Tartary buckwheat flavonoids relieve the tendency of mammary fibrosis induced by HFD during pregnancy and lactation

Abstract

Mammary gland fibrosis is a chronic and irreversible disease. Tartary buckwheat flavonoids (TBF) are a natural product of flavonoid extracts from buckwheat and have a wide range of biological activities. The purpose of this experiment was to explore whether HFD during pregnancy and lactation induces fibrosis of the mammary tissue and whether TBF alleviates the damage caused by HFD, along with its underlying mechanism. The HFD significantly increased the levels of TNF-α, IL-6, IL-1β, and MPO; significantly damaged the integrity of the blood-milk barrier; significantly increased the levels of collagen 1, vimentin and α-SMA, and reduced the level of E-cadherin. However, these effects were alleviated by TBF. Mechanistic studies showed that TBF inhibited the activation of AKT/NF-κB signaling and predicted the AKT amino acid residues that formed hydrogen bonds with TBF; in addition, these studies not only revealed that TBF promoted the expression of the tight junction proteins (TJs) claudin-3, occludin and ZO-1 and inhibited the activation of TGF-β/Smad signaling but also predicted the Smad MH2 amino acid residues that formed hydrogen bonds with TBF. Conclusion: HFD consumption during pregnancy and lactation induced the tendency of mammary fibrosis. TBF alleviated the tendency of mammary fibrosis by inhibiting the activation of AKT/NF-κB, repairing the blood-milk barrier and inhibiting the activation of TGF-β/Smad signaling.

Keywords: AKT/NF-κB; blood-milk barrier; fibrosis; high-fat diet; inflammatory microenvironment.

Figure 1

Effects of Tartary buckwheat flavonoids (TBF) on mammary gland tissues of mice induced by high fat diet (HFD) during pregnancy and lactation. 1 g/L TBF was dissolved in 0.1% carboxymethylcellulose sodium solution and was ingested by mice by drinking water. Drinking water administration began on the first day of mating and stopped one week after delivery, and then mammary tissue was collected. The mice mammary tissue was made into paraffin sections, which were used for H&E staining and Masson staining. (A–D) Mice mammary tissue; (E–H) H&E staining result of mammary tissue; (I–L) Masson result of mice mammary, blue represents collagen fiber, red represents muscle fiber; (M) Mammary gland damage score; The arrow represents the lesion of mammary tissue; (N) Schematic diagram of the mice experiment cycle; The arrow represents the lesion of mammary tissue; H&E and Masson are the results of 200× magnification, Scale bar: 100 μm. The data error was based on SEM, three independent repeated experiments were performed; ^#p < 0.01 vs. No treatment group (NT) group; ^∗∗p < 0.01 vs. HFD group.

Figure 2

The effect of TBF on inflammatory levels induced by HFD during pregnancy and lactation. 1 g/L TBF was dissolved in 0.1% carboxymethylcellulose sodium solution and was ingested by mice by drinking water. Drinking water administration began on the first day of mating and stopped one week after delivery, and then mammary tissue was collected. After grinding and centrifugation, the supernatant of mice mammary tissue was obtained, and then the levels of MPO and pro-inflammatory cytokines in mammary tissue were determined. (A) Changes in MPO levels in mammary tissue; (B) Changes in IL-6 levels in mammary tissue; (C) Changes in IL-1β levels in mammary tissue; (D) Changes in the level of TNF-α in mammary tissue; The data error was based on SEM. Three independent repeated experiments were performed; ^#p < 0.01 vs. NT group; ^∗∗p < 0.01 vs. HFD group.

Figure 3

The effect of TBF on AKT/NF-κB signal induced by HFD during pregnancy and lactation. 1 g/L TBF was dissolved in 0.1% carboxymethylcellulose sodium solution and was ingested by mice by drinking water. Drinking water administration began on the first day of mating and stopped one week after delivery, and then mammary tissue was collected. (A) Protein bands of p-AKT, p-P65, p-IκB, AKT, P65, IκB, β-Tublin; (B) Analysis of relative density value of p-AKT protein; (C) Analysis of relative density value of p-P65 protein; (D) Analysis of the relative density value of p-IκB protein. The data error was based on SEM, three independent repeated experiments were performed; ^#p < 0.01 vs. NT group; ^∗∗p < 0.01 vs. HFD group.

Figure 4

Molecular dynamics simulation of TBF and AKT protein. (A) The spatial binding mode of Rutin and AKT protein. (B) Amino acid residues where Rutin and AKT protein have hydrogen bonds, ALA-21, LYS-168, THR-51. (C) The spatial binding mode of Quercetin and AKT protein. (D) Quercetin and AKT protein have hydrogen bonding amino acid residues, VAL123, GLU-127, ASP-184, LEU-49. The 3D structure of the AKT protein was obtained from the PDB database with the number 3ow3. TBF contains 36.3% rutin and 58.2% quercetin. The 3D structure of rutin and quercetin came from PubChem, and then stored in PyMOL as pdbqt mode. AutoDock 4 was used to simulate the molecular docking of AKT with rutin and quercetin. The simulation was obtained by the software AutoDock 4, and the molecular docking pattern is obtained by the software PyMOL. The number of molecular docking simulations was performed 2.5 × 10⁶.

Figure 5

The effect of TBF on the blood-milk barrier induced by HFD during pregnancy and lactation. Fresh mice mammary tissue was immersed in 2 mg/mL FITC for 15 min, then the mammary tissue block was placed in liquid nitrogen, and finally the tissue block was frozen sectioned by a cryostat. After DAPI staining, the sections were sealed with anti-fluorescence quenching reagent, finally obtain the experimental results. FITC green fluorescent protein shuttle freely between the fresh mammary acinar interstitiumed and leaked into the damaged mammary acinar. In addition, FITC will show large green fluorescence in the mammary tissue with acinar atrophy matrix thickening. The lesion was shown by the arrow in the figure, and the scale bar is 100 μm. (A) Flow trajectory of FITC solution in mammary gland; (B) Occludin protein band and relative density analysis results; (C) Analysis results of the band and relative density value of Claudin-3 protein; (D) ZO-1 protein band and relative density analysis results; β-Tublin was used as a reference for protein control. The data error was based on SEM, three independent repeated experiments were performed; ^#p < 0.01 vs. NT group; ^∗∗p < 0.01 vs. HFD group.

Figure 6

The effect of TBF on fibrosis induced by HFD during pregnancy and lactation. 1 g/L TBF was dissolved in 0.1% carboxymethylcellulose sodium solution and was ingested by mice by drinking water. Drinking water administration began on the first day of mating and stopped one week after delivery, and then mammary tissue was collected. (A) Collagen 1, E-cadherin, α-SMA, Vimentin, β-Tublin protein bands; (B) Analysis of the relative density value of Collagen 1 protein; (C) Analysis of the relative density value of E-cadherin protein; (D) Analysis of relative density value of α-SMA protein; (E) Analysis of the relative density value of vimentin protein; β-Tubulin was used as a reference for protein control. The data error was based on SEM, three independent repeated experiments were performed; ^#p < 0.01 vs. NT group; ^∗∗p < 0.01 vs. HFD group.

Figure 7

The effect of TBF on TGF-β/Smad signal induced by HFD during pregnancy and lactation. 1 g/L TBF was dissolved in 0.1% carboxymethylcellulose sodium solution and was ingested by mice by drinking water. Drinking water administration began on the first day of mating and stopped one week after delivery, and then mammary tissue was collected. (A) Protein bands of TGF-β1, p-Smad2/3, Smad2/3, β-Tubulin; (B) Analysis of the relative density value of TGF-β1 protein; (C) Analysis of relative density value of p-Smad2/3 protein; β-Tublin was used as a reference of TBF-β1, Smad2/3 was used as a reference of p-Smad2/3. The data error was based on SEM, three independent repeated experiments were performed; ^#p < 0.01 vs. NT group; ^∗∗p < 0.01 vs. HFD group.

Figure 8

Molecular dynamics simulation of TBF and Smad 3 MH2 domain. (A) The spatial binding mode of Rutin and Smad3 MH2 domain protein. (B) Rutin and MH2 domain protein have hydrogen bonding amino acid residues, ALA-21, LYS-168, THR-51. (C) The spatial binding mode of Quercetin and MH2 domain protein. (D) Quercetin and MH2 domain protein have hydrogen bonding amino acid residues, VAL123, GLU-127, ASP-184, LEU-49. The 3D structure of the Smad 3 MH2 domain is obtained from the PDB database, and the number is 1MJS. TBF contains 53.6% rutin and 37.2% quercetin. The 3D structure of rutin and quercetin came from PubChem, and then stored in PyMOL as pdbqt mode. AutoDock 4 was used to simulate the molecular docking of AKT with rutin and quercetin. The simulation is obtained by the software AutoDock 4, and the molecular docking pattern is obtained by the software PyMOL. The number of calculations for molecular docking simulation is executed 2.5 × 10⁶.

Figure 9

The effect of TBF on alleviating mammary fibrosis tendency and its underlying mechanism induced by HFD during pregnancy and lactation. HFD during pregnancy and lactation caused inflammation of mammary tissue, destroyed the blood-milk barrier, and induced fibrotic lesions tendency in mammary. TBF significantly alleviates the inflammatory response caused by HFD, repairs the blood-milk barrier, and relieves the tendency of mammary tissue fibrosis. Potential protective mechanism of TBF: TBF inhibited the activation of the AKT/NF-KB signaling pathway, effectively inhibiting the inflammatory response to the induction of fibrosis; TBF promoted the expression of tight junction proteins claudin-3, occluding, and ZO-1, and repairs blood-milk barrier to reduce the risk of mammary tissue being exposed; TBF inhibited the activation of TGF-β/Smad signal and inhibits the occurrence of fibrosis tendency.