C34 is an orally active, selective Toll-like receptor 4 (TLR4) antagonist. C34 exerts anti-inflammatory effects by inhibiting the TLR4/MyD88/NF-κB signaling pathway, thereby reducing the production of pro-inflammatory cytokines[1-2]. C34 alleviates systemic inflammation in various disease models, including endotoxemia, necrotizing enterocolitis, ulcerative colitis, and acute kidney injury. TLR4-IN-C34 is applicable for research in immunology, inflammation, and related disorders[3-4].
In vitro, BV2 microglial cells were pretreated with C34 (10-100μM) for 2 hours, followed by LPS (200ng/mL) stimulation for 24 hours. C34 significantly inhibited LPS-induced activation of the TLR4/MyD88/NF-κB signaling pathway, downregulated the release of pro-inflammatory factors (TNF-α, IL-1β, IL-6, MCP-1, and NO), and reduced ROS generation and NLRP3 inflammasome activation[5]. Rat enteric glial cells were pretreated with C34 (50μM) for 1 hour, then exposed to Clostridioides difficile toxins TcdA (50ng/mL) or TcdB (1ng/mL) for 18 hours. C34 significantly suppressed toxin-induced TLR4/NF-κB p65 nuclear translocation and TNF-α protein expression, and decreased apoptosis[6].
In vivo, in an isoprenaline (ISO; 100mg/kg)-induced Wistar rat model, C34 (1mg/kg or 3mg/kg; administered intraperitoneally 1 hour before each of two ISO injections), C34 significantly reduced serum creatinine levels and ameliorated renal tissue inflammation, fibrosis, and apoptosis[7]. In a Pilocarpine (40mg/kg; i.p.)-induced status epilepticus Wistar rat model, C34 (1μg/rat; single intracerebroventricular injection) was administered 1 hour or 24 hours after pilocarpine injection. Administration at 1 hour post-induction, C34 significantly reduced hippocampal neuronal death, decreased levels of the apoptotic factor caspase-3 and inflammatory mediators TNF-α and NF-κB, whereas treatment at 24 hours, C34 showed a markedly diminished therapeutic effect[8].
References:
[1] Wipf P, Eyer BR, Yamaguchi Y, et al. Synthesis of anti-inflammatory α-and β-linked acetamidopyranosides as inhibitors of toll-like receptor 4 (TLR4). Tetrahedron Lett. 2015 Jun 3;56(23):3097-3100.
[2] Neal MD, Jia H, Eyer B, et al. Discovery and validation of a new class of small molecule Toll-like receptor 4 (TLR4) inhibitors. PLoS One. 2013 Jun 12;8(6):e65779.
[3] Wei K, Zou Y, Xu H, et al. TLR4-IN-C34 attenuates the progression of osteoarthritis through inhibiting inflammation, angiogenesis and pain. Cell Signal. 2025 Dec;136:112084.
[4] Filippova LV, Fedorova AV, Nozdrachev AD. Mechanism of Activation of Enteric Nociceptive Neurons via Interaction of TLR4 and TRPV1 Receptors. Dokl Biol Sci. 2018 Mar;479(1):44-46.
[5] Zhang SS, Liu M, Liu DN, et al. TLR4-IN-C34 Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses via Downregulating TLR4/MyD88/NF-κB/NLRP3 Signaling Pathway and Reducing ROS Generation in BV2 Cells. Inflammation. 2022 Apr;45(2):838-850.
[6] Barbosa MLL, Costa DVDS, de Pacífico DM, et al. Role of TLR4 in Enteric Glia Response to Clostridioides Difficile Toxins: Insights From In Vivo and In Vitro Studies. J Cell Mol Med. 2025 Nov;29(22):e70943.
[7] Abdelsalam HM, Helal MG, Abu-Elsaad NM. TLR4-IN-C34 protects against acute kidney injury via modulating TLR4/MyD88/NF-κb axis, MAPK, and apoptosis. Iran J Basic Med Sci. 2022 Nov;25(11):1334-1340.
[8] Varmazyar R, Naderi N, Javid H, et al. Neuroprotective Effects of Early TLR4 Blockade with Compound C34 in Temporal Lobe Epilepsy: Alleviation of Neuroinflammation and Apoptosis. Iran J Pharm Res. 2025 Mar 8;24(1):e159165.
C34是一种可口服的Toll样受体4(TLR4)选择性拮抗剂，C34通过抑制TLR4/MyD88/NF-κB信号通路，减少促炎细胞因子的产生，从而发挥抗炎作用[1-2]。C34可改善内毒素血症、坏死性小肠结肠炎、溃疡性结肠炎和急性肾损伤等多种疾病模型中的全身炎症，TLR4-IN-C34可用于免疫学、炎症及相关疾病的研究[3-4]。
在体外，C34（10-100μM）预处理BV2小胶质细胞2小时，随后在LPS（200ng/mL）刺激下培养24小时，C34显著抑制LPS诱导的TLR4/MyD88/NF-κB信号通路激活，并下调促炎因子TNF-α、IL-1β、IL-6、MCP-1和NO的释放，同时降低ROS生成和NLRP3炎症小体活化[5]。C34（50μM）预处理大鼠肠胶质细胞1小时，随后暴露于艰难梭菌毒素TcdA（50ng/mL）或TcdB（1ng/mL）18小时，C34显著抑制毒素诱导的TLR4/NF-κB p65核转位和TNF-α蛋白表达，并减少细胞凋亡[6]。
在体内，C34（1mg/kg或3mg/kg；每次ISO注射前1小时给药，共2次）腹腔注射处理经Isoprenaline（ISO；100mg/kg）诱导的Wistar大鼠，C34显著降低大鼠血清肌酐水平，改善肾组织炎症、纤维化和凋亡[7]。在Pilocarpine（40mg/kg；腹腔注射）诱导癫痫Wistar大鼠1小时或24小时后，进行侧脑室注射C34（1μg/只鼠；单次给药），在1小时注射C34显著降低小鼠海马神经元死亡、凋亡因子caspase-3及炎症介质TNF-α和NF-κB表达，而在24小时后给药的治疗效果显著减弱[8]。