To Explore The Mechanism Of Banxia Shumi Decoction In The Treatment Of Insomnia Based On Bioinformatics Data Mining Technology And The PI3K/AKT Pathway

In this study,UPLC-Q-Exactive-MS,network pharmacology and molecular docking technology were used to explore the mechanism of Banxia Shumi Decoction(BXSMD)in the treatment of insomnia based on the hippocampal PI3K/AKT pathway.Methods:1.Part I: The UPLC-Q-Exactive-MS technique was used to qualitatively analyze the main chemical components of BXSMD.The BXSMD active compounds and their potential targets were screened through the TCMSP database.Through the OMIM database and Gene Cards database,we mined the targets of insomnia,combined key targets of compounds and diseases,constructed a Chinese medicine compound-target-disease network diagram and PPI network,and performed GO function enrichment and KEGG pathway enrichment analyses.Using molecular docking technology and Py MOL 2.1 software,we predicted the binding ability and docking pattern of small molecules of Chinese medicine with key protein targets and performed visual analysis.2.Part II: We used PCPA intraperitoneal injection to prepare the insomnia models,and the experimental rats were randomly divided into 6 groups: normal group(N),model group(M),diazepam group(D)and low(ZL),medium(ZM)and high(ZH)BXSMD dose groups.After the end of intervention in each group,we observed the behavioral characteristics of rats by the open field experiment,the hippocampal structure by HE staining,the serum TNF-α,IL-1β,IL-2,IL-6,Orexin and 5-HT by ELISA technique,and RT–PCR detection of hippocampal orexin receptors(OX1R,OX2R)and 5-HT receptors(5-HT1 A,5-HT2A)m RNA changes,Western blot detection of hippocampal orexin receptors OX1 R,OX2R protein changes.3.The third part: The modeling and intervention steps for each group of rats were the same as in Part II,and then we used RT–PCR to detect the m RNA expression of the hippocampal PI3K/AKT pathway and Western blot to detect the expression of each protein of the hippocampal PI3K/AKT pathway.Results:1.Part I: UPLC-Q-Exactive-MS was used in positive and negative ion detection modes to qualitatively analyze the aqueous extracted components of BXSMD and obtain its total ion flow map.By accurate molecular weight calculation and ion fragmentation information,confirmed by reference standards,the main compound components including amino acids,small peptides,nucleotides,organic acids,flavonoids,fatty acids and lipids were identified by co-mass spectrometry.BXSMD contained 21 active ingredients corresponding to 38 targets of insomnia;the topological analysis and cluster analysis using PPI network obtained key targets FOS,AKT1,CASP-3,TP53,VEGFA,PRKCA,etc.These key targets involved 85 GO enrichment terms involved in biological processes such as G protein-coupled neurotransmitter receptor activity,serotonin binding,adrenoceptor activity,and catecholamine binding regulation.They were involved in the regulation of serotonergic pathways,neuroactive ligand–receptor interactions,calcium signaling pathways,dopaminergic synapses,apoptosis,PI3K/AKT and other KEGG pathways.The molecular docking of baicalein,beta-sitosterol,and stigmasterol was found to be strongly bound to four target proteins(AKT1,FOS,PRKCA,and VEGFA)with binding energies less than-5kcal/mol.2.Part II: In the sodium pentobarbital correction experiment,the sleep latency of insomnia model rats was significantly prolonged,and the sleep duration was significantly shortened.In the open field experiment,the total activity distance of the model rats within 5 minutes decreased;the distance,time and modification times of entering the central grid increased.HE staining showed that the morphological and structural damage to neurons in the hippocampal CA1 area of the M group was more serious,while the damage in the Chinese medicine groups with different doses was relatively relieved.ELISA showed that compared with the M group,the serum TNF-α,IL-1β,IL-2 and IL-6 levels of the rats in the D group and the ZL,ZM and ZH groups were significantly decreased(all P<0.05).The levels of orexin in the serum and hippocampus of rats were significantly decreased(all P<0.01),the levels of serum 5-HT in the ZL,ZM and ZH groups were decreased,and the level of 5-HT in the hippocampus was significantly increased(all P<0.01).RT–PCR showed that compared with the M group,the hippocampal OX1 R and OX2 R m RNA expression in each dose group of traditional Chinese medicine decreased(all P<0.05);compared with the M group,the ZL,ZM and ZH groups showed increased hippocampal 5HT1 A m RNA expression(all P>0.05),and hippocampal 5HT2 A m RNA expression was decreased in the ZL,ZM and ZH groups(all P>0.05).Western blot detection showed that compared with the M group,the expression of OX1 R in the hippocampus of the rats in the ZL,ZM and ZH groups decreased(all P<0.01),and the expression of OX2 R in the hippocampus of the D group and ZL group increased(all P<0.05).3.Third,RT–PCR showed that compared with that in the M group,the hippocampal PI3 K m RNA expression in the D,ZL,ZM and ZH groups increased(all P<0.05),the hippocampal AKT m RNA expression in the ZL and ZH groups increased(all P<0.05),and the hippocampal caspase-3 m RNA expression in the ZL,ZM and ZH groups increased(all P<0.05).Western blot detection showed that compared with that in the M group,the hippocampal PI3 K protein expression in the ZH group was significantly increased(P<0.05),and the hippocampal p-PI3 K protein expression in the ZH group was significantly increased(P<0.05).The hippocampal Bcl-2 protein expression in the ZM and ZH groups was significantly increased(all P<0.05).The hippocampal AKT protein expression in the ZH group was significantly increased(P<0.05);the hippocampal p-AKT protein expression in the ZL and ZH groups was significantly increased(P<0.05;P>0.05);the hippocampal Fas protein expression in the ZL,ZM and ZH groups was decreased(all P<0.01);and the hippocampal Bax protein expression in the ZL,ZM and ZH groups was decreased(all P<0.01).Compared with that in the M group,the hippocampal caspase-3 protein expression in the D and ZL groups was significantly increased(all P<0.01),and the caspase-3 protein expression in the rat hippocampus in the ZH group was decreased(P<0.05).Compared with the M group,the c-caspase-3 protein expression in the rat hippocampus of the ZL,ZM and ZH groups was significantly decreased(all P<0.01),the c-caspase-3 expression in the ZH groups was decreased(P<0.05),and the rat hippocampus p-P65/P65 ratio in the ZM and ZH groups was decreased(all P<0.01).Conclusion: In this study,for the first time,we used a combination of bioinformatics data mining techniques,such as UPLC-Q-Exactive-MS,herbal network pharmacology and molecular docking,to initially identify and match amino acids,small peptides,nucleotides,organic acids,flavonoids,fatty acids,lipids,and other chemical components of BXSMD and explored the visualized compounds and molecular associations between BXSMD and insomnia.The network predicted that the mechanism of action of Panicum italicum soup for insomnia may be associated with 5-HT,apoptosis,PI3K/AKT and other signaling pathways,and molecular docking showed that compounds baicalein,beta-sitosterol,stigmasterol and AKT1,FOS,PRKCA,VEGFA target proteins with good binding ability.Based on the hippocampal PI3K/AKT signaling pathway,it was verified and elucidated that the mechanism of action of BXSMD for treating insomnia may be through inhibition of hippocampal neuronal pro-apoptotic Fas,activation of PI3 K and AKT expression,increase of mitochondrial anti-apoptotic Bcl-2 expression and decrease of Bax expression,inhibition of caspase 9 and caspase-3 shear activation,decrease of NF-κB(p-P65/P65)phosphorylation,resulting in Fas-dependent and mitochondria-dependent apoptotic pathways in hippocampal neurons,and acting as a regulator of sleep-wake rhythms.