To Investigate The Effect Of Jingning Granules On Gastrointestinal Function In ADHD Based On The Brain-gut Axis And Its Mechanism

Background Attention deficit hyperactivity disorder(ADHD)is a common neurodevelopmental disorder in children and adolescents.It is characterized by hyperactivity,impulsivity and inattention.It is often accompanied by one or more comorbidities.The morbidity of ADHD in the world is increasing year by year,and the harm,as well as the pressure of prevention are also increasing.However,the specific pathogenesis of the disease is still unknown.Previous studies mainly focused on monoamine neurotransmitters such as dopamine(DA),noradrenaline(NE)and related signaling pathways.As brain-gut axis theory is put forward and enriched,and gut microbes have been confirmed playing a important role in more and more,especially in mental illness,it's of great significance to explore the mechanism of the brain-gut axis in the pathogenesis of ADHD and the regulatory effect of brain-gut peptide to expand the research field of the mechanism of ADHD and the idea of clinical treatment.Objective This study consists of two parts:clinical study and experimental study.Through clinical studies,the clinical efficacy of Jingning Granules in treating ADHD children with Qi and Yin deficiency syndrome was revaluated.Evaluate the gastrointestinal function problems of ADHD,and the improvement effect of Jingning Granules on the gastrointestinal symptoms accompanied by ADHD and the levels of serum GAS,MTL,Ghrelin,VIP,SP and intestinal microorganisms was verified.Animal experiments were conducted to explore the possible mechanism of brain-gut peptides in the pathogenesis of ADHD through the brain-gut axis,and to verify the correlation between gastrointestinal symptoms of ADHD and its core symptoms.In addition to participating in gastrointestinal function changes,brain-gut peptides may interact with intestinal microorganisms to affect the brain function through the brain-gut axis and cause the pathogenesis of ADHD.Methods Clinical study:In this study,30 children with ADHD aged 6-14 who met the inclusion and exclusion criteria were included and given Jingning Granules for oral treatment for 8 weeks.The clinical efficacy of Jingning Granules on ADHD children with Qi/Yin-deficiency syndrome was evaluated by TCM Syndrome Score Scale,SNAP-? scale,Digital Eliminating Test and Conners Children Behavior Scale(parents).The gastrointestinal function of ADHD children and the improvement effect of Jingning granules were evaluated by TCM Symptoms of Gastrointestinal Diseases Scale and serum GAS,MTL,Ghrelin,VIP and SP levels.The fecal samples of ADHD children before and after treatment with Jingning granules were detected to understand the changes in the diversity and richness of intestinal microorganisms.Experimental study:Fifty 4-week-old male SHR rats were randomly divided into model group,Jingning granules high-dose,medium-dose and low-dose groups,and atomoxetine group,and 10 male Wistar rats of the same age were used as blank group,and given corresponding drugs and deionized water intragastrically for 6 weeks,once a day.Six weeks later,open-field experiments were conducted.Execute the rats and calculate the gastric content residual rate and intestinal propelling rate.The levels of GAS,MTL,Ghrelin,VIP and SP in serum,the levels of GAS,MTL and their corresponding receptors in prefrontal lobes,and the diversity and richness of intestinal microorganism in each group were detected respectively.Results Clinical study:After 8 weeks of Jingning Granules treatment,the SNAP-?scale,Conners Children's Behavior Scale(parents),digital erasures test and TCM syndrome total score were significantly decreased in ADHD children(P<0.01).There was statistical significance in the improvement of the integral complexion of each subsymptom of TCM syndrome(P<0.05),and the improvement of the other symptoms was significant(P<0.01)The total effective rate of Jingning granules in treating ADHD children with Qi and Yin deficiency syndrome was 80.00%,and the total effective rate of TCM syndrome was 83.00%Jingning granules could significantly improve the scores of TCM symptom scale of gastrointestinal diseases in ADHD children(P<0.01),and significantly regulate the levels of GAS,MTL,Ghrelin,VIP and SP in serum(P<0.01).There were differences in the diversity and richness of intestinal microorganism in ADHD children before and after Jingning Granules treatment,and there was no significant difference in the dominant species at the genus level between the two groups.Blautia,Bifidobacterium,Faecalibacterium,Eubacterium_Hallii_group,Subdoligranulum and Bacteroides were the dominant species.The difference of sobs richness at OTU level between the two groups was statistically significant(P<0.05),and the difference of shannon diversity was significant(P<0.01).The difference test showed that there was statistical significance in Bacteroides(P<0.05).Clostridium innocuum_group,Norank_F_Norank_O_Clostridia_UCG-014 and Abiotrophia were also found to have significant differences before and after treatment.Experimental study:There was no significant difference in body weight between the groups at 0 and 6 weeks(P>0.05).The open field experiment results show that the model group total distance,central region movement time significantly higher than that of blank group(P<0.01),the total static and edge area movement time significantly lower than that of blank group(P<0.01),Chinese crude drug groups and ATX group compared with model group on the total distance,central region movement time significantly lower than that of model group(P<0.01).The total resting time and the movement time in the edge area were significantly higher than those in the model group(P<0.01).The residual rate of gastric contents in the model group was significantly higher than that in the blank group(P<0.01),the static height group was significantly lower than that in the model group(P<0.01),and the static medium group and ATX group were lower than that in the model group,the difference was statistically significant(P<0.05).The small intestine propulsion rate in model group was significantly lower than that in blank group(P<0.01),the small intestine propulsion rate in static height group,static medium group and ATX group was significantly higher than that in model group(P<0.01),and the static medium group was lower than that in ATX group,the difference was statistically significant(P<0.05),the static low group was significantly lower than that in ATX group and static height group(P<0.01).There were significant differences in serum levels of MTL,GAS,Ghrelin,VIP and SP in model group(P<0.01).Jingning granules high-dose and medium-dose groups and ATX groups could effectively improve these differences(P<0.01 or P<0.05).Jingning granules low-dose group only had improvement effect on MTL,Ghrelin and VIP(P<0.05).There were significant differences between model group and blank group in the levels of GAS and MTL in prefrontal cortex(P<0.01).Jingning granules high-dose,medium-dose and low-dose groups and ATX groups could significantly increase the levels of GAS and MTL in prefrontal cortex(P<0.01).Compared with the blank group,the expressions of GAS receptor(CCKBR)and MTL receptor in the prefrontal cortex of the model group were significantly lower(P<0.01).The expression of CCKBR in the high-dose and medium-dose Jingning granules groups and ATX groups were significantly higher than that in the model group(P<0.01).There was no statistical difference in the expression of MTL receptor in the treatment groups compared with the model group(P>0.05).Bar plot of the community showed that the dominant bacteria species in each group were basically the same,but there were great differences in relative abundance.The dominant species are Lactobacillus,Blautia,Allobaculum,Norank_F_Muribaculaceae,Unclassified_F_Lachnospiraceae,UCG-005.The Heatmap shows that there are significant differences in Desulfovibrio,Nonrank_F_Eggerthellaceae and Turicibacter among the groups.The community composition of the static medium group,static low group and ATX group was similar at the genus level,while the community composition of the static high group was more similar to that of the blank group.Significant difference test between groups at the genus level showed that Blautia,Allobaculum,Norank_F_Muribaculaceae,Unclassified_F_Lachnospiraceae,Romboutsia,Faecalibaculum,Lachnospiraceae_NK4A136_group,Dubosiella,Parabacetroides,Christensenellaceae_R-7_group was a significant difference.The significance of difference between groups was tested at the phylum level,and the final result showed that Bacteroidota,Actinobacteriota and Desulfobacterota were the target phylums.The most significantly different was Bacteroidota(P<0.001).Conclusion 1.Jingning granules can effectively improve the core symptoms of ADHD,such as hyperactivity,impulsiveness,and inattention,and significantly improve the scores of SNAP-?,digital erasure test,Conners Children's Behavior Scale(Parents),and TCM Syndrome Score Scale.2.ADHD children showed different degrees of gastrointestinal dysfunction in the TCM symptom rating scale of gastrointestinal diseases and in the levels of brain and intestinal peptides such as GAS,MTL,Ghrelin,VIP and SP,and Jingning Granules could effectively improve their gastrointestinal symptoms and reduce the scores of TCM symptom rating scale of gastrointestinal diseases and adjust serum levels of GAS,MTL,Ghrelin,VIP and SP.3.There were differences in the diversity and richness of intestinal microorganism in ADHD children before and after Jingning granules treatment,especially Bacteroides.Changes in gut microbiota played an important role in the incidence of ADHD,and Jingning granules had a good improvement effect on it.4.Jingning granules can significantly improve hyperactivity,impulsiveness and inattention behavior of SHR rats,and reduce excessive activities in open-field test.5.Compared with the control group,SHR rats showed differences in gastric content residual rate,small intestinal propulsation rate,serum and prefrontal cortex levels of GAS,MTL and other brain-intestinal peptides and related receptors.The drug treatment can effectively improve these differences,and brain-intestinal peptides and their receptors may play a role in the pathogenesis of ADHD through the brain-intestinal axis.6.There were significant differences in the diversity and richness of intestinal microorganism in each group.Jingning granules and ATX had a regulatory effect on intestinal microorganism in SHR rats,and brain-intestinal peptides may interact with other intestinal microorganism mainly Bacteroides to change the brain functional state through the brain-intestinal axis pathway and cause the onset of ADHD.