| Backgroud:Atherosclerosis(AS)is the main pathological basis of many cardiovascular and cerebrovascular diseases.It is a chronic pathological process characterized by atherosclerotic plaque.It belongs to the category of "turbid phlegm" and "blood stasis" in the theory of traditional Chinese medicine,which is within the indication scope of moxibustion.There have been a lot of reports on the clinical and mechanism of moxibustion to improve AS,which provides a scientific basis for the effectiveness of moxibustion against AS.Modern researches have proven that moxibustion can improve lipid metabolism,inflammatory,oxidative stress,platelet activation and endothelial cell function in the process of AS.Moxa smoke is one of the important factors of moxibustion effectiveness,and previous researches proved that it plays an irreplaceable role in anti-AS.Autophagy is an important survival promoting mechanism in human body,which is deeply related to various pathogenesis of AS.Acupuncture and moxibustion have been reported to regulate autophagy in the prevention and treatment of cardiovascular disease,but the direct observation of moxibustion regulating autophagy on AS is still in urgent need of research.Reports and our previous studies showed that moxibustion has the effect on autophagy pathway and its related autophagy proteins,and the pre-experimental results also exhibited the effect of moxibustion on autophagy in AS process.Based on the above research basis,we propose the hypothesis that moxibustion and moxa smoke can improve AS by regulating autophagy activity.Objective:To observe the effectiveness of moxibustion and moxa smoke on atherosclerosis from the aspects of pathological changes,lipid metabolism,plaque stability,and to explore the mechanism of moxibustion against AS by regulating autophagy activity and its related signaling pathway.Methods:Fifty-four ApoE apolipoprotein knockout mice aged 8 weeks were randomly divided into three groups according to the random number table:model group,moxibustion group and moxa smoke group.Eighteen C57BL/6 mice of the same age were used as blank control.Mice in the moxibustion group were captured and fixed daily,and were treated with moxibustion at CV 17 for 20 min,once a day,six times a week,for a total of 12 weeks.Mice in moxa smoke group were captured daily and exposed to moxa smoke after fixation.Mice in the model group and the blank control group were fixed daily without any intervention.At week 12,after all the interventions,the mice were executed.Biochemical tests were performed to examine the plasma lipid levels,VLDL,ApoAl and ox-LDL were detected by Elisa,and the aorta sections were observed by oil red "O" and HE staining,respectively.Aortic sections were treated with Masson staining,and plaque stability indexes such as ELA,CA,CD68,FCT and VI were calculated.Using immunohistochemical method to detect TNF alpha,P38MAPK,NF-?B protein expression,and MMP-2,MMP-9 and TIMP-1 were examined by Elisa method.Endothelial autophagosome was observed by transmission electron microscopy,and the expressions of Beclin-1,LC3-I,LC3-II,Atg5,Atg12 and P62 in aorta and liver of mice were detected by Western blot and immunofluorescence.The expressions of autophagy signaling pathways p-Akt,Akt,p-mTOR,mTOR and PI3K were observed by Western blot and RT-PCR,while apoptotic proteins Bcl-2 and Caspase-3 were detected by Western blot.Results:1.Pathological process and lipid level of ASAfter high-fat feeding,mice in the model group had obvious pathological changes of AS(lipid metabolism disorders,AS plaque).Compared with the blank group,serum TC(P<0.01),TG(P<0.01),LDL(P<0.01)and VLDL(P<0.01)were significantly increased,while the contents of ApoAl(P<0.05)and HDL(P<0.01)were significantly decreased.These indicators suggested the sign of successful modeling.Compared with the model group,after 12 weeks of intervention,the pathological changes of mice in moxibustion group were alleviated(aortic plaque became smaller and lipid metabolism disorders were ameliorated).In the moxibustion group,the serum TC(P<0.01),TG(P<0.01),LDL(P<0.05)and VLDL(P<0.01)decreased significantly,while the ApoAl and HDL levels of apolipoprotein increased,but there was no significant difference(P>0.05).Pathological changes of mice in moxa smoke group were alleviated(aortic plaque became smaller and lipid metabolism disorders were ameliorated).Serum TC(P<0.05),TG(P<0.05),and VLDL(P<0.05)of mice in moxa smoke group decreased significantly,and LDL showed a downward trend,but no significant difference was observed(P>0.05).ApoA1 and HDL contents of apolipoprotein increased,but there was no significant difference(P>0.05).2.Plaque stabilityCompared with the model group,after 12 weeks of intervention,the levels of CA(P<0.01)and FAT(P<0.01)in the moxibustion group were significantly increased,while ELA(P<0.01)and the plaque vulnerability index VI(P<0.05)were significantly decreased.The levels of TNF alpha(P<0.05),NF-?B(P<0.05),MMP-9(P<0.05),MMP-2(P<0.05)were significantly decreased,while the levels of TIMP-1(P<0.05)were significantly increased.The levels of CA(P<0.01)and FAT(P<0.01)in moxa smoke group were significantly increased,while ELA(P<0.01)and the vulnerable plaque index VI(P<0.05)were significantly decreased.The levels of inflammatory cytokines TNF alpha(P<0.05),MMP-9(P<0.05),MMP-2(P<0.05)decreased significantly,while the levels of TIMP-1 showed an increased trend,but no significant difference was observed(P>0.05).3.Autophagy levelCompared with the model group,after 12 weeks of intervention,the number of autophagosome and autophagolysosome in aorta of mice in moxibustion group were significantly increased.Western blot showed that Beclin-1(P<0.01),LC3-?/LC3-?(P<0.01)and Atg12(P<0.05)were significantly increased,while P62 were significantly decreased(P<0.05).Immuno:fluorescence showed that Beclin-1(P<0.05)and LC3-?(P<0.05)were significantly increased in the aorta of moxibustion group.Protein expressions of Beclin-1(P<0.05)and LC3-?(P<0.05)in the liver of moxibustion group were significantly increased,however there was no significant difference in P62(P>0.05).Compared with the model group,the number of autophagosomes and autophagolysosomes of aortic of mice in moxa smoke group were significantly increased.Western blot showed that Beclin-1(P<0.01),LC3-?/LC3-?(P<0.05)and Atg12(P<0.05)were significantly increased,while P62 were significantly decreased(P<0.01).Immnofluorescence showed that Beclin-1(P<0.05)and LC3-?(P<0.05)were significantly increased in the aorta of mice in moxa smoke group.The expressions of Beclin-1,LC3-? and P62 in the liver of mice in moxa smoke group were not significantly different from those in the model group(P>0.05).4.PI3K/Akt/mTOR signaling pathwayCompared with the model group,after 12 weeks of intervention,the expressions of p-Akt/Akt(P<0.01),p-mTOR/mTOR(P<0.01)in moxibustion group were significantly increased,while the expressions of PI3K showed no significant difference(P>0.05).Compared with the model group,the expressions of p-mTOR/mTOR(P<0.01)in moxa smoke group were significantly increased,while the expressions of p-Akt/Akt and PI3K showed no significant difference(P>0.05).RT-PCR showed that the results on mRNA were consistent with those on the protein level.5.Apoptotic proteinCompared with the model group,the Bcl-2 protein content in the aorta of moxibustion group was significantly increased(P<0.05)and the Caspase-3 protein content was significantly decreased(P<0.05).Compared with the model group,the content of Caspase-3 protein in the aorta of mice in the moxa smoke group decreased significantly(P<0.05)and the content of Bcl-2 protein increased,but there was no statistical difference(P>0.05).Conclusion:1.Both moxibustion and moxa smoke can improve the pathological progress in AS mice,alleviate the inflammatory reaction,improve dyslipidemia,and enhance the stability of plaque.2.Both moxibustion and moxa smoke can increase the level of autophagy in AS mice,and moxibustion to improve autophagy is a new target of moxibustion for the prevention and treatment of AS.3.Moxibustion and moxa smoke may inhibit active molecules on PI3K/Akt/mTOR signaling pathway,so as to enhance the level of autophagy in cells during AS process.4.By up-regulation of Bcl-2 and down-regulation of Caspase-3 protein expression,moxibustion and moxa smoke can inhibit cell apoptosis and showed a certain role in regulating autophagy/apoptosis balance.5.The regulating effects of moxibustion and moxa smoke intervention were basically the same,but moxibustion showed better results than moxa smoke in lipids indicators. |
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