Studies On The Effects On Ngf-Induced PC12Cells Neurite Outgrowth Of Some Small Molecular Compounds

Nerve growth factor (NGF), a neurotrophic polypeptide, is an indispensable pleiotrophicfactor in human and animals. NGF plays a crucial role in neuronal survival, differentiation,growth, axon growth, neuro-transmitter synthesis and cell apoptosis. The potential of NGF asa therapeutic agent for several diseases including neurodegenerative disorders, diabeticneuropathy, nerve injury, inflammation and certain types of cancers has been indicated byseveral investigators. It involves with the genesis and development of a variety of diseases,and the changes in level of NGF itself or the receptor and in their functions have an importantinfluence on the pathophysiology of certain diseases. Despite the amazing therapeuticpotential, NGF is not considered an ideal drug candidate. In fact, they show a poorpharmacokinetic behavior, short half-lives, instability in vivo, unable to cross the blood-brainbarrier to reach target tissues. And NGF binds two receptors (Trks and p75^NTR) which cancommand opposite actions, activate various signal pathways and produce different responses.Moreover, the NGF protein is relatively expensive to produce for medicinal applications, so itis difficult to large-scale application in neurodegenerative diseases, diabetic neuropathy andnerve injury. Therefore, efforts have been devoted to search for NGF mimics, NGF inducor orNGF agonist/antagonist activity compound with small molecule, better pharmacokinetics andless side effects.In order to find small molecules that can promote neuronal differentiation, we detectedthe NGF-dependent neurite outgrowth activity of a series of compounds isolated from thefruiting bodies of Sarcodon scabrosus (Fr.) karst, using PC-12cells as model. On the basis ofmorphological observation and statistical analysis of the cell differentiation, we chosecyathane-type diterpenoids and two protoberberines and their hydrogenation products asobject to study. Through morphological observation, cell counting, ELISA, Western blotting,we studied the structure-activity relationships and explained the differences on a molecularlevel. The main results were as follows:1. This is the first time to detect the NGF-induced PC12cell neurite outgrowth of20smallmolecules (including six new compounds and a synthetic compound). We reported firstlysarcodonin G and sarcodonin A have the NGF-dependent neurite outgrowth-promotingactivity in vitro. On the basis of the biological results such as detection of p-Trk A and p-ERK, statistics results of cell morphology and the structure of compounds, we initiallyclarified the structure-activity relationship on a molecular and mechanistic level and foundsome functional groups important for the NGF-dependent neurite outgrowth-promotingactivity. At the same time, we also confirmed they promoted the NGF-induced neuriteoutgrowth through NGF/Trk A and ERK1/2MAPK signal pathway in does-dependentmanner. The results provided the experimental base for further utilization of themushroom Sarcodon scabrosus (Fr.) karst and theoretical foundation for design of NGFagonist on the basis of discovery of bioactive lead compounds.2. We reported firstly scabronine M, a new compound has a suppressive effect onNGF-induced neuronal differentiation in vitro. We confirmed that it interacted with Trk A,partilly influenced the ERK1/2MAPK pathway to suppress the cell differentiationprocesses. The results provided the experimental base for further utilization of themushroom Sarcodon scabrosus (Fr.) karst and theoretical foundation for design of NGFagonist on the basis of discovery of bioactive lead compounds.3. We firstly reported tetrahydroberberine and tetrahydropalmatine, two hydrogenationproducts of plant origin protoberberines: bererine and palmatine, have the NGF-dependentneurite outgrowth-promoting activity in vitro. We compared the NGF-dependent neuriteoutgrowth-promoting activity of these four compounds through the lever of p-Trk A andp-ERK, and concluded that derivatives had better activity than their parent compoundsrespectively, the hydrogenation of protoberberines had a significant effect on their activity.The results provided a theoretical basis for the effective use of berberine-containing plantsand development of new drugs.