| Studies have shown that intestinal flora metabolites are closely related to body metabolism,and TMAO(trimethylamine oxide)is one of the main metabolites of choline through intestinal flora.Low levels of TMAO have positive effects such as stabilizing protein,nucleic acid structure,and cell volume.High concentrations of TMAO are obviously harmful and are considered to be the promoters of chronic diseases such as atherosclerosis and diabetes.Pyroptosis is a pro-inflammatory programmed cell death mediated by the Gsdermin family of proteins,occurring in most cells.Cancer cells and infected cells maintain the body’s homeostasis through pyroptosis,but generally,the inflammation accompanying the pyroptosis of somatic cells becomes a cause of disease.Adipocyte pyroptosis is accompanied by low-grade inflammation of adipose tissue,which may exacerbate the development of other related diseases.In addition,TMAO has been shown to promote the pyroptosis of vascular epithelial cells and activate the Nlrp3 inflammasome in a variety of cells,but there is still a lack of research on the role of TMAO in regulating adipocytes.In this study,mice were fed a high choline diet in vivo,and adipocytes were treated with high concentration of TMAO in vitro,and the mechanism of TMAO regulating adipocyte pyroptosis was explored by ELISA,WB,qPCR,IF and other techniques.Provide basic data for the role of TMAO in regulating energy metabolism and life activities of adipocytes,and provide new references for improving oxidative stress and low-grade inflammation in adipose tissue.It provides a new target for the role of intestinal flora metabolites in regulating adipocytes,and provides a new direction for meat quality improvement and high-quality pig breeding.The main results are as follows:1.A high-choline diet increases the concentration of TMAO in the body and promotes oxidative stress in adipocytes.High-dose(5%)choline diet increased the levels of TMAO in blood,liver and adipose tissue(P<0.05);up-regulated the expression levels of FMO3 and cut C/D,key factors of choline-TMAO metabolism(P<0.05);Gavage of Clostridium butyricum(C.)in mice increased the concentration of TMAO in blood and liver,and promoted the expression level of cut C/D(P<0.05).In addition,there were no obvious pathological changes in the organs of the mice in the high choline diet group,but the volume of adipose tissue was significantly reduced.Both high choline diet and 300 μM TMAO treatment in vitro significantly inhibited the m RNA and Sod2 protein levels of antioxidant enzyme genes Sod2,Gpx1,CAT in adipose tissue/adipocytes(P<0.05),and also down-regulated mitochondrial markers Ndufa9,Sdha and other m RNA and COXIV,ATP5A1 protein levels(P<0.05);at the same time decreased SOD enzyme activity(P<0.05),increased the content of peroxidation marker MDA(P<0.05);TMAO promoted the production of ROS in adipocytes and inhibited the production of ATP(P<0.05).In addition,similar results were detected in porcine primary adipocytes(P<0.05).The above data suggest that a high choline diet increases TMAO concentrations in vivo and promotes oxidative stress in adipocytes.2.High concentrations of TMAO lead to Nlrp3-dependent pyroptosis in adipocytes.HE staining of adipose tissue in the high choline diet group(HC)observed obvious coronary structures,and the expression levels of pro-inflammatory factors IL-6,TNF-α and i NOS were significantly increased,indicating that high choline diet caused adipose tissue inflammation.High-choline diet and high-concentration TMAO treatment significantly up-regulated the expressions of cleaved-Casp1,GSDMD and mature-IL-1β(P<0.05);increased the release of extracellular IL-1β and LDH(P<0.05);Nuclei were significantly fragmented;pretreatment of cells with the Nlrp3-specific inhibitor MCC950 significantly alleviated the above-mentioned changes caused by TMAO(P<0.05).Similar changes were detected in porcine primary adipocytes,including up-regulated expression of pyroptotic markers,increased extracellular release of IL-1β,and increased levels of up-regulated TNFα,i NOS,and IL-6(P<0.05).The above results show that high concentrations of TMAO lead to Nlrp3-dependent adipocyte pyroptosis.3.High concentrations of TMAO regulate adipocyte pyroptosis through the Nrf2/ROS/Nlrp3 inflammasome pathway.Pretreatment of cells with ROS scavenger Tempol significantly inhibited TMAO-induced oxidative stress(ROS production,ATP content,SOD enzyme activity,etc.)and Nlrp3 protein level(P<0.05).Both high choline diet and high concentration of TMAO significantly inhibited the expression of Nrf2(P<0.05);The DMF(Nrf2 activator)treatment significantly up-regulated Sod2,CAT etc.m RNA and Nrf2,Sod2 protein levels.Meanwhile,after drug activation of Nrf2,the level of Nlrp3,a key factor in pyroptosis,and the release of IL-1β were significantly reduced(P<0.05);on the contrary,drug inhibition of Nrf2 aggravated the above-mentioned effects of TMAO(P<0.05).In addition,overexpression of Gsta4 can alleviate oxidative stress and pyroptosis caused by TMAO,including increased levels of antioxidant enzyme genes Sod2,CAT,Gpx1,etc.(P<0.05),decreased ROS production(P<0.05),and key factors of pyroptosis.The expression levels of Nlrp3,cleaved-Casp1 and mature-IL-1β were down-regulated(P<0.05).These results demonstrate that TMAO regulates adipocyte pyroptosis through the Nrf2/ROS/Nlrp3 inflammasome pathway.Taken together,high choline diet induces oxidative stress in adipocytes by increasing the concentration of TMAO in vivo,which in turn leads to Nlrp3-dependent pyroptosis in adipocytes.Moreover,the increase of C.butyricum in intestinal tract can promote the production of TMAO by up-regulating the level of the choline lyase gene cut C/D.High concentrations of TMAO regulate adipocyte pyroptosis through the Nrf2/ROS/Nlrp3 inflammasome pathway,and increase the cellular antioxidant capacity to inhibit the above effects.These results provide new targets for the role of gut microbiota metabolites in regulating adipocytes and provide new references for meat quality improvement. |
