All chemical reagents used for synthesis were purchased from Acros Organics (United States), Alfa Aesar (United States), Sigma-Aldrich (United States), and Tokyo Chemical Industry (Japan) and were used without further purification. Solvents including acetonitrile (ACN), ethyl acetate (EA), dichloromethane (DMC), dimethyl sulfoxide (DMSO), and dimethylformamide (DMF) were used as received from TCI (Japan), Sigma-Aldrich (United States), Samchun Chemical Co., Ltd. (Korea), and Duksan Pure Chemicals Co., Ltd (Korea). 2-(Bromoacetyl)-naphthalene (1a) was purchased from Alfa Aesar (United States), and other starting compounds were prepared according to literatures: 1 b from 2-acetyl-1-naphthol (Zhang et al., 2010); 2a, 2b, 6a, 6b, and 6c from 1,4-naphthoquinone (Lee et al., 2021); and 2c and 4 from menadione (Jung et al., 2005; Kim et al., 2020). The reactions were monitored by thin-layer chromatography (TLC, silica gel 60 F₂₅₄, Merck), and column chromatography was performed using silica gel 60 (particle size 0.040–0.063 mm, Merck). The ¹H NMR and ¹³C NMR spectra were recorded on a Jeol 300 MHz spectrometer and Jeol 75 MHz spectrometer (Japan), respectively. Spectra are referenced relative to the chemical shift of tetramethylsilane (TMS). The melting points (m.p) were determined on a Barnsteand Electrothermal 9100 instrument (United Kingdom), and mass spectrometry (MS) spectra were recorded using Agilent 1260 Infinity models (United States).

The detail synthesis methods of KHAG-04 compounds are provided in the supplementary materials.

Raw 264.7 and HepG2 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Raw 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and HepG2 cells were cultured in RPMI-1640 medium, and both the cell culture medium were maintained, supplemented with 10% of fetal bovine serum (FBS) and 1% of penicillin/streptomycin, at 37°C in a humidified incubator containing 5% CO₂.

Cell viability was measured using the methyl thiazolyl blue tetrazolium bromide (MTT) assay. Raw 264.7 and HepG2 cells were seeded in 96-well plates. After incubation, cells were treated with several concentrations (0.1, 1, and 10 μM) of KHAG-04 for 24 h. After incubation for 24 h, MTT (0.5 mg/ml) solution was added to each well and the cells were incubated for 1 h. Then the medium was carefully suctioned, and DMSO was added to each well. The absorbance was taken at 570 nm using a microplate reader (Molecular Devices, CA, United States).

Raw 264.7 cells were seeded in 96-well plates and incubated for 24 h at 37°C in 5% CO₂. After incubation for 24 h, cells were pretreated with several concentrations (0.1, 1, and 10 μM) of KHAG-04 for 30 min, followed by treatment with 100 ng/ml LPS and MGO-AGEs (2 mg/ml) for 24 h. After the conditioned medium was carefully transferred to 96-well plates, an equal volume of Gries reagent was added to each well. Then the nitrite concentration was quantified by evaluating the absorbance at 540 nm in the microplate reader (Molecular Devices, CA, United States).

IL-1β, IL-6, and TNF-α secretion were evaluated using enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s protocol. Raw 264.7 cells were seeded in 24-well plates and incubated for 24 h at 37°C in 5% CO₂. After incubation, cells were pretreated with several concentrations (0.1, 1, and 10 μM) of KHAG-04 and KHAG-05 for 30 min, followed by treatment with 100 ng/ml LPS for 24 h; the conditioned medium was collected and centrifuged. Then IL-1β, IL-6, and TNF-α secretion were quantified using the supernatants via the respective ELISA kit (R&D Systems, MN, United States).

HepG2 cells were seeded in a 35-mm dish and incubated for 24 h at 37°C in 5% CO₂. After incubation, cells were pretreated with several concentrations (1 and 10 μM) of KHAG-04 for 1 h, followed by treatment with palmitic acid (0.5 mM), LPS (1 μg/ml), and MGO-AGEs (2 mg/ml) for 24 h. Then cells were fixed in 10% neutral buffered formalin (pH 6.8–7.2) for 1 h, washed with 60% isopropanol, and dried. Next, cells were stained with 0.5% oil red O solution for 20 min at 25°C in the shaker and washed with distilled water. After washing, the stained cells were observed under a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) at ×100 magnification. Lipid droplets’ accumulation was quantified via measuring the absorbance at 510 nm in the microplate reader (Molecular Devices, CA, United States).

Advanced glycation end products were prepared, following the procedure of Subedi et al. (2020), by incubating 5 mg/ml of BSA and 0.02% sodium azide (pH 7.4) with 10 mM of MGO/GO, which was diluted in PBS (1X), at 37 °C for 7 days. Samples were evaporated, filtered, and dialyzed by ZebaTM Spin Desalting Columns, 7K MWCO, 5 ml (Thermo Fisher Scientific, Waltham, MA), before further drying in a freeze dryer.

A TNBSA (2,4,6-trinitrobenzene sulfonic acid) assay was quantified to validate the effect of 13 compounds on MGO-AGEs breakdown, as described previously by Furlani et al. (2015), with slight modifications. In brief, 1 mg/ml GO-AGEs or MGO-AGEs was mixed with several concentrations (0.1 and 0.4 mM) of KHAG-04 or 15 compounds and incubated for 24 h at 37°C. After 24 h, TNBSA and NaHCO3 were added, and the ep-tubes were incubated for 2 h at 37°C. The chemical reaction was stopped by adding 10% SDS solution and 1M HCl to each ep-tube. Free amines were quantified by evaluating the absorbance at 335 nm in the microplate reader (Molecular Devices, CA, United States).

Raw 264.7 cells and hepatic tissue were harvested and homogenized with PRO-PREPTM protein extraction solution (iNtRON, Seoul, Korea) containing protease inhibitors. The cell and tissue lysates were centrifuged at 12,000 rpm for 30 min at 4°C. Then the protein concentration was measured by using the Bradford assay. An equal amount of protein was loaded on 6%–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to PVDF membranes, and then blocked using 5% skim milk for 1 h. Afterward, the membranes were incubated overnight at 4°C with the following primary antibodies: fatty acid synthase (FAS), SREBP1c, CCAAT/enhancer-binding proteins (C/EBPα), TNF-α (Cell Signaling Technology, Inc., Danvers, MA, United States, 1:500), carnitine palmitoyltransferase I (CPT1), acetyl-CoA carboxylase (ACC), phosphorylated ACC (p-ACC), PPARα (Santa Cruz Biotechnology, Santa Cruz, CA, United States, 1:200), α-tubulin (Sigma Aldrich, St. Louis, MO, United States 1: 4,000), anti-IL-6 (IL-6), and anti-C reactive protein (CRP) (Abcam, Cambridge, United Kingdom). After overnight incubation, the membranes were washed and incubated with the secondary antibody for 1 h at 20–25°C. The protein bands were detected using a ChemiDoc XRS+ imaging system (Bio-Rad, CA, United States) and Syngene GeneSnap (Syngene, Cambridge, United Kingdom).

RNA from cells was extracted using the TRIzol^® reagent (Invitrogen, CA, United States), following manufacturer instructions. Isolated RNA was used to synthesize cDNA using the PrimeScript™ RT reagent kit (Takara Bio, Otsu, Japan), according to the manufacturer’s protocol. cDNA was converted from 1 µg RNA to a volume of 20 µl. Quantitative polymerase chain reaction (qPCR) experiment was done using SYBR^® Premix Ex Taq™ (Takara Bio) on an Mx3005P qPCR system (Agilent Technologies). The thermocycling conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s and 60°C for 1 min. The primer sequences used for amplification are as follows: SREBP1c forward 5'-GCG​CCT​TGA​CAG​GTG​AAG​TC-3' and reverse 5'-GCC​AGG​GAA​GTC​ACT​GTC​TTG-3'; FAS forward 5'-CCC​CTG​ATG​AAG​AAG​GAT​CA-3' and reverse 5'-GCC​AGG​GAA​GTC​ACT​GTC​TTG-3'; and C/EBP-α forward 5'-TGG​ACA​AGA​ACA​GCA​ACG​AGT​A-3' and reverse 5'-ATT​GTC​ACT​GGT​CAG​CTC​CAG-3'. The mRNA levels were normalized to the values against α-tubulin, which was used as an internal control, and the data were justified by using the 2^−ΔΔCq method.

Several 4-week-old male C57BL/6 mice were purchased from Raon Bio (Gyeonggi-do, South Korea) and were maintained under controlled environmental conditions: temperature of 22 ± 1°C, humidity of 50 ± 5%, and 12-h light/12-h dark cycle. The mice were permitted ad libitum access to food and water. After 1 week of acclimation period, mice were randomly divided into three groups: control group (C; n = 6) which was fed a rodent diet (10% kcal fat, Research Diets, New Brunswick, NJ, United States), diabetic group (DMC; n = 6), and treatment group (KHAG; n = 6) which was fed with a high-fat–containing rodent diet (60% kcal fat, Research Diets, New Brunswick, NJ, United States). After 4 weeks of diet treatment, diabetic and treatment groups were twice injected with 60 mg/kg streptozotocin (STZ) into the peritoneum by a 3-week interval in citrate buffer (pH 4.5) to induce T2DM. Simultaneously, the control group was injected with only citric acid buffer. The mice groups are as follows: 1) control groups; 2) diabetic groups, 60% kcal high-fat diet (HFD) + 60 mg/kg STZ; and 3) treatment groups, HFD +60 mg/kg STZ +10 mg/kg KHAG-04. The distilled water or KHAG-04 was orally administrated daily for 12 weeks.

During supplementation, body weight, food intake, and fasting blood glucose levels from the tail vein were monitored once a week. At the end of the treatment of 12 weeks, the animals were anesthetized by inhalation with diethyl ether (Duksan, Seoul, South Korea). The blood samples were collected by using a heparin (Sigma Aldrich, St. Louis, MO, United States)-coated syringe by cardiac puncture and were centrifuged at 850 g at 4°C for 10 min to obtain plasma. For protein extraction, hepatic tissue was frozen in liquid nitrogen and stored at −80°C before the experiment.

The OGTT mice were fasted for 12 h, following which glucose solution was administrated at a 1 g/kg concentration. The blood glucose levels were measured at intervals of 0, 15, 30, 60, 90, and 120 min via administration of glucose with a glucometer from the mice’s tail vein. For the ITT, after a 12-h fasting period, mice were intraperitoneal injected with an insulin solution of 1.0 U/kg. Then blood glucose levels were measured as described in the OGTT method.

The glyoxal (GO) amounts were analyzed using high-performance liquid chromatography (HPLC, Waters Corp., MA, United States), according to the protocol by Dhar et al. (2009). The standard of unreacted MGO in the samples was detected by the ratio of the peak area of quinoxaline to that of 5-methylquinoxaline (5-MQ). It is known that o-phenylenediamine (o-PD) reacts with GO to form quinoxaline (doi; 10.5923/j.ajoc.20150501.03). Quinoxaline and 5-MQ were external and internal standards, respectively. The liver tissues were weighed and homogenized and incubated with 0.45M perchloric acid (PCA) and 10 mM o-PD for 24 h at 20–25°C in the dark. Then samples were centrifuged, filtered, and injected into the HPLC system. The samples were analyzed using a YMC-Triart C18 column (250 mm × 4.6 mm, 5 μm; YMC Co. Ltd. Japan) with 20% acetonitrile (ACN) as the mobile phase at a detection wavelength of 315 nm. The column temperature was maintained at 30°C with a flow rate of the mobile phase being 1 ml/min.

Animal liver and kidney tissues were fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned into 5-µm-thick sections. The sections were stained using hematoxylin and eosin (H&E), Masson’s trichrome (MT), and periodic acid–Schiff (PAS). Then the sections were stained using oil red O. The stained tissue slides were examined under a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) at ×100 magnification.

Before docking analysis, the crystal 3D structure of human protein SREBP1, FAS, and C/EBPα were retrieved from the Protein Data Bank (PDB ID: 1AM9, 4PIV, and 1NWQ). The structure of proteins rearrangement was obtained by using the Discovery Studio Visualizer. For docking, all water molecules were deleted and polar hydrogen atoms were added to the proteins. The modified proteins were saved in the PDB format. For ligand preparation, we used ChmeDraw software to draw and convert the 2D ligand structure into a 3D form. The 3D structure of the ligand (KHAG-04) was saved in the PDB format.

AutoDock (4.2.6) was used to conduct a molecular docking study between ligand and proteins. Initially, protein and ligand were uploaded in AutoDock Vina, following grid maps preparation. AutoGrid supplied with AutoDock Vina was used for grid box preparation at 126, 126, and 126 Å for X, Y, and Z, respectively. The output files after AutoGrid run were saved in the PDBQT format. Then these files were used for the AutoDock run. The Lamarckian genetic algorithm (LGA) was chosen for the best conformers. In the docking process, all other parameters were used as a default setting in AutoDock Vina. Windows 10 operating system was used for AutoDock 4.2.6 and AutoDock Vina. The interactions between protein and ligand were shown using the Discovery Studio Visualizer.

The data are expressed as means ± standard error of the mean (SEM). Statistical comparisons were performed between control and several groups using Bonferroni’s test for multiple comparisons of one-way analysis of variance (ANOVA) using GraphPad Prism 5.0 (GraphPad Software Inc., CA, United States). p values < 0.05 were considered statistically significant.

Novel naphthalene-2-acyl thiazolium derivatives (KHAG series) are designed to make analogs of alagebrium, which has a phenacyl thiazolium structure. A typical synthesis of target molecules (KHAG-01, KHAG-02, KHAG-03, and KHAG-04) includes the acylation of substituted naphthalene (Murakami et al., 1988; Boyer et al., 2000; Qandil and Fakhouri, 2012), α-bromination of 2-acylnaphthalene intermediate (Ran et al., 2016), and coupling reaction with 4,5-dimethylthiazole, as shown in Scheme 1.

On the other hand, Friedel-Crafts acylation reaction of compound 4 was attempted to synthesize 1,4-dimethoxy-3-methylnaphthalene-2-acyl compound KHAG-06, but intermediate 2c acylated at position 2 was not produced. Instead of the acyl group substitution at position 2, Friedel-Crafts acylation at position 7 was observed. Hence, we performed Friedel-Crafts acylation of 4 with chloroacetyl chloride to give an intermediate 5 and then coupled it with 4,5-dimethylthiazole to afford thiazolium salt KHAG-05. Thus, a thiazolium compound KHAG-06 containing the methyl group substituted at the C-3 position of the naphthalene ring was prepared from the starting compound 2c, which was prepared from 2-bromo-1,4-dimethoxy-2-methylnaphthalene (Jung et al., 2005; Kim et al., 2020). The α-bromination of 2c gave intermediate 3c, followed by a coupling reaction with 4,5-dimethylthiazole to provide the thiazolium salt KHAG-06.

4-Acetoxy-1-alkyoxy- or 1,4-dialkoxy-2-acylnaphthalen thiazolium salts, KHAG-07 to KHAG-13, were prepared in the same manner described in Scheme 1, except for o-alkylation methods (Scheme 2). In this synthesis route 4, a starting compound 6 was prepared by Fries rearrangement of 1,4-diacetoxy naphthalene, followed by o-alkylation (Paduraru et al., 2008). The bromination of 6a–d by TBA-Br₃ gave acylbromide intermediate 7a–d, and the following coupling reaction of 7a–d with 4,5-dimehtylthiazole provided KHAG-07, -08, -09, and -10, respectively. In addition, 1,4-dialkoxynaphthalene derivatives having different dialkoxy groups at positions 1 and 4 were also prepared from intermediate 6a–c. Hydrolysis of compounds 6a–c produced 4-monoalkoxy intermediate 7a–c, and the o-alkylation of intermediate 7a–c followed by α-bromination of the corresponding intermediate 8a–c gave the acylbromide intermediate 9a–c. Finally, coupling of intermediate 9a–c with 4,5-dimethylthiazole afforded KHAG-11, -12, and -13, respectively.

MGO/GO-AGEs breakdown potential of synthetic KHAG compounds was evaluated by measuring the level of free amines released following degradation of respective AGEs. The 13 synthetic KHAG compounds underwent screening, and among them, KHAG-04, which has a 1,4-dimethoxynaphthalene moiety, showed the highest free amines released percentage (Supplementary Table S1,2) against both of the AGEs types; hence, we selected this novel synthetic compound KHAG-04 for further experiments. As shown in Figure 2, MGO-AGEs or GO-AGEs (1 mg/ml) solution was reacted with several concentrations (0.1 and 0.4 mM) of KHAG-04, alagebrium, and aminoguanidine (1.0 mM) for 24 h at 37°C. KHAG-04 treatment significantly increased the breakdown of MGO and GO-AGEs in a dose-dependent manner (Figure 2). The AGE breakdown potential of KHAG-04 is almost 4- to 5-folds higher in both the treated concentration (0.1 and 0.4 nM) in relation to alagebrium. Similarly, the potential of 0.4 mM of KHAG-04 is higher or almost similar to that of 1 mM of aminoguanidine (positive control), suggesting that it has more than 2-fold higher potential than that of the commercial positive control. Altogether, the MGO/GO-AGEs breakdown potential of KHAG-04 is almost 4-fold higher than that of alagebrium (based on free amine released) and 2-fold higher than that of aminoguanidine (based on concentration).

The anti-inflammatory and cytotoxic effect of KHAG-04 was evaluated against LPS-activated macrophages by measuring the level of inflammatory biomarkers including nitrite (NO) production, TNF-α, IL-1β, IL-6, and cell viability. KHAG-04 did not show any cellular toxicity in the normal condition until 40 µM as well as LPS-activated microglia (Figure 3A). We used the three lowest concentrations of KHAG-04 to check its anti-inflammatory effects in RAW 264.7 cells. Treatment of KHAG-04 significantly lowered the NO production in the LPS-activated Raw 264.7 cells without cellular toxicity (Figure 3B) at 10 µM. LPS treatment dramatically induced the production of inflammatory cytokines including TNF-α, IL-1β, and IL-6 (Figures 3C–E). KHAG-04 treatment significantly lowered the production of TNF-α and IL-1β with high potency but did not alter the level of IL-6 against LPS-activated macrophages. All the treated concentrations even 0.1–10 μM of KHAG-04 showed a significant potency to lower the IL-1β production. In addition, we cheeked KHAG-05 effects on the same condition in Raw 264.7 cells. Though KHAG-05 showed inhibitory effects on nitrite production and inflammatory cytokines IL-1β, the results were not that promising as compared to KHAG-04 (Supplementary Figure S1). We also examined the cell toxicity and nitrite (NO) production in the presence or absence of MGO-AGEs in RAW 264.7 cells. MGO-AGEs showed no toxic effects on RAW 264.7 cells in the presence or absence of KHAG-O4 in a normal and activated state (Figure 3F). MGO-AGEs strongly induced NO production in a dose-dependent manner in RAW 264.7 cells (Figure 3G). KHAG-04 treatment remarkably reduced NO production in RAW 264.7 cells at 10 μM, while it has no damaging effects on cell viability (Figures 3H,I).

We investigated the effects of KHAG-04 against FFA-induced lipid droplets accumulation, its related factors proteins, and mRNA expression in HepG2 cells. FFA showed toxicity at 1 mM in Hep-G2 cells; we selected 0.5 mM of FFA for further study (Figure 4A). KHAG-04 did not show any toxicity until 80 µM (Figure 4B). Oil red O staining, for measuring lipid accumulation, indicated the FFA treatment markedly induced lipid droplets accumulation in HepG2 cells. KHAG-04 pre-treatment drastically reduced FFA-induced lipid accumulation only at 10 and 20 µM but lipid droplets were not reduced at 1 µM in HepG2 cells (Figures 4C,E). Moreover, KHAG-04 pre-treatment significantly reduced FFA-induced lipogenesis transcription factors (SREBP-1_C) and proteins’ (FAS and C/EBP-α) expression levels in Hep-G2 cells (Figures 4D,F). In addition, KHAG-04 seems to reduce FFA-induced mRNA expression levels of SREBP-1C, FAS, and C/EBP-α in Hep-G2 cells (Figure 4G).

Next, we were curious to see whether MGO-AGEs (AGEs) can induce any lipogenic effects in Hep-G2 cells and if KHAG-04 has any protective effects on that. First, cell toxicity of AGEs at different concentrations (0.25, 0.5, 1, 2, and 4 mg/ml) in Hep-G2 cells was assayed, and the cell death started at 1 mg/ml (Figure 5A). The lipid concentration was examined with similar concentrations of AGEs. AGEs significantly induced lipid accumulation in Hep-G2 cells in a dose-dependent manner (Figure 5B). From the aforementioned findings, we decided to use 0.5 mg/ml of AGEs for the next experiment. We investigated the effects of KHAG-04 against AGE-induced lipid droplets accumulation and its related factors proteins expression in HepG2 cells. KHAG-04 pre-treatment markedly ameliorates AGE-induced lipid accumulation only at 10 and 20 µM, but lipid droplets were not reduced at 1 µM in HepG2 cells (Figures 5C,D). In addition, KHAG-04 pre-treatment significantly attenuates AGE-induced lipogenesis transcription factors (SREBP-1_C) and proteins (FAS and C/EBP-α) expression levels in Hep-G2 cells (Figures 5E,F).

The body weight of T2DM groups (before treatment) was significantly increased compared with the control group (CON), whereas fasting blood glucose and food intake were not significantly increased. Body weight, liver weight, and food intake of the KHAG-04 treated group were noticed to be decreased in comparison to the T2DM/DMC treated group but it was not significant (Table 1).

OGTT and ITT were performed on HFD + STZ-induced T2DM mice. As shown in Figure 5, both DMC and KHAG showed higher glucose levels compared to the CON group, where the level of blood glucose level in both OGTT and ITT in the KHAG treated group was significantly lower than that of the DMC group at 120 min (Figures 6A,B). Similarly, the OGTT and ITT AUC of the DMC group were almost 2- to 3-fold higher than the CON group but it was significantly lower in the T2DM animals treated with KHAG-04 (Figures 6C,D) suggesting that KHAG-04 can take part in controlling the glucose homeostasis and insulin sensitivity of the T2DM animals in a positive way.

To analyze the KHAG-04 with the regulating abilities against HFD + STZ-induced T2DM mice, we measured the level of GO accumulation in the liver of the T2DM mice via HPLC analysis. The level of GO in the liver of the DMC group was significantly increased compared with the CON group (Supplementary Figure S2), but the KHAG group was significantly lower than the DMC group (Table 2). These data showed that KHAG treatment might regulate intracellular GO levels and hence less amount of GO-AGEs can form, and higher GO-AGEs can breakdown, and all these steps ultimately help to ameliorate GO and GO-AGE–related toxicities and secondary damage in T2DM animals.

To determine the effect of KHAG-04 against lipotoxicity in T2DM, we performed the H&E, oil red O, PAS, and MT staining in the liver tissue. The lipid droplet accumulation in the liver sections of the DMC group was remarkably increased compared with the CON group. Hepatic lipid deposition generated severe liver injury and it causes liver dysfunction by disturbing the lipid transport, uptake, synthesis, and ballooning function (Yan et al., 2015). These changes were also observed in our study. After treatment of animals with KHAG-04 for 12 weeks, liver histological damages were ameliorated, which was established via the significant improvement of steatosis and ballooning degeneration (Figures 7A–C). Furthermore, the collagen deposition of the liver tissues was checked by MT staining. However, the KHAG group significantly decreased collagen deposition compared with the DMC group (Figures 7A,D). Altogether, results revealed that KHAG-04 treatment can play an important role in ameliorating T2DM-mediated liver toxicity.

The protein expression levels of FAS, SREBP1c, and C/EBPα in the DMC group were significantly higher than those in the NC group. KHAG-04 treatment significantly lowered them compared to the DMC group at 10 mg/kg (Figures 8A,B). The protein levels of p-ACC and ACC were decreased, and the ratio of p-ACC/ACC was decreased in the DMC group compared to those of the NC group, but it was not normalized compared to the DMC group (Figures 8A,B).

To investigate the effects of KHAG-04 on NAFLD and pro-inflammatory cytokines in T2DM mice, we evaluated the protein levels of CRP, IL-6, and TNF-α in the experimented animal tissues. CRP is the biomarker of liver injury. The CRP protein expressions were increased compared to the CON group though it was not significant enough. The KHAG group showed a reduced level of CRP in comparison to the DMC group alone (Figures 8D,E). In the case of pro-inflammatory cytokines, the DMC group significantly increased the protein expression levels of IL-6 and TNF-α, but the protein expression of these inflammatory cytokines was significantly lower in the T2DM animal treated with KHAG-04 (Figures 8D,E). Altogether, these results suggested that KHAG-04 can lower the conditions of NAFLD and inflammation in the liver by reducing the level of NAFLD and pro-inflammatory biomarkers such as CRP, IL-6, and TNF-α.

The best docking site or mode of KHAG-04 and metformin with SREBP1_C, FAS, and C/EBPα is displayed in Figure 9. The docking analysis allowed us to identify several types of interactions between ligand and protein such as hydrogen bonding, Pi-Pi T-shaped bonding, Pi-sigma bonding, Pi-Alkyl bonding, salt bridge, and hydrophobic interactions. KHAG-04 interacted with amino residue HIS328, ARG325, ALA327, ILE 331, LYS324, and nucleotide DA60, DG13, DT15, DG61 of SREBP1_C, where it formed one hydrogen with HIS328, as it is highly conserved among helix-loop-helix protein, releasing the binding energy of −7.84 Kcal/Mol. Likewise, KHAG-04 interacted with amino residue ARG1387, SER1877, SER1382, LYS149, LEU 1388, LEU 1501 of FAS, where it formed one hydrogen with ARG1387, releasing the binding energy of −7.20 Kcal/Mol. Moreover, KHAG-04 only interacted with nucleotide DA4, DA5, DA6, DT3, DT4, DT6, and DG7 of C/EBPα releasing the binding energy of −7.32 Kcal/Mol. However, metformin formed two hydrogen bonds with nucleotides DG59 and DT58 of SREBP1_C, releasing the binding energy of −6.21 Kcal/Mol. Further analysis revealed that metformin interacted with amino residue ARG1255, ASP 1220, GLU 1211, PRO1213, and SER1216 of FAS, and formed two hydrogen bonds with ASP 1220, and GLU 1211. Last, metformin formed two hydrogen bonds with nucleotides DA8 and DC7 of C/EBPα. Metformin binding energy to FAS and C/EBPα was −5.17 Kcal/Mol and −6.10 Kcal/Mol, respectively. These results suggest that KHAG-04 has the lowest binding energy to lipogenic markers as compared to our standard compound metformin.