Study On The Mechanism Of Guanxinning Tablets In Anti-atherosclerosis And Safe Anti-thrombosis

Objective Atherosclerosis(AS)model induced by high fat diet in Tibet mini-pigs was used to observe the effects of Guanxinning tablets(GXN)on the formation of AS and gut microbiota and their metabolites,and to explore the anti-AS mechanism of GXN.And coronary artery thrombosis model and gastric ulcer model in rats,and cell experiment in vitro were used to observe the antithrombotic effect and bleeding risk of GXN,and to explore safe anti-thrombotic mechanism of GXN.Methods(1)18 male Tibet mini-pigs,aged 4-5 months and weighing 8-12 kg,were taken.According to the body weight,6 were selected,fed with normal chow,as the normal control group and another 12 were fed with high fat and cholesterol(HFC)chow until 16 weeks.Afterwards,12 mini-pigs were divided into the model group and the GXN group according to the body weight and blood lipid level.The model group continued to feed HFC chow,while the GXN group continued to feed HFC chow and were daily given GXN(162 mg/kg/d)for 12 consecutive weeks,that is,HFC was fed to 28 weeks.During the experiment,general observation was made.Blood samples were taken 0,4,8,and 12 weeks after administration to detect white blood cell classification and blood lipids(TC,TG,HDL-C,LDL-C).Blood samples were taken 0 and 12 weeks after administration to detect inflammatory factors such as ox-LDL,CRP,TNF-?,IL-1? and IL-6,oxidative stress indexes including SOD,MDA and GSH-Px,vascular endothelial injury related indexes such as vWF and ET-1,coagulation function indexes such as PT,TT,APTT,Fbg,and platelet aggregation rate.After administration was finished,doppler ultrasound was used to check the structure and function of the left ventricle of the heart.The feces of each group were collected,and the species and functions of gut microbiota were annotated via high-throughput metagenomic sequencing technology.The effects of GXN on the composition structure,functional gene composition and metabolites of the flora in AS model were analyzed.Based on the results of metagenomics sequencing,we selected gut microbiota metabolites closely related to the occurrence and development of AS,including trimethylamine N-oxide(TMAO),bile acids(BAs),and short chain fatty acids(SCFAs)for targeted metabolomics sequencing to analyze the regulating effects of GXN on the metabolites and look for metabolite markers.After the experiment was finished,the mini-pigs were euthanized,and the characteristic indicators(weight,BMI and body type index)and fat distribution were observed,and tissues including blood vessels,liver,fat were taken.HE staining was used to observe the histopathological changes of blood vessels,oil red "O" staining to observe lipid deposition,Sudan ? staining to observe aortic lipid deposition,Movat five-color overstaining to observe foam cells and collagen components in AS plaques,and immunohistochemistry to observe the expressions of NF-?B,ICAM-1 and MMP-9 proteins in blood vessels.(2)40 male SD rats,weighing 190-210 g,were randomly divided into sham-operated group(normal saline),model control group(normal saline),GXN low dose group(300 mg/kg GXN)and high dose group(600 mg/kg GXN),and aspirin group(50 mg/kg Aspirin)according to the body weight,8 rats for each group.Except for sham-operated group,rats in other groups were given corresponding doses of drugs(10 mL/kg)by oral gavage.1 h after administration,the model of coronary artery thrombosis in rats was established by photochemical induction.Then,the thrombosis was generally observed and the degree of thromboembolism was observed by HE staining.In addition,48 male SD rats,weighing 190-210 g,were randomly divided into sham-operated group(normal saline),model control group(normal saline),GXN low dose group(500 mg/kg GXN),GXN high dose group(1000 mg/kg GXN),aspirin low dose group(50 mg/kg Aspirin)and aspirin high dose group(100 mg/kg Aspirin)according to the body weight,8 in each group.Rats were given corresponding doses of drugs(10 mL/kg)by oral gavage.After continuous administration for 3 days,the acetic acid-induced gastric ulcer model was established and rats were continued to dosing for 5 days after operation.After the administration was finished,the stomach was taken to observe the general gastric tissue.Then,the gastric ulcer tissue was used for HE staining and ICAM-1 immunofluorescence staining,and the VEGF and ES levels were also measured.(3)CCK8 method was used to detect the effect of different concentrations of GXN on the viability of THP-1 cells in vitro cell experiment.THP-1 cells were treated with low,medium,and high doses of GXN(100,200,400 ?g/mL)and phorbol myristate acetate(PMA,1 ng/mL)before interacting with human platelets,and then effect on leukocyte-platelet aggregate was detected by flow cytometry.THP-1 cells were treated with low,medium and high doses of GXN and PMA(1 ng/mL),labeled with BCECF AM,and then seeded into a 96-well plate coated with GPIba protein,and the fluorescence intensity was detected by a continuous spectrum enzyme analyzer.After THP-1 cells were treated with low,medium and high doses of GXN and PMA(1 ng/mL),total RNA was extracted,and the mRNA expression level and activation level of Mac-1 was detected by PCR and flow cytometry,respectively.The THP-1 cells,treated with low,medium and high doses of GXN,were collected and interacted with human platelets,and flow cytometry was used to detect the expression of platelet activation markers CD41a and CD62p.Results(1)After 12 weeks of continuous GXN intervention,compared with the model group,1)in GXN group,the body type index decreased significantly(P<0.05),the total fat weight,total fat index and abdominal fat weight significantly reduced(P<0.01),and the back fat thickening in the front,middle and back was significantly inhibited(P<0.05,P<0.01).2)In GXN group,the white blood cell count,the neutrophil count,the lymphocyte count,and the eosinophil count decreased significantly at 8 weeks of administration(P<0.05,P<0.01),and the neutrophil count decreased significantly at 12 weeks of administration(P<0.05).3)In GXN group,the level of HDL-C increased significantly(P<0.01)and the AI index significantly reduced at 8 weeks of administration(P<0.01),and the level of LDL-C and AI index significantly reduced at 12 weeks of administration(P<0.05).4)ox-LDL,CRP,TNF-?,and IL-1? significantly reduced(P<0.05,P<0.01),serum SOD and GSH-Px significantly increased(P<0.01),and MDA content significantly reduced in GXN group(P<0.01).5)Serum ET and vWF levels significantly reduced in GXN group(P<0.05,P<0.01).6)serum Fbg level and platelet aggregation rate were both significantly lowered in GXN group(P<0.05,P<0.01).7)In GXN group,LVd mass,?Sd and LVPWd significantly reduced(P<0.05),while EF and FS significantly increased(P<0.05).8)In GXN group,vascular lipid deposits in the thoracic aorta,abdominal aorta,and common aorta significantly reduced(P<0.01),and vascular AS lesions significantly reduced.Quantitative analysis showed that the vascular IMT,NIA,NIA/MA and NIA/IELA ratios significantly reduced(P<0.05,P<0.01).The number of vascular endothelial foam cells significantly reduced,the deposition of collagen fibers in the inner and media layers was improved,the rate of vascular collagen fiber deposition significantly reduced(P<0.05,P<0.01),the rate of vascular lipid deposition significantly lowered(P<0.05),and the positive expression rates of vascular NF-?B,TNF-?,and MMP-9 significantly reduced(P<0.05,P<0.01).(2)The metagenomics sequencing results showed that compared with the model group,in GXN group,the relative abundance of proteobacteria significantly reduced(P<0.05),the relative abundance of enterobacteriales significantly reduced(P<0.05),the relative abundance of enterobacteriaceae significantly reduced(P<0.05),the relative abundance of prevotellaceae significantly increased(P<0.05),the relative abundance of escherichia significantly decreased(P<0.05),while the relative abundance of prevotella significantly increased(P<0.05),the relative abundance of escherichia coli significantly reduced(P<0.05),and the relative abundance of lactobacillus johnsonii significantly increased(P<0.05).Function analysis showed that in GXN group,genes of gut microbiota related to choline metabolism,carnitine metabolism,and acetate kinase production significantly reduced(P<0.05),while genes of gut microbiota related to short chain fatty acid transport,propionate kinase,and butyrate kinase production significantly increased(P<0.05).Bacterial genes related to the MAPK signaling pathway were significantly down-regulated(P<0.01),and bacterial genes related to the AMPK signaling pathway were significantly up-regulated(P<0.05).The relative abundance of bacteria related to Entner-Doudoroff pathway,lipopolysaccharide production,phosphoacetyltransferase-acetate kinase pathway,and choline production decreased significantly(P<0.05,P<0.01),while the relative abundance of bacteria related to pentose phosphate cycle and the hydroxypropionic acid pathway significantly increased(P<0.05).GO analysis showed that bacterial genes related to polysaccharide synthesis,propionate kinase activity and oxidoreductase activity increased significantly(P<0.05),while bacterial genes related to betaine biosynthesis,fatty acid biosynthesis and bile acid metabolism decreased significantly(P<0.05).The results of targeted metabolomics sequencing showed that compared with the model group,in GXN group,the contents of propionic acid and butyric acid in feces and serum significantly increased(P<0.05),and the content of total SCFAs in feces also significantly increased(P<0.05);the levels of choline in feces and liver significantly reduced(P<0.05),and the content of TMAO in serum and total TMA-related metabolites in feces also significantly reduced(P<0.05);CDCA in feces significantly reduced(P<0.05),and LCA-3S significantly increased(P<0.05).(3)The results of the coronary artery thrombosis in rats showed that compared with the model group,in GXN group,thrombosis reduced,and the area of thrombus also significantly reduced(P<0.01).The results of the gastric ulcer model in rats showed that compared with the model group,in GXN high and low dose groups,gastric mucosal ulcers and erosions were improved to varying degrees,and no obvious bleeding points were observed,and the area of ulcers was also reduced to varying degrees.While the ulcer area in aspirin high and low dose groups showed an increasing trend,and the ulcer area in aspirin high dose group was significantly increased(P<0.05).Pathological observation showed that the amount of bleeding in GXN high dose group significantly reduced,while the symptoms of hyperemia and hemorrhage in aspirin group increased significantly.There was no significant difference in ES and VEGF levels in GXN groups with low and high doses(P>0.05).While the expression of ES in aspirin high dose group was significantly increased(P<0.01),and the expression of VEGF was significantly decreased(P<0.01).The results of in vitro experiments showed that compared with the model group,the GXN groups with medium and high doses could significantly reduce the PLA(P<0.05,P<0.01),lower the adhesion rate of Mac-1 and GPIba(P<0.05,P<0.01),inhibit the expression of Mac-1 mRNA and the CD1lb activation(P<0.05,P<0.01)in THP cells,as well as the expression of platelet CD41a(P<0.05).Conclusions(1)GXN can reduce body fat and vascular lipid deposition by regulating blood lipids,inhibiting platelet aggregation,and improving cardiac dysfunction.And GXN has the effects of inhibiting vascular inflammation and lipid oxidation,inhibiting vascular intimal hyperplasia and collagen fiber proliferation,and protecting the vascular endothelium,thereby exerting anti-AS effect.(2)GXN can effectively improve the imbalance of gut microbiota in AS model of Tibet mini-pigs,and target to regulate functions of the flora such as Proteobacteria,Escherichia,Prevotell,and Lactobacillus johnsonii.Moreover,GXN can effectively increase the production of SCFAs of gut microbiota metabolites,promote the conversion of primary bile acids,and increase the excretion of secondary bile acids.Meanwhile,GXN can also reduce choline levels in feces and liver,serum TMAO content and total fecal TMA-related metabolite content,indicating that the anti-AS formation of GXN may be closely related to the regulation of microbial metabolites SCFAs,TMAO and BAs.(3)GXN can effectively inhibit thrombosis in rat model of coronary artery thrombosis without increasing the risk of gastrointestinal bleeding in gastric ulcer rat model,indicating that GXN had antithrombotic effect with low bleeding risk.At the same time,GXN can significantly inhibit leukocyte and platelet aggregation,inhibit leukocyte Mac-1 expression and aM I-domain activation,inhibit Mac-1:GPIba binding,and inhibit platelet activation index CD41a expression after leukocyte-platelet interaction.It is suggested that the safe antithrombotic mechanism of GXN may be related to the inhibition of leukocyte-platelet aggregation and its interaction target Mac-1:GPIba.