[Protective effect of Shenfu injection on the endothelium of severe sepsis by inhibiting CD11b+ cell paralysis induced by high mobility group protein B1]

Abstract

Objective: To investigate the relationship between the biochemical parameters, the pulmonary pathologic injury and the immune mechanism of severe sepsis in infant porcine, and the intervention effect of Shenfu injection.

Methods: Panamanian infant porcine (2-3 months old) were divided into sham operation group (Sham group; intravenous injection of normal saline), lipopolysaccharide (LPS) induced severe sepsis model group (LPS group; intravenous injection of LPS 1 mg/kg, and continuing at 0.5 mg×kg^-1×h^-1 for 12 hours), and Shenfu injection intervention group (SF group; intravenous injection of Shenfu injection 10 mL/kg at the same time of modeling, twice a day) according to the random number table method, with 5 in each group. Forty-eight hours after the challenge, the changes of C-reactive protein (CRP), procalcitonin (PCT), oxygenation index (PaO₂/FiO₂), base excess (BE), blood lactate (Lac) and other biochemical indexes were detected with blood sampling; the number of neutrophils [myeloperoxidase positive (MPO⁺)] and their activated subsets CD11b⁺CD64⁺, M1 macrophages (CD80⁺CD64⁺), CD4⁺ and CD8⁺ T cells were analyzed in peripheral blood by flow cytometry. The levels of plasma cytokines were detected by enzyme linked immunosorbent assay (ELISA). The pathological damage of lung tissue was observed by hematoxylin-eosin (HE) staining. The mRNA expression of lung injury related molecules and their receptors, chemokines, cytokines, vascular endothelial related molecules and tight junction protein were detected by reverse transcription-polymerase chain reaction (RT-PCR).

Results: Compared with Sham group, the levels of CRP, PCT and Lac in LPS group significantly increased, and PaO₂/FiO₂ and BE significantly decreased. In the peripheral blood, CD80⁺CD64⁺ macrophages, CD11b⁺CD64⁺ and MPO⁺ neutrophils, CD4⁺, CD8⁺ T cells and tumor necrosis factor-α (TNF-α) significantly decreased, but interleukin-10 (IL-10) and vascular endothelial cell growth factor (VEGF) significantly increased; the mRNA expressions of high mobility group protein B1 (HMGB1), receptor for advanced glycation end products (RAGE), angiopoietin 2/angiopoietin 1 (Ang2/Ang1) significantly increased, Toll like receptor 9 (TLR9), chemokines (CXCL9 and CXCL10), TNF-α, IL-27, Tek tyrosine kinase 2 (TIE2), vascular endothelial cadherin (VE-CAD) and Occludin significantly decreased in lung tissue. HE staining showed inflammatory cell infiltration and exudation in the alveolar cavity, alveoli consolidation, thickening of the alveolar interstitial layer and emphysema. Compared with LPS group, Lac in SF group significantly decreased (mmol/L: 4.2±1.0 vs. 6.3±1.1, P < 0.05), while BE and PaO₂/FiO₂ significantly increased [BE (mmol/L): -6.4±2.6 vs. -11.6±2.5, PaO₂/FiO₂ (mmHg, 1 mmHg = 0.133 kPa): 180±36 vs. 105±35, both P < 0.05]; the percentage of CD80⁺CD64⁺ macrophages, CD11b⁺CD64⁺ and MPO⁺ neutrophils significantly increased [CD80⁺CD64⁺: (7.13±2.01)% vs. (3.80±0.46)%, CD11b⁺CD64⁺: (8.33±2.55)% vs. (2.15±0.47)%, MPO⁺: (21.22±2.33)% vs. (8.31±0.46)%, all P < 0.05]; the mRNA expressions of HMGB1 and RAGE decreased to some extent [HMGB1 mRNA (2^-ΔΔCT): 1.81±0.45 vs. 2.23±0.85, RAGE mRNA (2^-ΔΔCT): 6.69±3.48 vs. 11.60±6.91, both P < 0.05], the mRNA expressions of CXCL9 and TIE2 increased to a certain extent [CXCL9 mRNA (2^-ΔΔCT): 1.06±0.63 vs. 0.50±0.12, TIE2 mRNA (2^-ΔΔCT): 1.42±0.68 vs. 0.27±0.16, both P < 0.05]; the pathological damage of lung tissue were significantly alleviated.

Conclusions: The increased abnormality of BE, Lac, and PaO₂/FiO₂, the immune exhausting and paralysis may be important factors leading to the fatal lung injury in infant porcine with severe sepsis. The possible mechanism is that the excessive activation of HMGB1 and its receptor RAGE, the suppression of TLR9 and corresponding chemokine and inflammatory factors lead to increased endothelial damage and decreased tight connection, which in turn induces capillary leakage. The intervention of immune disorders by Shenfu injection in the severe pneumonia accompanied by sepsis may be related to elevated M1 macrophages and activated neutrophils in the peripheral blood, inhibition of HMGB1 and its receptor RAGE mRNA expression, and elevated CXCL9 and TIE2 expression.