Exploring The Mechanism Of Action Of Qingzhitongmai Granules On Atherosclerosis Based On The Theory Of "Jueyin Qi Stagnation

Purpose:Guided by the interrelationship between the theory of"Jueyin qi stagnation"and atherosclerosis,we used molecular docking technology and molecular dynamics to explore the potential targets of Qingzhitongmai Granules and AS,discussed the association between S1PR1-LXR and TLR2/4-IRF3 pathways,and explored the regulation mechanism ofQingzhitongmai Granules on Apo E^-/-mice through cholesterol reversal transport and the interrelationship between the window structure of hepatic sinusoidal endothelial cells and liver detoxification function via animal experiment.Through animal experiments,we will explore the regulation mechanism of atherosclerosis in Apo E^-/-mice through cholesterol reversal and the interrelationship between the window structure of hepatic sinusoidal endothelial cells and the function of hepatic excretory function,so as to provide a new target and pathway for the clinical treatment of AS.Materials and Methods:The first part:theoretical discussionThrough reviewing the ancient and modern literature,we explored the interrelationship between"Jueyin qi stagnation"and atherosclerosis from the linkage of Chinese medicine’s disease name,etiology and pathogenesis with modern medicine’s physiology and pathology,and elucidated the foundation of the research on the preliminary study of Qingzhitongmai Granules and its shortcomings.Part two:Molecular mechanism network researchThe potential targets of Qingzhitongmai Granules in treating AS were explored by molecular docking and molecular dynamics.The active components of QingzhitongmaiGranules were obtained by TCMSP database,and the main active components obtained after TCMSP screening were supplemented by TCMID,BATMAN-TCM,Sym Map,CM-MESH database combined with the Chinese databases of China National Knowledge Network,Wanfang and Weipu.Based on the query resμlts of CTD database,five core targets were selected as docking objects according to the Inference Score,whether there is direct medical evidence,and relevant research hotspots.After the binding sites were determined,molecular docking was carried out with the active components of Qingzhitongmai Granules that met the screening criteria using Auto Dock Vina.The conformations with low binding energy and significance for further research in the docking results were further analyzed using molecular dynamics technology.Part three:Crosstalk of LR2/4-IRF3 signaling pathway with LXR protein regulates atherosclerosisTaking lipid metabolism and inflammatory response as an entry point,we will explore the crosstalk between LR2/4-IRF3 signaling pathway and LXR protein,which will provide new targets and pathways for clinical treatment of AS.Part four:experimental research Experiment 1:Seventy-five SPF Apo E^-/-mice were randomly divided into 5 groups,including normal group,model group,optimal dose group A,optimal dose group B,and western medicine treatment group,with 15 mice in each group.Apo E^-/-mice in the normal group were fed conventionally,and the other groups were fed with high-fat diet for 12 weeks（20 weeks due to the COVID-19 outbreak）after 1 week of adaptive feeding.Other conditions were completely the same.The formation of aortic plaque in mice was observed by optical microscope.After the successful modeling was confirmed,the mice were given the drug once a day for 60 days.Normal group and model group were given the same volμme of normal saline intragastric administration,optimal dose group A and optimal dose group B were given Qingzhitongmai Granules（single herbal medicine in the prescription according to the weight of one kilogram）,western medicine group was given atorvastatin calciμm tablets（according to the weight of one kilogram）.During gavage:The model group was still fed with high fat diet.Normal group,optimal dose group A,optimal dose group B and western medicine group were fed ordinary feed.Blood lipid（TC,TG,LDL-C,HDL-C）levels in five groups ofApo E^-/-mice were measured using an automatic biochemical analyser;HE staining and oil red O staining were used for pathological morphological observation of mouse aorta;electron microscopy was used to observe the differences in morphology and number of windowed pore structures of endothelial cells of mouse hepatic sinusoidal cells among groups;ELISA kits were used to determine the serum HDL-S1P levels,aortic S1PR1 levels by RT-PCR,liver tissue LXR levels by RT-PCR and aortic LXRɑ/βprotein levels by Western blot.Experiment 2:The feeding,grouping,modeling,dosage and feeding method of experimental animals,collection of serμm and tissue specimens are the same as experiment 1.HE staining,oil red O staining and blood lipid level determination were the same as experiment 1.TLR2 content in mouse aorta was detected by immunofluorescence method,TLR4 content in mouse aorta was detected by Western blot technique,serμm TLR2/4 level was detected by ELISA,and IRF3content in peripheral blood mononuclear cells was detected by fluorescence quantitative PCR kit.The contents of ABCA1 and ABCG1 in peripheral blood mononuclear cells were detected by Western blot.Statistical methods:Graphpad Prism8.0.2 software was used to draw charts and statistical analysis.Descriptive statistical data were expressed in the form of mean±standard deviation（x±s）.Student’st test was used to compare the mean of two groups,and one-way ANOVA analysis was used to compare the mean of mμltiple groups.When P<0.05,the difference was considered statistically significant.Results:The first part:theoretical discussion1.In traditional Chinese medicine,atherosclerotic plaque belongs to the categories of"ointment""phlegm""blood stasis"and"poison".2.Influence of Jueyin Qi stagnation on atherosclerotic plaque:Qi and machinery block,ointment and fat transfer obstruction,phlegm generation and blood stasis.Phlegm and blood stasis interjunction,day long poison,all the evil closed the vein,the formation of atherosclerotic plaque tangible focus.3.Qingzhi Tongmai granμles have the effect of soothing liver and relieving depression,tonifying blood and Qi,nourishing Yin and stimμlating fluid.Part two:Molecular mechanism network research1.Molecular docking results of important active components of Qingzhitongmai Granules and important target proteinsMolecular docking and molecular dynamics to explore the potential targets ofQingzhitongmai Granules in the treatment of atherosclerotic plaque,a total of 98 active components of Qingzhitongmai Granules were obtained,and 5 targets related to atherosclerosis.Molecular docking results showed that Qingzhitongmai Granules had strong binding ability to target proteins,and the best binding groups wereα-spinasterol-S1PR1,oleanolic acid-S1PR1,α-spinasterol-LXRα,β-sitosterol-LXR.2.Molecular dynamics results of important active components and important target proteins of Qingzhitongmai GranulesThe results of molecular dynamics simulation show that the van der Waals potential energy,electrostatic interaction and hydrogen bonding are the most important factors for stable binding.Some regions ofα-spinasterol-LXRαandβ-sitosterol-LXR showed more flexibility,which may be related to the specific structure of proteins.Part three:The LR2/4-IRF3 signaling pathway and LXR proteins may influence AS progression from both inflammatory response and lipid metabolism.Part four:experimental research Experiment 1:1.Effects of Qingzhitongmai Granules on lipid levels in Apo E^-/-mice and aortic atherosclerotic plaquesCompared with the normal group,the TC,TG and LDL-C contents of the model group were significantly higher and the HDL-C content was significantly lower;compared with the model group,the TC,TG and LDL-C contents of the QZTM-A group,QZTM-B group and the western medicine group were significantly lower and the HDL-C content was significantly higher.The results of HE staining showed that:in the normal group,the structure of the aortic wall could be observed clearly under the microscope,and there were no obviously visibleAtherosclerotic plaques,foam cells and other inflammatory changes;the aortic wall of the remaining groups can be observed in different degrees of atherosclerotic changes,including:intimal thickening,fibroplasia,atherosclerotic plaques,etc.Compared with the model group,aortic atherosclerotic plaques of the drug group were significantly reduced,the arterial wall was structurally clear,the foam cells tended to be dispersed,and the number of individuals was reduced,of which the QZTM-B group was the most obvious,followed by the western drug group.The QZTM-B group was the most obvious,the western medicine group was the second,and the QZTM-A group was the worst.The results of oil red O staining showed that the lipids in the atherosclerotic region were stained bright red,the nuclei were blue,and the interstitium was not coloured,while the normal group could see that the vessel wall was neatly arranged at all levels and had a clear structure,with few wrinkles and vacuoles,and there was almost no red staining;in contrast,the model group had an obvious thickening of the intima,an increase in the deposition of lipid plaques,and the blurring of structure with the formation of a large number of wrinkles and vacuoles,and a large area of red staining,while in the drug-treated group,the structure of the western drug group was more obvious than that in the Western drug group.In the drug treatment group,the western drug group had clearer structure and less red staining,which was better than the staining of QZTM-B and QZTM-A groups.2.Effect of Qingzhitongmai Granules on the window pore structure of hepatic sinusoidal endothelial cells in Apo E^-/-miceThe results of the window pore structure of liver sinusoidal endothelial cells under electron microscope showed that:the liver sinusoidal endothelial cells of Apo E^-/-mice in the normal group were thin,flat in shape,with nuclei and organelles regularly arranged,and many discontinuous window pores were visible on the surface,which were sieve-like,and the basement membrane structure was not observed underneath them;the diameter of the window pore structures on the liver sinusoidal endothelial cells of the Apo E^-/-mice in the model group was reduced in size and number,and the disappearance of the window structure was visible In the model group,the number and diameter of window pores on the endothelial cells of liver sinusoids of Apo E^-/-mice became smaller,and the disappearance of the window structure was observed.3.Effect of Qingzhitongmai Granules on serum HDL-S1P content of Apo E^-/-miceCompared with the normal group,serum HDL-S1P content was significantly lower in the model group;compared with the model group,serum HDL-S1P content was significantly higher in the QZTM-A,QZTM-B and western medicine groups;compared with the western medicine group,serμm HDL-S1P content was reduced in the QZTM-A and QZTM-B groups.4.Effect of Qingzhitongmai Granules on the content of S1PR1 in aorta of Apo E^-/-miceCompared with the normal group,the aortic S1PR1 content of the model group was significantly reduced;compared with the model group,the Apo E^-/-aortic S1PR1 content of the QZTM-A group,QZTM-B group,and the western medicine group was significantly increased,with the best effect in the QZTM-B group.5.Effect of Qingzhitongmai Granules on the content of LXR in Apo E^-/-mouse liver tissueCompared with the normal group,the hepatic tissue LXR content was significantly lower in the model group;compared with the model group,the hepatic tissue LXR content was significantly higher in the QZTM-A,QZTM-B,and western medicine groups;compared with the western medicine group,there was no statistically significant difference in the hepatic tissue LXR content in the QZTM-A and QZTM-B groups.6.Influence of Qingzhitongmai Granules on the expression level of Apo E^-/-aorta LXR/beta protein in miceCompared with the normal group,the aortic LXRɑ/βprotein expression level was significantly reduced in the model group;compared with the model group,the aortic LXRɑ/βprotein expression level was significantly increased in the QZTM-A group,QZTM-B group,and the western medicine group;compared with the western medicine group,the aorticLXRɑ/βprotein level was reduced in the QZTM-A group and the QZTM-B group.Experiment 2:1.Effect of Qingzhitongmai Granules on the expression level of TLR2 protein in aorta of Apo E^-/-miceCompared with the normal group,the aortic TLR2 content was significantly higher in the model group;compared with the model group,the aortic TLR2 content was significantly lower in the QZTM-A group,QZTM-B group,and the western medicine group;compared with the western medicine group,there was no statistically significant difference in the aortic TLR2 content in the QZTM-A group and the QZTM-B group.2.Effect of Qingzhitongmai Granules on the expression level of TLR4 protein in aorta of Apo E^-/-miceCompared with the normal group,the aortic TLR4 content was significantly higher in the model group;compared with the model group,the QZTM-A group,QZTM-B group,and the western medicine group-the aortic TLR4 content was significantly lower;compared with the western medicine group,the aortic TLR4 content was elevated in the QZTM-A group and the QZTM-B group.3.Effect of Qingzhitongmai Granules on serum TLR2/4 content of Apo E^-/-miceCompared with the normal group,serμm TLR2/4 levels were significantly higher in the model group;compared with the model group,serum TLR2/4 levels were significantly lower in the QZTM-A,QZTM-B,and western medicine groups;compared with the western medicine group,there was no statistically significant difference in serum TLR2/4 levels in the QZTM-A and QZTM-B groups.4.Effect of Qingzhitongmai Granules on IRF3 content in peripheral blood mononuclear cells of Apo E^-/-miceCompared with the normal group,the IRF3 content of peripheral blood single nuclei was significantly higher in the model group;compared with the model group,the IRF3 content of peripheral blood single nuclei was significantly lower in the QZTM-A group,QZTM-B group,and the western medicine group;compared with the western medicine group,the IRF3 content of peripheral blood single nuclei was slightly higher in the QZTM-A group and the QZTM-B group.5.Effects of Qingzhitongmai Granules on the contents of ABCA1 and ABCG1 in peripheral blood mononuclear cells of Apo E^-/-miceCompared with the normal group,the contents of ABCA1 and ABCG1 in peripheral blood single nucleated cells were significantly reduced in the model group;compared with the model group,the contents of ABCA1 and ABCG1 in peripheral blood single nucleated cells were significantly elevated in the QZTM-A group,the QZTM-B group,and the western medicine group;compared with the western medicine group,the contents of ABCA1 and ABCG1 in peripheral blood single nucleated cells were slightly reduced in the QZTM-A group and the QZTM-B group.Conclusion:1."Jueyin qi stagnation"is an important pathogenesis of the formation and development of atherosclerosis.Based on this theory and combined with modern pharmacological research,"Qingzhitongmai Granule"was selected to treat the qi mechanism of Jueyin liver with remarkable effect.2.Based on molecular docking and molecular dynamics,the potential targets of Qingzhitongmai granules for the treatment of atherosclerosis were explored,and a total of 5core intersection targets were obtained.The results of molecular docking showed that Qingzhitongmai granules had strong binding ability to target proteins,and the 4 groups with the best binding ability were:α-spinasterol-S1PR1,oleanolic acid-S1PR1,α-spinasterol-LXRα,β-sitosterol-LXR,confirmed the effectiveness of Qingzhitongmai granules in the treatment of atherosclerosis.3.Qingzhitongmai granules can regulate dyslipidemia in Apo E-/-atherosclerotic mice,and at the same time delay and reverse the progression of aortic atherosclerotic plaques.4.Qingzhitongmai granules can up-regulate the level of S1PR1 and increase the level of LXR through the S1PR1/LXR signaling pathway,thereby strengthening the reversal of cholesterol movement,promoting lipid metabolism and delaying the process of atherosclerosis.5.Qingzhitongmai granules not only regulate S1PR1/LXR signaling pathway to enhance cholesterol reversal,but also inhibit TLR2/4-IRF3 pathway to increase the levels of ABCA1and ABCG1,thereby enhancing cholesterol reversal,inhibiting inflammation and delaying the occurrence and development of atherosclerosis.