Tetramethylpyrazine mitigates lipopolysaccharide-induced acute lung injury by inhibiting the HMGB1/TLR4/NF-κB signaling pathway in mice

Abstract

Background: Tetramethylpyrazine (TMP) possesses anti-inflammatory and antioxidant properties and plays a crucial role in mitigating acute lung injury (ALI). However, the specific underlying mechanisms remain elusive. High mobility group box 1 (HMGB1), a pro-inflammatory factor, can bind to Toll-like receptor 4 (TLR4), activating downstream signaling pathways, which in turn activate nuclear factor kappa-B (NF-κB). This study aims to explore the preventive effects of TMP on lipopolysaccharide (LPS)-induced ALI and its influence on the HMGB1/TLR4/NF-κB signaling pathway.

Methods: C57BL/6 mice were randomly divided into a control group (CON group), a model group (LPS group), a heparin (Hep) group, and a TMP group. In the model group, mice received an initial intraperitoneal injection of LPS followed by a second airway injection three hours later to induce ALI. In the TMP group, LPS was administered 30 minutes before the first intraperitoneal injection and followed by intratracheal atomization of LPS. TMP was injected intraperitoneally 30 minutes after the second LPS administration. The Hep group received heparin at 200 IU/kg following the same schedule as the TMP group. Peripheral capillary oxygen saturation (SpO₂) was measured 24 hours after the initial intraperitoneal injection. Lung tissues were harvested for wet to dry weight ratio (W/D) calculation and pathological assessment via hematoxylin and eosin (HE) staining. Western blot analysis was performed to evaluate the protein levels of HMGB1, TLR4, and NF-κB p65 in lung tissues, along with the assessment of leukocyte content and protein concentration in bronchoalveolar lavage fluid (BALF).

Results: Compared to the control group, the model group showed reduced SpO₂ levels, increased inflammatory indicators, and elevated expression levels of HMGB1, TLR4, and phosphorylated p65 (p-p65) proteins. TMP treatment led to a decrease in the W/D, reduced alveolar exudation, improved alveolar structure, and decreased total white blood cell count and protein concentration in BALF. Notably, the protein levels of HMGB1, TLR4, and p-p65 were significantly reduced in the TMP and Hep groups, while there were no significant differences in p65 expression among the groups.

Conclusions: TMP can alleviate LPS-induced ALI in mice by reducing lung inflammatory response through inhibiting the HMGB1/TLR4/NF-κB pathway.

Keywords: HMGB1/TRL4/NF-κB signaling pathway; Tetramethylpyrazine (TMP); acute lung injury (ALI).