Functions Of GS On Basal-cortex Cholinergic System Of Aged Rats

PrefaceAnti - aging, as is called, though considered common wish of mankind, is more than a complex problem in current brain research. Magnocellular neurons of the primate basal forebrain provide a massive and widespread cholinergic input to cerebral cortex and hippocampal formation, which synthesized and secreted BDNF and supported the basal forebrain by retrograde transport. This basal- cortex system plays a very important role in learning and memory, and this system is subject to aging. Ginsenosides, an traditional anti — aging medicine, have excited function on central nerve system and could significantly prove the cognition of elder people. GS have been used in prevention and therapy of anti— aging. However, the mechanism remained poorly understood. In present stud-y, we decided to investigate the biomolecular mechanism of GS in anti - aging by studying its effects on the expression of free radicals, cholinergic fibers, neu-rotrophis and their receptors.Previous studies have shown that age - increased free radicals were mainly responsible for the cumulative damage underlying the aging process. The balance between free radicals and antioxidant defenses was damaged with aging: the increasing production of free radicals and the declining activity of the antixidant system. Ginsenosides are the major principles of ginseng. Presently, Maltol extracted from Ginsen was approved to have markable antioxidiant function. We have proved, in vitro, that GS could strengthen the antioxidiant ability of hippocampus and increase the SOD activity. To summarize previous reports, there was lack of the studies about the changes of free radicals and antioxidiant enzymes in basal - cortex system. We are going to discuss the age - associated changes of NO, MDA and SOD in this cholinergic system, as well as the protective function of ginsenosides.Basal forebrain cholinergic neurons provided the major cholinergic neurotro-phic source to hippocampus and cortex. Cholinergic hypothesis proposed that a-bility of learning and memory depended on the structural and functional integrality of cholinergic system. Learning ability was closely related to cholinergic fibers density in hippocampus: declining in learning ablity was companied with decreasing cholinergic fibers density in hippocampus and cortex. CNS of AD patients went on widerspreadly neurodegeneration, particularly the damage in basal fore-brain cholinergic neurons. Several studies showed that cholinergic deficit in AD patients had a significant correlation with declined cholinergic neuron activity. However most studies in the past focused on AD model animals, studies of normal aging still remained very limited. It has been proved that ginsenosides could promote mossy fiber sprouting in CA1 and CA3, and lengthen LTP in dentate granule cell. Experiment in animals also showed that ginsenosides could intensify the establishment of conditioned reflex and improve learning and memory ability of elder people. In present study, application of qualitative and quantitative research to cholinergic fibers density of hippocampus and cerebral cortex by chemical enzymatic methods with imaging analysis would test the anti - aging function of ginsenosides.The change in neuron morphology and function caused by defict of neurotro-phin and their recptors was the main reason of cognition dysfunction. Brain - derived neurotrophic factor, BDNF, is a necessary neruotrophic factorone for neurons and glia. Effects of BDNF are mostly mediated by the tyrosine receptor ki-nase, trkB, which are widely expressed in CNS. Opinion about the age - related changes of BDNF and its receptor trkB mRNA expression was controversial. Some thought BDNF /trkB mRNA expression in hippocampus and cerebral cortex was not associated with aging, others who had contrary opinion thought trkB mRNA expression declined with advanced age. In our present study, we determined to make sure the age - associated changes of BDNF and trkB mRNA expression in hippocampal formation and cerebral cortex, as well as the anti - aging function of ginsenosides.Method24 female Wistar rats in the same condition were randomly divided into 3 groups: young group (3-5 months) , aged groups (25 - 27 months) and GS -treated group (25 - 27 months). GS - treated group was feed with gensinosides from 17 to 25 -27 months.Radicalimmunoassay was to test the NO and MDA levels and SOD activity in basal forebrain, hippocampal formation and cerebral cortex. Enzymo - cytochemistry method was to qualify and quantitate AChE fibers in hippocampal formation and cerebral cortex. Immunohistochemestry and in situ hybridization with imaging analysis was to show and quantify the BDNF and its receptor trkB mR-NA expression in hippocampal, cerebral cortex and basal forebrain. Western blotting to quantitate the trkB protein levels in basal forebrain, hippocampal formation and cerebral cortex.Results1. Radicalimmunoassay showed that NO and MDA levels in basal forebrain, hippocampal formation and cortex of aged group increased compared with young group: NO levels increased 116% , 142% and 80% , and MDA increased 167% , 88% and 96% , respectively (P <0. 01). NO and MDA levels in GS group declined compared with aged group: NO levels declined 39% , 48% and 55% , and MDA levels declined 41% , 50% and 53% , respectively (P < 0. 01). In contrast, SOD activity in 3 brain areas of aged group decreased 61% , 66% and 64% compared with young group, respectively, and the activity in GS group increased 65% , 114% and 75% compared with aged group, respectively2. Chemical enzymatic methods showed obvious age - related changes of AChE fibers in hippocampal formation (CA3, CA1 and dentate gyrus) and cerebral cortex, which went on disordered, uncontinued, synapse decreasing and lighted stained. AChE fiber density decreased 49. 1% , 61. 5% 64. 8% and32% in CA3, CAl, dentate gyrus and cerebral cortex compared with young group, respectively. AChE fiber density in GS group increased 76. 5% , 31. 2% , 116.8% and 35.5% , respectively( P <0.01).3. Imunohistochemistry method showed BDNF positive granule stained brown mainly distributed in cell membrane, dentrite and axon of CA3, CAl, dentate gyrus and cortex. BDNF expression in CA3, CAl and cerebral cortex decreased 13. 3% , 10.4% ( P <0. 05) and 12. 1% ( P <0.01) , respectively. No change in BDNF levels in CA3 and CAl of GS group compared with aged group suggested that ginsenosides could It change the distribution of BDNF in these areas. In contrast BDNF expression in dentate gyrus and cortex increased 16.7% ( P<0.01) and 11.9% ( P<0.01) compared with aged group.4. In situ hybridization showed trkB mRNA positve granule stained brown mainly expressed in the plasma, not the nucleus, of pyramidal neurons in CAl and CA3, granule neurons in dentate gyrus and neurons in each lamine of cortex. TrkB mRNA expression in CA3, CAl, dentate gyrus and cortex of aged rats decreased 24. 2%, 13.4%, 50. 6% and 15. 5% compared with young group, respectively(P <0. 01) . Significant increase was found in CA3, dentate gyrus and cortex of GS group( 12.1% increase in CA3, 17.9% in dentate gyrus and 6.1% in cortex) , but no differece was found in CAl(P>0.05).5. Westen blotting test showed trkB protein bands of basal forebrain, hipp-ocampal formtion and cortex in young group were widest and darkest, which suggested the highest expression of trkB protein. Protein band of aged group was thinnest and lightest among 3 groups. Statistic analysis showed trkB protein expression in 3 brain areas of aged rats decreased 61% , 99. 7% and 57.7% , respectively ( P <0. 01). In contrast, the expression in GS group increased 65% , 78.5% and 22.6% compared with aged group, respectively( P <0. 01) .