Effects And Mechanisms Of Jingning Granules On Social Behavior And Spatial Memory In ADHD Model Rat

Attention deficit hyperactivity disorder(ADHD)is a group of behavioral and emotional disorders that occur in childhood and adolescence.The intelligence of children with ADHD is usually normal or nearly normal,but there are disadvantages on learning behavior and emotional aspects,mainly performance as impaired and short duration of concentration,hyperactivity and impulsive mood.Meanwhile often company with learning difficulties,cognitive disorder and conduct disorder etc.Epidemiological studies have confirmed that the prevalence of ADHD in school-age children is 5.29%,and the prevalence rate of boys is higher than girls.The proportion is about 4:1-9:1.Due to the symptoms of ADHD is always found in school-age children,it is considered to be a behavior disease limited to children and adolescents over the years.But recent years follow-up study of ADHD showed that about 10%-60%ADHD patients’ symptoms continued until youth and adult.Some even suffer from it all their life,demaging academic,family and social activities seriously.As a result,ADHD has become a growing concern for researchers.At present,the first-line drugs for clinical treatment of ADHD are mostly central stimulants,among which the most widely used is methylphenidate(Ritalin).It is ineffective or invalid for about 25%patients treated with ritalin,and what’s more some patients due to intolerable side effects such as sleep disorder,anorexia,convulsions,headache,as a result withdrawal,cannot control the clinical symptoms.In addition,because of the addictive nature of central stimulants,there is a potential risk of abuse,so some families refuse to take it.Therefore,it is of great significance to study the etiology and pathogenesis of ADHD,and to develop a new Chinese patent medicine for the treatment of ADHD.A suitable animal model is essential for drug research.An ideal animal model is able to simulate the pathogenesis and clinical symptoms of the disease,and all kinds of corresponding physiological and pathological changes are similar to the disease itself.In this study,juvenile spontaneously hypertensive rat(SHR)was used as an ADHD animal model.SHR rats was outbred originated from Wista Kyoto rats(WKY)by Okamoto and his co-worker.The SHR got its name for that it suffer hypertension spontaneously from 10 weeks of age.Since 4 weeks of age,The SHR rat began to exhibit hyperactivity,impulsivity,learning disabilities,and other similar ADHD clinical symptoms.It was positively related to similar ADHD between the difficulty level for finishing the task and stimulus delivery time.Studies have showed that the density of dopamine receptors in striatum and nucleus accumbens of SHR rats was higher than WKY rats,in accord with that of ADHD patients.Juvenile SHR rats have been widely used in ADHD model animals because of their advantages such as the clinical symptoms of ADHD,no need of artificial induction and the similarity of nervous system with human being.The etiology and pathogenesis of ADHD is unclear yet,but studies have found that the onset of ADHD relating with the prefrontal cortex and striatal monoaminergic neurotransmitter metabolic disorders.Researchers have proposed the dopamine dysregulation hypothesis.While a large number of experiments confirmed that ADHD is not a single neurotransmitter abnormity,but several kinds of neurotransmitter function disorder.Noradrenergic nerve played a very important role in regulating wakefulness,adaption to the environment,the whole brain excitability and vigilance state in the central nervous system.But there is seldom research relating noradrenergic nerve for ADHD disease currently.So this study explore the pathogenesis of ADHD from the point of the animal ethology and noradrenergic neura,resrarching the effect of Jingning Granule on ADHD model rat.1.Evaluation of ADHD model animals and screening of four different extraction processes of Jingning granulesObjective:1.Setting the WKY rats as normal control group,observe the social behavior of juvenile SHR rats and the ability of learning and memory,studying its rationality as ADHD animal model.2.According to the open-field test and social behavior test,meanwhile detecting the content of monoamine neurotransmitter in rat brain,compare the therapeutic effects among four different extraction process of Jingning particles on the ADHD rat model,choosing the best pharmacodynamic extraction process.Methods:The ADHD model,juvenile SHR rats,was taken drugs by intragastric administration respectively.The drug was Jingning granule extracted in four different process,they were Technology-1(extracting the Schisandra chinensis by ethyalcohol and other precipitation by water),Technology-2(extracting the total prescription by water),Technology-3(extracting the total prescription by water and then precipitating by ethyalcohol),Technology-4(the rhizome acori graminei was extracted the volatile oil and other precipitation was disposed in Technology-3 way).Set the methylphenidate(Ritalin)and Xiaoer Huanglong granule as positive control drug,and WKY(Wistar Kyoto rats)rats as normal control group.Detecting the autonomic activity index for each group of rats:the movement velocity and the total distance rat movement,the social aversion index:the cumulative time of the test rat stay in the strange side of olfactory region,the frequency of the test rat come into the strange smell region.Then detecting the content of single amine neurotransmittering in prefrontal by HPLC-ECD,to evaluate the therapeutic effect of each group on ADHD rats.Results:(1)In the spontaneous activity test,the rats in the model group movement velocity and the total distance were significantly increased compared with normal control group(P<0.05),Jingning granule in four different extraction process all can reduce the movement speed and distance of SHR rats,and the Technology-4 act best(P<0.05).(2)In social approach-avoidance test,the cumulative time and the the frequency of the test rat stay in the strange side of olfactory region of the model group was less than the control group.Compared with the model group,the cumulative time and the frequency of Jingning granule for each extraction process were longer,and among those four extraction process,the Technology-4 expand the cumulative time most significantly.(3)Compared with the control group,the dopamine(DA)and norepinephrine(NE)content in prefrontal of model group decreased.Compared with the model group,the dopamine(DA)and norepinephrine(NE)content in prefrontal in Jingning granule extracted in Technology-4 group increased most significantly.Conclusion:(1)Compare with the WKY rats in same age,the juvenile SHR rats showed hyperactivity,impulsivity,social disorder and other clinical symptoms similar with ADHD in the test of spontaneous activities and social behavior test.Which consistent with the clinical symptom of ADHD,so the juvenile SHR rats can be used as animal model in this study.(2)Jingning granule extracted in Technology-4(the rhizome acori graminei was extracted the volatile oil and other precipitation was extracted by water and then precipitating by ethyalcohol)is the best effect technology.2.Study on pharmacodynamics of Jingning granule in treating ADHDObjective:Set WKY as control rats,the juvenile SHR rats as ADHD model rats,observing the effect of Jingning Granule on ADHD model rat’s social behavior,the ability of learning and memory behavior,then studying the content of norepinephrine(NE),3-Methoxy-4-hydroxyphenylglycol(MHPG),serotonin(5-HT),5-hydroxyindoleacetic acid(5-HIAA)in prefrontal and striatal of ADHD model rat.Detecting the protein expression and gene expression of norepinephrine transporter(NET),alpha2A adrenergic receptor(ADR alpha 2A)to explore the biological mechanism of Jingning granule in treating ADHD model rat peeking for the may object target for the treatment of ADHD.Methods:Juvenile Spontaneously Hypertensive Rats(SHR)was treated with Jingning granule(20.25 g/kg,10.13 g/kg,5.06 g/kg),Ritalin(7.2 mg/kg),and Xiaoer Huanglong granule(1.02 g/kg)respectively by intragastric administration for continuous 17days.Additionally,set a model group,and WKY rats as control group.(1)From the eighth administration day,detecting the the lateral time the rat come into strange smell region(LTSS),the cumulative time the rat stay in the strange smell region(CTSS)and the frequency of the rat come into the strange smell region(FSS)in the 3-chamber social interaction test using the Noldus animal behavior tracking system.Evaluating the effect of Jingning granule in treating ADHD model rat on social behaviors aspects.(2)From eleventh day of administration,the rats were trained following Morris water maze test,it lasts 1 min per day for six day.On the seventh day tests were operated and testing the indicators including the escape latency,the times of crossing the platform,the cumulative time in the platform quadrant,to detect the effect Jingning granule acts on the ADHD model rats.(3)On the 17th day,after all the behavioral test,the rats were anesthetized and then decapitated,removed the prefrontal cortex and striatum quickly,then preservation in liquid nitrogen.Detecting contents of NE,MHPG,5-HT and 5-HIAA in prefrontal cortex and striatum in the brain tissue of rats using HPLC-ECD.Detecting the protein content of NET and ADRα2 A using Western-bolt test.And detecting the mRNA expression level of NET and ADRα2 A in prefrontal and striatal using the real time quantitative PCR method.So as to study the mechanism of Jingning granule on rat model of ADHD.Results:(1)In social behavior test,compared with control group,the sniffing time for the strange cage in model group shorter,the frequency increased,and the latency come into the strange area was prolonged.Compared with the model group,Jingning granule group in each dose can prolong the sniffing time in strange zone,reduce the frequency,and shorten the latency the rat come into a strange sniffing zone.(2)In the Morris water maze test,the cumulative time in the platform quarter of the model rats was shorter than that of WKY rats,and the times of cross platform zone increased,the escape latency was significantly higher than that in WKY rats(P<0.05,P<0.01);Compared with the model group,Jingning granules can shorten the escape latency,reduce the number of cross platform,extended platform quadrant time(P<0.05,P<0.01);(3)Compared with WKY group,the content of NE and 5-HT in prefrontal cortex and striatal disorders,the NE content reduced and 5-HT content increased of the rats in model group.Compared with the model group,the Jingning granule can increased the NE content and decreased the MHPG content.The Ritalin group,Jingning granule 20.25 g/kg group has the most significant impact(P<0.05);the content of 5-HT in Ritalin group and Jingning granule group was lower than model group,and 5-HIAA content increased.(4)Compared with the WKY rats,the ADRα 2A protein expression decreased in prefrontal cortex and striatal in the model group.The NET protein expression in prefrontal cortex was approximately equal with the WKY,while that in striatal rised slightly.Compared with the model rats,Jingning granules20.25 g/kg and 10.13 g/kg can suppress the ADR α 2A protein express in different degree,but it scarcely have a impact on the NET protein expression.(5)Compared with the normal group,the relative expression amount of NETmRNA in the prefrontal cortex and striatum of the model group was increased,and the expression of NETmRNA reduced in the Jingning granule group,and the 5.06 g/kg was the most significant dose.Compared with the normal group,the relative expression amount of ADR α 2AmRNA in the prefrontal cortex and striatum of rats in model group was decreased,and the relative expression of Jingning granule increased,and the 5.06 g/kg group was the most effective dose.Conclusion:(1)Jingning granule can improve the social behavior of ADHD model rats and promote the social interaction in depth with strange rats;(2)Jingning granule can prolong the cumulative time in the platform quadrant in ADHD model rats obviously,suggesting that Jingning granule can improve SHR rats learning and memory ability;(3)Jingning granule can rise the ratio of NE/MHPG,decline the ratio of 5-HT/5-HIAA in prefrontal cortex and striatal of ADHD model of rat,indicating that Jingning Granule can regulate the metabolism of on monoamine neurotransmitters,inhibit the metabolism of NE,so as to improve the function of neurotransmitter,that may be the mechanism of treatment on ADHD.(4)Jingning granule can decrease the expression of ADR α 2 A receptor protein,which may be a target for the treatment of ADHD.(5)Jingning granule has a slight reluate effect on the expression amount of NETmRNA and ADR alpha 2AmRNA,which may be the mechanism of regulation effect on noradrenergic nerves.