The Intervention Effect Of Bupleuri And Ramuli Cinnamomi Decoction In Fmr1Knockout Mice

Objective:To explore whether Fmrl mouse exhibits abnormal behavior as patients with Fragile X Syndrome (FXS).Method:Genotypes of Fmrl knockout mice (KO mice) and wild type mice (WT mice) were identified by PCR and Western Blot in Experiment Animal Center of Guangzhou Medical College. The morphology of testis and dendritic spine of the two groups were studied under HE and Golgi stain. Animals were tested for audiogenic seizure (AGS), for learning capability and memory through Morris water maze, passive avoidance test, step-down test, and for general locomotor activity and willingness to explore via open field test, elevated plus maze and inner open field test.Results:Genotype analysis revealed WT mice had the expected500bp DNA segments, whereas KO mice had800bp segments. What's more, KO mice exhibited macroochidism, abnormal morphology of dendritic spine and absent FMRP (Fragile X Mental Retardation Protein), and they were more susceptible to AGS (P<0.05). In the space navigation experiment of Morris water maze, they had longer latency and more platform-crossing activities than their WT counterparts (P<0.05); in space search experiment, KO mice stayed shorter than WT mice in the target quadrants (P<0.05). In passive avoidance test and step-down test, KO mice reacted swifter but made more mistakes (P<0.05). In open field test, they stayed longer in the central area and entered more constantly into the area than the WT mice (P<0.05); and in general, they traveled further and faster than WT mice (P<0.05). In elevated plus maze, they had longer duration, higher frequency and greater travel distance in the open area (P<0.05). In inner open field test, they traveled more often but stood less frequently (both P<0.05). Conclusion:Genotype, protein expression profile, morphological and behavioral aspects suggested that Fmrlgene knockout mice and patients of Fragile X syndrome behaved similarly, which are linked to the lost of FMRP expression. This study indicates that Fmrl KO mice can serve as animal model for FXS research. Objective:To observe the effect of bupleuri and ramuli cinnamomi decoction on abnormal behaviours in Fmrl knockout mice.Method:Bupleuri and ramuli cinnamomi decoction was extracted with supercritical carbon dioxide, whose components were assayed by GC-MS (Gas chromatography-mass spectrometry). Fmr1knockout (KO) and wild type (WT) mice of four-week old were randomized into three subgroups:treatment group (receiving intraperitoneal injection of bupleuri and ramuli cinnamomi decoction of different concentrations), solvent control group (receiving intraperitoneal injection of vegetable oil) and blank control group (receiving no injections). After treatments, animal behaviours were observed under open field test, elevated plus maze, inner open field test, step-down test, passive avoidance test and AGS test.Results:GC-MS identified28components in bupleuri and ramuli cinnamomi decoction, whose main ingredients included gingerol (31.94%), eugenol (18.01%), monoterpenoids (12.34%),3-Butyl-1(3H)-isobenzofuranone (9.34%) and cinnamon substances (8.03%).In open field test, the average speed, number of travels passing the central field and total traveling distance of KO and WT mice decreased with increased concentration of bupleuri and ramuli cinnamomi decoction. In elevated plus maze, travel duration, frequency and distance of KO treatment group in open arm decreased in comparison with KO solvent control group (P<0.05). In inner open field test, they moved less frequently but stood more often (P<0.05). In step-down test, their latency was prolonged and the number of errors was reduced (both P<0.05), whereas changes in those parameters were not significant among WT treatment group. In passive avoidance test, latency and errors extended and dwindled among KO treatment group (both P<0.05) but barely changed among WT mice (P>0.05). In audiogenic seizure experiment, latency delayed and severity of the seizure declined among KO treatment group with increased essential concentration (P<0.05) and seizure was suppressed under decoction of1.5mg/g. Conclusion:Bupleuri and ramuli cinnamomi decoction can be therapy to abnormal behaviour of KO mice by reducing audiogenic seizure and hyperactivity, and improving learning capability and memory. Objective:To identify the intervention mechanisms of bupleuri and ramuli cinnamomi decoction on Fmrl knockout mouse.Method:Experimental animals, KO and WT mice of four-week old, were randomized into three subgroups:treatment group (receiving intraperitoneal injection of bupleuri and ramuli cinnamomi decoction), solvent plus AGS group and solvent control group. Then, the first two groups underwent AGS induction whereas the third group was left untreated. We detected their levels of amino acid by high performance liquid chromatography and the expressions of GAD (glutamate decarboxylase) and other molecules in the hippocampus and cortex by immunohistochemistry. We assayed and compared the levels of superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO) respectively by pyrogallol autoxidation method, thiobarbituric method and nitrate reductase method. These efforts would lead to possible mechanisms of bupleuri and ramuli cinnamomi decoction's therapeutic effect on abnormal behaviour of KO mice.Results:KO mice possessed fewer neurons with GAD in the hippocampus and cortex than WT mice (P<0.05).KO mice in solvent control group expressed fewer y-aminobutyric acid (GABA) and glycine but more glutamate than WT solvent control group (P<0.05). After AGS induction, KO mice of solvent plus AGS group had a significant decrease in GABA level as compared with KO blank control group (P<0.05). The glutamate level of KO treatment group was lower than KO solvent plus AGS group and WT treatment group (P<0.05).SOD activity of KO solvent plus AGS group was lower but MDA and NO levels were higher than WT solvent plus AGS group (P<0.05). And KO treatment group manifested higher SOD activity and lower MDA and NO levels than KO solvent plus AGS and WT treatment groups (P<0.05). bupleuri and ramuli cinnamomi decoction did not alter spine length after1time treatment in4W KO mice (p>0.05).Conclusion:The intervention mechanisms of bupleuri and ramuli cinnamomi decoction on abnormal behaviours of Fmrl knockout mice may lie in the reduced ratio of excitatory amino acid to inhibitory amino acids, enhanced free radicals elimination, suppressed peroxide production and diminished NO neurotoxic effects.