Effect And Mechanism Of LW-AFC And Its Active Components On Protecting Corticosterone-induced Long-term Potentiation (LTP) Impairment

Stress can be defined as a brain-body reaction towards stimuli arising from the environment.The response to stress involves the activation of hypothalamic-pituitaryadrenal(HPA)axis and the release of glucocorticoids(in rat,corticosterone;CORT).Moderate stress could contribute to improve the adaptability to various environmental aspects,while excessive stress may cause persistent activation of HPA axis.Excess glucocorticoids,as induced by persistent activation of HPA axis,would lead to lower immune function and increased release of Glu,which would play a toxic role in the central nervous system.Excess glucocorticoids would also lead to hippocampal neuronatrophy,impairment of hippocampal structure,synaptic plasticity and cognitive function.Long-term potentiation(LTP)has been widely used for evaluating synaptic plasticity,both in physiological and pharmacological research.LW-active fraction combination(LW-AFC)is extracted from Liuwei Dihuang decoction(LW),which has been used clinically in the treatment of many types of diseases with “nourishing the kidney-Yin deficiency”.LW-AFC includes three active fractions,namely polysaccharide fraction(LWB-B),glycoside fraction(LWD-b)and oligosaccharide fraction(CA-30).The primary experiments have revealed that LWAFC could ameliorate the impairment of learning and memory and synaptic plasticity induced by acute multiple stress or CORT.Practitioners in our laboratory have made great progress in elaborating the pharmacological effects of LW-AFC on synaptic plasticity and cognitive function using various Alzheimer's disease(AD)mouse models,which was closely related to the Neuroendocrine immunomodulation(NIM)network.Up to now,the effects and mechanism of the three active fractions of LWAFC and its active components underlying the synaptic plasticity still remain unknown.Therefore,this study will focus on the effects and mechanism of the three active fractions of LW-AFC and its active components on synaptic plasticity.Part ?: Effects of LW-AFC and its active components on protecting CORTinduced LTP impairment1.Effects of LW-AFC on protecting CORT-induced LTP impairmentOur results showed that LW-AFC(0.4-3.2g/kg),given by i.g.for seven days,could protect CORT-induced LTP impairment significantly in a dose-dependent manner.LW-AFC in 0.8,1.6 and 3.2g/kg showed positive effects,while LW-AFC in 0.4 g/kg did not.2.Effects of the three active fractions on protecting CORT-induced LTP impairmentTo help clarify,the effects of the three active fractions on CORT-induced LTP impairment were further investigated.The results showed that LWB-B(0.16g/kg)and LWD-b(0.12g/kg),given by i.g.for seven days,could not protect CORT-induced LTP impairment significantly,while CA-30(0.52g/kg)did.However,LWB-B(0.32g/kg),LWD-b(0.24g/kg)and CA-30(1.04g/kg),given by i.g.for seven days,could protect CORT-induced LTP impairment significantly.3.Effects of the active components on protecting CORT-induced LTP impairmentThen the effects of the active components on CORT-induced LTP impairment were further investigated.The results showed that loganin(LOG,50mg/kg),morroniside(MOR,50mg/kg),paeoniflorin(PF,50mg/kg)and stachyose(STA,50 mg/kg),given by i.g.for seven days,could ameliorate CORT-induced LTP impairment significantly.This part of study focused on the effects of LW-AFC,the three active fractions and its active components on CORT-induced LTP impairment.The results suggested that all of them could ameliorate CORT-induced LTP impairment significantly.Part ?: Pathway of LW-AFC and its active components on protecting CORTinduced LTP impairmentThe first part of study had proved that LW-AFC,the three active fractions and its active components could protect CORT-induced LTP impairment significantly.However,whether they will affect the central nervous system directly or indirectly remains unknown.Thus,we investigated the possible different effects of active components through different drug-delivery cycle and ways.1.Influence of different drug-delivery cycle and ways on the effects of drugs on CORT-induced LTP impairmentWe applied three different drug-delivery ways,namely intracerebroventricular injection(i.c.v.),intragastric administration(i.g.)and intraperitoneal injection(i.p.).The results showed that single administration(i.c.v.,i.g.,i.p.)of any of the components had no effect on CORT-induced LTP impairment.Unlike single administration,multiple administration(i.g.or i.p.,seven days)of the active components protected CORT-induced LTP impairment.This indicated that the active components may be unlikely to improve the LTP by a direct effect on the central nervous system(CNS),but an indirect pathway.2.Influence of intestinal microbiota and immune function on the effects of drugs on CORT-induced LTP impairmentPrevious studies have proved that LW-AFC could regulate the immune function.LWB-B is mainly used to improve B lymphocyte function,LWD-b is mainly used to improve T lymphocyte function,while CA-30 is mainly used to improve the intestinal microbiota.Firstly,intragastric administration(i.g.)of STA for seven days protected CORTinduced LTP impairment,while intraperitoneal injection(i.p.)of STA for seven days didn't.This result indicated that STA might display improving effects on synaptic plasticity indirectly through intestinal microbiota.Then we tried to disrupt the intestinal microbiota by oral administration of non-absorbable antibiotics cocktail,wefound that the intestinal microbiota disorder inhibited the effect of STA on LTP.The antibiotics even inhibited the effect of CA-30.The results further proved that STA(including CA-30)might work indirectly on CNS,but through intestinal microbiota.Moreover,we also found that the antibiotics had little influence on LWB-B and LWD-b,suggesting that they might display improving effects through other pathways.Finally,after oral administration of LW-AFC for seven days,together with immunosuppressor cyclophosphamide(CPA),the effects of LW-AFC on CORTinduced LTP impairment was abolished.This indicated that immune function might be the common indirect pathway.In conclusion,this part of study suggested that the active components of LWAFC may be unlikely to improve the LTP by a direct effect on the central nervous system(CNS),but an indirect pathway.CA-30 and STA might display improving effects on synaptic plasticity indirectly through intestinal microbiota.Immune function might be the common indirect pathway.Part ?: Mechanism of LW-AFC and its active components on protecting CORT-induced LTP impairment1.Effects of LW-AFC and its active components on intestinal microbiota and immune functionThe sesults showed that Bacteroidetes,Firmicutes and Cyanobacteria were dominant bacteria taxa in the control group at the phylum level.However,the dominant bacteria taxa in the CORT group were Bacteroidetes,Firmicutes and Deferribacteres.Compared with the group group,the level of Bacteroidetes decreased significantly,while the level of Deferribacteres increased significantly.The results of PCA further confirmed that there was significant difference in intestinal microbiota between the control group and the CORT group.LW-AFC,CA-30 and STA could regulate the intestinal microbiota to a certain extent,which further demonstrated that CA-30 and STA might display their effects on synaptic plasticity indirectly through intestinal microbiota.There was a declining trend of the level of SIg A with no significant difference in CORT group.LW-AFC,CA-30 and STA also had the trend to protected the decreased SIg A level with no significant difference.2.Effects of LW-AFC and active components on amino acid neurotransmitterThe results showed that CORT induced the accumulation of Glu.LW-AFC,CA-30 and STA could decrease the Glu level significantly.The other active fractions and active components showed the trend of decreasing the Glu level with no significant difference.CORT induced the increase of GABA significantly.LW-AFC,LWB-B,CA-30 and STA showed the trend of decreasing the GABA level with no significant difference.CORT had little influence on L-Ser and D-ser,while LW-AFC could increase the D-ser level significantly.CA-30 and STA showed the trend of increase the D-ser level with no significant difference.The antibiotics cocktail inhibited the effects of CA-30 and STA on amino acid neurotransmitters,while had little influence on the effects of LW-AFC.3.Effects of LW-AFC and active components on the metabolic pathway of D-serThe research results show that CORT could lead to the decrease of glucose,3-phosphoglycerate dehydrogenase(PHGDH),Na+-independent alanine–serine–cystein transporter-1(Asc-1)and vesicle-associated membrane protein 2(VAMP2).LW-AFC and its active components could significantly increase their levels.CORT could lead to the increase of the level of serine racemase(SR),and LWAFC and its active components could significantly decrease the SR level.In conclusion,on the one hand,this part of study suggested that CORT could influence the intestinal microbiota,but had no influence on the intestinal SIg A level.LW-AFC,CA-30 and STA could regulate the intestinal microbiota.On the other hand,excess CORT,which binding to its glucocorticoid receptors,induced the accumulation of Glu and GABA,then resulted in Glu/GABA balance disorder.The Glu/GABA balance disorder played a crucial role in excitotoxicity and resulted in decreased NMDA receptor function.LW-AFC and part of its active components could protect against CORT-induced increasing of Glu and GABA levels,reduced the excitotoxicity in hippocampus.In addition,LW-AFC could also increase the D-ser level,and then promoted the NMDA receptor function.Further,our results showed that CORT could influence the metabolic pathway of D-ser.CORT decreased the levels of glucose,PHGDH,Asc-1 and VAMP2,while increased the levels of SR.LW-AFC and its active components could regulate the metabolic pathway of D-ser,promoted the NMDA receptor function,protected CORT induced-synaptic plasticity impairment.Conclusions:1.There was no significant effect on corticosterone-induced LTP impairment after the single administration of LW-AFC and its active components,while the multiple administrations of LW-AFC and its active components had the protecting effects on corticosterone-induced LTP impairment.The immunosuppressor could block the protecting effects of LW-AFC.These results indicated that LW-AFC and its active components may improve the LTP by regulating the immune function.2.The antibiotics could significantly inhibit the protecting effects of CA-30 and STA,and had no influence on other components.The results indicating that CA-30 and STA might display improving effects on synaptic plasticity by regulating the intestinal microbiota.3.Our results showed that corticosterone could induce LTP impairment by influence the key factors of the metabolism and translocation pathway of D-serine,and then reduce the NMDA receptor function.LW-AFC and its active components could regulate the function of he metabolism and translocation pathway of D-serine,then finally protect against the corticosterone-induced LTP impairment.The study have provided experimental basis for the further research of LW-AFC on the material basis and compatibility of herbs,and provided reference for future TCM formula research.In this study,we elaborated that one important mechanism which corticosterone induced LTP impairment was regulating the metabolism and translocation pathway of D-serine.Our research could provide a potential target for therapeutic intervention and strategy for prophylaxis and treatment of stress,and had great significance in science and potential applications.