Study On The Antidepressant Mechanism Of The Petroleum Ether Fraction Of Radix Bupleurum Based On Transcriptome

Rationales: Depression is a kind of mental disease with obvious depression as the main clinical characteristic.It is predicted that by 2020,it will become the second largest burden source of disease.Because of large side effects of western medicine,many scholars have turned to the field of traditional Chinese medicine(TCM),among which the research on Xiaoyao San,a famous prescription of "soothing the liver and relieving depression",is the most popular.In our previous research,which studying on the petroleum ether fraction of XYS and guiding by serum drug chemistry found that the polyacetylene of Chaihu is easy to enter blood and the pharmacokinetic parameters are suitable.Then,the polyacetylene components of Radix Bupleuri are extracted to the research about antidepressant activity.The result demonstrated that polyacetylenes potently inhibit monoamine of rat brain synaptosomes,with a better potency than their corresponding specific inhibitors.But polyacetylene in Radix Bupleuri can not fully represent the whole fraction of petroleum ether of Radix.Therefore,the antidepressant effect / toxicity of Radix Bupleuri is further studied,which was found that the petroleum ether fraction of Radix Bupleuri had good antidepressant activity.Neither the best dose of its antidepressant activity would be determined,nor the specific mechanism was clear,so further study was needed.Objective: To study the antidepressant mechanism of the petroleum ether fraction of Radix Bupleuri,and screen the optimum antidepressant dose based on the previous work.In order to study the mechanism at the level of transcriptome and molecular and provide a scientific evidence for the development of antidepressant drugs.Methods: 1.After analyzing the behavioral indicators in Fang Yuan's paper,we selected the following effective groups to conduct in-depth research: D0(CM?ZK),DY(CY),D4(C4?Z4),D5(C5?Z5),D6(C6?Z6),D7(C7?Z7).UHPLC-MS/MS quantitative analysis method of eight neurotransmitters(5-HT?GABA?Glu?ACH?NE?DA?5-HIAA ? HVA)was established,and ELISA method was used to detect the HPA axis index.By measuring the contents of 8 kinds of neurotransmitters and CRH,ACTH,CORT studied the mechanism of antidepressant activity of the petroleum ether fraction of Radix Bupleuri.Based on the previous work,the best antidepressant dose of Radix Bupleuri petroleum ether was obtained.2?Using Illumina HiSeq technique analysed CUMS model group(CM),blank group(ZK),D6(C6,Z6)dosages of Radix Bupleuri petroleum ether in CUMS rats / normal rats.Two pairs of samples from different groups were compared,and two pairs of differentially expressed genes were analyzed statistically.Then the significant differentially expressed genes were analyzed by GO and KEGG analysis.The main biological functions and pathways were discussed and the key genes were screened.3?The expression levels of the 32 genes were analyzed by qPCR technique.Results:1?The fraction of petroleum ether from Radix Bupleuri had a different degree of callback to the significant changes of 5-HT 5-HIAA and GABA induced by CUMS in serum,and the best doses were D6 and D7.The significant changes of 5-HT,Glu and Ach induced by CUMS in hippocampal tissue were corrected in varying degrees,and the best dose was D6.The increase of ACTH and CORT induced by CUMS in serum can be corrected to some extent.2?The transcriptome of rat liver tissue was successfully constructed by RNA-seq technique.The significant differentially expressed genes were analyzed among two pairs of samples,and the differentially expressed genes in each pair of samples were analyzed by GO and KEGG.The main biological functions and pathways were discussed.Depression induced by CUMS model might be associated with Circadian rhythm,p53 signaling,Steroid hormone synthesis,Retinol metabolism,Glycine metabolism,etc.The antidepressant effects of the petroleum ether fraction of Radix on CUMS model rats might be related to Bile secretion and synthesis,Steroid hormone synthesis,Metabolism of xenobiotics by cytochrome,Drug metabolism,Retinol metabolic,etc.The antidepressant effects of the petroleum ether fraction of Radix on normal rats might be related to Steroid hormone synthesis,Retinol metabolism,Drug metabolism,Circadian rhythm and p53 signal pathway,etc.Finally,according to the results of studies on neurotransmitters and HPA axes,32 genes involved in the circadian rhythm of 5-hydroxytryptaminergic synapses,bile secretion and synthesis,metabolic-related and other pathways needed to be verified were screened by combining the differentially expressed transcriptional genes and their KEGG pathways with related annotation genes.3?Using qPCR studied the 32 genes expression levels.CUMS model could induce the overexpression of the clock genes such as Npas2,Cry1,Arntl,Rora and Rorc,and decrease the expression of Bhlhe41,Nr1d1,Per1,Per2 and Per3 in vivo;could induce the overexpression of Cyp7b1 and Kcnn2 genes involved in bile secretion and synthesis,as well as the decrease of Nceh1 expression;could induce the overexpression of Prlr in neuroactive ligand receptor pathway;could induce the decrease of Cyp26a1 gene expression in multiple pathways.The fraction of petroleum ether from Radix Bupleuri significantly corrected the expression of circadian clock genes with Npas2?Per2 and Rorc induced by CUMS model,of whose callback effect on Npas2 was close to the control group;and marked callbacks on the expression of Cyp7b1?Nceh1 and Kcnn2 in the pathway related to bile secretion and synthesis induced by CUMS model.The expression of Akr7a3?Gsta3 and Ephx1 in the model/normal groups in the fraction of petroleum ether from Radix Bupleuri were significantly higher than the control group.Conclusion: 1?The depression caused by CUMS model is related to the disorder of circadian rhythm system and neurotransmitter changes.2?The fraction of the petroleum ether fraction of Radix has activity,which may regulate the emotional disturbance caused by excessive secretion of acth and cort by regulating monoamine,amino acids and acetylcholine neurotransmitters,and decreasing the overexpression of Npas2 and Rorc,which are involved in circadian rhythm,the expression of Cyp7b1,Nceh1 and Kcnn2 involved in bile secretion and synthesis,and the antidepressant effect by decreasing the expression of Prlr in the interaction of neuroactive ligands and receptors.In addition,its metabolism in liver tissue may be related to detoxification.3?Most of the regulatory pathways of the petroleum ether fraction of Radix site to model and normal animals were similar,indicating that the effects were basically the same.