Anxiolytic Mechanism Of Iridoids Of Roots And Rhizomes Of Valeriana Jatamansi Jones Based On The Agonism Of MGluR2 And MGluR3

Objective Studies on anxiolytic mechanism of iridoids of roots and rhizomes of Valeriana jatamansi Jones based on the agonism of metabotropic glutamate receptors in group II?mGluR2 and mGluR3?.Method 1.Vero-Ga15-m GluR2 cells and Vero-Ga15-mGluR3 cells were constructed by lipofection,and Real-Time PCR was used to verify the expression of the target genes-GNA15,mGluR2 and mGluR3.2.CCK-8 assay was used to determine the effects of different concentrations of total iridoids of roots and rhizomes of Valeriana jatamansi Jones?TIV?,Valjatrate E,mGluR2 and mGluR3 agonists?LY354740?and Glutamate?Glu?on the survival of Vero-Ga15-mGluR2 cells and Vero-Ga15-mGluR3 cells in order to determine the drug concentration.High-concentration drug screening system was used for detection the intracellular Ca²⁺intensities of different concentrations of TIV and Valjatrate E on Vero-Ga15-mGluR2 cells and Vero-Ga15-mGluR3 cells by Fluo-3/AM as Ca²⁺fluorescent probe.3.Rat cerebral cortical synaptosomes were prepared by gradient centrifugation.The microscopic morphology of synaptosomes was identified by transmission electron microscopy,and the activity of lactate dehydrogenase?LDH?in synaptosomes was measured.Flow cytometry was used to investigate the effects of TIV and Valjatrate E on the Ca²⁺fluorescence intensity in rat synaptosomes.4.Rat stress anxiety model was used to to evaluate the anxiolytic effect of TIV by the behavior experiments of elevated plus maze tests and open test.The activity of adenylate cyclase?AC?,cyclic adenosine monophosphate?cAMP?content,glutamic acid?Glu?content were tested by enzyme-linked immunosorbent assays,and the protein expression of PKA?/?/?,mGluR2,and mGluR3 protein was studied by Western Blot in rat cerebral cortex.Result 1.Vero-Ga15-mGluR2 and Vero-Ga15-mGluR3 were successfully constructed,which were consistent with the cells experiments,and the expression levels of the target genes GNA15,mGluR2,and mGluR3 were high through verification.2.The effect of TIV and Valjatrate E on Calcium Flow in Vero-Ga15-mGluR2 Cells and Vero-Ga15-mGluR3 Cells were studied by high-content drug screening system.For Vero-Ga15-mGluR2 cells,compared with the normal group,the Ca²⁺fluorescence mean values of positive drug group were significantly increased?P<0.05?;Glutamate group,3?g/m L and 12?g/m L TIV,10?M and 20?M Valjatrate E showed enhanced fluorescence values?P<0.01?;For Vero-Ga15-mGluR3cells,compared with the normal group,the Ga²⁺fluorescence mean values of positive drug group and 12?g/mL TIV was significantly increased?P<0.01?;Glutamate group,6?g/mL TIV,10?M Valjatrate E showed enhanced fluorescence values?P<0.05?.3.The prepared synaptosomes had typical morphological features by electron microscopy,the activity of LDH in synaptosomes was measured,and the total activity of LDH in the suspension and the activity in the supernatant were maintained a certain proportion.Flow cytometry was used to detect the effect of mGluR2 and mGluR3 receptor drugs on synaptic calcium.Compared with normal group,the Ca²⁺fluorescence mean values of positive drug group,6?g/m L TIV,20?M Valjatrate E were significantly reduced?P<0.01?,that of 10?M Valjatrate E was decreased significantly?P<0.05?.4.The rat stress anxiety model was successfully established.In the two anxiety model behavioral experiments,animals of model group had an anxiety state,while the positive group and the TIV groups?5,10,and 20 mg/kg?was improved.Further anxiolytic mechanism study results showed that the AC activity of high and low TIV groups,the cAMP content of the middle TIV group,and the Glu content of the high and middle TIV group were significantly decreased compared with the model group?P<0.01?.The c AMP content of middle TIV group was significantly decreased?P<0.05?;the expression levels of mGluR2 of high and low TIV groups and the expression of mGluR3 of middle and low TIV groups increased?P<0.01?.The expression levels of PKA?/?/?in the middle and low TIV groups decreased?P<0.01?.Conclusion 1.The iridoids of roots and rhizomes of Valeriana jatamansi Jones had an agonistic effect on mGluR2 and mGluR3.2.The anxiolytic mechanism of TIV was related to activate mGlu R2 and mGluR3,down-regulate AC-cAMP-PKA signaling pathway,and thus inhibited the release of glutamate by negative feedback.