| Background:The effect of moxa combustion products(MCP)is one of the mechanisms of moxibustion.MCP refers to the product generated in the process of moxa combustion,and moxa fume is the most important part.MCP contains a certain amount of particulate matter(PM).Studies show that the average mass concentration of PM10 in moxibustion clinic is 3.54 mg/m3,which exceeds the maximum limit value(0.15 mg/m3)specified in the health standard of waiting room(GB9671-1996).The existing certain proportion of PM2.5 in MCP particles causes concern while whether the particles in MCP are the key factors of moxibustion efficacy has not been studied.In recent years,the medical market has also seen the treatment and alternative methods and even medical equipment of moxa,such as acupoint daubing with moxa leaf essential oil,acupoint hot ironing with moxa floss,filtering moxa smoke,etc.Thus,we extract MCP products in different stages of moxa floss combustion process,to explore the key effective link of moxibustion,and to study whether the particles in moxa smoke are the key material of moxibustion.Atherosclerosis(AS)is the main pathological basis of cardiovascular and cerebrovascular diseases.Endothelial dysfunction(ECD)is the initial link of AS,and a key factor affecting the progress of AS.Nitric oxide(NO)is the most important vasoactive substance in ECD.The synthesis of NO is regulated by the activated endothelial nitric oxide synthetase(eNOS).The research shows that moxibustion and moxa can adjust the balance of NO/ET-1 and PGI2/TXA2,correct the abnormal hemorheology,improve the function of endothelial cells,and reduce the pathological changes of AS.Therefore,it is necessary to study the regulation of AS by eNOS activation in order to explore the mechanism of moxibustion.Confirmed by our research team in the early stage,could AS in apolipoprotein E-knockout(ApoE-/-)mice could be prevented and treated by the intervention of moxibustion or moxa smoke,by benign regulating lipid metabolism,reducing inflammatory response and endothelial damage.Objectives:To observe the effectiveness of MCP processed under different conditions(moxa smoke,filtered moxe smoke,volatile components of moxa floss and essential oil of Artemisia Argyi)on AS.The aim is to explore the effective link of moxibustion and the mechanism when treating atherosclerosis,in terms of the effect on vascular endothelial function from the signal pathway of eNOS activation,and to explore whether the particles in MCP are the key components of moxibustion.Methods:60 male 8-week-old ApoE-/-mice were randomly divided into 5 groups:moxa smoke group,filtered moxa smoke group,volatile components of moxa floss group,essential oil of Artemisia Argyi group,model group and 12 male 8-week-old C57BL/6 mice as the control group.The mice in the control group and model group were regularly grabbed and fixed;the mice in moxa smoke group were exposed to 2%concentration of moxa smoke,burning about 1.5g of moxa floss;the mice in filtered moxa smoke group were exposed to filtered moxa smoke environment,burning about 1.5 g of moxa floss;the mice in volatile components of moxa floss were exposed to volatile components of 1.5 g moxa floss heating.The mice in essential oil of Artemisia Argyi were exposed to 3.75 ul of Artemisia Argyi essential oil(1.5g of the volatile oil in moxa floss)and 16mL of distilled water.All interventions were carried out in the cabinet,20 mins per day,6 days per week,for 14 weeks.Serum TC,TG,HDL-C,LDL-C and NO were measured by biochemical method;serum ox-LDL,ApoA-I,ET-1,PGI2,TXA2,VEGF and vWF were measured by ELISA method;pathological changes of aortic root and thoracic aorta were observed by HE staining and oil red "O" staining;The expression of AMPK,PI3K,AKT and eNOS protein,the expression of p-AMPK、p-PI3K、p-AKT、p-eNOS-Thr495、p-eNOS-Ser1177 protein were detected by western blot and the expression of Ampk-mRNA,Pi3k-mRNA,Akt-mRNA,eNos-mRNA were detected by RT-PCR.Results:1.Lipids levelThe contents of TC,TG,HDL-C,LDL-C and ox-LDL in the model group were significantly higher than those in the control group(P<0.01 or P<0.05),and there was no significant difference in ApoA-I between the model group and the normal group(P>0.05).The contents of TC,TG,LDL-C and ox-LDL in plasma of mice in moxa smoke group were significantly lower than those in model group(P<0.01 or P<0.05),and the contents of HDL-C were significantly higher than those in model group(P<0.01),The contents of APOA-I was not significantly different from those in the model group(P>0.05).The contents of TC,TG and LDL-C in filtered moxa smoke group were significantly lower than those in the model group(P<0.01 or P<0.05),and the contents of HDL-C were significantly higher than those in the model group(P<0.01).The contents of APOA-I and ox-LDL were not significantly different from those in the model group(P>0.05).The content of TC in the plasma of mice in volatile components of moxa floss group was significantly lower than that in the model group(P<0.01).There was no significant difference in the contents of TG,HDL-C,LDL-C,apoA-I and ox LDL between the two groups(P>0.05).There was no significant difference in the contents of TC,TG,HDL-C,LDL-C,ox-LDL and ApoA-I between the model group and the essential oil of Artemisia Argyi group(P>0.05).Compared with the MCP groups,the effects of moxa smoke group,filtered moxa smoke group,volatile components of moxa floss group and the essential oil of Artemisia Argyi group in lowering plasma TC level decreased in turn,and there were statistical differences between the groups.In addition to TC,there was no significant difference between moxa smoke group and filtered moxa smoke group in regulating other blood lipid levels;moxa smoke group and filtered moxa smoke group were significantly better than essential oil of Artemisia Argyi group in reducing plasma TG and HDL-C levels;moxa smoke group was significantly better than volatile components of moxa floss group and essential oil of Artemisia Argyi group in reducing plasma LDL-C levels;moxa smoke group was significantly better than essential oil of Artemisia Argyi group in reducing plasma ox-LDL levels.2.Aortic pathologyNo obvious abnormality was found in the control group;obvious pathological changes of AS were found in the model group(plaque formation);moxa smoke group,filtered moxa smoke group and volatile components of moxa floss group were significantly improved compared with the model group(plaque formation is significantly reduced),and the essential oil of Artemisia Argyi group was not significantly improved compared with the model group(plaque formation).3.Endothelial functionThe level of NO,NO/ET-1 and PGI2 in the model group was significantly lower than that in the control group(P<0.01 or P<0.05),the level of ET-1,TXA2,TXA2/PGI2(T/P)in the model group was significantly higher than that in the control group(P<0.01 or P<0.05),and there was no significant difference in VEGF and vWF between the model group and the control group(P>0.05),The levels of NO,PGI2 and NO/ET-1 were significantly higher in moxa smoke group than those in model group(P<0.01),the contents of ET-1 and T/P were significantly lower than those in model group(P<0.01),and the contents of TXA2,VEGF and vWF were not significantly different from those in model group(P>0.05).The level ofNO and NO/ET-1 in filtered moxa smoke group was significantly higher than that in the model group(P<0.05),the content of ET-1 and T/P were significantly lower than that in the model group(P<0.01),and the content of TXA2,PGI2,VEGF and vWF were not significantly different from that in the model group(P>0.05).The level of NO and NO/ET-1 in the plasma of the mice in volatile components of moxa floss group was significantly higher than that in the model group(P<0.05),the content of ET-1 and T/P in the plasma of the mice was significantly lower than that in the model group(P<0.01,P<0.05),and the content of TXA2,PGI2,VEGF and vWF in the model group had no significant difference(P>0.05).The content of PGI2 in plasma of mice in essential oil of Artemisia Argyi group was significantly higher than that in model group(P<0.05).The ratio of T/P in plasma was significantly lower than that in model group(P<0.01).The content of NO,ET-1,NO/ET-1,TXA2,VEGF and vWF in plasma was not significantly different from that in model group(P>0.05).Compared within the MCP groups,the increase of NO in filtered moxa smoke group was significantly higher than that in essential oil of Artemisia Argyi group,the ET-1 in filtered moxa smoke group and volatile components of moxa floss group was significantly lower than that in the moxa smoke group,filtered moxa smoke group and the volatile components of moxa floss group,the increase of NO/ET-1 in the moxa smoke group,filtered moxa smoke group and volatile components of moxa floss group was significantly higher than that in essential oil of Artemisia Argyi group,the increase of PGI2 in moxa smoke group was significantly higher than that in filtered moxa smoke group and volatile components of moxa floss group,the decrease of T/P ratio in the aiyan group was significantly lower than volatile components of moxa floss group.There was no significant difference in TXA2,VEGF and vWF between the four groups.4.eNOS activation related signaling pathwaysignal path.p-AKT,p-AKT/AKT of aorta in model group were significantly higher than those in control group(P<0.01)and p-eNOS-Ser1177,p-eNOS-Ser1177/eNOS,p-AMPK,p-AMPK/AMPK,p-PI3K,p-PI3K/PI3K,Ampk-mRNA,PI3K-mRNA,eNos-mRNA were significantly higher than those in normal group(P<0.01 or P<0.05).p-eNOS-Thr1177,p-eNOS-Thr1177/eNOS,p-AMPK,p-AMPK/AMPK,Ampk-mRNA,eNos-mRNA of aorta in moxa smoke group were significantly higher than those in model group(P<0.01 or P<0.05).p-eNOS-Thr495,p-eNOS-Thr495/eNOS,p-AKT,p-AKT/AKT of aorta in moxa smoke group were significantly lower than those in model group(P<0.01).p-eNOS-Thr1177,p-eNOS-Thr1177/eNOS,p-AMPK,p-AMPK/AMPK,Ampk-mRNA,eNos-mRNA of aorta in filter moxa smoke group were significantly higher than those in model group(P<0.01 or P<0.05).p-eNOS-Thr495,p-eNOS-Thr495/eNOS,p-eNOS-Ser1177,p-eNOS-Ser1177/eNOS,p-AKT,p-AKT/AKT of aorta in filter moxa smoke group were significantly lower than those in model group(P<0.01).Ampk-mRNA,eNos-mRNA of aorta in volatile components of moxa floss group were significantly higher than those in model group(P<0.05).p-eNOS-Thr495,p-eNOS-Thr495/eNOS,p-eNOS-Thr1177,p-AKT,p-AKT/AKT of aorta in volatile components of moxa floss were significantly lower than those in model group(P<0.01).p-eNOS-Thr495,p-eNOS-Thr495/eNOS,p-AKT,p-AKT/AKT of aorta in essential oil of Artemisia Argyiere were significantly higher than those in model group(P<0.O1 or P<0.05).Conclusions:1.Moxa smoke and filtered moxe smoke can regulate lipid metabolism and improve endothelial cell function,so as to slow down the progress of AS,and they have the similar effect.It is suggested that the particles in moxa smoke have no significant effect in this process,and may not be the key component of moxibustion.2.Compared to moxa smoke,a certain concentration of volatile components of moxa floss and essential oil of Artemisia Argyi is not effective in regulating lipid metabolism,alleviating aortic disease and improving endothelial cell function,which suggests that the burning process of moxa floss may be the key link of moxibustion.3.One of the mechanisms of improving endothelial function of moxa smoke and filtered moxe smoke may be through activating the phosphorylation of AMPK protein,promoting the phosphorylation of Ser1177 and dephosphorylation of Thr495 of eNOS protein,and improving the level of NO to regulate vasoactivity. |
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