| Alzheimer’s disease (AD) is a central nervous system degenerative disease in old people characterized by progressive learning and memory impairment and affective disorder. β-amyliod protein (Aβ) deposition is one of the main pathological features of an AD-suffered brain, in which the transduction of multiple signal pathways become abnormal. Excessive Aβ produces from results of metabolic disturbance of Aβ precursor protein (APP) by three key enzymes known as α, β and γ-secretase. The activities of α, β and γ-secretase are mainly regulated by protein kinase C (PKC) and c-jun N-terminal kinase (JNK) signal pathways. Through protein kinase A (PKA), Extra cellular signal regulated kinase (ERK) and calmodulin kinase Ⅱ(CaMKⅡ) signal transduction pathways, Aβ can impact cAMP responsive elements binding protein (CREB) phosphorylation, inhibit production of brain-derived neurotrophic factor (BDNF), and result in nerve repair disorder and learning and memory impairments. Therefore, inhibition of Aβ formation and correction of abnormal signal transduction pathways is an effective intervening approach to AD prevention and treatment.Panax Notoginseng Saponins (PNS) is the chief active ingredient in pseudo-ginseng roots. Our previous studies show that PNS can inhibit expression of APP protein by intervening APP gene transcription, and protect the function of learn and memorize by reducing Aβ formation via regualting the activity of α, β and γ-secretase. In this project, we further explored the specific signal pathway mechanism underlying regulation of the activity of α, β and γ-secretase by PNS, and disclosed the signal transduction pathways mechanisms underlying the protection effects of PNS on Aβ neurotoxicity, which contributed to its important function of improvements of behavior and memory.ObjectiveTo determine whether PNS can impact the activity of α, β and γ-secretase by intervening the PKC and/or JNK signal transduction pathways, which would inhibit the production of Aβ protein in the brain of AD model mice SAMP8, and to further explore whether PNS can improve learning and memory by anti-Aβ neurotoxicity through intervening the PKA and/or ERK and/or CaMK Ⅱ signal transduction pathways, so as to recognize the mechanisms underlying the protection effects of PNS on learning and memory impairments, and provide fundamental experimental bases for prevention and treatment of AD and other degenerative diseases in the central nervous system by PNS.Methods1. Sixty male AD model SAMP8 mice were randomly divided into the Model group, Low Dose PNS (L-PNS) group, High Dose PNS (H-PNS) group and Huperzine A Positive Control (Hup-A) group,15 ones in each group. The L-PNS group was administered at 100mg/kg bw; the H-PNS group at 200mg/kg bw; the Hup-A group at 0.3mg/kg bw; and the Model group was subjected to intragastric administration of double distilled water of the same volume as the other groups. Intragastric administration was performed once per day, for 2 successive months, followed by the test of the following indexes.2. We investigated the effect of PNS on SAMP8 mice’s spatial learning and memory impairment, Aβ production and hippocampal nerve damage by using Morris Water Maze (MWM) to detect mice’s spatial learning and memory, applying Neutral Red Nissl Staining to the cerebral hippocampus of the mice for pathological and morphometric analyses, and employing ELISA to detect the level of protein Aβ in the brain.3. We detected the activity of kinases PKC and JNK 1/2/3 within the brain with Spectrophotometry, determined the expression level of proteins PKC (cPKC-βⅡ) and phospho-p46/54 MAPK (p-JNK 1/2/3) with Western Blotting, quantitatively determined the transcription level of PKC (cPKC-βⅠ/Ⅱ) mRNA and p46 MAPK (JNK1) mRNA with Real-time reverse transcription quantitative polymerase chain reation (Real-time RT-PCR). Meanwhile, we detected the activity of α, β and γ-secretase with Fluorescence Resonance Energy Transfer (FRET), determined the expression level of proteins ADAM 10 (α-secretase), BACE1 (β-secretase) and Aph-1 (γ-Secretase) with Western Blotting, quantitatively determined transcription level of ADAM 10, BACE1 and Aph-1 mRNAs with Real-Time RT-PCR, so as to explore the specific signal pathway mechanisms underlying the benefit effects of PNS on α, β and γ-secretase which inhibit the production of Aβ protein in the brain of AD model mice SAMP8.4. We determined the activity of kinases PKA, ERK1/2 MAPK and CaMK Ⅱ within the brain of SAMP8 with Spectrophotometry, determined the expression level of proteins PKARIα, phospho-p44/42 MAPK (p-ERK1/2) and p-CaMKⅡα/β with Western Blotting, quantitatively determined the transcription level of PKARIa mRNA、p42 MAPK (ERK2) mRNA、CaMKⅡα mRNA with Real-Time RT-PCR. Meanwhile, we determined the expression level of proteins p-CREB and BDNF with Western Blotting, quantitatively determined the transcription level of CREB and BDNF mRNAs with Real-Time RT-PCR, so as to explore the signal transduction pathways mechanisms underlying the protection effects of PNS on Aβ neurotoxicity, which contributed to improvements of behavior and memory.Results1. The protection effects of PNS on Aβ protein formation, hippocampal neuro-pathology and spatial learning and memory impairments.Our research showed that PNS significantly lowered the level of protein Aβ1-42 in the brain tissue of SAMP8 mice, reduced the pathological damage to hippocampal neurons, increased the quantity of neurons, and effectively improved the spatial learning and memory impairments in SAMP8 mice. The results indicate that PNS was effective in preventing nerve damage and cognitive dysfunction probably by intervention to Aβ formation in the brain. The details as follows:The level of Aβ1-42 within the brain of SAMP8 was significantly higher in the Model group than in other groups (P<0.01), showed no significant difference between the H-PNS and Hup-A groups (P>0.05), and that of both the latter groups were lower than that of the L-PNS group. The amount of hippocampal neurons was significantly lower in the Model group than in other groups (P<0.01), and in the L- and H-PNS groups than in the Hup-A group (P<0.01), that of the H-PNS group notably higher than that of the L-PNS group (P<0.01). In terms of escaping latency in the MWM place navigation test, the Model and L-PNS groups were significantly higher than the H-PNS and Hup-A groups (P<0.01), the Model and L-PNS groups showed no significant difference (P>0.05), and the H-PNS and Hup-A groups also exhibited no significant difference (P>0.05). In terms of swimming duration in the target quadrant, the Model group was significantly lower than the other groups (P<0.01), the L-PNS group was markedly lower than the H-PNS and Hup-A groups (P<0.01), and no significant difference existed between the H-PNS and Hup-A groups (P>0.05).2. The research on PKC and JNK signal transduction pathways mediated the mechanisms underlying the protection effects of PNS on Aβ formation.Our research showed that PNS significantly elevated the levels of cPKC-βⅠ/Ⅱ mRNA, cPKC-βⅡ protein and activity of kinase PKC, and enhanced the levels of ADAM 10 mRNA, ADAM 10 protein, activity of a-Secretase, meanwhile significantly decreased the levels of BACE1 mRNA, BACE1 protein, activity of β-Secretase. In addition, we found that PNS notably reduced the levels of p46 MAPK (JNK1) mRNA, phospho-p46 MAPK (p-JNK1) protein and activity of kinase JNK1, meanwhile significantly decreased the levels of Aph-1 mRNA, Aph-1 protein, activity of y-Secretase. Our experimental results implicated that PNS could produce a benefit regulation effect on a,p,y-Secretase by intervention with PKC and JNK signal pathways, e.g. activation of the PKC signal pathway, and inhibiting the JNK signal pathway, which contribute to the protection effects of PNS on AP formation. The details as follows:The Model group had significantly lower PKC activity than the H-PNS and Hup-A groups (P<0.01), and no significant was between the Model group and the L-PNS group and between the Hup-A group and the H-PNS group in terms of such activity (P>0.05). The Model group had a significantly lower cPKC-βⅠ/Ⅱ mRNA level than other groups (P<0.01), and no significant difference was between the H-PNS and Hup-A groups (P>0.05). In terms of the expression level of cPKC-βⅡprotein, the Hup-A and H-PNS groups were significantly higher than the Model and L-PNS groups (P<0.01), and no significant difference existed between the Hup-A group and the H-PNS group (P> 0.05) and between the Model and L-PNS groups (P>0.05).The Model group had significantly higher JNKl activity than the H-PNS and Hup-A groups (P<0.01), and no significant difference was between the L-PNS and Model groups (P>0.05) and between the H-PNS and Hup-A groups (P>0.05). No significant discrepancy existed with respect to inter-group comparison among the four groups in terms of the activity of JNK2 and JNK3 (P>0.05). The Model group had a significantly higher p46 MAPK(JNK1)mRNA level than other groups (P<0.01), the L-PNS group was significantly higher than the H-PNS and Hup-A groups (P<0.01), and no marked difference between the H-PNS and Hup-A groups (P>0.05). The Model group had a significantly higher phospho-p46 MAPK (p-JNK1) expression level than other groups (P<0.01), and no significant difference was between the Hup-A and H-and L-PNS groups (P>0.05).The Model group had significantly lower a-Secretase activity than the H-PNS and Hup-A groups (P<0.01) and significantly higher β and γ-Secretase activity than other groups (P<0.01), and no significant difference was between the H-PNS and Hup-A groups in terms of the activity of those proteins. The Model group had a significantly lower ADAM 10 mRNA level than the H-PNS and Hup-A groups (P<0.01), and no significant difference was between the L-PNS and Model groups (P>0.05) and between the H-PNS and Hup-A groups (P>0.05). The Model group had a significantly higher BACE1 mRNA level than the L-PNS group (P<0.01) and the H-PNS and Hup-A groups (P<0.01), and that in the L-and H-PNS groups was significantly higher than that in the Hup-A groups (P<0.05). The Model group had a significantly higher Aph-1 mRNA level than other groups (P<0.01), and no significant difference was between the H-PNS and Hup-A groups (P>0.05). In terms of the expression level of protein ADAM 10, the Hup-A and H-PNS groups were significantly higher than the Model and L-PNS groups (P<0.01), the Hup-A group was higher than the H-PNS group (P<0.01), and no significant difference existed between the Model and L-PNS groups (P>0.05). In terms of the expression level of proteins BACE1 and Aph-1, the Hup-A and H-PNS groups were significantly lower than the Model and L-PNS groups (P<0.01), and no significant difference existed between the Hup-A group and the H-PNS group (P> 0.05) and between the Model and the L-PNS groups (P>0.05).3. The research on PKA, ERK, and CaMKⅡ signal transduction pathways mediated the mechanisms underlying the protection effects of PNS against the neuron toxicity induced by Aβ.Our research showed that PNS significantly enhanced the activity of kinases PKA, ERK 1/2 MAPK and CaMKⅡ, and increased mRNA transcription and protein expression levels of factors PKARⅠα, p-ERK2, CaMKⅡα, p-CREB and BDNF. It is suggested that PNS could activate CREB, facilitate synthesis of BDNF and effectively prevent nerve damage and cognitive impairment as induced by protein Aβ through intervening the PKA, ERK, CaMKⅡ signal transduction pathways. The details as follows:The Model group had significantly lower PKA activity than the L-PNS group (P< 0.05) and the H-PNS and Hup-A groups (P<0.01), the L-PNS group was significantly lower than the H-PNS and Hup-A groups (P<0.01), and no marked difference between the H-PNS and Hup-A groups (P>0.05). The Model group had a significantly lower PKARⅠα mRNA level than other groups (P<0.01), the L-PNS group was significantly lower than the H-PNS and Hup-A groups (P<0.01), and no marked difference between the H-PNS and Hup-A groups (P>0.05). In terms of the expression level of protein PKARIa, the Hup-A and H-PNS groups were significantly higher than the Model and L-PNS groups (P<0.01), and no significant difference existed between the Hup-A group and the H-PNS group (P> 0.05) and between the Model and L-PNS groups (P>0.05).The Model group had significantly lower activity of kinase ERK 1/2 MAPK than other groups (P<0.01), and no significant discrepancy existed with respect to inter-group comparison among H- and L-PNS and Hup-A groups (P>0.05). The Model group had a significantly lower p42 MAPK(ERK2)mRNA level than other groups (P<0.01), the L-PNS group was significantly lower than the H-PNS group (P<0.01) and the Hup-A group (P< 0.05), and no marked difference between the H-PNS and Hup-A groups (P>0.05). The Model group had a significantly lower phospho-p44/42 MAPK (p-ERK1/2) expression level than other groups (P<0.01), the Hup-A group was significantly higher than the L-PNS group (P<0.01) and the H-PNS group (P<0.05), and the H-PNS group was notably higher than the L-PNS group (P<0.01).In term of the activity of kinase CaMKⅡ, the Model group was significantly lower than other groups (P<0.01), the L-PNS group was lower than the H-PNS and Hup-A groups (P<0.01), and no marked difference between the H-PNS and Hup-A groups (P>0.05). The Model group had a significantly lower CaMKⅡα mRNA level than other groups (P<0.01), and no significant difference between the H-PNS and Hup-A groups (P>0.05). In term of the expression level of p-CaMK Ⅱα/β, the Hup-A and H-PNS groups were significantly higher than the Model and L-PNS groups (P<0.01), and no significant difference existed between the Model and L-PNS groups (P>0.05).The Model group had a significantly lower level of p-CREB than the H-PNS group (P<0.01) and the Hup-A group (P< 0.05), and no significant difference between the L-PNS and Model groups (P>0.05) and between the H-PNS and Hup-A groups (P>0.05). In terms of the level of CREB mRNA, the Model group was significantly lower than other groups (P<0.01), the L-PNS group was lower than the H-PNS and Hup-A groups (P<0.01). No significant discrepancy existed between the H-PNS and Hup-A groups (P>0.05).In terms of the level of BDNF, the Hup-A and H-PNS groups were significantly higher than the Model and L-PNS groups (P<0.01), and no significant difference existed between the Hup-A group and the H-PNS group (P>0.05) and between the Model and L-PNS groups (P>0.05). The Model group had a significantly lower BDNF mRNA level than other groups (P<0.01), the L-PNS group was significantly lower than the H-PNS group and the Hup-A group (P<0.01), and no marked difference between the H-PNS and Hup-A groups (P>0.05).Conclusions1. PNS exhibits marked protection effects against hippocampal nerve damage and can effectively improve spatial learning and memory ability in SAMP8 model mice. These benefit functions are closely relevant to the ability of PNS against Aβ formation and against Aβ induced neurotoxicity.2. The promotion effects of PNS on mRNA transcription, protein expression, and enzyme activity of a-Secretase are one of key mechanisms underlying the inhibition effect of PNS against Aβ protein formation. These benefit functions are closely relevant to the activation of a-Secretase associated PKC signal pathway enhanced by PNS.3. The prohibition effects of PNS on mRNA transcription, protein expression, and enzyme activity of β-Secretase are another one of key mechanisms underlying the inhibition effect of PNS against Aβ protein formation. These benefit functions are closely relevant to the activation of β-Secretase associated PKC signal pathway enhanced by PNS.4. The prohibition effects of PNS on mRNA transcription, protein expression, and enzyme activity of y-Secretase are another one of key mechanisms underlying the inhibition effect of PNS against Aβ protein formation. These benefit functions are closely relevant to the inhibition of y-Secretase associated JNK signal pathway interrupted by PNS.5. The promotion effects of PNS on p-CREB, BDNF mRNA transcription and protein expression are one of key mechanisms underlying the protection effects of PNS against hippocampal neuron damages, and would contribute to its benefit effects of improving the Long-term potentiation (LTP) and learning and memory function.6. The promotion effects of PNS on p-CREB, BDNF mRNA transcription and protein expression are closely relevant to the activation of upstream associated signal transduction pathway enhanced by PNS, e.g. PKA, ERK, CaMK Ⅱ signal pathway.7. The protection effects of PNS against Aβ induced neurotoxicity are closely relevant to the activation of PKA, ERK, CaMK II signal transduction pathway enhanced by PNS, which followed by promotion of p-CREB, BDNF mRNA transcription and protein expression.8. The protection effects of PNS against AD like neuropathology are mediated by intervention of PNS with a series of signal transduction pathways and its regulation to associated proteins expression, which would account for the impressive effection of the drug in AD like neural degenerated diseases’treatments. |
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