Studies Of Ocs On Its Anti-Parkinsonian Pharmacodynamics And Mechanism In Vivo And In Vitro

Parkinson disease (PD) is a common neurodegenerative disease among aging populations, and the incidence tends to increase year by year. The etiopathogenisis of PD is not clear up to now, but many factors such as oxidative stress, mitochondria disorders, apoptosis and hereditary factors, may be involved in the initiation of the disease, which caused the degeneration of dopaminergic neuron in nigrostriatum system to initiate clinical symptoms.Since the 1960s, when Levodopa (L-Dopa) was developed and showed marked curative effects on PD patients, much headway has been made in anti-PD medicine development. Various dopamine receptors agonists, all kinds of enzyme inhibitors, acceptor block agents, neuro-protective medicine, and neuro-recovery medicine were brought into existence and proved to play an important part in alleviating PD patients' symptoms and prolonging their life spans. Nevertheless, it is estimated that the near future will see only a limited progress in anti-PD medicine development because of the growing R & D expenses, increasing research cycles and more demanding evaluation on medicine safety. At present, most treatment of PD is based on chemical medicine with limited curative effect and much side effect. Therefore, it is urgent to search for new therapeutic tools and methods.Policosanol (Ocs) is a compound of fatty alcohol of high relative molecule mass distilled from sugarcane wax, its main component is octacosanol. Different from statin drug, it is a new non-prescription drug. As a drug, it adjusts fat, reduces serum cholesterol level, counters lipid overoxidation, restrains blood platelets from agglomeration, checks hyperplasia of smooth muscle cells in endodermium of blood vessels, and protects patients with atherosclerosis caused by exogenous hypercholesterolemia. And as a health care product, it increases one's strength, energy and stamina. Currently, it is being sold in the markets of Cuba and South American countries, and its main component octacosanol is used to manufacture sports beverage in Japan.Ocs pharmacokinetic study with a gas chromatography approach shows that the absorption of ocs is very fast-the first peak appeared in the first hour after drug taking and the second in the fourth hour. Ocs is eliminated from one's body through bile and dejecta instead of kidney. According to toxicological study, no related toxic effect was found after one took a dose of 500mg-kg-1 of ocs(this amount is 1724 times of that of the treating maximum) for a long term. An initial clinical experiment made by Snider and his colleges indicates that certain amount of ocs had curative effect on PD patients. Since then, however, there have been no related research reports published on this subject. And this study is to fill in the blank by observing how ocs treats PD animal models and Mes 23.5 cells through in vivo and in vitro experiments, and by discussing the possible working mechanism of Ocs.Mitogen-activated protein kinases (MAPKs) is a widely expressed serine/tyrosine kinase which plays a crucial role in various signal transduction pathways of mammals' cells. It has three major families, namely, ERKs, JNKs and p38MAPKs. The MAPK signal transduction pathway exists in most cells, transmits ecto-spikes to the cell and the cell nuclear, and causes cytobiological reaction. According to study, this signal pathway plays a critical role in the apoptosis of neurocells. This study was designed to observe, at the level of animal brain, the expression of each MAPKs cell factor in PD models, the changes induced by MPTP (1-methyl-4phenyl-1,2,3,6-tetrahydropyridine), and the influence brought about by Ocs intervention.Neurotrophic factors (NTF) plays an important part in neroprotection and neurogenesis. Though it has being a widely agreed conclusion that the NTF has neurotrophy, the proNGF is found abundant in the patients' brain either injured or old with neuro-degenerative diseases like Alzheimer's disease (AD). The related study shows that a certain proportion of ligands (NGF:proNGF) and receptors (TrkA:p75NTR:Sortilin) will determine the survival of neurocells in AD models to a large extent. With this background, this study plans to observe, in the animal's brain, how proNGF, NGF and their acceptors express in the PD models, what changes the 6-OHDA incurs and what results the intervention generates. Part I:Effects of Ocs on its anti-Parkinsonian pharmacodynamics and mechanism in PD mouse induced by MPTP1. PD model was successfully established by injecting MPTP (i.p.,15mg/kg for 4 times, with 2h interval) on male C57BL/6 mice. Compared with sham group, both the rotarod time (at 16 rpm) and spontaneous movement times of model group were significantly reduced (P<0.05). Ocs-H treatment increased both indexes compared to model mice(P<0.05).2. Histomorphological observation showed the neurons in striatum zone of model mice were obviously damaged, which were manifested by severely decrease of Nissl body, condensed cellular nucleus and cytoplasm vacuolar degeneration. Ocs treatment can partly reversed the changes.3. Western blotting results showed expression of p-p38 MAPK and p-JNK which play an important role in cell apoptosis significantly increased in model group compared to sham group (P<0.01or P<0.001). While their levels in Ocs-M or Ocs-H group significantly decreased compared to model mice(P<0.01 or P<0.001). Besides, although p-Erkl/2 in model group in charge of cell survival was significant lower than sham group(P<0.001), Ocs treatment didn't show obvious changes compared to model group.In summary, the present research demonstrated that Ocs treatment showed its potential anti-Parkinsonian pharmacodynamics in MPTP animal model, which may probably affect the MAPKs pathway, especially levels of p-p38 MAPK and p-JNK.Part II:Effects of Ocs on its anti-Parkinsonian pharmacodynamics and mechanism in PD rats induced by 6-OHDA1. Rats were first divided randomly into three groups:normal, sham, and pre-model group. Then unilateral striatal lesions were performed in pre-model group.4w after operation, depending on the APO-induced rotation test, pre-model group were divided into Ocs-L (17.5mg/kg/d), Ocs-M (30mg/kg/d), Ocs-H (70mg/kg/d), and model group (vehicle only). Sham and normal group were also given vehicle only. Ocs treatment lasted 2w.2. Compared to sham group, both the contralateral rotation in 30min and escape latency in MWM of model group were significantly prolonged (*P<0.05). So did the Latency and total time of the narrow beam test in model group (*P<0.05).Ocs treatment can decrease the index of impaired behavior compared to model rats (#P<0.05).3. In the biochemical test, compared to sham group,6-OHDA treatment significantly elevated MDA levels and reduced the activities of SOD and GSH-Px (P<0.05) in the lesioned striatum. Ocs treatment can partly reversed the changes. However, there were no changes of antioxidant defense system during the each group in the healthy striatum.4. Histomorphological observation showed the neurons in striatum of model rats were obviously damaged, which were manifested by severely decrease of Nissl body and cytoplasm vacuolar degeneration, the values of optical density decreased compared to control group mice (P<0.01). Ocs administration could significantly increase the Nissl body, the values of optical density increased compared to model mice (P<0.05). It means that Ocs protected neurons against the neurotoxicity of 6-OHDA.5. Immunohistochemistry showed 8-oxo-dG, a biomarker of DNA oxidative damage, increased and the expression of MTH1, an oxidized purine nucleoside tiphosphatase which play an important role in base excision repair mechanism, decreased in model group (P<0.05). Results of TH staining showed significant reduction of dopaminergic neuron in the substantia nigra and their terminal fiber in 6-OHDA-lesioned nigrostriatal system(P<0.05). Hoechst 33258 staining and TUNEL assay showed apoptotic body appearance and neurons apoptosis in model group. Ocs treatment can elevate TH expression, the marker of dopaminergic neurons (P<0.05), furthermore it can protect neurons in striatum from 6-OHDA induced apoptosis.6. Western blotting results showed levels of proNGF and its dual-receptor sortilin and p75NTR which can form pro-apoptotic trimer were significantly increased in model group (P<0.05). At the same time, expression of the pro-apoptotic factors including p-JNK, p-53, Bad, Bax were also obviously increased in model group(P<0.05). So did levels of factors including cyt C, caspase-9, caspase-3 from the intrinsic apoptotic pathway. The anti-apoptotic Bcl-2 were significantly decreased in model group. Then we investigated the NGF-pTrkA-pAkt signaling pathway, their expression were coincidently decreased in model group (P<0.01). Depending on the dose, Ocs treatment can partly antagonize the increased contents of these pro-apoptotic factors and promote the expression of proteins from cell survival pathway including NGF, p-TrkA and p-Akt, compared to model group (P<0.05). In conclusion, the present study demonstrated that Ocs treatment showed its anti-Parkinsonian pharmacodynamics in 6-OHDA animal model, which maybe related to reduce the production of ROS. And its possible mechanism may be involved in proNGF-induced apoptotic and NGF-mediated anti-apoptotic pathway.Part HI Effects of Ocs on [2,5,6-3H]dopamine uptake of Mes23.5 induced by MPP+Mes23.5 cells were cultured for 24h, and then preincubated with Ocs at different concentrations for 12h. And then add 100μM MPP+ for 12h. After [3H]dopamine uptake test, we found [3H] dopamine uptake were significantly decreased in model group (P<0.001) while 10'6M and 10-7 M Ocs could partly recover the ability of dopamine uptake of Mes23.5 cells (P<0.05). Our results showed that Ocs can protect Mes23.5 cells from MPP+ toxicity.