Acupoint Catgut-embedding Therapy On Dazhui Point For Controlling Epilepsy Model In Mice Through The Pathway Of Egr1-T Type Ca²⁺-Limk1-cofilin

ObjectiveThe aims of this study are to assess the efficacy and safety of acupoint catgut-embedding therapy on Dazhui point for controlling epilepsy in clinical by systematic review,and to explore the potential mechanism on the pathway of Egr1-T type Ca2+-Limk1-cofilin for controlling epilepsy in mice,and to offer a possible therapeutic regimen and reliable evidence for clinical practices.MethodsIn the systematic review section,we included all the randomized controlled trials of acupoint catgut-embedding therapy for epilepsy to assess its efficacy and/or safety in the clinical practice by meta-analysis.Two reviewers confirmed the included trials by inclusion criteria after searching electronic databases and manual searching.The primary outcome was clinical effectiveness,and the secondary outcomes included the related assessments with epilepsy.All the data were analysied by RevMan5.3 software.In the animal research section,we explored the potential mechanism on the pathway of Egr1-T type Ca2+-Limk1-cofilin for controlling epilepsy in mice.Firstly we intraperitoneal injected different dosages(8mg/kg,10mg/kg,and 12mg/kg)of kainic acid(KA)to confirm the successful epileptic mice model for this study;then we divided 150 mice whom adaptive feeding for 7 days into 6 groups randomly by random number table:blank group(BG),model group(MG),catgut-embedding group(CEG),whole dose group(WG),catgut-embedding+whole dose group(CE+WG),and catgut-embedding+half dose group(CE+HG).Nothing to do for the mice in BG except for weighing every day;after injected by KA building the model successfully,nothing to do for the mice in MG except for weighing every day;acupoint catgut-embedding therapy at Dazhui point once by surgical absorbable sutures(the length was 3mm)for mice in CEG lasting 7 days;treated by Oxcarbazepine(30mg/kg)twice per day(7a.m.and 7p.m.)by intragastric gavage lasting 7 days for the mice in WG;the mice in CE+WG were treated by the same method in CEG plus WG;and mice in CE+HG were treated by the same method in CEG plus half dose(15mg/kg)of Oxcarbazepine.The outcomes included weight,open-field test,HE staining,Nissl's staining,Fluoro-Jade B staining,PET/CT and the expression of target proteins including Egr1,CACNA1H,Limk1,cofilin and p-cofilin.Statistical analysis was by SPSS23.0 software package,and P<0.05 meant statistical significance.Results1.The results of efficacy and safety from meta-analysisAcupoint catgut-embedding therapy(ACE)plus antiepileptic drugs(ADs)or Chinese herbal medicine(CHM)showed better clinical efficacy and lower epilepsy score when compared with only using ADs or CHM.Compared with ADs,using ACE alone showed no significant difference in the results of clinical efficacy(RR 1.03,95%CI[0.95,1.12])and epilepsy score(MD-2.26,95%CI[-5.08,0.56]).Compared with the control groups,no significant difference showed in the result of reducing seizure frequency in the ACE group(MD-0.46,95%CI[-1.48,0.56]).No serious safety events were identified on ACE therapy itself in this systematic review.2.The dosage for the successful modelThe rates of successful modeling of 8mg/kg,10mg/kg and 12mg/kg injected were 0%,71.4%and 25%respectively,which showed significant difference(P<0.01).So the dosage of 10mg/kg for intraperitoneal injection once by KA would be an ideal dosage for epileptic modeling in this study.3.Acupoint catgut-embedding therapy on Dazhui point for behaviorAfter injected by KA,the weights of mice reduced when compared with BG(P<0.05),and the weights showed gain by the interventions of acupoint catgut-embedding,whole dosage of Oxcarbazepine,acupoint catgut-embedding plus whole dosage of Oxcarbazepine and catgut-embedding plus half dosage of Oxcarbazepine(P<0.05).From the second treatment day,the weights of mice in the groups treated by Oxcarbazepine began to gain,and the rising tendency showed from the third treatment day of the mice treated by ACE alone.After injected by KA,mice showed anxiety with reducing numbers of climbing wall,distance and the time staying in the central area of the open-field test.The interventions of ACE,whole dosage of Oxcarbazepine,ACE plus whole dosage of Oxcarbazepine,and ACE plus half dosage of Oxcarbazepine could reduce anxiety(P<0.01),while the effectiveness in CEG was the worst compared with other three treatments(P<0.01),CE+WG was the best compared with other three treatments(P<0.01),and WG and CE+HG in the between with no significant difference between the two groups(P>0.05).4.Acupoint catgut-embedding therapy on Dazhui point for neurons in the hippocampus and glucose metabolism in the brainThe results of HE staining:in the BG,neurons in the hippocampal CA3 area were arranged,structures of cells were neat,and organelles were intact;in the MG,neurons in the hippocampal CA3 area were not arranged with a large number of apoptosis,structures of cells lost the form,and cells were incomplete heavily.After interventions,the number of apoptosis cells reduced and the structures of cells were neater than MG.The effectiveness in CEG was the least significant,the most was in CE+WG,and WG and CE+HG in the between with no significant difference.The results of Nissl's staining:after injected by KA,numbers of nissl body in the hippocampal CA3 area were reduced compared with BG(P<0.01).After treatments of the four interventions,the numbers of nissl body in the hippocampal CA3 area were increased compared with MG(P<0.01),while the effectiveness in CEG was the worst compared with other three treatments(P<0.05),CE+WG was the best(P<0.05),and WG and CE+HG in the between with no significant difference(P>0.05).The results of FJB staining:after injected by KA,numbers of the positive FJB cells(FJB(+))were increased compared with BG(P<0.01).After treatments of the four interventions,the numbers of FJB(+)were reduced compared with MG(P<0.01),while the effectiveness in CEG was the worst compared with other three treatments(P<0.01),CE+WG was the best(P<0.01),and WG and CE+HG in the between with no significant difference(P>0.05).The highest uptake of 18F-FDG showed in the brain of mice in BG,the lowest uptake showed in MG,and others in the between.From the results of SUVmax,the value of SUVmax were reduced after treated by KA compared with BG(P<0.01),and the value in the four treatment groups were increased compared with MG(P<0.01).The effectiveness in CEG was the worst compared with other three treatments(P<0.05),CE+WG was the best(P<0.01),and WG and CE+HG in the between with no significant difference(P>0.05).5.Acupoint catgut-embedding therapy on Dazhui point for the pathwayThe targeted proteins in the pathway included Egr1,CACNA1H,Limk1 and cofilin/p-cofilin which all low-expression in the hippocampus of mice in BG,while high-expression after treated by KA(P<0.01).After four different interventions,the expression showed reduced compared with MG(P<0.01).In the four treatment groups,there was the highest expression in CEG(P<0.01),the lowest in CE+WG(P<0.05),and the other two in the between with no significant difference between CE+HG and WG(P>0.05).Conclusions1.ACE therapy on Dazhui point could improve the clinical effectiveness and no serious safety events were identified on ACE therapy itself in the meta-analysis.2.The dosage of 10mg/kg for intraperitoneal injection once by KA was an ideal dosage for epileptic modeling in this study.3.ACE therapy on Dazhui point could improve the epileptic state and the co-morbidity anxiety of epileptic mice.4.ACE therapy on Dazhui point could improve the apoptosis of neurons in the hippocampus through reducing the expression of related proteins in the Egr1-T type Ca2+-Limk1-cofilin pathway,and could improve glucose metabolism in the brain of epileptic mice.5.ACE therapy combined with Antiepileptic(Oxcarbazepine)could have higher effectiveness compared with Oxcarbazepine monotherapy,or combined with half dosage of Oxcarbazepine still shows a satisfactory effective.