Pink Lotus Essential Oil and Alleviates on Free Fatty Acid Induced Steatosis in HepG2 Cells via PI3K/Akt and NF- κ B Pathways

tetra-Abstract: Pink lotus essential oil (PLEO) is the volatile components extracted from lotus flowers and there are few relevant research. The purpose of this study was to observe the effect of PLEO on NAFLD in vitro model and its possible mechanism. The ingredients of PLEO were determined by gas chromatography–mass spectrometry (GS-MS) and its lipid-lowering and hepatoprotective activities were investigated. HepG2 cells were treated with free fatty acid (FFA) to establish a cell model of NAFLD. Cell viability was evaluated by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Total cholesterol (TC), triglyceride (TG), tumor necrosis factor- α (TNF- α ), interleukin-1 β (IL-1 β ), and interleukin-6 (IL-6) were determined by Enzyme-Linked Immune Sorbent Assay (ELISA). Oil red O staining was performed to observe the lipid accumulation in the HepG2 cells. Lipid metabolism enzymes including fatty acid synthase (FAS), acetyl-coA carboxylase (ACC), stearoyl-CoA desaturase 1 (SCD-1), and carnitine palmitoyltransferase-1 (CPT-1), insulin signaling pathways including phosphatidylinositol 3 kinase (PI3K) and protein kinase B Akt, inflammatory signaling pathways such as nuclear factor kappa-B (NF-κB), were determined by Western blotting. There were 46 components determined in PLEO with many terpenoids compounds. PLEO decreased TC and TG contents in the FFA-treated HepG2 cells. Furthermore, PLEO inhibited TNF- α , IL-6 and IL-1 β excretion, decreased NF-κB, FAS, ACC and SCD-1 while increased phosphorylation of NF-κB, PI3K, Akt, and CPT-1 expression. It is the first time to reveal that PLEO alleviates FFA-induced steatosis in HepG2 cells by regulating lipid metabolism, inhibiting inflammatory response, and improving insulin sensitivity.

chloride CCl 4 in mice 8 . Nonedible lotus pods is a source of natural antioxidants and anticancer agents 9 . Lotus seedpod extracts have anti-inflammatory effects on lipopolysaccharide LPS induced liver inflammation 10 . However, there has been little research on lotus flowers. Pink lotus essential oil PLEO is a volatile component extracted from lotus flowers that gives it a special aroma. PLEO might have a variety of activities due to its complex composition. Therefore, it is necessary to explore the composition and its pharmacological activity for further development.
In the present study, we explored the protective effects of PLEO on lipid accumulation and inflammation induced by FFA in HepG2 cells. Furthermore, the potential molecular mechanisms involving in the inflammation and insulin signal pathway were discussed as well.

Chemicals and reagents
PLEO was obtained from Healzone Co. Ltd. Qingdao, China . Fetal bovine serum FBS , Dulbecco s modified Eagle s medium DMEM , penicillin-streptomycin solution were purchased from Gibco Carlsbad, CA, USA . The HepG2 cells were purchased from Fuheng Biology, Co. Ltd.

Gas chromatography-mass spectrometry
Coupled gas chromatography-mass spectrometry GC-MS analysis was performed on a Shimadzu system/GC 2010 coupled to a self-gun Shimadzu/AOC-5000 and mass detector Shimadzu MS 2010 Plus . The column used in the present study was HP-5, and the carrier gas was high purity nitrogen. The injection port temperature was 280 . The programmed warming process of the column was as follows: initial temperature was 50 maintaining for 2 min, and then programmed warming at 10 per minute until 280 for 10 min. The column pressure was 10 psi, the shunt injection ratio was 20:1, and the injection volume was 1 μL. Mass spectrum conditions were as follows: the ion source was electron bombardment ion, the electron energy was 70 eV, the interface temperature was 250 , the mass scanning range was m/z 50-600, and the scanning interval was 1.0 s.

Cell culture and drug treatments
Human hepatic carcinoma cell line HepG2 was cultured in DMEM medium supplemented with 10 FBS and 1 penicillin-streptomycin solution. The culture was maintained at 37 in a humidified 5 CO 2 incubator. When the cells reached 50-70 confluence, the MTT assay and the lipid-lowering experiment were conducted. PLEO was dissolved in DMEM medium containing 0.1 DMSO at a concentration of 0.01, 0.1, 1 μg/mL, respectively. In the lipidlowering experiment, HepG2 cells were treated with FFA mixture composed of oleate acid OA and palmitate acid PA 2:1 for 48 h to induce the hepatic steatosis model in vitro. After that, the experiment group was added 100 μL different concentration of PLEO. The control group and FFA group were added the same volume of DMEM after treated without or with FFA, respectively. Subsequently, the lipid and cytokines in the cell culture supernatant, lipid accumulation, the expression of lipid metabolism associated enzyme and PI3K/Akt and NF-κB p65 were determined by ELISA, oil red O staining and Western blotting analysis, respectively.

MTT assay
Cell viability was measured using the MTT assay. The cells 2 10 4 cells/well were seeded in a 96-well cell culture dish, and then treated with different concentrations of 100 μL PLEO 0.01, 0.1, 1 μg/mL for 48 h. The control group was treated with the same volume of DMEM containing 0.1 DMSO. Every group was set five duplicated wells. After incubation, the medium was discarded, and the cells were treated with 100 μL MTT solution 1 mg/ mL for 1 h. The solution was then removed, and DMSO was added to dissolve the insoluble formazan crystals. The absorbance at 570 nm was measured using a full-wavelength Microplate Reader Infinite m plex, Tecan, Switzerland . The cell viability was calculated as the following: cell viability OD sample OD control 2.5 ELISA for quanti cation of lipids and cytokines After treatment, the HepG2 cells 2 10 5 cells per well in a 24-well plate supernatants were centrifuged at 10,000 g at 4 for 10 min to remove debris. The contents of TC, TG, TNF-α, IL-6, and IL-1β in the supernatants were detected with ELISA kits according to the manufacturer s instructions.

Oil Red O staining
The treated HepG2 cells were washed with PBS and fixed in 4 paraformaldehyde for 30 min. After washed with distilled water, the cells were added 200 μL 5 oil red O and stained at room temperature for 30 min. Then the cells were washed with tap water and dyed with hematoxylin. The staining was observed with a microscope BX53, Olympus, Japan and quantified with Image J software v1.44, Bethesda, Rockville, MD, USA .

Western botting analysis
The HepG2 cells 1 10 6 cells per well in a 6-well plate were collected and the total protein was extracted using a RIPA buffer containing protease inhibitors or phosphatase inhibitors. Bicinchoninic acid BCA protein assay was used to determine the protein concentrations, and 30 μg of protein was loaded per sample. The total protein was separated by SDS-PAGE 10 separating gel-5 concentrating gel and transferred to PVDF membranes. The membranes were blocked with 5 nonfat skim milk for 2 h at room temperature. After washed with TBST for 3 times, the membranes were incubated with specific antibodies for Akt, p-Akt, PI3K, p-PI3K, NF-κB p65, p-NF-κB p65, GAPDH, FAS, SCD-1, CPT-1, ACC-1 1:1,000 for 2 h. The membranes were then incubated with goat anti-rabbit HRP secondary antibodies 1:5,000 for 1.5 h. The chemiluminescence detection was performed using an ECL reagent Thermo Scientific, Massachusetts, America and bands were developed with a gel imager TANON-4600 Chemiluminescence Imager, Shanghai Tianneng Technology Co., Ltd. Shanghai, China . Specific bands were detected, analyzed, and quantified by Image J Software v1.44, Bethesda, Rockville, MD, USA .

Statistical analysis
Statistical analysis was performed using SPSS 23.0. Data were expressed as mean standard deviation SD . The one-way ANOVA followed with multiple comparison was used to determine significant differences among groups. Values of P less than 0.05 were considered to be significant.

GC-MS analysis of PLEO
There were 46 components in the PLEO identified by

Effects of PLEO on cell viability of HepG2 cells
The effects of PLEO treatment on HepG2 cell viability were examined and shown in Fig. 2. When the cell viability is above 80 , the drug can be thought to have no significant cytotoxicity on cell growth. As expected, cell viability after treated with PLEO maintained at above 80 . Otherwise, it is amazing that the cell viability increased with the increase of PLEO concentration.

Effects of PLEO on FFA-induced lipid accumulation in
HepG2 cell The effects of PLEO treatment on lipid content in the FFA pre-treated HepG2 cells were examined and shown in Fig. 3. The results indicated that FFA pretreatment dramatically increased the intracellular TC and TG contents compared with the control Fig. 3, p 0.01 . Meanwhile, PLEO treatment dose-dependently decreased TC and TG contents in the HepG2 cells compared with the FFA group     There was no significant change of cell viability in HeG2 cells after treated with PLEO, even at the highest concentration of 1 μg/mL.

Discussion
The liver, the body s largest solid organ, plays a key role in nutrient storage, metabolism, synthesis of new molecules, purification of toxic chemicals and other physiologi-cal processes 11 . In recent decades, NAFLD has become the most common cause of chronic liver dysfunction. NAFLD represents a wide spectrum of disease stages, extending from simple steatosis triglycerides accumulation in the liver , nonalcoholic steatohepatitis steatosis with inflammation to fibrosis, which may eventually progress to cirrhosis and hepatocellular carcinoma 12 . Therefore, it is urgent to develop new drugs for NAFLD treatment. FFA treated HepG2 cells is an in vitro model of steatosis commonly applied to study the hepatocellular consequences of lipid accumulation. HepG2 cells have the advantages of high availability, simple operation, almost infinite life, stable phenotype and so on 13 . The functional properties of HepG2 cells have been extensively studied, expressing many different liver functions, such as synthesis and secretion of plasma protein and bile acid, metabolism of cholesterol, triglyceride and lipoprotein, glycogen synthesis or insulin signaling 14,15 . In the present study, the mixture of  OA and PA 2:1 was employed to induce liver cellular steatosis in the HepG2 cells. As expected, the contents of TC and TG increased significantly after treatment of FFA, indicating a lipid accumulation occurred. Oil red O staining exhibited a typical pathomorphologic changes of steatosis. Furthermore, the contents of TNF-α, IL-6 and IL-1β increased significantly, indicating that there accompanied by inflammation after FFA mixture induced lipid accumulation. All the above changes in the FFA treated HepG2 cells were similar with the key pathological characters of NAFLD in humans, which was an ideal in vitro model of NAFLD. Subsequently, we detected the effect of PLEO on lipid accumulation and inflammation induced by FFA mixture. The results revealed that PLEO significantly decreased TG, TC, TNF-α, IL-6 and IL-1β contents and alleviated cellular steatosis induced by FFA, showing a potential therapeutic effect on NAFLD. There were four groups of ingredients in PLEO recognized by GC-MS, including aliphatic aldehydes, terpenoids, lipids and aromatic hydrocarbons. 2-phenylmethylene -Octanal was the most abundant aliphatic compound detected. The identified terpenoids mainly included isolongifolol 5.83 , L-alphaterpineol 4.56 , citronellol 3.63 , geraniol 2.64 , linalool 2.45 , etc. Terpenoids represent the largest group of ingredients consisting of essential oil which account for its pharmaceutical activity 16 . Alpha-terpineol is a monoterpenoid widely found in essential oil with various bioactive such as antioxidant and anti-inflammatory activity, accounting for 4.56 in LEO. Recent report suggested that alpha-terpineol improved the insulin sensibility and reduced serum TNF-α and IL-1β levels in rats fed a high-fat diet 17 . Geraniol is an acyclic isoprenoid monoterpene isolated from LEO and accounts for 2.64 . Geraniol is potentially applied as a drug based on its various pharmacological properties, including anti-inflammatory, anticancer, antimicrobial, antioxidative, cardioprotective, hepatoprotective and neuroprotective activities 18 . Linalool, a monoterpene alcohol, accounts for 2.45 in LEO. Linalool inhibited the production of TNF-α and IL-6 induce by LPS both in vitro and in vivo 19 . Furthermore, linalool significantly inhibited lipid production, decreased cholesterol and maintained body weight through multiple mechanisms 20,21 . Based on these reports, it was speculated that terpenoids in LEO, especially L-alpha-terpineol, geraniol and linalool, were the key potent part contributing to free fatty acid induced steatosis. However, the exact active ingredients and detailed possible mechanisms need for further research. The accumulation of a large amount of fat in the liver leads to steatosis of liver parenchymal cells, which is an important inducer of NAFLD. FAS, ACC, and SCD-1 are three important enzymes participate in fatty acid synthesis. ACC catalyzes malonyl-CoA, an intermediate metabolite of lipid formation and oxidation 22 . FAS is a rate-limiting enzyme to catalyze the final step of lipid synthesis in the liver 23 . SCD-1 is another key enzyme involving in lipid synthesis, which catalyzes fatty acid elongation and desaturation 24 . At present, there are two signal pathways involving in insulin signaling that up-regulate the expression of these enzymes. One is the sterol regulated element-binding protein-1C SREBP-1C , and the other is carbohydrate responsive element binding protein ChREBP 25 . CPT-1 is the rate-limiting enzyme of fatty acid oxidation. In the process of fatty acid oxidation, CPT-1 is a key regulatory site for fatty acid flow into mitochondria, and abnormal fatty acid oxidation process is also an important pathogenic factor of NAFLD. CPT-1 controls β-oxidation of fatty acids 26 . In our present study, the expression of FAS, ACC and SCD-1 in the HepG2 cells treated with FFA increased significantly, while the expression of CPT-1 decreased when compared with the control group. These changes resulted in lipid accumulation in the cells. After PLEO was applied, the expression of FAS, ACC and SCD-1 decreased, while the expression of CPT-1 increased, indicating that PLEO inhibited fatty acid synthesis and promoted its oxidation. The regulation of PLEO on the lipid metabolism associated enzymes might be involved in the therapeutic effect of PLEO on steatosis induced by FFA.
The PI3K/Akt signaling pathway is involved in the regulation of various cell functions, such as proliferation, apoptosis and autophagy. PI3Ks are members of the intracellular lipid kinase family that phosphorylate the hydroxyl groups of 3 -phosphatidylinositol and phosphoinositides 27 . Akt is a key downstream target of PI3K and a central mediator of the PI3K pathway. It plays a variety of downstream effects through phosphorylation of many substrates involved in cell survival, proliferation, metabolism and movement. Furthermore, PI3K/Akt is an important pathway involving in insulin signaling. Insulin activates PI3K and downstream enzymes via its receptor to regulate glucose and lipid metabolism 28 . Damage of PI3K/AKT results in insulin resistance, which increases de novo lipogenesis and promotes FFA flux to the liver, therefore leads to metabolic disorders such as NAFLD, obesity and type 2 diabetes 29,30 . Cumulative evidences suggest that the PI3K/Akt signaling pathway plays a role in lipid metabolism and liver injury which is related with insulin resistance 31 . The activation of PI3K/Akt can inhibit fat generation, delay excessive fat deposition, and reduce liver steatosis 32 . Phosphorylation of Akt up-regulates lipase production and promotes lipid metabolism in hepatocytes 33 . Therefore, PI3K/Akt has been an important target for ameliorating insulin resistance in the treatment of NAFLD 34 36 . Here we confirmed that there was a significant decrease in expression of Akt, PI3K and their phosphorylation levels in the FFA treated HepG2 cells. Moreover, PLEO significantly increased the expression of p-Akt and p-PI3K 37,38 , which resulted in alleviation of liver cellular steatosis through improving insulin resistance and was a new strategy for NAFLD treatment.
NF-κB is a major transcriptional regulator of inflammation and cell death in the development of chronic liver disease 39 . It is always activated in NAFLD in response to inflammation and regulates the functions of hepatocytes, Kupffer cells, and hepatic stellate cells by inducing the expression of inflammation-related genes, essentially promoting inflammation of the liver during the progression of NAFLD 40 . Our Western blotting analysis data showed that expression of NF-κB p65 increased while its phosphorylation decreased in the HepG2 cells treated with FFA, indicating that NF-κB p65 activation mediated the inflammatory injury in the cellular model of NAFLD. After treated with PLEO, p-NF-κB p65 expression increased significantly while NF-κB p65 expression decreased, indicating an inhibition of inflammatory injury induced by FFA.

Conclusion
In conclusion, PLEO is a complex essential oil extracted from pink lotus flowers. It decreased TC and TG contents and inhibited lipid accumulation in the FFA treated HepG2 cells, exhibiting a potential therapeutic effect on NAFLD. The underlying mechanism might be involved in its regulation on the lipid metabolism associated enzymes, inhibition of NF-κB signals and inflammatory mediators release, activation of PI3K/Akt signal to improve insulin resistance. PLEO is a promising natural plant essential oil deserving for further research and development.