| Objective: To study the antagonistic effect of Cinnamomi Ramulus(CR)to Ephedrine Herba(EH)on central nervous system side effects by investigating the effects compatibility of CR and EH on behavioral activity,brain tissue morphology,central dopamine neurotransmitter levels,oxidative stress factor and inflammatory protein in mice.The topic investigated the underline mechanism of CR’s protective effect on the central nervous system damage in mice induced by EH from the pharmacodynamic point of view.Methods: According to weight,40 KM mice were randomly assigned into saline group,EH group(10g/kg),EH and CR 3:2 group(10g/kg+6.67g/kg),EH and CR 3:1group(10g/kg+3.33g/kg)and CR(6.67g/kg)group.All mice received its corresponding drugs one times a day,the volume of each administration were0.2ml/10 g for 14 days.After the last administration one hour,mice were sacrificed and their brains were removed.First,respectively rinsing those brain in frozen phosphate buffered solution(PBS).Then quickly using the knife to divide the brain into left and right two prats,and the left hemisphere of the mice was cut into up and down two parts in the same way and respectively wrapped in foil stored in liquid nitrogen.Final,fixing the right mice brain in 4% paraformaldehyde.1.Open field experiments were conducted on the 7th night.The mice were placed in the open-field reaction box to adapt for 5 seconds,then observing and recording the resting time and the exercise time,as well as the movement distance to evaluate the autonomic activity of mice in each group.Subsequently,the first passive avoidance test was conducted on the 12 th night.After the mice were put in the darkroom about 5seconds,the experiment was started according to the procedure.The second test was repeated after 24 hours,in this test,the first time(the escape latency)that the mice were shocked into the darkroom and the total number of shocks(errors times)should be observed and recorded within 5 minutes.2.HE staining was used to observe the pathological morphology of brain tissue in mice.Removing the mice brain from 4% paraformaldehyde,and separating the hippocampus,cortex and striatum.The pathological changes in the hippocampus,cortex and striatum were observed under the microscope.3.The levels of dopamine and dopamine transporter in hippocampus,striatum,cerebral cortex of mice brain were detected by immunohistochemistry.4.Mice brain tissue was removed from liquid nitrogen to prepare 10% brain tissue homogenate,then using the homogenate to detect the superoxide dismutase(SOD)activity,malondialdehyde(MDA)levels,nitric oxide(NO)levels,Nitric oxide synthase(NOS)levels with chemical methods.5.Western blot was used to detect the expression of NOD-like receptor 3(NLRP3),Apoptosis-associated speck-like protein containing CARD(ASC)and Cysteinyl aspartate specific proteinase 1(Caspase-1)in brain tissue.Results:1.Effects compatibility of CR and EH on the open field experiments and the passive avoidance in KM mice In the open field experiments,compared with the saline group data,the autonomic activity in the EH group was increased,this phenomenon is manifested as a decrease in the resting time(P<0.01)and an increase in the movement time and the distance(P<0.01).However,data of CR group showed no significant difference compared with saline group(P>0.05).Compared with the EH group,EH and CR 3:2group could significantly increase the resting time(P<0.01),decrease errors times and errors numbers(P<0.01),as well as EH and CR 3:1 group could decrease the autonomic activity with statistical significance(P<0.05).In the repeated passive avoidance test,compared with the saline group data,the escape latency of EH group mice was reduced and errors times and errors numbers were increased evidently(P<0.01).And data of CR group showed no significant difference compared with saline group(P>0.05).Compared with the EH group,EH and CR 3:2 group and 3:1 group could significantly increase the escape latency(P<0.05,P<0.01),decrease errors times and errors numbers(P<0.05,P<0.01),with statistical significance.2.Effects compatibility of CR and EH on dopamine neurotransmitter expression in hippocampus,striatum and cortex of KM mice Compared with the saline group,the levels of dopamine and dopamine transporter in hippocampus,striatum and cerebral cortex of EH group mice brain were significantly decreased(P<0.01).And data of CR group showed no significant difference compared with saline group(P>0.05).Compared with the EH group,EH and CR 3:2 group and 3:1 group could increase the levels of dopamine and dopamine transporter in hippocampus,striatum and cerebral cortex of mice brain(P<0.01),especially in EH and CR 3:2 group.3.Effect of compatibility of CR and EH on the pathological morphology in hippocampus,striatum and cortex of KM mice Under light microscope,the vertebral cells in the hippocampus of the normal saline group were arranged orderly,the number and thickness of vertebral cells were normal and cannot observe neuronal necrosis and microglia proliferation.At the same time,the structure of striatum was normal,the intercellular space was not widened,and the proliferation of glial cells cannot seen.Also,it could be seen that the cortical structure was normal,the membrane was intact,and the cortical neurons were clear in structure and arranged neatly.And compared with the saline group,CR group showed no significant difference.As to EH group,the CA1 region of the hippocampus appeared more necrotic vertebral cells and proliferative microglia.Meanwhile,it could be seen that moderate edema,widened interstitial space and obvious proliferation of glial cells in the striatum.And the neurons of the cortical layer were disordered,the cells were fuzzy and the neurons are scattered.The phenomenon of necrosis of neurons and the phenomenon of phagocytosis by microglia could be observed.Compared with the EH group,the lesions in the hippocampus,striatum and cortex of the EH and CR 3:2group and 3:1 group were improved.4.Effect of compatibility of CR and EH on contents of SOD,MDA,NO and NOS in brain tissue of KM mice Compared with the saline group,the activity of SOD in the brain tissue of the EH group decreased significantly(P<0.01),and the contents of MDA,NO,TNOS and i NOS increased significantly(P<0.01).And data of CR group showed no significant difference compared with saline group(P>0.05).Compared with the EH group,the SOD activity in the EH and CR 3:2 group and 3:1 group was significantly increased(P<0.05),and the contents of MDA,NO,TNOS and i NOS were significantly decreased,and the difference was statistically significant(P<0.05).5.Effect of compatibility of CR and EH on expression of NLRP3 inflammasome in brain tissue of KM mice Compared with the saline group,the NLRP3,ASC,Caspase-1 protein expression was significantly increased in the brain tissue of EH group and there was a statistically significant difference(P<0.01).And data of CR group showed no significant difference compared with saline group(P>0.05).Compared with the EH group,the expression of NLRP3,ASC and Caspase-1 was significantly decreased in the EH and CR 3:2 group,with statistical significance(P<0.05),as well as the date of the EH and CR 3:1 group indicated that the expression of NLRP3,ASC and Caspase-1 were reduced to a certain extent,and the expression of Caspase-1 had statistical significance(P<0.05).Conclusion:1.CR could inhibit the central excitatory effect,improve the adverse effects of behavioral activities in mice and reduce the learning and memory of mice errors which caused by EH.Also,CR could improve pathological damage in hippocampus,striatum,cortex of the mice brain tissue,the existence of "compatibility fear kill between ephedra and cinnamon.A compatibility relationship between EH and CR is that “mutual suppression and mutual restraint”.2.CR could raise the dopamine and dopamine transporter content,increase antioxidant activity,reduce oxidative stress,inhibit the NLRP3 inflammasome up-regulation to antagonist central dopamine neurotoxicity which induced by EH.And this protective effect has a certain dose-effect relationship,with the increase in the proportion of CR,the effect was more effective. |
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