Investigation Of Neural Restoration And Regeneration In Papez’s Circuit Of Rats Model With Generalized Anxiety Disease:The Intervening Mechanism Of Smoothing Liver, Clearing Heat And Strengthening Spleen Therapy

Objective:in this study, Danzhi Xiaoyao Powder with the effect of smoothing liver, clearing heat and strengthening spleen was used to treat generalized anxiety disease (GAD) model rats, and changes in behavior, neurotrophic factors (NTFs) and receptors in Papez’s circuit, proliferation and differentiation of neural stem cells (NSCs) in dentate gyms (DG) of hippocampus were tested. It was expected to explore the pathological mechanism of GAD and intervening mechanism of Danzhi Xiaoyao Powder on neural restoration and regeneration and to further improve the theory of smoothing liver, clearing heat and strengthening spleen treatment for GAD.Methods:male wistar rats were randomly divided into normal control group (NC), GAD model group (M), Chinese medicine group (CM) and western medicine group (WM),10 rats in each group. The GAD model was established by using the stimulation of uncertain empty bottle for 21 days. Rats in CM and WM were respectively treated with intragastric administration of Danzhi Xiaoyao Powder and Buspirone Hydrochloride for 7 days, NC and M with distilled water. Open-field test and elevated plus maze test were conducted on 7,14, 21 d respectively. After removing rats brains, correlated indexes were detected as follow:① measuring expressions of BDNF, TrkB, bFGF, bFGFR and NGF in hippocampus, amygdaloid and anterior thalamic nucleus in brain through immunohistochemistry; ② checking number of positive cells marked by Brdu, Brdu/Nestin, Brdu/NeuN and Brdu/GFAP in DG by using immunofluorescence and double-labeling immunofluorescence respectively.Results:1 Observation of general conditions1.1 Appearance, active and spirit stateOn 21d, the appearance, active and spirit state of M were worse than NC manifested such symptoms as gaunt body, dull fur, significantly reduced action of hair combing and searching, inactive diet and easy irritation such as attacking and biting bottle; after 7 days’ treatment, the general states of rats in CM and WM were partly improved compared with M but still worse than NC.1.2 Weightweight of rats in M on 7d,14d and 21d were obviously lower than NC(P<0.01); after 7 days’ treatment, weight of rats in CM and WM were obviously higher than M on 21d (P<0.01).2 Observation of praxiology2.1 OFTOn 7d,14d and 21d, horizontal score (HS) and vertical score (VS) of rats in M were lower than NC (P<0.05, P<0.01); after 7 days’ treatment, HS and VS of rats in CM and WM on 21d were higher.than M (P<0.01, P<0.05).2.2 EPMOn 7d,14d and 21d, OE/TE and OT/TT of rats in M were lower than M (P<0.01、 P<0.05), after 7 days’ treatment, OE/TE and OT/TT of rats in CM on 21d were higher than M (P<0.05) while there was no obvious difference between WM and M (P>0.05).3 Tests for NTFs and receptors in Papez’s circuit3.1 BDNF and TrkB① hippocampus:IOD of BDNF and TrkB in CA1, CA2, CA3 and DG in M was lower than NC (P<0.01, P<0.05); IOD of BDNF and TrkB in these four zones in CM was higher than M (P<0.05, P<0.01), and IOD of BDNF in WM was higher than M only in CA3 (P<0.05), IOD of TrkB in WM was higher than M in CA3 and DG (P<0.05).② amygdaloid, anterior thalamic nucleus:IOD of BDNF and TrkB in amygdaloid in M was lower than NC (P<0.01, P<0.05); IOD in CM and WM was obviously higher than M (P<0.01). there was no difference among IOD of BDNF and TrkB in anterior thalamic nucleus of four groups (P>0.05).3.2 bFGF and bFGFR① hippocampus:IOD of bFGF and bFGFR in CA1, CA2, CA3 and DG in M was higher than NC (P<0.05, P<0.01); IOD of bFGF and bFGFR in these four zones in CM was much higher than M (P<0.05, P<0.01), while IOD in WM was not different from M (P>0.05).② amygdaloid, anterior thalamic nucleus:IOD of bFGF and bFGFR in amygdaloid in M was lower than NC (P<0.05); IOD in CM was higher than M (P<0.05). there was no difference among IOD of bFGF and bFGFR in anterior thalamic nucleus of four groups (P>0.05).3.3 NGF① hippocampus:IOD of NGF in CA1, CA2, CA3 and DG in M was lower than NC (P<0.05, P<0.01); IOD of NGF in these four zones in CM and WM was higher than M (P<0.01, P<0.05).② amygdaloid, anterior thalamic nucleus:IOD of NGF in amygdaloid in M was obviously lower than NC (P<0.01); IOD in CM and WM was clearly higher than M (P<0.01). there was no significant difference among IOD of NGF in anterior thalamic nucleus of four groups (P>0.05).4 Detection of NSCs proliferation and differentiation in DG4.1 number of Brdu positive cellsCompared with NC, Brdu positive cells in hippocampus in M increased clearly (P<0.01); Brdu positive cells in CM were much more than M(P<0.05), while there was no significant difference between WM and M (P>0.05).4.2 number of Brdu/Nestin positive cellsCompared with NC, Brdu/Nestin positive cells in DG in M increased significantly (P<0.01); Brdu/Nestin positive cells in CM were much more than M (P<0.05), there was no significant difference between WM and M (P>0.05).4.3 number of Brdu/NeuN positive cellsCompared with NC, Brdu/NeuN positive cells in hippocampus in M decreased (P<0.05); Brdu/NeuN positive cells in CM increased compared with M (P<0.05), there was no significant difference between WM and M(P>0.05).4.4 number of Brdu/GFAP positive cellsCompared with NC, Brdu/GFAP positive cells in hippocampus in M decreased (P<0.05); compared with M, Brdu/GFAP positive cells in CM and WM both increased obviously (P<0.01,P<0.05).Conclusion:1 Anxious behaviors and typical symptoms of liver stagnation with heat and spleen deficiency can be obviously induced on Wistar rats by stimulation of uncertain empty bottle which is an effective and stable method for GAD model building. The anxious behaviors, active and exploring capacities and other associated symptoms of GAD rats can be improved by Danzhi Xiaoyao powder with the effect of smoothing liver, clearing heat and strengthening spleen.2 Down-regulation of BDNF and NGF in hippocampus and amygdaloid could be one initial event of GAD pathogenesis and it’s closely related with neuron injury and apoptosis in Papez’s circuit. bFGF in hippocampus plays a positive and critical role in endogenic neural restoration and regeneration. Danzhi Xiaoyao powder can up-regulate the expression of BDNF, TrkB, bFGF, bFGFR and NGF, improve the micro-environment of injured region, enhance neuroplasticity and create ideal and necessary conditions for neural restoration.3 As a chronic psychogenic stress, the stimulation of uncertain empty bottle can excite compensatory proliferation of NSCs in SGZ of GAD rats while inhibit NSCs differentiation to neuron and astrocyte. Danzhi Xiaoyao powder can promote NSCs proliferation further and enhance NSCs capacity of differentiation in SGZ. Consequently, neural restoration and regeneration can be strengthened.4 During CNS injury, endogenic processes of neural restoration and regeneration do exist which are relatively faint, however. Danzhi Xiaoyao powder can promote neural restoration and regeneration by up-regulating NTFs expression, enhancing neuroplasticity, proliferation and differentiation of endogenic NSCs, which could be one critical pathway of therapeutic mechanism for GAD.