Discussion On Mechanism Of Kidney-tonifying Therapy In Anti-LOD Based On "Folate-Transport Of Choroid Plexus To Hippocampal Neurogenesis"

ObjectiveBy establishing a rat model of late-onset depression(LOD),this study aimed to explore the anti-LOD effect and underlying neuropharmacological mechanisms of Erxian Decoction,a Kidney-tonifying prescription in Chinese medicine,from the perspective of"choroid plexus(CP)folate transport-hippocampal neurogenesis".Meanwhile,this study aimed to enrich and innovate the theory of "Kidney-brain system" in Chinese medicine,and further to provide a scientific basis for the clinical application of the Kidney-tonifying therapy for the prevention and treatment of senile mental diseases.Methods1.Establishment of the rat model of depressionChronic unpredictable mild stress(CUMS)was used to establish the depression rat model.The LOD model was established using natural aging combined with CUMS.Rats were exposed to CUMS for 6 consecutive weeks.Stressors included food deprivation(12 h),water deprivation(12 h),continuous lighting at night(20:00-8:00 the next day,12 h),plantar electric shock(1 mA,2 s,every 30 s interval,10 electric shocks in total),white noise(85 dB,5 h),stroboscopic illumination(300 flashes/minute,5 h)thermal swimming(45℃,5 min),restraint(12 h),soiled cage(10 h),paired with four other stressed animals(10 h),food and water deprivation(24 h).Combined solitary-housed,rats were randomly exposed to 1-2 of these stimuli once a day for 6 weeks and the same stressors were not scheduled in three consecutive days.Natural aging rats were 20-month-old male Wistar rats.2.To explore the effects of Erxian Decoction on depressive symptoms and cognitive functions in LOD rats40 male Wistar rats aged 7-8 weeks were randomly divided into the youth normal group(Control)and youth depression group(CUMS).80 male Wistar rats aged 20 months were randomly divided into the naturally aged group(AGED),late-onset depression group(LOD),Erxian Decoction treatment group(EXD)and fluoxetine combined with folate treatment group(FLX+FOL).All rats received CUMS for 6 weeks except for the CON and AGED group.The EXD group was given Erxian Decoction(8 g/kg/d),the FLX+FOL group was given fluoxetine combined with folic acid(fluoxetine 10 mg/kg/d,folic acid 1.8 mg/kg/d),and the other groups were given an equal amount of pure water orally.After modeling,behavioral tests were performed to detect depressive symptoms and cognitive functions,including sucrose preference test used for anhedonia,open-field test for autonomous activity,forced swimming test for despair,T maze test for the cognitive ability of rats.3.To explore the effects of Erxian Decoction on against hippocampal neuron damage and neurogenesis dysfunction in LOD ratsWestern-blot was used to detect the protein expression of Nestin,doublecortin(DCX),and neuronal nuclei antigen(NeuN).Immunofluorescence was used to detect the number of Ki-67/Nestin double-positive cells and 5-bromodeoxyuridine(5-Bromo-2-deoxyUridine,BrdU)/DCX double-positive cells.4.To observe the effects of cerebrospinal fluid(CSF)of each group on the proliferation and differentiation of primary hippocampal neural stem cellsIn vitro,the high-corticosterone(CORT)concentration model was used to simulate the stress-induced high-corticosterone concentration damage in the depression model in vivo;the D-galactose(D-gal)aging model was used to simulate the changes related to aging;high-CORT concentration combined with D-gal model was used to simulates the changes of LOD-related stress and aging.(1)To observe the effect of CSF on the proliferation of neural stem cells(CCK8).The cells were divided into four groups:normal hippocampal neural stem cell group(7 subgroups including Group A,B,C,D,E,F,and G);hippocampal neural stem cell high-CORT concentration model group(2 subgroups including Group H and I);hippocampal neural stem cell D-gal model group(2 subgroups including Group J and K);neural stem cell high-CORT concentration combined with D-gal model group(4 subgroups including Group L,M,N,and O),a total of 14 subgroups.The specific treatment for each subgroup is as follows:Group A:cultured with neural stem cell growth medium for 72 h;Group B:cultured with neural stem cell growth medium containing 10%normal rat CSF for 72 h;Group C:cultured with neural stem cell growth medium containing 10%CUMS-CSF for 72 h;Group D:cultured with neural stem cell growth medium containing 10%AGED-CSF for 72 h;Group E:cultured with neural stem cell growth medium containing 10%LOD-CSF for 72 h;Group F:cultured with neural stem cell growth medium containing 10%EXD-CSF for 72 h;Group G:cultured with neural stem cell growth medium containing 10%FLX+FOL-CSF for 72 h.Group H:cultured with neural stem cell growth medium containing 120 μM CORT for 72 h;Group I:cultured with neural stem cell growth medium containing 10%CUMS-CSF+120 μM CORT for 72 h.Group J:cultured with neural stem cell growth medium containing 40 mg/mL D-gal for 72 h;Group K:cultured with neural stem cell growth medium containing 10%AGED-CSF+40 mg/mL D-gal for 72 h.Group L:cultured with neural stem cell growth medium containing 120 μM CORT+40 mg/mL D-gal for 72 h;Group M:cultured with neural stem cell growth medium containing 10%LOD-CSF+120 μM CORT+40 mg/mL D-gal for 72 h;Group N:cultured with neural stem cell growth medium containing 10%EXD-CSF+120 μM CORT+40 mg/mL D-gal for 72 h;Group O:cultured with neural stem cell growth medium containing 10%FLX+FOL-CSF+120 μM CORT+40 mg/mL D-gal for 72 h.(2)To observe the effect of CSF on the neuron directional differentiation of neural stem cells(the ratio of BrdU/DCX double positive cells detected by immunofluorescence).The cells were divided into four groups:normal hippocampal neural stem cell group(7 subgroups including Group A,B,C,D,E,F,and G);hippocampal neural stem cell high-CORT concentration model group(2 subgroups including Group H and I);hippocampal neural stem cell D-gal model group(2 subgroups including Group J and K);neural stem cell high-CORT concentration combined with D-gal model group(4 subgroups including Group L,M,N,and O),a total of 14 subgroups.The specific treatment for each subgroup is as follows:Group A:treated with neural stem cell differentiation medium;Group B:treated with neural stem cell differentiation medium containing 10%normal rat CSF;Group C:treated with neural stem cell differentiation medium containing 10%CUMS-CSF;Group D:treated with neural stem cell differentiation medium containing 10%AGED-CSF;Group E:treated with neural stem cell differentiation medium containing 10%LOD-CSF;Group F:treated with neural stem cell differentiation medium containing 10%EXD-CSF;Group G:treated with neural stem cell differentiation medium containing 10%FLX+FOL-CSF.Group H:treated with neural stem cell differentiation medium containing 120 μM CORT;Group I:treated with neural stem cell differentiation medium containing 10%CUMS-CSF+120 μM CORT.Group J:treated with neural stem cell differentiation medium containing 40 mg/mL D-gal for;Group K:treated with neural stem cell differentiation medium containing 10%AGED-CSF+40 mg/mL D-gal.Group L:treated with neural stem cell differentiation medium containing 120μM CORT+40 mg/mL D-gal for;Group M:treated with neural stem cell differentiation medium containing 10%LOD-CSF+120 μM CORT+40 mg/mL D-gal;Group N:treated with neural stem cell differentiation medium containing 10%EXD-CSF+120 μM CORT+40 mg/mL D-gal;Group O:treated with neural stem cell differentiation medium containing 10%FLX+FOL-CSF+120 μM CORT+40 mg/mL D-gal.5.To explore the effects of Erxian Decoction on the content of 5-Methyltetrahydrofolate(5-MTHF)in plasma,CSF and hippocampus of LOD ratsELISA was used to detect the 5-MTHF content in plasma,CSF and hippocampus tissue of rats in each group.Pearson’s correlation analysis was used to determine the correlation among 5-MTHF content in plasma,5-MTHF content in CSF,and 5-MTHF content in the hippocampus;the correlation between 5-MTHF content in CSF and the content of Nestin,DCX,NeuN protein in hippocampal tissue;and the correlation between 5-MTHF content in the hippocampus and the content of Nestin,DCX,NeuN protein in the hippocampus.6.To explore the effects of Erxian Decoction on the expression of CP folate transporter and tight junction protein in LOD rats(1)In vivo,western-blot and immunofluorescence were used to detect the protein expression of zonula occluden-1(ZO-1),folate receptor α(FRα),reduced folate carrier(RFC),and proton-coupled folate transporter(PCFT)in CP tissues.Pearson’s correlation analysis was used to determine the correlation between 5-MTHF content in CSF and FRα,RFC,PCFT protein expression in CP tissues,and the correlation between 5-MTHF content in CSF and ZO-1 protein content in CP tissues.Results(2)In vitro,the high-CORT concentration model was used to simulate the CORT damage of choroid plexus epithelial cells in depression.The D-gal aging model was used to simulate the choroid plexus epithelial cells of natural aging rats.The high-CORT concentration combined with D-gal model was used to simulate the cellular state of choroid plexus epithelial cells in LOD rats.Primary choroid plexus epithelial cells were divided into 7 groups:① control group(Control):treated with choroid plexus epithelial cell growth medium for 72 h;②high-CORT concentration model group(CORT):treated with choroid plexus epithelial cell growth culture medium containing 120 μM CORT for 72 h;③ D-gal aging model group(D-gal):treated with choroid plexus epithelial cell growth medium containing 40 mg/mL D-gal for 72 h;④ high-CORT concentration combined with D-gal model group(CORT+D-gal):treated with choroid plexus epithelial cell growth culture medium containing 120 μM CORT+40 mg/mL D-gal for 72 h;⑤ low-dose Erxian Decoction lyophilized powder group(EXDL):treated with choroid plexus epithelial cell growth culture medium containing 120 μM CORT+40 mg/mL D-gal+500mg/mL Erxian Decoction lyophilized powder for 72 h;⑥high-dose Erxian Decoction lyophilized powder group(EXDH):treated with choroid plexus epithelial cell growth culture medium containing 120 μM CORT+40 mg/mL D-gal+1000mg/mL Erxian Decoction lyophilized powder for 72 h;⑦ fluoxetine+folate group(FLX+FOL):treated with choroid plexus epithelial cell growth culture medium containing 120 μM CORT+40 mg/mL D-gal+55 μM fluoxetine+40 μg/mL folate for 72 h.Western-blot and immunofluorescence were used to detect the protein expression of ZO-1,FRα,RFC and PCFT,and qPCR was used to detect the expression of FR a,RFC and PCFT mRNA.The trans-epithelial electrical resistance of choroid plexus epithelial cells in each group was measured.Results1.Erxian Decoction relieves depressive symptoms and cognitive impairment in LOD ratsBoth CUMS rats and LOD rats showed depressive symptoms such as anhedonia,decreased spontaneous activity and despair,accompanied by learning and memory impairment.The learning and memory impairment of LOD rats was more significant than that of CUMS rats.Erxian Decoction could alleviate depressive symptoms such as anhedonia,decreased spontaneous activity and despair,as well as learning and memory impairment in LOD rats.2.Erxian Decoction ameliorates hippocampal neuron damage and neurogenesis dysfunction in LOD ratsBoth CUMS rats and LOD rats showed decreased expression of Nestin,DCX,and NeuN proteins in the hippocampus,and decreased Ki-67/Nestin double-positive cells and BrdU/DCX double-positive cells in the DG,especially in LOD rats.Erxian Decoction up-regulated the expression of Nestin,DCX and NeuN proteins in the hippocampus of LOD rats,and increased the number of Ki-67/Nestin double-positive cells and BrdU/DCX double-positive cells in the DG of LOD rats.3.The effect of CSF in each group of rats on the proliferation and differentiation of primary hippocampal neural stem cells(1)The effect of CSF in each group on the proliferation of neural stem cellsCompared with Group A,the proliferation rate of neural stem cells in Groups B,C,D,F,and G increased,while the proliferation rate of neural stem cells in Group E decreased.Compared with Group B,the proliferation rate of neural stem cells in Groups C and D decreased.Compared with Group A,the proliferation rate of neural stem cells in Groups H,J,and L decreased.Compared with Group H,the proliferation rate of neural stem cells in Group I did not change significantly.Compared with Group J,the proliferation rate of neural stem cells in Group K did not change significantly.Compared with Group L,the neural stem cell proliferation rate in Group M decreased significantly,and the proliferation rate in Groups N and O increased.(2)The effect of CSF in each group on the differentiation of neural stem cells into neuronsCompared with Group A,the ratio of BrdU/DCX double-positive cells in Groups B,D,F,and G increased,while that in Group E decreased;the difference between Groups A and C was not significant.Compared with Group B,the proportion of BrdU/DCX double-positive cells in Group C decreased.Compared with Group A,the proportion of BrdU/DCX double-positive cells in Groups H,J,and L decreased.Compared with Group H,the ratio of BrdU/DCX double-positive cells in Group I decreased.Compared with Group J,the ratio of BrdU/DCX double-positive cells in Group K increased.Compared with Group L,the ratio of BrdU/DCX double-positive cells in Group M decreased,while that in Groups N and O increased.4.Erxian Decoction increases the content of 5-MTHF in CSF and hippocampus of LOD rats(1)There was no significant difference in plasma 5-MTHF content of rats in each group.LOD rats showed decreased 5-MTHF content in CSF and hippocampus.The administration of Erxian Decoction reversed the decrease in 5-MTHF content in CSF and hippocampus of LOD rats.(2)The content of 5-MTHF in CSF was correlated with that in the hippocampus.There was a correlation between the 5-MTHF content in CSF and the protein expression of Nestin,DCX and NeuN in the hippocampus.The 5-MTHF content in the hippocampus was correlated with the protein expression of Nestin,DCX,and NeuN in the hippocampus.5.Erxian Decoction improves folate transport of CP and regulates tight junctions in CP(1)In vivo,the expression of FRα,ZO-1 proteins in CP tissues of CUMS rats and AGED rats decreased,and the expression of FRα,RFC,PCFT and ZO-1 proteins in CP tissues of LOD rats decreased.Erxian Decoction up-regulated FRα,RFC,PCFT,ZO-1 proteins in CP tissues of LOD rats.(2)Under the condition of high-CORT concentration combining D-gal,a reduction was observed in the expression of FRα,RFC,PCFT protein and mRNA,and in the expression of ZO-1 protein in primary choroid plexus epithelial cells.The intervention of Erxian Decoction lyophilized powder up-regulated the expression of FRα,RFC,PCFT protein and mRNA in choroid plexus epithelial cells,as well as the expression of ZO-1 protein.(3)The content of 5-MTHF in CSF was correlated with the expression of FRα,RFC,PCFT protein in CP tissues.The content of 5-MTHF in CSF is correlated with the expression of ZO-1 protein in CP tissues.Conclusion1.LOD rats showed significantly decreased spontaneous activity and severe learning and memory impairment,which represents the pathogenesis characteristics of insufficient kidney yang and inadequate marrow sea in Chinese medicine.Erxian Decoction,a representative prescription of the Kidney-tonifying therapy,could significantly ameliorate the behavioral abnormalities and cognitive impairment in LOD rats.2.Changes in CSF composition in LOD rats led to hippocampal neuron damage and neurogenesis dysfunction.Erxian Decoction could attenuate hippocampal damage and promote hippocampal neurogenesis by changing CSF composition in LOD rats.3.The impaired CP structure and dysfunctional folate transport in LOD rats significantly reduced the folate content in CSF,leading to neurogenesis dysfunction and neuron damage.Erxian Decoction could attenuate the structural damage of CP in LOD rats,regulate the expression of CP folate transporter,and increase the folate content in CSF.4.CP secreting CSF and transporting folate is one of the important mechanisms underlying the theory of "the Kidney Essence communicating the brain" in Chinese medicine.Changes in CSF composition may be one of the mediators of the theory of "the Kidney Essence communicating the brain" in Chinese medicine.