粒细胞集落刺激因子上调内毒素结合蛋白表达的分子机制及其在脓毒症中的作用

Granulocyte-colony stimulating factor (G-CSF) upregulates the innate immunity, and is frequently used in patients with severe neutropenia associated sepsis. However, clinical trials where G-CSF was used therapeutically to treat ongoing severe sepsis did not result in a clear benefit, and the reason was unknown. We observed previously that G-CSF pretreatment increases the expression of lipopolysaccharide (LPS) binding protein (LBP). In the LPS induced inflammatory response model, G-CSF pretreatment caused an overwhelming inflammatory response, which leading to the death of all the rats. This response could at least be partly attributed to the upregulation of LBP prior to the LPS challenge, as blocking of LBP by using an inhibitory peptide abrogated the effects of G-CSF pretreatment. Moreover, in rat poly-microbial sepsis model, blockade of LBP after G-CSF pretreatment was associated with an increased 72h survival rate (93% versus 56%, P<0.001). We hypothesize that the futile treatment of G-CSF in sepsis may due to upregulated LBP expression. Moreover, the activation of STAT-3 and NF-κB is the key signal pathway in mediating the inflammatory response. These evidences support our hypothesis that enhanced bacterial clearance via augmentation of the innate immunity together with a reduction of the LPS-induced inflammatory response may contribute to a better outcome of sepsis. This project is designed to explore the mechanism of the induction of LBP via G-CSF treatment by both in in-vitro and in-vivo studies. And we investigate further that interfering with the LBP-mediated inflammatory response to LPS using inhibitors of STAT-3 and NF-κB signal pathway might reduce the overwhelming inflammation and improve outcome after a septic insult in G-CSF treated rats. If our hypothesis holds, we can contribute to a better understanding of the pathomechanism of sepsis, and to achieve a more effective therapeutic strategy by reducing the inflammatory response, and promoting the bacterial clearance in sepsis.

粒细胞集落刺激因子（G-CSF）作为免疫调节剂用于脓毒症病人的辅助治疗，但对部分病人无效，机制尚不清楚。我们前期研究证实：1）G-CSF上调LPS结合蛋白（LBP）表达,增加大鼠对炎症反应敏感性; 2）使用LBP封闭肽治疗可降低LPS诱导的炎症反应，并提高大鼠生存率。另外，STAT-3和NF-κB信号通路在脓毒症炎症反应中起重要作用。我们推测：1)G-CSF通过激活STAT-3和NF-κB信号通路诱导LBP表达上调并介导过强炎症反应，成为辅助治疗无效的原因;2)使用G-CSF和LBP封闭肽联合治疗，可降低炎症反应，提高脓毒症的疗效。为验证该假说，本研究拟使用大鼠盲肠结扎穿孔模型、大鼠原代肝细胞和枯否氏细胞体外培养模型，在体内外阐明G-CSF上调LBP表达的分子机制，探索G-CSF与LBP阻断联合治疗对大鼠脓毒症的影响作用，为临床脓毒症的治疗提供新线索。

粒细胞集落刺激因子（G-CSF）作为免疫调节剂作用于脓毒症病人的辅助治疗，但对部分病人无效，机制尚不清楚。我们前期研究证实：（1）G-CSF上调内毒素（LPS）表达，增加大鼠对炎症反应敏感性；（2）使用LBP封闭肽治疗可降低LPS诱导的炎症反应，并提高大鼠生存率。因此，我们推测：（1）G-CSF通过增强LPS敏感性，介导过强的炎症反应，成为辅助治疗无效的原因；（2）使用G-CSF和LBP封闭肽联合治疗，可以降低炎症反应，提高脓毒症的治疗效果。为验证该假说，本研究拟使用大鼠腹膜炎模型，和细胞体外培养模型，在体内外阐明G-CSF上调LBP表达的分子机制，探索G-CSF与LBP阻断联合治疗对大鼠脓毒症的影响作用。结果显示，（1）G-CSF刺激可诱导淋巴细胞炎症因子的表达。使用IL-6和TNF-α刺激大鼠原代肝脏细胞后，可明显上调LBP表达。使用高浓度LBP（100 μg/ml）刺激大鼠枯否氏细胞，对STAT3的磷酸化有抑制作用。（2）在感染前使用高浓度LBP封闭肽（5mg/kg）处理能够显著的提高生存率（LBPK95A -2h vs. control = 95% vs. 36%）在感染后使用高浓度（5mg/kg）LBP封闭肽处理，却具有损伤作用，表现为降低的生存率（LBPK95A -2h vs. LBPK95A 0h = 95% vs. 10%）。这个结果提示我们，在感染后使用LBPK95A，可能会由于炎症反应过度降低，反而导致损伤。进一步结果显示，在感染后选择合适浓度的LBPK95A可以调节炎症反应强度，并能够达到最佳的治疗效果（5 mg/kg LBPK95A vs. 0.5 mg/kg LBPK95A vs. 0.05 mg/kg LBPK95A = 80% vs. 50% vs. 10%）。以上结果显示，G-CSF上调LBP表达，可能通过刺激炎症因子的释放，和通过STAT-3信号通路的活化实现。使用G-CSF提高机体免疫功能的同时通过影响LPS-LBP间相互作用调节炎症反应强度，能够有效提高脓毒症大鼠生存率。SIRS和脓毒症有发病率高，治疗难度大等特点。LBP对LPS诱导的炎症反应具有调控作用，是一个潜在的治疗靶点。本项目通过调节机体免疫功能和炎症反应强度的策略，提高脓毒症的治疗效果，并探索其作用的分子机制，为临床治疗提供新线索。