O allow cells to adjust. Then, 1 mg/ml LPS was added

O allow cells to adjust. Then, 1 mg/ml LPS was added to all groups except the control group to induce an inflammation model. After 24 h of stimulation with LPS, purified rHDL was added to each group at a final concentration of 5 mg/ml.Western Blot Analysis of the Inflammation Signaling Pathway in RAW264.7 CellsCells were scraped into 100 ml lysis buffer (50 mM Tris-HCl, pH 6.8, 2 SDS, and 10 glycerol) supplemented with 1 ml (1006) TA 01 site protein inhibitor cocktail at 12 h, 24 h and 48 h after treatment with different rHDLs, and samples were boiled at 100uC for 10 min and clarified by centrifugation at 12,000 rpm for 10 min at room temperature. Protein concentrations were determined by the BCA method. Equivalent protein amounts were separated on 10 SDS-polyacrylamide gels and transferred to Immobilon-P polyvinylidene fluoride membranes. The blots were then hybridized with specific primary antibodies, and antigen-specific signals were detected using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence. Gel analyses were performed to determine the gray value of the western blot bands using Image J.Immunohistochemical Detection of NF-kB p65 in Lung TissueAs shown in Figure 2, compared with the saline group (Figs. 2A and 2B), the lungs of mice receiving only LPS (Figs. 2C and 2D) had significant pathological changes: 1) congestion; 2) broadening of pulmonary interstitial tissue; 3) leukocyte infiltration, including monocytes and neutrophils; and 4) high NF-kB p65-positive expression. As shown in Figures 2E and 2F, the mice treated with rHDLwt exhibited only low NF-kB p65-positive expression compared with the saline group. In addition, the ability 18297096 of rHDL74 to block the LPS-induced NF-kB pathway in this septic mouse model was strongly supported by its immunohistochemical results (Figs. 2G and 2H), which were close to the saline group, and there was almost no positive expression of NF-kB p65 in the rHDL74 group. However, in lung sections (Figs. 2I and 2J) from mice treated with rHDL228, we observed aggravated NF-kB p65 expression compared with the LPS group.ApoA-I Cysteine Mutants and InflammationWestern Blot Analysis of the Inflammation Signaling Pathway in the RAW264.7 Inflammation Model Treated with rHDLTo investigate the potential mechanisms of different rHDLs on inflammation, we examined the signaling pathway in the RAW264.7 inflammation model treated with rHDLs. As shown in Figure 3, at 12 h after treatment with rHDLs, RAW264.7 cells treated with rHDL74 or rHDLwt had a significant decrease in p38 Iloprost activation compared with the LPS group. rHDL74 and rHDLwt also showed decreased JNK activation compared with the LPS group. Moreover, rHDL228 aggravated the activation of ERK. At 24 h after treatment with rHDLs, RAW264.7 cells treated with rHDL74 had a significant decrease in p38 activation compared with the LPS and rHDLwt groups. More importantly, rHDL74 strongly inhibited the activation of JNK; we observed little phosphorylation of JNK. We found that rHDL228 significantly decreased the activation of JNK. Additionally, although there was no statistical significance (p = 0.06.0.05 vs. LPS), rHDL228 aggravated the activation of p38.DiscussionOur previous studies showed that recombinant HDL74 exhibited higher anti-inflammatory and anti-angiosclerotic capabilities, while rHDL228 showed hyper-proinflammation. In this study, we sought to identify the different mechanisms of these two rHDLs in inflammation. Our results showed that.O allow cells to adjust. Then, 1 mg/ml LPS was added to all groups except the control group to induce an inflammation model. After 24 h of stimulation with LPS, purified rHDL was added to each group at a final concentration of 5 mg/ml.Western Blot Analysis of the Inflammation Signaling Pathway in RAW264.7 CellsCells were scraped into 100 ml lysis buffer (50 mM Tris-HCl, pH 6.8, 2 SDS, and 10 glycerol) supplemented with 1 ml (1006) protein inhibitor cocktail at 12 h, 24 h and 48 h after treatment with different rHDLs, and samples were boiled at 100uC for 10 min and clarified by centrifugation at 12,000 rpm for 10 min at room temperature. Protein concentrations were determined by the BCA method. Equivalent protein amounts were separated on 10 SDS-polyacrylamide gels and transferred to Immobilon-P polyvinylidene fluoride membranes. The blots were then hybridized with specific primary antibodies, and antigen-specific signals were detected using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence. Gel analyses were performed to determine the gray value of the western blot bands using Image J.Immunohistochemical Detection of NF-kB p65 in Lung TissueAs shown in Figure 2, compared with the saline group (Figs. 2A and 2B), the lungs of mice receiving only LPS (Figs. 2C and 2D) had significant pathological changes: 1) congestion; 2) broadening of pulmonary interstitial tissue; 3) leukocyte infiltration, including monocytes and neutrophils; and 4) high NF-kB p65-positive expression. As shown in Figures 2E and 2F, the mice treated with rHDLwt exhibited only low NF-kB p65-positive expression compared with the saline group. In addition, the ability 18297096 of rHDL74 to block the LPS-induced NF-kB pathway in this septic mouse model was strongly supported by its immunohistochemical results (Figs. 2G and 2H), which were close to the saline group, and there was almost no positive expression of NF-kB p65 in the rHDL74 group. However, in lung sections (Figs. 2I and 2J) from mice treated with rHDL228, we observed aggravated NF-kB p65 expression compared with the LPS group.ApoA-I Cysteine Mutants and InflammationWestern Blot Analysis of the Inflammation Signaling Pathway in the RAW264.7 Inflammation Model Treated with rHDLTo investigate the potential mechanisms of different rHDLs on inflammation, we examined the signaling pathway in the RAW264.7 inflammation model treated with rHDLs. As shown in Figure 3, at 12 h after treatment with rHDLs, RAW264.7 cells treated with rHDL74 or rHDLwt had a significant decrease in p38 activation compared with the LPS group. rHDL74 and rHDLwt also showed decreased JNK activation compared with the LPS group. Moreover, rHDL228 aggravated the activation of ERK. At 24 h after treatment with rHDLs, RAW264.7 cells treated with rHDL74 had a significant decrease in p38 activation compared with the LPS and rHDLwt groups. More importantly, rHDL74 strongly inhibited the activation of JNK; we observed little phosphorylation of JNK. We found that rHDL228 significantly decreased the activation of JNK. Additionally, although there was no statistical significance (p = 0.06.0.05 vs. LPS), rHDL228 aggravated the activation of p38.DiscussionOur previous studies showed that recombinant HDL74 exhibited higher anti-inflammatory and anti-angiosclerotic capabilities, while rHDL228 showed hyper-proinflammation. In this study, we sought to identify the different mechanisms of these two rHDLs in inflammation. Our results showed that.