Anesthetic Effects And Mechanisms Of Black Berry And White Thorn

Background:Anxiety disorder known as anxiety neurosis is a kind of neurological disorders mainly containing dysphoric mood.It is characterized by mental disorders such as anxiety,tension,and fear,and it is often accompanied by the symptoms such as autonomic nerve function disturbance,muscle tension and uneasiness.There are currently six recognized anxiety syndromes,such as generalized anxiety disorder(GAD),panic disorder(PD),social anxiety disorder(SAD),obsessive compulsive disorder(OCD),post-traumatic stress disorder(PTSD)and agoraphobia.The incidence of anxiety increased year by year,which has become a common disease and frequently encountered disease,and which seriously impacts on people's quality of life.The treatment and mechanism of anxiety gradually become a hot point of academic research.Fruits of Lycium ruthenicum Murray(LR),also called "black Chinese wolfberry" in China,have long been record in the Tibetan medical classic Four-Volume Medical Code and Jing Zhu Materia Medica.It is natured and sweet,often used for treating cardiovascular disease,gynaecopathia and irregular menstruation.Fruits of Nitraria tangutorum Bobr.(NT),is commonly used as an alternative of the black Chinese wolfberry because of the similar of regional distribution,shapes and taste with it.LR and NT are all rich in anthocyanins.The anthocyanins which as a kind of flavonoid possesses a variety of biological activities,such as free-radical scavenging,antioxidant,prevention of cardiovascular diseases neurodegenerative diseases,and improvement of learning and memory.Then,how are anti-anxiety effects of the LR and NT?What signaling pathways are associated with the action mechanism of LR and NT in addition to the above mentioned?These questions are worthy of our study.Objective:The purpose of this study was to determine and compare the anxiolytic-like effects of the extract of Lycium ruthenicum Murry(LRE)and the extract of Nitraria tangutorum Bobr(NTE)in mice and rats using several behavioral experiments such as elevated plus-maze test(EPM),light/dark box test(LDB),empty bottle stimulation experiment(EBS)and restraint stress model experiment(RS).Evaluating anti-anxiety function mechanism of LRE and NTE of effects on hypothalamic pituitary adrenal axis(HPA)on EBS rats,the BDNFexpression in cortex and hippocampus on RS rats,the effects of ERK/MAPK pathways,the Bcl-2 and Bax expression in hippocampus on RS rats,and the neuron regeneration of hippocampal dentate gyrus on RS rats.Through the above research,We determined the anti-anxiety effects of LRE and NTE and explore the mechanism of action of related,providing experimental and theoretical basis for safe and effective application of them,providing a new idea for the development of new natural anti-anxiety drugs.Methods and results:The main research contents and achieved results are as follow:1 The preparation of LRE and NTE1.1 The extraction of LRE and NTEThe dry fruits were immersed twice with 8 folds of 80%alcohol(2%formic acid)at 50?in the dark for 3 h.1.2 The purify of LRE and NTERemoving impurity and enrichment with AB-8 macroporous resin adsorption method.Recovering the solvents and freeze drying.The yield of LRE and NTE were 8.04%and 5.02%,respectively.1.3 Total anthocyanins content of LRE and NTETotal anthocyanins content was determined spectrophotometrically using the pH differential method.Results were further treated to express total anthocyanins content in mg cyanidin-3-O-glucoside equivalents per 100 g fruits.2 The Pharmacological Research2.1 The anti-anxiety effect of LRE and NTE in miceDetermine the anxiolytic-like effects of LRE and NTE by EPM test and LDB test.According the results of the EPM test in mice,compared with control groups,LRE at dose of 320 mg/kg/d and NTE at dose of 400 mg/kg/d could significantly increase OE%and OT%of mice in EPM.According the results of the LDB test in mice,compared with control groups,LRE at dose of 320 mg/kg/d and NTE at dose of 400 mg/kg/d could significantly increase the time spent and transitions between light and dark compartments.2.2 The anti-anxiety effect of LRE and NTE on rats with EBSDetermine the anxiolytic-like effects of LRE and NTE on rats with EBS model.In the EPM,the animals subjected to the empty bottles stimulation showed an excessive anxiety,since they exhibited a reduction in the percentage of time spent and entries on open arms.In the OFT,the animals subjected to the empty bottles stimulation showed an excessive anxiety,since they exhibited a reduction in the time spent in center area.2.3 The anti-anxiety effects of LRE and NTE on rats with RS.Determine the anxiolytic-like effects of LRE and NTE on rats with restraint stress.In the EPM,the animals subjected to restraint showed an excessive anxiety,since they exhibited a reduction in the percentage of time spent and entries on open arms.3 Mechanism Studies3.1 Effects of LRE and NTE on hypothalamic pituitary adrenal axis by EBS exposure.The level of serum hormones in hypothalamic-pituitary-adrenal axis(for short,HPA axis)of the rats were measured by ELISA methods.According to the results,the level of CRF,ACTH and CORT were increased in EBS rats compared to the control group.Compare with the control group,LRE at the doses of 100 mg/kg/d and 200 mg/kg/d as well as NTE at doses of 250 mg/kg/d could significantly decreased the level of CRF,ACTH and CORT.The result indicated that the anti-anxiety effects of LRE and NTE might be related to HPA axis.3.2 Effects of LRE and NTE on the MAPK/ERK pathway by stress exposureThe protein levels of ERK1/2,CREB,phosphorylated ERK1/2(p-ERK1/2),and phosphorylated CREB(p-CREB)in the hippocampus(HP)and prefrontal cortex(PFC)were examined by Western blot analysis.Our results showed that the rats exposed to stress resulted in the increase of p-ERK1/2 and p-CREB in the HP and PFC compared with the control group.In addition,LRE-100 and LRE-200 as well as NTE-250 decreased the ratio of p-ERK1/2 to ERK1/2 and the ratio of p-CREB to CREB compared with the stress group in the HP and PFC.Our study suggested that one potential way,by which LRE and NTE had anti-anxiety-like effects,might be mediated by activating the ERK/MAPK pathway in the brain of rats.3.3 Effects of LRE and NTE on the BDNF protein expression by stress exposure.The protein levels of BDNF in the hippocampus(HP)and prefrontal cortex(PFC)were examined by Western blot analysis.Our results showed that the rats exposure to stressresulted in the decrease of BDNF in the HP and PFC.In addition,LRE-100 and LRE-200 as well as NTE-250 increased the ratios of BDNF in the HP and PFC.Our study suggested that one potential way,by which LRE and NTE had anti-anxiety-like effects,might be mediated by activating the BDNF in the brain.3.4 Effects of LRE and NTE on the protein of Bcl-2/Bax expression by stress exposure.The protein levels of Bcl-2 and Bax in the hippocampus(HP)were examined by Western blot analysis.Our results showed that the rats exposure to stress resulted in the decrease of Bcl-2 meanwhile the increase of Bax levels in the HP.In addition,LRE-100 and LRE-200 as well as NTE-250 increased the ratios of Bcl-2 in the HP,and decreasing the level of Bax.Our study suggested that one potential way,by which LRE and NTE had anti-anxiety-like effects,might be mediated by influences on apoptosis of hippocampal neurons.3.5 Effects of LRE and NTE on neuron regeneration of hippocampal dentate gyrus of rats by stress exposure.Analyzed the BrdU positive level on hippocampal dentate gyrus of RS rats by the immunohistochemical method.Our results showed that the rats exposure to stress resulted in the decrease of the BrdU positive level compared with the control group.In addition,LRE-100 and LRE-200 as well as NTE-250 increased the BrdU positive level on hippocampal dentate gyrus on RS rats.Our study suggested that one potential way,by which LRE and NTE had anti-anxiety-like effects,might be mediated by activating the regeneration of hippocampal neurons.Conclusion:The pharmacological models were used to evaluate the anxiolytic effect of LRE and NTE.Our results showed that LRE and NTE were both effective anxiolytics.However,LRE suggested slightlt better anti-anxiety effects compared with NTE at the same dosage.The mechanism studies suggested that the anxiolytic effect of LRE and NTE might via regulating HPA axis,and increasing BDNF protein expression level,and influencing MAPK/ERK pathway activity,and regulating apoptosis related factors,and promoting the regeneration of the neuron.