A Biological Basis Study On The Therapeutic Effect Of Sini Suanzaoren Combination Prescription From The Perspective Of The Liver

Insomnia is a sleep disorder commonly seen in clinic,which seriously harms physical and mental health.There is significant effect of Traditional Chinese Medicine in insomnia treatment,with targeted treatment from liver,of which lots of theory,prescriptions and experience are accumulated,but lack of systematic arrangements.Meanwhile,the biological mechanism of the therapy is still unclear.ObjectiveIn this study,we intend to collate the reports in the past decade on insomnia treatment from the point of liver to analyze the core drugs,formula thoughts and prescription regularity within.Based on the analysis above and my tutor’s clinical practice,we choose Si Ni Suan Zao Ren decoction(SNSZR decoction for short)as an access point to explore the mechanism of insomnia treatment from liver,using network pharmacology method to interpret its overall efficacy mechanism from multiple angles and levels.Continuing with the study of insomnia and GABA ergic system before,we choose therapeutic targets in GABA anabolism and metabolism system for an in-vivo experiment verification to clarify the therapeutic targrt,followed by macroscopic efficacy evaluation from sleep,emotion and memory.The research aims to provide comprehensive understanding from the perspective of principle,method and prescription in insomnia treatment from liver,and to lay foundation for further clinical research.MethodLiterature Research1.Using NKCI,VIP,Wanfang database as the source of literature research,we screened and input the prescription herb data to analyze the frequency,property and taste,channel tropism and efficacy.Besides,we use Ancient and Modern Medical Case Cloud Platform to analyze the dose distribution of high-frequency herbs.On the basis above,we use the Chinese Medicine Inheritance Auxiliary Platform and SPSS software to carry out correlation analysis and cluster analysis of the whole herb set and high frequency herb set,to explore the herb association regularity and formula thoughts.Then we use Gephi software to build complex network to find core herbs in insomnia treatment from liver.2.First of all,TCMSP and TCMID database were applied to screen the active components of the decoction,predict their targets and carry out an enrichment analysis.Then the OMIM and GeneCards database were used to obtain therapeutic targets for insomnia,which was mapped onto the predictive targets to get potential insomnia treatment targets.On the basis above,the co-components and co-targets of each herb were analyzed.Then we use Cytoscape software to construct the protein-protein interation network and component-target-disease network,to find key effect components and core therapeutic targets of insomnia treatment through topological analysis.And pathway enrichment on the therapeutic targets were analyzed to explore the co-regulation effect of herbs.Experimental Study1.The insomnia rat model was established with PCPA abdominal injection,and evaluated by sodium barbiturate experiment,sucrose preference experiment and Morris water maze experiment.Then the insomnia rats were randomly divided into 1 model group and 4 theraputic groups,and respectively intragastric adiministered physiological saline,positive drug estazolam,Si Ni decoction,Suan Zao Ren decoction and SNSZR decoction,for continuously 7 days.Then we observed the rats’ behavior,and evaluated the theraputic efficacy through sleep latent period,sleep duration,sucrose preference rate,platform crossing times and other indicators from above-mentioned behavioral experiments.2.The bilateral hippocampi of each rat ware taken out and disposed,to detect the protein and mRNA expressions of GAD65,GAD67,GABA-T,GAT-1 and SSADH,using protein immunoprinting technology and real-time PCR technology.ResultLiterature Research1.Statistical results show that in prescriptions of insomnia treatment from liver,tonics,neroleptic herbs and heat-clearing herbs are most commonly used.Besides,property and taste analysis shows hot herbs are least used,while sweet,bitter and spicy herbs are mostly used,with channel tropism of liver,heart and spleen.A total of 33 herbs with frequency of not less than 30 are found out,and their herb efficacy,property and flavor distribution are nearly the same with the whole herb set,with a wide range of dose distribution.The correlation rules analysis to the whole herb set,with support degree≥50,and confidence degree>0.9 shows a strong association rule among Paeonia lactiflora Pall,Poria and Bupleuri Radix,out of 57 herb groups.When we alter the support degree≥40,and confidence degree≥0.9.117 groups and 6 association rules were obtained,all related to Bupleuri Radix,Paeonia lactiflora Pall,Poria.Glycyrrhiza uralensis Fisch and Ziziphi Spinosae Semen.Complex network analysis shows that the core herbs were Bupleuri Radix,Paeonia lactiflora Pall,Glycyrrhiza uralensis Fisch and Ziziphi Spinosae Semen.Cluster analysis found Bupleuri Radix-Poncitrustrifoliata Raf-Cyperi Rhizoma,Gardenia Ellis-Adenophora capillaris-Talcum and other 5 core groups,which indicate 5 more new fomula.2.From the database we retrieve a total of 144 active components from 8 herbs in the decoction,including 7 multi-herb co-component.There are 229 predicted active component targets,including 9 multi-herb co-action targets,and the KEGG pathway and GO items obtained from the enrichment are involved with 53 diseases,covering a wide range of bio-functional activities.From above a total of 70 potential targets for the insomnia treatment are screened,which concentrate in several immune inflammatory response pathways,low oxygen induction pathways,and GABA energy synaptic activity,dopamine synaptic activity pathways,among which neural activity receptor-ligand interaction,TNF signaling pathway,calcium-ion signaling pathway and dopamine synaptic co-acting pathway are the co-pathway for more than half of the herbs.On the basis above,through network analysis,18 core components from the insomnia treatment decoction,like Quercetum and flavone glycoside,and 8 core therapeutic targets,including IL6,TNF,VEGFA,FOS,IL1B,CREB1,CAT,and IL10 are obtained.Experimental Study1.The PCPA insomnia model is successfully constructed.The circadian rhythm of the model rats disappears,with their sleep time decrease,less intake,weight loss and less activity.Compared with normal rats,model rats’ sleep durationa significantly reduce(P<0.01),and sleep latent period are significantly longer(P<0.05),while sucrose water preference rate doesn’t change significantly(P>0.05).In the Morris water maze space exploration experiment,the number of times the model rats swimming across the platform and the ratio of the target quadrant to the total stay time are significantly lower than the normal rats(P<0.01,P<0.01).After drug intervention,the weight of the rats in each theraputic group increased,and the mental state and spontaneous activity turn normal.Compared with mode 1 rats,the sleep latent period significantly reduces(P<0.05),and the sleep duration increased significantly(P<0.05),especially in SNSZR decoction group and Si Ni group(P<0.01).There is no significant difference in the consumption of sucrose water in each group(P>0.05).In Morris water maze space exploration experiment,the search strategy of rats transforms from edge to random and tendentious.Compared to the model group,there is no significant difference from the the positive drug group and Si Ni decoction group rats in the number of platform acrossed and the target quadrant stay time(P>0.05),while a significant increase in the SNSZR decoction group and the Suan Zao Ren decotion group(P<0.05,P<0.01).2.In protein immunoprinting experiment,GAT-1.GAD67,GAD65,GABA-T protein expression decrease significantly in model rats’ hippocampi,compared to the normal rats(P<0.05.P<0.05,P<0.01.P<0.01),and there is no significant difference in SADH protein expression(P>0.05).Compared with the model group,there is significant increase in GAD65,GAT-1 protein expression in the hippocampi of the positive drug group rats(P<0.01,P<0.05),and no significant difference in SSADH,GABA-T and GAD67 protein expression(P>0.05).Compared with the model group,there is significant increase in GAD67,GAD65,GAT-1 protein expression in the hippocampi of the SNSZR decetion group rats(P<0.01,P<0.05,P<0.05),and no significant difference in SSADH,GABA-T protein expression(P>0.05).Compared with the model group,there is significant increase in GAT-1 protein expression in the hippocampi of Si Ni decoction group rats(P<0.05),and no significant difference in SSADH,GABA-T,GAD65,GAD67 protein expression(P>0.05).Compared with the model group,there is no significant difference of all indicators in the hippocampi of Suan Zao Ren decoction group rats(P>0.05).In the real-time PCR experiment,compared with the normal group,the mRNA expressions of GAT-1,GAD65,GAD67 and GABA-T of model rats significantly reduce(P<0.01,P<0.01,P<0.01,P<0.01),while there is no significant difference in the expression of mRNA in SSADH(P>0.05).Compared to the model rats,there is significant increase in mRNA expression of GAD65,GAD67 and GAT-1 in the positive drug group rats’ hippocampi(P<0.01,P<0.01,P<0.01),and no significant difference in SSADH and GABA-T expression(P>0.05).Compared to the model rats,there is significant increase in mRNA expression of GAD65,GAT-1,GAD67 and GABA-T in the SNSZR decoction group rats’ hippocampi(P<0.05,P<0.05,P<0.01,P<0.01),and no significant difference in SSADH expression(P>0.05).Compared to the model rats,there is significant increase in mRNA expression of GAT-1,GAD67,GAD65 and GABA-T in the Si Ni decoction group rats’ hippocampi(P<0.05,P<0.05,P<0.01,P<0.01),and no significant difference in SSADH expression(P>0.05).Compared to the model rats,there is significant increase in mRNA expression of GAD65 in Suan Zao Ren decoction group rats’hippocampi(P<0.01),and no significant difference in other indicator expression(P>0.05).ConclusionIn this study,we find that treatment from liver is commonly used in clinic,and mainly plots around blood nourishing,tranquillization,Qi promoting and liver soothing.Network pharmacology research shows SNSZR decoction has a phenomenon of herb concentration and target composition during insomnia treatment,and its efficacy mechanism may be associated with a number of immune inflammatory response pathways,low oxygen-induced pathways,GABA ergic synaptic activities and dopamine synaptic activities.PCPA insomnia rats are hard to get asleep,with shorter sleep time and memory loss,while the SNSZR decoction can effectively improve their sleep and memory ability.Through further verification of molecular biology experiments,it is found that the SNSZR decoction may affects by regulating the key targets of GABA anabolic system,promoting GABA synthesis,to promote its neuroprotective function in insomnia treatment,laying the foundation for clinical research.