PTEN Interference Promote The Expression Of Inflammatory Factor In Alveolar Macrophages

Objective:To observe the transfection of PTEN interference RNA(PTEN si RNA) in alveolar macrophages of rats(NR8383) induced the expression of tumor necrosis factor-α(TNF-α) by lipopolysaccharide(lipopolysaccharide, LPS), to investigate the possible regulatory effect and mechanism of PTEN on NR8383 inflammatory reactionand.Methods:1. In vitro culture of NR8383, cells were divided into two groups: the control group transfected with control si RNA, interference group were transfected with PTEN si RNA, 10 n M, 20 n M, 40 n M concentration were selected, 24 hours after transfected, using real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-q PCR) to detect the expression of PTEN m RNA in two groups of cells after transfected, using Western blotting to detect the expression of PTEN protein in two groups of cells after transfected. Optimization of transfection conditions, choose 20 n M as the optimal transfect concentration.2. NR8383 cells were divided into two groups: the control group transfected with control si RNA, interference group were transfected with PTEN si RNA, 24 hours after transfected, cells in two groups were stimulation of LPS with the final concentration of 1μg/ml after 6h, using RT-q PCR to detect the expression of TNF-αm RNA in two groups of cells, using Western blotting to detect AKT phosphorylation level in two groups of cells, the expression of TNF-α protein in supernatant were detected in two groups of cells by enzyme linked immunosorbent assay(ELISA).Results:1. Compared with the control group, in 10 n M, 20 n M and 40 n M interference group cells, PTEN m RNA expression were reduced to(0.33±0.01) fold(P < 0.01),(0.23±0.01) fold(P < 0.01),(0.21±0.01) fold(P < 0.01); the expression of PTENprotein decreased to(0.30±0.03)fold(P < 0.01),(0.14±0.02) fold(P < 0.01),(0.10±0.02) fold(P < 0.01); in the interference group, compared with the concentration of10 n M, the expression of PTEN protein in the concentration of 20 n M and 40 n M had significant difference(P<0.01). The expression of PTEN protein was no significant difference between the concentration of 20 n M and the concentration of40 n M(P = 0.08).2. Two groups of cells were stimulated by LPS after 6 hours, compared with the control group, the ratio of p-AKT/AKT protein increased from(0.26±0.02) to(0.52±0.03)(P < 0.01), TNF-α m RNA expression in interference group cells were increase to(1.56±0.18) fold(P<0.01). Control si RNA + LPS group the expression of TNF-α protein in the supernatant is 1632.56±52.05pg/ml, PTEN si RNA + LPS group the expression of TNF-α protein in the supernatant is 1834.07±57.00pg/ml(P<0.05).Conclusion:Transfection of PTEN si RNA into NR8383 cells, can upregulate the phosphorylation level of AKT, promote the expression of TNF-α in alveolar macrophages by LPS induced. Therefore, we hypothesized that PTEN may be through the PI3K-AKT pathway regulation that LPS induced alveolar macrophage inflammatory response.