TLR4 信号通路调节剂作为炎症和败血症的潜在治疗药物 （其二）

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TLR4 信号通路调节剂作为炎症和败血症的潜在治疗药物 （其二）

5. Peptide TLR4 Modulators 肽类 TLR4 调节剂

We focus now on antagonists of TLR-4 signaling which have a peptide structure. As we discussed above, for the TLR4-mediated signal transduction multiple protein-protein interactions are necessary. A valuable strategy for TLR4 antagonism would therefore be the use of short peptides that mimic protein epitopes and disrupt interactions of TLR4 with MD-2 or other adaptor proteins containing TIR domains. 我们目前关注具有肽结构的 TLR-4 信号通路拮抗剂。如上所述，TLR4 介导的信号转导需要多种蛋白质-蛋白质相互作用。因此，使用模拟蛋白质表位的短肽来干扰 TLR4 与 MD-2 或其他含有 TIR 结构域的适配蛋白的相互作用，将是 TLR4 拮抗的一个有价值的策略。

5.1. Peptides that Disrupt the TLR4/MD-2 Interaction 打断 TLR4/MD-2 相互作用的肽

A 17-residue peptide was projected in silico as a mimic of TLR4-binding region of human MD-2 (hMD-2) and synthesized [156]. This peptide (sequence: CRGSDDDYSFCRALKGE) bound to TLR4 with higher affinity than hMD2 (ΔG = −7.8 kcal/mol vs −5.5 kcal/mol). 一种由 17 个氨基酸组成的肽被计算机模拟为人类 MD-2（hMD-2）TLR4 结合区域的类似物，并已合成[156]。该肽（序列：CRGSDDDYSFCRALKGE）与 TLR4 的结合亲和力高于 hMD2（ΔG = −7.8 kcal/mol vs −5.5 kcal/mol）。

Han et al. [157] have constructed decoy protein, which comprised the structure motifs necessary for interaction with MD2 and thus a competitive inhibitor was obtained. The mutation experiments led to the decoy receptor variants possessing higher affinity to MD-2. Han 等人[157]构建了诱饵蛋白，其中包含了与 MD2 相互作用的必要结构基序，从而获得了竞争性抑制剂。突变实验导致具有更高亲和力的 MD-2 的诱饵受体变体。

5.2. Peptides that Disrupt TIR/TIR Interactions

TIRAP decoy peptides were designed and synthesized to disrupt the interaction of the TLR4-TIR domain with adapter proteins necessary for the downstream signaling [158]. It has been known that both TLRs and their adapter proteins have TIR domain that mediates the interactions necessary for the signal transduction [159,160,161]. TIRAP peptides blocked both MyD88-dependent and MyD88-independent cytokine genes induced by LPS, and consisted of 10–14 amino acid residues. Among them, five peptides were found to inhibit cytokine gene expression as well as MAPK after LPS stimulation [158]. In vivo, IL-6 and TNF-α concentrations were also attenuated in murine blood by the two most active peptides after administration of a sublethal LPS dose. TIRAP 诱饵肽被设计和合成，以破坏 TLR4-TIR 结构域与下游信号通路所需的适配蛋白之间的相互作用[158]。已知 TLRs 及其适配蛋白都具有 TIR 结构域，该结构域介导信号转导所需的相互作用[159, 160, 161]。TIRAP 肽阻断了由 LPS 诱导的 MyD88 依赖性和 MyD88 非依赖性细胞因子基因，由 10-14 个氨基酸残基组成。其中，五种肽被发现可以抑制 LPS 刺激后的细胞因子基因表达以及 MAPK[158]。在体内，通过给予亚致死剂量的 LPS 后，两种最活跃的肽在鼠血中降低了 IL-6 和 TNF-α的浓度。

The same research group has designed and prepared TRAM adapter TIR domain derived decoy proteins applying the same approach [162]. Two of eleven peptides, IVFAEMPCGRLHLQ and ENFLRDTWCNFQFY, were the most potent to inhibit expression and secretion of all cytokines considered (TNF-α, IL-1β, IL-6, RANTES, IFN-β) in vitro. The activation of MAPKs was also inhibited. Co-immunoprecipitation assays have shown that these peptides block adapter recruitment to TLR4. Six truncated variants of IVFAEMPCGRLHLQ were also synthesized. The pentapeptide IVFAE was the most active among them and more potent than precursor with longer sequence. When administrated to the C57BL/6J mice, IVFAEMPCGRLHLQ, ENFLRDTWCNFQFY, and IVFAEMPCG effectively suppressed LPS-induced cytokine induction and protected animals from lethal endotoxemia after sublethal LPS dose. Treatment of mice with 10 nmol/g of peptide before the injection of LPS on the LD100 level either rescued all the animals (IVFAEMPCGRLHLQ and IVFAEMPCG) or at least part of them (77%, ENFLRDTWCNFQFY). Reversing the order of administration of peptides and LPS similar results were obtained. 同一研究小组采用相同的方法设计并制备了 TRAM 适配器 TIR 结构域衍生的诱饵蛋白[162]。在 11 个肽中，IVFAEMPCGRLHLQ 和 ENFLRDTWCNFQFY 对抑制所有考虑的细胞因子（TNF-α、IL-1β、IL-6、RANTES、IFN-β）的表达和分泌（体外）最为有效。MAPKs 的激活也被抑制。共免疫沉淀实验表明，这些肽可以阻断适配器对 TLR4 的招募。还合成了 IVFAEMPCGRLHLQ 的六个截短变异体。其中，五肽 IVFAE 是最活跃的，比序列更长的前体更有效。当给予 C57BL/6J 小鼠时，IVFAEMPCGRLHLQ、ENFLRDTWCNFQFY 和 IVFAEMPCG 有效地抑制了 LPS 诱导的细胞因子诱导，并在亚致死剂量的 LPS 后保护动物免受致死性内毒素血症。在 LD100 水平注射 LPS 前用 10 nmol/g 的肽治疗小鼠，IVFAEMPCGRLHLQ 和 IVFAEMPCG 可以挽救所有动物，或者至少部分动物（77%，ENFLRDTWCNFQFY）。颠倒肽和 LPS 的给药顺序，可以得到类似的结果。

Another way to block TLR4 signaling is to inhibit the dimerization of TLR4 TIR domains, which occurs by the stimulation of agonist and is necessary for downstream signal transduction. 12 peptides (each was 9–14 amino acids long) reproducing different parts of TIR-domain were synthesized and tested in vitro on murine macrophages stimulated by LPS. Five of them were the most active ones to reduce cytokines expression (IL-1β, TNF-α, IFN-β, and RANTES mRNA). Among these five, three (LHYRDFIPGVAIAA, AGCKKYSRGESIYD, and HIFWRRLKNALLD) demonstrated the best affinity to TLR4-TIR as have been shown by time-resolved fluorescence spectroscopy [163]. The same group also has designed and synthesized TRIF TIR domain-derived decoy peptides [164]. They also were effective in vivo, decreasing LPS-induced cytokine response and improving survival of mice after LPS challenge. 另一种阻断 TLR4 信号传导的方法是抑制 TLR4 TIR 结构域的二聚化，这是通过激动剂刺激发生的，对于下游信号转导是必要的。合成了 12 种肽（每种长度为 9-14 个氨基酸），它们复制了 TIR 结构域的不同部分，并在体外对由 LPS 刺激的小鼠巨噬细胞进行了测试。其中五种在降低细胞因子表达（IL-1β、TNF-α、IFN-β和 RANTES mRNA）方面最为活跃。在这五种中，三种（LHYRDFIPGVAIAA、AGCKKYSRGESIYD 和 HIFWRRLKNALLD）通过时间分辨荧光光谱显示对 TLR4-TIR 具有最佳的亲和力[163]。同一研究小组还设计并合成了 TRIF TIR 结构域衍生的诱饵肽[164]。它们在体内也有效，可以降低 LPS 诱导的细胞因子反应，并在 LPS 挑战后提高小鼠的存活率。

Decoy peptides designed to reproduce the binding part of TIR-domain of TcpB/Btp1 protein from Brucella spp. were also tested as blockers of protein-protein interactions necessary for TLR4 signal transduction [165]. The assumption was that TcpB/Btp1 can interact with adaptors downstream of TLRs, prevent NF-κB activation, and consequently diminish cytokine production, which is important for innate antibacterial immune response [166]. In vitro, two of the twelve synthesized peptides significantly inhibited mRNA expression of both MyD88-dependent (TNF-α and IL-1β) or TRIF-dependent (IFN-β) cytokines as well as their secretion. In Balb/c mice, pre-treatment with peptides significantly decreased cytokine levels after LPS administration. 诱饵肽被设计用来复制布鲁氏菌属 TcpB/Btp1 蛋白 TIR 结构域的结合部分，也被测试作为 TLR4 信号转导所需的蛋白质-蛋白质相互作用的阻断剂[165]。假设 TcpB/Btp1 可以与 TLR 下游的适配器相互作用，防止 NF-κB 激活，从而减少细胞因子产生，这对先天抗菌免疫反应很重要[166]。在体外，十二种合成的肽中有两种显著抑制了 MyD88 依赖性（TNF-α和 IL-1β）或 TRIF 依赖性（IFN-β）细胞因子的 mRNA 表达及其分泌。在 Balb/c 小鼠中，预先用肽处理显著降低了 LPS 给药后的细胞因子水平。

It is worth to note that TIR homologue proteins occur in other bacterial species, including Salmonella enterica [167], Staphylococcus aureus [168]. Such proteins can modulate the host immune response [169]. Bacterial proteins containing TIR-domains are discussed in review by Rana et al. [170]. 值得注意的是，TIR 同源蛋白存在于其他细菌物种中，包括沙门氏菌[167]、金黄色葡萄球菌[168]。这类蛋白可以调节宿主免疫反应[169]。含有 TIR 结构域的细菌蛋白在 Rana 等人[170]的综述中进行了讨论。

One more TRAM-derived peptide inhibits the inflammatory response in mouse mammary epithelial cells and a mastitis model in mice [171]. Hines et al. [172] have prepared blood-brain barrier (BBB)-permeating peptides, 18 amino acids long, to inhibit TLR4-MyD88 interaction. At concentration 3 μM, TNF-α production in the murine brain tissue after LPS stimulation was significantly decreased (ex vivo). Prior to peptide injection, the mice were treated with LPS. After that, these peptides were tested in vivo using intraperitoneal administration. Then, TNF-α level in whole brain lysate were measured by ELISA method. In the brain tissue, ex vivo TNF-α level were also decreased after LPS stimulation as compared with LPS control (p = 0.030 and p < 0.001). A short peptide (11 amino acid residues) VIPER of vaccinia virus A46 protein on both murine (iBMDM) and human cells (THP-1 and PBMC) in vitro at concentrations significantly decreased TNF-α production after LPS-stimulation (E. coli). Inhibiting resulted from the blocking interaction of TLR4 with Mal (TIRAP) and TRAM adaptor proteins [173]. Epta-peptides derived from BB-loop region of Toll/IL-1 receptor (TIR) domain also inhibited homodimerization of MyD88 TIR domains in vitro [174]. TIR-signaling modulation has been described in a review [175]. Therapeutic targets belonging to the TLR4 signaling pathway are discussed by Roy et al. [176]. It was concluded that disrupting the TLR4/ NF-κB pathway is a promising strategy to treat inflammatory disorders but it should not be blocked for long time because of its huge role in the host immune response. 一种由 TRAM 衍生的肽抑制了小鼠乳腺上皮细胞和老鼠乳腺炎模型的炎症反应[171]。Hines 等人[172]已经制备了 18 个氨基酸长的血脑屏障（BBB）渗透肽，以抑制 TLR4-MyD88 相互作用。在 3 μM 的浓度下，LPS 刺激后小鼠脑组织中的 TNF-α产生显著降低（体外）。在肽注射之前，小鼠接受了 LPS 治疗。之后，这些肽通过腹腔注射在体内进行了测试。然后，通过 ELISA 方法测量了全脑裂解物中的 TNF-α水平。与 LPS 对照组相比，在 LPS 刺激后，脑组织中的体外 TNF-α水平也降低了（p = 0.030 和 p < 0.001）。一种短肽（11 个氨基酸残基）VIPER，来自痘病毒 A46 蛋白，在体外对小鼠（iBMDM）和人类细胞（THP-1 和 PBMC）的浓度显著降低了 LPS 刺激后的 TNF-α产生。抑制是由于 TLR4 与 Mal（TIRAP）和 TRAM 适配蛋白的相互作用被阻断[173]。 Epta-肽来自 Toll/IL-1 受体（TIR）域的 BB 环区域，也抑制了体外 MyD88 TIR 域的同源二聚化[174]。TIR 信号调节已在综述中描述[175]。Roy 等人讨论了属于 TLR4 信号通路的药物靶点[176]。结论是，破坏 TLR4/NF-κB 通路是治疗炎症性疾病的有希望策略，但由于其在宿主免疫反应中的巨大作用，不应长时间阻断。

6. Discussion and Conclusions

The TLR4 antagonists described in this review are synthetic or natural molecules belonging to different classes of compounds, from glycolipids to chalcone derivatives, to terpenoids, to peptides. 本综述中描述的 TLR4 拮抗剂是不同类别的化合物，从糖脂到查耳酮衍生物，到萜类化合物，到肽类。

These molecules have diverse modes of action, blocking the TLR4 signal at various stages and binding to their targets non-covalently as well as covalently. The exact targets of some TLR signaling pathway blockers are not clear yet and the hypotheses generated by docking studies need experimental validation. Many compounds that decreased pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-γ) concentration after LPS stimulation in cell-based assays were also active in vivo on murine model, improving survival and decreasing the cytokine levels. 这些分子具有多种作用方式，在各个阶段阻断 TLR4 信号，并以非共价和共价方式与靶点结合。一些 TLR 信号通路阻断剂的精确靶点尚不明确，由对接研究产生的假设需要实验验证。在基于细胞的实验中，许多降低促炎细胞因子（TNF-α、IL-6、IL-1β、IFN-γ）浓度的化合物在体内小鼠模型中也表现出活性，提高了生存率并降低了细胞因子水平。

Among all TLR4 antagonists so far developed, only TAK-242 and Eritoran reached clinical trials, but unfortunately failed to pass them and were not active in improving patients survival [124,129,130,131,132]. At phase 3 of clinical trials, Eritoran was not active in improving survival also for sepsis patients infected by Gram-negative flora (21.9 vs 22.3%, p = 0.89), although for this subgroup a positive effect was expected. The negative results in clinic were in contrast with successful applications of TLR4 antagonists in sepsis therapy in animal models. This “translational gap” is well known in a series of other drugs, in the case of sepsis it could derive from the difference in TLR4 signaling and in the expression of inflammatory pathway genes in humans and in mice. 在迄今为止开发的 TLR4 拮抗剂中，只有 TAK-242 和 Eritoran 进入了临床试验，但不幸的是，它们未能通过临床试验，并且未能有效提高患者的生存率[124, 129, 130, 131, 132]。在临床试验的第三阶段，Eritoran 对于由革兰氏阴性菌感染的败血症患者来说，也没有在提高生存率方面发挥作用（21.9% vs 22.3%，p = 0.89），尽管对于这个亚组原本预期会有积极效果。临床试验中的负面结果与 TLR4 拮抗剂在动物模型败血症治疗中的成功应用形成对比。这种“转化差距”在一系列其他药物中也是众所周知的，在败血症的情况下，这可能是由于人类和老鼠中 TLR4 信号传导和炎症途径基因表达的不同所导致的。

In a comparative review written by Vaure and Liu [177], the expression patterns of TLR4 in different tissues and mammal species were analyzed, and the relative sensibility of various animal species to LPS also considered. When compared to humans, mice are less susceptible to LPS, with the threshold dose required for minimal physiological changes is 0.5 mg/kg intraperitoneally for mice [178] (BALB/c strain) vs 1–5 ng/kg intravenously for humans [179]. This is a substantial difference even considering the diverse susceptibility across the mouse strains. One of the possible reasons for that is the difference in cytokine production patterns [177]. 在 Vaure 和 Liu [177]撰写的比较综述中，分析了不同组织和哺乳动物物种中 TLR4 的表达模式，并考虑了各种动物物种对 LPS 的相对敏感性。与人类相比，小鼠对 LPS 的敏感性较低，引起最小生理变化的阈值剂量为小鼠腹腔注射 0.5 mg/kg [178]（BALB/c 品系）与人类静脉注射 1-5 ng/kg [179]。即使在考虑小鼠品系间差异的敏感性时，这仍是一个显著差异。其中一个可能的原因是细胞因子产生模式的差异 [177]。

It is also known that after LPS stimulation in mouse neutrophils and peritoneal macrophages, a decrease in TLR4 expression occurs [180], in contrast, TLR4 expression in human monocytes is unaltered at least after low doses of lipopolysaccharide [181]. Cytokine production and NF-kB DNA-binding activity are also suppressed in mice [182]. Moreover, several mRNA isoforms of TLR4 in mice are known [183]. A notable one is smTLR4 (soluble mouse TLR4). This splicing variant consists of 122 amino acid residues, is not membrane-bound, and contains a part of the TLR4 extracellular domain responsible for the interaction with LPS. One possible reason can be the inhibition of TLR4/MD-2 interaction since MD-2 is known to be essential in mediating the LPS response. smTLR4 can therefore be an endogenous TLR4-antagonist acting on the early stages of TLR4 signaling. There is not an equivalent of such TLR4 isoform in humans. 小鼠中性粒细胞和腹腔巨噬细胞在 LPS 刺激后，TLR4 表达降低[180]，而人类单核细胞在至少低剂量脂多糖处理后，TLR4 表达未改变[181]。小鼠的细胞因子产生和 NF-kB DNA 结合活性也受到抑制[182]。此外，小鼠中已知存在 TLR4 的几种 mRNA 异构体[183]。其中值得注意的是 smTLR4（可溶性小鼠 TLR4）。这种剪接变体由 122 个氨基酸残基组成，不是膜结合的，并包含 TLR4 细胞外域的一部分，该域负责与 LPS 相互作用。一个可能的原因可能是抑制 TLR4/MD-2 相互作用，因为已知 MD-2 在介导 LPS 反应中是必需的。因此，smTLR4 可以作为一种内源性 TLR4 拮抗剂，作用于 TLR4 信号传导的早期阶段。人类中没有这种 TLR4 异构体的等价物。

It is also important to consider the similarity between animal sepsis models and human sepsis. As stated before, the CLP model mimics human sepsis the most but rarely was used in the studies reported in this review. The LPS injection model was often employed, which reproduces septic shock rather than sepsis. So for further development of TLR4-signaling modulators, their testing on CLP sepsis model is also recommended. 考虑动物败血症模型与人类败血症之间的相似性也很重要。正如之前所述，CLP 模型最接近模拟人类败血症，但在本综述中报道的研究中很少使用。LPS 注射模型经常被采用，它复制的是脓毒症休克而不是败血症。因此，为了进一步开发 TLR4 信号通路调节剂，建议在 CLP 败血症模型上进行测试。

Taken together, these factors underline the great differences between murine and human innate immune response to LPS and explain, at least in part, the clinical gap in the case of sepsis. 综合考虑，这些因素突出了小鼠和人类对 LPS 的先天免疫反应之间的巨大差异，并在一定程度上解释了败血症病例中临床差距的原因。

Because of the complexity of innate immunity regulation, and also because of the redundancy of pathways involved in TLR-triggered cytokine production, in the future combined therapies could be investigated and tested in clinic. 由于固有免疫调节的复杂性，以及 TLR 触发的细胞因子产生所涉及途径的冗余性，未来可以研究并测试在临床上的联合疗法。

One possibility could be to combine TLR4 antagonists with specific antibacterial agents. 一种可能性是将 TLR4 拮抗剂与特定的抗菌剂结合使用。

Wang et al. [111] have studied the effect of the TLR4 antagonist artemisinin alone and in combination with antibiotics on the mice survival after administration of live E. coli bacteria as a source of LPS. When ampicillin sodium or a 2:1 mixture of ampicillin sodium/sulbactam sodium were co-administrated to the bacterium, the survival increased to 33% and 67%, respectively. When administering antibiotics alone, a 0% and 33% survival increase was obtained. Conversely, when administrating Artemisin alone, no protection was obtained after the injection of live E. coli bacteria. 王等[111]研究了 TLR4 拮抗剂青蒿素单独及与抗生素联合使用对注射活 E. coli 细菌（作为 LPS 来源）后小鼠存活率的影响。当与细菌共同给予氨苄西林钠或氨苄西林钠/舒巴坦钠 2:1 混合物时，存活率分别增加到 33%和 67%。单独使用抗生素时，存活率分别增加 0%和 33%。相反，单独给予青蒿素时，在注射活 E. coli 细菌后没有获得保护。

Another case when TLR4 antagonist was given together with antimicrobial substance was described by Shirey and co-workers [51], albeit the disease was influenza and not sepsis. Applied together with Tamiflu™ (oseltamivir), Eritoran significantly improved the survival of C57BL/6J mice as compared with the group given oseltamivir alone, and protected them from lethal re-infection. 另一个案例中，Shirey 及其同事[51]描述了 TLR4 拮抗剂与抗菌物质同时给予的情况，尽管疾病是流感而非败血症。与单独给予奥司他韦（Tamiflu™）的组相比，与奥司他韦联合应用的 Eritoran 显著提高了 C57BL/6J 小鼠的存活率，并保护它们免受致命性再感染。

A second possibility is the combination of blockers of different TLRs. In a recent successful example, anti-TLR2 and anti-TLR4 monoclonal antibodies were combined [184]. When metronidazole and ceftriaxone have been co-administrated with TLR4-targeting immunoglobulins, the survival increased more than twice when comparing with anti-TLR4 antibodies alone. 第二种可能性是不同 TLR 的阻断剂的组合。在最近的一个成功例子中，抗-TLR2 和抗-TLR4 单克隆抗体被结合使用[184]。当甲硝唑和头孢曲松与针对 TLR4 的免疫球蛋白共同给药时，与单独使用抗-TLR4 抗体相比，存活率增加了两倍以上。

Concerning pathologies related to PAMP/TLR4 activation, in particular in the case of acute sepsis and septic shock, one can therefore conclude that, despite promising results on animal models, the clinical trials based on a single TLR4 antagonist has not led so far to a significant improvement in survival. We propose here combination therapies as a new strategy to overcome the limitations discussed above. 关于与 PAMP/TLR4 激活相关的疾病，特别是在急性脓毒症和脓毒症休克的情况下，因此可以得出结论，尽管在动物模型上取得了有希望的结果，但基于单一 TLR4 拮抗剂的临床试验迄今为止并未导致生存率的显著提高。我们在此提出联合疗法作为克服上述局限性的新策略。

The use of TLR4 modulators in pathologies caused by TLR4 activation by endogenous DAMPs, however, is still at a very early stage and promising results have been obtained at a preclinical stage. Some small-molecular TLR4 blockers displayed a lack of toxicity and were efficient in blocking DAMP/TLR4 signaling and cytokine production in an array of inflammatory and autoimmune diseases [185,186,187]. These comprise of neuropathic pain, ALS, rheumatoid arthritis, vascular inflammation, atherosclerosis, and other pathologies reviewed here. TLR4 调节剂在由内源性损伤相关分子模式（DAMPs）激活 TLR4 引起的疾病中的应用，然而，仍处于非常早期阶段，并在临床前阶段取得了有希望的结果。一些小分子 TLR4 阻断剂显示出无毒性，并在一系列炎症和自身免疫疾病中有效地阻断 DAMP/TLR4 信号传导和细胞因子产生[185, 186, 187]。这些包括神经性疼痛、肌萎缩侧索硬化症、类风湿性关节炎、血管炎症、动脉粥样硬化和其他在此处回顾的疾病。

We are therefore convinced that some modern day chronic inflammations and autoimmune diseases that still lack specific pharmacological treatment could be efficiently targeted by new drugs interfering with TLR(4) activation and signaling. 我们因此坚信，一些目前仍缺乏特异性药物治疗的中度慢性炎症和自身免疫性疾病，可以通过干扰 TLR（4）激活和信号传导的新药有效靶向。

Acknowledgments 致谢

Nikolay N. Kuzmich acknowledges Olga Petina (KRKA) and Roman Shutov (Vertex) for valuable critical remarks regarding the manuscript. FP acknowledges the H2020-MSC-ETN-642157 project TOLLerant and the Italian Ministry for Foreign Affairs and International Cooperation (MAECI). 尼古拉·N·库兹米奇感谢奥莉加·彼得娜（KRKA）和罗曼·舒托夫（Vertex）对稿件提出的宝贵批评意见。FP 感谢 H2020-MSC-ETN-642157 项目 TOLLerant 以及意大利外交部和国际合作部（MAECI）。

Author Contributions 作者贡献

Nikolay N. Kuzmich wrote the paper; Konstantin V. Sivak participated in writing of the introduction section and performed the literature search together with Vladimir N. Chubarev, Yury B. Porozov and Tatiana N. Savateeva-Lyubimova; Francesco Peri reorganized and updated the manuscript with new data. 尼古拉·N·库兹米奇撰写了这篇论文；康斯坦丁·V·西瓦克参与了引言部分的撰写，并与弗拉基米尔·N·丘巴列夫、尤里·B·波罗佐夫和塔蒂亚娜·N·萨瓦特耶娃-柳比莫娃一起进行了文献检索；弗朗西斯科·佩里根据新数据重新组织和更新了手稿。

原文在此

TLR4 信号通路调节剂作为炎症和败血症的潜在治疗药物 --- TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis

TLR4 信号通路调节剂作为炎症和败血症的潜在治疗药物 （其二）

5. Peptide TLR4 Modulators 肽类 TLR4 调节剂

We focus now on antagonists of TLR-4 signaling which have a peptide structure. As we discussed above, for the TLR4-mediated signal transduction multiple protein-protein interactions are necessary. A valuable strategy for TLR4 antagonism would therefore be the use of short peptides that mimic protein epitopes and disrupt interactions of TLR4 with MD-2 or other adaptor proteins containing TIR domains. 我们目前关注具有肽结构的 TLR-4 信号通路拮抗剂。如上所述，TLR4 介导的信号转导需要多种蛋白质-蛋白质相互作用。因此，使用模拟蛋白质表位的短肽来干扰 TLR4 与 MD-2 或其他含有 TIR 结构域的适配蛋白的相互作用，将是 TLR4 拮抗的一个有价值的策略。

5.1. Peptides that Disrupt the TLR4/MD-2 Interaction 打断 TLR4/MD-2 相互作用的肽

A 17-residue peptide was projected in silico as a mimic of TLR4-binding region of human MD-2 (hMD-2) and synthesized [156]. This peptide (sequence: CRGSDDDYSFCRALKGE) bound to TLR4 with higher affinity than hMD2 (ΔG = −7.8 kcal/mol vs −5.5 kcal/mol). 一种由 17 个氨基酸组成的肽被计算机模拟为人类 MD-2（hMD-2）TLR4 结合区域的类似物，并已合成[156]。该肽（序列：CRGSDDDYSFCRALKGE）与 TLR4 的结合亲和力高于 hMD2（ΔG = −7.8 kcal/mol vs −5.5 kcal/mol）。

Han et al. [157] have constructed decoy protein, which comprised the structure motifs necessary for interaction with MD2 and thus a competitive inhibitor was obtained. The mutation experiments led to the decoy receptor variants possessing higher affinity to MD-2. Han 等人[157]构建了诱饵蛋白，其中包含了与 MD2 相互作用的必要结构基序，从而获得了竞争性抑制剂。突变实验导致具有更高亲和力的 MD-2 的诱饵受体变体。

5.2. Peptides that Disrupt TIR/TIR Interactions

TIRAP decoy peptides were designed and synthesized to disrupt the interaction of the TLR4-TIR domain with adapter proteins necessary for the downstream signaling [158]. It has been known that both TLRs and their adapter proteins have TIR domain that mediates the interactions necessary for the signal transduction [159,160,161]. TIRAP peptides blocked both MyD88-dependent and MyD88-independent cytokine genes induced by LPS, and consisted of 10–14 amino acid residues. Among them, five peptides were found to inhibit cytokine gene expression as well as MAPK after LPS stimulation [158]. In vivo, IL-6 and TNF-α concentrations were also attenuated in murine blood by the two most active peptides after administration of a sublethal LPS dose. TIRAP 诱饵肽被设计和合成，以破坏 TLR4-TIR 结构域与下游信号通路所需的适配蛋白之间的相互作用[158]。已知 TLRs 及其适配蛋白都具有 TIR 结构域，该结构域介导信号转导所需的相互作用[159, 160, 161]。TIRAP 肽阻断了由 LPS 诱导的 MyD88 依赖性和 MyD88 非依赖性细胞因子基因，由 10-14 个氨基酸残基组成。其中，五种肽被发现可以抑制 LPS 刺激后的细胞因子基因表达以及 MAPK[158]。在体内，通过给予亚致死剂量的 LPS 后，两种最活跃的肽在鼠血中降低了 IL-6 和 TNF-α的浓度。

The same research group has designed and prepared TRAM adapter TIR domain derived decoy proteins applying the same approach [162]. Two of eleven peptides, IVFAEMPCGRLHLQ and ENFLRDTWCNFQFY, were the most potent to inhibit expression and secretion of all cytokines considered (TNF-α, IL-1β, IL-6, RANTES, IFN-β) in vitro. The activation of MAPKs was also inhibited. Co-immunoprecipitation assays have shown that these peptides block adapter recruitment to TLR4. Six truncated variants of IVFAEMPCGRLHLQ were also synthesized. The pentapeptide IVFAE was the most active among them and more potent than precursor with longer sequence. When administrated to the C57BL/6J mice, IVFAEMPCGRLHLQ, ENFLRDTWCNFQFY, and IVFAEMPCG effectively suppressed LPS-induced cytokine induction and protected animals from lethal endotoxemia after sublethal LPS dose. Treatment of mice with 10 nmol/g of peptide before the injection of LPS on the LD100 level either rescued all the animals (IVFAEMPCGRLHLQ and IVFAEMPCG) or at least part of them (77%, ENFLRDTWCNFQFY). Reversing the order of administration of peptides and LPS similar results were obtained. 同一研究小组采用相同的方法设计并制备了 TRAM 适配器 TIR 结构域衍生的诱饵蛋白[162]。在 11 个肽中，IVFAEMPCGRLHLQ 和 ENFLRDTWCNFQFY 对抑制所有考虑的细胞因子（TNF-α、IL-1β、IL-6、RANTES、IFN-β）的表达和分泌（体外）最为有效。MAPKs 的激活也被抑制。共免疫沉淀实验表明，这些肽可以阻断适配器对 TLR4 的招募。还合成了 IVFAEMPCGRLHLQ 的六个截短变异体。其中，五肽 IVFAE 是最活跃的，比序列更长的前体更有效。当给予 C57BL/6J 小鼠时，IVFAEMPCGRLHLQ、ENFLRDTWCNFQFY 和 IVFAEMPCG 有效地抑制了 LPS 诱导的细胞因子诱导，并在亚致死剂量的 LPS 后保护动物免受致死性内毒素血症。在 LD100 水平注射 LPS 前用 10 nmol/g 的肽治疗小鼠，IVFAEMPCGRLHLQ 和 IVFAEMPCG 可以挽救所有动物，或者至少部分动物（77%，ENFLRDTWCNFQFY）。颠倒肽和 LPS 的给药顺序，可以得到类似的结果。

Another way to block TLR4 signaling is to inhibit the dimerization of TLR4 TIR domains, which occurs by the stimulation of agonist and is necessary for downstream signal transduction. 12 peptides (each was 9–14 amino acids long) reproducing different parts of TIR-domain were synthesized and tested in vitro on murine macrophages stimulated by LPS. Five of them were the most active ones to reduce cytokines expression (IL-1β, TNF-α, IFN-β, and RANTES mRNA). Among these five, three (LHYRDFIPGVAIAA, AGCKKYSRGESIYD, and HIFWRRLKNALLD) demonstrated the best affinity to TLR4-TIR as have been shown by time-resolved fluorescence spectroscopy [163]. The same group also has designed and synthesized TRIF TIR domain-derived decoy peptides [164]. They also were effective in vivo, decreasing LPS-induced cytokine response and improving survival of mice after LPS challenge. 另一种阻断 TLR4 信号传导的方法是抑制 TLR4 TIR 结构域的二聚化，这是通过激动剂刺激发生的，对于下游信号转导是必要的。合成了 12 种肽（每种长度为 9-14 个氨基酸），它们复制了 TIR 结构域的不同部分，并在体外对由 LPS 刺激的小鼠巨噬细胞进行了测试。其中五种在降低细胞因子表达（IL-1β、TNF-α、IFN-β和 RANTES mRNA）方面最为活跃。在这五种中，三种（LHYRDFIPGVAIAA、AGCKKYSRGESIYD 和 HIFWRRLKNALLD）通过时间分辨荧光光谱显示对 TLR4-TIR 具有最佳的亲和力[163]。同一研究小组还设计并合成了 TRIF TIR 结构域衍生的诱饵肽[164]。它们在体内也有效，可以降低 LPS 诱导的细胞因子反应，并在 LPS 挑战后提高小鼠的存活率。

Decoy peptides designed to reproduce the binding part of TIR-domain of TcpB/Btp1 protein from Brucella spp. were also tested as blockers of protein-protein interactions necessary for TLR4 signal transduction [165]. The assumption was that TcpB/Btp1 can interact with adaptors downstream of TLRs, prevent NF-κB activation, and consequently diminish cytokine production, which is important for innate antibacterial immune response [166]. In vitro, two of the twelve synthesized peptides significantly inhibited mRNA expression of both MyD88-dependent (TNF-α and IL-1β) or TRIF-dependent (IFN-β) cytokines as well as their secretion. In Balb/c mice, pre-treatment with peptides significantly decreased cytokine levels after LPS administration. 诱饵肽被设计用来复制布鲁氏菌属 TcpB/Btp1 蛋白 TIR 结构域的结合部分，也被测试作为 TLR4 信号转导所需的蛋白质-蛋白质相互作用的阻断剂[165]。假设 TcpB/Btp1 可以与 TLR 下游的适配器相互作用，防止 NF-κB 激活，从而减少细胞因子产生，这对先天抗菌免疫反应很重要[166]。在体外，十二种合成的肽中有两种显著抑制了 MyD88 依赖性（TNF-α和 IL-1β）或 TRIF 依赖性（IFN-β）细胞因子的 mRNA 表达及其分泌。在 Balb/c 小鼠中，预先用肽处理显著降低了 LPS 给药后的细胞因子水平。

It is worth to note that TIR homologue proteins occur in other bacterial species, including Salmonella enterica [167], Staphylococcus aureus [168]. Such proteins can modulate the host immune response [169]. Bacterial proteins containing TIR-domains are discussed in review by Rana et al. [170]. 值得注意的是，TIR 同源蛋白存在于其他细菌物种中，包括沙门氏菌[167]、金黄色葡萄球菌[168]。这类蛋白可以调节宿主免疫反应[169]。含有 TIR 结构域的细菌蛋白在 Rana 等人[170]的综述中进行了讨论。

One more TRAM-derived peptide inhibits the inflammatory response in mouse mammary epithelial cells and a mastitis model in mice [171]. Hines et al. [172] have prepared blood-brain barrier (BBB)-permeating peptides, 18 amino acids long, to inhibit TLR4-MyD88 interaction. At concentration 3 μM, TNF-α production in the murine brain tissue after LPS stimulation was significantly decreased (ex vivo). Prior to peptide injection, the mice were treated with LPS. After that, these peptides were tested in vivo using intraperitoneal administration. Then, TNF-α level in whole brain lysate were measured by ELISA method. In the brain tissue, ex vivo TNF-α level were also decreased after LPS stimulation as compared with LPS control (p = 0.030 and p < 0.001). A short peptide (11 amino acid residues) VIPER of vaccinia virus A46 protein on both murine (iBMDM) and human cells (THP-1 and PBMC) in vitro at concentrations significantly decreased TNF-α production after LPS-stimulation (E. coli). Inhibiting resulted from the blocking interaction of TLR4 with Mal (TIRAP) and TRAM adaptor proteins [173]. Epta-peptides derived from BB-loop region of Toll/IL-1 receptor (TIR) domain also inhibited homodimerization of MyD88 TIR domains in vitro [174]. TIR-signaling modulation has been described in a review [175]. Therapeutic targets belonging to the TLR4 signaling pathway are discussed by Roy et al. [176]. It was concluded that disrupting the TLR4/ NF-κB pathway is a promising strategy to treat inflammatory disorders but it should not be blocked for long time because of its huge role in the host immune response. 一种由 TRAM 衍生的肽抑制了小鼠乳腺上皮细胞和老鼠乳腺炎模型的炎症反应[171]。Hines 等人[172]已经制备了 18 个氨基酸长的血脑屏障（BBB）渗透肽，以抑制 TLR4-MyD88 相互作用。在 3 μM 的浓度下，LPS 刺激后小鼠脑组织中的 TNF-α产生显著降低（体外）。在肽注射之前，小鼠接受了 LPS 治疗。之后，这些肽通过腹腔注射在体内进行了测试。然后，通过 ELISA 方法测量了全脑裂解物中的 TNF-α水平。与 LPS 对照组相比，在 LPS 刺激后，脑组织中的体外 TNF-α水平也降低了（p = 0.030 和 p < 0.001）。一种短肽（11 个氨基酸残基）VIPER，来自痘病毒 A46 蛋白，在体外对小鼠（iBMDM）和人类细胞（THP-1 和 PBMC）的浓度显著降低了 LPS 刺激后的 TNF-α产生。抑制是由于 TLR4 与 Mal（TIRAP）和 TRAM 适配蛋白的相互作用被阻断[173]。 Epta-肽来自 Toll/IL-1 受体（TIR）域的 BB 环区域，也抑制了体外 MyD88 TIR 域的同源二聚化[174]。TIR 信号调节已在综述中描述[175]。Roy 等人讨论了属于 TLR4 信号通路的药物靶点[176]。结论是，破坏 TLR4/NF-κB 通路是治疗炎症性疾病的有希望策略，但由于其在宿主免疫反应中的巨大作用，不应长时间阻断。

6. Discussion and Conclusions

The TLR4 antagonists described in this review are synthetic or natural molecules belonging to different classes of compounds, from glycolipids to chalcone derivatives, to terpenoids, to peptides. 本综述中描述的 TLR4 拮抗剂是不同类别的化合物，从糖脂到查耳酮衍生物，到萜类化合物，到肽类。

These molecules have diverse modes of action, blocking the TLR4 signal at various stages and binding to their targets non-covalently as well as covalently. The exact targets of some TLR signaling pathway blockers are not clear yet and the hypotheses generated by docking studies need experimental validation. Many compounds that decreased pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-γ) concentration after LPS stimulation in cell-based assays were also active in vivo on murine model, improving survival and decreasing the cytokine levels. 这些分子具有多种作用方式，在各个阶段阻断 TLR4 信号，并以非共价和共价方式与靶点结合。一些 TLR 信号通路阻断剂的精确靶点尚不明确，由对接研究产生的假设需要实验验证。在基于细胞的实验中，许多降低促炎细胞因子（TNF-α、IL-6、IL-1β、IFN-γ）浓度的化合物在体内小鼠模型中也表现出活性，提高了生存率并降低了细胞因子水平。

Among all TLR4 antagonists so far developed, only TAK-242 and Eritoran reached clinical trials, but unfortunately failed to pass them and were not active in improving patients survival [124,129,130,131,132]. At phase 3 of clinical trials, Eritoran was not active in improving survival also for sepsis patients infected by Gram-negative flora (21.9 vs 22.3%, p = 0.89), although for this subgroup a positive effect was expected. The negative results in clinic were in contrast with successful applications of TLR4 antagonists in sepsis therapy in animal models. This “translational gap” is well known in a series of other drugs, in the case of sepsis it could derive from the difference in TLR4 signaling and in the expression of inflammatory pathway genes in humans and in mice. 在迄今为止开发的 TLR4 拮抗剂中，只有 TAK-242 和 Eritoran 进入了临床试验，但不幸的是，它们未能通过临床试验，并且未能有效提高患者的生存率[124, 129, 130, 131, 132]。在临床试验的第三阶段，Eritoran 对于由革兰氏阴性菌感染的败血症患者来说，也没有在提高生存率方面发挥作用（21.9% vs 22.3%，p = 0.89），尽管对于这个亚组原本预期会有积极效果。临床试验中的负面结果与 TLR4 拮抗剂在动物模型败血症治疗中的成功应用形成对比。这种“转化差距”在一系列其他药物中也是众所周知的，在败血症的情况下，这可能是由于人类和老鼠中 TLR4 信号传导和炎症途径基因表达的不同所导致的。

In a comparative review written by Vaure and Liu [177], the expression patterns of TLR4 in different tissues and mammal species were analyzed, and the relative sensibility of various animal species to LPS also considered. When compared to humans, mice are less susceptible to LPS, with the threshold dose required for minimal physiological changes is 0.5 mg/kg intraperitoneally for mice [178] (BALB/c strain) vs 1–5 ng/kg intravenously for humans [179]. This is a substantial difference even considering the diverse susceptibility across the mouse strains. One of the possible reasons for that is the difference in cytokine production patterns [177]. 在 Vaure 和 Liu [177]撰写的比较综述中，分析了不同组织和哺乳动物物种中 TLR4 的表达模式，并考虑了各种动物物种对 LPS 的相对敏感性。与人类相比，小鼠对 LPS 的敏感性较低，引起最小生理变化的阈值剂量为小鼠腹腔注射 0.5 mg/kg [178]（BALB/c 品系）与人类静脉注射 1-5 ng/kg [179]。即使在考虑小鼠品系间差异的敏感性时，这仍是一个显著差异。其中一个可能的原因是细胞因子产生模式的差异 [177]。

It is also known that after LPS stimulation in mouse neutrophils and peritoneal macrophages, a decrease in TLR4 expression occurs [180], in contrast, TLR4 expression in human monocytes is unaltered at least after low doses of lipopolysaccharide [181]. Cytokine production and NF-kB DNA-binding activity are also suppressed in mice [182]. Moreover, several mRNA isoforms of TLR4 in mice are known [183]. A notable one is smTLR4 (soluble mouse TLR4). This splicing variant consists of 122 amino acid residues, is not membrane-bound, and contains a part of the TLR4 extracellular domain responsible for the interaction with LPS. One possible reason can be the inhibition of TLR4/MD-2 interaction since MD-2 is known to be essential in mediating the LPS response. smTLR4 can therefore be an endogenous TLR4-antagonist acting on the early stages of TLR4 signaling. There is not an equivalent of such TLR4 isoform in humans. 小鼠中性粒细胞和腹腔巨噬细胞在 LPS 刺激后，TLR4 表达降低[180]，而人类单核细胞在至少低剂量脂多糖处理后，TLR4 表达未改变[181]。小鼠的细胞因子产生和 NF-kB DNA 结合活性也受到抑制[182]。此外，小鼠中已知存在 TLR4 的几种 mRNA 异构体[183]。其中值得注意的是 smTLR4（可溶性小鼠 TLR4）。这种剪接变体由 122 个氨基酸残基组成，不是膜结合的，并包含 TLR4 细胞外域的一部分，该域负责与 LPS 相互作用。一个可能的原因可能是抑制 TLR4/MD-2 相互作用，因为已知 MD-2 在介导 LPS 反应中是必需的。因此，smTLR4 可以作为一种内源性 TLR4 拮抗剂，作用于 TLR4 信号传导的早期阶段。人类中没有这种 TLR4 异构体的等价物。

It is also important to consider the similarity between animal sepsis models and human sepsis. As stated before, the CLP model mimics human sepsis the most but rarely was used in the studies reported in this review. The LPS injection model was often employed, which reproduces septic shock rather than sepsis. So for further development of TLR4-signaling modulators, their testing on CLP sepsis model is also recommended. 考虑动物败血症模型与人类败血症之间的相似性也很重要。正如之前所述，CLP 模型最接近模拟人类败血症，但在本综述中报道的研究中很少使用。LPS 注射模型经常被采用，它复制的是脓毒症休克而不是败血症。因此，为了进一步开发 TLR4 信号通路调节剂，建议在 CLP 败血症模型上进行测试。

Taken together, these factors underline the great differences between murine and human innate immune response to LPS and explain, at least in part, the clinical gap in the case of sepsis. 综合考虑，这些因素突出了小鼠和人类对 LPS 的先天免疫反应之间的巨大差异，并在一定程度上解释了败血症病例中临床差距的原因。

Because of the complexity of innate immunity regulation, and also because of the redundancy of pathways involved in TLR-triggered cytokine production, in the future combined therapies could be investigated and tested in clinic. 由于固有免疫调节的复杂性，以及 TLR 触发的细胞因子产生所涉及途径的冗余性，未来可以研究并测试在临床上的联合疗法。

One possibility could be to combine TLR4 antagonists with specific antibacterial agents. 一种可能性是将 TLR4 拮抗剂与特定的抗菌剂结合使用。

Wang et al. [111] have studied the effect of the TLR4 antagonist artemisinin alone and in combination with antibiotics on the mice survival after administration of live E. coli bacteria as a source of LPS. When ampicillin sodium or a 2:1 mixture of ampicillin sodium/sulbactam sodium were co-administrated to the bacterium, the survival increased to 33% and 67%, respectively. When administering antibiotics alone, a 0% and 33% survival increase was obtained. Conversely, when administrating Artemisin alone, no protection was obtained after the injection of live E. coli bacteria. 王等[111]研究了 TLR4 拮抗剂青蒿素单独及与抗生素联合使用对注射活 E. coli 细菌（作为 LPS 来源）后小鼠存活率的影响。当与细菌共同给予氨苄西林钠或氨苄西林钠/舒巴坦钠 2:1 混合物时，存活率分别增加到 33%和 67%。单独使用抗生素时，存活率分别增加 0%和 33%。相反，单独给予青蒿素时，在注射活 E. coli 细菌后没有获得保护。

Another case when TLR4 antagonist was given together with antimicrobial substance was described by Shirey and co-workers [51], albeit the disease was influenza and not sepsis. Applied together with Tamiflu™ (oseltamivir), Eritoran significantly improved the survival of C57BL/6J mice as compared with the group given oseltamivir alone, and protected them from lethal re-infection. 另一个案例中，Shirey 及其同事[51]描述了 TLR4 拮抗剂与抗菌物质同时给予的情况，尽管疾病是流感而非败血症。与单独给予奥司他韦（Tamiflu™）的组相比，与奥司他韦联合应用的 Eritoran 显著提高了 C57BL/6J 小鼠的存活率，并保护它们免受致命性再感染。

A second possibility is the combination of blockers of different TLRs. In a recent successful example, anti-TLR2 and anti-TLR4 monoclonal antibodies were combined [184]. When metronidazole and ceftriaxone have been co-administrated with TLR4-targeting immunoglobulins, the survival increased more than twice when comparing with anti-TLR4 antibodies alone. 第二种可能性是不同 TLR 的阻断剂的组合。在最近的一个成功例子中，抗-TLR2 和抗-TLR4 单克隆抗体被结合使用[184]。当甲硝唑和头孢曲松与针对 TLR4 的免疫球蛋白共同给药时，与单独使用抗-TLR4 抗体相比，存活率增加了两倍以上。

Concerning pathologies related to PAMP/TLR4 activation, in particular in the case of acute sepsis and septic shock, one can therefore conclude that, despite promising results on animal models, the clinical trials based on a single TLR4 antagonist has not led so far to a significant improvement in survival. We propose here combination therapies as a new strategy to overcome the limitations discussed above. 关于与 PAMP/TLR4 激活相关的疾病，特别是在急性脓毒症和脓毒症休克的情况下，因此可以得出结论，尽管在动物模型上取得了有希望的结果，但基于单一 TLR4 拮抗剂的临床试验迄今为止并未导致生存率的显著提高。我们在此提出联合疗法作为克服上述局限性的新策略。

The use of TLR4 modulators in pathologies caused by TLR4 activation by endogenous DAMPs, however, is still at a very early stage and promising results have been obtained at a preclinical stage. Some small-molecular TLR4 blockers displayed a lack of toxicity and were efficient in blocking DAMP/TLR4 signaling and cytokine production in an array of inflammatory and autoimmune diseases [185,186,187]. These comprise of neuropathic pain, ALS, rheumatoid arthritis, vascular inflammation, atherosclerosis, and other pathologies reviewed here. TLR4 调节剂在由内源性损伤相关分子模式（DAMPs）激活 TLR4 引起的疾病中的应用，然而，仍处于非常早期阶段，并在临床前阶段取得了有希望的结果。一些小分子 TLR4 阻断剂显示出无毒性，并在一系列炎症和自身免疫疾病中有效地阻断 DAMP/TLR4 信号传导和细胞因子产生[185, 186, 187]。这些包括神经性疼痛、肌萎缩侧索硬化症、类风湿性关节炎、血管炎症、动脉粥样硬化和其他在此处回顾的疾病。

We are therefore convinced that some modern day chronic inflammations and autoimmune diseases that still lack specific pharmacological treatment could be efficiently targeted by new drugs interfering with TLR(4) activation and signaling. 我们因此坚信，一些目前仍缺乏特异性药物治疗的中度慢性炎症和自身免疫性疾病，可以通过干扰 TLR（4）激活和信号传导的新药有效靶向。

Acknowledgments 致谢

Nikolay N. Kuzmich acknowledges Olga Petina (KRKA) and Roman Shutov (Vertex) for valuable critical remarks regarding the manuscript. FP acknowledges the H2020-MSC-ETN-642157 project TOLLerant and the Italian Ministry for Foreign Affairs and International Cooperation (MAECI). 尼古拉·N·库兹米奇感谢奥莉加·彼得娜（KRKA）和罗曼·舒托夫（Vertex）对稿件提出的宝贵批评意见。FP 感谢 H2020-MSC-ETN-642157 项目 TOLLerant 以及意大利外交部和国际合作部（MAECI）。

Author Contributions 作者贡献

Nikolay N. Kuzmich wrote the paper; Konstantin V. Sivak participated in writing of the introduction section and performed the literature search together with Vladimir N. Chubarev, Yury B. Porozov and Tatiana N. Savateeva-Lyubimova; Francesco Peri reorganized and updated the manuscript with new data. 尼古拉·N·库兹米奇撰写了这篇论文；康斯坦丁·V·西瓦克参与了引言部分的撰写，并与弗拉基米尔·N·丘巴列夫、尤里·B·波罗佐夫和塔蒂亚娜·N·萨瓦特耶娃-柳比莫娃一起进行了文献检索；弗朗西斯科·佩里根据新数据重新组织和更新了手稿。

原文在此

TLR4 信号通路调节剂作为炎症和败血症的潜在治疗药物 --- TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis

本文标签： TLR4 信号通路调节剂作为炎症和败血症的潜在治疗药物 （其二）