Fuzi has been used to treat diabetic complications for many years in china. In a previous study, we have shown that Fuzi aqueous extract can attenuate Diabetic peripheral neuropathy (DPN) in rats and protect Schwann cells from injury. Thus, the protective effect of Fuzi polysaccharides (FPS) on high glucose-induced SCs and the preliminary mechanism were investigated. Firstly, the FPS were obtained and their monose composition was analyzed by the combination of pre-column derivatization and high performance liquid chromatography coupled with electrospray ionization multi-tandem mass spectrometry (HPLC/ESI-MS n ). The results witnessed the efficiency of this method and seven monosaccharides were tentatively identified, among which fucose was first reported. Simultaneously, m/z 215 can be considered as diagnostic ions to confirm the number of monosaccharides. Next, high glucose-induced SC model was applied and divided into model group, treated group of FPS, normal and osmotic control group. After treatment for 48 h, the data showed FPS could significantly decrease the intracellular ROS and apoptosis, which were determined by the corresponding fluorescent probes. Then, the expression of oxidative stress-related proteins in SCs were measured by Western blot. Furthermore, the protein tests found that FPS markedly up-regulated superoxide dismutase (SOD), catalase (CAT) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein level, but down-regulated NADPH oxidase-1 (Nox1) protein level. Moreover, FPS could also increase AMP-activated protein kinase (AMPK) activation significantly. Hence, we preliminary deduced that AMPK-PGC-1α pathway may play an important role in the protective effect of FPS against high glucose-induced cell damage.
Background
Mutationally activated Ras proteins are closely linked to a wide variety of human cancers. Hence, there has been an intensive search for anti-Ras therapies for cancer treatment. The sole Ras gene, which encodes LET-60, in
Caenorhabditis elegans
regulates vulval development. While the loss of
let
-
60
function leads to failure of vulva formation, the
let
-
60
(
n1046gf
) allele, which contains a missense mutation mimicking a Ras codon 13 mutation found in human cancers, results in extra vulval tissue, a phenotype named Muv (multiple vulvas).
Methods
By taking advantage of the easy-to-score Muv phenotype of
let
-
60
(
n1046gf
), we used a step-by-step screening approach (from crude extract to active fraction to active natural compound) to search for inhibitors of oncogenic Ras. Mutants of other key components in the Ras–mitogen-activated protein kinase (MAPK) pathway were used to identify other candidate targets.
Results
The natural compound harmine, isolated from the plant
Peganum harmala
, was found to suppress the Muv phenotype of
let
-
60
(
n1046gf
). In addition, harmine targets the hyper-activation of the Ras/MAPK pathway specifically caused by overexpression or mutated forms of LET-60/Ras and its immediate downstream molecule LIN-45/Raf. Finally, harmine can be absorbed into the worm body and probably functions in its native form, rather than requiring metabolic activation.
Conclusion
In sum, we have revealed for the first time the anti-Ras activity of harmine in a
C. elegans
model system. Our results revealed the potential anti-cancer mechanism of harmine, which may be useful for the treatment of specific human cancers that are associated with oncogenic Ras mutations.
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