Effect of NAC and vitamin D on pulmonary hypoplasia in rats with nitrofen-induced congenital diaphragmatic hernia

Abstract

Purpose: This study investigated the effects of N-acetyl-cysteine (NAC) and vitamin D, separately and in combination, on pulmonary hypoplasia in rats with nitrofen-induced congenital diaphragmatic hernia (CDH). Materials and methods: Pregnant Sprague-Dawley rats were divided into 5 groups: control (C), nitrofen (N), N+NAC, N+VitD, and N+NAC+VitD. Rats, except group C, received 200 mg nitrofen on day 9.5 of gestation. NAC and vitamin D were administered for four days starting on day 16. The lungs of rats born on day 21-22 were dissected and examined morphologically, histopathologically, and immunohistochemically (transforming growth factor β1 (TGF-β1) and surfactant protein A (SP-A)). Results: Lung/body weight was significantly lower in group N than in group C. The pulmonary epithelial type was mostly mature in the treatment groups and immature in group N (p<0,05). Alveolar thickness was significantly increased in group N compared to the others. TGF-β1 expression was significantly increased while SP-A was decreased in group N compared to the others. SP-A expression was significantly increased in groups C and N+NAC+VitD compared to groups N+NAC and N+VitD. Conclusion: Vitamin D and NAC reduced pulmonary hypoplasia in rats with CDH. Although increased SP-A expression suggests that their combined use is more effective, other results do not support this.

Keywords

• Congenital diaphragmatic hernia
• N-acetyl-cysteine
• vitamin D
• transforming growth factor β1
• surfactant protein A

Supporting Institution

Pamukkale University

Project Number

2012TPF018

Ethical Statement

The Experimental Ethics Committee of the University approved the protocol (PAUHDEK-2012/002).

Thanks

We thank Hande Senol, Department of Biostatistics, Pamukkale University Medical Faculty, for the statistical analysis. We also thank Barbaros Sahin, for technical assistance during the experimental procedures.

Nac ve D vitamini’nin nitrofen ile konjenital diyafragma hernisi oluşturulan ratlarda pulmoner hipoplazi’ye etkisi

Abstract

Amaç: Bu çalışmada nitrofen ile konjenital diyafragma hernisi (KDH) oluşturulan sıçanlarda N-asetil-sistein (NAC) ve D vitamininin ayrı ayrı ve birlikte pulmoner hipoplazi üzerine etkileri araştırıldı. Gereç ve yöntem: Gebe Sprague-Dawley sıçanlar kontrol (C), nitrofen (N), N+NAC, N+VitD ve N+NAC+VitD olmak üzere 5 gruba ayrıldı. C grubu dışındaki sıçanlara gebeliğin 9.5. gününde 200 mg nitrofen verilmiştir. NAC ve D vitamini 16. günden itibaren dört gün boyunca uygulandı. 21-22. günlerde doğan sıçanların akciğerleri diseke edildi ve morfolojik, histopatolojik ve immünohistokimyasal (transforming growth factor β1 (TGF-β1) ve sürfaktan protein A (SP-A)) olarak incelendi. Sonuçlar: Akciğer/vücut ağırlığı grup N'de grup C'ye göre anlamlı derecede düşüktü. Pulmoner epitel tipi tedavi gruplarında çoğunlukla matür iken grup N'de immatürdü (p<0,05). Alveolar kalınlık grup N'de diğerlerine kıyasla önemli ölçüde artmıştır. TGF-β1 ekspresyonu grup N'de diğerlerine kıyasla anlamlı olarak artarken SP-A azaldı. SP-A ekspresyonu C ve N+NAC+VitD gruplarında N+NAC ve N+VitD gruplarına kıyasla anlamlı olarak artmıştır. Sonuç: Vitamin D ve NAC, KDH'li sıçanlarda pulmoner hipoplaziyi azaltmıştır. SP-A ekspresyonundaki artış, birlikte kullanımlarının daha etkili olduğunu düşündürse de, diğer sonuçlar bunu desteklememektedir.

Keywords

• Konjenital diyafragma hernisi
• N-asetil-sistein
• D vitamini
• transforming growth factor β1
• sürfaktan protein A

Supporting Institution

Pamukkale Üniversitesi

Project Number

2012TPF018

Ethical Statement

Üniversitenin Deneysel Etik Kurulu protokolü onaylamıştır (PAUHDEK-2012/002).

Thanks

İstatistiksel analizler için Pamukkale Üniversitesi Tıp Fakültesi Biyoistatistik Anabilim Dalı'ndan Hande Şenol'a teşekkür ederiz. Ayrıca deneysel prosedürler sırasındaki teknik yardımları için Barbaros Şahin'e teşekkür ederiz.

References

1. Ameis, D., Khoshgoo, N., & Keijzer, R. (2017). Abnormal lung development in congenital diaphragmatic hernia. Seminars in pediatric surgery, 26(3), 123–128. https://doi.org/10.1053/j.sempedsurg.2017.04.011
2. Wright, J. C., Budd, J. L., Field, D. J., & Draper, E. S. (2011). Epidemiology and outcome of congenital diaphragmatic hernia: a 9-year experience. Paediatric and perinatal epidemiology, 25(2), 144–149. https://doi.org/10.1111/j.1365-3016.2010.01172.x
3. Miyake, Y., Tse, W. H., Wang, J. Q., Patel, D., Ozturk, A., Yamataka, A., & Keijzer, R. (2024). Microinjection With Nanoparticles to Deliver Drugs in Prenatal Lung Explants - A Pilot Study for Prenatal Therapy in Congenital Diaphragmatic Hernia. Journal of pediatric surgery, 59(5), 847–853. https://doi.org/10.1016/j.jpedsurg.2024.01.046
4. Fisher, J. C., Kling, D. E., Kinane, T. B., & Schnitzer, J. J. (2002). Oxidation-reduction (redox) controls fetal hypoplastic lung growth. The Journal of surgical research, 106(2), 287–291. https://doi.org/10.1006/jsre.2002.6461
5. Corduk, N., Abban, G., Yildirim, B., & Sarioglu-Buke, A. (2012). The effect of vitamin D on expression of TGF β1 in ovary. Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association, 120(8), 490–493. https://doi.org/10.1055/s-0032-1314858
6. Fischer, K. D., & Agrawal, D. K. (2014). Vitamin D regulating TGF-β induced epithelial-mesenchymal transition. Respiratory research, 15(1), 146. https://doi.org/10.1186/s12931-014-0146-6
7. Chen, G., Qiao, Y., Xiao, X., Zheng, S., & Chen, L. (2010). Effects of estrogen on lung development in a rat model of diaphragmatic hernia. Journal of pediatric surgery, 45(12), 2340–2345. https://doi.org/10.1016/j.jpedsurg.2010.08.028
8. Kluth, D., Kangah, R., Reich, P., Tenbrinck, R., Tibboel, D., & Lambrecht, W. (1990). Nitrofen-induced diaphragmatic hernias in rats: an animal model. Journal of pediatric surgery, 25(8), 850–854. https://doi.org/10.1016/0022-3468(90)90190-k

9. Coleman, C., Zhao, J., Gupta, M., Buckley, S., Tefft, J. D., Wuenschell, C. W., Minoo, P., Anderson, K. D., & Warburton, D. (1998). Inhibition of vascular and epithelial differentiation in murine nitrofen-induced diaphragmatic hernia. The American journal of physiology, 274(4), L636–L646. https://doi.org/10.1152/ajplung.1998.274.4.L636
10. Islam, S., Narra, V., Coté, G. M., Manganaro, T. F., Donahoe, P. K., & Schnitzer, J. J. (1999). Prenatal vitamin E treatment improves lung growth in fetal rats with congenital diaphragmatic hernia. Journal of pediatric surgery, 34(1), 172–177. https://doi.org/10.1016/s0022-3468(99)90251-8
11. Xu, C., Liu, W., Chen, Z., Wang, Y., Xiong, Z., & Ji, Y. (2009). Effect of prenatal tetrandrine administration on transforming growth factor-beta1 level in the lung of nitrofen-induced congenital diaphragmatic hernia rat model. Journal of pediatric surgery, 44(8), 1611–1620. https://doi.org/10.1016/j.jpedsurg.2008.09.021
12. Cigdem, M. K., Kizil, G., Onen, A., Kizil, M., Nergiz, Y., & Celik, Y. (2010). Is there a role for antioxidants in prevention of pulmonary hypoplasia in nitrofen-induced rat model of congenital diaphragmatic hernia?. Pediatric surgery international, 26(4), 401–406. https://doi.org/10.1007/s00383-010-2552-2
13. Ito, Y., Tsuda, H., Imai, K., Miki, R., Miura, M., Tachi, A., Tano, S., Hirako-Takamura, S., Moriyama, Y., Ushida, T., Kobayashi, T., Sumigama, S., Kajiyama, H., Kikkawa, F., & Kotani, T. (2021). Vitamin D improves pulmonary function in a rat model for congenital diaphragmatic hernia. Archives of biochemistry and biophysics, 700, 108769. https://doi.org/10.1016/j.abb.2021.108769
14. Nose, K., Kamata, S., Sawai, T., Tazuke, Y., Usui, N., Kawahara, H., & Okada, A. (2000). Airway anomalies in patients with congenital diaphragmatic hernia. Journal of pediatric surgery, 35(11), 1562–1565. https://doi.org/10.1053/jpsu.2000.18310
15. Guilbert, T. W., Gebb, S. A., & Shannon, J. M. (2000). Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development. American journal of physiology. Lung cellular and molecular physiology, 279(6), L1159–L1171. https://doi.org/10.1152/ajplung.2000.279.6.L1159
16. Matsuzawa, Y., Kawashima, T., Yamazaki, R., Yamaura, E., Makiyama, T., Fujino, H., & Murayama, T. (2011). Inhibitory effects of clinical reagents having anti-oxidative activity on transforming growth factor-β1-induced expression of α-smooth muscle actin in human fetal lung fibroblasts. The Journal of toxicological sciences, 36(6), 733–740. https://doi.org/10.2131/jts.36.733
17. Bartram, U., & Speer, C. P. (2004). The role of transforming growth factor beta in lung development and disease. Chest, 125(2), 754–765. https://doi.org/10.1378/chest.125.2.754
18. Shi, W., Xu, J., & Warburton, D. (2009). Development, repair and fibrosis: what is common and why it matters. Respirology (Carlton, Vic.), 14(5), 656–665. https://doi.org/10.1111/j.1440-1843.2009.01565.x
19. Zhao, J., Bu, D., Lee, M., Slavkin, H. C., Hall, F. L., & Warburton, D. (1996). Abrogation of transforming growth factor-beta type II receptor stimulates embryonic mouse lung branching morphogenesis in culture. Developmental biology, 180(1), 242–257. https://doi.org/10.1006/dbio.1996.0298
20. Leinwand, M. J., Zhao, J., Tefft, J. D., Anderson, K. D., & Warburton, D. (2002). Murine nitrofen-induced pulmonary hypoplasia does not involve induction of TGF-beta signaling. Journal of pediatric surgery, 37(8), 1123–1127. https://doi.org/10.1053/jpsu.2002.34456
21. Takahashi, T., Zimmer, J., Friedmacher, F., & Puri, P. (2017). Follistatin-like 1 expression is decreased in the alveolar epithelium of hypoplastic rat lungs with nitrofen-induced congenital diaphragmatic hernia. Journal of pediatric surgery, 52(5), 706–709. https://doi.org/10.1016/j.jpedsurg.2017.01.020
22. Asabe, K., Tsuji, K., Handa, N., Kurosaka, N., & Kajiwara, M. (1997). Immunohistochemical distribution of surfactant apoprotein-A in congenital diaphragmatic hernia. Journal of pediatric surgery, 32(5), 667–672. https://doi.org/10.1016/s0022-3468(97)90001-4
23. Asabe, K., Tsuji, K., Handa, N., Kajiwara, M., & Suita, S. (2003). Immunohistochemical distribution of surfactant apoprotein-A in congenital diaphragmatic hernia-II. Asian journal of surgery, 26(4), 205–209. https://doi.org/10.1016/S1015-9584(09)60304-3
24. Marin, L., Dufour, M. E., Tordet, C., & Nguyen, M. (1990). 1,25(OH)2D3 stimulates phospholipid biosynthesis and surfactant release in fetal rat lung explants. Biology of the neonate, 57(3-4), 257–260 https://doi.org/10.1159/000243200
25. Yang, X., Yang, P., Zhang, J., Yang, Y., Xiong, M., Shi, F., Li, N., & Jin, Y. (2022). Silica nanoparticle exposure inhibits surfactant protein A and B in A549 cells through ROS-mediated JNK/c-Jun signaling pathway. Environmental toxicology, 37(9), 2291–2301. https://doi.org/10.1002/tox.23596
26. Khalak, R., Huyck, H. L., & Pryhuber, G. S. (1999). Antagonistic effects of pyrrolidine dithiocarbamate and N-acetyl-L-cysteine on surfactant protein A and B mRNAs. Experimental lung research, 25(6), 479–493. https://doi.org/10.1080/019021499270088
27. Adams, L. E., Moss, H. G., Lowe, D. W., Brown, T., Wiest, D. B., Hollis, B. W., Singh, I., & Jenkins, D. D. (2021). NAC and Vitamin D Restore CNS Glutathione in Endotoxin-Sensitized Neonatal Hypoxic-Ischemic Rats. Antioxidants (Basel, Switzerland), 10(3), 489. https://doi.org/10.3390/antiox10030489
28. Jenkins, D. D., Moss, H. G., Brown, T. R., Yazdani, M., Thayyil, S., Montaldo, P., Vento, M., Kuligowski, J., Wagner, C., Hollis, B. W., & Wiest, D. B. (2021). NAC and Vitamin D Improve CNS and Plasma Oxidative Stress in Neonatal HIE and Are Associated with Favorable Long-Term Outcomes. Antioxidants (Basel, Switzerland), 10(9), 1344. https://doi.org/10.3390/antiox10091344
29. Lykkedegn, S., Sorensen, G. L., Beck-Nielsen, S. S., Pilecki, B., Duelund, L., Marcussen, N., & Christesen, H. T. (2016). Vitamin D Depletion in Pregnancy Decreases Survival Time, Oxygen Saturation, Lung Weight and Body Weight in Preterm Rat Offspring. PloS one, 11(8), e0155203. https://doi.org/10.1371/journal.pone.0155203
30. Turkmen, G. G., Timur, H., Tokmak, A., Yilmaz, Z., Kirbas, A., Daglar, K., Sanhal, C. Y., & Uygur, D. (2017). Levels of serum vitamin D and calcium in pregnancies complicated with fetal congenital diaphragmatic hernia and normal pregnancies. The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians, 30(8), 990–994. https://doi.org/10.1080/14767058.2016.1196662
31. Palacios, C., Trak-Fellermeier, M. A., Martinez, R. X., Lopez-Perez, L., Lips, P., Salisi, J. A., John, J. C., & Peña-Rosas, J. P. (2019). Regimens of vitamin D supplementation for women during pregnancy. The Cochrane database of systematic reviews, 10(10), CD013446. https://doi.org/10.1002/14651858.CD013446