Study On The Mechanism Of The Anxiolytic Extracts Of Cortex Albiziae And The Effect Of The Main Active Ingredient On PC12 Cell Apoptosis

Anxiety disorder is a chronic,long-term and recurrent mental illness,which can cause both physical and mental abnormalities and affect the quality of life of patients and their families.At present,there are some obvious side effects of clinical antianxietic.Therefore,it is of great theoretical significance and practical value to develop safe,efficient and low toxic natural antianxietic.Cortex Albiziae is the stem bark of the leguminous Plant Albizia julibrissin Durazz.,which is always used to treat depression and insomnia in traditional Chinese medicine.The pharmacological effects of Cortex Albiziae are extensive,such as sedative,anti-tumor,anti-fertility,anti-anxiety and PAF receptor antagonistic action.Modern pharmacological experimental study have shown that it can ameliorate the anxiety-like behavior of anxiety model mice,but there are no reports on the cellular and molecular mechanisms of its anxiolytic effects.The preliminary study of the project showed that lignans are the main anxiolytic effective substances.In this paper,the classical anti-anxiety activity evaluation model was used to study the anti-anxiety mechanism of the main anxiolytic extracts（S-75）and composition of lignans from Cortex Albiziae.The contents were divided into three parts:The effect of S-75 on the level of 5-HT_1A receptor,GABA_A receptor and related receptor signaling pathway in brain tissue of mice with elevated plus maze test were observed from the whole animal level.The results showed that S-75 could promote the expression of mRNA and protein of 5-HT_1AR and GABA_AR,increase the expression of PKA,CREB,BDNF,TRKB protein and the level of cAMP,which were related to the key part of 5-HT_1AR signaling pathway.The results suggested that the anti-anxiety mechanism of S-75 may be related to 5-HT_1AR signaling pathway.In order to verify the anti-anxiety mechanism of S-75,the study in vitro was carried out on the basis of the integral experiment.To observe the protective effect of S-75 on PC12 cells damaged by corticosterone and the effects of S-75 with the corresponding receptor blockers on 5-HT_1AR,GABA_AR and related receptor signaling pathways by establishing the model of PC12 cells damaged by 0.1 mmol·L^-1 corticosterone.The results showed that S-75 stimulated the proliferation of PC12 cells,promoted the expression of mRNA of 5-HT_1AR and GABA_AR in PC12 cells,elevated the expression of PKA,CREB,BDNF,TRKB protein and the level of cAMP in PC12 cells.When S-75 was applied with WAY-100635 of 5-HT_1AR receptor blocker in PC12 cells,the expression of PKA,CREB,BDNF,TRKB protein in PC12cells decreased and the level of cAMP decreased correspondingly.Therefore,it was further confirmed that the anti-anxiety mechanism of S-75 was closely related to 5-HT_1AR signaling pathway.The two parts of the preliminary experiment confirmed that the anti-anxiety mechanism of S-75 was closely related to 5-HT_1AR signaling pathway.To further screen out the main active ingredient of S-75,and to study the effect of the main active ingredient on PC12 cell apoptosis induced by corticosterone and mitochondrial apoptotic signaling pathway.In this experiment,the S-75 and its seven monomer compositions were used in the PC12 cells induced by corticosterone,and the seven monomer compositions were:4-O-syringaresinol,4-O-syringaresinol Ethyl Ether,syringaresinol-4-O-p-nitrob-enzoate,（-）-syringaresinol-4-O-β-D-apiofuranosyl-（1→2）-β-D-gluc--opyranoside,daidzein,（-）-syringaresinol-4-O-β-D-glucopyranoside,syringar--esinol-di-O-glucoside.The main active ingredients were screened by the survival rate of the PC12 cells.The release amount of lactic dehydrogenase（LDH）was measured using a LDH assay kit.PC12 cell apoptosis was tested by flow cytometry and TdT-mediated dUTP nick end labeling（TUNEL）assay kit,and intracellular Ca²⁺concentrations were examined by Fluo-3-Am fluorescent probe.The mitochondrial membrane potential（ΔΨm）of PC12cells was detected by Rhodamine 123 staining.Then,the expression of CREB,BDNF,Bcl-2,Bax,cytochrome C（Cyt-C）and caspase-3 protein were monitored by Western blot.The results showed that the seven compounds promoted the proliferation of PC12 cells,（-）-syringaresinol-4-O-β-D-glucopy--ranoside（SRG）with different concentrations promoted the proliferation of PC12 cells significantly and the dose effect relation was obvious,which could be the main active ingredient of Cortex Albiziae.SRG could inhibit the leakage of LDH,reduce apoptosis and protect mitochondrial membrane of PC12 cells,inhibit the release of[Ca²⁺],increase the expression of BDNF,CREB,Bcl-2 protein and decrease the expression of Bax,cytochrome C and caspase-3 protein,which were related to the key part of mitochondrial apoptosis signaling pathway.Through the above experimental study,we found that the anti-anxiety mechanism of S-75 of Cortex Albiziae was closely related to 5-HT_1AR signaling pathway.（-）-syringaresinol-4-O-β-D-glucopyranoside（SRG）could be the main active ingredient of Cortex Albiziae.And found that SRG could inhibit the apoptosis of PC12 cells induced by 0.1 mmol·L^-1 corticosterone.The mechanism was related to the inhibition of mitochondrial apoptosis in PC12 cells.