Study On The Efficacy And Mechanism Of Jingning Granules In The Treatment Of ADHD Based On The "gut Microbiome-HPA Axis" Pathwa

BackgroundAttention deficit hyperactivity disorder（ADHD）is a relatively common childhood behavior disorders.The incidence of ADHD in China is about 4.9%～6.6%.The incidence of ADHD in boys is three times than that of girls,and the prevalence rate has gradually increased in recent years.In the etiological study of this disease,dopamine（DA）system abnormality is an important cause.In recent years,it has been found that gut microbiota and neuroendocrine network function which represented by HPA axis play the important roles in the pathogenesis of ADHD.Previous studies of our group have found that there are different degrees of DA dysfunction and hypothalamic-pituitary-adrenal（HPA）axis hyporesponsiveness in children with ADHD.After the treatment of Jingning granule,in addition to the clinical symptoms,DA system,HPA axis related indicators and gastrointestinal symptoms of children have also been relieved.Therefore,the further study of the relationship among DA system,HPA axis function and gut microbiota structure will help to discover the new mechanism of Jingning granule in treating ADHD and provide strong support for the treatment of ADHD with Traditional Chinese Medicine.PurposeThis study could be divided into clinical part and experimental part,which aims to further verify the clinical efficacy of Jingning granule in the treatment of ADHD,and to understand the HPA axis function and intestinal flora structure characteristics.In order to provide more data support for the treatment of this disease,we explored the mechanism of Jingning granule in the treatment of ADHD model rats.MethodClinical studyThis part of the study is divided into ADHD group and normal control group.All the children who met the inclusion criteria were treated with Jingning granule for 8 weeks,during which the general data of the children were recorded,the serum dopamine,HPA axis function,gut microbiota and safety index were detected at the specified observation node,the ADHD related scale and TCM syndrome integral scale were filled in,and the adverse reactions were recorded.The serum dopamine,HPA axis function and gut microbiota were detected once in the normal control group.To investigate the difference between children with ADHD and normal children in serum dopamine,HPA axis function and gut microbiota structure,and to observe the clinical effect of Jingning granule on ADHD and the improvement of the above indexes.Experimental study50 male Spontaneously Hypertensive Rat（SHR）rats were divided into Jingning granule high-,medium-,and low-dose group,methylphenidate hydrochloride group and model group according to random numbers.At the same time,10 Wistar rats of the same age were set as the normal control group.Normal control group and model group were given 1mg/100g of normal saline;methylphenidate hydrochloride group was given 1mg/100g of methylphenidate hydrochloride suspension;Jingning granule was given lmg/100g of different concentrations of particle suspension according to different groups,the specific concentration was 23.14g/kg high dose,11.57g/kg medium dose,5.785g/kg low dose,once a day for six weeks.To observe the effect of Jingning granule depend on the improvement of Morris water maze,open field experiment and other behavioral indexes in ADHD model rats,as well as gut microbiota structure,HPA axis function and dopamine system in brain tissue.ResultClinical studyThe scores of the abolition test,SNAP-Ⅳ scale,and the Conners parental scale scores of children in the ADHD group were significantly improved after treatment（P<0.01）;the scores of the main syndromes in the TCM syndrome score scale of children in the ADHD group were significantly improved after treatment（P<0.01）,and except for stool,the rest of the scores of the second syndrome were significantly improved compared with before treatment（P<0.01）.The total effective rate of ADHD group was 78.26%before and after treatment and that of TCM syndrome was 78.26%;Compared with the normal control group,the serum levels of dopamine and COR in the ADHD group were significantly decreased（P<0.01）,and there was no significant difference in ACTH levels（P>0.05）.The serum levels of dopamine and COR in the ADHD group were increased after treatment（P<0.01 or P<0.05）,no difference in ACTH content before and after treatment（P>0.05）;There was no significant difference in gut mircobiota richness and diversity between the ADHD group and the normal group before and after treatment,but the composition of the flora was different.At the gate level:Bacteroidetes accounted for the highest proportion in the ADHD group before treatment,Actinobacteria accounted for the highest ratio after treatment,and Firmicutes and Proteobacteria accounted for the highest in the normal control group.At the genus level:Bifidobacterium,Blautia,and Subdoligranulum had higher abundance before treatment in the ADHD group,with relative abundances of 24.63%,13.02%,and 6.67%.Blautia,Bifidobacterium,and Eubacterium_hallii_group had higher abundance after treatment in the ADHD group and were relatively abundant.The abundances were 21.25%,15.20%,8.13%.Blautia,Bifidobacterium,Faecalibacterium had higher abundances in the normal control group,with relative abundances of 23.35%,10.26%,and 7.69%.In the group-level differences test,Blautia,Bifidobacterium,Bacteroides,and unclassified f Lachnospiraceae are different bacterial species.Experimental studyThe results of the water maze space exploration experiment show that the Jingning granule high-,medium-dose group and methylphenidate hydrochloride group cross the platform and the target quadrant residence time are higher than the model group（P<0.05）;The target quadrant movement distance of the Jingning granule high-dose group and methylphenidate hydrochloride group was higher than that of the model group（P<0.05）.The results of open field experiments showed that the Jingning granule high-,and medium-dose group and methylphenidate hydrochloride group had lower movement distance,movement speed and central zone time than the model group（P<0.05）,and the time in the peripheral zone was higher than the model group（P<0.05）;Jingning granules in the middle-dose group and methylphenidate hydrochloride group had lower exercise time than the model group（P<0.05）.Jingning granule high-dose group and methylphenidate hydrochloride group can increase dopamine content in striatum of ADHD model rats（P<0.01）;Jingning granule high-,medium-,low-dose groups and methylphenidate hydrochloride group can up-regulate mRNA and protein expression levels of dopamine receptors in striatum and hypothalamus（P<0.01 or P<0.05）,and the regulating effect of the Jingning granule high-dose group in the hypothalamus is better than that of the methylphenidate hydrochloride group;the Jingning granule high-and medium-dose group and the methylphenidate hydrochloride group could increase the serum ACTH content in ADHD model rats（P<0.01 or P<0.05）,and the Jingning granule high-and low-dose groups and the methylphenidate hydrochloride group could increase the serum COR content in the ADHD model rats（P<0.01）.Gut microbiota alpha diversity analysis showed that compared with the methylphenidate hydrochloride group and the model group,the bacterial diversity in the Jingning granule low-dose group was different,but there was no significant difference.Compared with the normal group,the bacteria richness was higher in the methylphenidate hydrochloride group and the Jingning granule high-,medium-,and low-dose groups than in the normal group;compared with the model group,the Jingning granule high-,and medium-dose group were significantly higher than the model group.The composition of intestinal flora in each group is different at the genera level:the normal group is mainly composed of norank_fMuribaculaceae,Alloprevotella,and Romboutsia,with relative abundances of 14.2%,10.0%,and 9.5%,respectively;the model group is composed of norank_fMuribaculaceae,Lactocilacelus,and Lachnospiraceae_NK4A136_group with relative abundances of 27.0%,10.9%,and 8.8%;the methylphenidate hydrochloride group was dominated by norank_fMuribaculaceae,and the relative abundance was 25.9%;the Jingning granule low-dose group was dominated by unclassified_fPrevotellaceae,norank_fMuribaculaceae,and unclassified_f Lachnospiraceae with relative abundances of 21.0%,15.4%,and 6.2%;the medium-dose Jingning granule group was dominated by unclassified_fPrevotellaceae,norank_fMuribaculaceae,and Alloprevotella,and the relative abundances were 11.4%,11.0%,and 10.6%;the high-dose Jingning granule group was Alloprevotella,Norank_fMuribaculaceae and unclassified_fPrevotellaceae,with relative abundances of 15.4%,12.0%and 6.4%.In the group-level difference test,norank_fMuribaculaceae、unclassified_fPrevotellaceae、Alloprevotella、Lachnospiraceae_NK4A136_group、Lactobacillus、Ruminococcus₁、Prevotella₉、Ruminococcaceae_UCG-014、Bacteroides、Ruminiclostridium₉、Ruminococcaceae_NK4A214_group、Phascolarctobacterium、Rikenellaceae_RC9_gut_group、Prevotellaceae_UCG-001、Prevotella₁ were different flora species.LDA linear regression analysis found that norank_fMuribaculaceae,Lactobacillus,Lachnospiraceae_NK4A136_group,Ruminococcus₁,Ruminococcaceae_UGG₀14,Anaerofustis,Rikenellaceae_RC9_gut_group,Bilophila,and unclassified_oCoriobacteriales were intestinal biomarkers at the genus level.Conclusion1.Jingning granules can effectively improve the core clinical symptoms and TCM symptoms of children with ADHD;2 Jingning granule can change the disorder state of ADHD children with deficiency of Qi and Yin,which includes the deficiency of serum dopamine,the low reactivity of HPA axis and the differential gut microbiota;3.Jingning granule can effectively improve the learning ability of ADHD model rats and control their hyperactivity and impulsive behaviors;4 Jingning granule can improve the disorder of dopamine system in striatum and hypothalamus,and better than that of methylphenidate in hypothalamus;5.ADHD model rats have HPA axis low reactivity and differential gut microbiota,Jingning granule can improve this disorder;6.Jingning granule may play a role in treating ADHD by improving the structure of the gut microbiota to activate the HPA axis function,thereby affecting the expression levels of D1 and D2 receptors in the striatum and hypothalamus,and increasing the dopamine content in brain tissue.