| Major depressive disorder(MDD)is the fourth most disabling disease worldwide,characterized by high prevalence,high relapse rates,high suicide mortality,high disability and high symptom heterogeneity,while the treatment process lacks specificity and has low cure rates.It is projected that MDD will be the leading cause of the global burden of disease by 2030.With the development of psychiatry,research on the mechanisms and treatment of MDD has evolved considerably,but still less than one-third of patients benefit from their first drug treatment.The reasons for this may be related to the unknown mechanism of MDD and the lack of individualization in treatment.Therefore,it is important to improve the research on the pathological mechanism of MDD and to find individualized and precise treatment plans.The γ-aminobutyric acidergic system may be involved in the pathogenesis of MDD,but most of the current studies have focused on its metabolism or receptor function,and little research has been done on its transporter.The role of hippocampal neuronal plasticity in the pathogenesis of MDD has attracted increasing attention,and it is believed that reduced hippocampal neuronal plasticity leading to impaired hippocampal structure and function is the main pathological feature of MDD.Study of syndromes of traditional Chinese medicine(TCM)in order to realize individualized precision medical treatment improved the potential clinical Chinese medicine study found that the main TCM syndrome types of MDD is:liver-stagnation and spleen-deficiency syndrome,heart and spleen deficiency syndrome,qi-stagnation and blood-stasis syndrome,liver-depression and qi-stagnation syndrome,heart-kidney disconnection syndrome and liver-depression and phlegm obstruction syndrome,among which liver-stagnation and spleen-deficiency syndrome appears most frequently.In recent years,the medical profession has conducted multi-system,multi-level and multi-indicator studies on liver-stagnation and spleen-deficiency syndrome from neurological,endocrine and immunological aspects,and there are not many studies on liver-stagnation and spleen-deficiency syndrome in MDD.It has been found that the clinical efficacy of prolotherapy is significant in liver-stagnation and spleen-deficiency symptoms of multiple diseases in multiple systems,and it can also achieve antidepressant effects by protecting and promoting hippocampal growth and improving hippocampal structure and function,and its therapeutic mechanism may be multi-target and multi-level.The establishment of animal models of disease with surface validity,structural validity and predictive validity is the guarantee of scientific and effective study of disease.The theory of "environment and genetics" is more consistent with the pathogenesis of the disease,i.e.,stress and fragile physiological functions act synergistically to cause liver stagnation and spleen deficiency in MDD,but there are few studies,especially those involving y-aminobutyric acid transporter 2,which have not been reported at home and abroad.In order to explore the influence of y-aminobutyric acid transporter 2 in the pathogenesis of MDD in Chinese medicine,our group used y-aminobutyric acid transporter 2 gene(slc6al3)knockout mice(KO)as the study object,and analyzed their physiological characteristics and the effect of stress modeling in comparison with homologous C57BL6 wild type(WT);using citalopram as a positive control drug,we studied the therapeutic effect and the effect of stress modeling in in vitro and ex vivo experiments.The therapeutic effects and mechanisms of prolotherapy were investigated;the significance of plasma characteristic indexes in patients with liver-stagnation and spleen-deficiency symptom of MDD was explored using healthy controls and patients with other TCM symptoms of MDD as controls.The five main parts of the study include the following.Part Ⅰ.Effects of slc6a13 gene on behavioral and physiological functions in miceOBJECTIVE:To explore the differences in epigenetic behavior plasma monoamine neurotransmitters,hippocampal neuronal PI3k/Akt/mTOR and hippocampal neuronal synaptic plasticity between y-aminobutyric acid transporter 2 gene(slc6a13)knockout(KO)mice and wild-type(WT)mice,and to gain a preliminary understanding of the effects of slc6a13 gene on mice.METHODS:Ten homozygous KO and WT mice,male and female,were observed and compared in the open field test(OFT),sucrose preference test(SPT),tail suspension test(TST),forced swimming test(FST),feeding and self-grooming behavioral activities;ELISA was used to detect BDNF,Pro-BDNF,5-HT,DA and NE in the peripheral plasma of both groups of mice concentrations in the peripheral plasma of the two groups of mice;IHC method to qualitatively analyze the expression of target proteins in hippocampal tissues;qPCR method to detect differences in mRNA expression of target proteins.RESULTS:Compared with WT mice,KO mice showed no statistically significant differences in OFT,SPT,TST,FST,feeding and self-grooming behavioral epistasis(P>0.05);KO mice showed no statistically significant differences in plasma BDNF,pro-BDNF,5-HT,DA and NE concentrations compared with WT mice(P>0.05);hippocampal neuronal PI3k/Akt/mTOR signaling pathway indicators,the expression of Akt in KO mice was lower than that in WT mice(P=0.03),and the differences in the expression of PI3k and mTOR between the two groups of mice were not statistically significant(P>0.05);the expression of MAP-2 and SYP in KO mice was lower than that in WT mice(P=0.03),while the expression of GAP-43 was higher than that in WT mice(P=0.03)CONCLUSION:slc6a 13 knockout mice did not differ significantly from wild-type mice in apparent behavior,plasma neurotrophic factor and its precursors,and plasma monoamine neurotransmitters,but had an effect on protein expression of the PI3k/Akt/mTOR signaling pathway in hippocampal neurons and synaptic plasticity-related protein expression,which may be a manifestation of their physiological vulnerability.Part Ⅱ Establishment and evaluation of slc6a13-/-mouse model of MDD with liver stagnation and spleen deficiency symptomOBJECTIVE:To establish and evaluate the effect of slc6a13-/-mice to create a disease model of liver stagnation and spleen deficiency symptom of MDD.METHODS:Using WT mice as controls,chronic stress was administered to slc6a13-/-mice to assess the model effects in terms of OFT,SPT,TST,FWT,food intake and self-grooming behavior,respectively,and to analyze the changes of BDNF,Pro-BDNF,5-HT,DA and NE concentrations in the plasma peripheral plasma of model mice,and the effects of modeling on mouse hippocampal neurons PI3k/Akt/mTOR signaling pathway indicators and neuroplasticity indicators MAP-2,GAP-43 and SYP expression.RESULTS:At the end of modelling,in OFT,the total distance travelled was significantly lower in the KO-D group than in the WT-C and WT-D groups(P<0.01),the activity in both WT-D and KO-D groups was significantly lower than in the WT-C group(P<0.01),and the central zone time was significantly lower in both WT-D and KO-D groups(P<0.01);in SPT,WT-D(P<0.05)and KO-D(P<0.01)groups both had significantly lower sucrose preference than the WT-C group;in the TST,the KO-D group had significantly higher hanging tail immobility time than the WT-C group(P<0.05),and in the FST,both the WT-D and KO-D groups had significantly higher forced swimming immobility time than the WT-C group(P<0.01);both the WT-D and KO-D groups had significantly lower body weight than the WT-C group(P<0.01),daily food intake was significantly lower in both the WT-D and KO-D groups than in the WT-C group(P<0.05),and grooming time was significantly lower in both the WT-D and(P<0.05)KO-D(P<0.01)groups than in the WT-C group.At 7 days post-modeling,in OFT,total distance was significantly lower in the KO-D group than in the WT-C(P<0.05)and WT-D groups(P<0.01),activity was significantly lower in both WT-D and KO-D groups(P<0.05),and central zone time was significantly lower in the KO-D group than in the WT-C(P<0.01)and WT-D groups(P<0.05);in SPT,the KO-D group had significantly lower sucrose preference than the WT-C and WT-D groups(P<0.01);in the TST,the KO-D group had a significantly higher time of hanging tail immobility than the WT-C group(P<0.05);in the FST,the KO-D group had a significantly higher time of forced swimming immobility than the WT-C and WT-D groups(P<0.01);and both the WT-D and KO-D groups had significantly lower body weight than the WT-C group(P<0.01),daily food intake in the KO-D group was significantly lower than that in both WT-C and WT-D groups(P<0.01),and grooming time in the KO-D group was significantly lower than that in both WT-C and WT-D groups(P<0.05).Plasma pro-BDNF was significantly lower in the KO-D group than in both the WT-C and WT-D groups(P<0.01),mRNA of PI3k was significantly lower in both the WT-D and KO-D groups than in the WT-C group(P<0.01),mRNA of Akt was significantly lower in both the WT-D(P<0.05)and KO-D(P<0.01)groups than in the WT-C group,and mRNA of Akt was significantly lower in both the WT-D and KO-D groups both had significantly lower mRNA for mTOR than WT-C group(P<0.01),both WT-D and KO-D groups had significantly lower mRNA for MAP-2 than WT-C group(P<0.01),KO-D group had significantly higher mRNA for GAP-43 than WT-C(P<0.01)and WT-D(P<0.05)groups,WT-D and KO-D groups The mRNA of SYP was significantly lower than that of the WT-C group in both groups(P<0.01).CONCLUSION:slc6a13-/-mice create a more stable and reliable disease model of MDD with liver stagnation and spleen deficiency symptom,and are a better model of chronic stress with liver stagnation and spleen deficiency symptom;the greater degree of changes in protein and plasticity-related indicators in the PI3K/AKT/mTOR signaling pathway in hippocampal neurons of KO mice suggests that these changes may be related to the pathological mechanism of disease development.Part Ⅲ Study on the effect and mechanism of prolotherapy on slc6al3-/-MDD in mice with liver stagnation and spleen deficiency symptom modelOBJECTIVE:To study the effect and mechanism of prolotherapy intervention in a mouse model of MDD with liver-stagnation and spleen-deficiency symptom.METHODS:The slc6al3-/-mouse model of MDD with liver-stagnation and spleen-deficiency symptom was studied,and citalopram hydrobromide was used as a positive control drug to compare and analyze the effects of different concentrations of prozac.The treatment effects were assessed in terms of characteristic behaviors and peripheral plasma indices of MDD and liver stagnation and spleen deficiency,respectively,and the mechanisms were explored in terms of PI3k/Akt/mTOR signaling pathway and hippocampal synaptic plasticity.RESULTS:At 7 d of gavage,in OFT,the total distance traveled was significantly higher in the CH,XS-M and XS-H groups than in the PW group(P<0.01),as shown by significantly higher activity in the XS-H group than in the PW group(P<0.05)and significantly higher central zone time in the CH,XS-L,XS-M and XS-H groups than in the PW group(P<0.01);in SPT,the XS-M and XS-H groups sucrose preference were significantly higher than those of the PW group(P<0.01),and in the TST,the time to immobility of the hanging tail was significantly lower in the XS-H group than in the PW group(P<0.05).At 14 d of gavage,in OFT,the total distance was significantly higher in the CH,XS-M and XS-H groups than in the PW group(P<0.01),the activity was significantly higher in the CH,XS-M(P<0.05)and XS-H groups(P<0.01)than in the PW group,and the central zone time was significantly higher in the CH,XS-M and XS-H groups than in the PW group(P<0.01);in SPT,CH,XS-M and XS-H groups all had significantly higher sucrose preference than the PW group(P<0.01);in TST,the time of hanging tail immobility was significantly lower in the CH,XS-M and XS-L groups than the PW group(P<0.05);the time of forced swimming immobility was significantly lower in the CH,XS-M and XS-H groups than the PW group(P<0.01);the body weight was significantly higher in the XS-H group than the PW group(P<0.05),food intake was significantly higher in both the CH and XS-H groups than in the PW group(P<0.01);grooming time was significantly higher in the CH,XS-M and XS-H groups than in the PW group(P<0.01).Plasma BDNF was significantly higher in the CH,XS-L,XS-M and XS-H groups than in the PW group(P<0.01),and plasma pro-BDNF concentrations were significantly higher in the XS-M and XS-H groups than in the PW group(P<0.01);MAP-2 expression was significantly higher in the CH(P<0.01)and XS-H(P<0.05)groups than in the PW group,and CH(P<0.05)and XS-H(P<0.(01)The expression of SYP in all groups was significantly higher than that in the PW group.CONCLUSION:High concentration of pro-BDNF had the same effect as citalopram hydrobromide to improve the apparent behavior in the mouse model of MDD with liver stagnation and spleen deficiency symptom,and citalopram hydrobromide and low/medium/high concentration of pro-BDNF significantly increased the plasma BDNF concentration in mice,and medium/high concentration of pro-BDNF significantly increased the plasma pro-BDNF concentration,and high concentration of pro-BDNF had the same effect as citalopram hydrobromide to increase hippocampal neuronal synaptic plasticity-associated protein MAP-2 and SYP expression,and did not show a similar effect on GAP-43.Part Ⅳ Study on the effect of prolotherapy-containing serum on hippocampal neurons in mice with liver stagnation and spleen deficiency in MDDOBJECTIVE:To study the effect of prolotherapy-containing serum on hippocampal neurons of depressed mice with liver-stagnation and spleen-deficiency symptom in vitro culture.METHODS:The hippocampal neurons of the model mice were isolated and cultured with the sera prepared in the pure water(PW),citalopram hydrobromide(CH)and prolotherapy(XS)groups,respectively,and the growth of hippocampal neurons was identified by immunofluorescence,and the expression of PI3k/Akt/mTOR signaling pathway and hippocampal synapses were detected by Western Blot.expression of plasticity-related proteins.RESULTS:PW serum culture resulted in significantly higher protein expression of PI3k in the KO-D group than in the WT-C(P<0.01)and KO-C groups(P<0.01),CH serum culture resulted in significantly higher protein expression of PI3k in the WT-C and KO-D groups than in the KO-C group(P<0.01),XS serum culture resulted in significantly higher protein expression of PI3k in the KO-C and KO-D groups than in the WT-C group(P<0.01),and XS serum culture resulted in significantly higher than the WT-C group(P<0.01).PW serum culture resulted in significantly higher protein expression of Akt than WT-C(P<0.05)and KO-D groups(P<0.01),CH serum culture resulted in significantly lower protein expression of PI3k in KO-C(P<0.01)and KO-D groups(P<0.01),XS serum culture resulted in significantly higher protein expression of PI3k in KO-C and KO-D groups higher than that of the WT-C group(P<0.01).CH serum culture resulted in significantly higher mTOR protein expression in the KO-D group than in the WT-C(P<0.05)and KO-C groups(P<0.01),and XS serum culture resulted in significantly higher mTOR protein expression in the KO-C(P<0.05)and KO-D(P<0.01)groups than in the WT-C group.PW serum culture resulted in significantly higher MAP-2 protein expression in WT-C and KO-C groups than in KO-D group(P<0.01),CH serum culture resulted in significantly higher MAP-2 protein expression in WT-C and KO-C groups than in KO-D group(P<0.01),WT-C group significantly higher than in KO-D group(P<0.01),XS serum culture resulted in significantly higher WT-C and KO-C groups with significantly higher MAP-2 protein expression than the KO-D group(P<0.01).PW serum culture resulted in significantly lower GAP-43 protein expression in WT-C and KO-C groups than in KO-D group(P<0.01),CH serum culture resulted in significantly lower GAP-43 protein expression in WT-C and KO-C groups than in KO-D group(P<0.01),KO-C group significantly lower than in WT-C group(P<0.01),XS serum culture resulted in significantly lower GAP-43 protein expression in WT-C and KO-C groups with significantly lower GAP-43 protein expression than the KO-D group(P<0.01)PW serum culture resulted in significantly higher SYP protein expression in WT-C and KO-C groups than in KO-D group(P<0.01),significantly higher in KO-C group than in WT-C group(P<0.05),CH serum culture resulted in significantly higher SYP protein expression in WT-C and KO-C groups than in KO-D group(P<0.01),significantly higher in KO-C group than in WT-C group(P<0.05),XS serum culture resulted in significantly higher SYP protein expression in WT-C and KO-C groups than in KO-D group(P<0.01),and in KO-C group than in WT-C group(P<0.01).Conclusion:CS significantly decreased the relative expression of MAP-2 and SYP in slc6a13-/-depressed mice with liver stagnation and spleen deficiency symptom,while the opposite was true for GAP-43;prolotherapy-containing serum and citalopram hydrobromide containing serum increased the relative expression of PI3k,Akt and mTOR proteins in hippocampal neurons of depressed mice with liver stagnation and spleen deficiency symptom;prolotherapy-containing serum and citalopram hydrobromide containing serum increased the relative expression of PI3k,Akt and mTOR proteins in hippocampal neurons of depressed mice with liver stagnation and spleen deficiency symptom.The relative expressions of MAP-2 and SYP in hippocampal neurons of mice with depressive liver-stagnation and spleen-deficiency symptoms were increased by prolotherapy serum and citalopram hydrobromide serum,but the effect of prolotherapy serum was weaker than that of citalopram hydrobromide serum in increasing the expression of SYP.The relative expression of GAP-43 in hippocampal neurons of depressed mice with liver-stagnation and spleen-deficiency symptom was reduced by both prolotherapy-containing serum and citalopram hydrobromide containing serum.Part V.Blood indicators and the value of GAT-2mRNA in MDD with liver-stagnation and spleen-deficiency symptomOBJECTIVE:To explore the diagnostic value of characteristic indicators and GAT-2mRNA in peripheral blood of depressed patients with liver-stagnation and spleen-deficiency symptom.METHODS:ELISA,biochemical assay and q-PCR were used to detect the expression of BDNF,pro-BDNF,5-HT,DA,NE,GABA,GAD65 and GAD67,GLU concentration and SLC6A13 mRNA in plasma.RESULTS:The differences between the three groups of subjects in terms of gender composition(x2=0.95,P=0.10)and age group(F=1.52,P=0.23)were not statistically significant,and the between-group differences in HAMD scores were statistically significant(F=53.33,P=0.00),as demonstrated by the significantly higher scores in both the D-OS and D-LSSD groups than in the HC group(P<0.01),while the D-OS and D-LSSD groups were not statistically significant(P>0.05);the difference between groups in BDNF plasma concentration was statistically significant(F= 168.76,P=0.00),showing that plasma BDNF concentration was significantly lower in both the D-OS and D-LSSD groups than in the HC group(P<0.01),and significantly lower in the D-LSSD group than in the D-OS group(P<0.01).The difference between groups in por-BDNF plasma concentrations was statistically significant(F=21.35,P=0.00),as demonstrated by the fact that plasma pro-BDNF concentrations were significantly lower in both the D-OS and D-LSSD groups than in the HC group(P<0.01),with no statistically significant difference between the D-OS and D-LSSD groups(P>0.05);the difference between groups in 5-HT plasma concentrations was statistically significant(F=195.07,P=0.00),as shown by the plasma 5-HT concentrations in both the D-OS and D-LSSD groups were significantly lower than those in the HC group(P<0.01),and the D-LSSD group was significantly lower than the D-OS group(P<0.01);the difference between groups in DA plasma concentrations was statistically significant(F=353.07,P=0.00),as shown by the D-OS plasma DA concentrations were significantly lower in both the D-OS and D-LSSD groups than in the HC group(P<0.01),and significantly lower in the D-LSSD group than in the D-OS group(P<0.01);the dif-ference between groups in NE plasma concentrations was statistically significant(F=108.76,P=0.00),showing that plasma NE concentrations were significantly lower in both the D-OS and D-LSSD groups than in the HC group(P<0.01),and The D-LSSD group was significantly lower than the D-OS group(P<0.01);the difference between groups in Glu plasma concentrations was statistically significant(F=4.20,P=0.00),as demonstrated by the plasma Glu concentrations in both the D-OS and D-LSSD groups being significantly lower than those in the HC group(P<0.01)and the D-LSSD group being significantly lower than those in the D-OS group(P<0.01);the GABA plasma concentrations were statistically significant between groups(F=62.32,P=0.00),showing that plasma GABA concentrations were significantly higher in both the D-OS and D-LSSD groups than in the HC group(P<0.01)and in the D-LSSD group than in the D-OS group(P<0.01);GAD65 plasma concentrations were statistically significant between groups(F=222.34,P=0.00),showing that plasma GAD65 concentrations were significantly higher in both the D-OS and D-LSSD groups than in the HC group(P<0.01)and in the D-LSSD group than in the D-OS group(P<0.01);the difference between groups in GAD67 plasma concentrations was not statistically significant(F=0.34,P=0.71);the difference between groups in relative SLC6A13 mRNA plasma expression was statistically significant(F=191.47,P=0.00),as shown by plasma SLC6A13mRNA expression in both the D-OS and D-LSSD groups was significantly lower than that in the HC group(P<0.01),and in the D-LSSD group was significantly lower than that in the D-OS group(P<0.01);in plasma samples from patients with other TCM symptom of MDD and patients with liver stagnation and spleen deficiency symptom of MDD Area under the curve(area under the curve,AUC)of relative expression of SLC6A13 mRNA:0.97;Cut-off value:29.06;Sensitivity:96.7%;Specificity:90.0%;Jorden index:86.7%.CONCLUSION:Plasma BDNF,monoamine neurotransmitters,Glu,GABA and GAD65 may be sensitive indicators of liver stagnation and spleen deficiency symptom in MDD,while SLC6A13 mRNA is a characteristic indicator. |
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