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. 2023 Apr 7;12(2):100948. doi: 10.1016/j.imr.2023.100948

Effect and mechanism of Magnolia officinalis pharmacopuncture for treating localized fat via network pharmacology and experimental study

Won Jun Choi 1,1, Mi Hye Kim 1,1, Nayoung Park 1, Jae Yoon Chung 1, Sang Jun Park 1, Woong Mo Yang 1,⁎
PMCID: PMC10173613  PMID: 37181416

Abstract

Background

Recently, for various reasons, the need for non-invasive treatment for localized fat has emerged. This study confirmed whether Magnolia officinalis (MO) pharmacopuncture reduces localized fat by promoting lipolysis and inhibiting adipogenesis.

Methods

The network was built using genes related to the active compound of MO and the mode of action of MO was predicted by the functional enrichment analysis. Based on the result from network analysis, 100 µL of 2 mg/mL MO pharmacopuncture was injected into the inguinal fat pad for 6 weeks in obese C57BL/6J mice. Normal saline was injected into the right-side inguinal fat pad as a self-control.

Results

It was expected that the ‘AMP-activated protein kinase (AMPK) signaling pathway’ would be affected by the MO Network. MO pharmacopuncture reduced the weight and size of inguinal fat in HFD-induced obese mice. The phosphorylation of AMPK along with the increases of lipases was significantly increased by MO injection. Also, the expression levels of fatty acid synthesize-related mediators were suppressed by MO injection.

Conclusion

Our results demonstrated that MO pharmacopuncture promoted the expression of AMPK, which has beneficial effects on activation of lipolysis and inhibition of lipogenesis. Pharmacopuncture of MO can be a non-surgical alternative therapy in the treatment of local fat tissue.

Keywords: Magnolia officinalis, Pharmacopuncture, Localized fat, Network pharmacology, AMPK signaling pathway

1. Introduction

Excessive accumulation of fat tissue, especially local areas such as the abdomen and thighs, causes chronic diseases, including cancer, cardiovascular diseases and diabetes as well as esthetic problems.1 As the lifestyle of consuming too much sugar, fat, and salt becomes widespread, an imbalance between energy intake and consumption is caused, resulting in the accumulation of localized fat.2 There has been a need to reduce fat tissue by improving lifestyle, however, it is not easy to alter lifestyle. People want to get rid of fat through surgery, medication, and other procedures.

There are five types such as obesity gene product inhibitors, digestion and absorption blockers, metabolic promoters, central appetite suppressants, and other drugs used as treatments for obesity.3 However, drugs prescribed for weight loss cause many side effects and become social problems. In particular, it has been reported to affect monoamine neurotransmitters and eventually induce drug abuse and drug dependence.4 In the case of shibutramine, it causes various side effects, including symptoms such as thrombosis, anorexia, insomnia, dry mouth, and constipation.5,6 Liposuction has become a common cosmetic and esthetic surgery based on the aspiration of the beauty and body shape. However, lethal complications including necrotizing fasciitis, toxic shock syndrome, fat embolism syndrome, and even death could be accompanied with the liposuction procedure.7 For that reason, there is an increasing need for new treatments to treat localized fat tissue without side effects. In this respect, there are herbs with a long history of use have little toxicity and have the effect of promoting appetite loss and weight loss. Commonly, treatments using natural substances have been regarded as a safer and more economical alternative to weight loss.7 Pharmacopuncture is a common acupuncture technique in traditional Korean Medicine.8 This technique has been used as a non-surgical alternative to treat a localized obesity.9

The magnolia cortical cortex (family name Magnoliaceae), named Hubak in Korea, has been used to strengthen the gastrointestinal tract, induce sedative effects and treat ulcers, asthma, headaches, muscular pain, and fever in traditional Korean Medicine.10 Magnolia bark has been reported to contain several bioactive ingredients such as methyl-honokiol, obovatal, magnolol, and honokiol, which have various functions.10 Recent studies have shown that magnolol and honokiol, which are bioactive components of Magnolia, improved insulin resistance and inhibited body fat accumulation in high-fat diet (HFD) mice.11 In this study, we studied the effectiveness of Magnolia officinalis Rehder & E. Wilson (MO) pharmacopuncture on inguinal fat pad of obesity-induced mice through the network pharmacology-based prediction result, which showed the possible molecular mechanism of MO. Then, the lipolytic effects of MO were investigated in the HFD-induced obese mice.

2. Method

2.1. MO network construction

To construct an MO network, compounds and genes related to MO were collected through open scientific databases, TM-MC database, which is a database of medical materials and chemical compounds in Northeast Asian traditional medicine. Among 147 compounds of MO, eliminating overlapped or uninformed ones, 54 active compounds were selected. With 54 compounds, genes with co-occurrence for each compound were gathered through PubChem (https://pubchem.ncbi.nlm.nih.gov/). Among 2598 genes associated with the 54 compounds, removing duplicates, 751 genes were sorted through STRING database (http://www.string-db.org/) with a score ≥ 0.7, which represents high confidence. In total, 751 genes were used to create a network of MO. With the 751 genes of MO, we constructed a MO network of 751 nodes and 5721 edges. The nodes showed genes of MO, the edges showed the connectivity between the genes.

2.2. Functional enrichment analysis

Functional enrichment analysis was conducted to analyze the MO network, using Cytoscape StringApp, based on KEGG 2021 pathway. Enrichment analysis is a method to identify a huge set of gene targets and to discover related pathways and mechanisms. Enrich is a website which contains a large collection of diverse gene set libraries and provide tools to analyze the network of target gene set. KEGG is an integrated database of systems, genomic, chemical and health information. The enrichment analysis was conducted to analyze the MO network, to predict potential mechanisms of MO.

2.3. Preparation of samples

MO (Lot. DY261S04H) was acquired from Dong-Yang Herb Inc. (Seoul, Korea). 20 g of MO was extracted with 300 mL of distilled water for 3 h at 100 °C. The extract was concentrated by rotary vacuum evaporator (Eyela, Japan) after decompression filtration using 10 µm filter paper (Hyundai micro, Korea). The concentrate of MO extract was powdered by freezing dryer (yield: 14.9%) (Ilshin Bio, Korea). Sample was stored at −20 °C until use.

2.4. Animal treatment

All experiments were conducted according to the guidelines of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by Committee on Care and Use of Laboratory Animals of the Kyung Hee Univ. (KHSASP-21–270). 5 week-old male C57BL/6 J mice were purchased from DBL Inc. (Chungbuk, Korea). During the experiment, mice were raised in under 23 ± 2 °C temperature and 50 ± 10% relative humidity. The number of C57BL/6 J mice were 8 per group. After 1 week of accommodation, HFD containing 60% fat were fed to induce obesity. After 6 weeks of HFD feeding, 2 mg/mL of MO pharmacopuncture was injected to left inguinal fat pad (sample), and normal saline was injected to right inguinal fat pad (vehicle) for self-control. The injection solution of the MO pharmacopuncture was adjusted to pH 7 ± 10%, by dissolving MO powder in normal saline solution in order to avoid changes in osmotic pressure and side effect such as shock. The injection solution was then filtered by sterile syringe filters with a 0.22 μm pore size (Merk Millpore, MA, USA). Injection volume MO solution and normal saline was 100 μL per mice, respectively. The injection was conducted 3 times per week for 6 weeks. Daily food intake and body weight were measured once a week through whole animal experiments. At the end of experiment, all mice were scanned using a total body scanner and sacrificed. Both side of the inguinal fat pad was excised separately and weighted. The regions to determine the weight of inguinal fat pad were from knee to tail based on line of ventral spine.

2.5. Dual X-ray absorptiometry

Whole body scan was conducted using a dual X-ray absorptiometry (DXA; InAlyzer, Medikors, Gyeonggi, Korea). Through a software in the device of InAlyzer, fat distribution was monitored by a mapping image. At the end of experiment, all animals under anesthesia were scanned. The weight of respective inguinal fat tissues indicated red color was measured by a software in the device.

2.6. Histopathological analysis

Both sides of inguinal fat pad, liver, kidney, spleen tissues acquired from mice were excised and fixed with 10% neutralized formalin for overnight. Then, inguinal fat pad tissues were dehydrated and embedded in paraffin wax. The paraffin blocks were dissected into 5 μm using a microtome (HM355S, Thermo Scientific, UK). After deparaffinization and rehydration of each tissue sections, tissues were stained with hematoxylin and eosin (H&E). All slides were observed under an optical microscope (Leica DM 500; Leica, Wetzlar, Germany). H&E-stained sections were randomly selected, and average diameter of adipocyte was measured using an automated analysis program Image J (National Institutes of Health, Bethesda, MD, USA).

2.7. RNA extraction and RT-PCR

Total riboNucleic acid (RNA) was isolated from inguinal fat tissue using the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). Chloroform was added to homogenized tissues to separate the fractions. The supernatants were mixed thoroughly with one volume of 2-propanol to precipitate the RNA pellets. Total RNA pellets were dissolved in RNase free water. Complementary DNA (cDNA) was synthesized from 1 μg RNA of inguinal fat tissue using Maxime RT premix kit (Invitrogen, Carlsbad, CA, USA). cDNA was amplified by specific primers using Maxime PCR premix kit (Invitrogen, Carlsbad, CA, USA) at 45 °C for 60 min and then at 95 °C for 5 min. Then, the expression of transcripts was analyzed using polymerase chain reaction (PCR) with Maxime PCR premix kit (Invitrogen Corp.). The PCR products were separated by gel-electrophorosis on 2% agarose gel and stained with ethidium bromide staining.

2.8. Western blotting

Total protein was extracted from inguinal fat tissue by the tissue protein extraction reagent (T-PER; Thermo, USA) with a protease inhibitor (Roche, Hoffmann, USA). The protein concentrations were calculated by a Bradford assay solution (Bio-Rad, CA, USA). Then, 10 μg of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electro-transferred to activated polyvinylidene fluoride membranes (Bio-Rad, CA, USA). The membrane was then blocked with 3% bovine serum albumin and incubated with primary antibodies (1:2000, Cell signaling, USA) overnight at 4 °C. The membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:4500, Cell signaling, USA) at room temperature for 1 h and the blots were analyzed with detected with an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden).

2.9. Measurement of serum toxicity

Serum samples were separated by centrifuging the blood at 17,000 rpm for 20 min. Serum biochemical indicators including aspartate transaminase (AST) and alanine transaminase (ALT) were analyzed by respective commercially available assay kits. Based on standard curve, serum AST and ALT was calculated.

2.10. Statistical analysis

All results were indicated as the means ± standard error of the mean (S.E.M.). For comparison with data, one-way ANOVA followed by Tukey's multiple tests was used in these groups. The p values were given as follows: *p < 0.05, ⁎⁎p < 0.01, ⁎⁎⁎p < 0.001 were considered statistically significant.

3. Results

3.1. Functional enrichment analysis of MO network

MO network was constructed with 751 genes related to MO. 751 genes were from the 54 active compounds including honokiol, caffeic acid, esculetin, phenol, and magnolol, etc. The Network is composed of 751 nodes, which are target genes, and 5721 edges, which show the connections between nodes. According to the degrees, which represent several edges of the node, 751 nodes were visualized in centralized order (Fig. 1A). Functional enrichment analysis was performed on MO network to predict the potential effects on obesity. Whole genes in network were analyzed based on KEGG 2021 pathway. Based on p value, Top 7 Pathways of MO Network in KEGG pathway were detected. ‘AMP-activated kinase (AMPK) signaling pathway’ was expected to be the most relevant pathway to MO, followed by ‘Peroxisome proliferator-activated receptor (PPAR) signaling pathway’, ‘Insulin signaling pathway’, ‘cyclic adenosine monophosphate (cAMP) signaling pathway’, ‘Regulation of lipolysis in adipocytes’, ‘Fatty acid metabolism’ and ‘Fat digestion and absorption’ (Fig. 1B). As a result, functional enrichment analysis of MO network indicates that MO is expected to have the potential effects in lipid metabolism related to ‘AMPK signaling pathway’.

Fig. 1.

Fig 1

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Functional enrichment analysis of MO network and Biological processes related to target genes of MO. (A) Network of MO with 751 nodes and 5721 edges. (B) Enrichment analysis extracted from KEGG of target genes of MO network. (C) Common genes of MO network and AMPK signaling pathway derived from KEGG pathway 2021 human. (D) Diagram of AMPK pathway derived from KEGG pathway 2021 human. Matched genes were marked in red box.

3.2. Comparison with the target genes of MO and AMPK signaling pathway

As a result to compare the target genes of MO and gene set of AMPK signaling pathway, 31 crossover genes were overlapped. ACACA, ACACB, AKT1, CCNA2, CCND1, CD36, CPT1A, CPT1B, EEF2, FASN, FOXO1, G6PC, HMGCR, INS, INSR, IRS1, IRS2, LEP, LIPE, MAP3K7, MTOR, PDPK1, PIK3CA, PIK3R1, PPARG, PPARGC1A, RPS6KB1, SCD, SIRT1, STK11, ULK1 were identified as common genes of MO and AMPK signaling pathway (Fig. 1C). On a KEGG Pathway 2021 human map of the AMPK signaling pathway, those common genes were marked in red box and the paths of marked genes were highlighted in green box (Fig. 1D).

3.3. Effects of MO on weight of inguinal fat tissues in obese mice

The line with blue dots indicated inguinal fat site and distribution in HFD-induced obese mice. The value of fat weight in the vehicle-injected side pad was 503.7 ± 45.4 mg, while that in the MO-injected side pad was 336.5 ± 26.8 mg. Injection of MO pharmacopuncture significantly reduced inguinal fat weight by about 33.1% (Fig. 2A). Inguinal fat tissue designated regions of inguinal fat was marked as yellow dots in DXA scan image. Fat tissue expressed by red intensity in body composition image was markedly decreased by MO injection. Inguinal fat pad weight of vehicle injected side was 809.6 ± 99.4 mg, while that of MO injected side was 619.3 ± 57.9 mg. Inguinal fat pad of MO-injected side showed significant decrease in fat weight by about 23.5% compared to that of vehicle-injected side (Fig. 2B).

Fig. 2.

Fig 2

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Change of inguinal fat mass in HFD-induced obese mice by treatment of MO. (A) Representative morphological images of inguinal fat tissues with relative weight ratio of inguinal fat collected from knee to tail based on line of ventral spine at the end of the experiment. Blue dots from knee to tail indicate the designated regions of inguinal fat. Relative ratio of fat weight after MO treatment (n = 8) was calculated when the saline‑treated fat weight was normalized to 1. (B) Representative mapping images of body composition of mice constructed using DXA software with relative ratio of fat weight measured by targeting regions of interest (the designated regions of inguinal fat pad) indicated by yellow dots in DXA software. Yellow dots from knee to tail indicate the designated regions of inguinal fat. Red, fat tissue; blue, lean tissue; white, bone tissue. The saline‑treated fat weight was normalized to 1 and the inguinal fat pad weight after MO treatment (n = 8) was calculated as the relative intensity. Quantitative data are presented as the mean ± standard error of the mean and Tukey's multiple comparison tests were used to determine the statistical significance. ⁎⁎⁎p < 0.001 compared with saline‑treated side (Vehicle). (C) Histological changes of inguinal fat size in HFD-induced obese mice by treatment of MO. Representative histological images of inguinal fat tissues (n = 8). Size of adipocyte were evaluated by H&E staining and section of inguinal fat tissues were shown at 400 × magnification. The fat diameter of adipocyte was quantified by Image J program. Quantitative data are presented as the mean ± standard error of the mean. ⁎⁎⁎p < 0.001 compared with saline‑treated side (Vehicle).

3.4. Effects of MO on histological change of inguinal fat tissues in obese mice

H&E staining showed the size of adipocyte in the inguinal fat pad. The lipid droplets in MO-injected side of inguinal fat were smaller than those in vehicle-treated side. The diameter of adipocyte in inguinal fat pad tissues was significantly decreased by about 31.4% by MO treatment (Fig. 2C).

3.5. Effects of MO on the phosphorylation of AMPK in the inguinal fat tissues of obese mice

The phosphorylated AMPK protein expression was investigated in the inguinal fat tissues. The expression of phosphorylated AMPK was significantly up-regulated by 122.8% by injection with 2 mg/mL of MO pharmacopuncture (Fig. 3A).

Fig. 3.

Fig 3

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Expression of phosphorylated AMPK signaling pathway with lipolytic factors in inguinal fat tissues of HFD-induced obese mice. (A) Expression of AMPK in inguinal fat tissues of HFD-induced obese mice. (B) Expression of lipolytic enzymes including ATGL, HSL and MGL in inguinal fat tissues of HFD-induced obese mice. Quantitative data are presented as the mean ± standard error of the mean. ⁎⁎p < 0.01 and ⁎⁎⁎p < 0.001 compared with saline‑treated side (Vehicle). (C) Expression of CGI-58 and perilipin in inguinal fat tissues of HFD-induced obese mice. Quantitative data are presented as the mean ± standard error of the mean and Tukey's multiple comparison tests were used to determine the statistical significance. ⁎⁎p < 0.01 and ⁎⁎⁎p < 0.001 compared with saline‑treated side (Vehicle).

3.6. Effects of MO on the expressions of lipolytic enzymes in inguinal fat tissues of obese mice

The protein expression of lipolytic enzymes, such as adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL) after 6 weeks with or without MO injection was analyzed in the inguinal fat tissues, respectively. Compared to saline-treated side, the expression level of ATGL was increased by 1194% in MO injection side. The expression levels of HSL in the HFD-induced adipose tissues significantly increased about 118.3% by treatment with MO, respectively. Likewise, MO treatment enhanced the expression level of MGL about 268.4% in inguinal fat pad (Fig. 3B).

3.7. Effect of MO on the expression of lipolysis-related factors in inguinal fat tissues of obese mice

The lipolysis-related factors including comparative gene identification-58 (CGI-58) and perilipin were measured in the inguinal fat tissues. Treatment of MO pharmacopuncture significantly increased the protein expression level of CGI-58 by 184.4% compared to normal saline-treated side of inguinal fat tissue. Perilipin expression levels were decreased by 43.9% by MO injection in comparison to Vehicle side (Fig. 3C).

3.8. Effect of MO on the expression of lipogenesis-related mediators in inguinal fat tissues of obese mice

RT-PCR were performed to investigate the effects of MO on the expression levels of lipogenesis-related mediators including adiponectin, Cebpg CCAAT/enhancer binding protein-1 (C/EBP-1), Sterol regulatory element-binding proteins-1c (SREBP-1c) and Peroxisome proliferator-activated receptor-γ (PPAR-γ) in the inguinal fat tissue. The expression of adiponectin was increased in the MO-treated left inguinal fat pad compared to the saline-injected right inguinal fat pad. The mRNA expression of adiponectin (ADIPOQ) in the MO-treated left inguinal fat pads was significantly increased by 352.2%. The mRNA expression of PPAR-γ was reduced by 96% in the MO-treated left inguinal fat pad compared to saline-injected right inguinal fat pad. By injecting MO, the mRNA expression levels of C/EBP-1 (C/EBP1) and SREBP-1c (SREBF1) were significantly decreased to 60.0% and 10.5%, respectively. The expression of PPAR-γ mRNA (PPARG) was decreased in the MO-treated left inguinal fat pad compared to the saline-injected right inguinal fat pad (Fig. 4A).

Fig. 4.

Fig 4

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Expression of lipogenic and fatty acid biosynthesis-related mediators in inguinal fat tissues of HFD-induced obese mice. (A) mRNA expressions of adiponectin, C/EBP, SREBP-1c and PPAR-γ. (B) Protein expressions of FAS and phosphorylated ACC. (C) Protein expressions of phosphorylated mTOR. Quantitative data are presented as the mean ± standard error of the mean and Tukey's multiple comparison tests were used to determine the statistical significance. *p < 0.05 and ⁎⁎⁎p < 0.001 compared with saline‑treated side (Vehicle).

The protein expressions of lipogenic enzymes, such as fatty acid synthase (FAS) and Acetyl-CoA carboxylase (ACC), key enzymes regulating cellular lipogenesis and lipid homeostasis, were measured in the inguinal fat pads. The phosphorylated expression level of ACC in MO-treated left fat pad was significantly increased to 62.5%, compared with to saline-injected right inguinal fat pad. Compared to saline-injected right inguinal fat pad, the expression level of FAS was decreased by 34.7% in MO-treated left fat pad (Fig. 4B).

In addition, the expression of mammalian target of rapamycin (mTOR) was decreased in the MO-treated left inguinal fat pad compared to the saline-injected right side. The protein expression level of mTOR in the MO-treated left inguinal fat pads was significantly decreased by 57.9%. The expression of PPAR-γ fall down in the MO-treated left inguinal fat pad compared to the saline-injected right side. The protein expression of PPAR-γ was markedly reduced by 58.2% in the inguinal fat tissues of MO-treated side (Fig. 4C).

3.9. Toxicity of MO on the serum, liver, kidney and spleen in obese mice

In order to examine the potential toxic effect of MO, AST and ALT was analyzed. The levels of AST and ALT in individual mice were in normal range known as 46–221 U/L for AST and 22–133 U/L for ALT.12 MO-treated mice showed no toxicities in the serum levels of AST and ALT in obese mice (Fig. 5A). Histopathologically, in MO-treated liver tissues, the morphology of healthy liver lobules could be distinguished. In MO-treated spleen tissues, was alternately composed of red pulp and white pulp. Similarly, numerous inflammatory cells could not be infiltrated the MO-treated tissues, which suggests that there is no toxicity of the MO (Fig. 5B).

Fig. 5.

Fig 5

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(A) Serum AST and ALT levels in HFD-induced obese mice. Results are presented as mean ± standard error of the mean. (B) Histological toxicity evaluation of major organs after MO treatment. H&E staining of the liver, kidney, spleen from MO 2 mg/mL-treated group of mice.

4. Discussion

Localized fat, an accumulation of fat in specific areas of the body, occurs due to the imbalance between energy consumption and intake. In most women, this accumulation mainly deposits on the abdomen, the hip, the thighs, and the upper arm.13 Recently, non-invasive treatment for localized fat reduction is necessary not only for esthetic purposes but also for therapeutic purposes.7 Based on the previous studies, MO shows a wide range of pharmaceutical activities, especially inhibiting effect against fat accumulation.7,14 Network pharmacology is an emerging bioinformatic tool in drug discovery and development.15 Via functional enrichment analysis, biological functions of traditional medicine containing numerous multi-targeted chemicals could be evaluated.16 In this study, we tried to verify the potential underlying mechanism of MO through the network pharmacology analysis and confirmed in animal experiments. It was found that the MO network is closely associated with the ‘AMPK signaling pathway’, ‘PPAR signaling pathway’, ‘Insulin signaling pathway’, ‘cAMP signaling pathway’, ‘Regulation of lipolysis in adipocytes’, ‘Fatty acid metabolism’ and ‘Fat digestion and absorption’ through KEGG Pathway, in human. Especially, 31 genes of 120 genes in gene sets of ‘AMPK signaling pathway’ were matched with the MO network. The overlapping genes include AMPK, HSL, SREBP1c, FAS, ACC1, ACC2, mTORC1 and PPARγ, eventually are related to lipolysis and suppression of fatty acid biosynthesis according to the KEGG Pathway 2021 human map of the ‘AMPK signaling pathway’. Through the MO network analysis, it can be seen that MO has a high effect on lipid metabolism.

Based on the results from network pharmacology analysis, we investigated the lipolytic effects of MO pharmacopuncture in obese mice with the underlying mechanism related to the AMPK signaling pathway. According to the previous literatures, main compounds of MO, honokiol and magnolol, improve liver dysfunction hepatic steatosis via AMPK-SREBP-1c pathway in HFD-induced mice.7 Magnolol has been studied to upregulate AKT, AMPK, and PPARα expression and suppress SREBP-1c generation.17 AMPK is a key factor in regulating lipid metabolism, and the AMPK pathway is associated with a fatty acid synthesis and cholesterol synthesis in the localized area.18 In a state of long accumulated energy imbalance, adipocytes convert and store extra energy into triacylglycerols, as a result, the number and size of adipocytes are gradually expanded.19 For that reason, we assumed that the remodeling effect can be confirmed by injecting MO into the local area and comparing the size and weight of adipocyte at the corresponding area. Injection of MO pharmacopunture into the inguinal fat pad of HFD-induced obese mice showed the fat weight and size reductions of localized fat. As shown in body composition image of DXA software, red colored-localized fat of inguinal region was markedly decreased in MO-injected side. In addition, MO treatment significantly inhibited the excessive expanded adipocyte diameter compared to Vehicle-treated side. The results that MO reliably reduced the size of adipocyte, and the weight of inguinal fat pad indicates that MO inhibits adipocyte hypertrophy, thereby reducing the mass and the weight of inguinal fat.

After confirming the inhibitory effects of MO against the localized fat accumulation in obese mice, we further analyzed the relevance of lipolytic effects and significant molecular pathways derived from network pharmacology analysis. AMPK is involved in lipid metabolism such as fat oxidation, lipogenesis, lipolysis by phosphorylation of proteins directly or regulating gene transcription.20 AMPK promotes glucose absorption by phosphorylation of thioredoxin-interacting protein and TBC domain family, member 1. In addition, AMPK has been known to regulate Glucose transporter4 (GLUT4) and GLUT1 involved in glucose transfer on the cell surface.21 Moreover, AMPK regulates lipid release and absorption by phosphorylating lipases such as ATGL and HSL.22 Lipolysis process is accompanied by an important enzyme to help with triacylglycerols (TAGs) decomposition. TAG is generated into generate diacylglycerol (DAG) through a hydrolysis process through a catalytic reaction of ATGL. As the next process, DAG is converted into monoacylglycerol (MAG) through hydrolysis, and HSL acts as a key enzyme. As a final step, MAG is decomposed into glycerol and free fatty acids (FFAs) by a hydrolysis process.23 Several lipid droplet–associated proteins are known to modulate rates of basal (non-stimulated) and stimulated lipolysis. Perilipin and CGI-58 are representative lipid droplet-associated proteins. Phosphorylation of perilipin is essential for the mobilization of fats tissue.24 In the present study, we determined whether MO sequentially upregulate lipase activation, including MGL, HSL, ATGL, and CGI-58 to facilitate the process of lipolysis in HFD-induced obese mice. As a result, MO could promote ATGL, HSL, MGL, and CGI-58 release and increase lipolysis in HFD-induced obese mice. Based on these results, it can be confirmed that MO injection induces the expression of AMPK signaling pathway in adipose tissue. AMPK signaling pathway was significantly increased in the experimental group subjected to MO treatment on the inguinal fat pad of the HFD-induced obese mice. Subsequently, fat reduction in intracellular is associated with inhibition of the lipogenesis process and an increase in the lipolysis process.

Various research has shown that this hormone inhibits ACC activity in the liver or muscles and promotes AMPK activity in adipose tissue.25 When adiponectin is secreted, ACC is phosphorylated, and AMPK, the cell's energy regulation sensor, is activated. Based on the experimental results, MO injection in HFD-induced obese mice activates AMPK pathway by promoting the expression of adiponectin. mechanistic target of rapamycin complex 1 (mTORC1) is a kinase that in humans is encoded by the MTOR gene. Additionally, the nuclear receptor PPAR-γ is known as a regulator of adipocyte differentiation.26 PPAR-γ has a critical role in lipid metabolism, promoting free fatty acid uptake and triacylglycerol accumulation in adipose tissue and liver.27 mTOR signaling pathway activates PPAR-γ to stimulate adipogenesis.28 MO injection inhibited the protein expressions of mTOR and PPAR-γ, thereby affecting lipogenesis. Furthermore, ACC is an important site of regulation within the fatty acid synthesis and oxidation pathways, as it can catalyze the carboxylation of acetyl-CoA to malonyl-CoA during the synthesis of fatty acids or allosterically inhibit CPT-1, a key enzyme for β-oxidation.29 Fatty acid synthase (FAS, encoded by FASN), the enzyme catalyzing de novo synthesis of fatty acids, is traditionally thought of as a housekeeping protein, producing fatty acids that can be used for energy storage, membrane assembly and repair, and secretion in the form of lipoprotein triglyceride.30 AMPK can further reduce hepatic lipid contents by suppressing SREBP1c expression through decreasing the activity of mammalian target of rapamycin complex (mTORC),31 an important mediator for the regulation of cellular metabolism and growth that may also promote SREBP-dependent fatty acid synthesis. AMPK suppresses SREBP1c expression through these targets, reducing FAS expression, and subsequently inhibiting lipid synthesis. In this study, we showed that MO was expected to be associated with lipid metabolism including lipolysis and lipogenesis. MO pharmacopunture inhibited fatty acid biosynthesis through AMPK signaling pathway. The levels of the fat-forming enzymes, ACC, FAS as well as intracellular fat accumulation were also significantly decreased by AMPK signaling pathway. These results suggest that MO injection activated AMPK, which was followed by the downregulation of mature SREBP 1c and fat-forming enzymes, leading to the inhibition of adipogenesis.

This study showed that MO is expected to be related to lipid metabolism, especially in lipolysis and lipogenesis, based on network analysis. In addition, lipogenesis related to SRERBP1c, FAS, ACC was also studied. These results suggest that MO injection causes the activation of lipolysis and the deactivation of lipogenesis in vivo and in vitro. Further research is needed to prove the lipolytic effects and lipogenic effects of MO injection and safety in humans. Nevertheless, this study shows important implications for the development of pharmacupuncture and clinical application.

Funding

This work was supported by a National Research Foundation of Korea Grant funded by the Korean Government (NRF-2019R1I1A2A01063598).

Ethical statement

The animal experiment was approved by Committee on Care and Use of Laboratory Animals of the Kyung Hee Univ. (KHSASP-21-270).

Data availability

The data associated with this article are provided within this article and as a supplementary material. The data can also be requested to the corresponding author.

CRediT authorship contribution statement

Won Jun Choi: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Mi Hye Kim: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Nayoung Park: Formal analysis, Investigation. Jae Yoon Chung: Formal analysis, Investigation. Sang Jun Park: Investigation. Woong Mo Yang: Conceptualization, Writing – review & editing, Supervision, Funding acquisition.

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.imr.2023.100948.

Supplementary 1. Magnolia officinalis in TM-MC database.

Supplementary 2. compounds and their related genes of Magnolia officinalis.

Supplementary 3. related genes of Magnolia officinalis.

Supplementary 4. sorted genes of Magnolia officinalis via cytoscape string app with confidence score 0.7.

Supplementary 5. Functional enrichment analysis of Magnolia officinalis.

Appendix. Supplementary materials

mmc1.xlsx (105.2KB, xlsx)

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Associated Data

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Supplementary Materials

mmc1.xlsx (105.2KB, xlsx)

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