Study On The Compatibility Of Radix Scutellariae In The Prescription Of Treating Depression And Its Mechanism Of Antidepressant Action

ObjectiveRadix Scutellariae(RS)as a traditional heat-clearing drug,has been proved to have anti-depression effects.At present,there are no reports on the research of the antidepressant prescription containing RS.But clearing the clinical application of RS compatibility in the prescription have theoretical guiding significance for the clinical application.This study is guided by the theory of traditional Chinese medicine,using data mining methods to explore the characteristics and syndrome of depression,and the main compatibility methods of RS antidepressant prescriptions.In addition,the antidepressant mechanism of RS was discussed on the perspective of neurogenesis in corticosterone(CORT)-induced depression model animals.Methods1.Data mining research.Collecting literatures containing RS prescription for the treatment of depression published in Chinese databases from 1986 to 2019.①SPSS Statistics 22.0 was used to conduct frequency on the name,pathogenesis(disease location and pathogenic syndrome),symptoms,tongue and vein,drugs and drug categories,dosage and proportion of RS and its compatibility drugs,total clinical effective rate and symptoms of adverse drug reactions of RS;②The Apriori algorithm of SPSS Modeler 14.1 software was used to analyze the association rules of the two and three syndromes,the two and three drugs in the antidepressant prescription containing RS.③Gephi 0.9.2 software was used to analyze the complex network between syndrome elements,symptoms and drugs exploring the rule of anti-depression prescription containing RS.2.Experimental study.①The mices were subcutaneously injected corticosterone for 6 weeks to build depression model.3 weeks later,the mice were divided into blank,model,escitalopram,high dose and low dose of baicalin,high dose and low dose of RS groups.After three weeks treatment of drugs,preference of sugar water,the immobility time in tail suspension and forced swimming tests,open field test,novel feeding behavior test,and contents of serum corticosterone were detected;②Immunohistochemical method was used to detect the number of Ki-67 and BrdU positive cells in the dentate gyrus of the hippocampus;Immunofluorescence method was used to detect the number of BrdU/DCX/NeuN positive cells in the dentate gyrus;Golgi staining was used to detect the morphology,dendrite length and dendrite spine density of neurons in the dentate gyrus;Western blot was used to detect the proteins expression of PI3K/AKT/GSK3β/β-catenin pathway;PI3K and AKT inhibitors LY294002 and Perifosine were used in TH-22 cells to detect the proteins expression of the above signal pathway after corticosterone and baicalin treatment.③HPLC experiment was used to determine the contents of baicalin,baicalein,wogonoside and wogonin in RS decoction.Reseults1.Data mining research.The number of collected and sorted out literatures was 464,including 249 clinical research literatures contain 9702 cases,122 medical record literatures contain 153 cases,and 93 clinical literatures in addition with RS.1.1 Clinical research literatures The number of the prescriptions containing RS was 90,which can be divided into 9 categories.The main categories of antidepressant prescriptions containing RS were reconciling agents,phlegm reducing agents,heat-clearing agents,qi regulating agents.Among these,Bupleurum decoctions are used the most(65.49%),and Bupleurum and Chaihu Jia Longgu Muli Decoction ranks first among all prescriptions(33.10%).The total effective rate of the Chinese medicine treatment group and the combined Chinese and Western medicine treatment group of this type prescription was significantly higher than that of the western medicine group(p<0.01),and the incidence of adverse reactions was lower(p<0.01).There are 21 syndromes types of RS-containing antidepressant prescription.The frequency of syndrome type ranked top 4 was liver stagnation and qi stagnation(18.72%),liver stagnation and phlegm-heat(12.34%),liver stagnation and phlegm obstruction 10.37%)and liver stagnation to transform fire(heat)(9.66%).In addition,there were liver qi stagnation clamped damp heat,liver qi blood deficiency,liver qi spleen deficiency,qi stagnation blood stasis and so on.The disease involves 11 kinds of syndrome elements.The first 4 sthenia elements were Qi stagnation(85.14%),heat(46.21%),phlegm(30.66%),followed by blood stasis,dampness,Yang hyperactivity,and water stagnation.Asthenia element is less,mainly including qi deficiency,blood deficiency.The disease involves 9 syndromes,and liver was the most common disease location(96.17%),followed by spleen(16.03%),heart(12.55%)and gallbladder(6.66%).In terms of compatibility,the core single drugs compatible with RS were Chaihu and Banxia,with the use frequency of 88.03%and 75.21%respectively.The main compatible drugs(with a usage frequency of 30%-60%)were Fulin,Muli,Longgu,Baishao,Dazao,Shengjiang,Guizhi,Dahuang.Except for Baishao,the above drugs were all the drugs of Chaihu-jia-Longgu-Muli-Decoction.Secondary compatibility drugs(with a usage frequency of 15%-30%)include Yujin,Shichangpu,Suanzaoren,Hehuanpi,Yuanzhi,Danggui,Chenpi,Renshen,Dangshen,Zhizi,Zhiqiao.Common two medicine association mainly includes Banxia-Chaihu,Fulin-Chaihu,Fulin-Banxia,etc;common three drugs association mainly includes Guizhi-Banxia-Chaihu,Dahuang-Banxia-Chaihu,Dahuang-Muli-Chaihu,Dahuang-Longgu-Muli,Dahuang-Longgu-Chaihu,Guizhi-Muli-Chaihu,shown as the formation of connection between the core drugs and main drug.The main types of drugs compatible with RS were qi regulating drugs(93.38%),tonic drugs(86.02%),transhepatic drugs(83.59%),phlegmatic drugs(79.15%),and heat clearing drugs(57.06%).The secondary types of drugs compatible with RS were invigorating blood circulation and removing blood stasis,opening up and relieving diarrhea.The average dosage of RS and Banxia were10.50±2.26g and 10.35±2.54g;the average dosage of Chaihu was 13.45±4.87g.The dosage ratio of RS and Chaihu,RS and Banxia was mainly 1:1,and the dosage ratio of three drugs was also 1:1:1.1.2 Medical records literaturesThe number of the prescriptions containing RS was 46,which can be divided into 8 categories.The statistical results of the prescriptions usage frequency,the type of main syndromes,the disease location,the core compatible drugs and the dosage of compatible drugs were basically consistent with the results of clinical research literatures.There were 92 common symptoms of the prescriptions,involving 15 kinds of tongue and 10 kinds of pulse.Among them,the main symptoms(>30%frequency)in terms of mental performance were depression,irritability,irritability;physical symptoms were poor sleep,poor appetite,thoracolumbar discomfort;and tongue performance were red,pink,moss yellow,white,greasy,thin;pulse performance were string,thin.Secondary symptoms(frequency of 8.5-30%)in terms of mental performance were sadness and tears,few words,world-weariness,memory decline,thinking too much,anxiety,nervousness,loss of interest;physical symptoms include weakness,bitter mouth,palpitation,constipation,dry mouth,dizziness,pharyngeal discomfort,epigastric discomfort,sweating,abdominal distension,loose stools,headache,sighing,abnormal sensation of cold and heat;the tongue performance showed dark,fat big tooth mark,thick coating,pulse performance were fast,slippery,heavy.Common associations between two symptoms were depression-string,poor appetite-insomnia,poor appetite-string,irritability-insomnia,moss yellow-string,moss greasy-insomnia,red tongue-string;common associations between three symptoms were agitated-insomnia-irritability,moss yellow-depression-string,moss greasy-poor appetite-insomnia,agitated-irritability-insomnia,red tongue-insomnia-string,irritability-string-insomnia,poor appetite-depression-string,fatigue-insomnia-poor appetite.The results showed tha they were all the correlations between the main symptoms and secondary symptoms.In the research of the relationship between pathogenesis,symptoms and drugs,we outlined 3 common connections.① Liver stagnation and qi stagnation,liver stagnation and spleen deficiency syndromes:involving symptomatic depression,discomfort in the chest and hypothermia,reluctance to speak,abnormal feelings of cold and heat,despairing of life,unresponsiveness,stomach discomfort,fatigue,anorexia,abdominal distension,Sighing,belching,weight loss,sadness and crying,loose stools,thin moss,white moss,thin pulse,representative prescriptions are Chaihu-plus-Longgu-Muli-Decoction and Xiaoyao-San.②Liver stagnation,phlegm-heat,gallbladder-heat,phlegm disturbance:Involve symptoms,nervousness,poor sleep soundness,hallucinations,thoughtfulness,inattention,decreased memory,heavy head,cold limbs,hiccups,greasy fur,Slippery pulse,the representative prescription is Wendan-Decoction.③Liver depression transforms fire,liver fire disturbs the heart:it involves symptoms of irritability,irritability,fear,mental tension,restlessness,pain in the mouth,dry mouth,sweating,tinnitus,headache,abdominal pain,body pain,Yellow urine,short urine,constipation,red tongue,yellow coating,pulse number,pulse string,representative prescriptions are Huanglian-Jiedu-Decoction and Longdan-Xiegan-Decoction.1.3 Clinical literatures in addition with RSThe number of the prescriptions was 60.Studies have shown that when clinicians use a formula that does not contain RS for depression,the main basis for adding RS is:the pathogenesis of the main therapeutic syndromes mainly included heat,liver depression and fire,phlegm and heat,and the symptoms mainly included bitter mouth,upset,dry or obstructed stool,irritable,yellow and greasy tongue coating,and dry mouth.The main drugs used together with RS include Zhizi,Huanglian,Longdan,Chaihu,Gualou,Mudanpi,Dananxing which were the drugs for clearing heat,regulating qi and reducing phlegm.The usual dosage range of RS is 6-20g,with an average dosage of about 10.35 ±2.54g.Combined with the above research results and traditional Chinese medicine theory,we can know RS plays the following roles in antidepressant prescriptions:① Clearing heat and eliminating fire;②Clearing qi and decrease internal heat to remove phlegm and heat;③Preventing smurf-based heat;④Regulating qi compatible with Chaihu and Banxia;⑤Heat with cold,to prevent warming medicine to help the fire.2.Experimental research.2.1 Effects of Baicalin and RS on behaviors and serum corticosterone in CORT-induced depression mice.After subcutaneous injection of 40 mg/kg corticosterone for 6 weeks,the model animals’ sugar water preference rate decreased,the immobility time in tail suspension and forced swimming test increased,food consumption increased and feeding consumption decreased in NFST(p<0.01),showing obviously depression-like behaviors;and the serum CORT content increased(p<0.01).While baicalin 60mg/kg and RS 1.5g/kg can significantly improve CORT-induced depression-like behaviors and reduce serum CORT content(p<0.05,p<0.01).2.2 Effects of Baicalin and RS on neurogenesis in CORT-induced depression mice based on PI3K/AKT/GSK3β/β-catenin pathway.The number of Ki-67 and BrdU positive cells in model animals was significantly decreased(p<0.01),while RS and baicalin could increase the number of positive cells(p<0.01).Immunofluorescence showed a significant decrease in the number of immature neurons labeled DCX+/NeuN+/BrdU + and mature neuron labeled DCX-/NeuN +/BrdU+positive cells in the model animals(p<0.01),while baicalin 60mg/kg and RS 1.5g/kg significantly increased the number of surviving mature and immature neurons(p<0.01).Golgi staining showed that the dentate gyrus of the model group had few branches and simple morphology,and the total length of dendrites was significantly reduced(p<0.01),while the treatment of baicalin at 60mg/kg and RS 1.5g/kg could significantly increase the complexity of dendrites and the total length of dendrites(p<0.05,p<0.01).However,neither CORT nor baicalin nor RS administration had any effect on the density of total dendritic spines and mushroom dendritic spines(p>0.05).Protein western blot showed that the expression of PI3K/AKT/GSK3β/β-catenin signaling pathway proteins and PSD95 and Synapsin 1 in the model group were decreased(p<0.01),and the expression of the above relative proteins were up-regulated after baicalin 60mg/kg of and RS 1.5g/kg treatments(p<0.05,p<0.01).After the administration of PI3K and AKT inhibitors LY294002 and Perifosine in cort-induced ht-22 cells,the effect of baicalin increased PI3K/AKT/GSK3β/β-catenin pathway protein expression was offset(p>0.05).2.3 Determination of main components in RS water extract.The water extract of RS root mainly contained baicalin,wogonoside,baicalein,wogonin,and the content of baicalin was the highest(217.77±1.96mg/g).Conclusion1.The main syndrome types of the prescription containing RS are liver depression and qi stagnation,liver depression and phlegm heat,liver depression and phlegm obstruction,and liver depression and fire(heat).Common disease elements are qi stagnation,heat,phlegm,the locations are mainly the liver(gallbladder),followed by the heart,spleen(stomach).2.The main symptoms of the prescription containing RS Symptoms in the mental aspects are depression,agitated,irritability;physical performance are poor sleep,poor appetite,and chest discomfort.Secondary symptoms in the mental aspects are grief to cry,few words,world-weariness to die,memory decline,etc;physical performance are weakness,bitter mouth,palpitation,constipation,dry mouth,dizziness,pharynx discomfort,yuan-pi discomfort,etc.Tongue performances mainly are red tongue,light red tongue,thin,white,greasy moss,and pulse are string,fine.3.Among the prescription containing RS,the core single drugs that combined with RS are Chaihu and Banxia.The average dosage of the three drugs is 10g,13g and 10g.The dosage ratio of the two drugs is 1:1,and the dosage ratio of the three drugs is mostly 1:1:1.4.In addition to Chaihu and Banxia,the drugs compatible with RS mainly include Fulin,Muli,Longgu,Baishao,Dazao,Shengjiang,Guizhi and Dahuang,as well as Yujin,Shichangpu,Suanzaoren,Hehuanpi,Yuanzhi,Danggui,Chenpi,Renshen,Dangshen,Zhizi,Zhqiao and so on.The main types of drugs compatible with RS include regulating qi,calming the mind,tonic,eliminating phlegm and clearing heat drugs,as well as promoting blood circulation and removing blood stasis,awaking and relieving constipation drugs.5.Studies on the relationship between common diseases and prescription selection showed that:the diease of liver-qi stagnation,liver-qi stagnation and spleen-qi deficiency used the representative prescriptions of Chaihu-Jia-Longgu-Muli-Detoction and Xiaoyao-San;the diease of lliver-depression-phlegm-heat and gallbladder-heat-phlegm-disturbance used the representative prescriptions of Wendan-Decoctions;the diease of liver-qi stagnation changes fire and liver-fire disturbs the heart used the representative prescriptions of Huanglian-Jiedu-Decoction and Longdan-Xiegan-Decoction.6.RS and baicalin can upregulate PI3K/AKT/GSK3β/β-catenin signaling pathways to promote neurogenesis in the dentate gyrus of CORT-induced depression model mice to play an antidepressant effect.