Protective effects of combined use of atorvastatin and low molecular weight heparin on the inflammatory reaction and pulmonary functions in rats with sepsis

Objective To investigate the influence of combined use of atorvastatin (ATO) and low molecular weight heparin (LMWH) on the inflammatory reaction and pulmonary protection functions in rats with sepsis. Methods A total of 122 healthy male Sprague-Dawley (SD) rats were divided into five groups using a random number table: sham-operated group (sham group, n = 10), sepsis group (n = 10), ATO group (n = 34), LMWH group (n = 34), and ATO combined with LMWH group (ATO+LMWH group, n = 34). The rat model of sepsis was reproduced by cecal ligation and puncture (CLP), while in sham group, rats were only subjected to laparotomy without cecum ligation and puncture. The rats of each pretreatment group received relevant therapies for 5 days, either gastric perfusion with ATO 20 mg/kg or subcutaneous injection with LMWH 100 U/kg or both before operation. The sepsis severities of the model animals were scored according to the modified sepsis severity assessment standards of experimental animals. Ten rats in each group were calculated the 7-day cumulative mortality rate. Blood samples from 6 rats in each group were collected to determine the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and high mobility group protein box-1 (HMGB1) contents in plasma using enzyme linked immunosorbent assay (ELISA) before operation (0 hour) and 4, 8, 12, and 24 hours post operation. The lung tissue was harvested 24 hours after operation, and the pulmonary pathology was assayed by hematoxylin and eosin (HE) staining using optical microscope. Results ① The sepsis severity grades of sepsis group were significantly higher than those of sham group at 4 hours after operation (score: 12.2±2.0 vs. 7.2±0.5, P < 0.05 ). Furthermore, they displayed a gradually increasing tendency, with the 7-day cumulative mortality rate being 90% (9/10). The sepsis severity grades in ATO group, LMWH group, and ATO+LMWH group showed a significant decrease compared with sepsis group at 8 hours after operation (12.2±2.0, 11.2±2.2, 10.0±1.7 vs. 16.6±2.5, all P < 0.05). The 7-day cumulative mortality rates in ATO group, LMWH group, and ATO+LMWH group were 60% (6/10), 60% (6/10), and 40% (4/10), respectively, all of which was significantly lower than that of sepsis group (all P < 0.05). ② The levels of TNF-α, IL-1β and HMGB1 have not shown much variations in the sham group after operation; the levels of pro-inflammatory cytokines in other 4 groups were significantly increased after operation compared with those before operation; the levels of TNF-α, IL-1β, and HMGB1 reached peak at 4, 8, and 24 hours, respectively. The levels of pro-inflammatory cytokines in sepsis group were significantly higher than those in the sham group. However, the levels of pro-inflammatory cytokines in ATO group, LMWH group, and ATO+LMWH group were significantly lower than those in sepsis group [4-hour TNF-α (ng/L): 668.3±124.6, 536.5±118.5, 496.5±108.5 vs. 783.8±134.7; 8-hour IL-1β (ng/L): 2 476.7±137.8, 2 460.4±171.2, 2 090.0±151.2 vs. 2 873.9±295.6; 24-hour HMGB1 (μg/L): 654.4±154.4, 659.0±134.6, 609.4±90.5 vs. 859.3±167.5, P < 0.05 or P < 0.01]. ③It was showed by optical microscopy that the pulmonary tissue morphology was normal in sham group and that the damage of pulmonary pathology was relatively severe in sepsis group. Compared with sepsis group, the damage of pulmonary pathology in ATO group, LMWH group, and ATO + LMWH group was alleviated obviously, and the most obvious improvements were found in ATO + LMWH group. Conclusions Either ATO or LMWH could decrease sepsis severity, suppress the release of plasma pro-inflammatory cytokines at the early and late stages, alleviate the damage of pulmonary pathology, and reduce the 7-day cumulative mortality rate. Therefore, the combined treatment of sepsis using both ATO and LMWH resulted in better outcomes than implemented individually. Key words: Sepsis; Cecal ligation and puncture; Atorvastatin; Low molecular weight heparin; Pro-inflammatory cytokine