Mechanism Of Electroacupuncture Regulating TGF-β1 Associated Pathway To Improve Cognitive Dysfunction In VD Mice

ObjectiveThe aim of this study is to systematically review the animal literature on acupuncture for vascular dementia and explore the mechanism of acupuncture in improving cognitive dysfunction in VD mice.Through the application of meta-analysis,the efficacy of acupuncture treatment in animal models of vascular dementia was elucidated,and optimal modeling methods and acupuncture prescriptions were summarized to provide evidencebased medical evidence for future animal studies.Vascular dementia model mice was established through bilateral common carotid artery occlusion and reperfusion.Following electroacupuncture treatment,the learning and memory abilities of vascular dementia mice were assessed along with the pathological changes in their hippocampus.Subsequently,the molecular mechanism underlying the improvement of vascular dementia in mice through electroacupuncture was investigated,focusing on the TGF-β1 related pathways associated with neuroinflammation and apoptosis.These findings would provide a scientific foundation for the clinical prevention and treatment of vascular dementia.Methods1.Evidence-based medicine research.This study search Pubmed.Embase.and Web of Science(including Medline)databases to identify relevant literature.The SYRCLE bias risk tool was employed to assess the quality of the included studies.Additionally,this study summarized important experimental factors.such as the frequency of acupoint use,frequency of acupoint combination,acupuncture method,treatment course,success rate of various surgical modeling,and postoperative rest time.The network meta-analysis of escape latency,number of crossing the original platform.time spent in target quadrant,and swimming speed was conducted using Addis 1.16.8.Furthermore,subgroup analysis of distinct acupuncture prescriptions(i.e.,acupoint combination,acupuncture method,course of treatment)and surgical modeling methods was conducted using Review Manager 5.3.The outcomes derived from this investigation provide significant backing for the subsequent stages of animal experimental protocols.2.Animal experimental studyA total of 60 C57BL/6 mice,ten were chosen at random to be included in the sham operation group(S2VO),while the remaining mice underwent bilateral common carotid artery occlusion and reperfusion to be included in the surgical model group(2VO).Subsequently.the mice were divided into four groups,namely the electroacupuncture nonacupoint group(SEA),electroacupuncture group(EA),donepezil hydrochloride group(DON),and the 2VO group.Following surgery.the mice were allowed a period of rest for 7 to 8 days before commencing a 14-day treatment regimen on day 9.The electroacupuncture group received treatment at Baihui and bilateral Zusanli points while under isoflurane respiratory anesthesia.The electroacupuncture parameters employed in this study consisted of dense waves,1-2 mA,and a duration of 20 minutes.In the electroacupuncture nonacupoint group,bilateral hypochondria located 10 mm from the iliac spine were selected under isoflurane respiratory anesthesia,and the electroacupuncture parameters utilized were dense waves.1-2 mA,for a duration of 20 minutes.The group administered with donepezil hydrochloride received a combination of 20 mg of the drug and 5 ml of 0.5%sodium chloride injection,which was intragastrically administered at a dosage of 1 ml per 100 g.Following treatment,respiratory anesthesia was administered for a duration of 20 minutes.The sham operation and surgical model groups were subjected to the same respiratory anesthetic treatment,but no additional interventions were performed.Following the treatment regimen,the spatial memory and cognitive ability of mice were assessed through the utilization of the Morris Water Maze and Novel Object Recognition test.Additionally,the quantity of Nissl bodies,neuronal status,and neuronal count were observed through Nissl staining,while the number of apoptotic neurons was observed through TUNEL staining.Western blotting was used to detect the protein level expression of TGF-β1,p-TGFβR1,SMAD2/3.p-SMAD2/3,Bax.Bcl-2,and cleaved caspase-3 proteins,and qPCR was used to detect the mRNA level of TGF-β1 to assess the effect of TGF-β-related pathways on cognition in VD mice.The expression of TGF-β1 and Iba-1 in each group of mice was detected by immunofluorescence,and the protein and mRNA expression levels of IL-1β,IL-6,TNF-α,IL-4,and IL-10 were detected by ELISA kit and qPCR.Results1.Evidence-based medicine results①This meta-analysis comprises 39 articles,29 of which were in English and 10 in Chinese,published between June 2004 and October 2021 in China,involving a total of 1,361 animals.The meta-analysis encompassed 4 surgical modeling methods,namely 21 using 2VO.7 using 4VO.3 using EO.and 8 using MCAO.Of the included literature,22 clearly explicated the total number of modeling and the number of successful modeling.Based on statistical data,the MCAO modeling exhibited a success rate of 87.86%,while the 2VO modeling demonstrated a success rate of 81.52%.The EO modeling.on the other hand.yielded a success rate of 78.75%,whereas the 4VO modeling resulted in a success rate of 72.41%.Among the reviewed literature,36 articles provided information on the rest period following the modeling procedure,while three articles did not report such information.The rest period after surgery was predominantly observed to be 7 days(22.22%),followed by 3 days(30.56%)and 10 days(13.89%).②Summarizing the frequency of acupoint use,Baihui(GV20),Dazhui(GV14)and Zusanli(ST36)had the highest frequency of use.The combination of acupoints was summarized,and the combination of Baihui+Zusanli(GV20+ST36),Baihui+Dazhui(GV20+GV14),and Baihui+Shenting(GV20+GV24)had the highest frequency of use,28.21%,15.38%,and 15.38%,respectively.Fifteen articles incorporated sham acupuncture or nonacupoint studies,with nine of them utilizing bilateral hypochondria,10 mm above iliac crest,three articles using 2 cm above anterior superior iliac spine,two articles utilizing bilateral hypochondria,3 mm above iliac crest,and one article using thoraco-abdominal junction of the first and second lumbar vertebrae.The treatment time of each acupuncture and the total course of treatment were summarized,with 38 literatures providing clear explanations of the treatment time,while the remaining literature did not provide a clear explanation.The primary treatment time was 20 min(23.68%),30 min(28.95%),30 s(21.05%),and 10 min(18.42%).All articles stated the number of days for the entire course of treatment,mainly 14 days(46.15%),15 days(10.26%),and 10 days(10.26%).③The Morris Water Maze network meta-analysis yielded results indicating that the acupuncture group exhibited superiority over the impaired group,but inferiority to both the normal and sham impaired groups.Furthermore,the impaired group displayed inferiority to the normal,sham impaired,and medicine groups.The normal and sham impaired groups demonstrated superiority over the non-acupoint group,while no significant statistical difference in swimming speed was observed among all groups.It is noteworthy that the acupuncture and medicine groups did not exhibit any discernible differences.④The results of the subgroup analysis on escape latency suggest that acupuncture at Baihui and Zusanli acupoints(GV20+ST36),manual acupuncture(MA).and a 14-day treatment regimen demonstrate greater heterogeneity and Z value in comparison to other subgroups.This approach may be deemed suitable for animal studies.⑤The findings of the subgroup analysis on diverse surgical techniques in escape latency indicate that distinct surgical modeling approaches can generate animal models of vascular dementia.Additionally,the efficacy of acupuncture was observed in VD rats across various modeling techniques.2.Animal experimental study results(1)Behavioral results of electroacupuncture at Baihui and Zusanli in improving cognitive dysfunction in VD mice①Results from the Morris Water Maze included escape latency,number of original platform crossings,time spent in target quadrant,and swimming speed.In the results of escape latency,the test showed differences on the 5th day in some groups of mice,in which the escape latency was shortened in the S2VO group compared with the 2VO group(P<0.05),the escape latency was shortened in the S2VO group compared with the SEA group(P<0.01),the escape latency was shortened in the EA group compared with the 2VO group and the DON group(P<0.05),the escape latency was shortened in the EA group compared with the SEA group(P<0.01),and the escape latency was shortened in the DON group compared with the SEA group(P<0.05).In the number of original platform crossings,the number of platform crossings increased in the S2VO group compared with the SEA group(adj.P=0.025),and increased in the EA group compared with the SEA group(adj.P=0.005).In the time spent in target quadrant,there were differences among S2VO,2VO,EA and DON groups compared with SEA group(P<0.05).In swimming speed.there was no difference between all groups(adj.p=0.539).②In the Novel Object Recognition experiment,there was no difference among S2VO,2VO,EA,SEA and DON groups(P>0.05),however,the novel object recognition outcomes in the S2VO.EA and DON was higher than that in the other two groups.(2)Electroacupuncture at Baihui and Zusanli Inhibits Apoptosis of Hippocampal Neurons in VD Mice Based on TGF-β1 Related Pathways①Electroacupuncture at Baihui and Zusanli Reverses Hippocampal Neuronal Injury in VD MiceThe EA group exhibited a statistically significant increase in the number of Nissl positive neurons in the CA1 region of the hippocampus when compared to the 2VO group(adj.P=0.039).Similarly,the S2VO,SEA,EA,and DON groups demonstrated a significant increase in the number of Nissl positive neurons in the CA3 region of the hippocampus when compared to the 2VO group(P<0.05).②Electroacupuncture at Baihui and Zusanli Inhibits Apoptosis of Hippocampal Neurons in VD MiceThe S2VO group exhibited a significant reduction in TUNEL-positive neuronal cells in the CA1 region compared to the 2VO.SEA.and DON groups(P<0.01).Similarly,the EA group demonstrated a significant decrease in TUNEL-positive neuronal cells in the CA1 region compared to the 2VO and DON groups(P<0.05).However,no significant difference was observed in TUNEL-positive neuronal cells in the CA3 and DG regions among the groups of mice(adj.P>0.05).③Electroacupuncture at Baihui and Zusanli regulates TGF-β1-related pathwayCompared with the 2VO group,the protein expression of Bax in the hippocampus of S2VO,EA,and DON was significantly reduced(P<0.01),and the EA and DON groups still reflected differences when compared with SEA(P<0.05).Compared with 2VO and SEA groups,the protein expression of Bcl-2 was increased in S2VO,EA and DON hippocampal tissues(P<0.05),and the protein expression of Bcl-2 was significantly increased in EA and DON groups compared with SEA(P<0.01),and the advantages of S2VO,EA and DON groups were also reflected in cleaved caspase-3(P<0.05).Compared with the 2VO group.the protein expression of TGF-β1,p-TGFβR1,and pSMAD2/3 was reduced in the hippocampal tissues of the S2VO,EA,and DON groups(P<0.05),and in addition,the protein expression of TGF-β1 was reduced in the hippocampal tissues of the EA and DON groups compared with the SEA group(P<0.05).There was no difference in the comparison of the above three proteins between the S2VO.EA,and DON groups(P>0.05).Compared with 2VO group,S2VO group and DON group could decrease the expression of TGF-β1 mRNA(P<0.05),and there was no difference among 2VO,SEA and EA groups(P>0.05).(3)Electroacupuncture at Baihui and Zusanli inhibits hippocampal neuroinflammation①The expression of IL-1β in the S2VO.EA,and DON groups exhibited a significant decreasing trend in comparison to the 2VO group(P<0.05).Moreover,the expression of IL-1β was significantly lower in the DON group than in the EA group(P<0.05).Regarding IL-6,the expression in the S2VO and EA groups displayed a significant decreasing trend relative to the 2VO group(P<0.05).However,no significant differences were observed in TNF-α among all groups(adj.P>0.05).With respect to IL-4,the expression in the S2VO,EA,and DON groups was significantly increased in comparison to the 2VO group(P<0.01).Additionally,the EA and DON groups exhibited a significant increase in expression compared to the SEA group(P<0.01).The expression of the S2VO.EA,and DON groups on IL-10 exhibited a significant increase in comparison to both the 2VO and SEA groups(P<0.01).Furthermore,the DON group’s expression demonstrated a significant increase in comparison to the SEA group(P<0.05).②The mRNA expression of IL-1β exhibited a decrease in the EA,and DON groups when compared to the 2VO and SEA groups(P<0.05),the expression of S2VO was significantly decreased(P<0.01).Similarly,the mRNA expression of IL-6 demonstrated a difference in the S2VO and EA groups in comparison to the 2VO and SEA group(P<0.05),but no difference was observed between the DON group and both the 2VO and SEA groups.Notably,no significant differences were observed in TNF-α among all groups(P>0.05).Furthermore.the mRNA expression of IL-4 and IL-10 did not exhibit any differences between all groups(P>0.05).③The CA1 region of the hippocampus in the 2VO group exhibited a notable increase in the expression of TGF-β1(green)and Iba-1(red),while the CA3 and DG regions displayed equivalent TGF-β1(green)expression across all groups.However,the CA3 and DG regions of the hippocampus in both the 2VO and SEA groups demonstrated a marked increase in lba1(red)expression.Conclusion1.The effectiveness of acupuncture treatment in an animal model of vascular dementia surpassed that of non-acupuncture interventions.Specifically,in the 2VO model,a 14-day treatment course at Baihui and Zusanli acupoints following a 7-day post-surgical rest period yielded superior efficacy.2.Electroacupuncture at the acupoints of Baihui and Zusanli could enhance cognitive function and mitigate neuronal pathological alterations in mice models of vascular dementia.3.Electroacupuncture at Baihui and Zusanli points could mitigate neuronal damage in mice with vascular dementia.This effect was achieved through the suppression of neuroinflammation and apoptosis in the CA1 region of the hippocampus,and was likely mediated by the inhibition of TGF-β1 pathways that were prone to excessive activation.