Polydatin up-regulates Clara cell secretory protein to suppress phospholipase A2 of lung induced by LPS in vivo and in vitro

Abstract

Background: Lung injury induced by lipopolysaccharide (LPS) remains one of the leading causes of morbidity and mortality in children. The damage to membrane phospholipids leads to the collapse of the bronchial alveolar epithelial barrier during acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Phospholipase A2 (PLA2), a key enzyme in the hydrolysis of membrane phospholipids, plays an important traumatic role in pulmonary inflammation, and Clara cell secretory protein (CCSP) is an endogenous inhibitor of PLA2. Our previous study showed that polydatin (PD), a monocrystalline extracted from a traditional Chinese medicinal herb (Polygonum cuspidatum Sieb, et Zucc), reduced PLA2 activity and sPLA2-IIA mRNA expression and mitigated LPS-induced lung injury. However, the potential mechanism for these effects has not been well defined. We have continued to investigate the effect of PD on LPS-induced expression of CCSP mRNA and protein in vivo and in vitro.

Results: Our results suggested that the CCSP mRNA level was consistent with its protein expression. CCSP expression was decreased in lung after LPS challenge. In contrast, PD markedly increased CCSP expression in a concentration-dependent manner. In particular, CCSP expression in PD-pretreated rat lung was higher than in rats receiving only PD treatment.

Conclusion: These results indicated that up-regulation of CCSP expression causing inhibition of PLA2 activation may be one of the crucial protective mechanisms of PD in LPS-induced lung injury.

Figure 1

The chemical structure of trans-polydatin (3,4',5-trihydroxystilbene-3-β-single-D-glucoside).

Figure 2

Effects of polydatin on the expression of CCSP mRNA in rat lung. A. Relative expression of CCSP, quantified by real-time PCR. B. Electrophoresis showing the RT-PCR results. I: sham operation group, injected with normal saline; II: endotoxic shock group, injected with LPS (10 mg·kg^-1 body weight); III: PD treatment group, injected with LPS (10 mg·kg^-1 body weight), then a 0.5% PD solution (0.2 ml·kg^-1 body weight) 1 hour later; IV: PD pretreatment group, injected with a 0.5% PD solution (0.2 ml·kg^-1 body weight), then LPS (10 mg·kg^-1 body weight) 0.5 hours later; V: PD control group. ^cP < 0.01 vs. group I; ^eP < 0.05 and ^fP < 0.01 vs. group II; ⁱP < 0.05 vs. group III.

Figure 3

The dose-effect relationship between polydatin and CCSP mRNA expression in rat lung. A. CCSP mRNA expression in PD treatment groups with different doses by real-time PCR (A-1) and RT-PCR(A-2). B. CCSP mRNA expression in PD pretreatment groups with different doses by real-time PCR (B-1) and RT-PCR(B-2). ^bP < 0.05 vs. 1 mg·kg^-1 PD group, ^eP < 0.05 vs. 5 mg·kg^-1PD group, and ^jP < 0.01 vs. 10 mg·kg^-1 PD group.

Figure 4

Effects of polydatin on the expression of sPLA₂/cPLA₂ mRNA in rat lung. A. Relative expression of sPLA₂, quantified by real-time PCR. B. Relative expression of cPLA₂, quantified by real-time PCR. I: sham operation group, injected with normal saline; II: endotoxic shock group, injected with LPS (10 mg·kg^-1 body weight); III: PD treatment group, injected with LPS (10 mg·kg^-1 body weight), then a 0.5% PD solution (0.2 ml·kg^-1 body weight) 1 hour later; IV: PD pretreatment group, injected with a 0.5% PD solution (0.2 ml·kg^-1 body weight), then LPS (10 mg·kg^-1 body weight) 0.5 hours later; V: PD control group. ^bP < 0.05 and ^cP < 0.01 vs. group I; ^fP < 0.01 vs. group II; ⁱP < 0.05 and ^jP < 0.01 vs. group III.

Figure 5

The dose-effect relationship between polydatin and sPLA₂ mRNA expression in rat lung. A. sPLA2 mRNA expression in PD treatment groups with different doses by real-time PCR and RT-PCR. B. sPLA2 mRNA expression in PD pretreatment groups with different doses by real-time PCR and RT-PCR. ^cP < 0.01 vs. 1 mg·kg^-1 PD group, ^eP < 0.05 and ^fP < 0.01 vs. 5 mg·kg^-1PD group, and ⁱP < 0.05 vs. 10 mg·kg^-1 PD group.

Figure 6

The dose-effect relationship between polydatin and cPLA₂ mRNA expression in rat lung. A. cPLA₂ mRNA expression in PD treatment groups with different doses by real-time PCR. B. cPLA₂ mRNA expression in PD pretreatment groups with different doses by real-time PCR. ^cP < 0.01 vs. 1 mg·kg^-1 PD group, ^eP < 0.05 and ^fP < 0.01 vs. 5 mg·kg^-1PD group, and ⁱP < 0.05 vs. 10 mg·kg^-1 PD group.

Figure 7

Effects of polydatin on the expression of CCSP protein in rat lung. CCSP protein expression was examined by western blot. I: sham operation group; II: endotoxic shock group; III: PD treatment group; IV: PD pretreatment group; V: PD control group. A. The electrophoretogram of CCSP protein expression. B. Relative ratio of CCSP protein expression. ^bP < 0.05 and ^cP < 0.01 vs. group I; ^eP < 0.05 and ^fP < 0.01 vs. group II; ⁱP < 0.05 vs. group III.

Figure 8

Dose-effect relationship between polydatin and CCSP protein expression in rat lung. A.CCSP protein expression in PD treatment groups with different doses. B.CCSP protein expression in PD pretreatment groups with different doses. ^bP < 0.05 vs. 1 mg·kg^-1 PD group, ^eP < 0.05 and ^fP < 0.01 vs. 5 mg·kg^-1PD group, and ⁱP < 0.05 vs. 10 mg·kg^-1 PD group.

Figure 9

Effects of polydatin on serum CCSP level in different groups of rats. I: Sham operation group; II: endotoxic shock group; III: PD treatment group; IV: PD pretreatment group; V: PD control group. ^bP < 0.05 vs. group I; ^fP < 0.01 vs. group II.

Figure 10

Dose-effect relationship between polydatin and serum CCSP level in rat. A.Serum CCSP level in PD treatment groups with different doses. B. Serum CCSP level in PD pretreatment groups with different doses. ^bP < 0.05 and ^cP < 0.01 vs. 1 mg·kg^-1 PD group, ^fP < 0.01 vs. 5 mg·kg^-1PD group, and ⁱP < 0.05 vs. 10 mg·kg^-1 PD group.

Figure 11

Effects of polydatin on CCSP mRNA expression in LPS-induced BEAS-2B cells. A. Relative expression of CCSP quantified by real-time PCR. B. RT-PCR analysis shown by electrophoresis. ^bP < 0.05 vs. normal control group; ^eP < 0.05 and ^fP < 0.01 vs. LPS group; ⁱP < 0.05 vs. LPS+PD group.

Figure 12

Effects of polydatin on CCSP protein levels in LPS-induced BEAS-2B cells. A. The electrophoretogram of CCSP protein expression. B. Relative ratio of CCSP protein expression. ^cP < 0.01 vs. normal control group; ^eP < 0.05 and ^fP < 0.01 vs. LPS group; ⁱP < 0.05 vs. LPS+PD group.