Curcumin Attenuates PD-L1-Positive Neutrophil-Induced T-Lymphocyte Apoptosis and Alleviates Lung Injury During Sepsis in Rats

Abstract

Objective: This study aimed to investigate the effects of curcumin (Cur) on programmed cell death 1 ligand 1 (PD-L1) expression in neutrophils from septic rats and its regulatory influence on T-lymphocyte apoptosis and lung injury in a rat sepsis model.

Methods: Cecum ligation and puncture (CLP) experiments were conducted to establish a rat sepsis model, with the subsequent grouping of rats based on curcumin administration. Rats were monitored for 7 days to assess the 7-day survival rate. Serum, lung tissues, and thymus tissues were collected. Flow cytometry and immunohistochemistry were utilized to assess the number of PD-L1-positive neutrophils and PD-L1 positivity in both blood and lung tissues. Hematoxylin and eosin (HE) staining and Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) histochemistry were employed to examine pathological changes and cell apoptosis in lung and thymus tissues. Furthermore, a kit was employed to measure the activity of myeloperoxidase (MPO), a marker of neutrophil activation, in lung tissues. Enzyme-linked immunosorbent assay (ELISA) was utilized to determine plasma levels of inflammatory factors. Neutrophils were extracted and co-cultured with normal T lymphocytes. TUNEL assays were used to evaluate T-lymphocyte apoptosis, and Western blotting was performed to analyze the expression of PD-L1 and programmed cell death 1 (PD-1).

Results: In in vivo experiments, septic rats exhibited a markedly low 7-day survival rate of 12.5%, significantly elevated PD-L1 expression and positivity in blood and lung tissues, severe lung and thymus tissue damage, and significant cell apoptosis. Additionally, they had increased plasma concentrations of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), and decreased plasma concentration of interleukin 10 (IL-10) compared to normal and sham-operated rats (p < 0.05). Curcumin-treated septic rats demonstrated significantly improved 7-day survival, reduced PD-L1 expression and positivity in blood and lung tissues, mitigated lung and thymus tissue injury and cell apoptosis, lower plasma concentrations of TNF-α and IL-6, and higher plasma concentrations of IL-10 (p < 0.05). In vitro experiments showed that co-culture of T lymphocytes with neutrophils from septic rats resulted in a significantly higher rate of T cell apoptosis and increased expression of PD-L1 and PD-1 compared to co-culture with neutrophils from sham-operated rats (p < 0.05). Neutrophils from curcumin-treated rats exhibited a significantly lower rate of apoptosis in co-cultured T lymphocytes and decreased expression of PD-L1 and PD-1 (p < 0.05). The addition of PD-L1 antibodies to co-cultured neutrophils and T lymphocytes in septic rats significantly reduced T lymphocyte mortality (p < 0.05).

Conclusion: Curcumin effectively mitigates lung and thymus injury during sepsis and attenuates the apoptosis of rat T lymphocytes by down-regulating PD-L1 expression in centrocytes, both in vivo and in vitro.

Keywords: PD-L1; T-lymphocyte apoptosis; cecum ligation and puncture; curcumin; lung injury; neutrophil; sepsis.