ABSTRACT
AIM:To investigate the impact of palmitate-stimulated macrophages on the invasion and migration of HepG2 cells and to explore the underlying mechanism .METHODS:Human acute monocytic leukemia cell line THP-1 were induced to macrophages by phorbol myristate acetate and were stimulated with palmitate (0.16 mmol/L).The culture supernatants were collected and used to incubate HepG 2 cells.The effect of palmitate on migration of the macrophages was detected by Transwell chamber assay .The mRNA expression of target genes was measured by RT-qPCR.The invasion and migration of the HepG 2 cells were assessed by invasion assay and scratch test .RESULTS:Palmitate promoted the migra-tion of the macrophages and increased the mRNA levels of interleukin -1β( IL-1β) , interleukin-6 ( IL-6 ) , tumor necrosis factor-α(TNF-α) and monocyte chemotactic protein-1 (MCP-1) in the macrophages.The invasion and migration of the HepG2 cells incubated with conditioned media from palmitate-stimulated macrophages were greater than those of the HepG 2 cells incubated with conditioned media from macrophages without palmitate .The media of palmitate-stimulated macrophages up-regulated the mRNA expression of cytokines and N-cadherin, and down-regulated the mRNA expression of E-cadherin in the HepG2 cells.CONCLUSION:Palmitate-stimulated macrophages promote the invasion and migration of HepG 2 cells through paracrine/endcrine loop.
ABSTRACT
AIM: To investigate the role of fatty acid translocase/CD36 (FAT/CD36) in adipose tissue in-flammation induced by a high-fat diet.METHODS:C57BL/6J mice were fed with a normal-chow diet ( NCD) or a high-fat diet ( HFD) for 14 weeks.The content of free fatty acid ( FFA) in the serum was measured by ELISA.The expression of CD36, cytokines and chemokines at mRNA and protein levels in the adipose tissues was determined by real-time poly-merase chain reaction and Western blotting.Immunohistochemical staining was used to examine the macrophages infiltration in the adipose tissues.The inflammatory responses in CD36 knockout mice and wild type mice with high-fat diet were ana-lyzed.RESULTS:The levels of FAT/CD36 were higher in HFD group than that in NCD group.HFD feeding enhanced the mRNA and protein expression of IL-1β, IL-6, TNF-α, MCP-1 and MIP-1, as well as promoted macrophage infiltration in the adipose tissues.Interestingly, as fed with HFD, the expression of cytokines/chemokines and macrophage infiltration were significantly reduced in adipose tissues of the CD36 knockout mice, compared with the wild type mice.CONCLU-SION:High-fat diet promotes adipose tissue inflammation in the mice in a FAT/CD36-dependent manner.
ABSTRACT
The experiment was designed to investigate the function of SREBP cleavage-activating protein (SCAP) mutant (D443N) by constructing an eukaryotic expressive vector using a smooth muscle specific promoter SM22 (pGL3-SM22-SCAP(D443N)). SM22 promoter (pSM22) was amplified from genome DNA of mice by nested PCR, and then cloned into pMD-T vector. The SM22 promoter fragment released from the vector by Kpn I and Hind III digestion was sub-cloned into pGL3-control-Luc vector, to form pGL3-SM22-Luc. The activity of pSM22 in human vascular smooth muscle cells (VSMCs) was tested using Dual-Luciferase Reporter System. SCAP(D443) mutant amplified from plasmid pTK-HSV-SCAP(D443N) and pSM22 from mice liver were cloned into pGL3-control vector to construct pGL3-SM22-SCAP(D443N) which was transfected into Chinese hamster ovary cells (CHO) to test SCAP(D443) expression by real-time PCR and Western blot. The sequence and construction of pGL3-SM22-SCAP(D443N) were correct. SM22 promoter activity initiated the expression of luciferase in VSMCs and also drove SCAP(D443) expression in transfected CHO cells. The pGL3-SM22-SCAP(D443N) eukaryotic expression vector was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of SCAP and also in producing vascular smooth muscle specific SCAP transgenic mice.
Subject(s)
Animals , Cricetinae , Humans , Mice , CHO Cells , Cricetulus , Genetic Vectors , Genetics , Intracellular Signaling Peptides and Proteins , Genetics , Physiology , Membrane Proteins , Genetics , Physiology , Mice, Transgenic , Microfilament Proteins , Genetics , Muscle Proteins , Genetics , Mutant Proteins , Genetics , Promoter Regions, Genetic , Genetics , RNA, Messenger , Genetics , Recombinant Proteins , Genetics , TransfectionABSTRACT
ObjectiveTo clarify why accelarated atheroslcerosis is complicated in chronic kidney disease patients, and to investigate whether endoplasmic reticuhm (ER) stress can be observed in acetylated low density lipepmtein (ACLDL)-induced apoptosis in THP-1 macrophages differentiated by 160 nmol/L PMA for five days. Methods Hoechst 33258 stain and caspase 3,7 assay were used to detect apeptosis. Oil red O was used to examine the lipid droplet. High performance liquid chromatography was used to measure intracelhlar free cholesterol (FC) and cholesterol ester (CE). Western blot was applied to demonstrate the protein level of acylcoenzyme A cholesterol acyltransferase(ACAT)1, growth arrest and DNA damage(CHOP) and Bcl-2. Real-time PCR was used to detect the changes of mRNAs. Results ACLDL could induce THP-1 macrophages apoptosis in time-and dose-dependent manner. After exposure to 100 mg/L ACLDL for 24 hours, the level of free cholesterol and cholesterol ester mass had a significant increment by 1.5-and 2.4-fold respectively (P＜0.01). CHOP increased and Bcl-2 decreased both in protein and mRNA levels. ACLDL loading also resulted in an increase of ACAT1 protein without any change in mRNA level. ConclusionIn THP-1 macrophages foam cell, apoptosis can be induced by ACLDL accompanied by ER stress pathway activation.