Study On The Neuroprotective Effects Of Panaxadiol Saponins Fraction

Objectives:The degeneration or loss of brain function in various neurodegenerative diseases (including Parkinson's disease) mostly due to the loss or injury of neurons, and excitotoxicity is a key cause of neuronal damage. Parkinson's disease (PD) is a common but refractory neurodegenerative disease. Although basic and clinical researches on PD have been conducted deeply and comprehensively for more than the last half century, the principal treatment for PD remains reliant on dopamine replacement therapy, particularly as the dopamine precursor, L-DOPA. However, patients will develop involuntary movements known as "L-dopa-induced dyskinesias" (LID) during L-DOPA treatment and fluctuations in motor symptom control after 3-5 years of treatment. Ginsenosides are the major components in ginseng and have comprehensive pharmacologic activities on the central nervous system. Our previous studies demenstrated that panaxadiol saponins fraction (Rb) had better efficacy than total saponins or any individual panaxadiol saponin, indicating a possibility of Rb in the prevention and treatment for neurodegenerative diseases. Therefore, in this study, we use two rat models of neuronal injury or neurodegeneration to further reveal this possibility:a model of excitotoxicity neuronal injury induced by the gluatmate receptor agonist kainic acid (KA) is established to study the protective effects of Rb on hippocampal neurons, glia and the ability of spatial learning and memory; the other model of PD induced by rotenone is used to study whether Rb could prevent PD or is able to enhance L-DOPA efficacy and reduce its toxicity in treatment of PD.Methods:1. Neuronal injury in the hippocampus was induced by intracerebroventricular injection of kainic acid (KA). Rb or saline as normal control was administrated intraperitoneally 40 min before KA. After 48h (acute) or 3 weeks (chronic), rats were perfused with ice-cold 4% PFA in PBS and brains were removed and sliced. The brain sections were processed for FJC and Nissl staining to identify degenerative and survival neurons in the hippocampus, respectively. Additionally, immunohistochemical staining of glutamic acid decarboxylase 67 (GAD67) or parvalbumin (PV) protein expression was performed to observe the changes of the number of GABAergic interneurons in the hippocampus, immunohistochemical staining of glial fibrillary acidic portein (GFAP) and Iba-1 was performed to identify astrocytes and microglia, respectively. The rats that were perfused 3 weeks after administration of KA was tested by Morris water maze to evaluate their ability of spatial learning and memory.2. Rats were given increasing doses of rotenone to induce experimental Parkinsonism. Two sets of experimental animals included the following five groups, respectively: nai've control group, model group, Rb group, L-DOPA group and Rb+L-DOPA group. The drugs were administered twice daily 1 hour prior to rotenone injection. The animals in control group and model group were received the same volume of saline. During the experimental period, a baterry of tasks i.e. open field test, rearing teat, hole-board test, stepping test, grip strength test and rotarod test were performed in one set of animals to assess their spontaneous motor activity, exploratory activity, physical strength and coordinate ability in different time. Until 19th day when PD symptoms in model rats were severe, rotenone and drugs were withdrawed for next 2 days. Rats were again subjected to rearing test, grip strength test and rotarod test before perfused.Then the rats were perfused with ice-cold 4% PFA in PBS and brains were removed and sliced for identification of dopaminergic neurons in striatum and substantia nigra. Fresh cortex and striatum were separated from the other set of animals on the same time point to determine the content of GSH, NO and MDA, which indicated the severity of oxidative stress.Results:1. Rb protects hippocampal neurons, glia and impaired learning and memory in the model of excitotoxicity neuronal injury induced by KAKA injection produced significant acute hippocampal injury, including pyramidal neuron degeneration and paralbumin (PV)-positive GABAergic interneuron loss in both CA1 and CA3 regions of the hippocampus, GAD67-positive GABAergic interneuron loss in CA1, and damaged astrocytes accompanied with reactive microglia in CA1 and CA3. There was a delayed loss of GAD67-positive interneurons in CA1, CA3, hilus and the dentate gyrus, and microgliosis also became more severe 21 days later. Accordingly, KA injection resulted in impaired hippocampus-dependent learning and memory. Strikingly, the pretreatment with Rb fraction at 30 or 40 mg/kg significantly protected the pyramidal neurons and GABAergic interneurons against KA-induced acute excitotoxicity and delayed injury, and also protected the learning and memory. Furthermore, astrocytes were also protected from KA-induced acute excitotoxicity, and microglia activation, especially the delayed microgliosis, was inhibited by Rb fraction. As astrocytes can protect neurons, astrocyte loss or dysfunction will be a potential cause of neuronal degeneration; microgliosis has been proven to be responsible for neuronal death particularly the delayed hippocampal neuronal death following KA-induced excitotoxicity. Therefore, it is reasonable to believe that the protected astrocytes or maintained glial homeostasis by Rb fraction at least partially contributes to its protection of hippocampal neurons against KA-induced neuron death.2. Rb protects dopaminergic neurons and motor function in PD model induced by rotenoneThe animals in rotenone model group showed characteristic of impaired motor function as PD patients which was particularly indicated by bradykinesia and decreased spontaneous motor activity, balanced capacity, physical coordination, forelimb rearing and grip strength as well as exploratory activity. The motor symptoms became more severe over time. Consistently, the animals in model group lost most of dopaminergic neurons in striatum, but dopaminergic neurons in substantia nigra were not affected by rotenone. Pretreatment of Rb or L-DOPA during rotenone injection could protect motor function to a certain extent. Strikingly, combination of the two completely prevented the motor impairment induced by rotenone. Notably, Rb and combination of Rb and L-DOPA could still exert efficacy but L-DOPA had no effect after drug withdrawl. This indicated that Rb might protect neurons to play good efficacy, but L-DOPA could only be converted to dopamine to supply neurotransmitter but could not protect neurons. Results from immunohistochemistry demenstrated the suspect:half of animals in model group lost their dopaminergic neurons in striatum,30% animals in L-DOPA group were lesioned and the lesion volume of some individual was even bigger than that of model rats, only 12.5% of animals in Rb group were lesioned and all animals in Rb+L-DOPA group were intact, indicating the combination of Rb and L-DOPA could exert better neuroprotective effect than Rb itself. These results strongly demonstrate that Rb, particularly combined with L-DOPA, has a promising potential and special value in prevention and treatment of PD, as no drugs for PD treatment exist that could protect dopaminergic neurons.However, results from biochemistry showed that there were no differences between the groups in the contents of GSH, NO and MDA, which reflect the severity of oxidative stress. This indicates another unknown mechanism involved which need to be further determined.Conclusions:Rb is protective against KA-induced excitatory acute and delayed neurodegeneration as well as hippocampal-dependent cognitive function. As excitotoxicity plays a key role in the onset of various neurodegenerative diseases, results from this study provide a support for the promising potential of Rb in the prevetion and treatment of neurodegenerative diseases. The effects of protecting astrocytes and inhibiting microgliosis further indicate the application prospect of Rb. Rb, particularly combined with L-DOPA, significantly prevent the PD symptoms induced by rotenone, and the protection on dopaminergic neurons is of great value to the prevention and treatment of PD. The results reveal a promising potential of Rb, particularly combined with L-DOPA, in the treatment of PD.The clinical relevance of these observations will be of interest to explore further.