Effect of miR-21-3p on lung injury in rats with traumatic hemorrhagic shock resuscitated with sodium bicarbonate Ringer's solution

Abstract

Background: Restricted fluid resuscitation is the most important early method for treating traumatic hemorrhagic shock (THS). This study sought to explore whether micro ribonucleic acid (miR)-21-3p affected resuscitated THS rats by regulating the glycocalyx and inflammation.

Methods: MiRNAs extracted from the lung tissues were analyzed by miRNA microarray assays. A rat model of THS was induced by hemorrhage from a left femur fracture. The pathological change in the lung tissues and glycocalyx structure was observed by hematoxylin and eosin staining and a transmission electron microscope examination. The miR-21-3p expression in the lung tissues and serum was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The levels of glycocalyx-related factors and inflammation-related factors were determined by enzyme linked immunosorbent assays. The expression of glycocalyx-related proteins, cell junction-related proteins, and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor kappa B (NF-κB) signaling pathway-related proteins was analyzed by Western blot.

Results: After RT-qPCR verification, the variation trend of miR-21-3p was in line with expected trends. The mean arterial pressure (MAP) and heart rate (HR) were decreased, and the lung injury and damage to the glycocalyx were all aggravated in the THS rats resuscitated with sodium bicarbonate Ringer's solution (BRS) or sodium lactate Ringer's solution (LRS). The expression of miR-21-3p was decreased in the THS rats resuscitated with BRS and increased in the THS rats resuscitated with LRS, and the upregulation of miR-21-3p further decreased the MAP and HR, and increased the levels of syndecan-1 (SDC-1), heparanase-1 (HPA1), interleukin (IL)-6, IL-1β, and tumor necrosis factor alpha (TNF-α) in the serum of the THS rats resuscitated with BRS. The upregulation of miR-21-3p also increased the expression of SDC-1, HPA1, β-catenin, matrix metallopeptidase (MMP)2, and MMP9, but decreased the expression of E-cadherin (E-cad) and activated the PI3K/Akt/NF-κB signaling pathway in the THS rats resuscitated with BRS and transfected with miR-21-3p compared to that of the THS rats resuscitated with BRS and transfected with miR-negative control (NC).

Conclusions: miR-21-3p promoted inflammation and glycocalyx damage by activating the PI3K/Akt/NF-κB signaling pathway, thereby aggravating the lung injury in the THS rats resuscitated with BRS.

Keywords: MiR-21-3p; lung injury; sodium bicarbonate Ringer’s solution (BRS); traumatic hemorrhagic shock (THS).

Figure 1

MiR-21-3p was chosen as the differentially expressed gene of the THS rats after different treatments. The expression of 10 differentially expressed miRNAs in the lung tissue of the THS rats receiving BRS or LRS were verified by RT-qPCR. *, P<0.05 vs. sham; ^#, P<0.05 vs. model. THS, traumatic hemorrhagic shock; BRS, bicarbonate Ringer’s solution; LRS, lactate Ringer’s solution; RT-qPCR, real-time quantitative polymerase chain reaction.

Figure 2

MiR-21-3p decreased the MAP and HR of the BRS-treated THS rats. (A) MAP; (B) HR. **, P<0.01; ***, P<0.001, vs. model. ^#, P<0.05; ^###, P<0.001, vs. BRS + miR-NC. MAP, mean arterial pressure; HR, heart rate; BRS, bicarbonate Ringer’s solution; LRS, lactate Ringer’s solution; THS, traumatic hemorrhagic shock; NC, negative control; BPM, beats per minute.

Figure 3

MiR-21-3p caused damage to the lung tissues of the BRS-treated THS rats. A pathological change in the lung tissues of the BRS-treated THS rats transfected with lentivirus-mediated miR-21-3p was observed by hematoxylin and eosin staining. THS, traumatic hemorrhagic shock; BRS, bicarbonate Ringer’s solution; LRS, lactate Ringer’s solution; NC, negative control.

Figure 4

MiR-21-3p caused damage to the glycocalyx of the BRS-treated THS rats. The glycocalyx structure of pulmonary vascular endothelial cells in the BRS-treated THS rats transfected with lentivirus-mediated miR-21-3p was observed by transmission electron microscope examination. THS, traumatic hemorrhagic shock; BRS, bicarbonate Ringer’s solution; LRS, lactate Ringer’s solution; NC, negative control.

Figure 5

MiR-21-3p expression was increased in the lung tissues and serum of the THS rats treated with BRS. The expression of miR-21-3p in the lung tissues (A) and serum (B) of the THS rats treated with BRS was detected by RT-qPCR. ***, P<0.001, vs. sham. ^#, P<0.05; ^##, P<0.01; ^###, P<0.001, vs. model. ^&, P<0.05; ^&&&, P<0.001, vs. BRS + miR-NC. THS, traumatic hemorrhagic shock; BRS, bicarbonate Ringer’s solution; LRS, lactate Ringer’s solution; NC, negative control; RT-qPCR, real-time quantitative polymerase chain reaction.

Figure 6

MiR-21-3p destroyed the glycocalyx and induced inflammation in the THS rats treated with BRS. The levels of SDC-1, HPA1 (A), and IL-6, IL-1β, and TNF-α (B) in the serum of the BRS-treated THS rats transfected with lentivirus-mediated miR-21-3p were determined by their respective ELISA kits. *, P<0.05; **, P<0.01; ***, P<0.001, vs. sham. ^#, P<0.05; ^##, P<0.01; ^###, P<0.001, vs. model. ^&, P<0.05; ^&&, P<0.01; ^&&&, P<0.001, vs. BRS + miR-NC. THS, traumatic hemorrhagic shock; BRS, bicarbonate Ringer’s solution; LRS, lactate Ringer’s solution; NC, negative control; SDC-1, syndecan-1; HPA1, heparanase-1; IL, interleukin; TNF-α, tumor necrosis factor α.

Figure 7

MiR-21-3p regulated the PI3K/Akt/NF-κB signaling pathway. The related protein expression of the glycocalyx, cell junction, and PI3K/Akt/NF-κB signaling pathway was detected by Western blot. ***, P<0.001, vs. sham. ^###, P<0.001, vs. model. ^&, P<0.05; ^&&, P<0.01; ^&&&, P<0.001, vs. BRS + miR-NC. ac, acetylated; p, phosphorylated; BRS, bicarbonate Ringer’s solution; LRS, lactate Ringer’s solution; NC, negative control; SDC-1, syndecan-1; HPA1, heparanase-1; MMP, matrix metallopeptidase; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.