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Atorvastatin increases lipopolysaccharide-induced expression of tumour necrosis factor-α-induced protein 8-like 2 in RAW264.7 cells.

Liu MW, Su MX, Zhang W, Wang L, Qian CY - Exp Ther Med (2014)

Bottom Line: The cells were harvested, and the mRNA and protein expression levels of TIPE2, macrophage migration inhibitory factor (MIF), IκB and nuclear factor (NF-κB)-κB were analysed using quantitative polymerase chain reaction and western blotting analysis, respectively, the expression of NOS, COX-2 and HO-1 protein were essayed by western blotting analysis, NO and ROS activities were determined.Meanwhile, LPS enhanced the expression of NOS and COX-2 protein, blocked HO-1 protein expression, increased NO and PGE2 production and ROS activity in a dose dependent manner in RAW264.7 cells.Atorvastatin significantly increased LPS induced expression of TIPE2, downregulated the expression of NOS, COX-2, MIF and NF-κB and the production of PGE2, NO, IL-6 and TNF-α in a time and dose dependent manner, and increased HO-1 protein expression, reduced ROS production in a dose dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Emergency, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China.

ABSTRACT
RAW264.7 cells are one of the major sources of productive inflammatory biomediators, including tumour necrosis factor-α (TNF-α) and interleukin (IL)-6. TNF-α-induced protein 8-like 2 (TIPE2) is an essential negative regulator of Toll-like and T-cell receptors, and the selective expression in the immune system prevents hyper-responsiveness and maintains immune homeostasis. The aim of the present study was to investigate whether atorvastatin upregulates the expression of TIPE2 and further regulates the inflammatory response and oxidation emergency response in RAW264.7 cells. RAW264.7 cells were incubated in Dulbecco's modified Eagle's medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. Following incubation, the medium was collected and the levels of TNF-α and IL-6 were measured using an enzyme-linked immunosorbent assay. The cells were harvested, and the mRNA and protein expression levels of TIPE2, macrophage migration inhibitory factor (MIF), IκB and nuclear factor (NF-κB)-κB were analysed using quantitative polymerase chain reaction and western blotting analysis, respectively, the expression of NOS, COX-2 and HO-1 protein were essayed by western blotting analysis, NO and ROS activities were determined. The results revealed that LPS increased the mRNA and protein expression levels of TIPE2, MIF and NF-κB, as well as the production of IL-6 and TNF-α, in a dose and time dependent manner in RAW264.7 cells. Meanwhile, LPS enhanced the expression of NOS and COX-2 protein, blocked HO-1 protein expression, increased NO and PGE2 production and ROS activity in a dose dependent manner in RAW264.7 cells. Atorvastatin significantly increased LPS induced expression of TIPE2, downregulated the expression of NOS, COX-2, MIF and NF-κB and the production of PGE2, NO, IL-6 and TNF-α in a time and dose dependent manner, and increased HO-1 protein expression, reduced ROS production in a dose dependent manner. The observations indicated that atorvastatin upregulated LPS induced expression of TIPE2 and consequently inhibited MIF, NF-κB, NOS and COX-2 expression and the production of NO, PGE2, TNF-α and IL-6, increased HO-1 expression, and inhibited ROS activity in cultured RAW264.7 cells.

No MeSH data available.


Related in: MedlinePlus

Effect of TIPE2 on NF-κB p65 protein expression in LPS induced RAW264.7 cells. TIPE2 siRNA and control RAW264.7 cells were incubated with various concentrations of LPS for 9 h, or 10 ng/ml LPS for various time periods. NF-κB p65 protein expression was measured using western blot analysis. Values are expressed as the mean ± standard deviation of three independent experiments. Groups: 1, TIPE2 siRNA RAW264.7 cells; 2, RAW264.7 cells; C, control. (A) Dose and (B) time dependent effects of LPS on NF-κB p65 protein expression in RAW264.7 cells and TIPE2 siRNA RAW264.7 cells. (A) P<0.05, group 1 (5, 10 and 15 mg/ml) vs. group 2 (5, 10 and 15 mg/ml); group 1: *P<0.05, **P<0.01, vs. control; group 2: #P<0.05, ##P<0.01, vs. control. (B) P<0.05, group 1 (6, 9 and 12 h) vs. group 2 (6, 9 and 12 h); group 1: *P<0.05, **P<0.01, vs. control; group 2: #P<0.05, ##P<0.01, vs. control. TIPE2, tumour necrosis factor-α induced protein 8 like 2; NF-κB, nuclear factor κB; LPS, lipopolysaccharide.
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f1-etm-08-01-0219: Effect of TIPE2 on NF-κB p65 protein expression in LPS induced RAW264.7 cells. TIPE2 siRNA and control RAW264.7 cells were incubated with various concentrations of LPS for 9 h, or 10 ng/ml LPS for various time periods. NF-κB p65 protein expression was measured using western blot analysis. Values are expressed as the mean ± standard deviation of three independent experiments. Groups: 1, TIPE2 siRNA RAW264.7 cells; 2, RAW264.7 cells; C, control. (A) Dose and (B) time dependent effects of LPS on NF-κB p65 protein expression in RAW264.7 cells and TIPE2 siRNA RAW264.7 cells. (A) P<0.05, group 1 (5, 10 and 15 mg/ml) vs. group 2 (5, 10 and 15 mg/ml); group 1: *P<0.05, **P<0.01, vs. control; group 2: #P<0.05, ##P<0.01, vs. control. (B) P<0.05, group 1 (6, 9 and 12 h) vs. group 2 (6, 9 and 12 h); group 1: *P<0.05, **P<0.01, vs. control; group 2: #P<0.05, ##P<0.01, vs. control. TIPE2, tumour necrosis factor-α induced protein 8 like 2; NF-κB, nuclear factor κB; LPS, lipopolysaccharide.

Mentions: To investigate the effect of TIPE2 on NF-κB p65 expression and the secretion of TNF-α and IL-6, RAW264.7 cells or TIPE2-siRNA RAW264.7 cells were incubated with LPS (10 ng/ml) for various time periods. The expression of NF-κB p65 and the secretion of IL-6 and TNF-α in the two groups increased with LPS at each time point; however, when compared with the RAW264.7 macrophages, the NF-κB p65 expression and secretion of IL-6 and TNF-α were significantly elevated in the TIPE2-siRNA RAW264.7 macrophages at 6, 9 and 12 h (Figs. 1B and 2C and D). Thus, TIPE2 downregulated the expression of NF-κB p65 and decreased the production of IL-6 and TNF-α in a time-dependent manner (Figs. 1B and 2C and D). To determine the concentration-dependent effects of LPS, RAW264.7 cells or TIPE2-siRNA RAW264.7 cells were incubated at various concentrations of LPS for 9 h. LPS increased the expression of NF-κB p65 and the production of IL-6 and TNF-α in a concentration-dependent manner (Figs. 1A and 2A and B). However, when compared with the RAW264.7 macrophages, the NF-κB p65 expression and secretion of IL-6 and TNF-α were significantly elevated in the TIPE2-siRNA RAW264.7 macrophages treated with 5, 10 and 15 ng/ml LPS.


Atorvastatin increases lipopolysaccharide-induced expression of tumour necrosis factor-α-induced protein 8-like 2 in RAW264.7 cells.

Liu MW, Su MX, Zhang W, Wang L, Qian CY - Exp Ther Med (2014)

Effect of TIPE2 on NF-κB p65 protein expression in LPS induced RAW264.7 cells. TIPE2 siRNA and control RAW264.7 cells were incubated with various concentrations of LPS for 9 h, or 10 ng/ml LPS for various time periods. NF-κB p65 protein expression was measured using western blot analysis. Values are expressed as the mean ± standard deviation of three independent experiments. Groups: 1, TIPE2 siRNA RAW264.7 cells; 2, RAW264.7 cells; C, control. (A) Dose and (B) time dependent effects of LPS on NF-κB p65 protein expression in RAW264.7 cells and TIPE2 siRNA RAW264.7 cells. (A) P<0.05, group 1 (5, 10 and 15 mg/ml) vs. group 2 (5, 10 and 15 mg/ml); group 1: *P<0.05, **P<0.01, vs. control; group 2: #P<0.05, ##P<0.01, vs. control. (B) P<0.05, group 1 (6, 9 and 12 h) vs. group 2 (6, 9 and 12 h); group 1: *P<0.05, **P<0.01, vs. control; group 2: #P<0.05, ##P<0.01, vs. control. TIPE2, tumour necrosis factor-α induced protein 8 like 2; NF-κB, nuclear factor κB; LPS, lipopolysaccharide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4061217&req=5

f1-etm-08-01-0219: Effect of TIPE2 on NF-κB p65 protein expression in LPS induced RAW264.7 cells. TIPE2 siRNA and control RAW264.7 cells were incubated with various concentrations of LPS for 9 h, or 10 ng/ml LPS for various time periods. NF-κB p65 protein expression was measured using western blot analysis. Values are expressed as the mean ± standard deviation of three independent experiments. Groups: 1, TIPE2 siRNA RAW264.7 cells; 2, RAW264.7 cells; C, control. (A) Dose and (B) time dependent effects of LPS on NF-κB p65 protein expression in RAW264.7 cells and TIPE2 siRNA RAW264.7 cells. (A) P<0.05, group 1 (5, 10 and 15 mg/ml) vs. group 2 (5, 10 and 15 mg/ml); group 1: *P<0.05, **P<0.01, vs. control; group 2: #P<0.05, ##P<0.01, vs. control. (B) P<0.05, group 1 (6, 9 and 12 h) vs. group 2 (6, 9 and 12 h); group 1: *P<0.05, **P<0.01, vs. control; group 2: #P<0.05, ##P<0.01, vs. control. TIPE2, tumour necrosis factor-α induced protein 8 like 2; NF-κB, nuclear factor κB; LPS, lipopolysaccharide.
Mentions: To investigate the effect of TIPE2 on NF-κB p65 expression and the secretion of TNF-α and IL-6, RAW264.7 cells or TIPE2-siRNA RAW264.7 cells were incubated with LPS (10 ng/ml) for various time periods. The expression of NF-κB p65 and the secretion of IL-6 and TNF-α in the two groups increased with LPS at each time point; however, when compared with the RAW264.7 macrophages, the NF-κB p65 expression and secretion of IL-6 and TNF-α were significantly elevated in the TIPE2-siRNA RAW264.7 macrophages at 6, 9 and 12 h (Figs. 1B and 2C and D). Thus, TIPE2 downregulated the expression of NF-κB p65 and decreased the production of IL-6 and TNF-α in a time-dependent manner (Figs. 1B and 2C and D). To determine the concentration-dependent effects of LPS, RAW264.7 cells or TIPE2-siRNA RAW264.7 cells were incubated at various concentrations of LPS for 9 h. LPS increased the expression of NF-κB p65 and the production of IL-6 and TNF-α in a concentration-dependent manner (Figs. 1A and 2A and B). However, when compared with the RAW264.7 macrophages, the NF-κB p65 expression and secretion of IL-6 and TNF-α were significantly elevated in the TIPE2-siRNA RAW264.7 macrophages treated with 5, 10 and 15 ng/ml LPS.

Bottom Line: The cells were harvested, and the mRNA and protein expression levels of TIPE2, macrophage migration inhibitory factor (MIF), IκB and nuclear factor (NF-κB)-κB were analysed using quantitative polymerase chain reaction and western blotting analysis, respectively, the expression of NOS, COX-2 and HO-1 protein were essayed by western blotting analysis, NO and ROS activities were determined.Meanwhile, LPS enhanced the expression of NOS and COX-2 protein, blocked HO-1 protein expression, increased NO and PGE2 production and ROS activity in a dose dependent manner in RAW264.7 cells.Atorvastatin significantly increased LPS induced expression of TIPE2, downregulated the expression of NOS, COX-2, MIF and NF-κB and the production of PGE2, NO, IL-6 and TNF-α in a time and dose dependent manner, and increased HO-1 protein expression, reduced ROS production in a dose dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Emergency, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China.

ABSTRACT
RAW264.7 cells are one of the major sources of productive inflammatory biomediators, including tumour necrosis factor-α (TNF-α) and interleukin (IL)-6. TNF-α-induced protein 8-like 2 (TIPE2) is an essential negative regulator of Toll-like and T-cell receptors, and the selective expression in the immune system prevents hyper-responsiveness and maintains immune homeostasis. The aim of the present study was to investigate whether atorvastatin upregulates the expression of TIPE2 and further regulates the inflammatory response and oxidation emergency response in RAW264.7 cells. RAW264.7 cells were incubated in Dulbecco's modified Eagle's medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. Following incubation, the medium was collected and the levels of TNF-α and IL-6 were measured using an enzyme-linked immunosorbent assay. The cells were harvested, and the mRNA and protein expression levels of TIPE2, macrophage migration inhibitory factor (MIF), IκB and nuclear factor (NF-κB)-κB were analysed using quantitative polymerase chain reaction and western blotting analysis, respectively, the expression of NOS, COX-2 and HO-1 protein were essayed by western blotting analysis, NO and ROS activities were determined. The results revealed that LPS increased the mRNA and protein expression levels of TIPE2, MIF and NF-κB, as well as the production of IL-6 and TNF-α, in a dose and time dependent manner in RAW264.7 cells. Meanwhile, LPS enhanced the expression of NOS and COX-2 protein, blocked HO-1 protein expression, increased NO and PGE2 production and ROS activity in a dose dependent manner in RAW264.7 cells. Atorvastatin significantly increased LPS induced expression of TIPE2, downregulated the expression of NOS, COX-2, MIF and NF-κB and the production of PGE2, NO, IL-6 and TNF-α in a time and dose dependent manner, and increased HO-1 protein expression, reduced ROS production in a dose dependent manner. The observations indicated that atorvastatin upregulated LPS induced expression of TIPE2 and consequently inhibited MIF, NF-κB, NOS and COX-2 expression and the production of NO, PGE2, TNF-α and IL-6, increased HO-1 expression, and inhibited ROS activity in cultured RAW264.7 cells.

No MeSH data available.


Related in: MedlinePlus