Study on the Effect and Mechanism of Ganoderic Acid C 2 on Lipid Metabolism of Hepatocytes by Regulating S6K/SREBPs Signaling Pathway / 中国药房

OBJECTIVE：To stu dy in vitro lipid-lowering effect of ganoderic acid C 2（GAC2），and to investigate its potential mechanism on the basis of S 6K/SREBPs signaling pathway. METHODS ：Using human liver cells HL- 7702 as objects ，MTT assay was used to test relative cell viability after treated with low ，medium and high doses （5，10，20 μmol/L，hereinafter）of GAC 2. Using lovastatin as positive control ，ELISA method was used to detect the contents of TC and TG in cells after treated with low ， medium and high doses of GAC 2. Nile red staining was used to observe the accumulation of lipids in cells. After transfected SREBPs report gene plasmid ，using 25-HC as positive control ，relative viability of SREBPs luciferase in cells were determined by luciferase assay after treated with low ，medium and high doses of GAC 2. Using 25-HC as positive control ，real-time fluorescent quantitative PCR was used to measure the mRNA expression of SREBPs and their downstream genes in cells after treated with medium and high doses of GAC 2. Using SREBPs inhibitor （25-HC）and S 6K inhibitor （rapamycin）as control ，Western blotting assay was adopted to determine the expression of SREBP- 1 and SREBP- 2（in the case of n-SREBPs ），relative expression ratio of phosphorylated S 6K to S 6K（p-S6K/S6K ratio ）. AutoDock 4.0 and other softwares were used for molecular docking of S 6K and GAC2. RESULTS ：There was no significant effect of low ， 0.05）. Compared with blank control group ，the content of TC qq.com in lovastatin group and GAC 2 high-dose group as well as thecontent of TG in lovastatin group ， GAC2 medium- and 床应用。电话：0371-65962746。E-mail：whui3697@126.com high-dose groups were decreased significantly （P＜0.05 or P＜ 0.01）；the number of lipid droplets in the cells of all medication groups decreased. Compared with blank control group ，relative viability of SREBPs luciferase in 25-HC group ，GAC2 low-，medium- and high-dose groups were decreased significantly ；mRNA expression of HMGCS1，MVK，SCD，HMGCR gene in 25-HC group and GAC 2 medium-，high-dose groups ，mRNA expression of DHCR7 gene in 25-HC group ，mRNA expression of SREBP-2 gene in GAC- 2 high-dose group as well as mRNA expression of DHCR24 and MSMO2 gene in 25-HC group and GAC 2 high-dose group were all decreased significantly ；relative protein expression of n-SREBP- 1 in 25-HC group ，GAC2 low-，medium- and high-dose groups ，relative protein expression of n-SREBP- 2 in 25-HC group and GAC 2 high-dose group as well as p-S 6K/S6K ratio in rapamycin group and GAC 2 groups were decreased significantly （P＜0.05 or P＜0.01）. The molecular docking results showed that GAC 2 could bound to amino acid residues Arg 335，Arg330 and Ala332 of S 6K via hydrogen bond. CONCLUSIONS ：GAC2 can reduce the lipid level of HL- 7702 cells，which may be associated with inhibiting the expression of S 6K/SREBPs signaling pathway.

Network pharmacology analysis of the anti-cancer pharmacological mechanisms of Ganoderma lucidum extract with experimental support using Hepa1-6-bearing C57 BL/6 mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (GL) is an oriental medical fungus, which was used to prevent and treat many diseases. Previously, the effective compounds of Ganoderma lucidum extract (GLE) were extracted from two kinds of GL, [Ganoderma lucidum (Leyss. Ex Fr.) Karst.] and [Ganoderma sinense Zhao, Xu et Zhang], which have been used for adjuvant anti-cancer clinical therapy for more than 20 years. However, its concrete active compounds and its regulation mechanisms on tumor are unclear. AIM OF THE STUDY: In this study, we aimed to identify the main active compounds from GLE and to investigate its anti-cancer mechanisms via drug-target biological network construction and prediction. MATERIALS AND METHODS: The main active compounds of GLE were identified by HPLC, EI-MS and NMR, and the compounds related targets were predicted using docking program. To investigate the functions of GL holistically, the active compounds of GL and related targets were predicted based on four public databases. Subsequently, the Identified-Compound-Target network and Predicted-Compound-Target network were constructed respectively, and they were overlapped to detect the hub potential targets in both networks. Furthermore, the qRT-PCR and western-blot assays were used to validate the expression levels of target genes in GLE treated Hepa1-6-bearing C57 BL/6 mice. RESULTS: In our work, 12 active compounds of GLE were identified, including Ganoderic acid A, Ganoderenic acid A, Ganoderic acid B, Ganoderic acid H, Ganoderic acid C2, Ganoderenic acid D, Ganoderic acid D, Ganoderenic acid G, Ganoderic acid Y, Kaemferol, Genistein and Ergosterol. Using the docking program, 20 targets were mapped to 12 compounds of GLE. Furthermore, 122 effective active compounds of GL and 116 targets were holistically predicted using public databases. Compare with the Identified-Compound-Target network and Predicted-Compound-Target network, 6 hub targets were screened, including AR, CHRM2, ESR1, NR3C1, NR3C2 and PGR, which was considered as potential markers and might play important roles in the process of GLE treatment. GLE effectively inhibited tumor growth in Hepa1-6-bearing C57 BL/6 mice. Finally, consistent with the results of qRT-PCR data, the results of western-blot assay demonstrated the expression levels of PGR and ESR1 were up-regulated, as well as the expression levels of NR3C2 and AR were down-regulated, while the change of NR3C1 and CHRM2 had no statistical significance. CONCLUSIONS: The results indicated that these 4 hub target genes, including NR3C2, AR, ESR1 and PGR, might act as potential markers to evaluate the curative effect of GLE treatment in tumor. And, the combined data provide preliminary study of the pharmacological mechanisms of GLE, which may be a promising potential therapeutic and chemopreventative candidate for anti-cancer.

Ganoderic acid C1 isolated from the anti-asthma formula, ASHMI™ suppresses TNF-α production by mouse macrophages and peripheral blood mononuclear cells from asthma patients.

Asthma is a heterogeneous airway inflammatory disease, which is associated with Th2 cytokine-driven inflammation and non-Th2, TNF-α mediated inflammation. Unlike Th2 mediated inflammation, TNF-α mediated asthma inflammation is generally insensitive to inhaled corticosteroids (ICS). ASHMITM, aqueous extract of three medicinal herbs-Ganoderma lucidum (G. lucidum), Sophora flavescens Ait (S. flavescens) and Glycyrrhiza uralensis Fischer (G. uralensis), showed a high safety profile and was clinically beneficial in asthma patients. It also suppresses both Th2 and TNF-α associated inflammation in murine asthma models. We previously determined that G. uralensis flavonoids are the key active compounds responsible for ASHMITM suppression of Th2 mediated inflammation. Until now, there are limited studies on anti-TNF-α compounds presented in ASHMITM. The objective of this study was to isolate and identify TNF-α inhibitory compounds in ASHMITM. Here we report that G. lucidum, but not the other two herbal extracts, S. flavescens or G. uralensis inhibited TNF-α production by murine macrophages; and that the methylene chloride (MC)-triterpenoid-enriched fraction, but not the polysaccharide-enriched fraction, contained the inhibitory compounds. Of the 15 triterpenoids isolated from the MC fraction, only ganoderic acid C1 (GAC1) significantly reduced TNF-α production by murine macrophages (RAW 264.7 cells) and peripheral blood mononuclear cells (PBMCs) from asthma patients. Inhibition was associated with down-regulation of NF-κB expression, and partial suppression of MAPK and AP-1 signaling pathways. Ganoderic acid C1 may have potential for treating TNF-α mediated inflammation in asthma and other inflammatory diseases.

Chemical constituents from fruiting bodies of Ganoderma tsugae (Ⅱ) / 中草药

Objective To study the chemical constituents from the fruiting bodies of Ganoderma tsugae. Methods To isolate the compounds by silica gel and Sephadex LH-20 column chromatography and to elucidate their structures by means of spectral analyses. Results Eight triterpenoids were obtained from EtOAc fraction of EtOH extract and identified as ganoderiol A (Ⅰ), ganodermanontriol (Ⅱ), ganodermatriol (Ⅲ), ganoderic acid C (Ⅳ), ganoderic acid A (Ⅴ), lucidone A (Ⅵ), lucidenic acid C (Ⅶ), and lucidenic acid LM1 (Ⅷ). Conclusion Compounds Ⅰ-Ⅷ are all isolated from G. tsugae for the first time.