Clinical Research And Mechanism Of Jingning Recipe In The Treatment Of ADHD Based On Ca²⁺-CaM-CaMKⅡ Pathway

Background:Attention deficit hyperactivity disorder(ADHD)is the most common chi-ldhood psychological behavior disorder.The clinical manifestations are attention deficit,hyp-eractivity and impulsiveness that are not consistent with the age level,and the disease is com-plex and easy to repeat,which has a certain degree of damage to the school life,social beha-veior and cognitive function of children,and has become one of the important diseases affect-ting the physical and mental health of children.In recent years,the overall prevalence of Atte-ntion deficit hyperactivity disorder among children and adolescents in China has been incre-asing year by year,while the etiology and pathogenesis of ADHD are not yet clear.The first-line use of methylphenidate and tomoxetine can improve most of the core symptoms of Atten-tion deficit hyperactivity disorder and achieve good efficacy,but a few children will have ob-vious side effects,dependence or long-term late-stage disease.Traditional Chinese medicine has certain unique advantages in the treatment of ADHD.Jingning Formula has been applied in clinical practice for many years with remarkable curative effect and less side effects,and has obtained the national invention patent(patent number:201510303541.6).Objective:To explore the molecular mechanism of Jingning formula in the treatment of attention deficit hyperactivity disorder(ADHD)by network pharmacology.To evaluate the clinical efficacy of Jingning formula in the treatment of attention deficit hyperactivity disorder and its effect on serum calcium level.To explore the mechanism of Jingning formula regulating the Ca2+-CAM-CaMKII signaling pathway in ADHD model rats.Methods:TCMSP database was used to retrieve the active components and correspond-ing targets of Jingning formula.TTD,Drugbank,Genecards and DisGeNET databases were used to retrieve ADHD associated with targets.Venn diagram was used to extract the interse-ction targets of Jingning formula and ADHD.The string database was used to construct the PPI network of key targets.The Cytoscape 3.8.2 was used to construct the network of "key targets-active ingredients-herbs" and the David database was used to conduct GO analysis and KEGG analysis of key targets.The experimental method of case before and after control was used in clinical study.The clinical effect of Jingning formula on attention deficit hyperactivity disorder was observed for 8 weeks.After 0 weeks,4 weeks and 8 weeks of treatment,the chil-dren were evaluated by SNAP-Ⅳ scale,Conners parental questionnaire,eliminate test and ADHD TCM syndrome score scale.Before and after treatment,serum calcium level,blood routine examination,routine urine test,liver and kidney function were detected.Adverse events were recorded throughout the clinical observation phase.In animal experiment,fifty SPF 4-week-old male SHR rats were randomly divided into model group,Jingning high-dose,medium-dose and low-dose groups,and methylphenidate group.Ten male Wistar rats of the same age were used as normal control group.The daily intragastric dose of Jingning granules group was 23.14g/kg for high dose,11.57g/kg for medium dose,5.785g/kg for low dose,1.5 mg/kg for methylphenidate hydrochloride group,respectively.The normal group and the mo-del group were given 1m1/1 00g of deionized water by gavage.All rats were gavaged once a day for 6 weeks.The changes of body weight of rats were monitored.The open field experi-ment was conducted to evaluate the behavioral characteristics of rats.The content of calcium in the striatum,the expression of IP3 protein and mRNA in the striatum,the expression of CaM and CaMKII protein and mRNA in the hippocampus were detectedResults:There were 82 active components of Jingning formula,corresponding to 224 target genes.And 103 key targets were obtained by drawing Venn diagram with ADHD target genes,including AkT1,FOS,IL6,TP53,TNF,VEGFA,etc.These genes mainly involved in calcium signaling pathway,PI3K-Akt signaling pathway,cGMP-PKG signaling pathway,cAMP signaling pathway,HIF-1 signaling pathway.JAK-STAT signaling pathway and so on.The results of clinical study showed that the total effective rate of Jingning prescription in the treatment of ADHD was 84%,and the total effective rate of TCM syndromes evaluation was 84%.After 8 weeks of Jingning formula treatment,there were significant improvements in SNAP-Ⅳ scale test,Number Cancellation Test and Conners Parent Symptom Questionnaire,which were statistically significant.The main symptoms and secondary symptoms in the TCM syndrome scale were significantly improved compared with those in week 0,with statistical difference.After 8 weeks of Jingning formula treatment,serum calcium level was higher than that before treatment(P<0.05).Animal experiment results showed that compared with normal group,the total distance of movement in model group was significantly increased(P<0.05);Compared with model group,the total distance of exercise in methylphenidate group,Jingning formula high-dose,medium-dose and low-dose groups was significantly decreased(P<0.05);Compared with methylphenidate group,the total distance of exercise in static height group was significantly decreased(P<0.05).The total movement distance of normal group,methylphenidate group,Jingning formula high-dose and medium-dose groups was shorter,and the trajectory showed peripheral distribution,while the central movement trajectory was less.Compared with the other groups,the total distance of locomotion in model group and static low group was increased,and the characteristics of trajectory distribution were not clear,while the central trajectory was increased.The effect of Jingning formula on the calcium content of striatum:the calcium content of striatum in model group was significantly higher than that in normal group(P<0.05);After treatment,compared with model group,methy lphenidate group and Jingning high-dose,medium-dose and low-dose groups could all down-regulate the calcium content in the striatum(P<0.05).The calcium content in striatum of the statically high group was decreased compared with that of the methylphenidate group,but there was no statistical significance between the two groups(P>0.05).The effect of Jingning formula on the protein expression of IP3 in striatum,CaM and CaMKII in hippocampus:the protein expression level of IP3 in striatum in model group was significantly lower than that in normal group(P<0.05);After treatment,compared with model group,methylphenidate group,Jingning high-dose and medium-dose groups could significantly up-regulate the expression level of IP3 protein in striatum(P<0.05).Compared with the model group,the static low group had an increasing trend,but there was no statistical significance between the two groups(P>0.05).Compared with the methylphenidate group,the expression of IP3 protein in the static height group was increased,but there was no statistical significance between the two groups(P>0.05).The CaM protein expression level in the hippocampus of model group was significantly higher than that of normal group(P<0.05).After treatment,compared with model group,methylphenidate group,Jingning high-dose,medium-dose and low-dose groups could down-regulate the expression level of CaM protein in hippocampus(P<0.05).Compared with the methylphenidate group,the expression of CaM protein in the hippocampus of the statically high group was decreased,but there was no statistical significance between the two groups(P>0.05).The protein expression of CaMKII in the hippocampus of model group was significantly lower than that of normal group(P<0.05).After treatment,compared with model group,the protein expression of CaMKII in hippocampus was significantly up-regulated in methyl phenidate group,Jingning high-dose,medium-dose and low-dose groups(P<0.05).The protein expression of CaMKII in the statically high group was increased,but there was no statistical significance between the two groups(P>0.05).The effect of Jingning formula on the mRNA expression of IP3 in striatum,CaM and CaMKII in hippocampus:the mRNA expression level of IP3 in striatum in model group was significantly lower than that in normal group(P<0.05);After treatment,compared with the model group,the expression of IP3 mRNA in striatum of the methylphenidate group and Jingning high-dose,medium-dose and low-dose groups were significantly up-regulated(P<0.05).The expression of CaM mRNA in the static hypertensive group was higher than that in the methylphenidate group,but there was no statistical significance between the two groups(P>0.05).The expression level of CaM mRNA in hippocampus of rats in model group was significantly higher than that in normal group(P<0.05).After treatment,compared with model group,the expression level of CaM mRNA in hippocampus of methylphenidate group and Jingning high-dose,medium-dose and low-dose groups were significantly decreased(P<0.05).Compared with the methylphenidate group,the expression of CaM mRNA in the stably high group was decreased,and the difference between the two groups was significant(P<0.05).The mRNA expression level of CaMKII in the hippocampus of model group was significantly lower than that of normal group(P<0.05).After treatment,compared with model group,the mRNA expression level of CaMKII in hippocampus of methyl phenidate group and Jingning high-dose,medium-dose and low-dose groups was significantly increased(P<0.05).The mRNA expression level of CaMKⅡ in the hippocampus of the static-high group was significantly higher than that of the methylphenidate group,and there was a significant difference between them(P<0.05).Conlusion:(1)Jingning formula have the characteristics of multi-component,multi-target and multi-signal pathway,and play a therapeutic role by regulating neurotransmitter metabolism,central nervous system regulation and immune regulation.(2)Jingning formula can improve the core symptoms of the children with Qi and Yin deficiency ADHD,at the same time,it can be benefit to the diet and sleep of the children.(3)Jingning formula can improve the serum calcium level of ADHD children,(4)Jingning formula treated for 6 weeks had no obvious effect on the weight gain of SHR rats,and could effectively improve the hyperactivity behavior of SHR rats.(5)Jingning prescription can affect the release of DA and play a therapeutic role in ADHD by regulating the Ca2+-CAM-CAMK II signaling pathway.