Anat Cell Biol.  2021 Dec;54(4):448-464. 10.5115/acb.21.068.

Role of nuclear factor-kappa B in bleomycin induced pulmonary fibrosis and the probable alleviating role of ginsenoside: histological, immunohistochemical, and biochemical study

• El-Bassouny DR
• Omar NM
• Khalaf HA
• Al-Salam RAA

• KMID: 2523576
• DOI: http://doi.org/10.5115/acb.21.068

Abstract

Bleomycin (BLM) is one of anti-cancerous drugs. One of its limitation is the development of pulmonary fibrosis during therapy So, we proposed to examine the outcome of BLM take on the light and electron microscopic design of rat lung. Along with, assessment the probable protecting role of ginsenoside on BLM induced pulmonary changes. In this study, thirty adult male albino rats were comprised and were classified to four clusters; Negative & positive control group, BLM treated group and BLM& ginsenoside treated group. The lung was treated for histological and immunohistochemical (anti-p65) studies. Light microscopic examination of H&E stained sections of BLM treated group showed huge distortion of the lung building. Mallory trichrome stain of this group showed evident deposition of collagen fibers in the markedly thickened interalveolar septa and around intrapulmonary bronchi, bronchioles and blood vessels. Moreover, strong positive staining for nuclear factor (NF)-κB in the wall of bronchiole as well as the thickened interalveolar septa were observed. Ultrastructural inspection of lung of this group revealed muddled lung planning. Marked improvement of the lung structure and marked reduction in NF-κB immunoexpression was appeared in BLM and ginsenoside treated group. So, we concluded that coadministration of ginsenoside with BLM significantly enhanced the histological and morphometric image of the lung.

Keyword

Bleomycin; Ginsenosides; Pulmonary fibrosis; Nuclear factor-kappa B

Figure

• 

  Fig. 1 Figure showed photos of H&E-stained lung sections. (A, B) Control sections: (A) The lung section shows alveoli (A), alveolar sacs (AS), bronchioles (B) with folded mucosa, blood vessels (V), thin (arrows) and thick (arrow heads) portions of the interalveolar septum. (B) The intrapulmonary bronchus is formed of mucosa (M) lined by pseudostratified columnar ciliated epithelium with goblet cells (Ep), is surrounded by smooth muscle layer (SM) and hyaline cartilage (HC) with normal chondrocytes (thick arrow) in the adventitia (Ad). (C, D) Bleomycin (BLM) treated sections: (C) Collapsed alveoli (A) is seen with emphysematous ones (E). It separated by thick interalveolar septa (arrows) with mononuclear cellular infiltration (asterisks). The bronchioles (B) lining epithelium (zigzag arrow) is distorted with the appearance of intraluminal cellular debris (thick arrow), its smooth muscle layer (SM) is thickened and focally interrupted (arrowhead) by massive peribronchiolar lymphocytic infiltration (LI). Note, the presence of congested, thickened and dilated blood vessels (V). (D) Massive distortion and stratification (arrows) of the lining epithelium of the intrapulmonary bronchi with intraluminal cellular debris (curved arrows). Mononuclear cellular infiltration (asterisks) in the lamina propria (LP). Hyaline cartilage (HC) in the adventitia (Ad) is degenerated with absent nuclei of its chondrocyte (thick arrow). (E, F) BLM & ginsenosides treated sections: (E) The lung section shows inflated alveoli (A), intact bronchioles (B) with intact lining and blood vessels (V). The interalveolar septum is thin (arrows) with mild thickened parts (arrow heads). (F) The intrapulmonary bronchi show intact epithelial lining (arrows), mild mononuclear cellular infiltration (asterisks) in the lamina propria (LP). Thin smooth muscle layer (SM) and intact hyaline cartilage (HC) with normal chondrocytes (thick arrows) in the adventitia (Ad). (A, C, E) ×100; (B, D, F) ×400.

• 

  Fig. 2 Figure showed photos of mallory trichrome-stained lung sections. (A, B) Control sections: (A) Fine collagen fibers is observed in the interalveolar septa (arrows), around bronchioles (B) and blood vessels (V). (B) Fine collagen fibers (arrows) is detected in the lamina propria and adventitia of the intrapulmonary bronchi. (C, D) Bleomycin (BLM) treated sections: (C) Massive deposition of collagen fibers in the thickened interalveolar septa (arrows), in the wall of bronchioles (B) and blood vessels (V) and around them. (D) Massive deposition of collagen fibers (arrows) in the lamina propria and adventitia of the intrapulmonary bronchi and around the blood vessel (V). (E, F) BLM & ginsenosides treated sections: (E) Mild deposition of collagen fibers in the interalveolar septa (arrows), around bronchioles (B) and blood vessels (V). (F) Minimal deposition of collagen fibers (arrows) in the lamina propria and adventitia of the intrapulmonary bronchi show. (A, C, E) ×100; (B, D, F) ×400.

• 

  Fig. 3 Figure showed photos of anti-nuclear factor (NF)-κB (anti p65) stained lung sections (×400). (A) Control sections show negative immune-expression of anti NF-κB stain in the bronchioles (B) and very minimal expression in the interalveolar septa (arrows). (B) Bleomycin (BLM) treated sections show strong positive anti NF-κB stain expression in the interalveolar septa (arrows) and in the mucosa, musculosa& adventitia (arrows) of the bronchial wall (B). (C) BLM & ginsenosides treated sections show mild anti NF-κB stain expression in the interalveolar septa (arrows) and in the bronchioles (B).

• 

  Fig. 4 Figure showed Photos of Semithin stained lung sections (×1,000). (A) Control sections show lung alveoli (A) lined by type I (PI) and II (PII) pneumocytes. Interalveolar septa is formed of thin (arrows) and thick portions (zigzag arrows) and show few macrophage (M) with the presence of blood capillaries (C) lined by endothelial cells (E). (B) Bleomycin (BLM) treated sections show collapsed alveoli (A), thick septa (arrow) and congested blood capillaries (C). Mononuclear cellular infiltration including alveolar macrophage (M), lymphocytes (L) and neutrophils (N). Few type I pneumocytes (PI) & numerous type II (PII). (C) BLM and ginsenosides treated sections show inflated alveoli (A) lined by type I (PI), II (PII) pneumocytes and some macrophage (M). They are separated by slightly thickened interalveolar septa (arrows) with mild congested blood capillaries (C).

• 

  Fig. 5 Photos of ultrathin sections of lung sections. (A, B) Control sections: (A) Lung alveoli (A) are lined by type I (PI) and type II (PII) pneumocytes. The alveoli are separated by interalveolar septa, is formed of thin (arrows) & thick portions (zigzag arrow) with the presence of blood capillaries (C) lined by endothelial cells (E). (B) Lung alveoli (A) separated by interalveolar septa (arrow) with minimal collagen fiber (CO) and blood capillaries (C). (C, D) Bleomycin (BLM) treated sections: (C) Collapsed alveoli (A), thickened interalveolar septa (arrows) with congested blood capillaries (C) and distorted endothelial lining (E) are seen. (D) Collapsed lung alveoli (A) are separated by thickened interalveolar septa (arrows) with massive collagen fiber deposition (CO). (E, F) BLM & ginsenosides treated sections: (E) Intact & inflated alveoli (A) separated by thin interalveolar septa (arrows) with mild congested blood capillaries (C). The alveoli are lined by type I (PI) & type II (PII) pneumocytes with its characteristic lamellar bodies (LB). (F) Inflated Lung alveoli (A) separated by interalveolar septa (arrows) with mild collagen fiber (CO) deposition. (A, C, E) ×6,000; (B, D, F) ×20,000.

• 

  Fig. 6 Photos of ultrathin sections of lung sections. (A, B) Control sections: (A) Type I pneumocyte has large oval euchromatic nuclei (N) and little cytoplasm contain few mitochondria (M) and rough endoplasmic reticulum (rER) (arrows). (B) Type II pneumocyte has large rounded euchromatic nuclei (N) with prominent nucleolus (n) and cytoplasm contain mitochondria (M) and rER (arrows), Golgi apparatus (tailed arrows) and lamellar bodies (LB) with their characteristic closely packed concentric lamellae. It covered by intact short microvilli (curved arrow). (C, D) Bleomycin (BLM) treated sections: (C) Degenerated type I pneumocyte with thickened basement membrane (BM) by collagen deposition (CO), it has irregular nuclei (N) and little cytoplasm contain degenerated mitochondria (M), slightly dilated rER (arrow) and vacuolations (V). (D) Distorted type II pneumocytes with damage of its surface microvilli (curved arrow). It has heterochromatic nucleus (N), its cytoplasm contains degenerated mitochondria (M) with absent cristae, excess collagen fiber deposition (CO) and degenerated lamellar bodies (LB) with loss of its concentric lamellae. (E, F) BLM and ginsenosides treated sections: (E) Type I pneumocyte has large flat regular euchromatic nuclei (N) and little cytoplasm contain few mitochondria (M) and rER (arrow). Thin interalveolar septa (thick arrow) with mild collagen deposition (CO) is seen. Note, the presence of intact blood air barrier (Br). (F) Type II pneumocyte has large rounded euchromatic nuclei (N), its cytoplasm contains intact mitochondria (M) rER (arrow), and lamellar bodies (LB) with their characteristic closely packed concentric lamellae. It covered by intact short microvilli (curved arrow). (A, C, E) ×25,000; (B, D, F) ×20,000.

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  Fig. 7 Morphometric analysis of the studied groups. (A) Thickness of the interalveolar septa of the studied groups. (B) % area of collagen fibers of the studied groups. (C) % area of nuclear factor (NF)-κB immunoreaction of the studied groups. (D) Mean number of type II pneumocytes of the studied groups. *Statistically significant (P<0.0001).

• 

  Fig. 8 Oxidative markers of the studied groups. (A) Mean malondialdehyde (MDA) levels in the lung tissue of the studied groups. (B) Mean catalase (CAT) levels in the lung tissue of the studied groups. (C) Mean superoxide dismutase (SOD) levels in the lung tissue of the studied groups. *Statistically significant (P<0.0001).

Reference

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