Study On The Mechanism Of Jiedu Tongluo Granules In The Treatment Of Post-stroke Depression, Qi Deficiency And Blood Stasis Syndrome

Background and purpose:Poststroke depression(PSD)is the most common neuropsychiatric complication in patients with stroke.The cumulative incidence of PSD within 5 years after stroke is 39-52%.The pathogenesis of PSD has not yet been fully clarified,and is involved in many mechanisms such as neurotransmitters,neuroanatomy,neurotrophic and inflammatory reactions.In recent years,the role of glutamate(Glu)ergic nervous system in depression and PSD are increasingly prominent.At present,there is a lack of clinical therapeutic drugs for PSD,mainly anti-depressants related to monoamine neurotransmitters,but these drugs have certain side effects.Therefore,it is very necessary to find a safe and effective method for treating PSD clinically.In Chinese medicine,PSD falls into the categories of "stroke" and "depression".Qi deficiency is the pathological basis of post stroke depression,and qi depression,phlegm and blood stasis are the main pathological changes.Jiedu Tongluo Granules(Patent No.:201510419571.3)is made of ginseng,Ginkgo biloba,Hypericum perforatum,Rhizoma Chuanxiong,Gastrodia elata,Radix Astragali and Radix Astragali,which is an effective compound for the treatment of PSD with qi deficiency and blood stasis syndrome.Jiedu Tongluo Granules with the function of Yiqi Huoxue,Jiedu Tongluo and Shugan Jieyu.Based on previous studies,combining animal models with clinical samples,our study explored the neuroprotective mechanism of Jiedu Tongluo granules from the perspective of Glu excitotoxicity in both system biology and molecular mechanism,in order to provide further evidence for Jiedu Tongluo Granules for the treatment of PSD.Methods:(1)Animal models:A rat model of post-stroke depression with qi deficiency and blood stasis syndrome was prepared using multiple cerebral infarction combined with sleep deprivation.50 rats were randomly divided into control group,model group,fluoxetine group(10mg/kg),low dose of Jiedu Tongluo group(2g/kg),high dose of Jiedu Tongluo group(4g/kg),a total of 5 Groups,10 rats in each group,medicine taking were started on the first postoperative day,and were continuously fed for 28 days.Sleep deprivation was started on the third postoperative day.The body weights were measured at 1 day before surgery,14 days and 28 days after surgery.Neurological function scores were performed 1 day,14 days,and 28 days after operation.Open field tests,suspension tests,and sugar preference experiment were performed preoperative and 28 days postoperative.The RGB value and pulse amplitude of tongue were measured 28 days after surgery.The changes of ethology and TCM syndrome of rats with qi deficiency and blood stasis syndrome were observed by detecting these indexes.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to detect the change of protein expression in brain tissue of rats in the control group,model group,and high-dose group of Jiedu Tongluo group,GO function annotation and KEGG analysis were performed.HE stainning and electron microscopy were used to observe the morphology of rat hippocampal cells.Immunohistochemical staining and Western blot were used to detect the expression of NMDAR2B and PSD95 proteins.The protein and mRNA expression of CaMKIV,CREB,BDNF and TrkB were detected by Western blot and qPCR.(2)Clinical sample:Five patients with effective treatment were selected and total RNA of venous blood nucleated cells before and after treatment was extracted.Real-time Quantitative PCR array gene chip was used for miRNA detection,and the differential genes obtained were clustered and analyzed.GO function annotation and KEGG pathway analysis.Results:(1)Behavioral results:Compared with the control group,the rats in the model group had reduced body weight,decreased horizontal activity and upright activity in the open field,reduced sugar consumption,increased tail-sustained time,increased neurological deficits,and reduced RGB value of the tongue surface.Pulse amplitude decreased(P<0.001 or P<0.05).Compared with the model group,Jiedu Tongluo granules can increase the body weight of rats,increase the level of activity and upright activities in the open field,increase the sugar consumption rate,shorten the tail suspension time,improve the neurological deficit,increase the RGB value of the tongue and pulse amplitude(P<0.01 or P<0.05).(2)Proteomics analysis:The results of proteomics showed that 3237 proteins were detected in the control group,the model group,and the high-dose of Jiedu Tongluo group,861 kinds of proteins with a 1.5-fold difference in folds.The GO annotations were performed on three aspects of differential proteins from molecular functions,biological pathways,and protein types.The results showed that the effect of Jiedu Tongluo granules on neuroprotection mainly involved eight kinds of molecular functions,such as signal transduction activity,receptor activity,transport activity,antioxidant activity,etc.23 types of protein molecules such as skeleton protein,transporter,cell adhesion molecule cytoskeleton proteins etc..KEGG signaling pathway analysis revealed that the differential proteins are mainly enriched in metabolic pathways related to neurotransmitters,neurotrophins,and signal transduction,among which glutamatergic synapses,gamma-aminobutyric acid synapses,and neurotrophin pathways MAPK signaling pathway,cAMP signaling pathway and other pathways may be related to the therapeutic effect of Jiedu Tongluo Granules on post-cerebral infarction depression.(3)Pathological and ultrastructural observation of brain tissue:Through HE staining and electron microscopy observation of the brain tissue,the CA3 neurons of the control group were normal in morphology and structural integrity;the nucleus under the electron microscope was round or oval and the chromatin was nuclear.Uniform distribution,clear and complete mitochondria.In the model group,HE stainning showed the intercellular space in the CA3 region of the hippocampus increased,the number of cells decreased,the number of cells decreased,and the phenomenon of degeneration and necrosis appeared;the nuclear condensation,chromatin marginal aggregation,mitochondrial deformation and swelling,and sacral rupture were observed under the electron microscope.Jiedu Tongluo Granules and fluoxetine can improve the structural damage of neurons in the hippocampus.(4)Glu、GABA test:Compared with the control group,the Glu level in the brain tissue of the model group was significantly increased(P<0.01),and the GABA level was significantly decreased(P<0.05).After 4 weeks of treatments,compared with the model group,the levels of Glu in the low-dose and high-dose of Jiedu Tongluo groups and fluoxetine group were significantly decreased(all P<0.05),and GABA levels were significantly increased(all P<0.05).(5)Immunohistochemistry:Compared with the control group,the positive expression of NMDAR2B in the model group was significantly increased(p<0.05),and the positive expression of PSD95 was significantly reduced and loosely arranged(P<0.05).Compared with the model group,the positive expression of NMDAR2B in the low-dose and high-dose of Jiedu Tongluo groups and the fluoxetine group were significantly reduced(all P<0.05),and the positive expression of PSD95 was significantly increased(all P<0.05).(6)WB results:Compared with the control group,the expression level of NMDAR2B in the model group was significantly higher(P<0.05),and the expression levels of PSD95,CaMKⅣ,CREB,BDNF and TrkB were significantly decreased(P<0.05);Compared with the model group,the expression of NMDAR2B in Jiedu Tongluo groups and the fluoxetine group were significantly lower(all P<0.05);the expression levels of PSD95,CaMKⅣ⑧ CREB,BDNF and TrkB were increased in the Jiedu Tongluo high dose group and fluoxetine group(all P<0.05).The expression levels of PSD95,CaMKIV,BDNF,and TrkB in Jiedu Tongluo low-dose group had a significant increase(all P<0.05).(7)qPCR results:Compared with the control group,the expression levels of CaMKⅣ,CREB,BDNF and TrkB mRNA were significantly reduced in the model group(p<0.05 or p<0.01);compared with the model group,the expression of CaMKⅣ,CREB,BDNF and TrkB mRNA in the low-dose and high-dose Jiedu Tongluo groups and the fluoxetine group were significantly increased(p<0.05 or p<0.01).(8)miRNA expression microarray expression:Microarray analysis showed that a total of 18 miRNAs with higher difference were identified,including 11 down-regulated genes,7 up-regulated genes,miR-181d-5p,miR-502-3p and hsa-miR-550a-5p are identified with more target genes.Based on the KEGG pathway analysis of these target genes,it was found that the targets of differential genes were mainly concentrated on signaling pathways such as glutamatergic synapses,GABAergic synapses,serotonin synapses,MAPK signaling pathways,and cGMP-PKG signaling pathways.These pathways are mainly related to neural plasticity and cell adhesion,cell proliferation and apoptosis and other functions.Conclusion:(1)Multiple cerebral infarction combined with sleep deprivation can successfully replicate the model of post-stroke depression with qi deficiency and blood stasis syndrome.It manifests as weight loss,reduced open field activities,lack of anhedonia,prolonged tail suspension time,and reduced RGB tongue value,lower pulse amplitude.etc..(2)Jiedu Tongluo granules can improve neurobehavior of rats,via increasing body weight,sugar consumption rate and open field activity,reducing immobility time,improving tongue RGB,increasing pulse amplitude,and promoting nerve function recovery,which has a certain therapeutic effect.(3)Jiedu Tongluo Granules can regulate Glu and GABA levels in brain tissue,and activate NMDAR2B downstream CaMKⅣ/CREB/BDNF pathway,thereby inhibiting excitatory neurotoxicity after cerebral ischemia and exerting neuroprotective effects.(4)Animal proteomics and clinical miRNA chip detection,from the overall perspective to verify the neuroprotective mechanism of Jiedu Tongluo granule,which may be associated with multiple pathways.