Licochalcone A Protects the Blood-Milk Barrier Integrity and Relieves the Inflammatory Response in LPS-Induced Mastitis

Abstract

Background/Aims: Mastitis is an acute clinical inflammatory response. The occurrence and development of mastitis seriously disturb women's physical and mental health. Licochalcone A, a phenolic compound in Glycyrrhiza uralensis, has anti-inflammatory properties. Here, we examined the effect of licochalcone A on blood-milk barrier and inflammatory response in LPS-induced mice mastitis. Methods:In vivo, we firstly established mice models of mastitis by canal injection of LPS to mammary gland, and then detected the effect of licochalcone A on pathological indexes, inflammatory responses and blood-milk barrier in this model. In vivo, Mouse mammary epithelial cells (mMECs) were treated with licochalcone A prior to the incubation of LPS, and then the inflammatory responses, tight junction which is the basic structure of blood-milk barrier were analyzed. Last, we elucidated the anti-inflammatory mechanism by examining the activation of mitogen-activated protein kinase (MAPK) and AKT/NF-κB signaling pathways in vivo and in vitro. Result: The in vivo results showed that licochalcone A significantly decreased the histopathological impairment and the inflammatory responses, and improved integrity of blood-milk barrier. The in vitro results demonstrated that licochalcone A inhibited LPS-induced inflammatory responses and increase the protein levels of ZO-1, occludin, and claudin3 in mMECs. The in vivo and in vitro mechanistic study found that the anti-inflammatory effect of licochalcone A in LPS-induced mice mastitis was mediated by MAPK and AKT/NF-κB signaling pathways. Conclusions and Implications: Our experiments collectively indicate that licochalcone A protected against LPS-induced mice mastitis via improving the blood-milk barrier integrity and inhibits the inflammatory response by MAPK and AKT/NF-κB signaling pathways.

Keywords: AKT/NF-κB; MAPK; blood–milk barrier; licochalcone A; mMECs; mastitis.

Figure 1

Structure of licochalcone A.

Figure 2

Effects of licochalcone A on pathological injury of mammary gland in LPS-induced mice mastitis. Mammary gland tissues from each experimental group (n = 10) were obtained at 24 h after LPS administration, sectioned and stained with H&E (original magnification 100 ×). Mammary gland tissues of (A) control group, (B) LPS group, (C) LPS + licochalcone A (5 mg/kg) group, (D) LPS + licochalcone A (10 mg/kg) group, and (E) LPS + licochalcone A (15 mg/kg) group. (F) Mean injury scores of mammary glands were determined according to a previously described three-point scale. Values are presented as means ± SD (**p < 0.01 and **** p < 0.0001 vs. LPS group).

Figure 3

Effects of Licochalcone A on inflammatory response in LPS-induced mice mastitis. Mammary gland tissues from each experimental group (n = 10) were obtained at 24 h after LPS administration. (A) Myeloperoxidase (MPO) activity assay. The protein levels of IL-1β (B), TNF-α (C), and IL-6 (D) were detected using ELISA. Western blot assay of inducible nitric oxide synthase (iNOS) (E,F) and cyclooxygenase-2 (COX-2) (E,G), and the relative protein levels were quantified by scanning densitometry and normalized to β-tubulin. Values are presented as means ± SD (*p < 0.05, **p < 0.01, and ****p < 0.0001 vs. LPS group).

Figure 4

Effects of licochalcone A on mitogen-activated protein kinase (MAPK) signaling pathway in LPS-induced mice mastitis. Mammary gland tissues from different experimental groups were obtained 24 h after LPS administration. The tissue lysates were prepared and subjected to western blot by using p-ERK1/2 (A,B), p-JNK1/2 (A,C), p-P38 (A,D) antibodies, respectively. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin level. Values are presented as means ± SD (****p < 0.0001 vs. LPS group).

Figure 5

Effects of licochalcone A on AKT/NF-κB signaling pathway in LPS-induced mice mastitis. Mammary gland tissues from different experimental groups were obtained 24 h after LPS administration and total protein. The tissue lysates were prepared and subjected to western blot by using p-AKT (A,B), p-IκBα (A,C), and p-P65 (A,D) antibodies, respectively. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin level. Values are presented as means ± SD (*p < 0.05, ***p < 0.001, and ****p < 0.0001 vs. LPS group).

Figure 6

Effects of licochalcone A on the permeability of the alveolar epithelium. Mammary gland tissues from different experimental groups were obtained 24 h after LPS administration and immersed in FITC-albumin containing 0.5 mM CaCl₂ and 0.5 mM MgCl₂-containing phosphate-buffered saline (mPBS), and localization of FITC-albumin observed after cutting frozen sections. Green and blue colors represent FITC-albumin and nuclei (4′,6-diamidino-2-phenylindole, DAPI), respectively. FITC-albumin was observed in the alveolar lumen of LPS-injected mammary glands.

Figure 7

Protein levels change in claudin-3, occluding, and ZO-1 after LPS or LPS + licochalcone A injection. Protein levels was measured using ImageJ software (http://imagej.nih.gov/ij/) and normalized to that of β-tubulin. (A–D) Results of western blot analysis of claudin-3, occludin, ZO-1, and β-tubulin in mammary glands after LPS and LPS + licochalcone A injection. (B–D) Protein levels of claudin-3, occludin and ZO-1 normalized to that of β-tubulin. (E) Immunostaining images of ZO-1 (green) and nuclear staining with DAPI (blue) in mammary glands treated with LPS and licochalcone A. Values are presented as means ± SD (n = 10 in each group) (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. LPS group).

Figure 8

Immunostaining images of claudin-3 (green), occludin (red) and nuclear staining with DAPI (blue) in mammary glands treated with LPS and licochalcone A. Weak fluorescence intensity was observed for claudin-3 and occludin in the LPS group. With increasing doses, the fluorescence intensities of claudin-3 and occludin were gradually increased.

Figure 9

Effects of Licochalcone A on LPS-induced inflammatory response in mouse mammary epithelial cells (mMECs). Cells were cultured with different concentrations of licochalcone A (1.2, 1.8, 2.4, and 3 μg/mL) for 4 h and viability determined with the CCK8 assay. (A) The effect of licochalcone A and licochalcone A + LPS were determined by CCK8 assay. mMECs were pretreated with Licochalcone A (1.2, 1.8, 2.4, and 3 μg/mL) for 1 h and then stimulated with LPS for 4 h, protein and mRNA levels were determined by qRT-PCR and western blot. The mRNA levels of IL-6 (B), IL-1β (C), and TNF-α (D), and the relative mRNA level was normalized to β-actin mRNA. The protein levels of COX-2 (E,G) and iNOS (E,F), and the relative protein levels were quantified by scanning densitometry and normalized to β-tubulin. Values are presented as means ± SD (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. LPS group).

Figure 10

Effects of licochalcone A on LPS-induced activation of AKT/NF-κB and MAPK signaling pathways in mMECs. (A–E) Total protein in mMECs was collected after 4 h of LPS stimulation. Licochalcone A was added 1 h before LPS stimulation. Protein levels of p-AKT, p-JNK1/2, p-ERK1/2, and p-P38 were detected via western blot and quantitatively assessed via densitometry using β-tubulin as an internal control. Protein levels were measured using ImageJ software (http://imagej.nih.gov/ij/) and normalized to that of β-tubulin. (F–I) MMECs were divided into LPS (1 μg/mL) or LPS + licochalcone A (2.4 μg/mL) groups. After adding LPS to serum-free medium for 1 h, licochalcone A was added. Protein was collected after 0, 15, 30, 45, and 60 min. (F) Western blot analysis of p-IκBα, IκBα, p-P65, and P65 in mMECs were treated with LPS + licochalcone A. (G) Western blot analysis of p-IκBα, IκBα, p-P65, and P65 in mMECs were treated with LPS only. (H–I) The phosphorylation of P65 and IκBα at different time-points under LPS and LPS + licochalcone A treatment were detected via western blot. Values are presented as means ± SD (n = 3) (**p < 0.01 vs. LPS, ****p < 0.0001 vs. LPS).

Figure 11

Effects of licochalcone A on the protein levels of ZO-1, Occludin, and claudin3 in mMECs. mMECs were serum-starved for 3 h before treatment with Licochalcone A (1.8, 2.4 μg/mL) for 24 h (A). The protein levels of ZO-1 (B), claudin3 (C), and Occludin (D) were determined by western blot, and the relative protein levels were quantified by scanning densitometry and normalized to β-tubulin. Values are presented as means ± SD (n = 3) (**p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. Control group).

Figure 12

Schematic diagram of the pathways by which licochalcone A inhibits LPS-induced mastitis.