Pachymic acid (purity >97%) was purchased from Yuanye Biological Technology Co. Ltd. (Shanghai, China).

Male C57BL/6J mice (6-8 weeks) were purchased from the Model Animal Research Center of Guangzhou University of Chinese Medicine. Hepatocytes SIRT6 deficiency mice (6-8 weeks) were kindly provided by Professor Jinhan He (Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China) and Professor Yongsheng Chang (National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China) and used as previously described (24).

Male C57BL/6J mice and hepatocytes SIRT6 deficiency mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium 80 mg/kg and mouse hepatocytes (MPHs) were isolated and cultured in a RPMI-1640 medium, as previously described (7). Briefly, sterilize the body surface of mice with 75% ethanol, cut the skin, open the abdominal cavity, separate the inferior vena cava, place a catheter at the distal end of the inferior vena cava, inject 1-2 ml of heparin immediately after the blood return, and then inject 50 ml of perfusion solution I (1,000 ml Krebs Ringer with Glucose + 2 ml 50 mM EGTA), after the injection of heparin, and cut the open vein to bleed, and rapidly perfusion for about 3 min clarify the blood of the mouse was removed by this procedure. During this period, 0.015 g collagenase was dissolved in 30 ml of perfusate II (1,000 ml Krebs Ringer with Glucose + 1372 µl 2 M CaCl₂), mixed well and maintained at 37˚C. Then perfusion liquid II was infused, and the perfusion was stopped at a slow speed for about 6 min until the liver collapsed and cracked. The whole liver was removed and placed in a culture dish. After adding 20 ml of basic culture medium, it was transferred to the ultraclean table for operation. Tear the liver, filter it with a 70 µm cell net, collect the filtrate, 800 rpm/min, centrifuge for 3 min, and discard the supernatant. Then, Freshly prepared MPHs were suspended in RPMI-1640 medium supplemented with 10% fetal bovine serum, and plated in 6-well culture plates at 0.5x106 cells/well. After attachment, MPHs were washed with PBS, and media was replaced with RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin-streptomycin. Then, MPHs were exposed to 200 µM OA and 100 µM PA (OA&PA), and OA&PA with different densities of Pac.

MPHs were seeded in 96 well plates, where five wells were repeated. After culturing the MPHs in a cell incubator for 12 h, the cells were placed in a new medium, containing Pac. Blank and control wells were incubated for 24 h after administration of the Pac medium. The medium was aspirated, added, and incubated with the pre-mixed medium containing CCK-8 (100 ml 1640 medium and 10 ml CCK-8 solution), and the OD value was then measured at 450 nm, using a microplate reader.

MPHs were harvested after a 24-h incubation and assayed for triglycerides (TG) and total cholesterol (TC) levels using the commercially available enzymatic assay kits (Jiancheng Co.) according to the manufacturer's instructions.

The MPHs were stained with Oil Red O to determine their differentiation. After washing with phosphate buffered saline (PBS) and fixing with 4% paraformaldehyde, for 30 min, MHPs were then washed twice with PBS, stained with 60% saturated Oil Red O for 10-15 min, and washed with 60% isopropanol. Finally, adipocytes were imaged using a light microscope (Nikon x200).

For Cellular ROS determination, Cells were seeded in 12-well plates and treated as above, followed by incubation with DHE 5 µM (KeyGEN Co.) for 30 min at 37˚C in the dark. Then, cells were washed with PBS for three times and visualized under a fluorescence microscope (Nikon x200).

MPHs were seeded in six-well plates and treated as described above. MPHs were washed by a 1640 basic medium and stained with BODIPY 581/591 C11 and BODIPY 493/503 (Invitrogen; Thermo Fisher Scientific, Inc.) to investigate hepatic lipid accumulation and lipoperoxidation, respectively. Cell nuclei were stained with DAPI and visualized using fluorescence microscopy (Leica x200).

Total mRNA of liver tissues or MPHs was extracted with a TRIzol reagent. Reverse transcription was performed using a high-capacity cDNA reverse-transcription kit (Applied Biological Materials Inc.). cDNA was subjected to qPCR analysis with the PowerUp™ SYBRTM Green Master Mix (Abclonal Co.). All genes expression were standardized with β-actin and specific primer sequences were shown in Table SI.

Total protein and nuclear protein were extracted from cultured cells according to the manufacturer's instruction (Beyotime Co.). The concentrations were determined by BCA assay kit (Beyotime Co.). In total, equal amounts of the protein (20-60 µg) were fractionated by 10% SDS-polyacrylamide gel, and separated proteins were transferred onto PVDF membranes. The membranes were incubated overnight at 4˚C with various primary antibodies including anti-SIRT6 (13572-1-AP, Proteintech), anti-PPARα (A18252, Abclonal), anti-CPT1a (A20746, Abclonal), anti-H3K9ac (A7256, Abclonal), anti-H3K56ac (A2391, Abclonal), anti-Nrf2 (16396-1-AP, Proteintech), anti-SOD2 (A19576, Abclonal), anti-HO-1(A19062, Abclonal), and anti-β-ACTIN (Ac026, Abclonal) and followed by an incubation with a secondary antibody. Finally, the blots were observed using BIO-RAD Gel Doc XR from Science and Technology Innovation Center of Guangzhou University of Chinese Medicine.

The processing and optimization of molecular docking is completed by Glide module in Schrödinger Maestro software. Protein processing uses the SIRT6 Preparation Wizard module (25,26). The Pac structure was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and optimized using the molecular mechanics program Minimize to get the most stable structure. The three-dimensional crystal structure of the SIRT6 protein was downloaded from the RCSB PDB database (https://www.rcsb.org/structure/5MF6). Preconditioning, optimization and minimization of receptors (constraint minimization using OPLS3e force field). The compound structure is prepared according to the default settings of the LigPrep module. When screening in Glide module, the prepared receptor is introduced. According to the protein structure original ligand as the docking site (x=115.08, y=26.49, z=-22.42), the docking box is set to 20x20x20Å. Finally, molecular docking and screening were carried out by standard precision (SP) method.

The data are analyzed using GraphPad Prism (Version 8.0) and presented as means ± SEM. Statistical analysis was performed using the one-way analysis of variance followed by post hoc Tukey test for comparisons. Value of P<0.05 was considered statistical significance.

To investigate the role of Pac in abnormal lipid metabolism, OA&PA and Pac-incubated MPHs were used. According to our hypothesis, Pac displays lower cytotoxicity (Fig. 1A). In addition, Pac treatment effectively reduced intracellular TG levels in OA&PA-incubated MPHs, in a dose-dependent manner (P<0.05, Fig. 1B). Subsequent Oil Red O staining revealed decreased lipid deposition in OA&PA-incubated MPHs, following treatment with Pac (Fig. 1C).

Accumulating evidence indicates that oxidative stress damages the structure of cell membranes and leads to cell death-important mechanisms in the development of NAFLD (27). Thus, BODIPY 581/591 C11 and BODIPY 493/503 staining were used to test the levels of lipid accumulation and lipoperoxidation in OA&PA and Pac-incubated MPHs. Treatment with Pac decreased intracellular oxidative stress (due to lipid deposition) in a dose-dependent manner (Fig. 2). Collectively, these data suggest a potential role of Pac in promoting lipid metabolism.

Mechanistic studies have revealed that free fatty acids enter hepatocytes and provide energy through mitochondrial β-oxidation, or TG formation stored in hepatocytes. As NAFLD progresses, the β-oxidation of fatty acids is inhibited, leading to excessive lipid accumulation in hepatocyte (4). Further mechanistic analyses showed that administration of Pac (50 µM) significantly increased the fatty acid oxidation rate in OA&PA-incubated MPHs (P<0.01, Fig. 3B), while lipogenesis was only slightly affected by this administration (Fig. 3A). Pac treatment significantly upregulated the genes involved in fatty acid oxidation, along with a slight suppression of lipogenic genes (Fig. 3C), which led to decreased lipid deposition in OA&PA-incubated MPHs (P<0.05, P<0.01, Fig. 3D). Western blot analyses also indicated that Pac treatment increased the expression of PPAR-α and Cpt1a (Fig. 3E-F). Overall, these data suggest that Pac functions as a potent positive regulator of fatty acid oxidation and hepatic steatosis.

Previous studies have shown that SIRT6 can improve the β-oxidation of fatty acids by activating PPAR-α and also inhibit liver damage caused by ROS, where these effects are observed when SIRT6-Nrf2 interactions increase (14,28). To verify whether SIRT6 plays an important role in lipid accumulation in MPHs treated with Pac, we conducted docking analyses between Pac and SIRT6. Our data indicated notable binding affinity via hydrophobic interactions (Fig. 4A). We further performed qPCR analysis to confirm that Pac increased SIRT6 expression in vitro (P<0.05, Fig. 4B).

SIRT6 normally functions as a transcriptional repressor by deacetylating H3K9 and H3K56 on histones that bind to gene promoters. Therefore, we tested the expression of H3K9 and H3K56 in OA&PA and Pac-cultured MPHs-with or without Pac treatment. As expected, Pac increased the deacetylase activity of SIRT6 by inhibiting H3K9 and H3K56 expression (P<0.05, P<0.01, Fig. 4C-D). Overall, these data indicate that Pac can potentially activate SIRT6 by altering its expression and enzyme activity. This interaction may increase PPAR-α to mediate fatty acid oxidation and increase Nrf2, thereby reducing lipid peroxidation.

To further confirm the effects of SIRT6 in regulating Pac-induced therapeutic effects, MPHs isolated from liver-specific SIRT6-deficient mice were cultured in an OA&PA containing medium for 24 h, followed by co-treatment with Pac (50 µM) for another 24 h. Interestingly, Pac failed to reduce TG content in MPHs with a SIRT6-deficient state (P<0.05, Fig. 5A). Moreover, SIRT6 deficiency abrogated Pac-induced therapeutic effects on lipid accumulation, as shown by Oil Red O staining and BODIPY 581/591 C11 and BODIPY 493/503 staining in MPHs (Figs. 5B and 6). These data suggest that the effects of Pac are SIRT6-dependent during OA&PA-induced cellular damage in MPHs.

Multiple studies have indicated that long-term, high-fat diet exposure can lead to oxidative stress and ROS overproduction (29). Therefore, we investigated the effects of Pac on OA&PA-induced oxidative stress. Pac treatment significantly increased protein levels associated with antioxidant activity (including that of SOD2, HO1, and NRF2) in OA&PA-incubated MPHs derived from wild-type mice (P<0.05, Fig. 7A and B). Meanwhile, Pac treatment also reduced ROS levels in OA&PA and Pac-incubated MPHs, however, in MPHs derived from SIRT6-deficient mice (KO) Pac treatment did not alter ROS levels (Fig. 7C). Moreover, Pac failed to reduce the expression of NRF2, SOD2 and HO1 in SIRT6-deficient MPHs (Fig. 7D). Overall, these data suggest that Pac counteracts OA&PA-induced oxidative stress in MPHs, via the activation of SIRT6.