3-Methyl indole induced pulmonary emphysema in rats

의학과/박사[한글] 인체폐기종의 원인과 발생기전은 폐장의 잠재적인 복잡한 인자 때문에 아직도 해결되지 않고 있다. 그러나 실험동물에 폐기종을 유발시킬 수 있다면 폐기종에 관여할 것으로 추측되는 개개의 요인에 대한 연구분석에 많은 도움이 될 것으로 생각한다. 1965년 Gross 등이 식물성 단백분해효소인 papain으로 실험적 폐기종을 유발시킨 이후 여러종류의 실험동물에서 papain 투여로 초래되는 실험적 폐기종이 보고되고 있으나 (Gross등, 1965; Palecek등, 1967; Goldring등, 1968; Macro등, 1969; Pushpakom등, 1970; Park등, 1970; Mackelem등, 1970; Caldwell, 1970; Johanson등 1971), 폐포관 (alveolar duct) 및 페포확장등의 변화가 papain의 직접적인 elastin분해 작용 때문인지 (Johanson등, 1971) 혹은 papain은 단지 염증세포의 침윤을 자극할 뿐, 이세포로부터 유리되는 단백분해효소에 의하여 조직이 손상되는지 (Lieberman, 1972)는 확실하지 않다. 단백분해효소에 대하여 억제적으로 작용하는 혈청 α^^1-antitrypsin이 결핍된 경우에 인체폐기종이 잘 발생한다는 것 (Eriksson, 1964; Gordon등, 1972)과 인체의 중성백혈구로부터 유래하는 elastase의 작용이 α^^1-antitrypsin으로 억제된다는 보고 (Keuppers 및 Bearn, 1966; Janoff, 1972; Kibelstis 및 Resnick, 1972)들을 종합하면, 탄력섬유를 함유한 폐장조직이 어떤 내적인 단백분해효소에 의하여 파괴도기 때문에 폐기종이 초래된다고 추측할 수 있으며, 이들 단백분해효소는 흑종의 원인 또는 원인물질에 의하여 폐장에 침윤된 염증세포에서 기원할 가능성이 크다. Carlson등 (1968, 1972)은 소와 염소같은 반추동물에 자연발생하는 급성우형폐기종 (acute bovine pulmonary emphysema)이 L-tryptophan의 장관 발효최종산물인 3-methyl indole 및 기타 중성 indole 유도체에 의하여 발생한다고 주장하였고, Nakon-eczna등 (1969)은 관절부에 3-methyl indole을 투여할 때 염증반응이 초래되는 것을 관찰하면서 3-methyl indole은 조직의 지방성분과 친화력이 강하므로 세포의 막성구조에 작용하여 lysosome의 효소가 유리되기 때문이라고 보고한 바 있다. 중성백혈구의 이러한 조직파괴성 효소는 lysosome에서 유리되며 (de Duve 및 Wattiaux, 1966) steroid와 같은 lysosome 안정요소에 의하여 그 유리가 억제될 뿐 아니라 (Thomas 등, 1962; Weissmann 및 Thomas, 1963; Nam, 1971), 확실하게 밝혀지지 않은 어떤 기전에 의하여 steroid는 항염증작용을 가지고 있다 Sandberg, 1964). 그럼에도 불구하고 steroid가 인체폐기종에 미치는 영향에 관하여는 아직도 정설이 없다. 담배의 성분중에는 3-methyl indole이 포함되어 있다. 만성 습관성 흡연자의 경우 폐장에 염증세포가 침윤되는 것을 흔히 볼 수 있으며, 흡연을 하지 않는 사람에 비하여 폐기종 발생율이 높다는 것도 잘 알려져 있다. 몇가지 반추동물에서는 투여방법에 관계없이 3-methyl indole 및 기타 indole 유도체 투여에 의한 실험적 폐기종이 보고되었으나 (Dickinson, 1970; Carlson등, 1972) 기타 동물에서의 실험연구는 아직 보고된 바 없다. 저자는 소수의 백서를 사용한 예비실험에서 3-methyl indole이 염증세포의 침윤을 수반하는 폐기종의 조직소견을 초래함을 관찰하였기에 본 연구에서는 그 세부조직 변화와 폐장의 부피 및 elastin 함량의 변화를 조사하여 하나의 실험 model로서의 폐기종을 제시함과 아울러, 그 발생기전을 추구하고져 소염작용이 있는 cortison acetate를 투여하여 3-methyly indole 투여에 의한 백서폐장의 변화에 미치는 영향을 조사한 바 다음과 같은 결과를 얻었다. 1. 3-Methyl indole 투여로 백서폐장의 단위 무게당 부피는 증가되었으며 elastin량은 현저하게 감소하였다. 2. 3-Methyl indole 투여로 백서폐장은 폐포벽에 중성백혈구와 단해구를 위주로 하는 염증세포 침윤, 폐포관 및 폐포의 확장 폐포격벽(alveolar septae)의 단절 및 소실을 보여주었으며, 전자현미경상 폐포상피는 endoplasmic reticulum의 확장, 세포내부종 및 공포의 출현을 위주로 하는 퇴행성변화를 보였다. 3. 3-Methyl indole 투여에 의한 폐장의 변화들은 cortsone acetate 전처치에 의하여 다소 억제되었다. 이상의 소견을 종합하면 백서는 3-methyl indole 경구투여로 폐포벽과 격벽에 염증세포의 침윤을 수반하는 폐기종이 유발되며, 이러한 소견이 소염작용이 있는 cortisone acetate 전처치에 의하여 다소 억제되는 것으로 미루어 보아, 3-methyl indole 투여에 의한 폐 기종은 폐포벽에 침윤되는 염증세포에서 분리되는 파괴성 효소때문인 것으로 사료되었다. [영문] The significance of individual factors in the etiology of human emphysema is obscured by the diverse nature of potential lung insults. Characterization of animal models of emphysema may help deliniate the importance of these individual factors. Recently, the proteolytic enzyme, papain, has been used to produce a pulmonary lesion which resembles emphysema in a variety of experimental animals (Gross et al., 1965; Palecek et al., 1967; Goldring, et al., 1968; Marco et al., 1969; Pushpakom et al., 1970; Park et al., 1970; Macklem et al., 1970; Caldwell, 1970; Johanson et al., 1971). Whether administered by intreatracheal injection or by aeroslation, papain has been reported to cause dilation of the alveolar ducts and alveloar sacs in association with a minimal inflammatory response. But whether the papain induced emphysems is produced by the direct elastolytic action of papain (johanson et al., 1971), or papain acts only as a stimulus for leukocyte and monocyte accumulation and these then release the damaging proteases (Lieberman, 1972) is still controversial. It is well known that human pulmonary emphysema occurs more frequently when serum apha 1-antitrypsin, the protease inhibitory factor, is deficient than when it is sufficient (Eriksson, 1964; Gordon et al., 1972). Alpha 1-antitrypsin is thought to inhibit the human granulocyte elastase (Keuppers and Bearn, 1966; Janoff, 1972; Kibelstis and Resnick, 1972). It has been suggested that human emphysema could be caused by an endogenous proteloytic destruction of pulmonary tissue including elastic fibers and that the proteolytic enzyme is released from inflammatory cells accumulated within the lung due to the action of some stimulating agents. Carlson et al. (1968, 1972) observed that an acute bovine pulmonary emphysema could be caused by 3-methyl indole and other neutral indole derivatives which were ruminal fermentation products of L-tryptophan. Nakoneczna et al. (1969) reported that 3-methyl indole administration resulted in an inflammatory stimulation, which, he thought, was due to the lipophilic activity of this substance interacting with the cellular membrane structure and releasing lysosomal contents. 3-Methyl indole is known to be one of the components of cigarette smoke. Chronic human pulmonary emphysema with an accompanying inflammatory response is obseved with increased frequency in chronic cigarette smokers (Wydner and Hoffmann, 1967; Carlson et al., 1972). 3-Methyl indole induced pulmonary lesions have been reported in some ruminal animals (Dickinson, 1970; Carlson et al., 1972), but was not in other animal species. It was therefore the aim of this study to ascertain whether or not 3-methyl indole administration could induce pulmonary emphysema in rats, and to research the effect of cortisone acetate pretreatment on 3-methyl indole induced pulmonary changes, for some information about the pathogenic mechani sm of the latter. Materials and Methods Male albino rats weighing around 200gm were used for the experiment and divided into three groups and treated as follows; Grou Ⅰ: Control A. Normal control (12 rats) B. Cortisone acetate treated (12 rats) Group Ⅱ: 3-Methyl indole treated (36 rats) Group Ⅲ: Cortisone acetate and 3-methyl indole treated (36 rats) 3-Methyl indole was administered orally in a single dose of 15mg dissolved in 1cc of 50% propylene glycol per 100gm of body weight, and cortisone acetate givenintramuscularly in a dose of 5mg per 100gm of body weight. In group Ⅲ, animals were pretreated with cortisone acetate for from five days prior to 3-methyl indole administration until the day before sacrifice. Control and experimental animals were sacrificed serially after the treatment in periods ranging 6 hours to 7 days. The animals were anesthetized with intraperitoneal secobarbital, the external iliac vein was exposed and then 3cc of 4% glutaraldehyde was infused in an attempt to fix the lungs vitally. In half of the control and experimental animals, the right lungs were dissected free from the attached tissues. After measuring the weight the volume was determined by water displacement. The dry weight of lung elastin was measured using the technique of Lowry et al. (1941). In brief, this procedure is gravimetric and depends upon the insoluble nature of fibrous protein when it is placed in a tube with 0.1N NaOH and placed in a boiling water bath. The remaining half of the ainmals were used for morphological examination. For light microscopic examination, the lungs were fixed in 5% neutral formalin followed by paraffin embedding and then stained with hematoxylin-eosin. In addition, Gomori's aldehyde fuchsin and Weigert's reticulum stains were also applied to examine the elastic fiber and reticulin mesh work of the lung respectively. For electron microscopic study, the lung was cut in 1mm**3 in size and fixed in 1% osmium tetraoxide in phosphate buffer pH7.4, and embedded in Epon 812. The sections were cut with glass knife in 400 to 500 A thickness and stained with uranyl acetate and lead hydroxide. Observations are made using a Hitachi HU-11E model electron microscope. Results and Summary The changes of rat lung produced by oral administration of 3-methyl indole were studied. The effects of cortisone acetate on 3-methyl indole induced pulmonary changes were also observed and interpreted. The results are summarized as follows: 1. With 3-methyl indole administration, volume per weight of the lung was greatly increased and the elastin content of the lung was significantly decreased. 2. Infiltration of the inflammatory cells, predominantly neutrophils and monocytes, in alveolar walls and septae was present in the rats given oral doses of 3-methyl indole. In addition, disruption of the alveolar epithelial cells, dilatation of the alveolar ducts and alveolar sacs, and shortening or loss of alveolar septae were noted. The important alterations of the alveolar epithelial cells observed under the electron microscope were distension, or rupture of endoplasmic reticulum, the presence of cytoplasmic vesicles and of intracellular edema. 3. All of the 3-methyl indole induced pulmonray changes were inhibited somewhat but not completely, by the treatment with cortisone acetate. In summary, the oral administration of 3-methyl indole induced pulmonary emphysema with infiltration of inflammatory cells in alveolar walls and septae in rats. Because these changes were inhibited when the rats treated with the anti-inflammatory agent, cortisone acetate, the 3-methyl indole induced emphysema is thought to be produced by a damaging proteolytic enzyme which is released from the early infiltrating inflammatory cells in the alveolar walls and septae.restrictio