Study on the Mechanism of Rehmanniae Radix Praeparata in Promoting the Synthesis of γ-Aminobutyric Acid in the Brain of Yin Deficiency Rats by Up-regulating the Xylosyltransferase Ⅰ Signaling Pathway
Objective To investigate the regulatory mechanism of Rehmanniae Radix Praeparata and its active components rehmannioside D and catalpol on γ-aminobutyric acid(GABA)in the brain based on the xylosy-transferase I(xylt-1)signaling pathway.Methods(1)SD rats were randomly divided into normal group,model group and treatment group(Rehmanniae Radix Praeparata group,30 g·kg-1),with eight rats in each group,half male and half female.The rat model of yin deficiency was established by intragastric administration of traditional Chinese medicine compound(30 g·kg-1)with acrid-warm nature and dampness-drying and dampness-draining,once in the morning and once in the evening,for 10 consecutive days.After modeling,the treatment group was given Rehmanniae Radix Praeparata decoction by gavage,once in the morning and once in the evening,for 10 consecutive days.The body mass of rats was measured and recorded,and the activity,crossing times and total distance were detected by open field behavior experiment.The levels of serum gonadotropin-releasing hormone(GnRH),thyrotropin-releasing hormone(TRH),corticotropin-releasing hormone(CRH),luteinizing hormone(LH)and follicle-stimulating hormone(FSH)in rats were detected by ELISA.The mRNA and protein expression levels of xylt-1,multiligand proteoglycan 1(SDC-1),early growth response factor 1(EGR1)and glutamate decarboxylase 1(GAD67)in rat brain tissue were detected by qPCR and automatic capillary Western Blot.The content of GABA in brain tissue was detected by HPLC.(2)The xylt-1 gene of rat well-differentiated adrenal pheochromocytoma cells(PC12)was silenced by siRNA.The cells were divided into normal group,negative control group,silencing group,silencing+rehmannioside D(10 μmol·L-1)group,silencing+catalpol(10 μmol·L-1)group.The expression levels of xylt-1,SDC-1,EGR1 and GAD67 mRNA in cells were detected by qPCR.The content of GABA in intracellular and extracellular fluid was detected by HPLC.The expression level of SDC-1 in cells was detected by immunofluorescence.(3)PC12 cells were transfected with lentivirus to overexpress xylt-1 gene.The cells were divided into normal group,negative control group(empty vector group)and overexpression group.The mRNA expression levels of xylt-1,SDC-1,EGR1 and GAD67 in cells were detected by qPCR.Results(1)Compared with the normal group,the body mass of the rats in the model group was significantly decreased(P＜0.01),the activity was significantly increased(P＜0.01),and the number of crossings and the total distance were significantly increased(P＜0.01).The levels of serum LH,FSH,GnRH,CRH and TRH were significantly increased(P＜0.01).The content of GABA in hippocampus and cerebral cortex was significantly decreased(P＜0.05,P＜0.01).The expression of xylt-1,SDC-1,EGR1 and GAD67 mRNA(hippocampus,cerebral cortex)and protein(hypothalamus,cerebral cortex)in brain tissue were significantly down-regulated(P＜0.05,P＜0.01).Compared with the model group,the body mass of the rats in the treatment group was significantly increased(P＜0.05),the activity was significantly decreased(P＜0.01),and the total distance was significantly shortened(P＜0.05).The levels of serum LH,FSH,GnRH,CRH and TRH were significantly decreased(P＜0.01).The content of GABA in hippocampus and cerebral cortex was significantly increased(P＜0.05,P＜0.01).The content of GABA in hippocampus and cerebral cortex was significantly increased(P＜0.05,P＜0.01).The expressions of xylt-1,SDC-1,EGR1 and GAD67 mRNA(hippocampus,cerebral cortex)and protein(hypothalamus,cerebral cortex)in brain tissue were significantly up-regulated(P＜0.05,P＜0.01).(2)Compared with the negative control group,the mRNA expressions of xylt-1,SDC-1,EGR1 and GAD67 in the silencing group was significantly down-regulated(P＜0.01).The content of GABA in intracellular and extracellular fluid was significantly decreased(P＜0.05,P＜0.01).The expression of SDC-1 in cells was significantly down-regulated(P＜0.01).Compared with the silencing group,the expression of xylt-1 mRNA in the silencing+rehmannioside D group was up-regulated,but the difference was not statistically significant(P＞0.05).The mRNA expression of xylt-1 in the silencing+catalpol group was significantly up-regulated(P＜0.05).The mRNA expressions of SDC-1,EGR1 and GAD67 in the silencing+rehmannioside D group and the silencing+catalpol group was significantly up-regulated(P＜0.05,P＜0.01),the content of GABA in the intracellular and extracellular fluid was significantly increased(P＜0.01),and the expression of SDC-1 in the cells was significantly up-regulated(P＜0.05,P＜0.01).(3)Compared with the normal group and the negative control group,the mRNA expression of xylt-1 in the overexpression group was significantly up-regulated(P＜0.01).Compared with the negative control group,the mRNA expressions of SDC-1,EGR1 and GAD67 in the overexpression group was significantly up-regulated(P＜0.01),and the GABA content in the intracellular and extracellular fluid was significantly increased(P＜0.01).Conclusion There may be a xylt-1/SDC-1/EGR1/GAD67 pathway that regulates GABA synthesis in the brain.Rehmanniae Radix Praeparata may increase GABA levels in the brain by up-regulating this pathway.
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