Protective effects of the notoginsenoside R1 on acute lung injury by regulating the miR-128-2-5p/Tollip signaling pathway in rats with severe acute pancreatitis

Abstract

Notoginsenoside R1 (NG-R1), the extract and the main ingredient of Panax notoginseng, has anti-inflammatory effects and can be used in treating acute lung injury (ALI). In this study, we explored the pulmonary protective effect and the underlying mechanism of the NG-R1 on rats with ALI induced by severe acute pancreatitis (SAP). MiR-128-2-5p, ERK1, Tollip, HMGB1, TLR4, IκB, and NF-κB mRNA expression levels were measured using real-time qPCR, and TLR4, Tollip, HMGB1, IRAK1, MyD88, ERK1, NF-κB65, and P-IκB-α protein expression levels using Western blot. The NF-κB and the TLR4 activities were determined using immunohistochemistry, and TNF-α, IL-6, IL-1β, and ICAM-1 levels in the bronchoalveolar lavage fluid (BALF) using ELISA. Lung histopathological changes were observed in each group. NG-R1 treatment reduced miR-128-2-5p expression in the lung tissue, increased Tollip expression, inhibited HMGB1, TLR4, TRAF6, IRAK1, MyD88, NF-κB65, and p-IκB-α expression levels, suppressed NF-κB65 and the TLR4 expression levels, reduced MPO activity, reduced TNF-α, IL-1β, IL-6, and ICAM-1 levels in BALF, and alleviated SAP-induced ALI. NG-R1 can attenuate SAP-induced ALI. The mechanism of action may be due to a decreased expression of miR-128-2-5p, increased activity of the Tollip signaling pathway, decreased activity of HMGB1/TLR4 and ERK1 signaling pathways, and decreased inflammatory response to SAP-induced ALI. Tollip was the regulatory target of miR-128-2-5p.

Keywords: Notoginsenoside R1; Toll interacting protein; acute lung injury; miR-128-2-5p; pancreatitis; rats.

Figure 1.

Transfection efficiency of the mimics or inhibitor. MiR-128-2-5p mimics, miR-128-2-5p mimic–NC, miR-128-2-5p inhibitor, and miR-128-2-5p inhibitor–NC were mixed with Lipofectamine 2000 (Invitrogen) and added to the A549 cell culture. The cultures were transfected for 24 h, and the miR-128-2-5p level was measured using qRT-PCR. The data obtained from quantitative densitometry are presented as mean ± SD of three independent experiments. The data from three independent experiments are presented as mean ± SD. *P < 0.05 vs control mimic. #P < 0.05 vs control inhibitor.

Figure 2.

Effect of miR-128-2-5p on the tollip signaling pathway in RAW264.7 macrophages. MiR-128-2-5p mimics, miR-128-2-5p mimic–NC, miR-128-2-5p inhibitor, and miR-128-2-5p inhibitor–NC were mixed with Lipofectamine 2000 (Invitrogen) and added to the cell culture. The cultures were transfected for 24 h. (A–C) Expression levels of NF-κB65, p-IκB-α, TLR4, and Tollip were measured using Western blot. (D–E) IL-6 and TNF-α expression levels in RAW264.7 macrophages were measured using ELISA. (F) The growth inhibition rate was measured using the MTT assay. The data obtained from quantitative densitometry are presented as mean ± SD of three independent experiments. *P < 0.05 vs control mimic. ^#P < 0.05 vs control inhibitor.

Figure 3.

Effect of NG-R1 on miR-128-2-5p and the tollip signaling pathway in RAW264.7 macrophages. RAW264.7 macrophages were incubated with LPS (1 mg/l) and various concentrations of NG-R1 (10, 20, 30, 40, and 50 μM) for 24 h to analyze the effects of NG-R1 on LPS-induced miR-128-2-5p and the Tollip signaling pathway in RAW264.7 macrophages. The miR-128-2-5p expression was measured using qRT-PCR. The expression levels of Tollip, NF-κB65, p-IκB-α, and TLR4 were measured using Western blot. The data obtained from quantitative densitometry are presented as mean ± SD of three independent experiments. **P < 0.01, *P < 0.05 vs blank control group. ^#P < 0.05 vs LPS group.

Figure 4.

Effect of NG-R1 on cellular inflammation and cell viability of LPS-induced RAW264.7 macrophages. RAW264.7 macrophages were incubated with LPS (1 mg/l) and various concentrations of NG-R1 (10, 20, 30, 40, and 50 μM) for 24 h to analyze the effect of NG-R1 on cellular inflammation and cell viability of LPS-induced RAW264.7 macrophages. IL-6 and TNF-α levels were measured using ELISA. The cell viability was measured using the CCK-8 assay. Data are expressed as mean ± SD of three replicates. **P < 0.01 vs the blank control group; ^#P < 0.05 vs the LPS group.

Figure 5.

Effect of miR-128-2-5p knockdown on SAP-induced ALI. The miR-128-2-5p-knockdown construct was delivered to rats through intravenous tail administration of anti-miR-128-2-5p oligonucleotides, and SAP-induced ALI was induced through intraperitoneal taurocholate challenge. The rat lungs were histopathologically examined. The lung injury score was measured. (A) Representative images of H&E-stained lung sections from three experimental groups (400 × magnification). (B) Lung injury score. Data are expressed as mean ± SD of three experiments. **P < 0.01 vs SO or SO treatment group; ^##P < 0.01 vs SAP group.

Figure 6.

Effect of miR-128-2-5p knockdown on miR-128-2-5p and Tollip/TLR4 signaling pathway in SAP-induced ALI. The miR-128-2-5p-knockdown construct was delivered to rats through intravenous tail administration of anti-miR-128-2-5p oligonucleotides, and SAP-induced ALI was established through intraperitoneal taurocholate challenge. The rat lungs were histopathologically examined. (A–B) Representative qRT-PCR images showing the miR-128-2-5p expression level from four experimental groups. (C–E) Representative Western blots and statistical summary of the densitometric analysis of the expression levels of NF-κB65, p-IκB-α, HMGB1, TLR4, and Tollip in rat lungs with acute pancreatitis at 24 h after administering NG-R1 treatment. Data were expressed as mean ± SD of three experiments. **P < 0.01 vs SO group; ^#P < 0.05 vs SAP group.

Figure 7.

Tollip as a direct target of miR-128-2-5p. (A–B) Tollip mRNA levels increased with miR-128-2-5p down-regulation in RAW264.7 macrophages (P < 0.05). (C–D) The Tollip protein levels decreased with miR-128-2-5p overexpression in RAW264.7 macrophages compared with vector controls (P < 0.05). MiR-128-2-5p bound to Tollip-3′-UTR-wt, whereas binding was blocked by Tollip-3′-UTR-mt. (F) Dual luciferase reporter assays confirmed that the miR-128-2-5p mimic bound to Tollip-3′-UTR-wt but not to Tollip-3′-UTR-mt (P < 0.05).

Figure 8.

Effect of the notoginsenoside R1 treatment on the gene and the protein expression levels of miR-128-2-5p, ERK1, Tollip, HMGB1, TLR4, IκB, and NF-κB from the acute pancreatitis-induced lung tissue. (A) Representative qRT-PCR images showing the miR-128-2-5p, IκB, ERK1, Tollip, HMGB1, TLR4, and NF-κB genes expression level in rats' lungs at 48 h after the notoginsenoside R1 administration. (B–C) Statistical summary of the densitometric analysis of the expression levels of miR-128-2-5p, ERK1, Tollip, HMGB1, TLR4, and NF-κB genes in rats. (D) Representative Western blots showing the expression levels of TLR4, p-IRAK1, MyD88, Tollip, p-ERK1, NF-κB65, IκB, p-IκB-α and HMGB1 in rats' lungs with acute pancreatitis at 24 h after the notoginsenoside R1 administration. (E–H) Statistical summary of the densitometric analysis of the expression levels of TLR4, p-IRAK1, MyD88, Tollip, p-ERK1, NF-κB65, IκB, p-IκB-α, and HMGB1 in rat lung tissues. Data were presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. the SO group; #P < 0.05, ##P < 0.01 vs. the SAP group.

Figure 9.

Attenuation of MPO activity and systemic inflammation in the lungs and pancreas in acute pancreatitis-induced lung injury by NG-R1 treatment. (A) The MPO activity in the lungs and pancreas were measured at 48 h after NG-R1 administration. (B–F) The BALF TNF-α, IL-1β, IL-6, and ICAM-1 levels were determined using ELISA at 48 h after NG-R1 administration. Data are expressed as mean ± SD. ** P < 0.01 vs SO group; ^#P < 0.05 vs SAP group.

Figure 10.

Effect of NG-R1 on the activation of NF-κB65 and TLR4 in acute pancreatitis-induced lung tissue observed using immunohistochemical staining. (A) Representative immunohistochemically stained graphs of NF-κB65- and TLR4-positive expression (400 × magnification). (B) Statistical analysis of NF-κB65- and TLR4-positive expression. Data are expressed as mean ± SD. **P < 0.01 vs SO group; ^#P < 0.05 vs SAP group.

Figure 11.

Effect of NG-R1 treatment on the lung wet/dry mass ratio and the BALF protein concentration in rats with taurocholate-induced acute pancreatitis. SAP was induced using intraperitoneal injection of 5% sodium taurocholate. Subsequently, the rats were administered with either 15 mg/kg NG-R1 injection or 300 μl aqueous saline via tail vein injection for 48 h. Lung wet/dry mass ratio and BALF protein concentration were measured. Data are expressed as mean ± SEM. **P < 0.01 vs control group; ^#P < 0.05 vs SAP group. Data are expressed as mean ± SD. * P < 0.05 vs SO group; ^#P < 0.05 vs model group.

Figure 12.

Effect of NG-R1 treatment on the changes in lung pathology and lung injury score in rats with taurocholate-induced acute pancreatitis. SAP was induced using intraperitoneal injection of 5% sodium taurocholate. Subsequently, rats were administered with either 15 mg/kg NG-R1 injection or 300 μl aqueous saline via tail vein injection for 48 h. (A) Representative images of H&E-stained lung sections from four experimental groups (200 × magnification). (B) Statistical analysis of lung injury score. Data are expressed as mean ± SD. **P < 0.01 vs control group; ^#P < 0.05 vs paraquat group. Data are expressed as mean ± SEM. * * P < 0.01 vs SO group; ^#P < 0.05 vs SAP group.