【作者】 吴强；

【导师】 菅向东；

【作者基本信息】 山东大学 ， 急诊医学（专业学位）， 2018， 博士

【摘要】 第一部分一例百草枯中毒死亡患者肺部组织免疫组化病理研究和文献回顾一、前言百草枯中毒后会引起患者肺部出现炎性细胞的浸润,并发生明显的炎症反应。百草枯中毒后的免疫机制激活可能是重要的致病机制之一。但是具体有哪些炎性细胞可以浸润到肺组织内目前缺乏相应的研究。我们对一例死亡的百草枯患者的肺组织进行了 MPO,CD4,CD8,CD21,CD56,CD68,CD79,SMA免疫组化的染色,来明确浸润细胞的类型。为证明免疫激活在百草枯中毒中的作用提供病理学依据。二、方法和材料材料伦理学:尸体解剖的取样和分析经家属同意,并经医院伦理委员会审核通过。符合现行的中国法律,遵循赫尔辛基宣言的道德规范。病例报告和尸体解剖:2014年12月,患者,石某某,女,38岁,因“口服百草枯100毫升4小时”入院,一周后死亡。尸体保存在2-3摄氏度尸体箱内,于死后的第三天进行解剖。留取心、肺、肾标本。为进一步明确百草枯中毒后免疫细胞的浸润情况,2016-08我们对死者的肺部组织蜡块再次切片,进行HE染色和应用MPO,CD4,CD8,CD21,CD56,CD68,CD79,SMA 免疫组化分析。三、实验结果3.1肺部HE染色HE染色下的肺组织呈现典型的ARDS样改变,包括炎性细胞浸润、弥漫性肺泡损伤(diffuse alveolar damage,DAD)、透明膜形成和纤维化。相对于正常的肺组织(图7),镜下可见肺部组织结构紊乱,肺实变明显,炎性细胞(以中性粒细胞为主)广泛的浸润到肺间质及肺泡腔内(图1,图2)。可见到肺组织的灶性坏死,坏死灶内的正常结构消失,充填大量炎性细胞(以中性粒细胞为主)、红细胞、纤维蛋白沉积(图3,图4)。部分肺组织呈现纤维化样表现(图5),镜下见到胞核较大,核仁明显,胞质丰富的成纤维样细胞,细胞周边纤维增生,炎性细胞浸润。肺泡塌陷,大量的肺泡内有明显出血,肺泡内充满浆性渗液,并含有大量纤维素,有肺泡上皮细胞的脱落和大量炎性细胞的浸润。肺泡表面可见到明显的透明膜形成(图6)。肺间质水肿,肺泡壁增厚,肺泡旁毛血管明显充血、扩张。3.2中性粒细胞中性粒细胞是在肺内浸润最多的炎性细胞。镜下可见大量胞质深染的MPO阳性中性粒细胞浸润到肺间质和肺泡腔内(图8)。在血管旁的密度较大,可见到部分中性粒细胞正在穿过血管壁(图9)。3.3单核/巨噬细胞单核/巨噬细胞是在肺内浸润的数量仅次于中性粒细胞。镜下可见肺组织内有大量的CD68阳性的巨噬细胞分布(图10)。在出血坏死灶内的巨噬细胞明显增多,染色不均一,染色深的考虑是已经吞噬粉尘和组织碎片的巨噬细胞,染色较浅的考虑是新招募的还未吞噬颗粒的单核巨噬细胞(图 11)。3.4辅助T细胞CD4阳性的T细胞为Th细胞,在镜下也明显增多(图12),这些胞膜深染的Th细胞多聚集在一起,分布在出血灶内(图13),肺间质内(图14)。3.5杀伤T细胞杀伤T细胞的免疫组化CD8呈阳性反应,在镜下也明显增多(图15),这些胞膜深染的杀伤性T细胞多聚集在一起,分布在出血灶内(图16),肺间质内(图17)。3.6 NK细胞CD56是NK细胞表面表达的标识分子。NK细胞数量较少。镜下可见胞膜深染的NK细胞散在分布在肺间质内(图18,图19)。3.7 CD21阳性和CD79阳性的B细胞镜下可见CD21(图20)和CD79(图21,图22)阳性的B细胞胞膜深染,主要分布在血管内部。肺间质内很少出现。3.8成纤维细胞/肌成纤维细胞SMA是平滑肌的α的肌动蛋白,可以分布在血管平滑肌和肌成纤维细胞胞质内,在该患者的免疫组化中发现,SMA高表达,广泛分布在肺血管和肺间质内(图23,图25),并出现泛染的迹象。我们在肺泡毛细血管和肺泡上皮细胞(图24)中也发现存在SMA的高表达,在这些区域SMA在这些区域通常不会表达。四、结论通过HE和免疫组化染色,该患者的肺部存在严重的炎症反应,固有免疫和适应性免疫都参与到了这种严重的炎症反应中。在固有免疫中以中性粒细胞和单核巨噬细胞的浸润为主。在适应性免疫中Th细胞和细胞毒性的T细胞增多的明显。SMA的免疫组化显示患者肺部间质、肺泡上皮细胞和肺泡毛细血管的上皮细胞中存在高表达,为早期纤维化的倾向。第二部分实验一:DAMPs、无菌性炎症通路、中性粒细胞参与百草枯中毒介导的肺损伤一、前言百草枯中毒是临床上较为棘手的一种中毒性疾病。目前的治疗方法没有从总体上改善患者的病死率。研究发现百草枯中毒后免疫系统主动参与了病情的发展及转归,我们的尸体解剖报告和初步动物试验证实固有免疫在百草枯中毒中可能起到重要作用。百草枯中毒的免疫致病机制实质上可能是DAMPs触发的无菌性炎症。但是目前没有相关研究。对百草枯中毒后免疫反应的始动环节(DAMPs释放)、炎性通路的激活、炎性效应细胞和肺损伤的关系的研究具有一定创新性。基于以上理由我们在本研究中提出以下假说:百草枯中毒后大量的ROS产生后,各种细胞膜性结构的破坏,使得细胞坏死,释放出大量的DAMPs,DAMPs作用于细胞上的PPR,激活无菌性的炎性通路(以TLR4为代表的胞膜途径,以NLRP3为代表的胞内途径),产生一系列的炎性因子和趋化因子招募中性粒细胞为代表的炎性细胞到达损伤部位,从而产生无菌性炎症。二、试验方法将36只SD大鼠随机分为3组,轻度组(百草枯25mg/Kg腹腔注射)、重度组(百草枯50mg/Kg腹腔注射)和空白组(生理盐水2ml腹腔注射),每组12只。在腹腔注射百草枯后第12.24.72h分别取出4只,麻醉后处死,收集左肺的肺泡灌洗液,右肺组织,血液。生化仪检测左肺的肺泡灌洗液和血清中尿酸(DAMP),左肺的肺泡灌洗液和血清中促炎因子(IL-1β,IL-18,TNF-α,TGFβ1)。提取右肺上叶 RNA,qPCR 检测 TLR4、myD88、NF-κB、mtDNA(ND2,DAMP)、NLRLP3、ASC、capase-1、mRNA 表达情况。提取部分右肺中叶蛋白Western Blot检测TLR4、myD88、ASC、capase-1、NF-κB蛋白表达情况。右肺中叶部分肺组织进行ROS检测。右肺下叶部分组织包埋切片,HE染色进行组织学肺损伤评分。(染色+拍照+评分),免疫组织化学方法检测中性粒细胞(MPO)、HMGB1(DAMP)。(n=3)右肺下叶部分组织无水乙醇固定、包埋、切片。Gomori六胺银溶液染色观察尿酸。三、实验结果3.1百草枯中毒导致DAMPs的释放。3.1.1 HMGB1结果发现:百草枯可以导致HMGB1的升高。相对于对照组,各时间点的不同剂量染毒组的HMGB1明显升高(除24小时轻度组,与对照组比较有上升趋势,无统计学意义),P<0.05,且随着时间的延长成升高趋势。3.1.2尿酸发现:百草枯可以导致尿酸的升高。六胺银染色中的尿酸成棕黑色,随着剂量和和时间的增大,棕黑色的面积和深度明显增强。在血清和肺泡灌洗液的生化检测中,相对于对照组,各时间点的不同剂量染毒组的尿酸均明显升高,P<0.01,且随着时间的延长成升高趋势。3.1.3mtDNA结果发现:百草枯可以导致mtDNA的升高。相对于对照组,各时间点的不同剂量染毒组的mtDNA明显升高,P<0.001,且随着时间的延长成升高趋势。3.2TLR4途径的激活3.2.1 TLR4的转录和表达3.2.1.1 TLR4 的转录结果发现:百草枯可以导致TLR4的转录增强。相对于对照组,各时间点的不同剂量染毒组的TLR4明显升高,P<0.01,且随着时间的延长成升高趋势。3.2.1.1 TLR4 的表达结果发现:百草枯可以导致TLR4的表达增强。相对于对照组,各时间点的不同剂量染毒组的TLR4有升高(12小时和24小时有统计学差异,P<0.001,72小时的无明显统计学差异),且随着时间的延长成升高趋势。3.2.2MyD88的转录和表达结果发现:百草枯可以导致MyD88的转录和表达增强。相对于对照组,各时间点的不同剂量染毒组的MyD88明显升高,P<0.05,除在72小时轻度组MyD88的表达升高无统计学意义。3.2.3 NF-κ B的转录和表达结果发现:百草枯可以导致NF-κB的转录和表达增强。相对于对照组,各时间点的不同剂量染毒组的NF-κB明显升高,P<0.01,且随着时间的延长成升高趋势。3.3NLRP3途径的激活3.3.1 NLRP3 的转录结果发现:百草枯可以导致NLRP3的转录增强。相对于对照组,各时间点的不同剂量染毒组的NLRP3明显升高,P<0.001,且随着时间的延长成升高趋势。3.3.2 ASC的转录和表达结果发现:百草枯可以导致ASC的转录和表达增强。相对于对照组,各时间点的不同剂量染毒组的ASC明显升高,P<0.01,且随着时间的延长成升高趋势。3.3.3 Caspase-1的转录和表达结果发现:百草枯可以导致Caspase-1的转录和表达增强。相对于对照组,各时间点的不同剂量染毒组的Caspase-1明显升高,P<0.001,且随着时间的延长成升高趋势3.4 IL-1β和IL-18的分泌结果发现:百草枯可以导致Il-1β、11-18的水平升高。相对于对照组,各时间点的不同剂量染毒组的I1-1β、11-18明显升高,P<0.05,(除血清中的Il-1β在轻度中毒组12和24小时有升高趋势,无统计学意义),且随着时间的延长成升高趋势。3.5其他细胞因子的分泌结果发现:百草枯可以导致TNF-α、TGFβ1的水平升高。相对于对照组,各时间点的不同剂量染毒组的TNF-α、TGFβ1明显升高,P<0.05,(除血清中的TNF-α,在轻度组24小时有升高趋势无统计学意义),且随着时间的延长成升高趋势。3.6中性粒细胞的计数及功能3.6.1中性粒细胞在MPO肺内免疫组化染色及计数结果发现:百草枯可以导致中性粒细胞的在肺内增加。相对于对照组,各时间点的不同剂量染毒组的中性粒细胞数量明显升高,P<0.05,且随着时间的延长成升高趋势。3.6.2 R0S结果发现:百草枯可以导致ROS的在肺内增加。相对于对照组,各时间点的不同剂量染毒组的ROS水平明显升高,P<0.01,且一直维持在较高的水平,未见明显的波动。3.7肺病理评分结果发现:大体观察:con的肺组织大小重量正常,肺组织呈粉红色,弹性良好。轻度组和重度组的肺组织均出现明显肿胀,重量增加。肺组织色泽较暗,质地较硬,有明显的出血灶,出血的范围随着时间和剂量对的加大明显加重。由于时间较短,未观察到明显的纤维化征象。HE染色:con正常;轻度组与重度组表现出不同程度的肺充血,水肿,炎性细胞浸润,纤维化(72小时候明显)。百草枯可以导致肺损伤增加。相对于对照组,各时间点的不同剂量染毒组的肺损伤评分明显升高,P<0.01,且随着时间的延长成升高趋势。3.8肺部浸润的中性粒细胞和肺损伤之间相关性性我们采用直线相关分析72小时段MPO和肺损伤评分。结果发现:MPO与肺损伤评分成正相关,r=0.908,P<0.01。四、结论百草枯中毒早期(72小时内),大量的ROS产生后,释放出大量的DAMPs(HMGB1,尿酸,mtDNA),DAMPs作用于细胞上的PPR,激活无菌性的炎性通路(以TLR4-MyD88-NFκB为代表的胞膜途径,以NLRP3炎性小体为代表的胞内途径),产生了炎性因子IL-1β和IIL-18,招募以中性粒细胞为主的炎性细胞在肺部浸润增加,产生无菌性炎症造成肺损伤。百草枯引发的炎症还使得TNF-α、TGFβ1的产生增多。在百草枯中毒的早期,炎症反应的严重程度(IL-1β、IL-18、TNF-α、TGFβ1和肺损伤评分)和百草枯在一定程度上成时间和剂量成正相关趋势。72小时段,肺部中性粒细胞的数量和肺损伤评分成正相关。第三部分实验二从无菌性炎症的角度研究环磷酰胺对百草枯中毒治疗作用的机制一、前言我们在实验一中证明了百草枯导致无菌性炎症中,中性粒细胞起到重要作用。DAMPs招募中性粒细胞的正反馈效应可能是百草枯中毒过度的无菌性炎症反应的关键。环磷酰胺是治疗百草枯的基础药物。早在1986年E.A DDO就阐述了了利用环磷酰胺治疗百草枯中毒的理由:环磷酰胺作为免疫抑制剂可以有效地减少中性粒细胞,减轻炎症反应,可能对后期的纤维化有一定的治疗效果。后期的临床研究也发现患者血常规中的白细胞计数可以作为百草枯中毒的预后指标。是否可以从抑制无菌性炎症的角度,来阐述环磷酰胺的治疗百草枯中毒的机制,目前没有相关的研究。我们从无菌性炎症的角度来验证E.A DDO等人提出的假说:中性粒细胞在百草枯中毒介导的肺损伤中起到重要作用。中性粒细胞缺乏可能减轻百草枯造成的肺损伤。本研究使用环磷酰胺预处理大鼠,造成粒细胞减少的免疫抑制模型,研究环磷酰胺与无菌性炎症的关系,比较干预组的大鼠和正常染毒组的大鼠之间炎症通路激活和肺部炎症发生的差异性。二、方法和材料根据预实验及实验一结果,将48只SD大鼠随机分为4组,重度染毒组(百草枯50mg/Kg腹腔注射)、干预组(环磷酰胺+染毒组,实验前48、24h,100mg/kg环磷酰胺腹膜内注射,百草枯50mg/Kg腹腔注射)、空白对照组(生理盐水2ml腹腔注射),免疫抑制组(实验前48、24h,100mg/kg环磷酰胺腹膜内注射),每组12只。在腹腔注射百草枯后第12.24.72h分别取出4只,麻醉后处死,收集左肺的肺泡灌洗液,右肺组织,血液。生化仪检测左肺的肺泡灌洗液和血清中尿酸(DAMP),左肺的肺泡灌洗液和血清中促炎因子(IL-1β,IL-18,TNF-α,TGFβ1)。提取右肺上叶 RNA,qPCR 检测 TLR4、myD88、NF-κB、mtDNA(ND2,DAMP)、NLRLP3、ASC、capase-1、mRNA 表达情况。提取部分右肺中叶蛋白Western Blot检测TLR4、myD88、ASC、capase-1、NF-κB蛋白表达情况。右肺中叶部分肺组织进行ROS检测。右肺下叶部分组织包埋切片,HE染色进行组织学肺损伤评分。(染色+拍照+评分),免疫组织化学方法检测中性粒细胞(MPO)、HMGB1(DAMP),n=3。右肺下叶部分组织无水乙醇固定、包埋、切片。Gomori六胺银溶液染色观察尿酸(染色+拍照)。三、结果3.1环磷酰胺对百草枯中毒介导的DAMPs释放的影响3.1.1 HMGB1免疫组化染色:深染的HMGB1的细胞在染毒组、干预组、环磷酰胺组中均明显增多,其中染毒组和干预组的增多最为明显。定量分析:相对于对照组,HMBG1阳性的细胞数在染毒组、干预组、环磷酰胺组中均明显升高。与染毒组相比较,干预组在24小时明显高于染毒组,P<0.05,其余未见明显差异。3.1.2尿酸六胺银染色的肺组织:六胺银染色中的尿酸成棕黑色,相对于对照组,染毒组、干预组、环磷酰胺组棕黑色的面积和深度明显增强,以干预组最为明显。生化仪监测尿酸水平:在血清和肺泡灌洗液中的表现较为一致。相对于对照组,HMBG1染毒组、干预组、环磷酰胺组都明显升高,其中干预组升高最为明显,P<0.001。与染毒组相比较,干预组的尿酸明显高于染毒组,P<0.001。各时间点趋势基本相同。3.1.3mtDNA相对于对照组,各时间段的mtDNA在染毒组、干预组、环磷酰胺组都明显升高,其中以环磷酰胺组最高,染毒组次之,干预组最低,P<0.001。且随时间的延长各组有增高的趋势。与染毒组相比较,干预组的mtDNA明显小于染毒组,P<0.01。且随时间的延长各组有增高的趋势。3.2环磷酰胺对百草枯中毒介导的TLR4途径激活的影响3.2.1 TLR4的转录和表达3.2.1.1 TLR4 的转录相对于对照组,各时间段TLR4的转录在染毒组、干预组、环磷酰胺组都明显升高,P<0.01,其中以环磷酰胺组最高,染毒组次之,干预组最低。且随时间的延长各组有增高的趋势。与染毒组相比较,干预组的TLR4转录明显小于染毒组,P<0.01,除24小时无统计学意义。各时间点趋势基本相同。3.2.1.2 TLR4 的表达相对于对照组,各时间段TLR4的表达在染毒组、干预组、环磷酰胺组都明显升高,P<0.05(除72小时,仅环磷酰胺组有统计学差异,干预组和染毒组有升高趋势,无统计学差异),其中以环磷酰胺组最高,染毒组次之,干预组最低。且随时间的延长各组有增高的趋势。与染毒组相比较,干预组的TLR4表达明显小于染毒组,P<0.001(除72小时的无统计学差异)。3.2.2MyD88的转录和表达3.2.2.1 MyD88 的转录相对于对照组,各时间段MyD88的转录在染毒组、干预组、环磷酰胺组都明显升高,其中以环磷酰胺组最高,染毒组次之,干预组最低,P<0.01,除12小时干预组有升高趋势,无统计学意义。与染毒组相比较,干预组的MyD88转录明显小于染毒组,P<0.05。各时间点趋势基本相同。3.2.2.2 MyD88 的表达相对于对照组,各时间段MyD88的表达在染毒组、干预组、环磷酰胺组都明显升高,P<0.05,其中以环磷酰胺组最高,染毒组次之,干预组最低。与染毒组相比较,干预组的MyD88表达小于染毒组,12小时和24小时有统计学差异,P<0.05,72小时无统计学差异。3.2.3 NF-κ B的转录和表达3.2.3.1 NF-κB 的转录相对于对照组,各时间段NF-κB的转录在染毒组、干预组、环磷酰胺组都明显升高,P<0.01,其中以环磷酰胺组最高,染毒组次之,干预组最低。且随时间的延长各组有增高的趋势。与染毒组相比较,干预组的NF-κB转录均明显小于染毒组,P<0.001。3.2.3.2 NF-κ B 的表达相对于对照组,各时间段NF-κB的表达在染毒组、干预组、环磷酰胺组都明显升高,P<0.01,其中以环磷酰胺组最高,染毒组次之,干预组最低。与染毒组相比较,干预组的NF-κB表达明显小于染毒组,P<0.001。3.3环磷酰胺对百草枯中毒介导的NLRP3途径激活的影响3.3.1 NLRP3 的转录相对于对照组,各时间段NLRP3的转录在染毒组、干预组、环磷酰胺组都明显升高,其中以环磷酰胺组最高,染毒组次之,干预组最低,P<0.001。且随时间的延长各组有增高的趋势。与染毒组相比较,干预组的NLRP3转录明显小于染毒组,P<0.001(72小时无显著统计学差异)。3.3.2ASC的转录和表达3.3.2.1 ASC 的转录相对于对照组,各时间段ASC的转录在染毒组、干预组、环磷酰胺组都明显升高,P<0.001,其中以环磷酰胺组最高,染毒组次之,干预组最低。且随时间的延长各组有增高的趋势。与染毒组相比较,干预组的ASC转录均明显小于染毒组,P<0.01。3.3.2.2 ASC 的表达相对于对照组,各时间段ASC的表达在染毒组、干预组、环磷酰胺组都明显升高,P<0.01,其中以环磷酰胺组最高,染毒组次之,干预组最低。与染毒组相比较,干预组的ASC表达明显小于染毒组,P<0.001。3.3.3 Caspase-1的转录和表达3.3.3.1 Caspase1 的转录相对于对照组,各时间段Caspase1的转录在染毒组、干预组、环磷酰胺组都明显升高,P<0.001,其中以环磷酰胺组最高,染毒组次之,干预组最低。且随时间的延长各组有增高的趋势。与染毒组相比较,干预组的Caspase1转录明显小于染毒组,P<0.001。3.3.3.2 Caspase-1 的表达彩图见3.2环磷酰胺对百草枯介导相关炎性通路蛋白表达的影响,WB定量见图 3.3.3.2结果发现:相对于对照组,各时间段Caspase-1的表达在染毒组、干预组、环磷酰胺组都明显升高,P<0.001,其中以环磷酰胺组最高,染毒组次之,干预组最低。与染毒组相比较,干预组的Caspase-1表达均小于染毒组,12小时和24小时无统计学差异,72小时的下降有统计学意义,P<0.05。各时间点趋势基本相同。3.4 环磷酰胺对百草枯中毒介导的炎性通路下游炎性因子(IL-1β和IL-18)的影响3.4.1 I L-1 β血液中的Il-1β相对于对照组,各时间段I1-1β的水平在染毒组明显升高,P<0.01;干预组在12小时和24小时高于对照组,在72小时低于对照组,但均无统计学差异。环磷酰胺组在12小时和24小时低于对照组,但无统计学差异,72小时明显降低,有统计学差异。与染毒组相比较,在各时间段,干预组的I1-1β水平均明显小于染毒组,P<0.01。肺泡灌洗液中的Il-1β相对于对照组,各时间段Il-1β的水平在染毒组明显升高,P<0.01;干预组和对照组无明显差异;环磷酰胺组各时间段Il-1β的水平均明显降低,P<0.05。与染毒组相比较,在各时间段,干预组的I1-1β表达明显小于染毒组,P<0.001。3.4.2 IL-18血清中的IL-18相对于对照组,各时间段11-18的水平在染毒组、干预组明显升高,P<0.01;环磷酰胺组在24小时明显降低,12小时和72小时有升高趋势,无统计学差异与染毒组相比较,在各时间段,干预组的11-18水平均明显小于染毒组,P<0.001。肺泡灌洗液中的IL-18相对于对照组,各时间段11-18的水平在染毒组均明显升高P<0.05;干预组在12小时低于对照组,无统计学差异,在24、72小时干预组明显高于对照组P<0.01;环磷酰胺组均小于对照组,仅12小时的降低有统计学差异P<0.05。与染毒组相比较,在各时间段,干预组的I1-18水平均明显小于染毒组P<0.001。3.5环磷酰胺对百草枯中毒介导的其他细胞因子(TNF-α和TGFβ)的影响TNF-α为了确定环磷酰胺对百草枯中毒介导的TNF-α的影响,我们对血液和肺泡灌洗液中的TNF-α进行了 ELISA检测。血清中的TNF-α相对于对照组,各时间段TNF-α的水平在染毒组明显升高,P<0.05;干预组较对照组有升高趋势,仅72小时有统计学差异,P<0.01,12、24小时无统计学差异,环磷酰胺组在12小时升高趋势,但无统计学差异,24小时和72小基本持平,无统计学差异。与染毒组相比较,在12小时和24小时时间段,干预组的TNF-α水平明显小于染毒组,P<0.01。72小时后,TNF-α水平高于染毒组,无统计学差异。肺泡灌洗液中的TNF-α相对于对照组,各时间段TNF-α的水平在染毒组、干预组明显升高,P<0.01;环磷酰胺组均略低于对照组,无统计学差异。与染毒组相比较,在各时间段,干预组的TNF-α水平均明显小于染毒组,P<0.01。TGFβ血清中的TGFβ相对于对照组,各时间段TGFβ的水平在染毒组、干预组、环磷酰胺组明显升高,P<0.05。其中染毒组最高,干预组次之,环磷酰胺组最低。与染毒组相比较,各时间段,干预组的TGFβ水平均明显小于染毒组,P<0.001。肺泡灌洗液中的TGFβ相对于对照组,各时间段TGFβ的水平在染毒组、干预组明显升高,P<0.001;环磷酰胺组趋势不明显。与染毒组相比较,各时间段,干预组的TGFβ水平明显小于染毒组,P<0.01。3.6环磷酰胺对百草枯中毒介导的中性粒细胞的计数及功能的影响3.6.1中性粒细胞在MPO肺内免疫组化染色及计数免疫组化:深染的MPO阳性的细胞在染毒组、干预组中明显增多,环磷酰胺组相对于其他各组有减少。定量分析:相对于对照组,各时间段MPO阳性的细胞数的比例的水平在染毒组、干预组明显升高,P<0.05;环磷酰胺组在12小时、24小时明显低于对照组,P<0.01,在72小时高于对照组,但无统计学差异。与染毒组相比较,在干预组在各时间段的MPO均明显低于染毒组,P<0.05。3.6.2 ROS相对于对照组,各时间段的ROS在染毒组、干预组、环磷酰胺组都明显升高,P<0.001,其中以环磷酰胺组最高,染毒组次之,干预组最低。与染毒组相比较,干预组各时段的ROS小于染毒组,但仅有72小时有统计学差异P<0.01。3.7肺病理评分大体观察:con组的肺组织大小重量正常,肺组织呈粉红色,弹性良好。环磷酰胺组的肺组织较为苍白,弹性差。部分肺组织出现出血灶。染毒组和干预组的肺组织均出现明显肿胀,重量增加。肺组织色泽较暗,质地较硬,有明显的出血灶,出血的范围随着时间延长有加重趋势。与染毒组相比较,干预组肺组织的充血范围并不均一,存在较为正常的肺组织,残存相对正常的肺组织范围随时间也逐渐缩小。由于时间较短,未观察到明显的纤维化征象。HE染色:con正常;染毒组和干预组表现出不同程度的肺充血,水肿,炎性细胞浸润,纤维化(72小时候明显)。与染毒组相比较干预组的炎症表现相对较轻。环磷酰胺组有出血表现,相对于其他各组,炎性细胞浸润明显减少。肺损伤评分:相对于对照组,各时间段的肺损伤评分在染毒组、干预组、环磷酰胺组都明显升高,P<0.05,(除12小时环磷酰胺组,无统计学差异)其中以染毒组最高,干预组次之,环磷酰胺组最低。与染毒组相比较,干预组的肺损伤评分明显小于染毒组,P<0.05。3.8在环磷酰胺作用下,肺部浸润的中性粒细胞和肺损伤之间相关性性MPO与肺损伤评分成正相关,r=0.969,P<0.01。见图3.8四、结论环磷酰胺预干预后,在百草枯中毒早期(72小时内),可以减少中性粒细胞浸润,减轻肺损伤。72小时段,肺部浸润的中性粒细胞与肺损伤评分成正相关。环磷酰胺并不是治疗百草枯中毒非常理想的药物。相对于对照组,它本身增加ROS、DAMPs的释放及无菌性的炎性通路的激活(以TLR4-MyD88-NF-κB为代表的胞膜途径,以NLRP3-ASC-Caspase1为代表的胞内途径),但并不增加下游的炎性因子IL-1β和IL-18(部分呈下降趋势),不增加TNF-α的分泌,可增加TGFβ1的产生。在环磷酰胺干预后百草枯中毒的大鼠中,相对于染毒组,虽然不减少DAMPs的释放,但可以减轻ROS的释放(72小时有意义),减轻无菌性的炎性通路的激活,以及下游产生炎性因子IL-1β和IL-18。TNF-α、TGFβ1的产生也相应减少。其机制除了减少中性粒细胞外,还可能和环磷酰胺干扰DNA功能,NF-κB无法帮助下游炎症因子翻译顺利进行等机制有关。更多还原

【Abstract】 Part OneImmunohistochemical pathological and literature review of lung tissue A fatal case caused by paraquat poisoning1 IntroductionParaquat poisoning can induce infiltration of inflammatory cells and a significant inflammatory response in the lungs of patients.Immune activation after paraquat poisoning may be one of the important pathogenic mechanisms.However,there are currently no specific studies on which inflammatory cells can infiltrate into lung tissue.However,there are no relevant studies on which inflammatory cells can infiltrate into lung tissue currently.We dissected a case of dead paraquat poisoning patients.The lung tissue was stained with immunohistochemistry of MPO,CD4,CD8,CD21,CD56,CD68,CD79,SMA to determine the type of infiltrating cells.Our study provided a pathological evidence for demonstrating the role of immune activation in paraquat poisoning.2 Method and materialEthics:Sampling and analysis of autopsy was approved by family members and the hospital ethics committee.Medical behavior is in accordance with current Chinese laws and follows the ethics of the Helsinki Declaration.Case report and autopsy:In December 2014,the patient,female,38 years old,was admitted to the hospital because of "oral intake of 100 ml of paraquat for 4 hours".She died after a week of admission.The corpse was kept in a cadaver box with 2-3 ℃ and dissected on the third day after death.Tissue specimens of organs such as heart,lung,kidney are retained.To further clarify the infiltration of immune cells after paraquat poisoning,at 2016-08-12,we sliced the lung tissue wax block again.The lung tissue was performed HE staining and immunohistochemical staining of MPO,CD4,CD8,CD21,CD56,CD68,CD79,SMA.3 Results3.1 HE staining in the lungsTypical histological presentation of this patient involves infiltration of inflammatory cells,diffuse alveolar damage,hyaline membrane formation in alveolar walls and fibrosis.Compared with normal lung tissue,In Fig.1 and Fig.2,there is a disorder and consolidation of lung tissue.Inflammatory cells(mainly neutrophils)are extensively infiltrated into the pulmoriary interstitial and alveolar spaces.In Fig.3 and Fig.4,focal necrosis of the lung tissue was observed,and the normal structure in the necrotic foci disappeared,filling a large number of inflammatory cells(mainly neutrophils),red blood cells,and fibrin deposition In Fig.5,part of the lung tissue showed fibrosis.Fibroblast-like cells with large nucleus and cytoplasm-rich,cellular peripheral fibrosis and inflammatory cell infiltration were seen under the microscope.In Fig.6,alveolar collapse,A large number of alveoli have obvious bleeding.Alveolar filled with pulpy exudate,and contains a lot of cellulose,alveolar epithelial cells shedding and a large number of inflammatory cells infiltration.Significant hyaline membrane formation was seen on the alveolar surface.Pulmonary interstitial edema,thickening of the alveolar wall,and obvious hyperemia and expansion of the alveolar capillaries can also be observed.3.2 NeutrophilsNeutrophils are the most infiltrating inflammatory cells in the lungs.In Fig.8,microscopically,a large number of neutrophils with nucleus deeply stained infiltrated into the pulmonary interstitial and alveolar spacesIn Fig.9,The density of neutrophils adjacent to the blood vessels is significantly increased.Some neutrophils are passing through the vessel wall.3.3 monocytes/macrophagesThe number of monocytes/macrophages infiltrated in the lungs is second to neutrophils.Immunohistochemical Microscopically,a large number of CD68-positive macrophages were distributed in the lung tissue.The macrophages in the hemorrhagic necrosis were significantly increased。The staining of macrophage was uneven.The deep staining was considered to be macrophages that had already phagocytized dust and tissue fragments.The lighter staining consideration was the newly recruited monocytes that had not yet phagocytized the particles.3.4 T helper cellsMarker of immunohistochemistry of T helper cells is generally labeled with antibodies to CD4.In Fig.12,CD4-positive T cells also increased significantly under the microscope,and Th cells deeply stained by the membrane were mostly clustered together in the hemorrhage(Fig.13)and in the pulmonary interstitial(Fig.14).3.5 Cytotoxic T cellThe immunohistochemical CD8 of cytotoxic T cells was positive and increased significantly under the microscope(Fig.15).The cytotoxic T cellsdeeply stained by the membrane were clustered together and distributed in the hemorrhage(Fig.16)and pulmonary interstitial(Figure 17).3.6 Natural killer cellCD56 is a marker molecule for surface expression of NK cells.Microscopically.the number of NK cells was less than the above cells.Deep-stained NK cells were scattered in the pulmonary interstitial(Fig.18,Fig.19).3.7 B cellsCD21 and CD79 are marker molecules on the B cell cell membrane.Microscopically,CD21(Fig.20)and CD79(Fig.21,Fig.22)positive B cell membranes were deeply stained,they were mainly distributed inside the blood vessels and rarely appeared in the pulmonary interstitial.3.8 Fibroblasts/myofibroblastsSMA is the a-actin of smooth muscle,which can be distributed in the cytoplasm of vascular smooth muscle and myofibroblasts.In this patient’s immunohistochemistry,SMA is highly expressed and widely distributed in pulmonary vessels and pulmonary interstitium(Fig.23,Figure 25),We also found high expression of SMA in alveolar capillaries and alveolar walls(Figure 24).In these areas,SMA is usually not expressed.4 ConclusionThrough HE and immunohistocchemical staining,there is a severe inflammatory response in the patient’s lung.Both innate and adaptive immunity are involved in this serious inflammatory response.In innateimmunity,the infiltration of neutrophils and mononuclear macrophages is predominant.In adaptive immunity,Th cells and cytotoxic T cells are significantly increased.The patient’s lung has a severe inflammatory response,both innate and adaptive immunity are involved in this serious inflammatory response..The increase in Th cells and cytotoxic T cells is evident in adaptive immunity.Immunohistochemistry of SMA showed high expression in the lung interstitial cells,alveolar epithelial cells and capillary epithelial cells,which was a tendency to early fibrosis.Part twoExperiment 1:DAMPs,sterile inflammatory pathway and neutrophils involved in paraquat-mediated lung injuryⅠ.IntroductionParaquat poisoning is a thorny toxic disease.Current treatments do not generally improve the mortality.The studys found that the immune system may actively participate in the development and outcome of paraquat poisoning.Our autopsy report and preliminary animal experiment confirmed that innate immunity may play an important role in paraquat poisoning.The immunopathogenic mechanism of paraquat poisoning may be substantially sterile inflammation triggered by DAMPs.However,there is currently no research on the release of DAMPs after paraquat poisoning.Our research on the initiation of the immune response after paraquat poisoning has been innovative.For the above reasons,we propose the following hypothesis in this study:Because a large amount of ROS is produced after paraquat poisoning,the destruction of various cell membrane structures causes cell necrosis and releases a large amount of DAMPs.Sterile inflammatory pathways(the membrane pathway represented by TLR4,the intracellular pathway represented by NLRP3)are activated when DAMPs are combined with PPR.2.MethodThirty-six SD rats were randomly divided into 3 groups:mild group(paraquat 25 mg/Kg intraperitoneal injection),severe group(paraquat 50 mg/Kg intraperitoneal injection)and blank group(normal saline 2 ml intraperitoneal injection),12 in each group.Four rats were taken out at 12.12.72h after intraperitoneal injection of paraquat.After anesthesia,they were sacrificed.The alveolar lavage fluid of the left lung,right lung tissue and blood were collected.Biochemical analyzer detects uric acid(DAMP)in alveolar lavage fluid and serum,alveolar lavage fluid in the left lung and serum cytokines(IL-1β,IL-18,TNF-α,TGFβ1).RNA was extracted from the right upper lobe,and the expression of TLR4,myD88,NF-κB,mtDNA(ND2,DAMP),NLRLP3,ASC,capase-1 and mRNA was detected by qPCR.In order to detect the expression of TLR4,myD88,ASC,capase-1 and NF-κB by Western Blot,proteins in the part of right middle lobe were extracted.Part of the right middle lobe was tested for ROS.Part of the right lower lobe were embedded and sectioned.HE staining was performed for histological lung injury score,(staining + photographing + scoring).Immunohistochemistry was used to detect neutrophils(MPO)and HMGB1(DAMP).Part of the right lower lobe were fixed,embedded and sliced with absolute ethanol.The uric acid was observed by staining Gomori hexamine silver solution.3 Results3.1 Paraquat causes the release of DAMPs.3.1.1 HMGB1Paraquat can cause an increase in HMGB1.Compared with the control group,HMGB1 was significantly increased in different doses at each time point,and increased with time.3.1.2 uric acidParaquat can cause an increase in uric acid.The uric acid in the hexamine silverstaining is brownish black.As the dose and time increase,the area and depth of brownish black are significantly enhanced.In the biochemical test,compared with the control group,the uric acid in the different dose groups at each time point increased significantly,and increased with time.3.1.3mtDNAParaquat can cause an increase in mtDNA.Compared with the control group,the mtDNA of the different dose groups at each time point increased significantly and increased with time.3.2 Activation of the TLR4 pathway3.2.1 Transcription and expression of TLR4Paraquat can result in increased transcription and expression of TLR4.Compared with the control group,the TLR4 of the different dose groups at each time point increased significantly and increased with time.3.2.2 Transcription and expression of MyD88Paraquat can cause an increase in the transcription and expression of MyD88.MyD88 was significantly elevated in the different dose groups at each time point relative to the control group.However,the transcription of MyD88 decreased at 72 hours compared with 24 hours.3.2.3 Transcription and expression of NF-κBParaquat can lead to increased transcription and expression of NF-κB.Compared with the control group,NF-κB was significantly increased in different doses at each time point,and increased with time.3.3 Activation of the NLRP3 pathway3.3.1 Transcription of NLRP3Paraquat can cause transcriptional enhancement of NLRP3.Compared with the control group,NLRP3 in the different dose groups at each time point was significantly increased,and it increased with time.3.3.2 ASC transcription and expressionParaquat can cause increased transcription and expression of ASC.Compared with the control group,the ASC of the different dose groups at each time point increased significantly and increased with time.3.3.3 Caspase-1 transcription and expressionParaquat can lead to increased transcription and expression of Caspase-1.Compared with the control group,Caspase-1 was significantly increased in different doses at different time points,and increased with time.3.4 I L-1β and IL-18 secretionParaquat can cause an increase in the levels of Il-1β and Il-18.Compared with the control group,Il-1β and Il-18 were significantly increased in different doses at different time points(Il-1β only in serum,there was an increasing trend in the mild poisoning group at 12 and 24 hours,no statistical significance)and increased with time.3.5 Secretion of other cytokinesParaquat can cause elevated levels of TNF-α,TGFβ1.Compared with the control group,TNF-α and TGFβ1 were significantly increased in different doses at different time points(only TNF-a in serum,there was no statistically significant increase in the mild group at 24 hours),and increased with time.3.6 Neutrophil count and function3.6.1 Immunohistochemical staining and counting of neutrophils by MPO in lungParaquat can cause an increase in neutrophils in the lungs.Compared with the control group,the number of neutrophils in the different dose groups at each time point increased significantly,and increased with time.3.6.2 ROSParaquat can cause an increase in ROS in the lungs.Compared with the controlgroup,the ROS levels of the different dose groups at each time point were significantly increased,and remained at a high level.There are no significant fluctuations were observed.3.7 Lung pathologyGross observation:The size and weight of the lung tissue in the control group were normal,the lung tissue was pink,and the elasticity was good.The lung tissues inf both the mild and severe groups showed significant swelling and increased weight.The lung tissue is darker in color,harder in texture,and has obvious hemorrhagic foci.The range of bleeding is significantly aggravated with time and dose.Due to the short time,no significant signs of fibrosis were observed.HE staining:con normal;mild and severe groups showed different degrees of pulmonary congestion,edema,inflammatory cell infiltration,fibrosis(obvious at 72 hours).Paraquat can cause an increase in lung damage.Compared with the control group,the lung injury scores of the different dose groups at each time point were significantly increased,and increased with time.3.8 Correlation between neutrophils and lung injury infiltrating the lungThe 72-hour MPO and lung injury scores were analyzed by linear correlation.The results showed that MPO was positively correlated with lung injury score,r=0.908,P<0.01.4 ConclusionIn the early days of paraquat poisoning(within 72 hours),a large amount of DAMPs(HMGB1,uric acid,mtDNA)were released after a large amount of ROS was produced.DAMPs act on PPR on cells and activate a sterile inflammatory pathway(the membrane pathway represented by TLR4-MyD88-NF-κB,the intracellular pathway represented by NLRP3-ASC-Caspasel).Activation of the inflammatory pathway produces the inflammatory factors IL-1β and IL-18,and recruits inflammatory cells dominated by neutrophils to the lungs.Therefore,paraquat mediates sterile inflammation resulting in lung damage.Inflammation caused by paraquat also increases the production of TNF-a and TGFβ1.In the early days of paraquat poisoning,the severity of the inflammatory response(IL-1β,IL-18,TNF-α,TGFβ1,and lung injury scores)and paraquat were somewhat dependent on time and dose.In the early days of paraquat poisoning,the severity of the inflammatory response(IL-1β,IL-18,TNF-α,TGFβ1 and lung injury scores)and paraquat were positively correlated with time and dose to some extent.During the 72-hour period,the number of lung neutrophils was positively correlated with the lung injury score.Part threeExperiment 2:The mechanism of cyclophosphamide on the treatment of paraquat poisoning from the perspective of sterileinflammationⅠ.IntroductionIn Experiment 1,we demonstrated that neutrophils play an important role in paraquat leading to sterile inflammation.The positive feedback effect of DAMPs recruiting neutrophils may be the key to excessive sterile inflammatory responses in paraquat poisoning.Cyclophosphamide is the basic drug for the treatment of paraquat.As early as 1986,E.A DDO reported the use of cyclophosphamide to treat paraquat poisoning.The reason for its treatment is that cyclophosphamide as an immunosuppressive agent can effectively reduce neutrophils and reduce inflammation,and may have a certain therapeutic effect on late fibrosis.Later clinical studies have also found that granulocyte counts in patients with blood routine can be used as prognostic indicators for paraquat poisoning.Whether the mechanism of cyclophosphamide treatment of paraquat poisoning can be elucidated from the standpoint of inhibiting sterile inflammation,currently,there is no relevant research to be available.In our study,We validated the hypothesis proposed by E.A DDO et al.from the perspective of sterile inflammation:Neutrophil deficiency can alleviate the damage caused by paraquat.In this study,rats were pretreated with cyclophosphamide,resulting in an immunosuppressive model of neutropenia.The relationship between cyclophosphamide and sterile inflammation was studied.Differences in inflammatory pathway activation and pulmonary inflammation between the rats in the intervention group and in the exposure group were compared.2.methods and materialsAccording to the results of the preliminary experiment and experiment one,48 SD rats were randomly divided into 4 groups:severely exposed group(paraquat 50 mg/Kg intraperitoneal injection),Intervention group(cyclophosphamide + exposure group,48,24 h before the experiment,100 mg/kg cyclophosphamide intraperitoneal injection,then paraquat 50 mg/Kg intraperitoneal injection),Blank control group(2 ml of saline was injected intraperitoneally),Immunosuppressive group(48,24 h before the experiment,100 mg/kg cyclophosphamide intraperitoneal injection),12 in each group.Four rats were taken out at 12.12.72h after intraperitoneal injection of paraquat.After anesthesia,they were sacrificed.The alveolar lavage fluid of the left lung,right lung tissue and blood were collected.Biochemical analyzer detects uric acid(DAMP)in alveolar lavage fluid and serum,alveolar lavage fluid in the left lung and serum cytokines(IL-1β,IL-18,TNF-α,TGF-β1).RNA was extracted from the right upper lobe,and the expression of TLR4,myD88,NF-κB,mtDNA(ND2,DAMP),NLRLP3,ASC,capase-1 and mRNA was detected by qPCR.In order to detect the expression of TLR4,myD88,ASC,capase-1 and NF-κB by Western Blot,proteins in the part of right middle lobe were extracted.Part of the right middle lobe was tested for ROS.Part of the right lower lobe were embedded and sectioned.HE staining was performed for histological lung injury score,(staining + photographing + scoring).Immunohistochemistry was used to detect neutrophils(MPO)and HMGB1(DAMP).Part of the right lower lobe were fixed,embedded and sliced with absolute ethanol.The uric acid was observed by staining Gomori hexamine silver solution.3.Results3.1.1 HMGB1Immunohistochemical staining:The cells deeply stained with HMGB1 were significantly increased in the exposed group,the intervention group and the cyclophosphamide group,and the increase was most obvious in the exposed group and the intervention group.Quantitative analysis:Compared with the control group,the number of HMBG1-positive cells was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Compared with the exposure group,the intervention group was significantly higher than the exposure group at 24 hours,and the rest did not show significant differences.3.1.2 uric acid Hexamine silver staining in lung tissue:The uric acid with the hexamine silver staining became brownish black.Compared with the control group,the area and depth of the brown-black color in the exposed group,the intervention group and the cyclophosphamide group were significantly enhanced,which was the most obvious in the intervention group.Biochemical analyzerThe performance in serum and alveolar lavage fluid was consistent.Compared with the control group,HMBG1 exposure group,intervention group and cyclophosphamide group were significantly increased,and the intervention group was the most obvious.Compared with the exposed group,the uric acid in the intervention group was significantly higher than that in the exposed group.3.1.3mtDNACompared with the control group,the mtDNA in each time period was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the mtDNA of the intervention group was significantly lower than that of the exposed group.3.2 Effect of cyclophosphamide on the activation of TLR4 pathway mediated by paraquat poisoning3.2.1 Transcription and expression of TLR43.2.1.1 Transcription of TLR4Compared with the control group,the transcription of TLR4 in each time period was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the TLR4 transcription of the intervention group was significantly lower than that of the exposed group(no statistical difference at 24 hours).3.2.1.2 Expression of TLR4Compared with the control group,the expression of TLR4 was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the TLR4 expression in the intervention group was significantly lower than that in the exposed group(no statistical difference at 72 hours).3.2.2 Transcription and expression of MyD883.2.2.1 Transcription of MyD88Compared with the control group,the transcription of MyD88 in each time period was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest(no statistical difference at 12 hours in the intervention group).Compared with the exposed group,the MyD88 transcription of the intervention group was significantly smaller than that of the exposed group.3.2.2.2 Expression of MyD88Compared with the control group,the expression of MyD88 was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.Compared with the exposed group,the expression of MyD88 in the intervention group was lower than that in the exposed group,and there was statistical difference between 12-hours and 24 hours.There was no statistical difference at 72 hours.3.2.3 Transcription and expression of NF-κB3.2.3.1 Transcription of NF-κBCompared with the control group,the transcription of NF-κB in each time period was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the NF-κB transcription of the intervention group was significantly smaller than that of the exposed group.3.2.3.2 Expression of NF-κBCompared with the control group,the expression of NF-κB was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.Compared with the exposed group,the expression of NF-κB in the intervention group was significantly lower than that in the exposed group.3.3 Effect of cyclophosphamide on the activation of NLRP3 pathway mediated by paraquat poisoning3.3.1 Transcription of NLRP3Compared with the control group,the transcription of NLRP3 in each time period was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the NLRP3 transcription in the interventiongroup was significantly smaller than that in the exposed group(no statistically significant difference at 72 hours).3.3.2 ASC transcription and expression3.3.2.1 ASC transcription Compared with the control group,the transcription of ASC in each time periodwas significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the ASC transcription of the interventiongroup was significantly smaller than that of the exposed group.3.3.2.2 Expression of ASCCompared with the control group,the expression of ASC was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.Compared with the exposed group,the ASC expression of the intervention group was significantly smaller than that of the exposed group.3.4 Effect of cyclophosphamide on downstream inflammatory factors(I L-1β and IL-18)mediated by paraquat poisoning 3.4.11 L-1βIl-1β in serumCompared with the control group,the level of Il-1β was significantly increased in the exposed group at each time period;the intervention group was higher than the control group at 12 hours and 24 hours,and was lower than the control group at 72 hours,but there was no statistical difference.The cyclophosphamide group was lower than the control group at 12 hours and 24 hours,but there was no statistical difference,and the 72 hours were significantly lower,with statistical difference.Compared with the exposed group,the Il-1β level of the intervention group was significantly smaller than that of the exposed group at each time period.Il-1β in alveolar lavage fluidCompared with the control group,the level of Il-1β was significantly increased in the exposed group at each time interval;there was no significant difference between the intervention group and the control group;the level of II-1β was significantly decreased in the cyclophosphamide group.Compared with the exposed group,the expression of Il-1β in the intervention group was significantly lower than that in the exposed group.3.4.2 11-1811-18 in serumCompared with the control group,the level of 11-18 was significantly increased in the exposed group and the intervention group at each time period;the cyclophosphamide group was significantly decreased at 24 hours,and there was an increasing trend at 12 hours and 72 hours,no statistical difference.Compared with the exposure group,the Il-18 level of the intervention group was significantly smaller than that of the exposure group at each time period.I L-18 in alveolar lavage fluidCompared with the control group,the level of Il-18 in the time group was significantly higher in the exposed group;the intervention group was lower than the control group at 12 hours,no statistical difference,and the intervention group was significantly higher than the control group at 24 and 72 hours.The cyclophosphamide group was smaller than the control group,and the decrease of only 12 hours was statistically different.Compared with the exposure group,the IL-18 level of the intervention group was significantly smaller than that of the exposure group at each time period.3.5 Effects of cyclophosphamide on other cytokines(TNF-α and TGF-β)mediated by paraquat poisoningTNF-αTNF-α in serumCompared with the control group,the levels of TNF-α were significantly increased in the exposed group at each time period;the intervention group had an increasing trend,and there was a statistical difference in only 72 hours.the cyclophosphamide group increased at 12 hours,but there was no statistical difference,and the 24 hours and 72 hours were basically the same.There was no statistical difference.Compared with the exposed group,the TNF-a level in the intervention group was significantly lower than that in the exposure group at 12 hours and 24 hours.After 72 hours,the level of TNF-a was significantly higher than that of the exposed group.TNF-α in alveolar lavage fluidCompared with the control group,the levels of TNF-a were significantly increased in the exposed group and the intervention group at each time point;the cyclophosphamide group was slightly lower than the control group,and there was no statistical difference.Compared with the exposed group,the TNF-a level of the intervention group was significantly smaller than that of the exposed group at each time period.TGF-βTGF-p in serumCompared with the control group,the level of TGF-β was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group at each time period.Among them,the exposure group was the highest,the intervention group was the second,and the cyclophosphamide group was the lowest.Compared with the exposure group,the TGF-β level of the intervention group was significantly smaller than that of the exposure group at each time period.TGFβ in alveolar lavage fluidCompared with the control group,the level of TGFP was significantly increased in the exposed group and the intervention group at each time period;the trend of the cyclophosphamide group was not obvious.Compared with the exposed group,the TGFP level of the intervention group was significantly lower than that of the exposed group at each time period.3.6 Effect of cyclophosphamide on the counting and function of neutrophils mediated by paraquat poisoning3.6.1 Immunohistochemical staining and counting of neutrophils in MPO lungImmunohistochemistry:Deeply stained MPO-positive cells were significantly increased in the exposed group and the intervention group,and the cyclophosphamide group was reduced compared with the other groups.Quantitative analysis:Compared with the control group,the proportion of the number of MPO-positive cells in each time period was significantly higher in the exposed group and the intervention group;the cyclophosphamide group was significantly lower than the control group at 12 hours and 24 hours,higher than 72 hours.Control group,but no statistical difference.Compared with the exposure group,the intervention group was significantly lower than the exposure group at each time period.3.6.2 ROSCompared with the control group,ROS in each time period was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the infected group was the second,and the intervention group was the lowest.Compared with the exposed group,the ROS of the intervention group was significantly smaller than that of the exposed group.3.7 lung pathology scoreGross observation:The size and weight of the lung tissue in the con group were normal,the lung tissue was pink,and the elasticity was good.The lung tissue in the cyclophosphamide group was pale and poorly elastic.Hemorrhagic foci appear in some lung tissues.The lung tissues in the exposed group and the intervention group showed obvious swelling and increased weight.The lung tissue is darker in color,harder in texture,and hasobvious hemorrhagic foci.The range of bleeding increases with time.Compared with the exposed group,the bleeding range of the lung tissue of the intervention group was not uniform,and there was a relatively normal lxrng tissue.The range of residual normal lung tissue gradually decreased with time.Due to the short time,no significant signs of fibrosis were observed.HE staining:normal was normal;the exposed group and the intervention group showed different degrees of pulmonary congestion,edema,inflammatory cell infiltration,and fibrosis(obvious at 72 hours).The inflammatory performance of the intervention group was relatively light compared with the exposure group.Hemorrhagic manifestations were observed in the cyclophosphamide group,and inflammatory cell infiltration was significantly reduced relative to the other groups.Lung injury score:Compared with the control group,the lung injury scores in each time period were significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the drug-treated group was the highest,the intervention group was the second^ and the cyclophosphamide group was the lowest.Compared with the exposure group,the lung injury score of the intervention group was significantly smaller than that of the exposure group.3.8 Correlation between neutrophils infiltrating the lung and lung injury pretreated with cyclophosphamideMPO was positive更多还原