| Atherosclerosis(AS)is the main pathological basis of many cardiovascular and cerebrovascular diseases.It is a chronic pathological process characterized by atherosclerotic plaque and belongs to the category of "phlegm" and "blood stasis" in traditional Chinese medicine.Moxibustion has unique advantages in the prevention and treatment of AS because of its characteristics of Warming Yang,activating blood circulation and removing blood stasis.Modern research has proved that moxibustion can improve lipid metabolism,inflammatory response,oxidative stress,platelet activation and endothelial cell function,and play a role in multi-target treatment of AS.Moxa smoke is one of the important factors for moxibustion to take effect.The previous research of our research group also proved the role of moxa smoke in anti AS.Rho/ROCK signaling pathway is widely distributed in the human body.Abnormal activation of this pathway can activate platelets,damage endothelial function,promote macrophage foam,inhibit reverse cholesterol transport,aggravate inflammatory reaction and oxidative stress,and have a deep connection with the pathogenesis of AS.It is also a new target and focus in anti AS therapy in recent years.The previous study of the research group found that moxibustion can improve the reverse cholesterol transport and inflammation.Therefore,the study of moxibustion intervention on Rho/ROCK signal pathway,is to further deepen the previous results of the research group,and it is of great significance to reveal the mechanism of moxibustion prevention and treatment of AS.Based on the above,we propose a hypothesis that moxibustion and moxa smoke may play a role in the prevention and treatment of AS by regulating Rho/ROCK signal pathway,regulating lipid metabolism and reverse cholesterol transport,and alleviating inflammatory response.Objective:To observe the effect of moxibustion and moxa smoke on the AS routine indexes of ApoE-/-mice,such as lipid metabolism and thoracic aorta pathology,and to verify the anti AS effect of moxibustion and moxa smoke by comparing the positive drug simvastatin.From the Rho/ROCK signal pathway as a starting point,the potential mechanism of moxibustion and moxa smoke improving as was discussed by comparing with the ROCK inhibitor fasudil.Methods:In the first two chapters,60 8-week-old male ApoE-/-mice were randomly divided into four groups:model group,moxibustion group,moxa smoke group and simvastatin group,which were fed with high-fat diet.15 non transgenic C57BL/6 mice of 8-week-old male were used as the blank control group and fed with common feed.The mice in the blank group and the model group were fixed with fixators,which were grabbed and fixed for 20 minutes every day;the mice in the moxibustion group were fixed with fixators,which were moxibustion in the Danzhong(CV17)point for 20 minutes;the mice in the moxa smoke group were fixed with fixators,which were put into the moxa smoke environment of 5-15 mg/m3 for 20 minutes;the mice in the simvastatin group were given 2.8 mg/kg each day,and each group was intervened for 6 days per week.After 14 weeks of continuous intervention,the serum TG,TC,LDL-C,HDL-C lipid metabolism related indexes were detected by biochemical method,ApoAl,ox-LDL and other lipoproteins were detected by ELISA,the pathological changes and structural changes of thoracic aorta plaques were observed by HE staining and oil red "O" staining.In the latter two chapters,the third intervention method was changed from simvastatin to fasudil,and the other mice strains,feeding,grouping and intervention were consistent with the former two chapters.The intervention method and experiment period are consistent with the former two chapters.Immunohistochemistry,Western blot and RT-PCR were used to detect the positive area ratio,protein expression level and mRNA expression of Rho,ROCK2,PPAR ?,LXR ? and ABCA1 in thoracic aorta,and ELISA was used to detect the levels of TGF-?1,IL-6,IL-10 and IL-17 and other inflammatory factors.Result:1.Lipids and lipoproteinsThe contents of TC,TG,LDL-C,HDL-C and ox-LDL in the model group were significantly higher than those in the blank group(P<0.01),and the contents of ApoA1 in the model group were significantly lower than those in the blank group(P<0.01);the contents of TC,LDL-C and ox LDL in the moxibustion group were significantly lower than those in the model group(P<0.01),and the contents of HDL-C in the moxibustion group were significantly higher(P<0.01),and the contents of ApoAl were higher(P<0.05).The contents of TC,LDL-C and ox LDL in the serum of mice in moxa smoke group were significantly lower than those in the model group(P<0.01),the contents of TG were lower but no significant difference(P>0.05),the contents of HDL-C were higher(P<0.05),the contents of ApoAl were significantly higher(P<0.01);the contents of TC,TG and LDL-C in the serum of mice in simvastatin group were significantly lower than those in the model group(P<0.01),the content of ox-LDL decreased but no statistical difference(P>0.05),HDL-C increased but no statistical difference(P>0.05),ApoAl increased significantly(P<0.01).2.Pathological observation of thoracic aortaHE and oil red "O" staining showed that atherosclerotic plaques were the most serious lesions in the model group,and the lumen of aorta was narrowed.Foam cells,inflammatory cell infiltration,cholesterol crystallization and intimal hyperplasia were observed in the plaques.The pathological improvement of moxibustion group and simvastatin group was relatively significant.Compared with the model group,the area of atherosclerotic plaque was smaller,the infiltration of inflammatory cells was less,and the proliferation of vascular smooth muscle in the middle membrane was lighter.There was no significant difference between the two groups.The aorta lumen of mice in moxa smoke group was narrowed,and atherosclerotic plaques were seen.Infiltration of foam cells and inflammatory cells was observed in plaques.3.Rho/ROCK signal pathwayImmunohistochemical results showed that the expression and positive area of Rho and ROCK2 in aorta of model group increased compared with that of blank group(P<0.01);compared with model group,the expression and positive area of Rho and ROCK2 in moxibustion group,moxa smoke group and fasudil group decreased(P<0.01).Western blot showed that the expression of Rho and ROCK2 protein in the model group was significantly higher than that in the blank group(P<0.01);compared with the model group,the expression of Rho and ROCK2 protein in the moxibustion group,moxa smoke group and fasudier group was significantly lower(P<0.01).RT-PCR results showed that the mRNA expression of Rho and ROCK2 in the model group was significantly higher than that in the blank group(P<0.01);compared with the model group,the mRNA expression of Rho and ROCK2 in the moxibustion group,moxa smoke group and fasudier group was significantly lower(P<0.01).4.Reverse cholesterol transport and inflammatory factorsThe results of immunohistochemistry showed that the expression and positive area of PPARy and LXRa in the model group were significantly lower than those in the blank group(P<0.05),and ABCA1 was decreased but no statistical difference(P>0.05);compared with the model group,the expression and positive area of PPARy and LXRa in the moxibustion group,moxa smoke group and fasudier group were significantly increased(P<0.01),ABCA1 increased but there was no significant difference(P>0.05).The results of Western blot showed that PPARy and LXRa protein content in the model group was significantly lower than that in the blank group(P<0.01),and ABCA1 protein expression was significantly higher than that in the blank group(P<0.01);compared with the model group,PPARy and LXRa protein expression in the moxibustion group,moxa smoke and fasudier group were significantly higher(P<The expression of LXRa protein in the aortas of mice in moxibustion group,moxa smoke group and fasudier group increased significantly(P<0.01).There was no significant difference in ABCA1 protein expression between moxibustion group and fasudil group(P<0.01).RT-PCR showed that the mRNA expression of PPARy,LXRa and ABCA1 in the model group was significantly lower than that in the blank group(P<0.01);compared with the model group,the mRNA expression of PPARy,LXRa and ABCA1 in the moxibustion group,moxa smoke group and fasudier group was significantly higher(P<0.01).The content of TGF-?1 and IL-10 in the model group was lower than that in the blank group(P<0.05),the content of IL-10 was significantly lower than that in the blank group(P<0.01),the contents of IL-6 and IL-17 was significantly higher than that in the blank group(P<0.01,P<0.05),the contents of TGF-?1 and IL-10 in the moxibustion group was significantly higher than that in the model group(P<0.01),the content of IL-6 and IL-17 was significantly lower than that in the model group(P<0.01),The contents of TGF-?1 and IL-10 in serum of mice in moxa smoke group was higher than that in model group(P<0.05),the content of IL-6 was lower than that in model group(P<0.01),the content of IL-17 was lower but no statistical difference(P>0.05);the contents of TGF-?1 and IL-10 in serum of mice in fasudier group was higher than that in model group(P<0.01),the content of IL-6 was lower than that in model group(P<0.05),compared with the model group,IL-17 content decreased significantly(P<0.01).Conclusion:1.Moxibustion and moxa smoke can slow down the occurrence and development of AS by regulating the disorder of lipid metabolism and improving the pathological condition of aortic atherosclerosis.2.Moxibustion and moxa smoke can down regulate the expression of protein and mRNA related to Rho/ROCK signal pathway.3.Moxibustion and moxa smoke can up regulate the related indexes of reverse cholesterol transport,and regulate the level of inflammatory factors in two ways,so as to slow down the pathological process of AS.4.The mechanism of moxibustion and moxa smoke regulating the reverse cholesterol transport and inflammatory response may be related to the regulation of Rho/ROCK signal pathway. |
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