Butyric Acid Regulates The Phenotypic Transition Of Vascular Smooth Muscle Cells By Inhibiting M1 Type Macrophage Polarization In Abdominal Aortic Aneurysms

Objective: Abdominal aortic aneurysm(AAA)is a fatal disease that often results in the patient’s death due to sudden rupture of the aneurysm.According to statistics,the prevalence of AAA in men over 60 years old is about 4-8%,and about 0.5-1.5% in women.Surgery is usually the recommended treatment for AAA larger than 5 cm in diameter.The most common treatments are conventional AAA resection with artificial vessel replacement and minimally invasive AAA endoluminal repair,but neither have significantly improved patients’ survival rate or their quality of life.Early detection and treatment of AAA can reduce the risk of rupture and mortality.So far,we haven’t seen any medicine that can effectively control the progression of AAA in clinical practice.Therefore,it is of tremendous and far-reaching clinical significance to explore AAA’s pathogenesis and find new targets for medical therapy.In recent years,many scholars have tended to believe that inflammatory response is a critical factor in the pathogenesis of abdominal aortic aneurysms.During AAA formation,pathological vascular remodeling is driven by macrophage infiltration.During vascular remodeling,monocytes are recruited into the aortic tissue,which differentiates into macrophages that participate in the inflammatory response of the vessel wall.Classically,macrophages can be divided into 2 types: pro-inflammatory Type M1 and anti-inflammatory Type M2.The former secretes pro-inflammatory cytokines,including IL-6,IL-1β,and TNF-α,involved in the vascular wall inflammatory response leading to aneurysm formation,while the latter secretes anti-inflammatory substances,such as IL-10,involved in extracellular matrix remodeling and playing a role in suppressing aneurysms.Activated inflammatory cells can also secrete various inflammatory stimuli which act on vascular endothelial cells and smooth muscle cells by autocrine and paracrine means,causing their phenotypic changes,inducing immune responses,and prompting smooth muscle cell apoptosis.Therefore,exploring the specific pathogenesis of inflammation in AAA and the impact of interactions between macrophage and vascular smooth muscle cell on AAA provides an essential theoretical basis for the clinical discovery of new medicine.Current studies have demonstrated the relevance of gut microbes and their metabolites to the development of cardiovascular disease.Our group has shown that AAA patients suffer dysbiosis more than the average population,with the elevated abundance of thick-walled phylum and decreased abundance of Rhodobacter spp.Trimethylamine N-oxide(TMAO),a major metabolite of the thick-walled phylum,accelerates the progression of AAA.Roseburia spp.,as a major microorganism in the intestinal tract,has multiple physiological functions such as anti-inflammation,intestinal immunity regulation,metabolic syndrome,etc.Butyric acid is its specific metabolite.Butyric acid is one member of the short-chain fatty acid family,which are essential metabolites of intestinal flora.Other members in the family include acetic acid,propionic acid,etc.Butyric acid has multiple physiological functions such as protecting the intestinal barrier,regulating body immunity,regulating intestinal flora,anti-tumor,anti-inflammation,and anti-oxidation.Research has proved that a lack of butyric acid is associated with the development of several cardiovascular diseases,such as coronary heart disease,chronic heart failure,myocardial infarction,and atherosclerosis.Studies have shown that butyric acid can inhibit the inflammatory response of the vessel wall and slow down the progression of atherosclerosis by reducing macrophage adhesion and migration and increasing plaque stability.This is similar to the disease progression in AAA.However,we haven’t seen any report on whether butyric acid is involved in the development and progression of AAA.Therefore,this study aims to explore the pathogenesis of AAA and provide new pharmacological targets for clinical prevention of AAA progression by investigating the changes of butyric acid as well as inflammation levels in patients with AAA and by exploring the role and possible mechanisms of butyric acid in the development and progression of the rat elastase AAA model.Methods:(1)First,we collected blood and stool specimens from patients with AAA and an healthy population.We then examined the changes in stool and serum butyric acid concentrations and the differences in butyric acid-producing bacteria Roseburia spp.between the two groups.Differences in tumor necrosis factor-α(TNF-α)and IL-6,indicators of inflammation,in circulating serum from AAA patients and normal healthy subjects were also examined.Immunofluorescence staining of clinically obtained AAA aneurysm walls and normal controls was also performed to observe the M1-type infiltration of macrophages in the vessel wall and VSMC phenotype transition.(2)To explore the inhibitory effect of exogenous supplementation of butyric acid on the rat elastase AAA model: changes in the maximum diameter of the abdominal aorta were recorded,while the extent of damage to the integrity of the elastic fibers of the vessel wall by butyric acid was observed by HE and EVG staining of rat AAA specimens.Finally,changes in the expression of macrophage M1 marker CD86,inflammatory factors TNF-α and IL-6 in rat serum and vessel wall were investigated by Elisa and protein immunoblotting assays;changes in the expression of VSMC synthetic marker OPN,contractile marker α-SMA,and MMP-9 in rat vessel wall were detected.(3)We investigated the interrelationship between the PI3K/AKT pathway in macrophages and abdominal aortic aneurysm and butyric acid using a bioinformatics approach for data mining on the mechanism of action of butyric acid and abdominal aortic aneurysm.Then,the effect of butyric acid on M1-type polarization of macrophages and PI3K/AKT signaling pathway and the effect of macrophage conditioned culture supernatant on VSMC phenotype transition were verified by cellular experiments.Finally,the PI3K/AKT pathway expression was verified by immunohistochemistry and protein immunoblotting experiments in clinically obtained human aneurysm specimens and rat aneurysm specimens.Results:(1)AAA patients had a significantly lower abundance of butyric acid-producing Roseburia spp compared with the average population.Serum butyric acid concentrations were lower in patients with AAA than in the average population(P < 0.01),and serum inflammatory factor expression levels were higher than in the average population(P < 0.01).There was increased infiltration of M1-type macrophages in the vessel wall tissue of patients with AAA;the VSMC synthetic phenotype was increased,and the contractile phenotype was decreased.This suggests that butyric acid may be associated with the development and progression of AAA.(2)In the AAA rat elastase model,compared with the sham group,the diameter of arteries in the AAA group was significantly dilated,accompanied by the degradation of elastic fibers.At the same time,the administration of butyric acid significantly inhibited the dilatation of AAA aneurysm diameter and reduced the degradation of elastic fibers.ELISA detected the expression levels of serum inflammatory stimuli TNF-α and IL-6 in rats,and administration of butyric acid significantly reduced the expression of inflammatory factors compared with the AAA group;RT-PCR and WB detected the expression of CD86,TNF-α,IL-6,and the expression of OPN,α-SMA,and MMP-9 in the vessel wall,and found that butyric acid could inhibit the expression of CD86,TNF-α,and IL-6 expression.Butyric acid could inhibit the transition from contractile to synthetic VSMC in AAA,and the inhibitory effect was more substantial at a high concentration of butyric acid.The experimental results suggest that butyric acid may inhibit the development of aneurysms by suppressing the inflammation of the vessel wall.(3)Bioinformatics analysis predicted that butyric acid is involved in AAA development through PI3K/AKT signaling by regulating macrophage polarization.In cellular experiments,butyric acid was found to inhibit macrophage M1 polarization and reduce the expression of inflammatory stimuli TNF-α and IL-6.Western Blotting results showed that butyric acid inhibited macrophage M1-type polarization by suppressing the expression of the PI3K/AKT signaling pathway.We found that treatment of VSMC with M1-type macrophage conditioned culture supernatant promoted VSMC transition from contractile to synthetic phenotype,but this effect was reversed after administration of butyric acid.Finally,we verified the expression of the PI3K/AKT pathway in human aneurysm specimens and rat aneurysm specimens.Conclusion:Serum butyric acid level is lower in AAA patients compared with the healthy population,and the abundance of butyrate-producing bacteria Roseburia spp.in their intestinal flora is also lower.Butyric acid has inhibited elastase-induced dilation of diameter and inflammatory response of the vessel wall in a rat AAA model.Butyric acid has also inhibited the expression of serum inflammatory factors in a rat AAA model.The possible mechanism by which butyric acid inhibits AAA development is through the inhibition of PI3K/AKT signaling pathway activation,which inhibits macrophage M1-type polarization,attenuates atheroma wall macrophage infiltration,and regulates the atheroma wall macrophage infiltration VSMC phenotypic transition,and exerts an inhibitory effect on AAA development.