ABSTRACT
OBJECTIVE: To investigate the effect of minimally modified low-density lipoprotein (mmLDL) on ETA receptor of mesenteric artery (endothelin type A receptors, ETA) in mice for the first time and whether autophagy is involved in this process. METHODS: Mice were injected mmLDL in the tail vein and intraperitoneally with Class III PI3K autophagy pathway inhibitor 6-amino-3-methylpurine (3-MA) to explore the role of autophagy in mmLDL treated mice. The changes of vasoconstriction curve of mesenteric artery induced by ET-1 (endothelin 1) in mice were observed by a sensitive myograph system. ETA receptor was detected by RT-PCR quantitative mRNA and Western blot. The protein levels of Class III PI3K, Beclin-1, LC3-Ⅱ/, p62 and p-NF-κB, NF-κB were detected by Western blot. RESULTS: The contractility curve of ET-1 induced by mmLDL was significantly enhanced, showing that the Emax value increased from the nomal saline (NS) group (184.87±7.46)% to (319.91±20.31)% (P < 0.001), the pEC50 increased from NS group (8.05±0.05) to (9.11±0.09) (P<0.01). mmLDL up-regulated Class III PI3K,beclin-1,LC3-Ⅱ/ and down-regulated p62 protein level, at the same time, it also caused the ETA receptor mRNA level, protein expression increased significantly, up-regulated the protein level of p-NF-κB; intraperitoneal injection of 3-MA inhibits these effects of mmLDL. CONCLUSION: mmLDL can activate autophagy and down-stream NF-κB pathway through Class III PI3K/Beclin-1 pathway to up-regulate ETA receptor.
ABSTRACT
OBJECTIVE: To investigate the effect and mechanism of TLR4 monoclonal antibody (TLR4mAb) on mmLDL induced impairment of endothelium-dependent vasodilatation in mouse mesenteric artery. METHODS: The experiment established three groups of normal saline group, mmLDL treatment group and TLR4mAb intervention group. The concentration of IL-1β and TNF-α in plasma was determined by enzyme-linked immunosorbent assay (ELISA). Measurement of endothelium-dependent vasodilatation was achieved by microvascular tension mapping. Western blot and RT-PCR were used to investigate the expression level of protein and mRNA expressions in vascular tissues. In addition, ultra-structure of mesenteric artery endothelial cells was observed by transmission electron microscope. RESULTS: TLR4mAb could improve the damage of mmLDL induced impairment of endothelium-dependent vasodilatation in a dose-dependent manner. Besides, TLR4mAb obviously up-regulated protein expressions in KCa3.1-channel and KCa2.3-channel, and down-regulated the expression of inflammatory factors TNF-α and IL-1β. Furthermore, the improvement of mmLDL impaired vascular endothelial cells and endothelium-dependent vasodilatation might be correlated with its competitive antagonism of mmLDL-activated TLR4 signal transduction pathway and its downstream NF-κBp65 and p-38 MAPK pathway. CONCLUSION: Administration of TLR4mAb in advance can alleviate the impairment of endothelial cells and the decrease of endothelium-dependent vasodilatation induced by mmLDL, and inhibit the overexpression of inflammatory factors. Regulation of TLR4 pathway as well as its downstream NF-κBp65 and P-38 MAPK pathways may be effective targets for the prevention and treatment of cardiovascular diseases.