PTPRO knockdown protects against inflammation in hemorrhage shock-induced lung injury involving the NF-κB signaling pathway

Abstract

Background: Hemorrhage shock (HS) is characterized by decreased tissue oxygenation and organ damage due to severe blood loss. Protein tyrosine phosphatase receptor type O (PTPRO) is abnormally up-regulated in the rat lungs after trauma/HS.

Methods: To elucidate the regulatory mechanism of PTPRO in lung inflammation following HS, we established a rat model of HS via withdrawing blood by a catheter inserted into the femoral artery followed by resuscitation. The rats were infected with lentivirus harboring short hairpin RNA (shRNA) targeting PTPRO by intratracheal instillation.

Results: PTPRO was significantly up-regulated in rat lungs after HS. PTPRO knockdown enhanced epithelial integrity and reduced capillary leakage by up-regulating tight junction proteins zonula occludens-1 (ZO-1) and occludin (OCC) in the lungs. Besides, HS-induced myeloperoxidase activity and inflammatory cell infiltration was mitigated by PTPRO knockdown. The expression of inflammatory cytokines/chemokines (TNF-α, IL-6, MIP-2, MCP-1, and KC) in the lungs and bronchoalveolar lavage fluid was regressed after PTPRO knockdown. The nuclear factor kappa B (NF-κB) pathway was involved in HS-induced lung inflammation. PTPRO down-regulation inhibited the NF-κB pathway activation by suppressing the phosphorylation of NF-κB and its translocation from the cytoplasm into the nucleus in HS.

Conclusion: Taken together, we demonstrated that PTPRO knockdown may contribute to attenuating inflammation in HS-induced lung injury via inhibiting NF-κB pathway activation.

Keywords: Hemorrhage shock (HS); Inflammation; Lung injury; Protein tyrosine phosphatase receptor type O (PTPRO); The NF-κB signaling pathway.

Fig. 1

a Increased expression of PTPRO in the serum of HS patients and lungs of HS rats. RT-qPCR analysis of the expression of PTPRO in the serum of HS patients (n = 7) versus healthy participants (n = 30). b Timeline of the experiments. Rats were subjected to bloodletting by a catheter inserted into the femoral artery. Mean arterial pressure was maintained at 40–50 mmHg for 1 h (shock-period) and then the rats were resuscitated. Three hours after resuscitation, all rats were euthanized for tissue collection. For PTPRO knockdown, the rats were instilled with lentivirus harboring short hairpin RNA (shRNA) targeting PTPRO (LV-shPTPRO) or negative control shRNA (LV-shNC) 48 h before hemorrhage. c, d HS strongly induces the expression of PTPRO in rat lungs. c The relative mRNA expression of PTPRO in the lung of HS rats was detected by RT-qPCR. d Representative blot image and relative PTPRO protein expression in rat lungs were detected by Western blot. e Immunohistochemical staining for PTPRO in rat lungs. Magnification: 200 ×. Scale = 100 μm. f Double immunofluorescence staining of PTPRO and beta IV tubulin or keratin 19. Magnification: 400 × . Scale = 50 μm. White arrows show the representative co-localization. The values represent the mean (n = 6) ± SD. **P < 0.01

Fig. 2

Kockdown of PTPRO reduces capillary leakage and attenuates HS-induced lung injury. a Lung histopathology in the HS model was assessed by H&E staining. Magnification: 200 × . Scale = 100 μm. b Histologic score corresponding to H&E staining for lung injury assessment. c Micrograms of Evans blue (EB) per gram of lung tissue and per microlitre of bronchoalveolar lavage fluid (BALF) were used to assess lung permeability in HS model. d Relative mRNA levels of tight junction proteins zonula occludens-1 (ZO-1) and occludin (OCC) in the lung tissues were determined by RT-qPCR. e, f Representative blot image e. and relative protein expression f of ZO-1 and OCC in the lung tissues were detected by Western blot. Values are means (n = 6) ± SD. **P < 0.01

Fig. 3

Down-regulation of PTPRO decreases inflammatory cell infiltration and related cytokine/chemokine levels in HS. a Representative images of myeloperoxidase (MPO) immunofluorescence staining in the lung of HS rats were shown. Magnification: 400 × . Scale = 50 μm. b MPO activity in the lung tissues of HS rats was detected with the MPO kit. c Relative mRNA levels of pro-inflammatory cytokines TNF-α and IL-6 were detected by RT-qPCR. d The levels of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were detected with ELISA. e Relative mRNA levels of chemokine MCP-1, MIP-2, and KC in the lung tissues. f The levels of MCP-1, MIP-2, and KC in BALF were detected with ELISA. g BALF cell counts including total leukocyte, neutrophils, and macrophage. Values are means (n = 6) ± SD. *P < 0.05, **P < 0.01

Fig. 4

Knockdown of PTPRO contributes to the suppression of the NF-κB pathway activation in HS. a Immunofluorescence staining showed the intracellular localization of NF-κB P65 in the lung tissues. Magnification: 400 × . Scale = 50 μm or 25 μm. White arrow heads show the representative intracellular localization of NF-κB P65. b Expression levels of total IKKα/β and phosphorylated IKKα/β in the rat lung were detected by Western blot and the ratio of p-IKKα/β to total IKKα/β was calculated. c, d Western blot and expression quantification of phosphorylated NF-κB P65. c and nuclear NF-κB P65. d In the lungs were performed. Values are means (n = 6) ± SD. **P < 0.01

Fig. 5

Diagram of the potential regulatory mechanism of PTPRO in HS-induced lung inflammation. PTPRO is up-regulated in the lungs after HS, which may contribute to NF-κB P65 phosphorylation and the translocation of NF-κB from the cytoplasm into the nucleus. Activated NF-κB in the nucleus likely initiates the transcription of proinflammatory genes including cytokine and chemokine, thus inducing lung inflammation following HS. PTPRO knockdown alleviates the HS-induced inflammation in the lungs by reducing the NF-κB pathway activation