大黄酚促进巨噬细胞M2型极化缓解脓毒症大鼠肺损伤

Chrysophanol alleviates lung injury of sepsis rats by promoting macrophage M2 polarization

目的本研究旨在探究大黄酚(Chrysophanol,CP)对脓毒症模型大鼠肺损伤、巨噬细胞极化以及对核因子κB p65(NF-κB p65)、细胞外调节蛋白激酶1/2(ERK1/2)通路的影响,为治疗脓毒症提供理论依据。方法将大鼠随机分为空白组、模型组、低、中、高剂量CP组。HE染色观察各组大鼠肺组织损伤情况并进行肺损伤评分。称取大鼠右肺湿质量、干质量,计算湿/干(W/D)比值。其次,通过RT-PCR和Western blot检测肺组织中裂解型半胱天冬酶cleaved Caspase-3、cleaved Caspase-9的表达情况;通过流式细胞术分选大鼠外周血中诱导型一氧化氮合酶(iNOS)+、CD16/32+M1型和CD206+、CD10+M2型巨噬细胞。通过Western blot和ELISA检测大鼠肺巨噬细胞中iNOS和CD10的表达。Western blot检测大鼠肺组织中NF-κB p65、ERK1/2通路磷酸化情况。结果与对照组相比,模型组大鼠肺泡结构破坏严重,而在低、中、高剂量CP组中,随CP剂量增加肺脏损伤程度降低。与对照组相比,模型组大鼠肺损伤评分和W/D比值升高(P<0.05),且cleaved Caspase-3、cleaved Caspase-9表达量升高(P<0.05),外周血中iNOS+、CD16/32+M1型巨噬细胞数增多(P<0.05),肺巨噬细胞中iNOS表达量增多(P<0.05),CD10表达量降低(P<0.05);与模型组相比,中、高剂量CP组大鼠肺损伤评分和W/D比值降低(P<0.05),且cleaved Caspase-3、cleaved Caspase-9表达量降低(P<0.05),CD206+、CD10+M2型巨噬细胞数增多(P<0.05),iNOS表达量降低(P<0.05),CD10表达量增多(P<0.05)。最后,模型组中NF-κB p65、ERK1/2磷酸化水平较对照组显著升高(P<0.05);中、高剂量CP组中NF-κB p65、ERK1/2磷酸化水平较模型组显著降低(P<0.05)。结论大黄酚可促进模型大鼠巨噬细胞向M2型极化进而缓解脓毒症大鼠模型肺损伤,下调大鼠肺组织中cleaved Caspase-3、cleaved Caspase-9表达量,抑制NF-κB p65、ERK1/2通路活性,有望成为脓毒症的治疗药物。

This study was designed to investigate the effects of chrysophanol(CP)on lung injury,macrophage polarization and the pathways of nuclear factor kappa-B(NF-κB)p65 and extracellular regulated protein kinases1/2(ERK1/2)in sepsis rats,and to provide theoretical basis for the treatment of sepsis.Rats were randomly divided into blank group,model group,low dose CP group,medium dose CP group and high dose CP group.HE staining was used to observe the lung injury of rats in each group and to evaluate the lung injury score.The right lung wet weight and dry weight of rats were weighed,and the weight/dry(W/D)ratio was calculated.Then,the expression of cleaved Caspase-3 and cleaved Caspase-9 in lung tissue were detected by RT-PCR and Western blotting;inducible nitric oxide synthase(iNOS)+,CD16/32+M1 and CD206+,CD10+M2 macrophages in peripheral blood of rats were sorted by flow cytometry;Western blot and ELISA were also used to detect the expression of iNOS and CD10 in rat alveolarmacrophage;the phosphorylation of NF-κB p65 and ERK1/2 pathways in rat lung tissue were detected by Western blotting.Compared with the control group,the pulmonary alveolar structure of sepsis model group was severely damaged,while in low,medium and high dose CP groups,the degree of lung injury gradually decreased with the increase of the dose.Compared with the control group,the lung injury score and W/D ratio of the model group were increased(P<0.05),the expression of cleaved Caspase-3 and cleaved Caspase-9 increased(P<0.05),the number of iNOS+,CD16/32+M1 macrophages in the peripheral blood increased(P<0.05),the expression of iNOS in pulmonary macrophages increased,(P<0.05),and the expression of CD10 decreased(P<0.05).the middle and high dose CP can reverse these changes metioned above in model goup.Finally,the phosphorylation levels of NF-κB p65 and ERK1/2 in the model group were significantly higher than those in the control group(P<0.05),which also could be reversed by the middle and high dose CP.In conclusion,chrysophanol can promote the polarization of macrophage to M2 type and alleviate lung injury in rats with sepsis,and also can down-regulate the expression of cleaved caspase-3 and cleaved Caspase-9 in rat lung tissue and inhibit NF-κB p65,ERK1/2 pathway activity,thus could be expected to become a therapeutic drug for the treatment of sepsis.