Study On Anti-atherosclerosis Mechanism Of Shenhongtongluo Formula In Improving Macrophage Efferocytosis Based On PPAR/Ca²⁺ Pathway

Objective:Under the guidance of the theory that Tanyufuluo,to study the effect of Shen-Hong-Tong-Luo formula on atherosclerosis,improve the efferocytosis in the atherosclerosis process,explain the regulatory effect of Shen-Hong-Tong-Luo formula on PPAR/Ca2+signal pathway through in vivo experiments.To provide experimental basis for the clinical application of Shen-Hong-Tong-Luo formula.Methods:Experiment 1:The establishment of animal Model:six-week-old male Apo E^-/-mice were randomly divided into Shen-Hong-Tong-Luo formula Low,middle and High dose groups（SHTL-l group,SHTL-m group,SHTL-h Group）and Model group（Model group）,24 mice each;Six-week-old male C57BL6/J mice were used as Control group（24 mice）.After1 week of adaptive feeding,the Apo E^-/-mice were induced to arteriosclerosis with purified high-fat Diet from Diet Research,several mice were randomly selected after 7 weeks of continuous feeding,Apo E^-/-was observed by tissue staining.After plaque formation,gastric lavage was performed.The low,middle and high dose groups of traditional Chinese medicine were given 9·g^-1·kg^-1·d^-1,18·g^-1·kg^-1·d^-1,36·g^-1·kg^-1·d^-1 doses respectively,in the Model group and the Control group,the mice were fed with the same volume of sterilized water,and their body weight and food intake were recorded regularly during the experiment.At the end of 8 weeks,the experimental animals were euthanized,the serum was extracted,the heart and aorta were separated,and the follow-up experiments were carried out.The components of Shenhong Tongluo Prescription were analyzed by HPLC,and the core target of Shenhong Tongluo Prescription was predicted by network pharmacology.Experiment 2:To observe the effect of Shen-Hong-Tong-Luo formula on pathomorphology of Apo E-^/-mouse aortic plaque:the aorta of mice was stained with oil red O,the general situation of plaque formation was observed,the size of plaque necrosis core was counted by Image J,apoptosis in aortic root was detected by TUNEL method.Experiment 3:The Untargeted Relative Quantitative Lipidomics based on UPLC-Orbitrap mass spectrometry system was adopted,and the lipid Search software was combined to conduct lipid identification and data pretreatment on the extracted serum,and the effect of Shenhong Tongluo Formula on serum lipidomics was summarized,so as to reveal the mechanism of improving cell burial and anti-atherosclerosis.Experiment 4:To observe the effect of Shenhong Tongluo Formula on efferocytosis related protein in Apo E^-/-mouse aortic pathological tissue,F4/80 was used to label macrophages in pathological section,and Mertk,CD47,LRP1 were detected by immunohistochemistry,the expression of CAMKIIγ,CRT and SRB1 in the pathological sections of the aortic roots of the mice in each group was compared.Experiment 5:On the basis of network pharmacology,the signal pathway of PPAR-γ/LXR-α/ABAC1 was activated by Shenhong Tongluo in vitro,and MERTK,CD47,CRT,LRP1,SRB1 were detected by Western Blot in vivo,expression of PPAR-γ,lxr-α,CA2+homeostasis associated protein CAMKIIγin PPAR signaling pathway.Results:Experiment 1:The seven main ingredients of Shenhong Tongluo formula were salidroside,luteolin chlorogenic acid,luteolin paeoniflorin,ferulic acid and ginsenoside Rg1,and has a good repeatability of quality control.At the 7th week of Apo E^-/-mice induced by high fat diet,the aorta tissues of the mice were stained with oil red O and HE at random.The results showed that the arteriosclerosis model was successfully prepared and there was no significant difference between the groups The aorta of C57BL6J mice in the Control group had no atherosclerosis plaque formation;after 8 weeks of gavage,the specimens were harvested and stored according to the standard method.Experiment 2:（1）Oil red O staining results:in model group and Shen-Hong-Tong-Luo formula Low,middle and high dose group,the atherosclerosis plaques were bright and blood red,the border was clearly different from the blood vessel wall;The plaque area ratio was 75.00±6.48%in model group,40.00±2.94%,38.50±3.11%,50.75±3.86%in low,middle and high treatment groups,respectively The plaque area ratio in the middle dose group was smaller than that in the high dose group and smaller than that in the low dose group（p<0.01）;（2）HE staining results:in the model group,there were obvious plaques in the aorta,necrotic nuclei were formed,and the intima were thickened significantly,including macrophages,foam cells,inflammatory cells,fibrous tissue proliferation,and atrophy of the media,the arrangement of smooth muscle cells was disordered,the elastic fibreboard was wavy,the gap between boards was enlarged,and some of them were broken.Shen-Hong-Tong-Luo formula group had different degrees of improvement.（3）Necrotic core statistics:the necrotic core ratio of model group was 17.19±0.71%,the low,middle and high necrotic core ratios of Shen-Hong-Tong-Luo formula were13.08±0.28%,7.11±0.49%and 7.6±0.26%,respectively,there was significant difference（p<0.01）between middle-dose group and high-dose group,（4）TUNEL method was used to detect the apoptosis of aortic root cells,the ratio of apoptotic cells in Shenhong Tongluo Fang group was 6.05±0.35%,3.69±0.30%and 5.44±0.25%respectively,and the ratio of apoptotic cells in Shenhong Tongluo formula group was significantly lower than that in model group（p<0.01）.Experiment 3:Shenhong Tongluo formula may promote efferocytosis by intervening the metabolism of glycerol phospholipid sphingolipid glyceride to anti-AS role.Experiment 4:The results of immunohistochemistry showed that Shenhong Tongluo formula could up-regulate the expression of Mertk,LRP1,SRB1,CRT and down-regulate the expression of CAMKIIγand CD47.Experiment 5:On the basis of network pharmacology,the signal pathway of PPAR/lxr-α/ABAC1 was activated by Shenhong Tongluo in vitro,shenhong Tongluo decoction could up-regulate the expression of Mertk,PPAR,LXR,LRP 1,SRB1,CRT,down-regulate the expression of CD47 and CAMKII.Conclusion:1.Shenhong Tongluo formula identified a total of seven major components with good repeatability of quality control.2.Shenhong Tongluo formula can effectively improve Apo E^-/-mouse aortic lesion,reduce plaque formation,reduce the formation of necrotic nuclei,reduce the proportion of apoptotic cells,play an anti-arteriosclerosis role;2.Shenhong Tongluo can up-regulate the expression of“Eat me”signal,down-regulate the expression of“Don’t eat me”signal CD47,up-regulate the expression of LRP1,SRB1,CRT,so as to improve the phagocytosis and digestion of macrophages,finally,it can promote efferocytosis.3.Shenhong Tongluo formula can improve the effect of efferocytosis by down-regulating the expression of many kinds of lipid molecules in SM,CER,PE,TG,DG,PC and LPC classes.3.Shenhong Tongluo can activate the PPAR signal pathway,down-regulate the expression of CD47,down-regulate the expression of effeorocytosis related receptor,and down-regulate the expression of CAMKIIγ,which inhibits the output of macrophages in the Ca2+pathway,and regulate the intracellular calcium homeostasis.