Study On The Mechanism Of Different Treatment Methods Of Moxa Combustion Products In Intervening Reverse Cholesterol Transport In ApoE^-/- Mic

Background:atherosclerosis(AS)is the primary cause of cardiovascular and cerebrovascular diseases such as coronary heart disease,heart failure,and stroke,and has become a hotspot in the cardiovascular field.Atherosclerosis is a kind of chronic inflammatory disease characterized by lipid deposition in the arterial wall.Therefore,the increase of cholesterol content in the blood is an important pathological basis for the occurrence and development of AS.Reverse Cholesterol Transport refers to the process in which cholesterol flows out of peripheral histiocytes and is transported to the liver through blood for metabolism.It is an important pathway for regulating lipid levels in the body and a key step to maintain cholesterol homeostasis.Therefore,reverse cholesterol transport-related genes are important targets for anti-atherosclerosis.The moxa combustion products refer to the products of moxa floss after burning,which also known as moxa smoke.Moxa smoke is one of the effective factors of moxibustion.Our research team previously found that moxa smoke can regulate lipid metabolism and improve aortic pathology in AS mice,and the mechanism may be relevant to the regulation of reverse cholesterol transport by moxa smoke.However,the active ingredients of moxa smoke against atherosclerosis remain unclear.In this study,the moxa combustion products were treated with different methods to form different components,namely conventional moxa smoke,filtered moxa smoke,and moxa floss volatilization.The effect of improving atherosclerosis in different moxa smoke groups was observed to elucidate the main effective components of moxa combustion products.Objectives:to observe the effects of moxa combustion products processed under different conditions on blood lipid levels,pathological morphology of liver and thoracic aorta,and indicators related to reverse cholesterol transport in ApoE-/-mice,and to compare the therapeutic effects of conventional moxa smoke,filtered moxa smoke,and moxa floss volatilization,in order to explore the forms of effective components in moxa smoke.Methods:ten 8-week-old C57BL/6 mice were used as the blank group,and 40 ApoE-/mice of the same age were randomly divided into the model group,conventional moxa smoke group,filtered moxa smoke group,and mugwort floss volatilization group.The blank and model group mice were not intervened except for grasping and fixing.The mice in the conventional moxa smoke group were exposed to moxa smoke at concentrations ranging from 10 mg/m3 to 15 mg/m3.The mice in the filtered moxa smoke group were exposed to the moxa smoke which was pre-filtered with Cambridge filters.The mice in the mugwort floss volatilization group were exposed to an environment containing the volatilization of heating mugwort floss.All interventions were conducted in an exposure cabinet,20 minutes per day,6 days per week,for a total of 14 weeks.After the experiment,serum TC,TG,HDL-C,and LDLC levels were measured using the biochemical method;the Elisa method was used to measure the levels of serum ox-LDL,ApoA1,LCAT,and PLTP;HE staining,transmission electron microscopy,and oil red "O" staining were used to observe the pathological morphology of the aorta and liver.RT-PCR and immunohistochemical methods were used to detect the mRNA and protein expression levels of the PPARγ-LXRα-ABCA1/ABCG1 signaling pathway in the aorta and liver of mice in each group,as well as the mRNA and protein expression levels of liver SRB1,LDLR,ABCG5,ABCG8,and CYP7A1,which are related to liver cholesterol metabolism.Results:1.Body weight and blood lipid levels of miceBefore the intervention,compared with the blank group,the weight of mice in the model group,conventional moxa smoke group,filtered moxa smoke group,and mugwort floss volatilization group was increased significantly(P<0.05).After 14 weeks of intervention,compared with the blank group,the model group mice showed a significant increase in body weight(P<0.05).Compared with the model group,the weight of mice in the conventional moxa smoke group was significantly decreased(P<0.05).Compared with the blank group,the serum TC,HDL-C,LDL-C,and ox-LDL-C contents in the model group were significantly increased(P<0.05),while the serum ApoAl content in the model group was significantly reduced(P<0.05).There was no statistically significant difference in TG content(P>0.05).Compared with the model group,the serum levels of TC,TG,and LDL-C in the conventional moxa smoke group were significantly reduced(P<0.05),while the ApoA1 content was significantly increased(P<0.05).There was no statistically significant difference in the levels of HDL-C and ox-LDL(P>0.05).Compared with the model group,the serum TC and LDL-C content of the mice in the filtered moxa smoke group were significantly decreased(P<0.05),while the ApoA1 content was significantly increased(P<0.05).There was no statistically significant difference in TG,HDL-C,and ox-LDL content(P>0.05).Compared with the model group,the serum TC content of the mice in the mugwort floss volatilization group was significantly reduced(P<0.05),while the ApoAl content was significantly increased(P<0.05).There was no statistically significant difference in other indicators(P>0.05).Compared with the conventional moxa smoke group,the serum ApoAl content of mice in the filtered moxa smoke group and the mugwort floss volatilization group was significantly decreased(P<0.05).There was no statistically significant difference in other indicators(P>0.05).Compared with the filtered moxa smoke group,there was no statistically significant difference in all indicators in the mugwort floss volatilization group mice(P>0.05).2.Pathological morphology of aorta and liver in miceThe morphology of the aorta and liver cells in the blank group mice showed no obvious abnormalities.In the model group,there were obvious AS pathological changes in the aorta,such as lipid deposition,plaque formation,intimal thickening,lumen stenosis,etc.,and significant steatosis and lipid deposition were observed in the liver.The pathological morphology of the aorta and liver in the conventional moxa smoke group,filtered moxa smoke group,and mugwort floss volatilization group showed varying degrees of improvement compared to the model group.The improvement ranked from high to low as the conventional moxa smoke group,filtered moxa smoke group,and mugwort floss volatilization group.3.Indicators related to reverse cholesterol transport in miceThe mRNA and protein expression levels of PPARγ-LXRα-ABCA1/ABCG1 signaling pathway in the aorta and liver of mice in each group:Compared with the blank group,the mRNA expression of ABCA1 and ABCG1 in the aorta of the model group mice was significantly increased(P<0.05),and the mRNA expression of ABCA1 and ABCG1 in the liver was significantly increased(P<0.05).There was no statistically significant difference in other indicators(P>0.05).Compared with the model group,the mRNA expression of PPARγ,LXRα,ABCA1,ABCG1 in the aorta in the conventional moxa smoke group mice was significantly increased(P<0.05),while the protein expression of LXRα,ABCA1 and ABCG1 was significantly increased(P<0.05).The mRNA expression of liver LXRa and ABCG1 was significantly increased(P<0.05),and the protein expression of ABCGI was significantly increased(P<0.05).There was no statistically significant difference in other indicators(P>0.05).Compared with the model group,the mRNA expression of PPARy,ABCA1 and ABCG1 in the aorta of filtered moxa smoke group mice was significantly increased(P<0.05),and the protein expression of ABCA1 was significantly increased(P<0.05).There was no statistically significant difference in other indicators(P>0.05).Compared with the model group,there was no statistically significant difference in any indicators of the mice in the mugwort floss volatilization group(P>0.05).Compared with the conventional moxa smoke group,the protein expression of ABCA1 and ABCG1 in the aorta of mice in the filtered moxa smoke group was significantly reduced(P<0.05),and the mRNA expression of liver LXRa was significantly decreased(P<0.05),and there was no statistically significant difference in other indicators(P>0.05).Compared with the conventional moxa smoke group,the mRNA expression of ABCA1 and ABCG1 in the aorta of mice in the mugwort floss volatilization group was significantly reduced(P<0.05),while the protein expression of LXRα,ABCA1 and ABCG1 was significantly decreased(P<0.05).In Liver,the mRNA expression of LXRα was significantly decreased(P<0.05),while the protein expression of ABCG1 was significantly decreased(P<0.05),and there was no statistically significant difference in other indicators(P>0.05).Compared with the filtered moxa smoke group,the protein expression of ABCA1 in the aorta of mice in the mugwort floss volatilization group was significantly reduced(P<0.05),while there was no statistically significant difference in other indicators(P>0.05).Serum LCAT and PLTP levels in each group of mice:Compared with the blank group,the model group mice showed a significant decrease in serum LCAT content(P<0.05),and a significant increase in PLTP content(P<0.05).Compared with the model group,the serum LCAT content of the conventional moxa smoke group mice was significantly increased(P<0.05),while the PLTP content was significantly reduced(P<0.05).Compared with the model group,the serum LCAT content of mice in the filtered moxa smoke and mugwort floss volatilization group was significantly increased(P<0.05),while the difference in PLTP content was not statistically significant(P>0.05).Besides,there was no statistically significant difference in the content of LCAT and PLTP in any groups of moxa combustion products(P>0.05).The mRNA and protein expression levels of liver cholesterol metabolism-related indicators SRB1,LDLR,ABCG5,ABCG8,and CYP7A1 in each group of mice:Compared with the blank group,the mRNA expression of LDLR in the liver of the model group mice was significantly increased(P<0.05),while there was no statistically significant difference in other indicators(P>0.05).Compared with the model group,the mRNA expression of SRB1,LDLR,ABCG8,and CYP7A1 in the liver of mice in the conventional moxa smoke group was significantly increased(P<0.05),while the protein expression of SRB1,LDLR,ABCG8,and CYP7A1 was significantly increased(P<0.05).There was no statistically significant difference in other indicators(P>0.05).Compared with the model group,the mRNA expression of SRB 1,ABCG8,and CYP7A1 in the liver of filtered moxa smoke mice was significantly increased(P<0.05),while the protein expression of SRB1,LDLR,ABCG8,and CYP7A1 was significantly increased(P<0.05).There was no statistically significant difference in other indicators(P>0.05).Compared with the model group,there was no statistically significant difference in any indicators of the mice in the mugwort floss volatilization group(P>0.05).Compared with the conventional moxa smoke group,there was no statistically significant difference in any indicators in the filtered moxa smoke group mice(P>0.05).Compared with the conventional moxa smoke group,the mRNA expression of SRB1,LDLR,ABCG8,and CYP7A1 in the liver of mice in the mugwort floss volatilization group was significantly reduced(P<0.05),while the protein expression of LDLR,ABCG8,and CYP7A1 was significantly reduced(P<0.05).There was no statistically significant difference in other indicators(P>0.05).Compared with the filtered moxa smoke group,the expression of ABCG8 mRNA and CYP7A1 protein in the liver of the mugwort floss volatilization group mice were significantly reduced(P<0.05),while there was no statistically significant difference in other indicators(P>0.05).Conclusions:1.The moxa combustion products processed under different conditions can regulate blood lipid levels,improve the pathological morphology of the aorta and liver,and regulate the expression of reverse cholesterol transport related indicators in atherosclerosis mice to varying degrees.2.The efficacy of conventional moxa smoke and filtered moxa smoke is similar,indicating that the filtering particulate matter has no significant impact on the efficacy of moxa smoke.3.The effect of conventional moxa smoke is better than that of mugwort floss volatilization,indicating that some key effective substances are generated after moxa floss combustion.