| Attention deficit hyperactivity disorder(ADHD)is a common mental behavior disorder in children.Its clinical manifestations are mainly inattention,hyperactivity and impulsivity,and are often accompanied with neuropsychological defects.Such as memory disorder,conduct disorder,oppositional defiant disorder,etc[1].According to the clinical manifestations of this disease,it belongs to the category of "manic","restless","forgetfulness" and "deaf" in traditional Chinese medicine.In traditional Chinese medicine,it is believed that the children with this disease have more yin deficiency than yang hyperactivity.Therefore,it is believed that there are more children with the syndrome of yin deficiency and yang hyperactivity[2].The Xiaoer Huanglong Granule,which is product by Chongqing Hilan Pharmaceutical Co,Ltd,is the only Chinese patent medicine approved by the State Food and Drug Administration for the treatment of ADHD.The key medicine of the prescription is Paeoniae,cooked Rehmannia radix and Ophiopogonis.The three medicines have the synergistic effect of nourishing yin and replenishing blood,calming the mind and stabilizing the will,and calming the liver and relieving yang.Although Xiaoer Huanglong Granule has significant clinical efficacy in the treatment of ADHD,its pharmacodynamic characteristics and pharmacological mechanism are not clear yet,and there is no research on the model about combining ADHD disease with syndrome.Therefore,this topic conducts experiments on the above problems,including the following three parts:1 Construction of an animal model of ADHD in children with yin deficiency and yang hyperactivityObjective:To provide a new method for constructing a syndrome combination model of yin deficiency and yang hyperactivity in children with ADHD,so as to promote the development of new drugs for the treatment of ADHD.Method:Young spontaneously hypertensive rats(SHR)were used as ADHD model rats.16 healthy SHR rats were randomly divided into SHR group and BZSHR group according to body weight.WKY group and Wistar group were also set up(this group was only used as the control group of behavioral experiment),with 8 animals in each group.The BZSHR group were injected subcutaneously with L-Thyroxin sodium(300 ug/kg)daily,and the other groups were injected subcutaneously with normal saline for 14 consecutive days.The diet volume,water volume,defecation volume,urine volume,heart rate,respiratory rate and specific values of the tongue image red vector(R),green vector(G)and blue vector(B)were used as the evaluation indicators of yin deficiency and yang hyperactivity syndrome.The degree of autonomous activity,social preference,anxiety,irritability,and the ability of spatial memory and attention set transfer of each group of rats were observed as the evaluation indexes of ADHD behavior,and the model was diagnosed and evaluated.Results:(1)Results of the syndrome with yin deficiency and yang hyperactivity:There was no significant difference between the SHR group and the WKY group in metabolic indexes such as body weight,diet volume,water volume,defecation volume and urine volume,while the metabolic indexes of the BZSHR group were higher than that of the SHR group(P<0.05).The heart rate and respiratory rate of the BZSHR group were significantly higher than that of the SHR group(P<0.05),and the values of tongue image R,G and B were significantly higher than that of the WKY group(P<0.05).(2)Results of degree of autonomic activity:Compared with WKY and Wistar groups,the moving distance,moving speed,the distance in the central region and activity continuity of rats in SHR and BZSHR groups were significantly increased(P<0.01);the distance in the central region of the BZSHR group was significantly higher than that of the SHR group(P<0.05).(3)Results of degree of social preference:In terms of social time,all indexes of SHR group,BZSHR group and WKY group were significantly decreased compared with Wistar group(P<0.01),and there was no significant difference between BZSHR group and SHR group.In terms of social distance,compared with WKY group,the distance and b/a value in area b and a+b were significantly increased in BZSHR group(P<0.01),while the a+b/a+b value and b/B value in Wistar group were significantly decreased(P<0.01),and the distance in area b and a+b in SHR group were significantly increased(P<0.05).In terms of social frequency,the a+b/A+B,b/B and b/a value in BZSHR group were significantly decreased compared with WKY group(P<0.05),the a+b/a+B value in Wistar group was significantly decreased(P<0.05),and the frequency in a+b area in SHR group was significantly increased compared with SHR group(P<0.05),while the b/B and b/a values were decreased.(4)Results of spatial memory ability:Latent period of rats in SHR group and BZSHR group was reduced compared with that in WKY group and Wistar group,but the times of error was increased,with no statistical significance.The latent period and error times of BZSHR group were higher than that of SHR group,and the difference was not significant.(5)Results of degree of anxiety:The OT and OE ratios of the SHR group and BZSHR group were higher than that of WKY group,but lower than Wistar group,and the difference was not statistically significant;there was no significant difference in OT and OE ratio between BZSHR group and SHR group.(6)Results of degree of irritation:In terms of aggressive behavior,the times of all kinds of aggressive behaviors and the total times of aggressive behaviors in SHR group and BZSHR group were higher than that of WKY group,especially the total times of biting target,clawing target,exploring target and aggressive behavior in BZSHR group were significantly increased(P<0.05);compared with Wistar group,the total times of aggressive behaviors in SHR group was significantly increased(P<0.01),and the total times of biting,clawing,delayed biting and aggressive behaviors in BZSHR group was significantly increased(P<0.05).Compared with SHR group,BZSHR group showed an increase in all other behaviors except target tracking and delayed biting.In terms of defensive behavior:the times of avoidance behaviors in BZSHR group was significantly lower than that of Wistar group(P<0.05),but there was no significant difference in the times of various defensive behaviors among all groups.The total times of defensive behaviors in SHR and BZSHR groups was higher than that in WKY group but lower than that in Wistar group,with no statistical significance.(7)Results of attention set transfer ability:Except RL reverse learning stage,the error frequency and error rate of rats in SHR group and BZSHR group were higher than those in WKY group and Wistar group in the five-phase test.Conclusion:By subcutaneous injection of L-Thyroxin sodium in young SHR rats,a more stable and reliable model of ADHD syndrome with yin deficiency and yang hyperactivity can be obtained.2.Influence of Xiaoer Huanglong Granule on the general signs and behaviors of ADHD model rats and the ADHD model rats combined with yin deficiency and yang hyperactivityObjective:To observe the intervention effect of Xiaoer Huanglong Granule on general signs and behaviors of ADHD model rats and the ADHD model rats combined with yin deficiency and yang hyperactivity.Method:A total of 56 healthy SHR rats were randomly divided into 7 groups according to body weight.SHR group(SHR),SHR+Xiaoerhuanglong Granules low-dose group 1.88 g/kg(SHR+XEHLL),SHR+Xiaoerhuanglong granules high-dose group 3.75 g/kg(SHR+XEHLH),disease combined syndrome model group(BZSHR),disease combined syndrome+Xiaoerhuanglong granules low-dose group 1.88 g/kg(BZSHR+)XEHLL),disease combined syndrome+Xiaoer Huanglong Granule high-dose group 3.75 g/kg(BZSHR+XEHLH),methylphenidate group(PJZ),and 8 WKY rats and 8 Wistar rats were set as normal control group.BZSHR,BZSHR+XEHLL and BZSHR+XEHLH groups were injected subcutaneously with Levothyroxine sodium(300 ug/kg)every day,and other groups were injected subcutaneously with normal saline for 2 weeks.From the 11th day,SHR+XEHLL,SHR+XEHLH,BZSHR+XEHLL and BZSHR+XEHLH groups were given Xiaoer Huanglong granules according to body weight,PJZ rats were given methylpiperidol,and other groups were given the same amount of drinking water intragastically for 21 consecutive days.Diet voume,water volume,defecation volume,urine volume,heart rate,respiratory rate and specific values of the tongue image red vector(R),green vector(G)and blue vector(B)were used as indicators of yin deficiency and yang hyperactivity.The degree of autonomous activity,social preference,anxiety,irritability,and the ability of spatial memory and attention set transfer of each group of rats were observed as the evaluation indexes of ADHD behavior,the improvement effect of Xiaoer Huanglong Granules on ADHD model rats and ADHD model rats combined with yin deficiency and yang hyperactivity was observed.Results:(1)Results of the syndrome with yin deficiency and yang hyperactivity:Xiaoer Huanglong Granules could relieve the mental tension and irritability of rats in SHR group and BZSHR group;It had little effect on body weight,diet volume,water volume,defecation volume,urine volume and tongue image,but significantly reduced the heart rate and respiratory rate of BZSHR group(P<0.01).(2)Results of degree of autonomic activity:Each dose of Xiaoer Huanglong granule and methylphenidate reduced the moving distance,moving speed and movement continuity of SHR group to varying degrees;all doses of Xiaoer Huanglong granules could reduce all indexes in BZSHR group,especially high dose of Xiaoer Huanglong granules could significantly reduce the moving distance,moving speed and movement continuity of rats in BZSHR group(P<0.01).(3)Results of degree of social preference:Methylphenidate significantly affected the social time and social distance of SHR group(P<0.05),low dose of Xiaoer Huanglong granules significantly increased the social time of SHR group(P<0.05),and significantly increased the a+b/A+B,b/B distance proportion of SHR group(P<0.05).High dose of Xiaoer Huanglong granules could significantly increase the times in area b,a+b and the regional time proportion a+b/A+B of rats in BZSHR group(P<0.05),and significantly increase the a+b/a+b,b/B distance proportion of rats in area B(P<0.05).(4)Results of degree of anxiety:Each dose of Xiaoer Huanglong granule and methylphenidate could increase OT and OE proportion of SHR group,but the difference was not significant;the OT and OE ratios of rats in BZSHR group were lower than those in SHR group,and each dose of Xiaoer Huanglong granules could improve the OT and OE ratios of rats in BZSHR group,and the difference was not statistically significant.(5)Barnes maze results:Low dose of Xiaoer Huanglong granules and methylphenidate could reduce latent period and error times of SHR rats,but the effect was not significant;high dose of Xiaoer Huanglong granules could reduce latent period and error times of BZSHR group,but the difference was not statistically significant.(6)The results of degree of irritation:Methylphenidate could significantly reduce the total times of aggressive behavior in SHR group(P<0.05);All doses of Xiaoer Huanglong granules could reduce all kinds of aggressive behaviors in SHR group and BZSHR group,and significantly reduce the total times of aggressive behaviors(P<0.05),but had little effect on defensive behaviors.(7)Results of attention set transfer ability:Xiaoer Huanglong Granules can reduce the times of error,error rate and total times of excavation in SHR group and BZSHR group to varying degrees,and its therapeutic effect on both groups is similar.Conclusion:Xiaoer Huanglong Granules can improve the general signs and behavior on ADHD model rats and ADHD model rats with yin deficiency and yang hyperactivity,especially can significantly improve the syndrome indications in ADHD model rats with yin deficiency and yang hyperactivity,and significantly improve the degree of autonomous activity,social ability and irritability.3 The influence of Xiaoer Huanglong Granule on the monoaminergic nerves of ADHD model rats and ADHD model rats with yin deficiency and yang hyperactivityObjective:To observe the effect of Xiaoer Huanglong Granules on the monoaminergic nerves of ADHD model rats and ADHD model rats with yin deficiency and yang hyperactivity,so as to analyze the mechanism of effect of Xiaoer Huanglong Granules.Method:Selecting the upper part of the sample,use the ultra-high performance liquid chromatochromato-mass spectrometry(UPLC-MS/MS)to detect the levels of dopamine(DA),vanillic acid(HVA),norepinephrine(NE)and 3-methoxy-4-hydroxyphenylate glycol(MHPG)in the prefrontal lobe and striatum of rats.The mRNA and protein expression levels of dopamine receptor 1(DRD1),dopamine receptor 2(DRD2),tyrosine hydroxylase(TH)and dopamine transporter(DAT)in the prefrontal lobe and striatum of rats were detected by polymerase chain reaction(PCR)and Western Blot(WB).Results:(1)WB results:The protein expressions of DRD1,DRD2,DAT and TH in the prefrontal lobe and striatum of rats in SHR and BZSHR groups were decreased compared with those in WKY and Wistar groups,while the positive drug methylphenphenate could increase the protein expressions of DRD1,DRD2,DAT and TH in the prefrontal lobe and striatum of rats in SHR and BZSHR groups to some extent.Xiaoer Huanglong granules can increase the protein expressions of DRD1,DRD2,DAT and TH in the prefrontal lobe and striatum of rats in SHR and BZSHR groups.(2)PCR results:The expressions of DRD1,DRD2,DAT and THmRNA in the prefrontal lobe of SHR and BZSHR groups were decreased compared with those of WKY and Wistar groups,while the positive drug methylpiperidol could increase the expressions of DRD1,DRD2,DAT and THmRNA in the prefrontal lobe of SHR and BZSHR groups to some extent.Xiaoer Huanglong granules can increase the mRNA expressions of DRD1,DRD2,DAT and TH in the prefrontal lobe of rats in SHR and BZSHR groups.(3)UPLC-MS/MS results:DA and HVA detection results:HVA content in prefrontal lobe of rats in SHR and BZSHR groups was significantly increased compared with Wistar rats(P<0.05),and HVA content in striatum of rats in BZSHR group was significantly increased compared with Wistar rats(P<0.05);High dose of Xiaoer Huanglong granules could significantly reduce the content of DA in prefrontal lobe of SHR rats(P<0.05),and reduce the content of HVA,while methylphenidate could significantly increase the content of DA in prefrontal lobe of SHR rats(P<0.05).Compared with SHR rats,HVA content in striatum of BZSHR group was significantly increased(P<0.05),low dose of Xiaoer Huanglong granules could significantly increase DA content in prefrontal lobe of BZSHR group(P<0.05),and reduce HVA content(P<0.05).NE and MHPG detection results:Compared with Wistar rats,NE content in prefrontal lobe and striatum of SHR and BZSHR groups decreased to a certain extent.High dose of Xiaoer Huanglong granules and methylphenidate significantly increased the NE content in prefrontal lobe of SHR rats(P<0.05).Each dose of Xiaoer Huanglong granules could increase the content of NE in the prefrontal lobe and striatum of BZSHR group and reduce the content of MHPG,but the effect was not significant.Conclusion:The mechanism of Xiaoer Huanglong Granule in treating ADHD model rats and ADHD model rats with yin deficiency and yang hyperactivity may be related to the improvement of monoaminergic nerve function. |
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