Extra virgin olive oil mitigates lung injury in necrotizing enterocolitis: Effects on TGFβ1, Caspase-3, and MDA in a neonatal rat model

Abstract

Background: Necrotizing enterocolitis (NE), which is common in premature babies, has been associated with lung damage. Our aim is to explore the effect of enterally administered extra virgin olive oil (EO) with rich polyphenol content on clinical parameters, histopathological score, Transforming growt factor beta-1 (TGFβ1), Caspase 3 and Malondialdehyde (MDA) levels in NE-related lung injury of neonatal rats.

Methods: Three groups (control, NE, NE+EO) were created, with 8 neonatal rats in each group. NE was induced by hypoxia-hyperoxia-hypothermia and formula feeding. EO was given to the treatment group by orogastric probe for 3 days. Intestinal and lung tissue were excised for analysis.

Results: TGFβ1 expression levels, TGFβ1 and MDA concentration levels were higher in the NE compared to NE + EO and control groups (p < 0.001), and their levels decreased after EO treatment compared to the NE group (p < 0.001). It was determined that EO treatment significantly reduced the histopathological damage and the caspase-3 (CASP3) expression level in the lung (p < 0.001).

Conclusion: Our findings emphasize that TGFß1 has an crucial function in NE-related lung injury and that EO has therapeutic potential in NE-related lung injury.

Fig 1. Illustrative examples of our study.

Three main groups rats as control, NE, and NE+EO each of them with 8 members were included in the study. Both TGFβ1gene expression and concentration levels, MDA concentration levels, histopathological scoring and Casp-3⁺ cells count/unit area of groups were detected. As a response to the cellular damage caused by NE, the expression and translation levels of the different genes may be changed in the nucleus of cells. Depending on the NE, the lung tissue concentration level of MDA significantly increased (a). Also the histopathological scoring (H&E scored 0–12) (b) and Casp-3⁺ cells count/unit area (c) in lung tissue significantly increased depending on the NE injuries. The TGFβ1gene is transcribed TGFβ1 mRNA, and TGFβ1 proteins are translated from mature TGFβ1 mRNA. There was statistically significant differences among the groups in terms of TGFβ1 gene expression levels. The expression level of TGFβ1 gene significantly increased depending on the NE injuries (d). Also depending on the NE, the lung tissue concentration level of TGFβ1 significantly increased (e). It may be said that, the EO has an important protective effect against lung tissue damage caused by NE injuries.

Fig 2. Histological evaluation of the lung in neonatal rats from each experimental group.

A. Control group, Bar: 100µm, B. NE group showing increased neutrophil infiltration and severe lung tissue damage, C. Treatment group. Haemotoxylene-Eosin staining (H-E) Bar: 50µm.

Fig 3. Caspas-3 staining in lung tissue from neonatal rats in all groups.

A. Negative control. B. Control group. C. NE group, D. Treatment group. Arrowhead: Casp-3⁺ negative cells. Arrows: Casp-3⁺ cells. It is noteworthy that Casp-3⁺ cells were more numerous in the NE group than in the treatment group. Streptavidin-biotin-peroxidase method, Bar: 50µm.

Fig 4. Relation between the TGFβ1 concentration levels in lung tissue and clinical parameters of the groups.

There were statistically significant relation between TGFβ1 concentration levels and all of last day weight (b), clinical disease score(c), bowel macroscopy(d), MDA concentration levels (e), Caspase-3 positive cell count/unit (f) and histopathological scoring (H&E scored 0–12) (g). Conversly the relation between TGFβ1 concentration and birth day was not significant (a).

Fig 5. Relation between the MDA concentration levels in lung tissue and clinical parameters of the groups.

There were statistically significant relation between MDA concentration levels and all of last day weight (b), clinical disease scor(c), bowel macroscopy(d), Caspase-3 positive cell count/unit (e) and histopathological scoring (H&E scored 0–12) (f). Conversly the relation between MDA concentration and birth day was not significant (a).

Fig 6. Relation between the TGFβ1 expression levels in lung tissue and clinical parameters of the groups.

There were statistically significant relation between TGFβ1 expression levels and all of last day weight (b), clinical disease scor(c), bowel macroscopy(d), MDA concentration levels (e), Caspase-3 positive cell count/unit (f), histopathological scoring (H&E scored 0–12) (g) and TGFβ1 concentration levels (h). Conversly the relation between TGFβ1 expression levels in lung tissue and birth day was not significant (a).