大鼠肢体缺血再灌注后肺组织BAX基因表达上调

目的探讨在体大鼠肢体缺血再灌注(LIR)后肺组织BAX和BCL-2基因表达的变化。方法在大鼠肢体缺血再灌注(LIR)损伤动物模型上,用TUNEL法、电泳法及免疫组织化学等技术观察LIR后肺损伤发生过程中,肺组织细胞凋亡变化以及BAX和BCL-2蛋白质表达的改变。结果大鼠LIR后,肺血管内皮细胞及附壁的炎细胞凋亡明显增加;肺组织BCL-2表达的变化不大,但BAX蛋白质表达明显上调,DNA断链率升高,活性氧(ROS)含量增加,且与肺组织细胞凋亡的增加相一致。结论肺组织细胞凋亡以及BAX和BCL-2表达的变化可能参与LIR后肺损伤的发生。     

大鼠肢体缺血再灌注后肺组织一氧化氮合酶的变化

目的:研究正常大鼠肺组织内一氧化氮合酶(NOS)的分布及肢体缺血再灌注(LIR)后肺组织内NOS分布及活性的变化。方法:用止血带复制肢体缺血再灌注模型,利用β-NADPH-d组织化学方法、计算机图像分析系统及分光光度法,观察对照组大鼠肺内NOS的分布及LIR组肺内NOS分布及活性的变化。结果:组织学上显示,对照组大鼠呼吸道包括支气管、细支气管、终末细支气管、肺泡管的上皮细胞和血管内皮细胞NOS表达均阳性,肺泡上皮细胞NOS表达阴性;LIR组上述肺组织阳性部位NOS表达增强,且出现血管平滑肌细胞、肺泡上皮细胞NOS表达阳性;生化测定结果显示,LIR组与对照组比较,NOS活性增强,NO₂^-/NO₃^-水平增多。结论:一氧化氮不仅参与肺的生理过程,而且在LIR后急性肺损伤(ALI)病理生理过程中可能发挥重要作用。     

内质网应激诱导的细胞凋亡在大鼠肢体缺血再灌注后肺损伤中的作用及牛磺酸的影响

目的: 探讨内质网应激诱导的细胞凋亡在大鼠肢体缺血再灌注(LIR)后肺损伤中的作用及牛磺酸的影响。方法: 成年雄性SD大鼠40只,随机分成4组:(1)对照组(control);(2)LIR组;(3)牛磺酸处理组(LIR+taurine);(4)生理盐水处理组(LIR+saline)。采用生物化学方法检测血气变化及肺组织超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和丙二醛(MDA)含量;采用原位末端标记(TUNEL)法观察肺组织细胞凋亡情况;采用蛋白质印记方法(Western blotting)及实时定量PCR(real-time PCR)方法观察LIR后肺损伤发生过程中CCAAT增强子结合蛋白(C/EBP)同源蛋白(CHOP)及活化的转录因子4(ATF4)和X-盒结合蛋白-1(XBP1)基因表达的改变;光镜下观察肺脏组织的形态学改变。结果: 与对照组相比,LIR组的动脉血氧分压(PaO₂)和动脉血二氧化碳分压(PaCO₂)明显下降,肺组织中MDA水平明显升高,SOD和CAT活性明显降低,细胞凋亡数增多,CHOP蛋白表达上调,XBP-1、ATF4和CHOP mRNA表达水平明显上调;而牛磺酸能明显减轻LIR后肺损伤;肺呼吸功能明显改善,肺组织MDA的含量减少,SOD和CAT活性增加,细胞凋亡明显减少,CHOP蛋白表达下调,ATF4、XBP1和CHOP基因表达下调。结论: 内质网应激诱导的细胞凋亡参与LIR后肺损伤,牛磺酸对LIR后肺组织的保护作用与其减轻内质网应激诱导的细胞凋亡有关。     

牛磺酸对大鼠肢体缺血再灌注后肺损伤时黏附分子ICAM-1表达的影响

目的：探讨牛磺酸对大鼠肢体缺血再灌注致肺损伤时黏附分子ICAM-1表达的影响。方法：复制大鼠肢体缺血再灌注(LIR)损伤模型,采用流式细胞技术、逆转录聚合酶链反应（RT-PCR）和Western blotting观察LIR后肺损伤发生过程中，血液中CD18阳性的多形核白细胞（PMN）百分率、肺系数（LI）与肺通透指数（LPI）、肺组织形态学的改变、肺组织ICAM-1 在mRNA和蛋白水平的变化及牛磺酸对上述指标的影响。结果：大鼠肢体缺血再灌注后LI、LPI及CD18阳性细胞数均明显增加，肺组织髓过氧化物酶（MPO）活性增加，ICAM-1表达明显升高，而提前给予牛磺酸可以明显抑制这些变化，从而对肺起一定保护作用。结论： 肺组织细胞ICAM-1表达上调及血液中CD18阳性细胞数的增加可能参与LIR后肺损伤的发生；牛磺酸可减少血液中CD18阳性细胞数，并且下调肺组织ICAM-1的表达，从而减轻肺损伤。     

肺缺血再灌注损伤中Fas/FasL系统的表达及川芎嗪的影响

目的： 探讨肺缺血再灌注损伤Fas/FasL系统的表达及其与肺泡上皮细胞凋亡的关系及川芎嗪的影响。方法: 采用在体兔单侧肺左肺门持续性阻断1 h，再灌注1、3、5 h缺血-再灌注损伤的动物模型。TMP干预组于缺血前1h静脉滴注川芎嗪60 mg/kg。用原位末端标记法(TUNEL)检测细胞凋亡的发生情况；用原位杂交的方法检测兔肺组织Fas/FasL mRNA的表达。结果: IR组与 TMP组在肺缺血再灌注后1、3、5 h发生明显的肺泡上皮细胞凋亡。TMP组各时相细胞凋亡指数明显低于IR组（P<0.01）；肺组织Fas/FasL mRNA的表达与肺泡上皮细胞凋亡呈显著正相关（r₁=0.900,r₂=0.869,均P<0.01）。 结论: Fas/FasL系统在肺缺血再灌注诱导的肺泡细胞凋亡中起着重要作用，川芎嗪因抑制Fas/FasL，而减轻由Fas/FasL系统激活导致的细胞凋亡，从而对缺血再灌注肺具有保护作用。     

肢体缺血再灌注后的肺损伤和细胞凋亡及NO的效应

目的：探讨肢体缺血再灌注（LIR）后肺的损伤性变化以及细胞凋亡在肺损伤发生中的作用；探讨一氧化氮（NO）对LIR后肺组织细胞凋亡的影响。 方法：采用本室常规方法复制大鼠LIR模型，给予外源性一氧化氮合酶底物（L-Arg）和一氧化氮合酶抑制剂(L-NAME)处理，采用原位末端标记法（TUNEL）检测缺血4 h再灌注4 h时各组动物肺组织细胞凋亡情况；采用放免法检测凋亡相关细胞因子TNF-α在肺组织的表达，结合计算机分析系统对结果进行定量分析；采用免疫组织化学方法检测Bcl-2、Bax、caspase-3、TNF-α蛋白表达情况，结合自动图像分析系统对其结果进行定量分析；在光镜下观察肺组织的形态学改变。结果：大鼠LIR后4 h，肺泡Ⅱ型上皮细胞、肺血管内皮细胞呈凋亡改变,肺组织TNF-α、caspase-3、Bax明显上调,Bcl-2表达下调。L-Arg处理组，凋亡细胞数明显减少，肺组织TNF-α、caspase-3、Bax的表达情况与IR组相比明显减弱，Bcl-2表达明显增强；L-NAME处理组动物肺组织TNF-α、caspase-3、Bax的表达情况与IR组相比明显增强，Bcl-2表达明显减弱。结论：细胞凋亡参与了大鼠LIR后急性肺损伤的发生，且与TNF-α有关；NO可通过减弱细胞凋亡相关因子TNF-α的表达，减轻LIR后肺组织的细胞凋亡。     

一氧化氮减轻大鼠肢体缺血再灌注后肺损伤

肢体缺血再灌注损伤(limb ischemia reperfusion injury,IJRI)是临床上常见的一种损伤现象,已有研究表明,严重的肢体缺血再灌注(limb ischemia reperfusion,LIR)可致急性肺损伤(acute lung injury,ALI).后者的病理变化主要为肺血管内皮和肺泡上皮广泛受损[1],但这类靶细胞受损的确切机制和损伤方式迄今仍不十分清楚.     

肢体缺血再灌注后肺损伤小鼠肺组织AT1R和Mas受体蛋白表达变化

目的:通过观察小鼠肢体缺血再灌注(LIR)后不同时点肺组织血管紧张素Ⅱ1型受体(AT1R)和Mas受体蛋白表达与肺损伤的变化,探讨局部组织AT1R和Mas受体蛋白表达失衡在LIR急性肺损伤(ALI)中的作用。方法:42只8周龄雄性ICR小鼠随机分为7组,每组6只,其中1组作为对照组,其余6组为再灌注0.5 h、1h、2 h、4 h、6 h和12 h模型组。模型组小鼠用橡皮圈结扎双后肢根部,缺血2 h后剪断橡皮圈,分别于再灌注后不同时点眼球取血处死小鼠。取肺组织计算脏器系数和湿/干重比;肺泡灌洗液细胞计数和蛋白浓度检测;肺组织病理切片常规HE染色观察肺组织形态变化并进行病理损伤评分;Western blot检测肺组织AT1R和Mas受体蛋白的表达。结果:模型组小鼠肺脏器系数、湿/干重比、肺泡灌洗液细胞计数和蛋白浓度在LIR后显著升高。病理学结果显示,LIR后不同时点小鼠肺组织出现肺泡壁毛细血管扩张和充血、间质和肺泡水肿、血管壁和支气管壁炎症细胞浸润、肺泡间隔增厚、炎症细胞浸润及肺气肿等不同程度的损伤变化,且随着再灌注时间的延长,肺损伤评分逐渐升高。Western blot结果显示,AT1R蛋白在再灌注0.5 h时开始升高,1 h达到最高,之后随再灌注时间的延长,AT1R表达逐渐降低;Mas受体蛋白随再灌注时间延长逐渐升高。结论:LIR引起急性肺损伤,并随再灌注时间的延长损伤逐渐加重;AT1R和Mas受体蛋白表达的变化可能与小鼠LIR后急性肺损伤有关。     

三七总皂甙对肺缺血再灌注损伤时细胞凋亡及Fas/FasL的影响

目的：探讨细胞凋亡与肺缺血再灌注损伤的关系以及三七总皂甙的作用及机制。 方法: 健康日本大耳白兔84只，随机分为对照组、肺缺血再灌注1、3、5 h组和三七总皂甙干预1、3和5 h组。复制肺缺血再灌注损伤模型。采用原位缺口末端标记（TUNEL）法观测肺组织细胞凋亡指数，免疫组化和原位杂交技术检测肺组织细胞Fas/FasL系统蛋白和基因表达的变化。 结果: 肺缺血再灌注组肺组织细胞凋亡指数和Fas/FasL蛋白及基因表达均显著高于对照组（均P<0.01）。三七总皂甙干预组Fas/FasL mRNA及其蛋白质的表达显著低于缺血再灌注组（P<0.01），肺组织细胞凋亡指数也显著低于缺血再灌注组（P<0.01）。肺组织细胞凋亡指数分别与Fas/FasL蛋白和Fas/FasL mRNA之间均呈显著正相关（r分别=0.540,0.658,0.668,0.686;均P<0.01）。 结论: Fas/FasL系统活化启动的肺组织细胞凋亡可能参与了肺缺血再灌注损伤的发生。三七总皂甙可能通过抑制Fas/FasL系统的激活，阻遏肺组织细胞凋亡，从而减轻肺缺血再灌注损伤。     

葛根素对肺缺血-再灌注损伤时Fas/FasL表达的影响

目的：探讨葛根素对肺缺血-再灌注损伤（PIRI）时Fas/FasL表达的影响。方法：采用在体兔单肺原位缺血-再灌注模型。实验兔70只，随机分为假手术对照组（sham，10只）、肺缺血-再灌注组（I-R，30只）和肺缺血-再灌注加葛根素组（Pur，30只）。每组又分为再灌注1 h、3 h、5 h 3个亚组，每个亚 组10只，分别于再灌注 1 h、3 h、5 h 3个时点取左肺组织，观察Fas/Fas配体（Fas/FasL）mRNA定位表达、凋亡指数（AI）、肺组织湿干重比（W/D）、肺损伤组织学定量评价指标（IQA）及光镜、电镜下的组织形态学改变。结果：肺再灌注1 h、3 h、5 h，Pur组Fas/FasL mRNA在肺小动脉内（外）膜、肺小静脉内膜、肺泡上皮及肺支气管上皮呈弱阳性表达，明显低于同一时点I-R组（P<0.05）；AI、W/D和IQA值显著低于I-R组（P<0.01和P<0.05）；肺组织形态学异常改变不同程度减轻。结论：葛根素可下调肺组织Fas/FasL mRNA的表达而减轻细胞凋亡，对PIRI发挥积极的防治作用。     

Fas and fas ligand are up-regulated in pulmonary edema fluid and lung tissue of patients with acute lung injury and the acute respiratory distress syndrome

Apoptosis mediated by Fas/Fas ligand (FasL) interaction has been implicated in human disease processes, including pulmonary disorders. However, the role of the Fas/FasL system in acute lung injury (ALI) and in the acute respiratory distress syndrome (ARDS) is poorly defined. Accordingly, we investigated both the soluble and cellular expression of the Fas/FasL system in patients with ALI or ARDS. The major findings are summarized as follows. First, the soluble expression of the Fas/FasL system was assessed in undiluted pulmonary edema fluid and simultaneous plasma. Pulmonary edema fluid obtained from patients with ALI or ARDS (n = 51) had significantly higher concentrations of both soluble Fas (27 ng/ml; median; P < 0.05) and soluble FasL (0.125 ng/ml; P < 0.05) compared to control patients with hydrostatic pulmonary edema (n = 40; soluble Fas, 12 ng/ml; soluble FasL, 0.080 ng/ml). In addition, the concentrations of both soluble Fas and soluble FasL were significantly higher in the pulmonary edema fluid of the patients with ALI or ARDS compared to simultaneous plasma samples (soluble Fas, 16 ng/ml; soluble FasL, 0.058 ng/ml; P < 0.05), indicating local release in the lung. Higher soluble Fas concentrations were associated with worse clinical outcomes. Second, cellular expression of the Fas/FasL system was assessed by semiquantitative immunofluorescence microscopy in lung tissue obtained at autopsy from a different set of patients. Both Fas and FasL were immunolocalized to a greater extent in the patients who died with ALI or ARDS (n = 10) than in the patients who died without pulmonary disease (n = 10). Both proteins were co-expressed by epithelial cells that lined the alveolar walls, as well as by inflammatory cells and sloughed epithelial cells that were located in the air spaces. Semiquantitative immunohistochemistry showed that markers of apoptosis (terminal dUTP nick-end labeling, caspase-3, Bax, and p53) were more prevalent in alveolar wall cells from the patients who died with ALI or ARDS compared to the patients who died without pulmonary disease. These findings indicate that alveolar epithelial injury in humans with ALI or ARDS is in part associated with local up-regulation of the Fas/FasL system and activation of the apoptotic cascade in the epithelial cells that line the alveolar air spaces.     

三七总皂甙降低兔肺缺血再灌注损伤和抑制细胞凋亡

 目的: 探讨细胞凋亡与肺缺血再灌注损伤的关系以及三七总皂甙的作用及机制。方法: 健康日本大耳白兔84只,随机分为对照组、肺缺血再灌注1、3、5h组和相应三七总皂甙干预组。复制肺缺血再灌注损伤模型。用原位缺口末端标记(TUNEL)法、聚丙烯酰胺凝胶电泳观测肺组织细胞凋亡,原位杂交技术检测肺组织细胞Fas/FasL系统和Caspase-3基因表达。结果: 肺缺血再灌注组肺组织细胞凋亡指数(肺缺血再灌注5h组:22.08±1.93;对照组:2.04±0.67)、Fas/FasL和Caspase-3基因表达(肺缺血再灌注5h组:0.241±0.029;对照组:0.121±0.015)均显著高于对照组(P<0.01),并出现电泳梯形条带结构;三七总皂甙干预组Fas/FasL mRNA及其Caspase-3的表达(三七总皂甙干预5h组:0.199±0.020;肺缺血再灌注5h组:0.241±0.029)显著低于缺血再灌注组(P<0.01),肺组织细胞凋亡指数(三七总皂甙干预5h组:12.58±1.82;肺缺血再灌注5h组:22.08±1.93)也显著低于缺血再灌注组(P<0.01),梯形条带结构基本消失。肺组织细胞凋亡指数分别与Caspase-3 mRNA及Fas/FasL mRNA之间均呈显著正相关(P<0.01)。结论:三七总皂甙可能通过抑制Fas/FasL系统的激活,阻遏肺组织细胞凋亡,从而减轻肺缺血再灌注损伤。     

脂多糖对吸烟大鼠肺泡巨噬细胞增殖细胞核抗原及肺泡Ⅱ型上皮细胞Fas/FasL系统表达的影响

目的:研究脂多糖(LPS)对吸烟大鼠肺泡巨噬细胞(AM)增殖细胞核抗原(PCNA)及肺泡Ⅱ型上皮细胞Fas/FasL系统表达的作用。方法:应用免疫组织化学SABC法和免疫荧光标记技术,检测不同时期LPS对吸烟大鼠肺泡巨噬细胞PCNA表达和肺泡Ⅱ型上皮细胞Fas/FasL系统表达的影响。结果:吸烟大鼠AM上PCNA表达在第3、4月达高峰,LPS刺激的各组PCNA表达明显高于不加LPS组(P＜0.01);肺泡Ⅱ型上皮细胞Fas/FasL系统表达在加入LPS组表达显著高于未加LPS组(P＜0.01),且与AM上PCNA表达的变化相平行。结论:吸烟引起气道AM增殖速率加快,AM在肺泡Ⅱ型上皮细胞损伤与修复过程中起重要作用。     

Seawater induces apoptosis in alveolar epithelial cells via the Fas/FasL-mediated pathway

Our previous study showed that seawater can cause lung tissue cell apoptosis; in the present study, the immunohistochemistry and Western blot analysis results demonstrated that Fas, FasL, and cleaved caspase-8 and caspase-3 were up-regulated in the rat lungs exposed to seawater. We found that seawater-induced human lung alveolar epithelial A549 cell apoptosis was concentration and time dependent. Moreover, seawater increased the expression of Fas, FasL, and cleaved caspase-8 and caspase-3 in A549 cells. The incubation of A549 cells in the presence of FasL-neutralising antibody (NOK-2) or caspase-8 inhibitor (Z-IETD-FMK) resulted in a decrease of seawater-induced cell apoptosis. NOK-2 inhibited Fas/FasL interaction and reduced the cleavage of caspase-8 and caspase-3, and Z-IETD-FMK blocked caspase-8 and caspase-3 activation. Seawater similarly produced a significant increase in rat alveolar type II cell apoptosis and expression of Fas and cleaved caspase-8. In summary, the Fas/FasL pathway involved in alveolar epithelial cell (AEC) apoptosis could be important in the pathogenesis of seawater-induced acute lung injury (SW-ALI).     

The role of apoptosis in the pathophysiology of Acute Respiratory Distress Syndrome (ARDS): An up-to-date cell-specific review

ARDS pathophysiology is characterized by complex mechanisms that involve cells of inflammation, lung tissue cells, cytokines, chemokines, as well as apoptosis activators and inhibitors. There are two important theories that link apoptosis with ARDS and suggest that epithelial cell apoptosis, as well as the accumulation of neutrophils in the lung, may contribute to a cascade of events and, finally, ARDS. The activation of the Fas/FasL pathway is an important mechanism of alveolar epithelial injury in the lungs of patients with ALI. In addition, neutrophilic inflammation in the alveolar spaces is characteristic of ALI in humans and in most animal models of ALI. The enhanced phagocytosis of apoptotic neutrophils could lead to resolution of inflammation and repair during ARDS. In this review, we will focus on elucidating the role of apoptosis in the pathophysiology of ARDS and the contribution of Fas-mediated inflammation in ARDS. Furthermore, we will give evidence that TNF-alpha, IL-1beta and IL-13 attenuate the pro-cell death effects of Fas/CD95 on A549 epithelial cells, at least partially, by the NF-kB and PI3-K pathways, suggesting that induction of the expression of antiapoptotic genes protects the epithelial cells from cell death.     

Dual role of TGF-β1 on Fas-induced apoptosis in lung epithelial cells

Recent evidence suggests that TGF-β1 has a dual role in regulating cell response to Fas/Fas ligand (FasL)-induced apoptosis. TGF-β1 may play a positive or negative role on cell sensitivity to apoptosis via Fas/FasL system, depending on cell types and their specific environment. TGF-β1 and the Fas/FasL system are also involved in pathological processes of acute lung injury (ALI) and interstitial lung diseases including early lung injury and subsequent tissue repair. However, it is not well understood how TGF-β1 regulates Fas/FasL mediated apoptotic signaling in lung epithelium. In this study, we found that TGF-β1 could affect the sensitivity of lung epithelial A549 cells to Fas/FasL mediated apoptosis in a time-dependent manner. Apoptosis of A549 cells could be enhanced significantly by co-treatment with TGF-β1 and FasL, or pretreatment with TGF-β1 followed by FasL exposure, as evidenced by markedly increased caspase-8 and JNK activities. However, prolonged exposure to TGF-β1 could result in an obvious inhibition of the Fas/FasL-induced apoptosis, accompanied by down-regulation of Fas and up-regulation of c-Flip. Our results also showed that the effect of TGF-β1 on cell sensitivity to Fas-mediated apoptosis was independent of Akt pathway activation. These findings suggest that timely interplay of TGF-β1 and the Fas/FasL system could determine the final outcomes of cell survival/death signaling, for example, switching cell death signaling to survival signaling during early injury and later repair process of lung epithelium.     

NF-κB在大鼠急性肺损伤模型肺组织中的表达及N-乙酰半胱氨酸的影响

目的 :检测NF κB在LPS诱导的急性肺损伤 (ALI)肺组织中的表达 ,以及N 乙酰半胱氨酸 (NAC)对ALI的抑制作用。方法 :采用免疫组化染色 (ABC法 )和Westernblot,检测NF κB在急性肺损伤大鼠气道和肺组织中的表达 ,以及NAC干预后活性NF κB表达的变化。结果 :正常对照组大鼠气道黏膜上皮和肺间质中 ,仅见少量散在的NF κB核阳性细胞 ;而LPS诱导ALI后 ,气道黏膜、肺间质、肺泡腔及血管内皮细胞中NF κB核阳性的细胞明显增多 (P <0 .0 1)。NF κB核阳性反应细胞主要为气道黏膜上皮细胞、浸润的炎症细胞、肺泡上皮细胞和血管内皮细胞。NAC治疗组NF κB核阳性细胞较LPS诱导的ALI组及对照组均明显减少 (P <0 .0 1)。Westernblot的结果显示 ,LPS诱导的ALI后不同时间点 ,NF κB的表达不同 ,于急性肺损伤 3h达高峰。各时间点NF κB的表达均较正常对照组高。结论 :LPS诱发的大鼠急性肺损伤的气道和肺组织内NF κB的表达增加 ,肺组织内的多数细胞参与了NF κB的激活。NAC可通过抑制NF κB的激活减轻急性肺损伤的炎症程度     

Induction of interleukin-8 secretion and apoptosis in bronchiolar epithelial cells by Fas ligation.

Epithelial cell injury is the common manifestation of lung injury. Contributing to such injury of epithelial cells is apoptosis. Although apoptosis is part of the normal process of epithelial renewal, in excess it is pathologic. We previously demonstrated the excessive apoptosis of lung epithelial cells and the upregulation of Fas and Fas ligand (FasL) in fibrosing lung diseases. We also showed that inhalation of anti-Fas antibody induced lung injury and fibrosis in mice. Interleukin (IL)-8 is one of the most important cytokines in the pathophysiology of acute lung injury and pulmonary fibrosis. In this study we investigated whether Fas ligation induces IL-8 secretion in addition to apoptosis in bronchiolar epithelial cells in vitro. Bronchiolar epithelial cells underwent apoptosis and also secreted IL-8 in response to tumor necrosis factor (TNF)-alpha or Fas ligation. New gene expression and protein synthesis were not necessary for Fas ligation- and TNF-alpha- mediated apoptosis, but were necessary for IL-8 secretion. We further found that Fas ligation induced activation of nuclear factor-kappa B. We conclude that the Fas/FasL pathway not only mediates apoptosis but also plays a proinflammatory role, and that stimulation of the Fas/FasL pathway in bronchiolar epithelial cells leads to IL-8 production, which may amplify the inflammatory cascade in lung injury and pulmonary fibrosis.