| PART 1Evaluation studies on the quantitative-effective relationship between two rosemary extracts on the antioxidant effects of 3MCC rat brainObjective(s): The 3MCC rat model was established to detect the changes of reactive oxygen species in hippocampal tissues of rats damaged by different doses of carnoic acid and rosmarinic acid,to explore whether the two drugs had a dose-response relationship in terms of reactive oxygen species clearance,and to find the optimal therapeutic dose of the two drugs.Methods:(1)Animal grouping: 52 adult male SD rats were randomly divided into13 groups of 4 animals each,namely group N(normal group),group 3MCC(model group),group DMSO(solvent group),group C1-C5(different doses of carnosic acid group)and group R1-R5(different doses of rosmarinic acid group).(2)Model replication: Except for group N,a metal single pendulum closed rat concussion replicator was applied to replicate the 3MCC model by hitting the top of the rat’s forehead every 24 hours for three consecutive days.(3)Pharmacological intervention:Within 3 hours of successful replication of the 3MCC model,the corresponding drugs were injected intraperitoneally by group for 14 d.(4)After 14 days,the brain was killed by execution and hippocampal tissue was isolated,and the reactive oxygen content of hippocampal tissue was measured by ELISA kit.(5)A dose-relationship curve was fitted according to the ELISA kit assay results.Results:(1)Hippocampal tissue reactive oxygen species assay results:Compared with the N group,the ROS content in the 3MCC,DMSO,C4 and C5 groups increased(p<0.05);the ROS content in the R2,R3,R4 and R5 groups decreased(p<0.05);the ROS content in the C1,C2,C3 and R1 groups did not change significantly(p>0.05).Compared with the DMSO group,there was no significant change in the ROS content in the 3MCC,C4 and C5 groups(p>0.05);the ROS content decreased in the C1,C2,C3 and R1-R5 groups(p<0.05).There was no difference in the comparison between the C1,C2 and C3 groups(p>0.05).There was no difference in the comparison between the R2 and R5 groups,and the R3 and R4groups(p>0.05).(2)Dose-relationship curves: The dose-effect curve of CA reactive oxygen species clearance rate was S-shaped,and the best effect was achieved with a dose of 1 mg/kg;the dose-effect curve of RA reactive oxygen species clearance rate was inverted U-shaped,and the best effect was achieved with a dose of 20 and 50mg/kg.Conclusion(s):(1)The content of reactive oxygen species in hippocampal tissue was significantly increased in rats with 3MCC.(2)10% DMSO as a solvent had no intervention effect on the change of reactive oxygen species content in hippocampal tissues of 3MCC rats.(3)98% CA at a dose of 1-50 mg/kg can significantly intervene to reduce the content of reactive oxygen species in hippocampal tissues 14 days after injury in 3MCC rats;98% of RA at a dose of 10-200 mg/kg significantly reduced the content of reactive oxygen species in hippocampal tissues in 3MCC rats 14 days after injury,and 20-50 mg/kg RA intervention had the best efficiency in clearing 3MCC rats hippocampal reactive oxygen species.(4)CA intervention in the change of reactive oxygen species content in 3MCC rats had a dose-dependent relationship;RA intervention 3MCC rats with reactive oxygen species content changes did not have a dose-dependent relationship.(5)The optimal dose range of the matching study: 98%CA: ≥1mg/kg,98% RA: Around 20mg/kg and 50mg/kg.PART 2 Evaluation studies on the synergistic effect of the compatibility of two rosemary extracts on brain protection in rats with 3MCCObjective: The 3MCC rat model was established to detect the improvement effect of changes in learning memory,emotional cognition,oxidative stress and inflammatory indexes,and whether the combination of the two drugs had a synergistic brain protective effect,explore the mechanism of action of the drug,and find the best therapeutic dose for 3MCC.Methods:(1)Animal grouping: 90 adult male SD rats were randomly divided into 9 groups of 10 animals each,namely group N(normal group),group 3MCC(model group),group DMSO(solvent group),group CA(carnosic acid group),group RL(rosmarinic acid low dose group),group RH(rosmarinic acid high dose group),group ML(mixed drug low dose group),group MH(mixed drug high dose group),group Y(edaravone group).(2)Model replication: Except for group N,a metal single pendulum closed rat concussion replicator was applied to replicate the 3MCC model by hitting the top of the rat’s forehead every 24 hours for three consecutive days.(3)Pharmacological intervention: Within 3 hours of successful replication of the 3MCC model,the appropriate drugs were injected intraperitoneally by group for 14 days.(4)The Morris Water Maze(MWM),Open Filed Test(OFT),High Plus Maze(HPM)and Forced Swimming Test(FST)were applied.The experiments tested the learning memory(including working memory and short-term memory),emotional-cognitive functions and the therapeutic effects of the drugs.(5)After 14 days,rats were killed and brain were taken,hippocampal tissue and prefrontal cortex were separated,and the ELISA kit detected hippocampal tissue ROS,MDA,SOD.Changes in TREM4,TLR4,NF-κB,TNF-α,IL-1β,IL-4,IL-10 in the prefrontal cortex.Results:(1)Behavioural experiments on learning memory in rats after injury:(1)Underwater platform experiment: Compared with group N,the escape latency of 3MCC group increased on days 1,2,3,4 and 5;the escape latency of DMSO group and RL group increased on days 1,2,3 and 4;the escape latency of CA group,RH group and ML group increased on day 1(p<0.05).Compared with the DMSO group,the escape latency was reduced in the CA,RL,RH,ML,MH and Y groups on day l;and reduced in the CA,ML,MH and Y groups on day 2(p<0.05).Compared with the Y group,the escape latency increased in both the CA and RH groups on day 1;the escape latency increased in the RL group on days 2 and 3(p<0.05).Compared with the ML group,there was an increase in escape latency in the RL group on day 2(p<0.05).Compared with the MH group,the escape latency increased in both the CA and RH groups on day 1;the escape latency increased in the RL group on days 2 and 4(p<0.05).(2)Water platform experiment: No difference in escape latency between groups(p<0.05).(3)No platform experiment: The dwell time in quadrant IV was reduced in both the 3MCC and DMSO groups compared to the N group(p<0.05).The residence time in quadrant IV was increased in the RH,ML,MH and Y groups compared to the DMSO group(p<0.05).The dwell time in quadrant IV was reduced in the RL group compared to the Y group(p<0.05).The residence time in quadrant IV was reduced in both the CA and RL groups compared to the ML group(p<0.05).(4)Inversion platform experiment: Day 1 of the inversion platform experiment: No difference in escape latency between groups(p>0.05);day 2 of the inversion platform experiment: Increased escape latency in both the 3MCC and DMSO groups compared to the N group(p<0.05).The escape latency was reduced in the CA,ML,MH and Y groups compared to the DMSO group(p<0.05).The trend of change in escape latency was the same on both days.(2)The results of post-injury emotional-cognitive-behavioural experiments in rats:(1)OFT experiment: Compared with the N group,the movement distance of the central area of the remaining groups was reduced;The number of grooming and exercise time in the surrounding area increased in the 3MCC group and DMSO group,while the number of standing and exercise time in the central area decreased.The number of grooming times and the exercise distance of the surrounding area in the ML group were reduced.The distance around the CA group,RL group and MH group decreased(p<0.05).Compared with the DMSO group,the number of standing times increased in the RL group,ML group and MH group,and the number of grooming times and the exercise distance in the surrounding area were reduced.The number of grooming decreased in the RH group and the Y group,and the number of standing times increased(p<0.05).Compared with the Y group,the distance of movement around the CA group and the MH group decreased,and the number of standing times increased in the ML group(p<0.05).Compared with the ML group,the number of grooming times increased in the RL group,RH group and MH group,and the exercise distance around the RL group increased(p<0.05).Compared with the ML,MH and Y groups,the number of standing times in the CA group was reduced,and the number of grooming and the distance of movement in the surrounding area were reduced compared with the DMSO group(p<0.05).(2)HPM experiment: The number of open arm entries was reduced in the 3MCC,DMSO,CA,RL,RH and Y groups compared to the N group;and the number of downward exploration was reduced in all groups except the MH group(p<0.05).Compared with the DMSO group,the ML and MH groups had increased open-arm dwell time,decreased closed-arm dwell time and increased number of downward explorations;the CA,RH and Y groups all had increased number of downward explorations(p<0.05).Compared with group Y,open-arm dwell time increased and closed-arm dwell time decreased in groups RH,ML and MH(p<0.05).The number of downward exploration was reduced in the CA,RL,RH,MH and Y groups compared to the ML group(p<0.05).The number of downward explorations was reduced in the CA and RL groups compared to the MH group(p<0.05).There was no difference in the number of closed arm entries between the groups(p>0.05).(3)FST experiment: The percentage of C behaviours increased in both the 3MCC and DMSO groups compared to the N group(p<0.05).The ratio of C behaviours was decreased in the CA,RL,RH,ML,MH and Y groups compared to the DMSO group(p<0.05).(3)The results of oxidative stress and inflammation index tests: Compared with the N group,the contents of ROS,MDA,TLR4,NF-κB,TNF-α and IL-1β in the 3MCC group and DMSO group increased significantly(p<0.05),the content of SOD,IL-4 and IL-10 decreased significantly(p<0.05),and the contents of CA group and IL-10 were significantly reduced(p<0.05),CA group.Compared with the DMSO group,the content <of ROS,MDA,TLR4,NF-κB,IL-1β,TNF-α content in ML,MH group and Y group increased(p<0.05),and the content of IL-10 in RH group decreased(p<0.05).Compared with the DMSO group,the content of ROS,MDA,TLR4,NF-κB,IL-1β,TNF-α in ML,MH and Y groups were significantly reduced(p<0.05);SOD,IL-4 in each dosing group,CA,RL,ML,the content of IL-10 in the MH group increased significantly(p<0.05);the SOD content in the RL group decreased(p<0.05)and the IL-10 content in the ML group increased(p<0.05).Compared with the Y group,and the ROS content in the RL group,RH group and MH group increased(p<0.05).Compared with the ML group,the content of MDA in RL group decreased(p<0.05),SOD content in RL group(p<0.05),IL-10 content in RH group(p<0.05),and IL-4 content in CA group and RL group decreased(p<0.05).Compared with N,DMSO,RL,RH and ML,the content of TREM2 in the CA group was lower(p<0.05).Conclusion:(1)Triple concussion(3MCC)rats had significantly reduced working memory and short-term memory function after injury,prominent anxiety and depression,and oxidative stress and inflammatory response.(2)10% DMSO as solvent intraperitoneal injection had no effect on the experimental results.(3)CA and RA intervention can improve the symptoms of memory loss,anxiety,depression and other symptoms caused by triple concussion,and reverse the oxidative stress and inflammatory response in the brain of rats after 3MCC injury.And the treatment effect of mixed drugs is better,to some extent surpassing edaravone.(4)CA and RA may regulate the inflammatory response through the TLR4/NF-κB pathway. |
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