Glaucocalyxin A alleviates lipopolysaccharide-induced inflammation and apoptosis in pulmonary microvascular endothelial cells and permeability injury by inhibiting STAT3 signaling

Abstract

Glaucocalyxin A (GLA), an ent-kauranoid diterpene derived from Rabdosia japonica var. glaucocalyx, possesses antibacterial, anti-oxidative and anti-neuroinflammatory properties. The present study aimed to investigate the potential mechanisms underlying GLA in the pathogenesis of pneumonia. Human pulmonary microvascular endothelial cells (hPMVECs) treated with lipopolysaccharide (LPS) were treated with GLA, followed by the detection of cell viability, inflammation, apoptosis and cell permeability. Furthermore, the protein expression levels of apoptosis- and permeability-associated proteins were determined using western blot analysis. Following treatment with a signal transducer and activator of transcription 3 (STAT3) activator, the protein expression levels of STAT3 and endoplasmic reticulum stress-associated proteins were determined, to confirm whether STAT3 signaling was mediated by GLA. Lastly, the mRNA expression level of inflammatory cytokines, apoptosis and permeability injury were also determined following treatment with a STAT3 activator. The results revealed that GLA ameliorated inflammation, apoptosis and permeability injury in LPS-induced hPMVECs. Following treatment with a STAT3 activator, the therapeutic effects of GLA on LPS-induced hPMVECs were abrogated. In conclusion, GLA alleviated LPS-induced inflammation, apoptosis and permeability injury in hPMVECs by inhibiting STAT3 signaling, which highlighted the potential therapeutic value of GLA in the treatment of pneumonia.

Keywords: STAT3; apoptosis; glaucocalyxin A; inflammation.

Figure 1

GLA ameliorates LPS-induced hPMVEC injury. Cell viability of (A) hPMVECs treated with different concentrations of GLA was determined by performing a Cell Counting Kit-8 assay and (B) hPMVECs treated with GLA after LPS induction. ^***P<0.001 vs. LPS (-) + GLA (µM) (-); ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. LPS (+) + GLA (µM) (-). hPMVECs, human pulmonary microvascular endothelial cells; GLA, glaucocalyxin A; LPS, lipopolysaccharide.

Figure 2

GLA ameliorates inflammation and apoptosis in LPS-induced hPMVECs. (A) mRNA expression level of inflammatory cytokines, (B) rate of apoptosis (magnification, x200) and (C) protein expression of apoptosis-related proteins in LPS-induced hPMVECs treated with GLA was analyzed using reverse transcription-quantitative PCR, TUNEL and western blot analysis, respectively. ^***P<0.001 vs. LPS (-) + GLA (µM) (-); ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. LPS (+) + GLA (µM) (-). hPMVECs, human pulmonary microvascular endothelial cells; GLA, glaucocalyxin A; LPS, lipopolysaccharide.

Figure 3

GLA ameliorates damage of cell permeability in LPS-induced hPMVECs. (A) Permeability of LPS-induced hPMVECs treated with GLA was detected using FITC-dextran. (B) Protein expression level of VE-cadherin, ZO-1 and claudin 5 in LPS-induced hPMVECs treated with GLA was analyzed using western blot analysis. ^***P<0.001 vs. LPS (-) + GLA (µM) (-); ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. LPS (+) + GLA (µM) (-). hPMVECs, human pulmonary microvascular endothelial cells; GLA, glaucocalyxin A; LPS, lipopolysaccharide; VE-cadherin, vascular endothelial cadherin; ZO-1, zonula occludens-1.

Figure 4

GLA reduces the STAT3-mediated expression of ERS-related proteins in LPS-induced hPMVECs treated with GLA. Protein expression level of (A) p-STAT3 and STAT3 and (B) ERS-related proteins in LPS-induced hPMVECs treated with GLA was analyzed using western blot analysis. ^***P<0.001 vs. LPS (-) + GLA (µM) (-); ^#P<0.05, ^##P<0.01, ^###P<0.00 vs. LPS (+) + GLA (µM) (-). hPMVECs, human pulmonary microvascular endothelial cells; GLA, glaucocalyxin A; LPS, lipopolysaccharide; p, phosphorylated, ERS, endoplasmic reticulum stress; GRP78, glucose regulated protein 78; ATF6, activating transcription factor 6; CHOP, C/EBP-homologous protein; STAT3, signal transducer and activator of transcription 3.

Figure 5

GLA reduces the release of ERS in LPS-induced hPMVECs by inhibiting STAT3 signal. The protein expression level of p-STAT3, STAT3 and ERS-related protein expression in LPS-induced hPMVECs co-treated with GLA and colivelin was analyzed using western blot analysis and subsequently quantified using densitometry. ^***P<0.001 vs. LPS (-) + GLA (µM) (-); ^###P<0.001 vs. LPS (+) + GLA (µM) (-) + Colivelin (µM) (-); ^∆∆P<0.01, ^∆∆∆P<0.001 vs. LPS (+) + GLA (µM) (10) + Colivelin (µM) (-). hPMVECs, human pulmonary microvascular endothelial cells; GLA, glaucocalyxin A; LPS, lipopolysaccharide; p, phosphorylated, ERS, endoplasmic reticulum stress; ATF6, activating transcription factor 6; CHOP, C/EBP-homologous protein; STAT3, signal transducer and activator of transcription 3; GRP78, glucose regulated protein 78.

Figure 6

GLA reduces the inflammation and apoptosis in LPS-induced hPMVECs by inhibiting STAT3 signal. (A) mRNA expression level of inflammatory cytokines, (B) rate of apoptosis (magnification, x200) and (C) protein expression level of apoptosis-related proteins in LPS-induced hPMVECs co-treated with GLA and colivelin was analyzed using reverse transcription-quantitative PCR, TUNEL and western blot analysis, respectively. hPMVECs, human pulmonary microvascular endothelial cells; GLA, glaucocalyxin A; LPS, lipopolysaccharide; STAT3, signal transducer and activator of transcription 3. ^***P<0.001 vs. LPS (-) + GLA (µM) (-); ^###P<0.001 vs. LPS (+) + GLA (µM) (-) + Colivelin (µM) (-). ^∆∆P<0.01 and ^∆∆∆P<0.001 vs. LPS (+) + GLA (µM) (10) + Colivelin (µM) (-).

Figure 7

GLA ameliorates the damaged permeability of LPS-induced hPMVECs by inhibiting STAT3 signal. (A) Permeability of LPS-induced hPMVECs co-treated with GLA and colivelin were detected using FITC-dextran. (B) mRNA levels of VE-cadherin, ZO-1 and claudin 5 in LPS-induced hPMVECs co-treated with GLA and colivelin were analyzed using reverse transcription-quantitative PCR analysis. (C) Protein expression level of VE-cadherin, ZO-1 and claudin 5 in LPS-induced hPMVECs co-treated with GLA and colivelin was analyzed using western blot analysis. ^***P<0.001 vs. LPS (-) + GLA (µM) (-); ^###P<0.001 vs. LPS (+) + GLA (µM) (-) + Colivelin (µM) (-); ^∆P<0.05, ^∆∆P<0.01 and ^∆∆∆P<0.001 vs. LPS (+) + GLA (µM) (10) + Colivelin (µM) (-). hPMVECs, human pulmonary microvascular endothelial cells; GLA, glaucocalyxin A; LPS, lipopolysaccharide; VE-cadherin, vascular endothelial cadherin; ZO-1, zonula occludens-1.