| ObjectiveTourette syndrome(TS)is a common neurobehavioral and neuropsychiatric disorder in children.The period of treatment on TS is long and easy to repeat.And the side effects of chemical agents are serious,at present which is a main clinical challenge.The latest study exhibted that the occurrence of nervous system diseases may be closely related to the imbalance of intestinal flora,neuroinflammatory and neurotransmitter imbalance.Therefore,in the study,based on the analysis of characteristics of intestinal flora between TS patients with healthy children using 16 S rRNA sequencing and Qinglong zhidong decoction(QLZDD)chemical compositions by performing LC-MS method qualitatively,we established IDPN-induced TS model mice to study the mechanism of affecting intestinal flora,neuroinflammation and neurotransmitters to anti-tic.Methods(1)Twenty-seven TS patients(TS group)and 30 healthy children(Control group)were collected from November 20,2019 to August 20,2020.The basic data and questionnaire information were filled out completely,and fresh fecal specimens were collected.Then total DNA and PCR of fecal bacteria were extracted and amplified.Based on Illumina Hi Seq sequencing platform,OTUs clustering,species annotation and inter-group difference analysis were carried out.(2)The chemical composition of QLZDD was qualitatively controlled and qualitatively analyzed by LC-MS.(3)Ninety healthy male KM(Kunming mice)mice were randomly divided into normal group(Control)and model group(Model).The TS model were established by intraperitoneal injection of IDPN at dosage of 350mg/kg.Then the model mice were randomly divided into haloperidol group,QLZDD-low dose group,QLZDD-middle dose group and QLZDD-high dose group.All groups were given intragastric administration for 4 weeks.The general condition(food intake,water intake,body weight,coat score)and tic-like behaviors(stereotyped behavior scores,space restriction behavior scores,autonomous activity behavior scores and head-body tics)were recorded weekly.After experiment,the mice were killed under anesthesia,and their brains and intestines were dissected.The pathological changes of colon were observed by HE staining,and the changes of intestinal flora were detected by 16 S rRNA sequencing.(4)The number of cortical neurons and striatum microglia were observed by HE staining.The content changes of oxidative stress factors(MAD,NO,SOD)in the striatum and inflammatory factors(TNF-α,IL-1β,IL-6)in the striatum and serum were detected by ELISA.(5)The levels of Glu,GABA,DA and 5-HT in serum and striatum were measured by ELISA.The mRNA expression of D1 R,D2R,DAT and GABAR in striatum were measured by RT-q PCR.The protein expression of D1 R,D2R,DAT and GABAR in striatum was measured by Western Blot.The protein expression of D1 R,D2R,DAT and GABAR in brain striatum and cortex was measured by IHC,further the correlation between intestinal microorganisms and environmental factors was investigated.Results(1)There was no significant difference in sex,age and BMI index between TS group and Control group.α diversity analysis increased significantly in TS group(P<0.05).β diversity analysis showed that there were significant differences between the two groups;In TS group,Prevotellaceae＿NK3B31＿group group increased mainly,while Acinetobacter and Trichococcus;decreased mainly in Bacteroides and Lactobacillus.(2)LC-MS analysis showed that a total of 291 chemical constituents were identified in the water extract of QLZDD,mainly alkaloids,flavonoids,terpenoids,etc.Main ingredients:(1)Hirsutine;(2)Isorhynchophylline;(3)Citric acid;(4)3-Hydroxy-5-isopropylidene-3,8-dimethyl-2,3,3a,4,5,8a-hexahydro-6(1H)-azulenone;(5)Albiflorin;(6)Parishin E;(7)Hesperetin;(8)Quinic acid;(9)methy(1S,5R,9S,13R)-5,9-dimethyl-14-methylidenetetracyclo;(10)Pyroglutamic acid.(3)The IDPN significantly decreased food intake,water intake,body weight and increased coat scores(P<0.05)(Control group vs model group).Compared with the model group,QLZDD significantly increased food intake,water intake,body weight(P<0.05),while significantly decreased coat scores(P<0.05).QLZDD significantly decreased tic-like behaviors(all P-value < 0.05).HE staining results showed that the colonic mucosal layer became thinner and the number of glands decreased in model group(compared with control group),while QLZDD-M and QLZDD-H groups were significantly thickener than the model group.16 S rRNA analysis: QLZDD significantly increased the relative abundance of Lactobacillus and Bacteroidetes,while significantly decreased the relative abundance of Akkermansia and Alloprevotella in IDPN-induced TS model mice.(4)The IDPN significantly increased the numbers of microglia in striatal(P<0.05),while significantly decreased the number of neurons in cortical(P<0.05).Compared with the model group,all QLZDD groups and haloperidol groups significantly decreased the numbers of microglia in the striatal(P<0.05),while significantly increased the numbers of neurons in the cortical.Compared with the control group,the IDPN significantly increased the contents of MDA and NO(P<0.05)in the striatum and significantly lifted the contents of IL-1 β,IL-6 and TNF-α in the striatum and serum(P<0.05).Compared with the model group,QLZDD and haloperidol significantly decreased the contents of MDA and NO(P<0.05)in the striatum,and significantly block the contents of IL-1 β,IL-6 and TNF-α(P<0.05)in the striatum and serum.(5)The IDPN significantly increased the levels of DA and Glu(P<0.05),while decreased the levels of GABA and 5-HT in the striatum and serum.Compared with the model group,all QLZDD dose groups and haloperidol significantly decreased the levels of DA(P<0.05),while increased the levels of GABA(P<0.05)in the striatum and serum.RT-PCR and WB results exerted,compared with the control group,the IDPN significantly increased the expression of mRNA and protein of D1 R and D2R(P<0.05),and significantly decreased the expression of mRNA and protein of DAT and GABAR in the striatum;compared with the model group,all QLZDD dose groups and haloperidol significantly decreased the mRNA and protein expression of D1 R and D2R(P<0.05),and significantly increased the mRNA and protein expression of DAT and GABAR(P<0.05)in the striatum.IHC results showed that the IDPN significantly decreased the protein expression of D1 R and D2R(P<0.05),and significantly increased the protein expression of DAT and GABAR(P<0.05)in the striatum and cortex.Conclusion(1)There were significant differences in intestinal flora between TS patients with healthy children.(2)QLZDD improved the tic-like behaviors of IDPN-induced TS model mice,and protected intestinal mucosa and regulated intestinal flora.(3)QLZDD reduced the numbers of microglia in striatum and increase the number of neurons in cortex,and reduce oxidative stress and inflammation.(4)QLZDD significantly decreased the levels of DA and Glu,while increased the levels of GABA and 5-HT;QLZDD significantly decreased the levels of the expression of mRNA and protein of D1 R,D2R,while increased the levels of DAT and GABAR.(5)QLZDD may play an anti-tic effect by regulating intestinal flora,reducing oxidative stress and neuroinflammatory reaction,and affecting the level of neurotransmitters through the "intestinal-brain axis". |
