RS-09

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.

This study evaluated shuxuetong injection's specific molecular mechanisms on flap necrosis.

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.

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.

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

Tuberculosis is a deadly infectious disease, and the current BCG vaccine being inconsistent in protecting against it, underscores the urgent need to develop better therapeutics. PPE65 is a surface-localized and highly immunogenic protein of Mycobacterium tuberculosis, and has been proposed as a potential vaccine target because of its ability to induce both humoral and cellular immunity. Hence, the present study outlines the systematic design and detailed computational and in vitro assessment of a novel multi-epitope vaccine against M. tuberculosis utilizing PPE65 protein as a core antigen. The designed vaccine construct (MtbPV), which integrates chosen immunodominant epitopes alongside TLR4 agonist RS-09 as an adjuvant, was found to be non-allergenic, non-toxic and highly antigenic. The 3D structure of MtbPV was constructed, and subsequent in silico docking and simulation analyses confirmed binding and steady interactions with human toll-like receptors. Computational disulfide engineering further enhanced structural stability, while immune simulations indicated strong activation of immune responses. Codon-adapted MtbPV sequence was cloned into pET-28a(+) expression vector, the protein was expressed in a bacterial system, and then purified using affinity chromatography. In vitro assays employing THP-1 macrophages revealed MtbPV to elicit proinflammatory cytokines and activation of NF-κB signaling pathway, where macrophage activation and maturation surface markers were also observed to be upregulated. Blocking of TLR2/TLR4 hampered the cytokine production that indicate involvement of these host receptors in MtbPV-mediated immune response. Altogether, a polyepitopic vaccine construct, based on a mycobacterial virulence protein, was successfully designed, highlighting its potential as a promising vaccine candidate to combat tuberculosis.