Effect of pinolenic acid on oxidative stress injury in HepG2 cells induced by H2O2

Abstract To investigate the effect and mechanism of pinolenic acid (PNA) on H2O2‐induced oxidative stress injury in HepG2 cells. Methods: PNA was used to regulate oxidative stress injury of HepG2 cells induced by H2O2. Quantification of cell survival rate, accumulation of intracellular reactive oxygen species (ROS), and expression levels of anti‐oxidation‐related genes were determined using MTT, fluorescent probe technology (DCFH‐DA), and real‐time quantitative reverse transcription polymerase chain technology (qRT‐PCR) method, respectively. Meanwhile, the activity of intracellular antioxidant enzymes was determined by biochemical methods. The results showed that PNA improved the survival rate of HepG2 cells induced by H2O2 (29.59%, high‐dose group), reduced the accumulation of intracellular ROS (65.52%, high‐dose group), and reduced the level of intracellular malondialdehyde (MDA; 65.52%, high‐dose group). All these results were dose‐dependent, which indicated that PNA can improve oxidative stress damage of cells. Furthermore, the mechanism of PNA regulating oxidative stress was investigated from the gene level. Results showed that under supplementation of PNA, the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH‐Px) had been improved (39.74%, 17.58%, and 23.83%, high‐dose group). Further studies on gene expression which controls the activity of antioxidant enzymes showed that under the regulation of PNA, the expression level of Keap1 gene was decreased, while Nrf2 gene was increased. The expression levels of HO‐1 and NQO1 in the downstream of Nrf2 were increased. Results indicated that under the regulation of PNA, Nrf2 was separated from Keap1, entered the nucleus, bound to ARE, and up‐regulated the expression levels of HO‐1 and NQO1 genes. Conclusion: PNA has a conspicuous improvement effect on oxidative stress damage induced by H2O2 in HepG2 cells. We also found the antioxidant mechanisms of PNA where it protected cells from oxidative stress damage by causing nuclear translocation of Nrf2 gene and up‐regulated the expression levels of antioxidant enzymes in the downstream. This shows that PNA prevented oxidative stress by mediating the Keap1/Nrf2 transcriptional pathway and down‐regulating enzyme activities.


| INTRODUC TI ON
Excessive reactive oxygen species (ROS) and other free radicals cause oxidative damage which further contribute to numerous diseases, such as aging, cancer, and neurodegenerative diseases, such as dementia and Alzheimer's disease (Wang, 2015). Hence, decreasing oxidative damage may help to prevent those diseases. Oxidative damage constantly occurs in vivo where damaged biomolecules must be repaired. Failure to replace the damaged biomolecules enhances oxidative damage and contributes to inflammation (Long et al., 2020;Yueming et al., 2020). Dietary antioxidants from natural sources play important roles by reducing oxidative damage that may help to reduce the severity of chronic diseases, as well as to extend the shelf life of food products (Chang et al., 2021;Poljsak & Milisav, 2012). Research on natural plant antioxidants has always been promising since it generates findings for practical applications in the field of human nutrition. Natural antioxidants have shown promising anti-inflammatory, anti-bacteria, skin-whitening, as well as disease preventive properties (Lourenço et al., 2019). PUFAs have been shown to function as antioxidants at the plasma membranes by regulating the antioxidant signaling pathways (Oppedisano et al., 2020).
Its chemical formula is C 18 H 30 O 2, and it is the isomer of linolenic acid (Xie et al., 2016). PNA is the major Δ5-unsaturated polymethyleneinterrupted fatty acid (Δ5-UPIFA) in pine nuts and their oil, which accounts for about 15% of the total fatty acids, and it can be as high as 17%-20% in red pine seed oil . The chemical structure of PNA is similar to γ-linolenic acid (GLA) and α-linolenic acid (ALA). GLA is an n-6 PUFA while ALA is an n-3 PUFA, and it is an isomer of GLA (Ryan et al., 2006). Pine nuts and pine nuts oil have been regarded as functional food in China, Korea, and Japan for many years (Zhang et al., 2019). Numerous studies demonstrated that PNA has various health benefits such as antioxidant, weight loss, lipid-lowering, anti-inflammation, appetite control, improving insulin sensitivity, cardio-protection, and anti-cancer (Le et al., 2012;Xie et al., 2016;Zhang et al., 2019). Previous study demonstrated that PNA has beneficial effect on antioxidant protective mechanisms in rats fed with high-fat diet (Chen et al., 2011). Recently, Zhang et al. (2019 demonstrated that PNA acts as an antioxidant by alleviating cellular oxidative stress which is beneficial to prevent nonalcoholic steatohepatitis. However, the antioxidative mechanisms of PNA have not been fully elucidated.
The objective of this study was to determine whether supplementation of PNA alleviates H 2 O 2 -induced oxidative stress in HepG2 cells. Besides, regulation of ROS production and related antioxidant enzymes defense system by PNA was also evaluated.
By detecting the MDA content and intracellular ROS accumulation of HepG2 cells, figure out whether PNA can regulate the oxidative stress response and improve the oxidative stress damage of the cells; by detecting the activity of intracellular antioxidant enzyme system in HepG2 cells and by detecting the expression levels of related antioxidant genes in HepG2 cells, explore the mechanism of which PNA regulates the expression levels of Kelch-like ECHassociated protein 1 (Keap1) and Nrf2 genes and study the variation of HO-1 and NQO1 gene expression levels. Based on the above test results, this experiment may clarify the mechanism of which PNA improves cellular oxidative stress injury and provides a theoretical basis for the development of antioxidant functional foods of PNA and Korean Pine Oil.

| Cell culture
HepG2 cells were maintained in DMEM with 10% heat-inactivated FBS (with 1% penicillin-streptomycin) and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2 based on the method of Hao (2018). The growth medium was prepared according to the procedures recommended by the American Tissue Cell Culture (Tang et al., 2021). In all experiments, 80%-90% confluent HepG2 cells were used before treatment. Five treatments were grouped as follows: Cells incubated with serum-free medium were treated as a control group: Cells incubated with serum-free medium followed by H 2 O 2 solution (0.2 mM) for 12 h and then cultured for 24 h were treated as a model group; Cells incubated with medium containing H 2 O 2 solution (0.2 mM) for 12 h and then incubated with medium containing PNA for 24 h were treated as the experimental groups. The experimental groups were divided into PNA low-dose (PNA-L), middle-dose (PNA-M), and high-dose groups (PNA-H) at 1, 5, and 10 µM, respectively.

| Preparation of pinolenic acid (PNA)
According to the previous experimental results of the research group, high purity pinolenic acid has been extracted from Korean pine seed oil through esterification embedding combined with secondary urea embedding method, and its fatty acid composition has been determined by GC-MS (Zhou, 2019). The GC-MS diagram is shown in Figure 1, and the component analysis results are shown in Table 1. Due to the poor solubility of pinolenic acid in culture medium, and considering that DMSO (dimethyl sulfoxide) has certain toxic effects on cells, ethanol was used to dissolve pinolenic acid first. In this test, the final concentration of ethanol in the mother liquor of the prepared sample should not exceed 2%. 9 µl pinolenic acid was added to 291 µl ethanol to prepare the sample mother liquor, which was mixed by vortex oscillation, filtered by 0.22 µm organic filter membrane to remove bacteria, and stored at −20°C.

| Cell viability assay
HepG2 cell viability was determined using MTT assay according to Ali Chiroma et al. (2020) with slight modifications. Briefly, HepG2 cells were inoculated at a density of 1 × 10 5 cells/ml per well in a 96well plate. After 24 h, the growth medium was removed and 10 µl of MTT (5 mg/ml) was added to each well and incubated with the cells for 4 hr at 37°C in a 5% CO 2 atmosphere. Subsequently, 200 µl of DMSO was added to each well with shaking for 10 min at room temperature to dissolve the formazan crystals. The absorbance value was measured using an enzyme-linked immunosorbent assay (ELISA) reader (BIO-TEK EL×800, USA) at 570 nm. Results were expressed as percentages of cell viability according to the formula below:

| Determination of intracellular ROS production
First, HepG2 cells (1.0 × 10 5 cells/ml) were seeded in 12-well plates and treated as designed. The plate containing the cells was incubated in a humidified incubator at 37°C with 5% CO 2 until further usage. After discarding the culture medium, each well was filled with DCFH-DA fluorescent probe (10 µM). After incubating at 37°C in the dark for 40 min, cells were washed twice using cool PBS before digesting the cells with trypsin. After centrifuging at 1000 rpm for 8 min, cells were resuspended in PBS, and transferred to the 96-well plate.
Intracellular ROS production was measured by the reactive oxygen species assay kit (Beyotime, China) using a fluorescence microplate reader at the excitation and emission wavelengths of 502 and 530 nm.

| Determination of intracellular MDA content
First, HepG2 cells (1.0 × 10 5 cells/ml) were seeded in 12-well plates and treated as designed. After 24 h of incubation, cells were collected and centrifuged at 3000 rpm for 10 min. Subsequently, the supernatant was removed and 300 µl of 10% medium followed by sonication in ice for 1 min. After removing the culture solution, 100 µl of pre-cooled PBS buffer was added to each well, and the cells were subjected to ultrasound. Cells were collected and stored at −20°C.

| Statistical analysis
Data were expressed as mean ± SD carried out at least in three independent replicates. One-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test was used to compare the experimental means. Data were analyzed using GraphPad Prism (version 7.0) with model of program. p < .05 was considered statistically significant.

| The Effect of PNA on the survival rate of HepG2 cells induced by H 2 O 2
HepG2 cell line is commonly used in cytotoxicity and antiproliferative activity assays for their liver-specific suppression responses toward phytochemicals. Hydrogen peroxide (H 2 O 2 ) acts as a cell signaling molecule under normal physiological conditions. However, excessive H 2 O 2 -induced toxicity in HepG2 cells eventually leads to oxidative stress (Han et al., 2018). Excessive accumulation of ROS causes an increase in intracellular free Ca 2+ which, in turn, leads to mitochondrial dysfunction, irreversible membrane damage and, finally, cell death (Zhang et al., 2018). Cell viability is the most intuitive indicator to reflect degree of cell damage in order to establish the oxidative stress model. Figure

| The effect of PNA on the intracellular ROS content of HepG2 induced by H 2 O 2
Exogenous H 2 O 2 penetrates the cell membrane easily and generates lots of free radicals which attacks the mitochondrial membrane,

| The effect of PNA on the antioxidant enzyme system of HepG2 induced by H 2 O 2
SOD, CAT, and GSH-Px are important components of the cellular antioxidant enzyme system. It is important as the antioxidant defense system to maintain the dynamic balance of oxidative stress (Niki, 2010). Under oxidative stress, these antioxidant enzymes will act as endogenous antioxidant to scavenge free radicals intracellularly (Musa et al., 2017). SOD catalyzes the decomposition of superoxide anion into H 2 O 2 and O 2 , which in turn scavenge free radicals.
CAT and GSH-Px catalyze the decomposition of H 2 O 2 into H 2 O to maintain redox balance of the organism by eliminating excessive free radicals produced in vivo. It can be seen from Figure 7 that the enzymatic activities of SOD, CAT, and GSH-Px in HepG2 cells reduced significantly (p < .05) by 35.04%, 21.15%, and 34.17%, respectively, compared with the control group after 24 hr incubation with H 2 O 2 . However, the enzymatic activity of SOD increased significantly (p < .05) by 21.8%, 29.6%, and 39.7%, respectively, after treatment using 1, 5, and 10 µM PNA (Figure 7a). The enzymatic activity of CAT increased by 4.95%, 16.6%, and 17.6%, respectively, while the enzymatic activity of GSH-Px increased by 11.1%, 23.6%, and 23.8%, respectively, after treatment using 1, 5, and 10 µM PNA HO-1 is known as a stimulation response protein that is induced in various stress conditions. HO-1 is one of the most representative ARE response enzyme regulated by Nrf2 (Loboda et al., 2016). As shown in Figure 8c,d, the expression levels of NQO1 and HO-1 genes reduced significantly (p < .01) by 29.8% and 39.9%, respectively, compared with the control group. However, treatment with 1, 5, and 10 µM PNA increased the expression level of HO-1 gene by 12.1%, 23.5%, and 37.8%, respectively, compared with the control group. In relation, treatment with 1, 5, and 10 µM PNA also increased the expression level of NQO1 gene by 13.5, 26.3, and 28.9, respectively.
However, cells treated with medium and high doses of PNA (5 and 10 µM) demonstrated significant increase (p < .05) of the expression levels of HO-1 and NQO1 genes compared with the model group.
It was well-known that down-regulation of Nrf2 expression level affected the cellular antioxidant defense responses, resulting in high F I G U R E 3 Effect of PNA on HepG2 cells viability induced by H 2 O 2 . HepG2 cells were induced by H 2 O 2 and regulated by PNA, and then the cell viability was measured. The data were expressed in x ± SD. Compared with Mod, **p < .01, ***p < .001. Excel 2010 was used for preliminary processing of the data ROS production that caused cell death (Yu et al., 2019). When HepG2 cells were treated with PNA, the expression levels of Nrf2, HO-1, and NQO1 increased at a dose-dependent manner. This event further activates the production of antioxidant enzymes, such as SOD, CAT, and GSH-Px. Hence, MDA production was decreased. All these events enhanced the antioxidant defense mechanisms, increasing the cellular antioxidant capacity to alleviate high ROS production. the expression level of Keap1 gene and up-regulating the expression level of Nrf2 gene; therefore, the expression levels of the antioxidant enzyme genes HO-1 and NQO1 genes in the downstream of Nrf2 were improved, so as to improve the activity of antioxidant enzymes and cellular oxidative stress.

ACK N OWLED G M ENTS
This work was supported by the 13th Five-Year National Key Research and Development Program (No. 2016YFD0600805-05) and national R & D plan.

CO N FLI C T O F I NTE R E S T
None of the authors have conflict of interests regarding this research.