), a plant hormone, is associated with plant growth, development, and stress responses. ABA can also affect the growth, metabolism, and physiological activities of different fungi and participates in the regulation of the tetracyclic triterpenes of some plants. Our findings indicated that ABA treatment promoted GT accumulation by regulating the gene expression levels (squalene synthase (sqs), 3-hydroxy-3-methylglutaryl-CoA reductase (hmgr), and lanosterol synthase (ls)), and also activated cytosolic Ca²⁺ channels. Furthermore, under ABA mediation, exogenous Ca²⁺ donors and inhibitors directly affected the cytosolic Ca²⁺ concentration and related gene expression in Ca²⁺ signaling. Our study also revealed that ABA-mediated cytosolic Ca²⁺ played a crucial regulatory role in GT biosynthesis, accompanied by antioxidant defense modulation with increasing superoxide dismutase (SOD) activity and ascorbate peroxidase (APX) activity, and the resistance ability of O₂^•- and glutathione (GSH) contents.
Reishi/metabolism* ; Triterpenes/metabolism* ; Abscisic Acid/metabolism* ; Antioxidants/metabolism*

Polysaccharide is among the main active components of Ganoderma lucidum for tumor prevention and treatment. Howe-ver, it remains unclear whether it has synergy with tumor immunotherapy. This study evaluated the effect of G. lucidum polysaccharides(GLP) on the infiltration of T lymphocytes into tumor and the underlying mechanism, in order to provide a reference for its application in tumor immunotherapy. GLP were prepared by water extraction and alcohol precipitation combined with Sevag method and then given(intraperitoneal injection) to the mice bearing B16-F10 cells at 25, 50 and 100 mg kg~(-1), respectively, to evaluate the effect on tumor growth. The infiltration of CD3~+ and CD8~+ T cells and the expression of intercellular cell adhesion molecule-1(ICAM-1) in tumor were detected by immunohistochemistry. EA.hy926 cells were treated with 50, 100 and 200 μg·mL~(-1) GLP, and the expression of ICAM-1 was determined by Western blot. The adhesion of EA.hy926 cells treated with GLP was measured with fluorescence-labeled Jurkat cells. To analyze the mechanism based on NF-κB pathway, this study determined the protein levels of nuclear factor kappa-B(NF-κB) p65, alpha inhibitor of NF-κB(IκBα), p-NF-κB p65 and p-IκBα by Western blot. The results showed that GLP can significantly inhibit the tumor growth in mice bearing B16-F10 cells, promote the infiltration of CD3~+ and CD8~+ T cells in tumor, and increase the expression of ICAM-1 in tumor. Meanwhile, GLP could also enhance the expression of ICAM-1 in EA.hy926 cells, thus strengthen the adhesion to Jurkat cells, induce phosphorylation and protein degradation of IκBα, and raise the expression and phosphorylation level of NF-κB p65. These results suggested that GLP could promote the expression of ICAM-1 through NF-κB pathway and further enhance the infiltration of T lymphocytes into tumor, thereby inhibiting tumor growth. This study lays a foundation for the further application of GLP in tumor immunotherapy.
Animals ; Endothelial Cells/metabolism* ; Intercellular Adhesion Molecule-1/genetics* ; Mice ; NF-kappa B/metabolism* ; Neoplasms ; Polysaccharides ; Reishi ; Signal Transduction ; T-Lymphocytes ; Tumor Necrosis Factor-alpha

Lanosterol synthase( LS) is a key enzyme involving in the mevalonate pathway( MVA pathway) to produce lanosterol,which is a precursor of ganoderma triterpenoid. And the transcriptional regulation of LS gene directly affects the content of triterpenes in Ganoderma lucidum. In order to study the transcriptional regulation mechanism of LS gene,yeast one-hybrid technique was used to screen the transcription regulators which interact withthe promoter of LS. The bait vector was constructed by LS promoter,then the vector was transformed yeast cells to construct bait yeast strain. One-hybrid c DNA library was constructed via SMART technology. Then the c DNA and p GADT7-Rec vector were co-transformed into the bait yeast strain to screen the upstream regulatory factors of the promoter region of LS by homologous recombination. Total of 23 positive clones were screened. After sequencing,blast was performed against the whole-genome sequence of G. lucidum. As a result,8 regulatory factors were screened out including the transcription initiation TFIIB,the alpha/beta hydrolase super family,ALDH-SF superfamily,60 S ribosomal protein L21,ATP synthase β-subunit,microtubule associated protein Cript,prote asome subunit β-1,and transaldolase. Until now,the regulation effect of these 8 regulatory factors in G.lucidum has not been reported. This study provides candidate proteins for in-depth study on the expression regulation of LS.
Gene Library ; Intramolecular Transferases/metabolism* ; Reishi/genetics* ; Saccharomyces cerevisiae ; Transcription Factors/metabolism*

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.
Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Caspase 9 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; E2F1 Transcription Factor ; genetics ; metabolism ; G1 Phase Cell Cycle Checkpoints ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Nucleosomes ; drug effects ; metabolism ; pathology ; Plant Extracts ; chemistry ; Prostate ; drug effects ; metabolism ; pathology ; Reishi ; chemistry ; Signal Transduction ; Triterpenes ; isolation & purification ; pharmacology

To study the effect and mechanism of the light quality acting on Ganoderma lucidum, and provide a theoretical basis for G. lucidum mycelium cultivation, we focused on growth and endogenous IAA metabolism of G. lucidum mycelium under different light-emitting diode (LED) condition. The growth index, endogenous levels of IAA and Enzymes related to IAA metabolism and Polysaccharides content were investigated in different growth periods. Results showed that blue light irradiation was the best from the viewpoint of steady growth and polysaccharides accumulation, red light irradiation improved endogenous IAA level and promoted growth of mycelium in early stage of cultivation, green light irradiation decreased growth rate and fresh weight of mycelium, but increased drying rate. Enzymes related to IAA metabolism also significantly influenced by light quality. The activity of indole acetic acid oxidase (IAAO), peroxidase (POD) and tryptophan synthetase with blue light irradiation were showed high level in early time, but decreased later, and the IAA content was consistently at lower level than that in other treatments, while mycelium irradiated with yellow light showed the highest activity of both IAAO and tryptophan synthetase, and medium level of IAA content. In conclusion, the light quality affects growth and regulation of the level of endogenous IAA of G. lucidum mycelium.
Fungal Polysaccharides ; analysis ; Indoleacetic Acids ; metabolism ; Light ; Peroxidases ; metabolism ; Reishi ; growth & development ; metabolism

OBJECTIVETo study the application of degrading multi-enzymes from Ganoderma lucidum in extracting effective constituents from fibrous roots of Salvia miltiorrhiza.

METHODEffective constituents were extracted from fibrous roots by degrading multi-enzymes of wood fiber. The enzymatic parameters were optimized by the orthogonal design.

RESULTThe extraction efficiencies of total tanshinones and total salvianolic acids in the extracts of fibrous roots of S. miltiorrhiza was obtained using optimum enzymolysis process reached 11.923%, 12.465%, respectively, which were 62.794%, 56.086% more than that by conventional non-enzymatic hydrolysis.

CONCLUSIONDegrading multi-enzymes of wood fiber can be used to fully extract effective constituents from fibrous roots of S. miltiorrhiza, which provides a new approach for recycling wastes of traditional Chinese medicines.

Alkenes ; isolation & purification ; metabolism ; Diterpenes, Abietane ; isolation & purification ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; metabolism ; Hydrogen-Ion Concentration ; Plant Roots ; chemistry ; Polyphenols ; isolation & purification ; metabolism ; Reishi ; enzymology ; Salvia miltiorrhiza ; chemistry ; Temperature ; Wood ; enzymology

Cytochrome P450 (CYP450) is a key element in the Ganoderma triterpenoid biosynthetic pathway. The catalytic reaction process for CYP450 requires NADPH / NADH for electron transfer. After searching the genome dataset of Ganoderma lucidum, the unique sequence encoding CYP450 and NADPH were discovered, separately. The open reading frames of GLCYP450 and GLNADPH were cloned separately using RT-PCR strategy from G lucidum. The appropriate restriction enzyme cutting sites were introduced at the 5' and 3' ends of gene sequence. The genes of GLCYP450 and GLNADPH were recombined into the yeast expression vector pESC-URA, leading to the formation of the yeast expression plasmid pESC-GLNADPH-GLCYP450. This study provides a foundation for researching Ganoderma triterpene biosynthesis using the approach of synthetic biology.
Amino Acid Sequence ; Cloning, Molecular ; Cytochrome P-450 Enzyme System ; genetics ; DNA, Complementary ; genetics ; metabolism ; Gene Expression Regulation, Fungal ; Genetic Vectors ; NADP ; genetics ; Open Reading Frames ; Plasmids ; Reishi ; enzymology ; genetics ; metabolism ; Synthetic Biology ; Triterpenes ; metabolism ; Yeasts ; genetics ; metabolism

OBJECTIVETo study the effect of Ganoderma lucidum polysaccharides on oxidative stress of hyperlipidemic fatty liver in rats.

METHODSeventy-two SD rats were randomly divided into six groups, namely the normal control group (NG), the model group (MG), the G. lucidum polysaccharides groups of low, middle and high dose (GLPs-LG, GLPs-MG, GLPs-HG) and the Simvastatin group (SV). The rats were fed with high fat diet to establish the model of hyperlipidemic fatty liver in rats. After administration for 12 weeks, rats in each group were tested with the following indexes: total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) in serum as well as the contents of SOD, MDA, GSH-Px and T-AOC in hepatic tissues. Histopathological changes of hepatic tissues were observed under light glass.

RESULTSThe contents of TC, TG and LDL-C were significantly increased in the model group (P < 0.01). Compared with the model group, both the GLPs-M group and the GLPs-H group showed significant decreases in TC, TG and LDL-C (P < 0.05 or P < 0.01), while the GLPs-H group showed a notable increase in HDL-C (P < 0.05). Compared with the model group, both the GLPs-M group and the GLPs-H group showed significant decreases in MDA (P < 0.05 or P < 0.01) and notable increases in SOD, GSH-Px, T-AOC (P < 0.05 or P < 0.01). The GLPs-M group and the GLPs-H group proved a remarkable alleviation in fatty degeneration of hepatic cells.

CONCLUSIONG. lucidum polysaccharides can significantly reduce the blood fat level of hyperlipidemic fatty liver in rats and effectively inhibit oxidant stress, showing the effect on preventing and treating hyperlipidemic fatty liver in rats to some extent.

Animals ; Antioxidants ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Fatty Liver ; drug therapy ; metabolism ; Glutathione Peroxidase ; metabolism ; Humans ; Male ; Malondialdehyde ; blood ; Oxidative Stress ; drug effects ; Polysaccharides ; administration & dosage ; Rats ; Rats, Sprague-Dawley ; Reishi ; chemistry ; Triglycerides ; blood

This study was aimed to investigate the inhibitory effects of sporoderm-broken ganoderma lucidum spores (GLS) on transplanted lymphoma in nude mice and its mechanism. The models of the subcutaneously transplanting tumor were established by N-methylnitrosourea-induced thymus T-cell lymphoma. The cellular apoptosis of tumor tissues in nude mice were observed by transmission electron microscopy (TEM), the mRNA expression of Bax, Bcl-2, Bcl-xl was determined by RT-PCR. The results indicated that the GLS had a certain anti-tumor effect, and the inhibitory rate was 45.8 with the dose of 4 g/kg (ig, once daily for 21 d). Apoptotic cells in lymphoma cells treated with GLS were observed by TEM. RT-PCR analysis showed that the expression of Bax was up-regulated, Bcl-2 and Bcl-xl were down-regulated in T-cell lymphoma cells. It is concluded that GLS can inhibit proliferation of lymphoma cells and induce the lymphoma cell apoptosis, the mechanism of which may be related with up-regulating the expression of Bax and down-regulating the expression of Bcl-2 and Bcl-xl.
Animals ; Apoptosis ; Gene Expression Regulation, Neoplastic ; Lymphoma, T-Cell ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Nude ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; Reishi ; Spores, Fungal ; Thymus Neoplasms ; metabolism ; prevention & control ; Transplantation, Heterologous ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism

OBJECTIVETo study the effect of light quality on growth, antioxidant enzyme activities of Ganoderma lucidum mycelium.

METHODG. lucidum mycelium was cultured under different light qualities by light emitting diodes (LED). The growth G. lucidum mycelium was observed and antioxidant enzyme activities was determined in different growth periods.

RESULTUnder the red LED, the blue LED and dark condition (CK), the mycelium grew faster than that under other light qualities. The white LED resulted in a largest increase in the amount of the mycelium and always kept the activities of CAT high level. Major fluctuations of POD activities emerged under the green LED, while enhanced severely in the late phase. Under the yellow LED, the activities of SOD appeared high level. However, SOD activities on dark (CK) raised obviously in late period. At the late stage, the content of mycelium polysaccharides was significant higher than that under the blue LED.

CONCLUSIONThe light quality could influence the growth and metabolism of G. lucidum mycelium.

Antioxidants ; metabolism ; radiation effects ; Catalase ; metabolism ; radiation effects ; Light ; Mycelium ; chemistry ; growth & development ; metabolism ; radiation effects ; Peroxidases ; metabolism ; radiation effects ; Plant Extracts ; chemistry ; metabolism ; radiation effects ; Plants, Medicinal ; chemistry ; growth & development ; metabolism ; radiation effects ; Polysaccharides ; metabolism ; radiation effects ; Reishi ; chemistry ; growth & development ; metabolism ; radiation effects ; Superoxide Dismutase ; metabolism ; radiation effects