| Alzheimer's disease (AD) is the most common form of progressive dementia in the elderly. The aetiology of AD remains unknown and there is lack of effective cure. Cholinesterase inhibitors are most widely used for the treatment of AD. However, current drugs do not significantly ameliorate the corruption of neurodegeneration, and only provide limited or transient benefit to many patients. The development of drugs with more effective and less adverse drug reaction is imminent.The discovery and validation of drug target are very important procedure of developing original drug. In the past time, drug therapy based on single-target-directed strategy seems inappropriate for the treatment of complex diseases, AD, that have multiple pathogenic factors and multiple dysfunctions. So the comprehensive treatment by means of multi-target is feasible to ameliorate therapy for AD. And the discovery, study and validation of one or one more groups of combinatorial targets associated with cognitive dysfunction and pathological abnormalities tightly will provide a new strategy for effective therapy for AD. Combining the techniques of genomics, proteomics, microarray and bioinformatics in drug discovery has been proposed as one of rapid and efficient approaches to search and find new potential drug targets.The main clinical symptoms of AD are primarily memory loss and cognitive impairments. The expression and transcription of many genes and their encoding protein are associated with occurrence and development of AD. At present, genes of amyloid precursor protein (APP), APOE4, presenilin-1 (PSEN1), PSEN2, transcription factor CP2 (TCFP2), Alpha-T-catenin (CTNNA3) and LRP1 are known as some of the reasons of AD, but for this multi-factorial disease, there may be many unknown genes that play important roles in AD. Hippocampus and cerebral cortex are important brain regains for learning and memory. The study on expression levels of gene in the brain may provide clues of some function of CNS and potential gene targets.The senescence-accelerated mouse (SAM) is a model of accelerated senescence that was established through phenotypic selection. The SAMP8 strain, a substrain of SAM, has been proposed as a good model for the study of aging of the brain and of deficits in learning and memory at gene and protein levels with the features of pathological abnormalities in the brain, learning and memory deficits. The study of gene expression change in hippocampus and cortex of SAMP8 will provide clues for discovery of genes about learning and memory in CNS and gene targets for AD.The traditional Chinese medicine and their effective components have properly their own inimitable predominance with their multi-factorial, multi-target and multi-functional action than chemical compound with a single-target. In fact, Dang-Gui-Shao-Yao-San (DSS), Kai-Xin-San, Liu-Wei-Di-Huang decoction (LW), Ba-Wei-Di-Huang decoction (BW) and Huang-lian-Jie-Du Decoction (HL) etc, traditional Chinese medicinal prescriptions, have many clinical pharmacological actions including enhancing the cognitive function of CNS and benefits to AD patients in China and Japan. In the past, the study in our lab showed the above Chinese medicine ameliorated learning and memory. According to described in the theory of traditional Chinese medicine, the effect and indication of above Chinese medicine are different, including purging the fire and detoxifying, invigorating the kidney, promoting blood flow etc. These diversified effects indicated the different pharmacological actions and mechanisms maybe result in the difference of gene expression change in CNS. The difference and similarity of differential expression genes treated with diversified Chinese medicines were analyzed to discover the specific and common differential expression genes. The common differential expression represented the similarity of pharmacological actions and mechanisms. But the specific differential expression genes to single Chinese medicine represented drug action or mechanism. In this paper, the gene expression patterns of hippocampus and cortex of SAMP8 treatment with LW, BW and HL were investigated to provide insight into the study of the gene targets for AD.This research consisted of two sections. One was the screening of differential expression genes in the period of aging and patterns of gene expression in hippocampus and cortex of SAMP8 through the technology of cDNA microarray. This aimed to discover a group of expression change genes associated with aging and drug action treatment with Chinese medicine. Meanwhile this aimed to establish the foundation of candidate gene targets discovery. The other was proposed a group of combinatorial candidate genes and primary validation. With the animal model of SAMP8, natural product HupA and Chinese medicine LW, BW, HL, DSS, TXF, the expression change of candidate genes in hippocampus and cortex of SAMP8 was carried out through the technology of real time quantitative RT-PCR. 1. The screening of gene targets for AD therapyIn order to investigate the potential gene targets, the differential month-old animal model SAMP8 and LW, BW and HL were applied in this study. The screening and analysis of differential gene expression in hippocampus and cortex were performed employing the cDNA microarray that designed of one's own.1.1 The differential gene expression in hippocampus and cortex of differential ageSAMP8The differential gene expression patterns in hippocampus and cortex of 2,6,12 month-old SAM were performed employing cDNA microarray and validation of real-time RT-PCR. The results showed there were 124 differential expression clones in hippocampus of 2-month SAMP8 and none in cortex of 2-month SAMP8 comparing with 2-month SAMR1. These clones were sequenced and 100 clones were sequenced successfully. The analysis of 100 differential expression clones by bioinformatics indicated these clones represented 45 genes. The results showed there were 20 differential expression clones in hippocampus of 6-month-old SAMP8 comparing with 6-month-old SAMR1. These clones were sequenced and 9 clones were sequenced successfully. These clones represented 5 genes. The results showed there were 31 differential expression clones in cortex of 6-month SAMP8 comparing with 6-month SAMR1. These clones were sequenced and 21 clones were sequenced successfully. The analysis of 21 differential expression clones by bioinformatics indicated that these clones represented 15 genes. The results showed there were 14 differential expression clones in hippocampus of 12-month-old SAMP8 comparing with 12-month-old SAMR1. These clones were sequenced and 7 clones were sequenced successfully. These represented 4 genes with distinct function. The results showed there were 146 differential expression clones in cortex of 12-month SAMP8 comparing with 12-month SAMR1. These clones were sequenced and 120 clones were sequenced successfully. These clones represented 36 genes. After the above results of differential expression genes were analyzed by bioinformatics, these represented distinct function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, regulation of transcription, protein metabolism, nervous system development, energy metabolism, cell growth and maintenance, immune response and many genes whose biological function and process were still unknown.SAMP8 is a good animal model of age-related cognitive decline with relevance to alterations of the gene expression and protein abnormalities in AD. In this study, the results of differential gene expression in 2, 6, 12-month-old SAMP8 with SAMR1 showed more and more differential expression genes in the period of ageing and hint these genes were associated with cognitive decline and accelerated senescence, especially genes of signal transduction, protein and energy metabolism. These genes maybe provide significant clues for aging and cognitive impairment, meanwhile provide important base for study of potential targets for AD therapy.1.2 Gene expression patterns of hippocampus and cortex of SAMP8 treated with LWLiu-Wei-Di-Huang decoction (LW), a traditional Chinese medicinal prescription, hasmany clinical pharmacological actions including enhancing the cognitive function of CNS. Recently, pharmacological studies of our lab demonstrate that the major effect of LW was improvement of cognition and memory of SAMP8, according to research of neuroendocrine immunological regulation (NIM). In order to get the message of mechanism of LW enhancing the cognitive function and discover target for therapy, cDNA microarray was adopted.The results showed there were 11 differential expression clones in hippocampus and 67 differential expression clones in cortex of SAMP8, after treated with LW (5g/kg). These clones were sequenced and 53 clones were sequenced successfully. They represented 28 genes with diversified biological function. The results showed there were 43 differential expression clones in hippocampus and 222 differential expression clones in cortex of SAMP8, after treated with LW (10g/kg). These clones were sequenced and 137 clones were sequenced successfully. They represented 60 genes with diversified biological function. The results showed there were 80 differential expression clones in hippocampus and 233 differential expression clones in cortex of SAMP8, after treated with LW (15g/kg). These clones were sequenced and 197 clones were sequenced successfully. They represented 55 genes with diversified biological function. After the above results of differential expression genes were analyzed by bioinformatics these genes treated with different dosage of LW represented similar biological function, including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, regulation of transcription, protein metabolism, nervous system development, energy metabolism, cell growth and maintenance, immune response, electron transport and many genes whose biological function and process were still unknown.These results demonstrated there was similar functional category of differential expression genes. But there were more expression change genes in group medium and high dosage than group of low dosage. Gene Ttc3 Oxr1 Slc17a7 Rps6ka1 C1qb Nsf Mast2 Rock1 Dusp12 Tac2 Def8 Mink1. Prr6, Acrbp 300002F03 AL845475.12 expression changed significantly. It hints the effect of LW about learning and memory maybe is related to above genes.1.3 Gene expression patterns of hippocampus and cortex of SAMP8 treated with BW and HL BW and HL, traditional Chinese medicinal prescription related to invigorating kidney-YANG and purging the fire and detoxifying respectively, have many clinical pharmacological actions including enhancing the cognitive function of CNS. Recently, pharmacological studies demonstrate that the effect of improvement of cognition and memory was one of their diversified effects. In order to get the message of mechanism of BW and HL enhancing the cognitive function and discover target for therapy, cDNA microarray and 6-month-old SAMP8 were adopted and real time PCR was performed to validate the results from microarray. We analyzed and compared with the different expression patterns of hippocampus and cortex treated with LW, BW and HL.The results showed there were 42 differential expression clones including 21 up-regulated clones and 21 down-regulated clones in hippocampus and 14 differential expression clones including 9 up-regulated and 5 down-regulated in cortex of SAMP8, after treated with BW (10.2g/kg). These clones were sequenced and 39 clones were sequenced successfully. They represented 14 genes with diversified biological function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, protein metabolism, cell growth and maintenance, and 5 genes whose biological function and process were still unknown.The results showed there were 143 differential expression clones including 29 up-regulated clones and 114 down-regulated clones in hippocampus and 93 differential expression clones including 9 up-regulated and 84 down-regulated in cortex of SAMP8, after treated with HL (5g/kg). These clones were sequenced and 168 clones were sequenced successfully. They represented 36 genes with diversified biological function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, protein metabolism, energy metabolism, cell growth and maintenance, regulation of transcription, and nerve system development and 25 genes whose biological function and process were still unknown.In order to analyze the patterns of gene expression treated with different Chinese medicine prescription, we investigated the differential gene expression treatment with LW meanwhile. The results showed there were 44 differential expression clones including 40 up-regulated clones and 4 down-regulated clones in hippocampus and 27 differential expression clones including 7 up-regulated and 20 down-regulated in cortex of SAMP8, after treated with LW (10g/kg). These clones were sequenced and 54 clones were sequenced successfully. They represented 12 genes with diversified biological function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, protein metabolism, energy metabolism, cell growth and maintenance, regulation of transcription, and nerve system development and 8 genes whose biological function and process were still unknown.Comprehending the above screening results of HL, BW and LW, we detected that the three Chinese medicine prescriptions possessed significant ability of regulation of gene expression, including common effective genes to all medicine and specific effective genes to specific medicine. The common effective genes were eight, including nucleic acid metabolism gene Fhit and Itm2c, protein metabolism gene Ube2d2, transport gene Slc17a7, signal transduction gene Rps6kal, ribosome biogenesis and assembly gene Rn18s and 18srRNA, functional unknown gene Rps19bp1. These common genes hint there is some similarity with the three Chinese medicine prescriptions.2. The propose and primary study of combinatorial candidate gene targets for AD therapy2.1 The propose of combinatorial candidate gene targets for AD therapyFrom the aggregate analysis the patterns of gene expression change in hippocampus and cortex in the period of aging and gene expression change in hippocampus and cortex treated with Chinese medicine prescription LW, BW and HL, there were 60 differential expression genes with clear biological function. Among the 60 genes with different function, signal transduction, nucleic acid metabolism, protein metabolism, energy metabolism was the major portion in ratio. This demonstrated these categories of biological function were mutually related to the occurrence and development of AD, pathway of drug action and drug targets. This is worth to further study. In addition, 45 genes with function unknown were interrelated to the occurrence and development of AD and possessed more significance of neurobiology. These results provided extensive prospect of research.After comparing and analyzing the differential expression genes that always in the period of aging, that treated with two or three Chinese medicine prescription of LW, BW, HL or that in a specific month-old and treated with one Chinese medicine prescription simultaneously, and combining the bioinformatics and references, we posed the combinatorial candidate gene targets for AD therapy, namely (1) nucleic acid metabolism gene, including Itm2c, Fhit; (2) signal transduction gene, including Rps6ka1, Rock1, Dusp12, Mink1, Rab26, Mast2, Camk2α, Ephb6, S100a11, Tac2, Clstn1, Strn4; (3) protein metabolism gene, including Amfr, Ube2d2, Ttc3, Prr6; (4) transporter gene, including Slcl7a7; (5) regulation of transcript gene, including D1Ertd161e, Ssu72; (6) energy metabolism gene, including Stub1, Uqcr, Ranbp5, Nsf; (7) cell growth and maintenance gene, including Ngrn; (8) nerve system development gene, including Trim3; (9) electron transport gene, including Ndufs2; (10) Neurogila cell differentiation gene, including Tspan2; (11) function unknown gene, including Rps19bp1, 300002F03.2.2 Primary study of combinatorial candidate gene targetsTo validate the above combinatorial candidate gene targets, we applied the real time PCR to assess the patterns of gene expression in the period of aging and treated with LW, BW, HL, DSS, TXF, HupA that benefited to AD therapy.The results showed there were 14 types of change of the 32 genes expression in the hippocampus of SAMP8 in the period of aging. And there were 13 types of change of the expression 31 genes in the cortex of SAMP8 in the period of aging, except for Ndufs2. After treated with Hup A, LW, BW, HL, DSS, TXF, there were all change of 32 genes expression in the hippocampus and cortex of SAMP8. In hippocampus, these changes of gene expression were defined 6-7 types. In cortex, these changes of gene expression were defined 5-7 types. After treated with 5 Chinese medicine prescriptions and HupA, gene Trim3, Tspan2, Ttc3, Rock1, Ube2d2, Mast2, Clstn1 in hippocampus all presented gene expression change, but gene Amfr, C1qb, Dusp12, Ephb6, Itm2c, Mink1, Tac2, Ndufs2, Ngrn, Nsf, Ranbp5, S100all, Fhit, Prr6, Rab26, 300002F03, Slc17a7 in hippocampus or cortex presented expression change for one or one more Chinese medicine prescriptions. Only gene Ssu72, Rpsl9bpl, Rps6kal in hippocampus presented no gene expression change.Through the primary validation of 32 genes, the expression of them presented regularity. This hint these genes were interrelated to the occurrence and development of AD. The similarity and difference of 32 genes expression demonstrated the characters of effects of Chinese medicine and HupA.The discovery and validation of drug targets is an arduous work, by no means achieve the goal through one paper or one research. But we believe the "combinatorial targets" is a helpful attempt. The results from this paper only provided clues and more complicated and hard works need our endeavor.
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