Shuxuetong injection promotes skin flap survival by inhibiting pyroptosis mediated by the TLR4/NF-κB/NLRP3 signaling pathway

Ethnopharmacological relevance: Shuxuetong (SXT) injection, formulated from Whitmania pigra Whitman (Leech) and Pheretima aspergillum E.Perrier (Dilong), possesses improving blood perfusion, anti-inflammation, and antioxidant properties. Although SXT is widely used for the treatment of ischemic diseases, its effect on skin FLAP survival is still unclear.

Aim of the study: This study evaluated shuxuetong injection's specific molecular mechanisms on FLAP necrosis.

Materials and methods: An improved McFarlane skin FLAP model was established. 48 male Sprague-Dawley rats were randomly assigned to four groups: control (equal volume of saline), low-dose (0.5 ml/kg/day), medium-dose (1.5 ml/kg/day), and high-dose (3 ml/kg/day). All groups received continuous intraperitoneal injections for 7 days. On the 7th day following surgery, the FLAP survival area was measured. Blood perfusion was measured via laser Doppler flowmetry, and oxidative stress levels were quantified with commercial assay kits. Histopathological status of the FLAP was assessed using hematoxylin-eosin staining, and immunofluorescence was adopted to detect inflammation-relevant proteins' expression. Additionally, a glucose-oxygen deprivation model was established using HUVECs, which were classified into five groups: control (equal volume of saline), low-dose (1 μL/ml), medium-dose (5 μL/ml), high-dose (10 μL/ml), and TLR4 Agonist (RS 09). Cell viability and migration were detected using CCK8 and transwell assays, respectively. The expression of proteins involved in the TLR4/NF-κB/NLRP3 signaling pathway and Pyroptosis was analyzed by Western blotting.

Results: SXT significantly alleviated OGD/R-induced endothelial injury in HUVECs. Mechanistically, SXT inhibited Pyroptosis by suppressing the TLR4/NF-κB/NLRP3 signaling pathway. The use of the TLR4 Agonist RS 09 further verified the pivotal role of this pathway in regulating Pyroptosis. In vivo, SXT enhanced FLAP blood perfusion, suppressed Pyroptosis and inflammation, thereby markedly improving the survival rate of ischemic skin flaps in rats.

Conclusions: Shuxuetong injection improves the survival of flaps primarily by inhibiting TLR4/NF-κB/NLRP3-mediated Pyroptosis, decreasing oxidative stress and inflammation.

Flap; Pyroptosis; RS 09; Shuxuetong injection; TLR4/NF-κB/NLRP3 signaling pathway.