| In combination with pharmacy, computer and information technology, the computer-aided controlling system for Step Down (SD) and Locomotor Activity (LA) to test learning and memory behaviour has been improved with rats and mice. Scopolamine induced learning and memory impairment mice model was applied to evaluate the different efficacy and characteristic of ginsengnoside Rg1 and Rb1 in ameliorating learning and memory impairment with SD and Morris water maze (MWM) test, the impact on neurotransmitters of Rg1 and Rb1, the Rgl’s metabolites Rhl and PPT also have been investigated. The effects of Rhl and PPT on cholinergic and monoamine neurotransmitters, CaMKII and CREB signalling pathway, and anti-oxidative stress injury has been partly elucidated.Two computer-aided controlling and real time analysis system, SD and LA for testing animal cognitive behaviour has been developed, which are considered to have high level of automatic and intellectual characteristic, with superiority of objective as well as our own independent intellectual property. The hard ware of SD device introduced infrared sensitive apparatus. It’s designed with spring screw ascending-descending and electric network sub-boxes control devices. Soft ware of this device introduced pseudo-dynamic scheduling and multipoint average signal recognize method, real time video camera tracing technique were applied in locomotor activity test. Combination of hard and soft ware makes it possible to capture and extract accurately the time axis and other multidimensional information when animals completing behavior task. With the long term accumulated experience of neuropharmacologists, an indicator evaluation analysis system for step down and locomotor activity tests has been successfully established. The system centralized various recognizing, analysis, collecting and data input-output of animal behavioral task signals, as well as the results processing and statistical data calculation. At the same time, system status, tracking of the animal behavior operations and activities of real-time monitoring, the labor work processing has been greatly improved. Experimental results of caffeine, diazepam, traditional Chinese herbal formula Kai Xin San (KXS) and other drugs in normal animals and scopolamine mice model showed the high stability and reliability of the system. It has been demonstrated that the accomplishment of this apparatus supply a animal behavior cognitive evaluation system which consist of step down and locomotor activity testing methods with high level of automatic and intellectual characteristic, with superiority of objective as well as our own independent intellectual property. The patent of "Computerized monitoring analysis processing system for rat and mouse" has been certified by the State Intellectual Property Office of the People’s Republic of China (Patent No. ZL200710188378.9), "An apparatus for the test of animal locomotor activity" has been in stage of patency. (200910177048.9).The different effects of ginsenosides Rgl and Rbl on memory improvement has been deeply determined in scopolamine-induced mice model. The effects of Rgl and Rb1 on scopolamine-induced dementia mice model were determined with the SD and MWM tests after 3-12 mg/kg of both Rgl and Rbl were administrated ip for 1 week. The results showed that both Rg1 and Rb1 can improve the impaired memory performance induced by scopolamine in SD and water maze tests. It is interesting to note that Rg1 was more effective than Rb1 in the escape acquisition in the water maze test, while there was no significant difference between Rgl and Rbl in the SD test. Although Rg1 and Rb1 have the same effects on increasing ACh content, Rg1 is superior to Rb1 on the inhibition of AChE activity in the hippocampus of mice, while Rb1 was more active than the same dose of Rg1 in increasing 5-HT and DA level in the hippocampus of scopolamine-induced dementia mice model. The difference between Rg1 and Rb1 in their effects on scopolamine induced memory deficits suggests that Rg1 might be more potent than Rbl to improve acquisition impairment, and the two ginsenosides act through different mechanisms to improving memory deficits induced by scopolamine.Rhl and PPT, the metabolites of Rgl showed stronger effects than Rgl in memory enhancement. The efficacy of Rgl, and Rgl’s metabolites Rhl and PPT in ameliorating learning and memory impairment has been evaluated in scopolamine-induced mice model. The results demonstrated clearly that the three compounds could improve learning and memory impairment, the beneficial impacts were no different when Rhl was given in 1/28 Rgl dosage, PPT was given in 1/2.8 Rg1 dosage, compared with Rgl equivalent dosage. This indicated that Rhl and PPT, as the metabolites of Rg1, have stronger pharmacological efficacy in learning and memory improvement.Rhl and PPT might be effects to regulate cholinergic and monoamine neurotransmitter, CaMKII and CREB signalling pathway, as well as to relieve oxidative stress damage. All above mentioned effects may contribute to the improvement of learning and memory. SD test demonstrated that Rhl (0.03-0.27μmol/kg) and PPT (0.9-2.7μmol/kg) significantly improved scopolamine-induced memory deficiency in mice. Results of brain tissue enzymes activity detection indicated that Rhl and PPT could reverse the abnormal high level of AChE and MAO, increase the activity of ChAT. More results from western blotting and immunohistochemistry showed the protein expression levels of CaMKII and CREB in mice brain could be changed to some extent when administered by Rhl and PPT. Moreover, these two compounds significantly increased the p-CaMKII expression level both in cerebral cortex and CA1, CA2, CA3, DG zones of hippocampus, also high level of phosphorylated CREB could be detected in cerebral cortex. However, high level of phosphorylated CREB has not been observed in the four hippocampal regions. ELISA and spectrophotometry experiments have been applied to evaluate the antioxidative damage effects of Rhl and PPT using cellular models. The results showed that they could improve the cell injury induced by H2O2 both in SH-SY5Y cell line and neurons. The minimum effective concentration of Rhl and PPT in SH-SY5Y was 1.25 nmol/L, while in primary cultured neurons, this figure for Rhl was 0.125 umol/L, and 1.25 nmol/L for PPT. Rhl 0.125-12.5μmol/L and PPT 1.25-125 nmol/L have been demonstrated to improve the increased ROS, NO and 3-NT (40 percent reversal) induced by H2O2 in SH-SY5Y cells, also the reduced mitochondrial membrane potential has been increased more than 120 percent.In conclusion, our results provide the computer-aided controlling and real time analysis systems for SD and LA with high level of automatic and intellectual characteristic, which are used to test animal cognitive behaviour, with our own independent intellectual property. There is difference between Rg1 and Rb1 in memory enhancement in MWM test, as well as the AChE activity,5-HT and DA in the hippocampus of mice, although both of Rg1 and Rb1 are effective to improve memory deficiency. Rhl and PPT, the metabolites of Rgl showed stronger effects than Rgl in memory enhancement. The remedy of learning and memory impairment manifested by Rh1 and PPT is realized by tuning up the balance of cholinergic and monoamine transmitter as well as the regulation of CaMKII and CREB signalling pathway. In addition, amelioration of oxidative stress damage indicators such as ROS, NO,3-NT and mitochondria membrane potential also takes part in this beneficial impact. This research provides solid experimental evidence for the comprehensive development and utilization of Panax Ginseng and the seeking of new noontropic drugs. |
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