Cholic Acid Alleviates Obesity And Metabolic Inflammation By Inhibiting NLRP3 Inflammasome In Mice

Background: Obesity has become a significant burden of global public health,and a series of metabolic diseases,such as type Ⅱ diabetes,atherosclerosis and metabolism-related fatty liver disease,have also become global health problems.Commonly,cells in the body are defended by the immune system.The immune system is a powerful system that recognizes and eliminates pathogens and maintains the homeostasis of the body’s tissues.The body’s nutritional status is crucial for the precise maintenance of this homeostasis,and changes in nutritional status can regulate systemic metabolism and the occurrence and development of associated diseases by affecting the metabolism of immune cells.Obesity caused by overnutrition and chronic inflammation in related tissues and organs(such as adipose tissue,liver)are important causes of insulin resistance.There is growing evidence that the degree of inflammation is closely related to the process of obesity,and macrophages are of great interest as vital immune cells to maintain local and systemic homeostasis.At present,the primary mechanism of the development of chronic inflammation during obesity-induced adipose tissue inflammation has not been fully solved,and how macrophages affect tissue and systemic energy metabolism needs more and deeper exploration.Therefore,studying the regulatory mechanism of immune cells,especially macrophages,in adipose tissue inflammation is of great significance for obesity-related metabolic diseases.Objective: This project aims to investigate whether cholic acid,as a cholesterol metabolite and pluripotent signaling metabolite,can inhibit the activation of NLRP3 inflammosomes to improve obesity and related metabolic diseases,and to explore the new molecular mechanism of targeting cholic acid in immune metabolism by NLRP3 inflammasomes.Methods: 1.Human samples and mouse experiments: In this project,clinical samples were collected to analyze the differences in NLRP3 gene expression levels in preperitoneal and subcutaneous adipose tissues of patients with BMI≤24 and BMI>24,and then the expression levels of visceral fat(eWAT)and inguinal fat(iWAT)NLRP3and IL-1β genes leptin receptor-deficient(db/db)mice.The NCBI gene bank was used to analyze the gene expression of NLRP3 and IL-1β in obese non-diabetic and obese patients with diabetes.WT mice were subsequently fed with a normal diet,a high-fat-diet and a high-fat-diet supplement with cholic acid(0.5%).Mice were monitored weekly for food intake and body weight.IPGTT and IPITT were performed four weeks after the cholic acid intervention to observe glucose metabolism in obese mice and after the bile acid intervention.After the experiment,mice were dissected and the heart,liver,visceral fat,subcutaneous fat,brown fat and quadriceps muscle were isolated for analysis.The extracted mouse eWAT and iWAT were divided into 4-part experiments:1)Adipose tissue(0.5g)were cultured in M199 medium for 24 h,and take the supernatants for TNF-α and IL-1β analyzed by ELISA inflammatory factors.2)Weigh an appropriate amount of tissue for RNA extraction and perform QPCR detection.3)Pathological sectioning experiment: an appropriate amount of adipose tissue was fixed,embedded,sectioned,and stained by Hematoxylin and eosin(HE)to observe the adipose tissue morphology.4)The tissue was digested to realize the separation of SVF and mature adipocytes,and the SVF was subsequently subjected to QPCR,flow cytometry and RNA-Seq analysis,and the mature adipocytes were analyzed by QPCR.At the same time,NLRP3 knockout mice were subjected to parallel modeling.2.In vitro experiments: Induced culture of peritoneal macrophages,stimulation of macrophages with LPS(500 ng/mL),subsequently add ATP(2 mM)induced activation of NLRP3 inflammasomes,intervention using cholic acid,protein extraction of cell culture medium supernatant and cell lysis layer,and detection of Caspase-1 and IL-1βexpression by Western blot experiment.The ELISA method measured the level of IL-1βand TNF-α secretion in cell culture media,and the intervention effect of cholic acid on ASC oligomerization was detected by chemical crosslinking.Results: 1.Human samples and mouse experiments: Statistical analysis of data from clinical samples revealed that NLRP3 expression was statistically differentially upregulated in adipose tissue of patients with BMI≤24 compared to patients with BMI>24.The results of QPCR showed that the expression of NLRP3 and IL-1β genes in db/db mice was upregulated compared with the control group,and NLRP3 and IL-1βwere significantly upregulated in obese patients with type Ⅱ diabetes compared with non-diabetic patients.Subsequently,WT mice were divided into a chow diet group,a high-fat-diet group,and a high-fat-diet supplement with cholic acid(0.5%).Fasting blood glucose,glucose tolerance test and insulin tolerance test all showed that cholic acid could improve hyperglycemia and insulin resistance caused by high-fat diet,and HE staining results of white adipose tissue in mice showed significant improvement in adipose tissue size and adipose tissue inflammation.Subsequently,the QPCR results and flow cytometry results of adipose tissue and stromal vascular fraction showed that cholic acid could improve adipose tissue inflammation and inhibit macrophage inflammatory activation in obese mice.The results of RNA-Seq analysis showed that cholic acid could inhibit the activated immune response pathway of obese mice under high-fat diet conditions,and significantly downregulate the expression of inflammatory factor IL-6 and tumor necrosis factor TNF superfamily.The NLRP3 knockout mice were subjected to high-fat diet modeling,cholic acid intervention,and the improvement of inflammatory factor gene expression and secretion levels of inflammatory factor genes from the fat tissue of NLRP3 knockout mice in vivo glucose metabolism and adipose tissue HE staining were significantly lower than those of wild-type mice.2.In vitro experiments: Experiments on WT mouse intraperitoneal macrophages were performed,Western blot experiments and ELISA experiments showed that cholic acid reduces the secretion of inflammatory factors IL-1β by inhibiting the activation of NLRP3 inflammasomes and the activation of their effector protein Caspase-1 and the nucleation and oligomerization of the linker protein ASC.Conclusion: The level of NLRP3 inflammasome in obese individuals is increased,and at the animal and cellular levels,cholic acid improves the occurrence and development of inflammation in vivo and in vitro by inhibiting NLRP3 inflammasome activation.