Abstract：
BACKGROUND: Paraquat (PQ) is a widely used herbicide that poisons human by accident or intentional ingestion. PQ poisoning causes systemic inflammatory response syndrome (SIRS) resulting in acute lung injury (ALI) with an extremely high mortality rate. Blood trematode Schistosoma japonicum-produced cystatin (Sj-Cys) is a strong immunomodulatory protein that has been experimentally used to treat inflammation related diseases. In this study, Sj-Cys recombinant protein (rSj-Cys) was used to treat PQ-induced lung injury and the immunological mechanism underlying the therapeutic effect was investigated.
METHODS: PQ-induced acute lung injury mouse model was established by intraperitoneally injection of 20 mg/kg of paraquat. The poisoned mice were treated with rSj-Cys and the survival rate was observed up to 7 days compared with the group without treatment. The pathological changes of PQ-induced lung injury were observed by examining the histochemical sections of affected lung tissue and the wet to dry ratio of lung as a parameter for inflammation and edema. The levels of the inflammation related cytokines IL-6 and TNF-α and regulatory cytokines IL-10 and TGF-β were measured in sera and in affected lung tissue using ELISA and their mRNA levels in lung tissue using RT-PCR. The macrophages expressing iNOS were determined as M1 and those expressing Arg-1 as M2 macrophages. The effect of rSj-Cys on the transformation of inflammatory M1 to regulatory M2 macrophages was measured in affected lung tissue in vivo (EKISA and RT-PCR) and in MH-S cell line in vitro (flow cytometry). The expression levels of TLR2 and MyD88 in affected lung tissue were also measured to determine their role in the therapy of rSj-Cys on PQ-induced lung injury.
RESULTS: We identified that treatment with rSj-Cys significantly improved the survival rate of mice with PQ-induced lung injury from 30 % (untreated) to 80 %, reduced the pathological damage of poisoning lung tissue, associated with significantly reduced levels of proinflammatory cytokines (IL-6 from 1490 to 590 pg/ml, TNF-α from 260 to 150 pg/ml) and increased regulatory cytokines (IL-10 from360 to 550 pg/ml, and TGF-β from 220 to 410 pg/ml) in both sera (proteins) and affected lung tissue (proteins and mRNAs). The polarization of macrophages from M1to M2 type was found to be involved in the therapeutic effect of rSj-Cys on the PQ-induced acute lung injury, possibly through inhibiting TLR2/MyD88 signaling pathway.
CONCLUSIONS: Our study demonstrated the therapeutic effect of rSj-Cys on PQ poisoning caused acute lung injury by inducing M2 macrophage polarization through inhibiting TLR2/MyD88 signaling pathway. The finding in this study provides an alternative approach for the treatment of PQ poisoning and other inflammatory diseases.
METHODS: PQ-induced acute lung injury mouse model was established by intraperitoneally injection of 20 mg/kg of paraquat. The poisoned mice were treated with rSj-Cys and the survival rate was observed up to 7 days compared with the group without treatment. The pathological changes of PQ-induced lung injury were observed by examining the histochemical sections of affected lung tissue and the wet to dry ratio of lung as a parameter for inflammation and edema. The levels of the inflammation related cytokines IL-6 and TNF-α and regulatory cytokines IL-10 and TGF-β were measured in sera and in affected lung tissue using ELISA and their mRNA levels in lung tissue using RT-PCR. The macrophages expressing iNOS were determined as M1 and those expressing Arg-1 as M2 macrophages. The effect of rSj-Cys on the transformation of inflammatory M1 to regulatory M2 macrophages was measured in affected lung tissue in vivo (EKISA and RT-PCR) and in MH-S cell line in vitro (flow cytometry). The expression levels of TLR2 and MyD88 in affected lung tissue were also measured to determine their role in the therapy of rSj-Cys on PQ-induced lung injury.
RESULTS: We identified that treatment with rSj-Cys significantly improved the survival rate of mice with PQ-induced lung injury from 30 % (untreated) to 80 %, reduced the pathological damage of poisoning lung tissue, associated with significantly reduced levels of proinflammatory cytokines (IL-6 from 1490 to 590 pg/ml, TNF-α from 260 to 150 pg/ml) and increased regulatory cytokines (IL-10 from360 to 550 pg/ml, and TGF-β from 220 to 410 pg/ml) in both sera (proteins) and affected lung tissue (proteins and mRNAs). The polarization of macrophages from M1to M2 type was found to be involved in the therapeutic effect of rSj-Cys on the PQ-induced acute lung injury, possibly through inhibiting TLR2/MyD88 signaling pathway.
CONCLUSIONS: Our study demonstrated the therapeutic effect of rSj-Cys on PQ poisoning caused acute lung injury by inducing M2 macrophage polarization through inhibiting TLR2/MyD88 signaling pathway. The finding in this study provides an alternative approach for the treatment of PQ poisoning and other inflammatory diseases.
摘要：
背景：百草枯（PQ）是一种广泛使用的除草剂，可通过意外或故意摄入对人中毒。PQ中毒引起全身炎症反应综合征（SIRS），导致急性肺损伤（ALI），死亡率极高。血液吸虫日本血吸虫产生的胱抑素（Sj-Cys）是一种强大的免疫调节蛋白，已在实验上用于治疗炎症相关疾病。在这项研究中,Sj-Cys重组蛋白(rSj-Cys)用于治疗PQ诱导的肺损伤,并研究其治疗作用的免疫学机制。
方法：腹腔注射百草枯20mg/kg建立PQ诱导的急性肺损伤小鼠模型。用rSj-Cys治疗中毒小鼠,与未治疗组相比观察到7天的存活率。通过检查受累肺组织的组织化学切片和肺的干湿比作为炎症和水肿的参数，观察PQ引起的肺损伤的病理变化。使用ELISA在血清和受影响的肺组织中测量炎症相关细胞因子IL-6和TNF-α以及调节细胞因子IL-10和TGF-β的水平，并使用RT-PCR在肺组织中测量其mRNA水平。将表达iNOS的巨噬细胞确定为M1,将表达Arg-1的巨噬细胞确定为M2。在体内受影响的肺组织（EKISA和RT-PCR）和体外MH-S细胞系（流式细胞术）中测量了rSj-Cys对炎性M1向调节性M2巨噬细胞转化的影响。还测量了TLR2和MyD88在受影响的肺组织中的表达水平,以确定它们在rSj-Cys治疗PQ诱导的肺损伤中的作用。
结果：我们发现，用rSj-Cys治疗可将PQ诱导的肺损伤小鼠的存活率从30％（未经治疗）提高到80％，减轻中毒肺组织的病理损伤，与促炎细胞因子水平显着降低相关（IL-6从1490到590pg/ml，TNF-α从260到150pg/ml）和增加的调节细胞因子（IL-10从360到550pg/ml，血清（蛋白质）和受影响的肺组织（蛋白质和mRNA）中的TGF-β为220至410pg/ml）。发现巨噬细胞从M1到M2型的极化参与rSj-Cys对PQ诱导的急性肺损伤的治疗作用,可能通过抑制TLR2/MyD88信号通路。
结论：我们的研究表明rSj-Cys通过抑制TLR2/MyD88信号通路诱导M2巨噬细胞极化对PQ中毒引起的急性肺损伤具有治疗作用。这项研究的发现为PQ中毒和其他炎症性疾病的治疗提供了一种替代方法。
方法：腹腔注射百草枯20mg/kg建立PQ诱导的急性肺损伤小鼠模型。用rSj-Cys治疗中毒小鼠,与未治疗组相比观察到7天的存活率。通过检查受累肺组织的组织化学切片和肺的干湿比作为炎症和水肿的参数，观察PQ引起的肺损伤的病理变化。使用ELISA在血清和受影响的肺组织中测量炎症相关细胞因子IL-6和TNF-α以及调节细胞因子IL-10和TGF-β的水平，并使用RT-PCR在肺组织中测量其mRNA水平。将表达iNOS的巨噬细胞确定为M1,将表达Arg-1的巨噬细胞确定为M2。在体内受影响的肺组织（EKISA和RT-PCR）和体外MH-S细胞系（流式细胞术）中测量了rSj-Cys对炎性M1向调节性M2巨噬细胞转化的影响。还测量了TLR2和MyD88在受影响的肺组织中的表达水平,以确定它们在rSj-Cys治疗PQ诱导的肺损伤中的作用。
结果：我们发现，用rSj-Cys治疗可将PQ诱导的肺损伤小鼠的存活率从30％（未经治疗）提高到80％，减轻中毒肺组织的病理损伤，与促炎细胞因子水平显着降低相关（IL-6从1490到590pg/ml，TNF-α从260到150pg/ml）和增加的调节细胞因子（IL-10从360到550pg/ml，血清（蛋白质）和受影响的肺组织（蛋白质和mRNA）中的TGF-β为220至410pg/ml）。发现巨噬细胞从M1到M2型的极化参与rSj-Cys对PQ诱导的急性肺损伤的治疗作用,可能通过抑制TLR2/MyD88信号通路。
结论：我们的研究表明rSj-Cys通过抑制TLR2/MyD88信号通路诱导M2巨噬细胞极化对PQ中毒引起的急性肺损伤具有治疗作用。这项研究的发现为PQ中毒和其他炎症性疾病的治疗提供了一种替代方法。
