Enoxaparin sodium bone cement displays local anti-inflammatory effects by regulating the expression of IL-6 and TNF-α.

OBJECTIVE

To explore the roles of Enoxaparin Sodium-Polymethyl methacrylate bone cement on inflammatory factors Interleukin-6 and Tumour Necrosis Factor-α in a rabbit knee replacement model. As well as the mechanisms underlying its potential effects on lipopolysaccharide-induced endothelial cell injury.

METHODS

A knee replacement model was established using New Zealand rabbits. Forty rabbits were randomly divided into four groups: PMMA, ES-PMMA, sham-operated, and blank control groups (n = 10 in each group). Local tissues around the incision were taken at the 30th, 60th, and 90th minute after the surgical implantation of the corresponding bone cement. Immunohistochemistry in the surgical field was used to measure the expression of local inflammatory factors IL-6 and TNF-α. In the in vitro experiments, 1 cm of bone cement was immersed in 3 mL of the medium for 24 h. The bone cement was discarded and diluted to 25% with normal medium. Pre-experiments were screened for the best LPS-inducing concentration of 100 mg/mL, and the most compatible LPS concentration was used for subsequent experiments simulating the primary cultures of rats' Inferior Vena Cava Endothelial Cells. The experiments were divided into four groups: blank control group, LPS induction group, PMMA + LPS group, and ES-PMMA + LPS group. The apoptosis rate was detected by flow cytometry, and the expression levels of TNF-α and IL-6 in the cells and supernatant were measured by ELISA, western blotting, and immunofluorescence.

RESULTS

According to immunohistochemical results, IL-6-positive cells were concentrated in the tissue interstitial space. In the PMMA and sham-operated groups, the number of IL-6-positive cells gradually increased over time. At all time points, IL-6 expression in the ES-PMMA group was much lower than in the PMMA and sham-operated groups. At 30 min, TNF-α positive cells in the ES-PMMA group expressed less than those in the PMMA and sham-operated groups, with no discernible difference between the PMMA and ES-PMMA groups at 60 or 90 min. Using ELISA and flow cytometry, the expression levels of IL-6 and TNF-α were improved and the apoptosis rate was magnified in the LPS-induced group (*** < 0.001) in contrast with the blank control group. Additionally, the expression levels of IL-6 and TNF-α were reduced in the ES-PMMA + LPS group compared with the LPS-induced group (* < 0.05) and the apoptosis rate was reduced (*** < 0.001), with statistically significant variations. Western blotting and immunofluorescence analysis confirmed that IL-6 and TNF-α protein expression in cells was upregulated in the LPS-induced group compared to the blank control group (*** < 0.001), and the mean fluorescence intensity was enlarged (*** < 0.001). Meanwhile, IL-6 and TNF-α expression in the ES-PMMA + LPS group were down-regulated (** < 0.01 or * < 0.05) compared with the LPS-induced group and PMMA + LPS crew protein expression, and the average fluorescence intensity of IL-6 and TNF-α was lowered in the ES-PMMA + LPS group compared to the LPS-induced group (*** < 0.001).

CONCLUSIONS

ES-PMMA bone cement reduced the expression levels of local inflammatory factors IL-6 and TNF-α in a rabbit knee model. ES-PMMA bone cement reduced the rate of LPS-induced endothelial cell apoptosis and diminished local inflammatory damage by regulating the secretion of inflammatory factors TNF-α and IL-6.