Activation of SIRT1 ameliorates LPS-induced lung injury in mice via decreasing endothelial tight junction permeability

Abstract

The integrity of the endothelial barrier is a determinant of the prognosis of lipopolysaccharide (LPS)-induced acute lung injury (ALI). In this study, we investigated whether and how Sirtuin 1 (SIRT1) maintained the vascular integrity during ALI. An experimental model of ALI was established in mice through intratracheal administration of LPS (10 mg/kg). LPS stimulation significantly increased the pulmonary permeability and decreased the expression of SIRT1 and tight junction proteins (TJs), including occludin, claudin-5, tight junction protein 1 and tight junction protein 2. Morphological studies showed that LPS induced obvious lung injury with inflammatory cell infiltration in the interstitial and alveolar space, hemorrhage, edema, and the thickened alveolar wall compared to the control mice. Intratracheal administration of the selective SIRT1 activator SRT1720 (6.25 mg/kg) significantly attenuated LPS-induced lung injury, lung hyper-permeability and increased TJs expression, whereas intratracheal administration of the selective SIRT1 inhibitor EX527 (6.25 mg/kg) aggravated LPS-induced ALI. Similar protective effects of SIRT1 on pulmonary cellular permeability were observed in primary human pulmonary microvascular endothelial cells treated with LPS (2 mg/mL) in vitro. We further demonstrated that the RhoA/ROCK signaling pathway was activated in SIRT1 regulation of tight junction permeability. The RhoA/ROCK inhibitor Y-27632 (10 μM) increased the expression of TJs and reversed LPS- or EX527-induced hyper-permeability. In conclusion, SIRT1 ameliorates LPS-induced lung injury via decreasing endothelial tight junction permeability, possibly via RhoA/ROCK signaling pathway. This finding may contribute to the development of new therapeutic approaches for lung injury.

Keywords: EX527; LPS; RhoA/ROCK; SIRT1 protein; SRT1720; Y-27632; capillary endothelial permeability; human pulmonary microvascular endothelial cells; lung injury; tight junction proteins.

Fig. 1

LPS decreased SIRT1 expression in mice. a Relative expression of SIRT1 mRNA in different groups of mice (control, 10 mg/kg LPS intratracheally induced mice, 6.25 mg/kg of EX527, 6.25 mg/kg of SRT1720 alone or with LPS treatment). b Representative images of SIRT1 expression in IHC staining in six different groups. c Statistical IHC score of SIRT1 calculated using the multiplicative quick score method (QS). d Histograms represented the quantitative densitometric ratio of SIRT1 signaling molecules. e Representative Western blot images revealed SIRT1 expression. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; n = 6 for each group. Similar results were observed for three independent experiments. LSD-t test was used in two comparisons between multiple data sets

Fig. 2

A regulator of SIRT1 could influence LPS-induced lung injury. a Representative images of hematoxylin and eosin (H&E)-stained lung sections from six different groups. b Pathologic score results of lung section in these six groups. *P < 0.05, ***P < 0.001. Data are expressed as the mean ± SEM. n = 6/group. Similar results were observed in three independent experiments. LSD-t test was used in two comparisons between multiple data sets

Fig. 3

SIRT1 regulated pulmonary permeability in LPS-induced ALI. a Lung wet/dry ratio of mice. b Bronchoalveolar lavage fluid (BALF) protein concentration measured using the BCA kit. c Evans blue index. *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SEM. n = 6/group. Similar results were observed in three independent experiments. LSD-t test was used in two comparisons between multiple sets of data

Fig. 4

SIRT1 regulated LPS-induced transmembrane permeability in vitro. a Value of TEER (ohm.cm²) index of HPMECs among the group of control and different stimulation. b Permeability index of Transwell-membrane measured after the addition of FITC-dextran at 0, 1, 2, 3, and 4 h. c Knockdown of SIRT1 by siRNA. d Value of TEER between negative and siSIRT1 with/without LPS stimulation. *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SEM. n = 6/group. Similar results were observed in three independent experiments. One-way ANOVA was used in multiple comparisons. LSD-t test was used in two comparisons between multiple data sets

Fig. 5

Effect of a SIRT1 regulator on tight junction (TJs) protein expression. Relative gene expression of occludin (a), ZO-1 (b), and ZO-2 (c). d Representative image of Western blot of GAPDH, occludin, ZO-1, and ZO-2. e Histograms represented the quantitative densitometric ratio of target protein compared with GAPDH. *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SEM. n = 6/group. LSD-t test was used in two comparisons between multiple data sets

Fig. 6

Decreased occludin expression in the lung of LPS-induced mice. a Representative images of occludin expression based on IHC staining in different six groups. b Statistical IHC score of occludin calculated using the multiplicative quick score method (QS). *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SEM. n = 6/group. Similar results were observed in three independent experiments. LSD-t test was used in two comparisons between multiple data sets

Fig. 7

SIRT1 regulates claudin-5 expression in the lung. a. Relative gene expression of claudin-5. *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SEM. Scale bar = 50 μm. n = 6/group. LSD-t test was used in two comparisons between multiple sets of data. b Representative images of claudin-5 expression based on IF staining from four different groups

Fig. 8

SIRT1 acts on tight junction permeability through the RhoA/ROCK signal pathway. a Relative RhoA mRNA expression. b Relative ROCK mRNA expression. c Representative Western blot images of β-actin and RhoA. d Histograms represented the quantitative densitometric ratio of target protein compared with β-actin. e, f Permeability index of Transwell-membrane measured after the addition of FITC-dextran at 0, 1, 2, 3, and 4 h. *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SEM. n = 6/group. Similar results were observed in three independent experiments. One-way ANOVA was used in multiple comparisons. LSD-t test was used in two comparisons between multiple data sets