Construction of yeast one-hybrid library and screening of transcription factors regulating FPS expression in Ganoderma lucidum / 中草药

Objective: To screen the upstream regulatory transcription factors of farnesyl diphosphate synthase (FPS) in the triterpenoid synthesis pathway in Ganoderma lucidum. Methods: In this study, the FPS promoter was cloned and connected to the pAbAi plasmid to construct bait vector pAbAi-FPS, which was transformed into Y1H yeast competent cells to construct bait yeast. The yeast one-hybrid cDNA library was constructed by using SMART technology, then the purified ds-cDNA and pGADT7-Rec were co-transformed into bait yeast strain to screen the upstream transcriptional regulatory factors of PFS. Results: The bait vector containing pAbAi-FPS was constructed and the bait strain was screened, the cDNA library was constructed and transformed to the bait strain. A total of 37 positive clones were screened and sequenced. The sequences of conserved domain were predicted and performed blast search against the whole-genome database to identify their function. As a result, a total of 18 upstream regulatory factors were screened out including three transcription factors, five ribosomal proteins, and 10 other transcription regulators. Conclusion: The results indicated that transcription factors GlSNF2, GlMHR, and GlZn2Cys6 were candidate genes for regulating the expression of FPS, and this study offered data for further study on the regulation mechanism of FPS expression.

Ganoderma triterpenes slow cyst growth in polycystic kidney disease / 中国药理学与毒理学杂志

OBJECTIVE Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease with a high morbidity around 1/1000-1/400, characterized by progressive enlargement of fluid-filled cysts derived from renal tubular epithelial cells. Massive cysts gradually compress renal parenchyma destroying normal renal structures and compromising renal functions. Unfortunately, it will cause end-stage renal disease in most of the patients but without effective therapy now, who have to live on hemodialysis or kidney transplantation. Based on this present situation, it is of great significance to find early intervention to inhibit renal cyst development. The projective of this study was to investigate whether Ganoderma triterpenes (GT) can inhibit renal cyst development and study the related mechanism. METHODS and RESULTS First, we used MDCK cyst model, cultivated MDCK cells in vitro to form fluid-filled cysts surrounded by monolayer cells. GT inhibited MDCK cyst formation significantly, and inhibited cyst enlargement dose-dependently proving GT cyst inhibition in vitro. Then we used an embryonic kidney cyst model, wile-type mice kidneys were taken out on embryonic day 13.5 to form renal cysts stimulated with 8-Br-cAMP. GT inhibited embryonic kidney cyst development significantly in a dose-dependent and reversible manner proving GT cyst inhibition at organ level. Furthermore, we used two ADPKD mouse models with severe cystic kidney disease phenotypes. GT dramatically inhibited renal cyst development, decreased ADPKD mouse kidney volume and the cyst index inside proving GT cyst inhibition in vivo. By Western blot, we proved GT down-regulated Ras/MAPK signal pathway without detectable effect on mTOR signal pathway both in MDCK cells and two ADPKD mouse kidneys. CONCLUSION GT retard renal cyst development both in vitro and in vivo significantly. The related mechanisms were involved in GT promoting renal tubular epithelial cell differentiation, down-regulating intracellular cAMP level and Ras/MAPK signal pathway.

Nanostructured lipid carrier based topical gel of Ganoderma Triterpenoids for frostbite treatment / 中国天然药物

The objective of this study was to prepare nanostructured lipid carrier (NLC)-based topical gel of Ganoderma Triterpenoids (GTs) and evaluate their effects on frostbite treatment. GT-NLCs was prepared by the high pressure homogenization method and then characterized by morphology and analyses of particle size, zeta potential, entrapment efficiency (EE), and drug loading (DL). The NLCs was suitably gelled for skin permeation studies in vitro and pharmacodynamic evaluation in vivo, compared with the GT emulgel. The GT-NLC remained within the colloidal range and was uniformly dispersed after suitably gelled by carbopol preparation. Transmission electron microscopy (TEM) study showed GT-NLCs was spherical in shape. The EE (%) and DL (%) could reach up to (81.84 ± 0.60)% and (2.13 ± 0.12)%, respectively. The result of X-ray diffractograms (XRD) showed that GTs were in an amorphous state in the NLC-gel. In vitro permeation studies through rat skin indicated that the amount of GTs permeated through skin of GT-NLCs after 24 h was higher than that of GT emulsion, and GT-NLCs increased the accumulative amounts of GTs in epidermis 7.76 times greater than GT emulsion. GT-NLC-gel was found to possess superior therapeutic effect for frostbite, compared with the GT emulgel. The NLC based topical gel of GTs could improve -their therapeutic effect for frostbite.

Preparation of Lingyi Formula multicomponent microemulsion and its anti-lung cancer activity / 中草药

Objective: To prepare Lingyi Formula multicomponent microemulsion and evaluate its anti-lung cancer activity. Methods: Lingyi Formula multicomponent microemulsion was prepared by aqueous titration method using polysaccharides solution of Ganoderma lucidum and Coix lachryma-jobi var. ma-yuen as aqueous phase and coix seed oil as oil phase, loading ganoderma triterpenes. The average particle size, Zeta potential, and stability were detected. The results of antitumor efficacy including tumor inhibitory rate, body weight change, immune organ index, and concentration of TNF-α and IL-6 were investigated. Besides, pathological section of tumor tissue and TUNEL labeling were conducted subsequently. Results: The prepared microemulsion displayed spherical surface with mean droplet size of (69.92 ± 8.43) nm, polydispersity (PDI) of 0.060 ± 0.008, and Zeta potential of (-11.30 ± 1.34) mV. Tumor inhibitory rate of microemulsion (57.25%) was significantly higher than that of suspension (45.89%), immune tissue index as well as the concentration of TNF-α and IL-6 were increased significantly. TUNEL labeling and pathological section of tumor tissue showed that the antitumor activity of microemulsion was significantly effective compared with that of suspensions. Conclusion: Lingyi Formula multicomponent microemulsion has a good anti-lung cancer activity.