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Inhibiting toll-like receptor 4 signaling ameliorates pulmonary fibrosis during acute lung injury induced by lipopolysaccharide: an experimental study.

He Z, Zhu Y, Jiang H - Respir. Res. (2009)

Bottom Line: Toll-like receptor 4 (TLR4) is essential in lipopolysaccharide (LPS)-induced fibroblast activation and collagen secretion in vitro.Overexpression of TLR4, type I procollagen, alpha-SMA, and p-AKT in murine pulmonary tissue after intraperitoneal injection of LPS at 72 hours and 28 days were detected.All of these changes were alleviated by intravenous infection with TLR4-shRNA lentivirus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China. hzyyy@163.com

ABSTRACT

Background: Toll-like receptor 4 (TLR4) is essential in lipopolysaccharide (LPS)-induced fibroblast activation and collagen secretion in vitro. However, its effects on the process of lung fibroblast activation and fibrosis initiation during LPS induced acute lung injury (ALI) remain unknown. The goal of the present study was to determine the effect of inhibiting TLR4 on LPS-induced ALI and fibrosis in vivo.

Methods: The ALI model was established by intraperitoneal injection of LPS in mice. TLR4-small hairpin RNA (shRNA) lentivirus was injected intravenously into the mice to inhibit TLR4 expression. mRNA and protein levels were detected by real-time PCR and Western-blot analysis, respectively. The contents of the C-terminal propeptide of type I procollagen (PICP) in bronchoalveolar lavage fluid (BALF) were detected by ELISA, and the degree of fibrosis was detected by van Gieson collagen staining, the hydroxyproline assay, and alpha smooth muscle actin (alpha-SMA) immunohistochemical staining.

Results: Overexpression of TLR4, type I procollagen, alpha-SMA, and p-AKT in murine pulmonary tissue after intraperitoneal injection of LPS at 72 hours and 28 days were detected. Moreover, the degree of fibrosis was shown to increase by ELISA analysis of PICP in BALF, van Gieson collagen staining, the hydroxyproline assay, and alpha-SMA immunohistochemical staining. All of these changes were alleviated by intravenous infection with TLR4-shRNA lentivirus.

Conclusions: Inhibiting TLR4 signaling could ameliorate fibrosis at the early stage of ALI induced by LPS.

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Inflammation and fibrosis in mouse lung tissue after LPS challenge. (A) The pathological changes in mouse lung tissue 72 hours and 28 days after intraperitoneal injection of LPS were observed by means of HE staining (HE, A, magnification ×200). Pulmonary fibrosis was observed by means of Van-Gieson staining (VG, magnification ×200). (B) The Ashcroft fibrosis score was used to compare the degrees of the pulmonary fibrosis 28 days after application of LPS in mice. The mice showed obvious inflammatory reactions in lung tissue 72 hours after LPS challenge. Typical pulmonary interstitial fibrosis appeared 4 weeks later. Infection with TLR4-shRNA lentivirus significantly inhibited the inflammatory reaction and fibrosis induced by LPS. BC: blank control group; NC: negative control group; PC: positive control group; TI: TLR4 inhibition group; TI+L: TLR4 inhibition group stimulated with LPS; 3d: specimens were collected 72 hours after LPS (or physiological saline) challenge. 4w: specimens were collected 28 days after LPS (or physiological saline) challenge. Each subgroup contains 6 specimens respectively (n = 6). Results were expressed as mean ± standard deviation indicated with column graph and error bar. Statistical significance was defined at p values < 0.05. *: P < 0.05 versus PC.
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Figure 1: Inflammation and fibrosis in mouse lung tissue after LPS challenge. (A) The pathological changes in mouse lung tissue 72 hours and 28 days after intraperitoneal injection of LPS were observed by means of HE staining (HE, A, magnification ×200). Pulmonary fibrosis was observed by means of Van-Gieson staining (VG, magnification ×200). (B) The Ashcroft fibrosis score was used to compare the degrees of the pulmonary fibrosis 28 days after application of LPS in mice. The mice showed obvious inflammatory reactions in lung tissue 72 hours after LPS challenge. Typical pulmonary interstitial fibrosis appeared 4 weeks later. Infection with TLR4-shRNA lentivirus significantly inhibited the inflammatory reaction and fibrosis induced by LPS. BC: blank control group; NC: negative control group; PC: positive control group; TI: TLR4 inhibition group; TI+L: TLR4 inhibition group stimulated with LPS; 3d: specimens were collected 72 hours after LPS (or physiological saline) challenge. 4w: specimens were collected 28 days after LPS (or physiological saline) challenge. Each subgroup contains 6 specimens respectively (n = 6). Results were expressed as mean ± standard deviation indicated with column graph and error bar. Statistical significance was defined at p values < 0.05. *: P < 0.05 versus PC.

Mentions: First, we determined the role of TLR4 in the activation of lung fibroblasts and collagen secretion at the tissue level. As shown in Figure 1, HE and VG staining of lung tissue stimulated with LPS revealed that 72 hours after the LPS challenge, the lung tissue showed obvious inflammatory reactions, and typical interstitial fibrosis occurred 28 days after LPS challenge. At the same time, the Ashcroft score increased significantly compared with the blank and negative control groups (P < 0.05).


Inhibiting toll-like receptor 4 signaling ameliorates pulmonary fibrosis during acute lung injury induced by lipopolysaccharide: an experimental study.

He Z, Zhu Y, Jiang H - Respir. Res. (2009)

Inflammation and fibrosis in mouse lung tissue after LPS challenge. (A) The pathological changes in mouse lung tissue 72 hours and 28 days after intraperitoneal injection of LPS were observed by means of HE staining (HE, A, magnification ×200). Pulmonary fibrosis was observed by means of Van-Gieson staining (VG, magnification ×200). (B) The Ashcroft fibrosis score was used to compare the degrees of the pulmonary fibrosis 28 days after application of LPS in mice. The mice showed obvious inflammatory reactions in lung tissue 72 hours after LPS challenge. Typical pulmonary interstitial fibrosis appeared 4 weeks later. Infection with TLR4-shRNA lentivirus significantly inhibited the inflammatory reaction and fibrosis induced by LPS. BC: blank control group; NC: negative control group; PC: positive control group; TI: TLR4 inhibition group; TI+L: TLR4 inhibition group stimulated with LPS; 3d: specimens were collected 72 hours after LPS (or physiological saline) challenge. 4w: specimens were collected 28 days after LPS (or physiological saline) challenge. Each subgroup contains 6 specimens respectively (n = 6). Results were expressed as mean ± standard deviation indicated with column graph and error bar. Statistical significance was defined at p values < 0.05. *: P < 0.05 versus PC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2803172&req=5

Figure 1: Inflammation and fibrosis in mouse lung tissue after LPS challenge. (A) The pathological changes in mouse lung tissue 72 hours and 28 days after intraperitoneal injection of LPS were observed by means of HE staining (HE, A, magnification ×200). Pulmonary fibrosis was observed by means of Van-Gieson staining (VG, magnification ×200). (B) The Ashcroft fibrosis score was used to compare the degrees of the pulmonary fibrosis 28 days after application of LPS in mice. The mice showed obvious inflammatory reactions in lung tissue 72 hours after LPS challenge. Typical pulmonary interstitial fibrosis appeared 4 weeks later. Infection with TLR4-shRNA lentivirus significantly inhibited the inflammatory reaction and fibrosis induced by LPS. BC: blank control group; NC: negative control group; PC: positive control group; TI: TLR4 inhibition group; TI+L: TLR4 inhibition group stimulated with LPS; 3d: specimens were collected 72 hours after LPS (or physiological saline) challenge. 4w: specimens were collected 28 days after LPS (or physiological saline) challenge. Each subgroup contains 6 specimens respectively (n = 6). Results were expressed as mean ± standard deviation indicated with column graph and error bar. Statistical significance was defined at p values < 0.05. *: P < 0.05 versus PC.
Mentions: First, we determined the role of TLR4 in the activation of lung fibroblasts and collagen secretion at the tissue level. As shown in Figure 1, HE and VG staining of lung tissue stimulated with LPS revealed that 72 hours after the LPS challenge, the lung tissue showed obvious inflammatory reactions, and typical interstitial fibrosis occurred 28 days after LPS challenge. At the same time, the Ashcroft score increased significantly compared with the blank and negative control groups (P < 0.05).

Bottom Line: Toll-like receptor 4 (TLR4) is essential in lipopolysaccharide (LPS)-induced fibroblast activation and collagen secretion in vitro.Overexpression of TLR4, type I procollagen, alpha-SMA, and p-AKT in murine pulmonary tissue after intraperitoneal injection of LPS at 72 hours and 28 days were detected.All of these changes were alleviated by intravenous infection with TLR4-shRNA lentivirus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China. hzyyy@163.com

ABSTRACT

Background: Toll-like receptor 4 (TLR4) is essential in lipopolysaccharide (LPS)-induced fibroblast activation and collagen secretion in vitro. However, its effects on the process of lung fibroblast activation and fibrosis initiation during LPS induced acute lung injury (ALI) remain unknown. The goal of the present study was to determine the effect of inhibiting TLR4 on LPS-induced ALI and fibrosis in vivo.

Methods: The ALI model was established by intraperitoneal injection of LPS in mice. TLR4-small hairpin RNA (shRNA) lentivirus was injected intravenously into the mice to inhibit TLR4 expression. mRNA and protein levels were detected by real-time PCR and Western-blot analysis, respectively. The contents of the C-terminal propeptide of type I procollagen (PICP) in bronchoalveolar lavage fluid (BALF) were detected by ELISA, and the degree of fibrosis was detected by van Gieson collagen staining, the hydroxyproline assay, and alpha smooth muscle actin (alpha-SMA) immunohistochemical staining.

Results: Overexpression of TLR4, type I procollagen, alpha-SMA, and p-AKT in murine pulmonary tissue after intraperitoneal injection of LPS at 72 hours and 28 days were detected. Moreover, the degree of fibrosis was shown to increase by ELISA analysis of PICP in BALF, van Gieson collagen staining, the hydroxyproline assay, and alpha-SMA immunohistochemical staining. All of these changes were alleviated by intravenous infection with TLR4-shRNA lentivirus.

Conclusions: Inhibiting TLR4 signaling could ameliorate fibrosis at the early stage of ALI induced by LPS.

Show MeSH
Related in: MedlinePlus