Studies On Depression And The Pharmacological Mechanisms Of Kai Xin San Using BDNF Gene Knockdown By RNAi

Brain-derived neurotrophic factor (BDNF) is the most widely distributedneurotrophin in the central nervous system (CNS), servicing many biological functionssuch as neural survival, differentiation, and plasticity. BDNF has been implicated in thepathophysiology of depression and has an important function in the mechanism ofantidepressant action. However, there is no direct evidence that the actual reduction ofthe BDNF can induce depressive-like behaviors. Therefore, we began a series of studiesto knockdown BDNF in dentate gyrus (DG) subregion of hippocampus by RNAinterference to examine the role of BDNF in depression and the pharmacologicalmechanisms of Kai Xin San(KXS).To induce a stable knockdown, we generated four lentiviral vector(LV)constructsexpressing sequences short hairpin RNAs(shRNAs) complementary to the coding exonof the rat BDNF gene, common to all isoforms of this gene. Then we tested whichsequence most effectively reduces BDNF levels in a C6rat glioma cell line, whichnaturally expresses the BDNF protein, with qPCR and western blot analysis, and weobserved a significant reduction in BDNF secretion in LVshBDNF-3.In order to further verify the efficiency of BDNF knockdown with LVshBDNF-3,primary hippocampal neurons of neonatal rats were cultured and the identification ofneurons and the purity calculation were done by immunofluorescence. After transfectingthe neurons by the lentivirus, the effects of KXS on the expressions of BDNF gene andprotein was detected by qPCR and western blot, respectively.We microinjected a lentiviral vector containing either a shRNA targeting BDNF(LVshBDNF-3) or a nonsilencing sequence bilaterally into the DG of the SD rat. To investigate the effects of BDNF knockdown in specific brain sites on behavior, oneweek after the last injection, a battery of behavioral tests associated with somesymptoms of depression were used, including sucrose preference, open field test andMorris water maze. One week following the last microinjection of lentiviral shRNA invivo, we observed a significant reduction in BDNF protein expression in thehippocampus, sucrose preference and the open field test. The BDNF knockdown in theDG also resulted in a delay in spatial learning as measured in the Morris water maze,but had no effect on performance in a subsequent memory probe test.Upon establishing the LV-shRNA silencing of BDNF in rat dentate gyrus induceddepression model, KXS were administrated via gastric intubation for14days, we thenexamined antidepressant effect of KXS and its antidepressant mechanism. The resultsthat were obtained from the behavioral experiment indicate that KXS has anameliorative effect on depression both in sucrose preference and in open-field test in therats. In Morris water maze, results showed that KXS could improve ability of learning,however, no such effect was observed in memory.Together with our finding of an inverse correlation between depression and BDNFexpression levels, these results implicate BDNF in the genesis and expression ofdepressive disorders. Our study strengthens the neurotrophic hypothesis of depression.Furthermore, KXS can reverse LVshBDNF-3induced reduction of BDNF onhippocampus and this effect is at least partly medicated by increasing expression ofincomplete BDNF gene knockdown. The KXS activate related signaling pathways andexert its antidepressantlike effects through a multi-components and.multi-targets way.