Squalene Loaded Nanoparticles Effectively Protect Hepatic AML12 Cell Lines against Oxidative and Endoplasmic Reticulum Stress in a TXNDC5-Dependent Way

Abstract

Virgin olive oil, the main source of fat in the Mediterranean diet, contains a substantial amount of squalene which possesses natural antioxidant properties. Due to its highly hydrophobic nature, its bioavailability is reduced. In order to increase its delivery and potentiate its actions, squalene has been loaded into PLGA nanoparticles (NPs). The characterization of the resulting nanoparticles was assessed by electron microscopy, dynamic light scattering, zeta potential and high-performance liquid chromatography. Reactive oxygen species (ROS) generation and cell viability assays were carried out in AML12 (alpha mouse liver cell line) and a TXNDC5-deficient AML12 cell line (KO), which was generated by CRISPR/cas9 technology. According to the results, squalene was successfully encapsulated in PLGA NPs, and had rapid and efficient cellular uptake at 30 µM squalene concentration. Squalene reduced ROS in AML12, whereas ROS levels increased in KO cells and improved cell viability in both when subjected to oxidative stress by significant induction of Gpx4. Squalene enhanced cell viability in ER-induced stress by decreasing Ern1 or Eif2ak3 expressions. In conclusion, TXNDC5 shows a crucial role in regulating ER-induced stress through different signaling pathways, and squalene protects mouse hepatocytes from oxidative and endoplasmic reticulum stresses by several molecular mechanisms depending on TXNDC5.

Keywords: Eif2ak3; Ern1; Gpx4; PLGA; TXNDC5; endoplasmic reticulum stress; liver; olive oil; oxidative stress; squalene.

Figure 1

Electron microscopy analysis, SEM, TEM and particle size histograms of PLGA nanoparticles with and without squalene.

Figure 2

Detection of the effect of different PLGA nanoparticles concentrations (with and without squalene) on the AML12 cell line. (A) 150 µM, (B) 60 µM, (C) 30 µM, (D) 15 µM and (E) untreated cells.

Figure 3

In vitro cellular uptake of squalene. Hepatic AML12 cells were incubated with 30 μM of PLGA-based squalene NPs and PLGA NPs for 72 h. (A) normal mouse AML12 cells (wild-type (WT)), (B) TXNDC5-deficient AML12 cells (knockout (KO)). Statistical analyses were done according to Mann–Whitney’s U-test; * p < 0.05.

Figure 4

Assessment of ROS production in normal mouse AML12 cells cell line (AML12 wild-type (WT)) and TXNDC5-deficient AML12 cells (AML12 knockout (KO)). (A,B) After treatment of cells with 30 µM of squalene NPs for 72 h, (A) ROS was measured in normal conditions, (B) oxidative stress circumstance by 25 mM of H₂O₂ for 3 h. (A1) potent reduction of ROS in squalene group in WT cells, and (A2) significant enhancement in KO cells were observed. (B1) Considerable decrement of ROS in squalene group in WT cells and (B2) remarkable increase in KO cells are indicated. Statistical analyses were done according to Mann–Whitney’s U-test; ** p< 0.01, *** p< 0.001.

Figure 5

Squalene loaded PLGA NPs increased viability against oxidative stress. MTT was applied to evaluate the cell viability. (A) Normal mouse AML12 cells (AML12 wild-type (WT)) were exposed to 30 µM PLGA based squalene NPs for 72 h had a significant increase in viability in presence of 20, 25 and 30 mM of H₂O₂, and (B) TXNDC5-deficient mouse hepatocyte cells (AML12 knockout (KO)) displayed a similar increment in viability. (C) Statistically, a significant difference of 16% and 21% was observed on average in viability enhancement of WT and KO cell lines, respectively, related to respective control in 20, 25 and 30 mM of H₂O₂. (D) TXNDC5 deletion can drastically lower the viability of the mouse hepatocyte in the absence of squalene at all concentrations tested; however, when the cells were treated with squalene, the viability of both cell lines increased. Although (D3) there were no significant differences between WT and KO cells at 30 mM H₂O₂, (D1,D2) a statistical difference was seen in samples treated with squalene loaded in PLGA nanoparticles in presence of 20 and 25 mM H₂O₂. Statistical analysis was carried out according to two-way ANOVA and Mann–Whitney’s U-test for pairwise comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Effect of 30 µM PLGA based squalene nanoparticles on the Gpx4 gene expression levels of oxidative-challenged (A,C) in wild-type mouse hepatic cells, and (B,D) knockout mouse hepatic cells. (A,B) Significant Gpx4 induction after 72 h treatment with squalene encapsulated by PLGA in WT and KO cell line, respectively, (C,D) H₂O₂ can significantly downregulate Gpx4 mRNA level after 30 min exposure to the 25 mM in the control group; whereas squalene encapsulated by PLGA prevented this reduction in both cell lines. The Mann–Whitney U-test was used in the statistical analysis; * p < 0.05.

Figure 7

The cell viability evaluation of mouse hepatocytes upon ER stress. After 72 h, treated with 30 µM of squalene-loaded PLGA nanoparticles, ER stress was induced by 18 nM of thapsigargin for 24 h. (A,B) PLGA NPs did not produce a substantial difference in cell viability when compared to the untreated cells. PLGA based squalene nanoparticles at the 18 nM of thapsigargin (A) produced an enhancement of approximately 6% on the cell viability in WT cells (cell viability is 2.60% in control); likewise, (B) similar result was observed in KO cells. Statistical analyses were conducted according to two-way ANOVA; *** p < 0.001, **** p < 0.0001.

Figure 8

The mRNA expressions of Atf6, Ern1 and Eif2ak3 in presence of squalene-PLGA nanoparticles under the ER stress challenge. After 72 h of treatment with 30 µM PLGA based squalene NPs, (A) the results of three genes (Atf6, Ern1 and Eif2ak3) represented a non-significant difference in WT cells. (B) Squalene with PLGA NPs induced a striking discrepancy in mRNA levels of Atf6, Ern1 and Eif2ak3 in TXNDC5 knockout cells. In order to evaluate the protective activity of squalene-PLGA NPs, when the cells were treated with 12.5 nM thapsigargin for 24 h to produce ER stress, (C) wild-type cells showed a significant reduction in Ern1 mRNA rate, but no significant decline in Atf6 or Eif2ak3 mRNA levels. (D) TXNDC5 knockout cells revealed that the presence of squalene reduced Eif2ak3 expression when exposed to a compound that generates ER stress, but there was no difference in Atf6 and Ern1 expression. The two-way ANOVA and Mann–Whitney’s U-test were used in the data analysis; * p < 0.05, ** p < 0.01.

Figure 9

Scheme showing the effects of squalene on cells involved in oxidative and ER stress in the presence and absence of TXNDC5. In presence of squalene, wild-type and TXNDC5 deficient AML12 cell lines were exposed to oxidative and ER stress. The scheme reveals that in absence of TXNDC5, ROS abundance and Gpx4 mRNA expression are enhanced, and Eif2ak3 expression is decreased; whereas, in presence of TXNDC5, the ROS amount and Ern1 mRNA level are reduced. This scheme was designed using Microsoft Publisher Document version 2010. ↑Increased,↓Decreased.