GGCLT has the following four botanical components, the following herbs were produced in an 8:3:3:2 weight ratio: (i) Puerariae Radix (roots of Pueraria lobata, Ge Gen, 480 g); (ii) Scutellariae Radix (roots of Scullellaria baicalensis, Huang Qin, 180 g); (iii) Coptidis Rhizoma (rhizomes of Coptis chinensis, Huang Lian, 180 g), and (iv) Glycyrrhizae Radix (roots of Glycyrrhiza uralensis, Gan Cao, 120 g). The 960 g of the herbal components were steeped in 5 liters of dH${}_2$O at 80 °C for 2 hours to create the dH${}_2$O extracts. Filtered and lyophilized (VirTis Freezemobile, Gardiner, NY, USA) extracts were produced with an estimated yield of 12.5% (120 g). These substances were freeze-dried, and store at –20 °C. Before being used in the in vivo tests, the GGCLT extract was dissolved in dH${}_2$O.

The six main index components of the GGCLT decoction were recognized by HPLC. A Waters HPLC System (Waters Corporation, Milford, Massachusetts, United States) with a 600 quaternary pump, a Sugai U-620 column temperature controller, a 717 plus autosampler, and a 996 photodiode array detector was used to perform the HPLC-UV study. Puerarin, baicalin, berberine, baicalein, glycyrrhizic acid, and wogonin in analytical-grade pure powder forms were used as standards for the HPLC assay. Puerarin, baicalin, baicalein, glycyrrhizic acid, and wogonin were purchased from Merck (Rahway, NJ, USA). Bererine was purchased from Selleckchem (Burlington, MA, USA). The analytical conditions were set as follows: gradient elution by the mixture of mobile phases A (mixture of 20 mM potassium dihydrogen phosphate and 0.0085% phosphoric acid), B (acetonitrile) and C (water) at 0–30 min with a ratio of 90–75% A and 10–25% B; 30–40 min with a ratio of 75–65% A and 25–35% B; 40–55 min with a ratio of 65–0% A, 35–75% B and 0–25% C; 55–60 min with a ratio of 75–10% B and 25–90% C; 60–65 min with a ratio of 0–90% A, 90–10% B and 90–0% C. The flow rate was kept constant at 1.0 mL/min. The column temperature was controlled at 35 °C, the post time was 15 min and the injection volume was 5 $\mu$L. Approximately 0.5 g of GGCLT (lyophilized) was weighed and put into 20 mL of 70% methanol solution as extractive solvent, ultrasonically vibrated for 15 min at room temperature, vibrated at 160 rpm for 20 min in a 40 °C water bath, centrifuged at 3000 rpm for 10 min, and the supernatant liquid was injected in HPLC-UV for quantitative analysis. The 200–400 nm UV wavelength was chosen for complete detection. As a guard column, a Lichrospher RP-18 end-capped column (particle size, 5 m; column size, 4.0 mm internal diameter $\times$ 10 mm; Merck KGaA, Rahway, NJ, USA) was employed. The analytical column was a Cosmosil 5C18MSII column (particle size, 4.6 mm internal diameter) $\times$ 250 mm. Puerarin, baicalin, baicalein, wogonin, berberine, and glycyrrhizic acid are represented by Puerariae Radix, Scutellariae Radix, Coptidis Rhizoma, and Glycyrrhizae Radix, respectively (Fig. 1).

Fig. 1.

Ger-Gen-Chyn-Lian-Tang (GGCLT) chromatogram determined by HPLC-UV. The panel shows the fingerprint of the HPLC fingerprint (detection wavelength 250 nm) (A) and three-dimensional structures of these six main index components (B). Structures of the compounds identified by HPLC-UV (C). Puerarin, baicalin, berberine, baicalein, glycyrrhizic acid, and wogonin, the six main index components of GGCLT.

The Traditional Chinese Medicine Integrative Database (TCMID) were used to screen the active compounds of GGCLT for herbal molecular mechanism analysis. Besides, literature analysis was performed to supplement the potential active ingredients and their pharmacological interactions. The fatty liver related genes were screened by Public Health Genomics and Precision Health Knowledge Base (v7.7). The intersection genes between targets of the active compounds of GGCLT and fatty liver related genes were overlapped by Venny 2.1.0.

The protein-protein interaction (PPI) network was constructed by Cytoscape 3.9.1 (Institute of Systems Biology, Seattle, Washington, USA). Network Analyzer was used for network diagrams. Construction of PPI network and screening of core targets fatty liver-related target proteins were analyzed by online STRING11.5 database to construct the PPI network. Protein type was set as “Homo sapiens” with the minimum interaction threshold of 0.4.

The Traditional Chinese Medicine Integrative Database (TCMID, Bioinformatics & Drug Design group in Department of Pharmacy National University, Singapore) was used to screen the GO function and KEGG pathway analysis that were performed to determine the function of candidate genes in the drug-compound-disease-target gene network. GO term, including biological process (BP), molecular functions (MF), cellular components (CC) and KEGG pathway with p 0.05 were considered as statistically significant.

The National Laboratory Animal Center (Taiwan) provided the C57BL/6J and BKS.Cg-m ${}^{+/+}$ Leprdb/JNarl (db/db) mice utilized in this investigation. The animal experiments were performed in accordance with the Chang Gung University Institutional Animal Care and Use Committee (CGU107-265). The mice were then matched into 4 groups (n = 5/group). (1) normal mice, (2) db/db mice; (3) db/db mice + GGCLT 50 mg/kg (oral, daily for 28 consecutive days); (4) db/db mice + GGCLT 150 mg/kg (oral, daily for 28 consecutive days). The raw components of Puerariae Radix (Pueraria lobata roots, Ge Gen), Scutellariae Radix (Scullellaria baicalensis roots, Huang Qin), Coptidis Rhizoma (Coptis chinensis rhizomes, Huang Lian), and Glycyrrhizae Radix (Glycyrrhiza uralensis roots, Gan Cao) were combined to create the powdere [20, 21]. The dried compound was subsequently dissolved in dH${}_2$O before use. After 28 days, animals were terminated by gradual fill isoflurane asphyxiation.

After a 6-hour fasting, a glucose load (1 g/kg of body weight) was administered by gavage or by intraperitoneal (i.p.) injection. Blood samples were collected from the tail at 0 (baseline), 15, 30, 60, 90 and 120 minutes after the glucose loads. Blood glucose levels were measured with a Blood Glucose Monitoring System (OneTouch® Ultra, Lifescan, Johnson & Johnson, USA). AUCs of glucose levels calculated according to Trapezoid rules with those from GTT.

After a 6-hour fasting, an insulin load (0.75 U/kg of body weight; Lilly, USA) was administered by gavage or by intraperitoneal (i.p.) injection. Blood samples were collected from the tail at 0 (baseline), 15, 30, 60, 90 and 120 minutes after the insulin loads. AUCs of glucose levels calculated according to Trapezoid rules with those from ITT. Plasma insulin levels were determined by ELISA kit.

The levels of fasting insulin and fasting glucose are used to compute the homeostatic model assessment of insulin resistance (HOMA-IR). Scores were calculated with the following equation: [fasting glucose (mmol/L) $\times$ fasting insulin (mIU/mL)] / 22.5 [22].

Prior to biochemical examination, plasma was collected by centrifugation and kept at –20 °C. The Randox ATL and AST test was used to assess the plasma concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Randox, Kearneysville, WV, USA).

Using the Randox triglycerides test and the NEFA Assay, the concentrations of plasma triglycerides (TG) and free fatty acids (FFA) were both directly assessed (Randox, Kearneysville, WV, USA).

Liver and EWAT were embedded in paraffin after being fixed overnight in 10% Neutral buffered formalin. Then, xylene was used to deparaffinize portions that were five micrometers thick, and a graded ethanol series was used to dehydrate them. Hematoxylin (2 g/L) was used to stain the tissue slices for 15 minutes, and eosin (0.1 percent in 0.0003 percent acetic acid) was used for 10 minutes at room temperature. Masson stain used by Trichrome Stain (Masson) Kit (abcam, Cambridge, USA). For immunohistochemistry, endogenous peroxidase activity was quenched with 3% H${}_2$O${}_2$ in water for 10 min. Non-specific staining was minimized with 5% normal goat serum for 50 min. Next, sections were subject to antigen retrieval, as needed, and incubated with primary and second antibodies for 2 hours at room temperature. The antibodies used in this study are shown in Table 1. Images were obtained using Olympus IX71 microscope.

Tissues were homogenized in T-PER™ Tissue Protein Extraction Reagent (0.30 mg tissue/200 $\mu$L; Thermo Fisher Scientific, MA, USA), NE-PER nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, MA, USA), or Mitochondria Isolation Kit (Thermo Fisher Scientific, MA, USA) containing proteinase inhibitors (1 $\mu$L/mL; Sigma-Aldrich; Merck KGaA, Rahway, NJ, USA). The proteinase inhibitors inhibit serine, cysteine, and acid proteases and aminopeptidases. The soluble proteins were quantified with the Bio-Rad Rapid Coomassie kit (Bio-Rad Laboratories, Hercules, California, USA), and then protein (60–80 $\mu$g/lane) was run on a 10% SDS-PAGE gel and subsequently transferred to a PVDF membrane (GE Healthcare, Chicago, Illinois, USA). Blocking was performed using Pierce™ Fast Blocking Buffer (Thermo Fisher Scientific, MA,USA), 1X TBS with 0.1% Tween-20 at room temperature for 1 h. For immunoblotting, membranes were incubated with diluted primary antibody in Pierce™ Fast Blocking Buffer at 4 °C with gentle shaking overnight. Following primary incubation, the secondary antibody was incubated at room temperature for 1 hour with gentle shaking in PierceTM Fast Blocking Buffer. The protein expression was detected using an enhanced chemiluminescence kit and quantified using ImageJ 1.53 software (National Institutes of Health, Maryland, USA). The primary and secondary antibodies used in this study are shown in Table 1. Restore™ PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific, MA, USA) was used for detection of proteins with comparable MW to $\beta$-actin protein (i.e., CCR7, p-p62, and p62). After washing the blot to remove the chemiluminescent substrate, incubate it in Restore Western Blot Stripping Buffer for 5 to 15 minutes at room temperature (or at 37 °C for high affinity antibodies).

Total RNA was extracted from 0.2 g liver and EWAT tissues using 1 mL of TRIzol (Thermo Fisher Scientific, MA, USA). The cDNA was reverse transcribed from RNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, MA,USA) according to the manufacturer’s instructions. The cDNA samples were amplified by qRT-PCR using SYBR Green I (Roche, Switzerland). For qPCR, the following thermocycling conditions were applied: Initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, and elongation at 72 °C for 0.5 min, before holding at 4 °C. By utilizing the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ approach to normalize each Ct result to either gene expression, relative mRNA expression levels were calculated. Table 2 (Ref. [3, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38]) displays the primer sequences applied in this research.

Crude mitochondrial fraction was extracted from liver and EWAT using Mitochondria Isolation Kit (Thermo Fisher Scientific, MA, USA) for Tissue followed by manufacturer’s instructions. With the assistance of the Bio-Rad Rapid Coomassie kit, the proteins were measured. For measurement of mitochondrial Complex I, III, and IV activity in liver and EWAT, using Mitochondrial Complex activity Assay Kit, respectively. For measurement of Citrate synthase activity and MDA using Colorimetric Assay kit followed by manufacturer’s instructions.

Examinations of serum TNF alpha, MCP-1 and IL-1$\beta$ were performed using mouse uncoated ELISA Kit mouse uncoated ELISA kit (Thermo Fisher Scientific, MA, USA).

All data are expressed as means $\pm$ SEM. Statistical analysis was performed with SPSS 21.0 software ((International Business Machines Corporation, Armonk, NY, USA). The experiments were repeated for three times. All data were analyzed using a one-way ANOVA followed by a Bonferroni’s post hoc test. p values less than 0.05 was considered to indicate a statistically significant difference.

A typical GGCLT HPLC-UV chromatography fingerprint profile was obtained and six major constituents were identified (Fig. 1). The well-separated peaks with retention times of 65 min and perentage of compound in GGCLT (lyophilizate, %) were identified as follows: (1) Puerarin (15.07 min, 28.90%); (2) Baicalin (33.55 min, 11.67%); (3) Berberine (40.15 min, 7.77); (4) Baicalein (49.81 min, 0.54%); (5) Glycyrrhizic acid (50.42 min, 1.22); (6) Wogonin (54.35 min, 0.28%) (Table 3). Puerarin is the bioactive ingredient of Puerariae Radix; baicalin, baicalein, wogonin are the bioactive ingredient of Scutellariae Radix; berberine is the bioactive ingredient of Coptidis Rhizoma; and glycyrrhizic acid is the bioactive ingredient of Glycyrrhizae Radix. Based on the results, Puerarin was found to be the most abundant compound of GGCLT (lyophilizate), followed by baicalin, berberine, glycyrrhizic acid, baicalein, and wogonin. In addition, the 3D-HPLC fingerprint and the structure of compounds are shown (Fig. 1).

A total of 26 genes overlapped between 512 target genes of the active compounds of GGCLT and 163 fatty liver related genes (Fig. 2A). Then, a PPI network was constructed based on 26 overlapped genes between target proteins and fatty liver relate genes (Fig. 2B). We found the network contained 26 nodes with interaction score $>$0.4, which represented the overlapped genes between target proteins of the active compounds of GGCLT and fatty liver related genes and their interaction relationship (Fig. 2C). In GO function and KEGG pathway enrichment analysis, we found that the top-regulated overlapped genes mainly enrich in response to regulation of lipid metabolism, such as long-chain fatty acid metabolic process, lipid metabolic process, and fatty acid metabolic process (Fig. 2C). KEGG pathway mainly enriched in such as insulin resistance, steroid hormone biosynthesis, Metabolic pathways, and adipocytokine signaling pathway (Fig. 2D).

Fig. 2.

GGCLT target prediction, creation of the PPI network, and analysis of enriched GO and KEGG pathways. Venn plot of the overlapped genes between targets of the active compounds of GGCLT and fatty liver related genes (A). The hub genes in the protein-protein interaction (PPI) network (B). PPI network of the 26 overlapped genes between target proteins of the active compounds of GGCLT and fatty liver related genes. Data show the top 5 remarkably enriched items in the cell component (CC), biological process (BP), molecular function (MF) (C). Data show the top 22 remarkably enriched items in the KEGG pathways (D).

When compared to normal mice, db/db animals had a higher body weight and liver weight/body weight ratio (Fig. 3A,B). GGCLT 150 mg/kg dramatically lowered body weight and the liver/body weight ratio in db/db mice (Fig. 3A,B). In addition, plasma ALT and AST were significantly decreased in db/db mice treated GGCLT compared with db/db mice (Fig. 3C,D). After 28 days GGCLT treatment, the glucose and GTT/ITT, fasting glucose, fasting insulin, and HOMA-IR were decreased compared with db/db mice (Fig. 3E–K), suggesting that GGCLT improved whole-body insulin sensitivity. GGCLT significant reduced plasma TG and FFA level compared with db/db mice (Fig. 3J,K), as well as reduced liver steatosis and fibrosis (Fig. 3L). As shown in Fig. 3L–O, the SREBP-1c and downstream target genes, Fas, Scd1 and Acc1 mRNA expression were significantly increased as well as fatty acid transporters (Cd36, Fatp, Cpt1 and Cpt2) were significantly increased in the liver of db/db mice (Fig. 3L–O). Moreover, GGCLT significantly reduced hepatic lipogenesis therefore decreased fatty acid uptake in NAFLD model.

Fig. 3.

GGCLT prevents NAFLD development in db/db mice. Body weight (g) (A). Liver weight/ body weight (%) (B). Plasma ALT (U/L) (C). Plasma AST (U/L) (D). Glucose tolerance test (E). Insulin tolerance test (F). Fasting glucose (mmol/L) (G). Fasting insulin (mIU/mL) (H). HOMA-IR (I). Plasma TG (mg/dL) (J). Plasma FFA (mmol/L) (K). Representative HE, Masson, SREBP-1c and CD36 staining of liver (L). Red arrow highlights the positive staining. Scale bar: 50 $\mu$m. Quantification of SREBP-1c and CD36 protein levels by Western blot of liver (M). Right graphs indicate quantification relative to Histone or $\beta$-actin. Quantification of Srebp-1, Fas, Scd1, Acc1, Cd36, Fatp, Cpt1 and Cpt2 by qRT-PCR (N,O). qRT-PCR indicates quantification relative to Gapdh. For each animal group, n = 5. All values represent the mean $\pm$ SEM. *p 0.05; normal vs. db/db. #p 0.05; db/db vs. db/db + GGCLT 50 mg/kg. ##p 0.05; db/db vs. db/db + GGCLT 150 mg/kg.

The serum and hepatic TNF-$\alpha$, MCP1, and IL-1$\beta$ content of db/db mice group were significantly higher than those of the normal control group (Fig. 4A,B). Tnf$\alpha$, interferon gamma (Ifn$\gamma$), Mcp1, and IL-1$\beta$ mRNA expression were significantly increased in liver of db/db mice compared with normal mice (Fig. 4C). The inflammasome factors, NACHT, LRR and PYD domains-containing protein 3 (Nlrp3), absent in melanoma 2 (Aim2), apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), and Caspase 1 mRNA expression were also higher in db/db mice compared with normal mice. GGCLT significantly decreased the levels of TNF-$\alpha$, MCP1, and IL-1$\beta$ in serum and liver tissue as well as reduced pro-inflammation cytokines and NLRP3 inflammasome in db/db mice (Fig. 4A–D). We next confirmed that GGCLT attenuated angiogenesis markers, hypoxia-inducible factor 1$\alpha$ (HIF1$\alpha$), vascular endothelial growth factor (VEGF), matrix metallopeptidase 9 (MMP9), and monocyte Chemoattractant Protein-1 (MCP-1) protein levels were increased in db/db mice, and GGCLT treatment significantly decreased angiogenesis markers (Fig. 4E,F).

Fig. 4.

GGCLT reduced hepatic inflammation and M1 polarization in db/db mice. Serum concentration of TNF$\alpha$, MCP1, and Il-1$\beta$ (A). Liver concentration of TNF$\alpha$, MCP1, and Il-1$\beta$ (B). Quantification of Tnf$\alpha$, Ifn$\gamma$, Mcp1, Il-1$\beta$, Nlrp3, Aim2, Asc and Caspase1 by qRT-PCR. qRT-PCR indicates quantification relative to Gapdh (C,D). Representative HIF1$\alpha$, VEGF, MMP9 and MCP-1 staining of liver (E). Red arrow highlights the positive staining. Scale bar: 50 $\mu$m. Quantification of HIF1$\alpha$, VEGF and MCP1 protein levels by Western blot of liver. Right graphs indicate quantification relative to Histone or $\beta$-actin (F). Representative F4/80, CD11b, CD11c, CCR7 and CD163 staining of liver (G). Red arrow highlights the positive staining. Scale bar: 50 $\mu$m. Quantification of CD11b, CD11c, CCR7 and CD163 protein levels by Western blot of liver (H). Right graphs indicate quantification relative to $\beta$-actin. Ratio of CD11c/CD206 and CCR7/CD163 in liver. For each animal group, n = 5. All values represent the mean $\pm$ SEM. *p 0.05; normal vs. db/db. #p 0.05; db/db vs. db/db + GGCLT 50 mg/kg. ##p 0.05; db/db vs. db/db + GGCLT 150 mg/kg.

F4/80-positive macrophage infiltration in the db/db mce was considerably enhanced in the liver, which corresponded to the level of serum inflammatory cytokine expression. Macrophage infiltration in the group administered GGCLT 150 mg/kg was significantly lower than that in the db/db mice (Fig. 4G,H). Next, we examined whether macrophagy polarization is involved in NAFLD in db/db mice by assesing M1/M2 marker expression. Immunohistochemistry analysis showed a significant reduction in F4/80, CD11b, CD11c, CCR7 and increase in CD163, CD206 protein level in db/db mice treated with GGCLT 150 mg/kg compared to db/db mice (Fig. 4G,H).

The SIRT1, PGC1$\alpha$, and UCP1 protein levels and mRNA expression in liver of db/db mice were significantly lower than normal mice, but GGCLT 150 mg/kg treatment improved the reduced markers in db/db mice (Fig. 5A–C). Also, the mitochondrial complex I (20kDa), II (Ip), III (Core II), IV (Cox2), and V (F1$\alpha$) in liver were determined. GGCLT enhanced the mitochondrial complexes mRNA expression compared to db/db mice (Fig. 5D) and decreased enzymes activity including NADH: coenzyme Q oxidoreductase (mitochondrial complex I), coenzyme Q-cytochrome c reductase (mitochondrial complex III), cytochrome c oxidase (mitochondrial complex IV) and, citrate synthase were observed in liver of db/db mice compared with normal mice as shown in Fig. 5E–H. Malondialdehyde (MDA) levels were found to be much higher in db/db mice compared to normal mice, and high dosage GGCLT significantly decreased MDA levels in db/db mice (Fig. 5I).

Fig. 5.

GGCLT improved hepatic mitochondrial biogenesis in db/db mice. Representative SIRT1, PGC1$\alpha$, and UCP1 staining of liver (A). Red arrow highlights the positive staining. Scale bar: 50 $\mu$m. Quantification of SIRT1, PGC1$\alpha$, and UCP1 protein levels by Western blot of liver (B). Right graphs indicate quantification relative to Histone or $\beta$-actin. Quantification of Sirt1, Pgc1$\alpha$, Ucp1, 20kDa/Complex I, Ip/Complex II, Core II/Complex III, Cox2/Complex IV and F1$\alpha$/Complex V by qRT-PCR (C,D). qRT-PCR indicates quantification relative to Gapdh. Mitochondrial complex I, III, and IV activity (percent of control) (E-G). Mitochondrial citrate synthase activity (H). Malondialdehyde (MDA) levels in liver (nM/mg protein) (I). For each animal group, n = 5. All values represent the mean $\pm$ SEM. *p 0.05; normal vs. db/db. #p 0.05; db/db vs. db/db + GGCLT 50 mg/kg. ##p 0.05; db/db vs. db/db + GGCLT 150 mg/kg.

Compared to normal mice, the protein expression levels of Parkin, LC3B, phospho (p)-p62, and mitofusin-1 (MNF1) in the liver of db/db mice were decreased but phospho-dynamin-related protein (p-Drp1) expression was increased (Fig. 6A,B). The GGCLT administration for 28 days, the opposite expression patterns were observed (Fig. 6A,B). Moreover, GGCLT effectively enhanced the expression of Parkin-dependent/independent mitophagy pathway, including Pink, calcium binding and coiled-coil domain 2 (Ndp52/Calcoco2), prohibitin 2 (Phb2), autophagy and beclin 1 regulator 1 (Ambra1), B-cell lymphoma 2 (Bcl2), BCL2 interacting protein 3 (Bnip3), FUN14 domain containing 1 (Fundc1) compared to db/db mice (Fig. 6C,D).

Fig. 6.

GGCLT enhanced hepatic mitophagy in db/db mice. Representative Parkin, LC3B, p-p62, MNF1, and p-DRP1 staining of liver. Red arrow highlights the positive staining (A). Scale bar: 50 $\mu$m. Quantification of Parkin, LC3B, p-p62, p62, MNF1, p-DRP1, and DRP1 protein levels by Western blot of liver (B). Right graphs indicate quantification relative to $\beta$-actin. Ration of p-p62/p62 and p-DRP1/DRP1 in live. Quantification of Pink, Ndp52, Ambra1, Phb2, Bnip3, Nix, Fundc1 and Bcl2 by qRT-PCR. qRT-PCR indicates quantification relative to Gapdh (C,D). For each animal group, n = 5. All values represent the mean $\pm$ SEM. *p 0.05; normal vs. db/db. #p 0.05; db/db vs. db/db + GGCLT 50 mg/kg. ##p 0.05; db/db vs. db/db + GGCLT 150 mg/kg. MNF1, mitofusin-1; Drp1, dynamin-related protein; Ndp52/Calcoco2, calcium binding and coiled-coil domain 2; Phb2, prohibitin 2; Ambra1, autophagy and beclin 1 regulator 1; Bcl2, B-cell lymphoma 2; Bnip3, BCL2 interacting protein 3; Fundc1, FUN14 domain containing 1.

The shape of EWAT and adipocyte diameter size were large in db/db compared with normal mice (Fig. 7A,B), and GGCLT 150 mg/kg significant reduced EWAT size and adipocyte diameter size. GGCLT resulted in dramatically reduced the SREBP-1c and target genes, Fas, SCD1, Scd1, as well as fatty acid transporters (Cd36, Fatp, Cpt1 and Cpt2) mRNA expression in EWAT of db/db mice (Fig. 7C–F). Moreover, GGCLT significantly reduced hepatic lipogenesis and fatty acid transporters, suggesting that GGCLT regulated lipid homeostasis in db/db mice.

Fig. 7.

GGCLT attenuated lipogenesis in EWAT of db/db mice. Representative pictures of EWAT in mice (A). Adipocyte diameter ($\mu$m) (B). Representative HE, Masson, SREBP-1c and CD36 staining of EWAT (C). Red arrow highlights the positive staining. Scale bar: 50 $\mu$m. Quantification of SREBP-1c and CD36 protein levels by Western blot of EWAT (D). Right graphs indicate quantification relative to Histone or $\beta$-actin. Quantification of Srebp-1, Fas, Scd-1, Acc1, Cd36, Fatp, Cpt-1 and Cpt-2 by qRT-PCR (E,F). qRT-PCR indicates quantification relative to Gapdh. For each animal group, n = 5. All values represent the mean $\pm$ SEM. *p 0.05; normal vs. db/db. #p 0.05; db/db vs. db/db + GGCLT 50 mg/kg. ##p 0.05; db/db vs. db/db + GGCLT 150 mg/kg. Srebp-1, sterol regulatory element-binding protein 1; Fas, fatty acid synthase; Scd-1, stearoyl-CoA desaturase-1; Acc1, acetyl-CoA carboxylase; Fatp, fatty acid transport protein; Cd36, cluster of differentiation 36; Cpt1, carnitine palmitoyltransferase 1; EWAT, epididymis white adipose tissue.

db/db mice had higher Tnf$\alpha$, Ifn$\gamma$, Mcp1, IL-1$\beta$, Nlrp3, Aim2, Asc, and Caspase 1 mRNA expression in EWAT, after GGCLT treatment 28 days, the proinflammatory cytokines and NLRP3 inflammasome were significantly reduced compared with db/db mice (Fig. 8A,B). HIF1$\alpha$, VEGF, MMP9, and MCP1 protein levels were increased in db/db mice, and GGCLT 150 mg/kg treatment significantly decreased angiogenesis markers in EWAT compared to db/db mice (Fig. 8C,D).

Fig. 8.

GGCLT reduced inflammation and M1 polarization in EWAT of db/db mice. Quantification of Tnf$\alpha$, Ifn$\gamma$, Mcp1, Il-1$\beta$, Nlrp3, Aim2, Asc and Caspase1 by qRT-PCR (A,B). Representative HIF1$\alpha$, VEGF, MMP9 and MCP-1 staining of EWAT (C). Red arrow highlights the positive staining. Scale bar: 50 $\mu$m. Quantification of HIF1$\alpha$, VEGF and MCP-1 protein levels by Western blot of EWA (D). Right graphs indicate quantification relative to Histone or $\beta$-actin. qRT-PCR indicates quantification relative to Gapdh. Representative F4/80, CD11b, CD11c, CCR7 and CD163 staining of EWAT (E). Red arrow highlights the positive staining. Scale bar: 50 $\mu$m. Quantification of CD11b, CD11c, CCR7 and CD163 protein levels by Western blot of EWAT (F). Right graphs indicate quantification relative to $\beta$-actin. Ratio of CD11c/CD206 and CCR7/CD163 in EWAT. For each animal group, n = 5. All values represent the mean $\pm$ SEM. *p 0.05; normal vs. db/db. #p 0.05; db/db vs. db/db + GGCLT 50 mg/kg. ##p 0.05; db/db vs. db/db + GGCLT 150 mg/kg. EWAT: epididymis white adipose tissue.

The EWAT dramatically boosted F4/80-positive macrophage infiltration in the db/db mce. Macrophage infiltration was considerably lower in the GGCLT group than in the db/db mice (Fig. 8E). Macrophages were increased in EWAT of db/db mice were due to an increase in CD11b, CD11c, and CCR7 compared with normal mice. GGCLT reduced macrophage infiltration (F4/80, CD11b) and M1 macrophage markers (CD11c, CCR7) in EWAT of db/db mice (Fig. 8E,F). On the contrary, GGCLT 150 mg/kg enhanced M2 macrophage markers (CD163, CD206) protein levels in EWAT of db/db mice (Fig. 8E,F).

Consistent with the histological features, GGCLT enhance mitochondria biogenesis and thermogenesis markers, SIRT1, PGC-1$\alpha$, UCP1, and transmembrane protein 26 (Tmem26) protein levels in EWAT of db/db mice as compared to db/db mice (Fig. 9A–C). GGCLT 150 mg/kg enhanced the mitochondrial complexes mRNA expression compared to db/db mice (Fig. 9D). Thermogenic genes, mitochondrial transcription factor A (Tfam), Cd137, Tmem26, PR domain containing 16 (Prdm16), cytochrome c oxidase polypeptide 7A1 (Cox7a1), Cox8b, Zinc finger protein 1 (Zic1), and Lxh8 mRNA expression were increased in db/db mice treatment GGCLT (Fig. 9E,F). Decreased enzymes activity including NADH: coenzyme Q oxidoreductase (mitochondrial complex I), coenzyme Q - cytochrome c reductase (mitochondrial complex III), cytochrome c oxidase (mitochondrial complex IV) and, citrate synthase were observed in EWAT of db/db mice compared with normal mice (Fig. 9G–J). MDA levels determined in the EWAT tissue were found to be significantly lower in db/db mice that treat with GGCLT (Fig. 9K).

Fig. 9.

GGCLT improved mitochondrial biogenesis in EWAT of db/db mice. Representative SIRT1, PGC1$\alpha$, and UCP1 staining of EWAT (A). Red arrow highlights the positive staining. Scale bar: 50 $\mu$m. Quantification of SIRT1, PGC1$\alpha$, and UCP1 protein levels by Western blot of EWAT (B). Right graphs indicate quantification relative to Histone or $\beta$-actin. Quantification of Sirt1, Pgc1$\alpha$, Ucp1, 20kDa/Complex I, Ip/Complex II, Core II/Complex III, Cox2/Complex IV and F1$\alpha$/Complex V by qRT-PCR (C–F). qRT-PCR indicates quantification relative to Gapdh. Mitochondrial complex I, III, and IV activity (percent of control) (G–I). Mitochondrial citrate synthase activity (J). Malondialdehyde (MDA) levels in EWAT (nM/mg protein) (K). For each animal group, n = 5. All values represent the mean $\pm$ SEM. *p 0.05; normal vs. db/db. #p 0.05; db/db vs. db/db + GGCLT 50 mg/kg. ##p 0.05; db/db vs. db/db + GGCLT 150 mg/kg.