Rong-Fang Mu 1,4 · Yan-Fen Niu 3 · Qian Wang 1,4 · Hui-Min Zhou 1,4 · Jing Hu 1 · Wan-Ying Qin 1,4 · Wen-Yong Xiong 1,2,4

Abstract

Keywords Eriocalyxin B · Adipocyte diあ erentiation · Cell cycle

Graphic Abstract

1 Introduction

With the rapid development of modern society, the epidemic of overweight and obesity has brought huge medical burden to our society. According to the World Health Organization, obesity is not only a physical trait but also a chronic disease, which is regarded as a high-risk factor for various diseases including diabetes, non-alcoholic fatty liver disease, cardiovascular disease, hypertension and cancers [ 1- 4]. Obesity is a disorder which is accompanied to the unbalance of energy intake and expenditure [ 5, 6]. As we are known that obesity is characterized by the increased size (hypertrophy) and number (hyperplasia) of adipocytes in adipose tissue, which results in excessive lipid accumulation in white adipose tissue [ 7, 8].

In theory, reducing calorie intake and increasing expenditure are the methods for overcome obesity. In addition, other approaches are used for the treatment of obesity, such as surgical treatment and medication. So far, the US FDA has approved six major therapeutic drugs for obesity treatment: phentermine, orlistat, lorcaserin, liraglutide, naltrexone/bupropion sustained release, and phentermine/topiramate extended release [ 9]. However, most of anti-obesity medications were withdrawn from the market due to their useless eあ ects or undesirable side eあ ects. Examples of adverse eあ ects include diarrhea, dry mouth, dyspepsia (orlistat), pulmonary hypertension (aminorex), stroke (phenylpropanolamine), and neuropsychiatric issues (rimonabant) [ 10, 11]. These undesirable experiences highlight the importance of safety assessments and necessitate the development of new drug development strategy to achieve more eき cient and safer obesity treatment options. Traditional medicinal plants have huge natural compound resources and better safety in drug development and application. At present, several studies revealed the eあ ective natural compounds of antiobesity are extracted from botany, including sulforaphene [ 12], daidzein [ 13], quercetin [ 14], hesperidin and capsaicin [ 15], methylated cereal fl avonoid [ 16], and dietary phenolic [ 17], indicating the natural products are a great choice as potential candidates for treatment of obesity.

In vitro, the 3T3-L1 cell line is considered as a wellestablished and classic cell line that was frequently used for study of adipocyte diあ erentiation, lipid metabolism, insulin signaling, regarding of its diあ erentiation process involving both hypertrophy and hyperplasia of obesity [ 18, 19]. The adipocyte diあ erentiation is regulated by several adipogenicspecifi c genes, such as peroxisome proliferator-activated receptorγ (PPARγ), CCAA/enhancer-binding protein (C/EBP) family members and fatty acid binding protein (FABP) [ 20- 23]. Moreover, the adipocyte diあ erentiation is extensively regulated through crosstalk between the cell cycle regulators and adipogenic transcription factors [ 12, 24, 25]. The process of adipogenesis is divided into several phases, including growth arrest, mitotic clonal expansion (MCE), lipid accumulation, and late phase of diあ erentiation [ 26]. Especially, MCE is an essential stage for terminal diあ erentiation, which results in an increase in cell numbers [ 27]. Additionally, a number of proteins participated in MCE, the progression of cell cycle are largely regulated by the activation of cyclin-dependent kinases (CDKs), and cyclins which are considered as critical determinants for early adipocyte diあ erentiation program [ 28]. Many studies show that MCE is a prerequisite for diあ erentiation of 3T3-L1 preadipocytes into adipocytes. A study demonstrated that Eriocalyxin B markedly arrested cell cycle at G2/M phase in a dosedependent manner [ 29]. CDKs are serine/threonine kinases that control cell cycle progression. CDKs are activated by cyclins and act as a regulatory subunit. These CDK/cyclin complexes are fundamental to the orderly progression of the cell [ 30].

Eriocalyxin B, isolated and identifi ed in 1982 by Handong Sun’s group, is the major component in Chinese plant Isodon eriocalyx (Dunn.) Hara (family Lamiaceae) showing many pharmacological activities, such as inhibiting infl ammatory response, regulating immune cell diあ erentiation, inhibiting tumor cells proliferation, causing cell cycle arrest aあ ecting angiogenesis and promoting cancer cells apoptosis [ 31- 33]. However, it is unknown whether Eriocalyxin B functions in adipocyte diあ erentiation. Therefore, in this work we aimed to elucidate whether Eriocalyxin B could prevent adipogenesis and address the potential mechanism.

2 Results

2.1 Eriocalyxin B Inhibited Adipocyte Diあ erentiation

The 3T3-L1 cells, a classic cell model for adipogenesis in vitro, were treated with a widely-used adipogenesis inducing cocktail for 7-day [ 34]. At the end of adipogenesis induction, the level of lipid accumulation in adipocytes was stained by Oil Red O and followed by OD measurements. As shown, the lipid accumulation of the cells were fully diあ erentiated after inducing (Ctrl), whereas the accumulations were blunted by the treatment of Eriocalyxin B (Fig. 1 b), which were quantifi ed by the OD values of

Oil Red O from the cells (Fig. 1 c-d). The half inhibitory concentration of Eriocalyxin B for blunting adipogenesis is about 2.745 μM (Fig. 1 d). These inhibiting results were insured by measuring the TG contents in these cells treated by the compound (Fig. 1 e), supporting that the compound blunted adipogenesis with a dose-dependent manner.

2.2 Eriocalyxin B Suppressed Key Regulators in Adipocyte Diあ erentiation

Since we have demonstrated the effect of eriocalyxin B in adipogenesis above, we next detected the expressions of several key regulator in adipogenesis, including C/EBPα, C/EBPβ, PPARγ and FABP4 [ 20- 23]. As expected, the regulators were all upregulated at the end of differentiation (Ctrl group), whereas these regulators were all gradually downregulated following the gradually increases of the compound’s concentrations (Fig. 2 a). The values were further quantified as following: C/EBPα (Fig. 2 b), C/EBPβ (Fig. 2 c), PPARγ (Fig. 2 d) and FABP4 (Fig. 2 e), supporting the effect of the compound in blunting the process of adipogenesis (Fig. 1).

2.3 Eriocalyxin B Blunted the Adipogenesis at the Early Stage

Next we aimed to investigate how eriocalyxin B aあ ect differentiation of adipocytes. Here we treated 3T3-L1 cells with eriocalyxin B (2.5 μM) at the diあ erent stages of the 7-day induction of adipogenesis (Fig. 3 a). Our data showed that adding eriocalyxin B to the induction mix during 0-3 days, 0-6 days and 0-7 days (protocol 1, 2, 3 respectively) of the adipogenesis process resulted in the blunting of adipogenesis signifi cantly, whereas the adding to the inducing mix after the 3rd (protocol 4-6) day of induction did not inhibited adipogenesis signifi cantly, suggesting that the compound functions in adipogenesis in the early stage of adipocyte diあ erentiation.

2.4 Eriocalyxin B Arrested Cell Cycle Progression During the MCE Period of Adipogenesis

During the earlier process of adipogenesis, the growtharrested 3T3-L1 preadipocytes go through two sequential rounds of mitosis in the initial 48 h of diあ erentiation which is termed as MCE. Above result has shown that Eriocalyxin B functioned in the early phase, including MCE of diあ erentiation, therefore we perform cell cycle assay to address whether Eriocalyxin B aあ ects the cell cycle progression during MCE.

As expected, by flow cytometric analysis, the undifferentiated cells did not undergo cell cycle progression because most of cells (～ 65%) resided in G0/G1 phase from 16 to 48 h, whereas in the diあ erentiated group (Ctrl), the percentage of cells in G2/M phase were 12.99%, 30.84%, 32.41% and 33.13% at four time points of 16, 24, 36 and 48 h (Fig. 4). Interestingly, when cells were treated with 2.5 μM Eriocalyxin B, the percentage of G2/M phase cells was 15.31%, 42.8%, 49.34%, and 51.74% respectively. The results displayed that the cells aggregated in the G2/M phase is compared with the Ctrl group, supporting that eriocalyxin B blocks the cell cycle progression by arresting the cells in G2/M phase during the MCE period of adipogenesis.

2.5 Eriocalyxin B Suppressed the Expressions of the Regulators for Cell Cycle

To further investigate the mechanism of the eriocalyxin B on the cell cycle arrest, we assessed the key cell cycle regulators in the G2/M phase, including CDK1, CDK2, Cyclin A, and Cyclin B1. The mRNA levels of CDK1, Cyclin A and Cyclin B1 were suppressed by eriocalyxin B as determined at 24 h after diあ erentiation (Fig. 5 a-c), which is consistent with result of cell cycle (Fig. 4). Moreover, in parallel with the result of mRNA levels of these regulators, the protein levels of CDK1, CDK2, Cyclin A and Cyclin B1 were also down-regulated by eriocalyxin B treatment (Fig. 5 d-i), confi rming the eriocalyxin B inhibited the cell cycle progression during MCE period of adipogenesis.

3 Discussion

Eriocalyxin B has been documented to possess a variety of pharmacological activities, which has the eあ ects of antiinfl ammatory, antiproliferative, anti-angiogenic, and the most common activity is anti-cancer [ 29, 35, 36]. In this work, we reported that eriocalyxin B is capable of inhibiting adipocyte diあ erentiation.

Experimental data has shown that eriocalyxin B exerts biological properties through arresting cell cycle, prompting apoptosis, and modulating cell signaling pathway, which modulates multiple cell signaling pathways including NF-κB, MAPK, JAK, STAT3, NOTCH, and Wnt pathways [ 36- 41]. Eriocalyxin B prevented the cancer cells proliferation by inhibiting the progression of cell cycle, which aあ ected cell cycle related regulators, including CDK, Cyclins and CKI [ 31, 41]. Our study further explored the molecular mechanism and found that eriocalyxin B suppressed adipocyte diあ erentiation by causing cell cycle arrest in 3T3-L1 adipocytes. The 3T3-L1 cells we use are normal tissue cells (adipocytes) rather than cancer cells. Therefore, we speculate that the inhibitory eあ ect of eriocalyxin B on adipogenesis may be a benign “side eあ ect” when it exerts anti-cancer function. Of course, this inference needs to be fully addressed in the future.

In conclusion, eriocalyxin B, a natural entkaurene diterpene compound isolated from Isodon eriocalyx, is able to inhibit the early stage of adipogenesis by suppressing the signaling proteins involved in cell cycle progression.

3.1 Experimental Section

3.1.1 Chemicals and Reagents

Adipocyte growth medium (DMEM) were purchased from Biological Industries (Israel). The fetal bovine serum (FBS) for cell culture was purchased from GIBCO BRL (Grand Island, NY, USA). Penicillin/streptomycin (P/S) and Calf serum (CS) were obtained from Biological Industries (Israel). Rosiglitazone (Rosi), dimethylsulfoxide (DMSO) and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma-Aldrich (St Louis, MO, USA). Insulin was from Roche (Switzerland), and dexamethasone (DEX) was from Adamas (Switzerland). Bovine serum albumin (BSA) and TRIzol reagent were from Shanghai Sangon Biotech (Shanghai, China). QuantiSpeed SYBR kit was from novopretion (China). Triglycerides Kit was purchased from Nanjing Jiancheng Bioengineering Institute. The primary antibodies against β-actin, FABP4, C/EBPβ, CDK2, C/EBPα and PPARγ were from Cell Signaling Technology (Beverly, MA, USA) and CDK1, Cyclin A, Cyclin B1 from Abcam (USA) and HuaBio (China).

3.1.2 Cell Culture and Diあ erentiation

The 3T3-L1 murine pre-adipocytes cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were routinely cultured in DMEM supplemented with 10% CS and 1% P/S at 37 ℃ in a 5% CO2atmosphere. Two days after confl uence (day 0), cells were induced for diあ erentiation with DMEM supplemented with 10% FBS, IBMX, DEX and insulin (MDI) as previously described (Jing Hu, Molecules 2019). On day 3, the medium was changed to medium containing 10% FBS and 1 μg/mL insulin for 2 days (day 5), and then insulin was removed from 10% FBS-DMEM for another 2 days. The cells were fully diあ erentiated into mature adipocytes on day 7.

3.1.3 Oil Red O Staining

After removing the culture medium, 3T3-L1 cells were washed three times with PBS and subsequently fi xed in 10% formaldehyde for 1 h at room temperature. After fi xing, the cells were washed with water twice and one time with 60% isopropanol then the cells were stained with Oil Red O working solution for 30 min at room temperature, washed with water three times, and then photos were taken under microscopy. To quantify the lipid accumulation, the stained 3T3-L1 cells were washed with 100% isopropanol and the absorbance was measured at 492 nm by using a microplate reader (Perkin Elmer Envision Multilabel reader).

3.1.4 Measurement of Intracellular Triglyceride

After treatment with eriocalyxin B, rinsed cells with 100% isopropanol. The intracellular lipid was collected by isopropanol to detect triglycerides (Triglycerides Kit, Biosino Bio-Technology and Science Incorporation, China).

3.1.5 Cell Cycle Assay

3T3-L1 cells were induced to diあ erentiate in DMI medium with or without eriocalyxin B for 16 h, 24 h, 36 h and 48 h. The cells were then harvested, washed with PBS, fi xed in 70% pre-cooling ethanol overnight at - 20 °C, washed with PBS, resuspended in 500μL of PBS containing 100 μg/mL RNase for 30 min at 37 °C and subsequently incubated with the nuclear stain PI at a fi nal concentration of 50 μg/mL for 15 min at 37 °C. The stained cells were analyzed by using a BD AccuriC6 fl ow cytometer (BD Biosciences, San Jose, CA, USA) and the cell cycle distribution was analyzed by using FlowJo software.

3.1.6 RNA Isolation and Real-time PCR

RNA was extracted from 3T3-L1 cells using TRIzol reagent (TIANGEN BIOTECH), and cDNA was synthesized using cDNA synthesis kits using total RNA (2 μg) (Applied Biological Materials Inc). Real-time PCR cDNA gene expression was detected using the SYBR Green Master kit and a spectrofl uorometric thermal cycler (Applied Biosystems). β-actin was used as a housekeeping gene to evaluate the relative expression of genes.

Gene Forward primer Reverse primer CDK1 AAG CCG CTT TTC CAC GGC GCT CCC CGG CTT CCA CTT CyclinA ACC TCA AAG CGC CAC AAC AT CAA AGC CGG CAG TCT TTC AC CyclinB GCT GGT CGG TGT AAC GGC GGC GAC CCA GGC TGA AGT β-actin CAC CCC AGC CAT GTA CGT GTC CAG ACG CAG GAT GGC

3.2 Western Blotting

Western blot analysis was performed for the expression of adipogenesis-associated proteins. Cells were lysed in RIPA extraction buあ er (Beyotime, Haimen, China) on ice for 30 min. The proteins were subjected to 10% SDSPAGE and transferred to a PVDF membrane (Merck Millipore, Billerica, MA, USA) for 90 min. After blocking with 5% skim milk, the membrane was incubated with a primary antibody at 4 °C overnight using the indicated commercial antibodies (PPARγ, FABP4, CEBPα, CEBPβ, CDK1, CDK2, Cyclin A, Cyclin B1, β-actin). Subsequently, the membranes were incubated with secondary antibodies and then developed using Western Lightning Chemilum-inescence Reagent (Perkin-Elmer Life Science, MA, USA). Finally, the immunoblots were quantifi ed using the Metamorph software.

3.3 Statistical Analysis

All data are presented as the means ± SEM of triplicate experiments. An analysis of variance (ANOVA) and Student’s t-test were used to determine the signifi cance of diあ erence means. P values less than 0.05 were considered to be statistically signifi cant.

Acknowledgements Thanks Dr. Jing Hu, Yan-Ting Lu and Fang Wang for discussing the work. This work was supported by National Key R&D Program of China, (2017YFC1700906) and Yunnan Provincial Science and Technology Department, China (2017FA044 and 2013HA023).

Compliance with Ethical Standards

Conflict of interest The authors declare no competing fi nancial interest.

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