Korean J Physiol Pharmacol.  2021 Nov;25(6):555-564. 10.4196/kjpp.2021.25.6.555.

Beneficial effects of naringenin and morin on interleukin-5 and reactive oxygen species production in BALB/c mice with ovalbumin-induced asthma

• Qi P
• Wei C
• Kou D

• KMID: 2521481
• DOI: http://doi.org/10.4196/kjpp.2021.25.6.555

Abstract

We investigated the effects of naringenin and morin on IL-5 and ROS production in PMA+ionomycin-treated EL-4 cells with the corroboration of their antioxidant and anti-inflammatory properties using an asthma-induced mouse model. The EL-4 cell line was used to study the outcomes of naringenin or morin, followed by cell viability studies. Western blot analysis and ELISA test were used to determine Th2 mediated cytokines. In vivo studies were carried out on BALB/c mice to induce allergic asthma using ovalbumin administered intraperitoneally. Intracellular ROS was determined using 2’,7’-dichlorodihydrofluorescein diacetate, followed by serum enzymatic (AST and ALT) estimations and inflammatory cell count in the bronchoalveolar lavage fluid (BALF) and lung tissues. Histopathological studies were conducted to examine lung tissue-stained architecture. Our findings suggested that naringenin and morin significantly suppressed IL-5 and ROS production via various pathways. Interestingly, by reducing NFAT activity, naringenin and morin stimulated HO-1 expression, thereby suppressing IL-5 secretion due to regulating the transcription factor Nrf2 via P13/Akt or ERK/JNK signalling pathways in EL-4 cells, demonstrating the involvement of HO-1 expression in inhibiting asthmatic inflammation. The increased inflammatory cells in the BALF were substantially decreased by both naringenin and morin, followed by inhibition in the elevated Th-2 cytokines levels. The TNF-α protein levels in an allergic asthma mouse model were significantly reduced by suppressing Akt phosphorylation and eosinophil formation. Recent findings confirmed that naringenin and morin possess the potential to control asthma-related immune responses through antioxidant and anti-inflammatory properties, indicating potential therapeutic agents or functional foods.

Keyword

Asthma; Ionomycin; Morin; Naringenin

Figure

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  Fig. 1 Regulation of IL-5 secretion and ROS generation in PMA + ionomycin-mediated EL-4 cells. (A) IL-5 and (B) ROS were stimulated by 0–10 μg/ml PMA and 500 nM ionomycin intervention for 24 h. (C) The viability of cells treated with 1 μg/ml PMA and 500 nM ionomycin for 24 h was determined. The data are shown as mean ± SD (n = 3). IL, interleukin; ROS, reactive oxygen species. Different letters indicate p < 0.05 when compared to the other groups.

• 

  Fig. 2 Naringenin (NGN) and morin inhibited IL-5 secretion and ROS generation in EL-4 cells induced with PMA+ionomycin. Naringenin or morin and PMA+ionomycin were co-treated for 24 h. ELISA assessed the levels of (A) IL-5 in the medium. (B) The fluorescent probe DCFH-DA (20 μM) was used to measure ROS levels in cells. (C) Cell viability was measured after the cells were treated with naringenin or morin for 24 h. The data is summarised as mean ± SD (n = 3). IL, interleukin; ROS, reactive oxygen species. Different letters indicate p < 0.05 when compared to every other group.

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  Fig. 3 Naringenin (NGN) and morin inhibited IL-5 secretion and ROS generation in EL-4 cells induced with PMA+ionomycin. Naringenin or morin, combined with PMA+ionomycin treatment, regulated cytoslic and nuclear fractions of NFAT protein expression in EL-4 cells for 24 h. The data are shown as mean ± SD (n = 3). NFAT, nuclear activated factor of T cell; IL, interleukin; ROS, reactive oxygen species. *Indicate p < 0.05 and **indicate p < 0.01 when compared to PMA+ionomycin without naringenin or morin respectively.

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  Fig. 4 Naringenin and morin decreased heme oxygenase-1 (HO-1) expression in EL-4 cells by virtue of the transcription factor Nrf2 and PPARγ respectively. The cells were incubated for 0, 2, 4, and 6 h with or without 100 μM of naringenin and morin respectively. Western blot analysis was used to measure the protein expression of transcription factors Nrf2 and PPARγ and HO-1. Data are summarized as mean ± SD (n = 3). *p < 0.05 and **p < 0.01 compared with 0-hr. The relative protein level is expressed as the fold changes to standardize against the control (β-actin or Lamin B1).

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  Fig. 5 Effect of naringenin (NGN) and morin on reactive oxygen species (ROS) in the bronchoalveolar lavage fluid of mice. The values are expressed as the mean ± SEM (n = 10). #Indicates, p < 0.05 compared to control; *p < 0.05 compared with asthma model.

• 

  Fig. 6 Effects of naringenin (NGN) and morin on Akt and MAPK phosphorylation (Erk1/2 and p38) in mouse lung tissue. Asthma mice; Naringenin + OVA-sensitized mice and Morin + OVA-sensitized mice. The levels of p-Akt, t-Akt, p-Erk1/2, t-Erk1/2, p-p38, and t-p38 were standardized to the levels of β-actin in each sample. The values are expressed as the mean ± SD (n = 10). Compared with every other group, different letters suggest p < 0.05.

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  Fig. 7 The effects of naringenin (NGN) and morin on the levels of cytokines in mice’s bronchoalveolar lavage fluid. Asthma mice; Naringenin + OVA-sensitized mice and Morin + OVA-sensitized mice. The values are expressed as the mean ± SD (n = 10). *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to asthma model.

• 

  Fig. 8 Naringenin and morin interfere with cell infiltration in lung tissue, resulting in the prevention of lung inflammation. (A) Control, (B) Asthma induced mice, (C) Naringenin + OVA-sensitized mice and (D) Morin + OVA-sensitized mice. Hematoxylin and eosin staining demonstrated histopathological changes in lung tissues. Asthma induced mice; Naringenin + OVA-sensitized mice; and Morin + OVA-sensitized mice. The scale bar, 100 μm.

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