Salubrinal protects against cigarette smoke extract-induced HBEpC apoptosis likely via regulating the activity of PERK-eIF2α signaling pathway.

BACKGROUND AND AIMS: Endoplasmic reticulum (ER) stress plays an important role in cigarette smoke extract (CSE)-induced apoptotic cell death, which is an important pathogenic factor of chronic obstructive pulmonary disease (COPD). The aim of this study was to explore the role of the PERK-eIF2 pathway in CSE-induced human bronchial epithelial (HBE) cell apoptosis and to evaluate the protective effects and possible mechanism of salubrinal (Sal) on CSE-induced HBE cell apoptosis. METHODS: Normal human bronchial epithelial cells (HBEpC) were cultured and then treated with CSE alone or together with Sal or preincubated with or without PERK siRNA. Expressions of p-PERK/PERK, p-eIF2α/eIF2α, and caspase 3 and 4 were detected with PCR, Western blot, and immunofluorescence. Apoptosis was detected using AnnexinV-PI flow cytometry. RESULTS: CSE induced apoptotic cell death and caused a dynamic change in PERK-eIF2α pathway activity following the course of CSE exposure. The knockdown of PERK suppressed the expression of both PERK and p-eIF2a and caused a great increase in cell apoptosis. Sal could eliminate the effects of PERK knockdown, protecting the cells against the CSE insult, and this protection was accomplished through maintaining the homeostasis of PERK- eIF2α pathway. CONCLUSIONS: PERK-eIF2α pathway mediates the CSE-induced HBE cell apoptosis. The intactness of PERK-eIF2α pathway is crucial for HBE cell survival under CSE insult. Sal can protect against CSE-induced HBE cell apoptosis, and this effect is likely achieved through maintaining the homeostasis of PERK- eIF2α pathway.

[Expression of secretory leukocyte proteinase inhibitor in human bronchial epithelial cell is downregulated by transforming growth factor-beta1/Smads pathway].

OBJECTIVE: To study the relationship between the downregulated expression of secretory leukocyte proteinase inhibitor (SLPI) in human bronchial epithelial cells and transforming growth factor (TGF)-beta1/Smads pathway. METHODS: Normal human bronchial epithelial cells of the line HBE were cultured and divided into 4 groups: TGF-beta1 stimulation group stimulated by TGF-beta1, interference group preincubated with Smad4 siRNA and then stimulated by TGF-beta1, interference control group preincubated with negative siRNA and then stimulated by TGF-beta1, and normal control group. Forty-eight hours later immunocytochemistry was used to observe the SLPI positive staining in the cells, and the protein and mRNA expression levels of Smad4 and SLPI were detected by Western blotting and RT-PCR respectively. RESULTS: Immunocytochemistry showed that the Smad4 staining was strongly positive in the TGF-beta1 stimulation group and interference control group, and the SLPI staining was strongly positive in the normal control group, positive in the interference group, and only weakly positive in the TGF-beta1 stimulation and interference control groups. The protein and mRNA expression levels of Smad4 in the HBE cells of the TGF-beta1 stimulation group were 1.18 +/- 0.17 and 1.33 +/- 0.16 respectively, both significantly higher than those of the normal control group (0.29 +/- 0.06 and 0.31 +/- 0.07 respectively, both P < 0.01) and interference group (0.27 +/- 0.08 and 0.34 +/- 0.09 respectively, both P < 0.01). The protein and mRNA expression levels of SLPI in the HBE cells of the TGF-beta1 stimulation group were 0.17 +/- 0.10 and 0.11 +/- 0.05 respectively, both significantly lower than those of the normal control group (1.29 +/- 0.21 and 0.83 +/- 0.13 respectively, both P < 0.01), and those of the interference group (1.22 +/- 0.18 and 0.81 +/- 0.11 respectively, both P < 0.01). There were no significant differences in the expression levels of Smad4 and SLPI between the TGF-beta1 stimulation group and interference control group. CONCLUSION: TGF-beta1 downregulates the SLPI expression in human bronchial epithelial cell via Smads pathway.

Downregulation of secretory leukocyte proteinase inhibitor in chronic obstructive lung disease: the role of TGF-beta/Smads signaling pathways.

BACKGROUND: Secretory leukocyte proteinase inhibitor (SLPI) is an important antileukoprotease in airway. The aim of the present study was to explore the expression of SLPI in the bronchi and lung tissues of chronic obstructive pulmonary disease (COPD) models and the regulative mechanism by transforming growth factor (TGF)beta(1)/Smads signal pathway in bronchial epithelial cell. METHODS: COPD rat model was established and was treated with or without TGFbeta1 monoclonal antibody. Spirometry was conducted, and expressions of TGFbeta(1), Smad4 and SLPI were examined by immunohistochemistry and reverse-transcription polymerase chain reaction (RT-PCR), respectively. The normal human bronchial epithelial cell (NHBE) was cultured, preincubated with or without siRNA (Smad4), and then stimulated with TGFbeta(1). Expressions of Smad4 and SLPI were detected by immunocytochemistry, Western blot and RT-PCR, respectively. RESULTS: As compared with the model group, after treatment with TGFbeta(1) monoclonal antibody, peak expiratory flow (PEF), forced expiratory volume in 0.3 sec (FEV(0.3)) and FEV(0.3)/forced vital capacity (FVC) in the TGFbeta(1) monoclonal antibody intervention group were all significantly improved. Expression of SLPI was also improved, but expression of Smad4 was significantly decreased. Expression of SLPI in NHBE cells was inhibited by TGFbeta(1) both at the mRNA level and the protein level. Furthermore, effect of TGFbeta(1)-inhibited expression of SLPI in NHBE cells was disengaged by siRNA (Smad4) both at the mRNA level and the protein level. CONCLUSIONS: Decreased expression of SLPI in the COPD rat model may be mainly caused by the increased expression of TGFbeta(1), and this process is probably related to the activation of Smads signal pathway.

Palmitic and linoleic acids impair endothelial progenitor cells by inhibition of Akt/eNOS pathway.

BACKGROUND: Endothelial progenitor cells (EPCs) are involved in adult neovasculogenesis and maintenance of vascular integrity. Scarce data have been provided for the individual effect of elevated free fatty acids (FFAs) on EPCs. This study was designed to investigate the association between Akt/eNOS signal pathway changes and the proliferation/function of EPCs in the presence of palmitic and linoleic acids. METHODS: After 14-day culture, EPCs were stimulated with different concentrations of palmitic and linoleic acids, with or without SNP, L-NAME, or LY294002. The proliferation and ability of adhesion, migration and tube structure formation of EPCs were observed and the level of phosphorylated Akt protein expression and eNOS protein expression were assayed. RESULTS: Incubation with palmitic and linoleic acids at concentrations of 0.2 muM or higher inhibited EPCs proliferation, significantly reduced migratory rate, reduced adhesion to fibronectin and impaired ability of EPCs to form tube structure in a dose-dependent manner. A simultaneous dose-dependent NO generation and Akt phosphorylation decrease as well as eNOS expression reduction at protein levels were also observed. However, all of the detrimental effects were attenuated by pretreating EPCs with SNP, NO donor. AKT and eNOS inhibitor, LY294002 and L-NAME, respectively, augmented palmitic and linoleic acids inhibitory effects on EPCs. CONCLUSIONS: These findings suggest that palmitic and linoleic acids downregulated AKT/eNOS signal pathway, which contributed to overall poor function and decrease proliferation of EPCs. These changes induced by palmitic and linoleic acids in signaling offer a novel explanation for the overall poor function of EPCs in diabetes mellitus.

[Expression of secretory leukocyte proteinase inhibitor in the bronchi and lung tissues of chronic obstructive pulmonary disease rat models and the regulatory mechanism by transforming growth factor beta(1)].

OBJECTIVE: To study the expression of secretory leukocyte proteinase inhibitor (SLPI) in the bronchi and lung tissues of chronic obstructive pulmonary disease (COPD) rat models and the regulatory mechanism by transforming growth factor beta(1) (TGF-beta(1)). METHODS: Rat COPD models were established by intratracheal instillation of lipopolysaccharide (LPS) twice and exposure to cigarette smoke daily. The drug intervention group received TGF-beta(1) monoclonal antibody 0.5 mg twice via tail venous injection. Spirometry was conducted and the pathological changes were observed. The concentrations of SLPI in bronchoalveolar lavage fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA), the expressions of TGF-beta(1), Smad4 and SLPI in the bronchi and lung tissues examined by immunohistochemistry, and the expressions of TGF-beta(1) mRNA, Smad4 mRNA and SLPI mRNA in the bronchi and lung tissues detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The SLPI positive coefficient, SLPI mRNA IOD value and the concentration of SLPI in BALF were significantly lower in the model group [(1.07), (0.17 +/- 0.01), (47 +/- 4) microg/L, respectively] as compared to the control group [(3.86), (0.84 +/- 0.10), (82 +/- 7) microg/L, respectively]. The TGF-beta(1) positive coefficient and the TGF-beta(1) mRNA IOD value were higher in the model group [(3.91), (0.71 +/- 0.09) respectively]than the control group [(1.12), (0.15 +/- 0.01), respectively]. After treated with TGF-beta(1) monoclonal antibody, the SLPI positive coefficient, SLPI mRNA IOD value and the concentration of SLPI in BALF were all significantly increased [(2.69), (0.59 +/- 0.05), (69 +/- 6) microg/L, respectively]. The PEF, FEV(0.3) and FEV(0.3)/FVC were all significantly improved in the drug intervention group [(28 +/- 6) ml/s, (4.4 +/- 1.3) ml, (80 +/- 10)%, respectively] as compared to the model group [(23 +/- 5) ml/s, (3.3 +/- 1.4) ml, (62 +/- 9)%, respectively]. CONCLUSION: The expression of SLPI in the COPD rat models significantly decreased, which may be caused by the increased expression of TGF-beta(1), and this process is probably related to the activation of Smads signal pathway.