Study on the Reduces Lipid Droplet Effect of Hypericin on 3T3-L1 and mBAT Adipocytes

Abstract

Objective: To investigate the effect of Hypericin (HYP) on lipid droplet accumulation during the differentiation of mouse preadipocytes 3T3-L1 and brown adipocytes mBAT of mouse.
Methods: Cell viability was assessed using the CCK-8 assay. An improved “cocktail” method was employed to induce differentiation and maturation of 3T3-L1 and mBAT adipocytes. Lipid droplet accumulation was visualized and quantified in vitro using Oil Red O and Bodipy staining. The mRNA expression of key lipid metabolism genes-including adipocyte Protein-2 (ap2), Uncoupling Protein-1 (ucp1), Adipose Triglyceride Lipase (atgl), Hormone-Sensitive Lipase (hsl), and Lipoprotein Lipase (lpl)-was measured by RT-qPCR.
Results: CCK-8 assays determined the maximum non-toxic concentrations of hypericin to be 2.00μmol/L for 3T3-L1 and 0.40 μmol/L for mBAT cells. Oil Red O staining and RT-qPCR revealed that 0.40 μmol/L hypericin significantly reduced lipid droplet accumulation in 3T3-L1 cells, downregulated ap2 and atgl expression, and upregulated ucp1 expression. In contrast, the same concentration increased lipid droplet accumulation in mBAT cells and downregulated atgl, hsl, and lpl expression.
Conclusion: Hypericin reduces lipid droplet content in white 3T3-L1 adipocytes and upregulates the browning marker ucp1, while increasing lipid storage in brown mBAT adipocytes. These dual effects suggest a coordinated enhancement of thermogenic potential in both cell types, collectively improving systemic lipid metabolism.

Keywords: Hypericin, white adipocytes, brown adipocytes, lipid droplets, browning

Introduction

Obesity has reached epidemic proportions globally. According to the latest World Health Organization report (2025), the prevalence of obesity is rising across all age groups and populations. Obesity adversely affects physical health and quality of life, while also imposing substantial socioeconomic and healthcare burdens. Excessive lipid accumulation in adipose tissue disrupts metabolic health, increasing the risk of chronic conditions such as cardiovascular disease, type 2 diabetes, and non-alcoholic fatty liver disease. Adipocytes, located primarily in subcutaneous and visceral depots, are broadly classified into white, beige, and brown adipocytes based on their function. White adipocytes store energy as triglycerides, whereas brown adipocytes dissipate energy as heat through uncoupled respiration. Beige adipocytes exhibit an intermediate, inducible thermogenic capacity. The balance between energy storage and expenditure in these cell types is critical for maintaining metabolic homeostasis.

Hypericin, a naphthodianthrone derived from Hypericum perforatum (Clusiaceae), is administered orally, topically, or intraperitoneally and is clinically used for its antidepressant properties [1-3]. Previous studies indicate that hypericin ameliorates non-alcoholic fatty liver disease and type 2 diabetes by reducing hepatocyte apoptosis and lipid accumulation [4,5]. However, its direct effects on adipocytes remain unclear. Adipocyte membranes, composed of a phospholipid bilayer, regulate the flux of substances into the cell. During differentiation, fatty acids are esterified into triglycerides and stored within cytoplasmic lipid droplets. Thus, the extent of lipid droplet accumulation and the expression of lipid metabolism genes serve as key indicators of adipocyte status.

This study investigates the effects of hypericin on lipid droplet accumulation and gene expression in mouse 3T3-L1 (white) and mBAT (brown) adipocytes, providing novel insights into its potential role in modulating adipocyte function and lipid metabolism.

Materials and Methods

Test Compound

Hypericin (HYP, 10 mg, HPLC ≥98%, CAS No.: 548-04-9, MCE).

Cell Lines

Mouse preadipocytes 3T3-L1 were purchased from the National Biomedical Experimental Cell Resource Bank (Catalog No.: 1101MOU-PUMC000155); Mouse brown adipocytes mBAT were kindly donated by the Center for Life Sciences, Purdue University, USA.

Reagents

Fetal bovine serum (PAN); 3-Isobutyl-1-methylxanthine (Sigma- Aldrich); BODIPY 493/503 (pythonbio); CCK-8 kit (Beyotime); Trizol (Thermo Fisher); Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR and Hieff UNICON® qPCR SYBR® Green Master Mix (antibody method, No Rox) (Yeasen); DMEM high-glucose medium, penicillin-streptomycin, 0.25% trypsin, PBS phosphatebuffered saline (gibco); Recombinant human insulin, rosiglitazone, dexamethasone, dimethyl sulfoxide (DMSO), 4% paraformaldehyde fixative (PFA), BODIPY, Oil Red O (Sudan Red), and Hoechst 33342 staining solution (Solarbio); Anhydrous ethanol, chloroform, isopropanol, etc. were of analytical grade (Tianjin Beilian).

Main Instruments

Synergy H1 multifunctional microplate reader (BioTek); Conventional PCR instrument (Applied Biosystems); Roche realtime quantitative PCR instrument (Roche); Nanodrop 2000c (Thermo Fisher); ECLIPSE Ts2 inverted microscope (Nikon).

Methods

Cell Culture

3T3-L1 and mBAT cells were thawed and cultured in 25cm² flasks containing 5 mL DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (DMEM+). Cells were maintained at 37°C in a 5% CO₂ atmosphere, with medium replenished every two days.

CCK-8 Assay for Cell Viability

At 80–90% confluence, cells were trypsinized, centrifuged, resuspended, and counted. Cells were seeded into 96-well plates at 5×10³cells/well. After 24h, cells were treated with various concentrations of hypericin in DMEM+ for 48 h (n=6 per group). CCK-8 reagent (10μL) was added to each well and incubated for 3h. Absorbance was measured at 450 nm, and cell viability was calculated.

Experimental Grouping

Hypericin was dissolved in DMSO to a 10 mmol/L stock solution and serially diluted to 2, 0.4, and 0.08 mmol/L. For CCK-8 assays, DMEM+ was used to prepare hypericin concentrations of 0, 0.08, 0.4, 2, and 10μmol/L. Based on viability results, the highest nontoxic concentration was selected as the high-dose, the next lower non-toxic concentration as the low-dose, and a solvent control (DMSO) was included for subsequent experiments.

Adipocyte Differentiation (“Cocktail” Method)

Cells were seeded into 12-well plates at 5×10⁴ cells/well (n=3 per group). At 80–90% confluence, contact inhibition was maintained for two days. Cells were then induced for four days in induction medium (DMEM+ containing 1μmol/L DEXA, 0.5 mmol/L IBMX, 10μg/mL insulin, and 100μmol/L rosiglitazone), with hypericin or DMSO vehicle added. Medium was changed every two days. This was followed by a four-day differentiation period in differentiation medium (DMEM+ with 10μg/mL insulin and 10 nmol/L triiodothyronine), with hypericin or DMSO refreshed at each medium change.

Oil Red O Staining

After differentiation, cells were washed with PBS, fixed with 4% PFA, and stained with Oil Red O for 30 min. After PBS washes, images were acquired under bright-field microscopy. Stained lipid droplets were dissolved in isopropanol, and absorbance was measured at 358 nm for quantification.

Bodipy and Hoechst Staining

Differentiated cells were washed with PBS and stained with Bodipy (1 mL/well) at 37°C for 30 min in the dark. After PBS washes, nuclei were counterstained with Hoechst 33342 (1 mL/ well) for 10 min at room temperature. Fluorescence images were captured using an inverted fluorescence microscope.

RT-qPCR

Total RNA was extracted with Trizol, and chloroform was added for phase separation. The aqueous phase was mixed with isopropanol to precipitate RNA. The pellet was washed with 75% ethanol, air-dried, and dissolved in nuclease-free water. RNA concentration and purity were determined (Nanodrop 2000). cDNA was synthesized using Hifair® III 1st Strand cDNA Synthesis SuperMix. RT-qPCR was performed using Hifair UNICON® qPCR SYBR® Green Master Mix. Mouse RPS18 served as the internal control. Primer sequences are listed in (Table 1).

Table 1: Primer Sequences for RT-qPCR.

Statistical Analysis

All experiments were performed in triplicate. Data are presented as mean ± standard deviation (x±s). Statistical analysis was conducted using GraphPad Prism 8.0.1, with group comparisons by t-test. A P-value < 0.05 was considered statistically significant.

Results

Hypericin Effects on Cell Viability

The CCK-8 assay measures the reduction of WST-8 to a formazan dye by cellular dehydrogenases, with absorbance at 450 nm proportional to viable cell number. Hypericin significantly reduced 3T3-L1 viability at 10μM (Figure1A) (P<0.01). Thus, subsequent 3T3-L1 experiments used 0.40μM (low) and 2.00μM (high) hypericin. For mBAT, viability was significantly impaired at 2.00μM (Figure 1B) (P<0.01), leading to selected doses of 0.08μM (low) and 0.40μM (high).

Hypericin Modulates Lipid Droplet Accumulation

A modified “cocktail” protocol, including rosiglitazone during induction and triiodothyronine during differentiation, enhanced the maturation of both adipocyte types. After 8 days, abundant lipid droplets were observed. Bodipy staining (green fluorescence) visualized lipid droplets, while Hoechst (blue) labeled nuclei. Oil Red O staining (orange-red) also revealed lipid droplets, which were quantified via isopropanol extraction. Hypericin (0.40μM) reduced lipid droplets in 3T3-L1 (Figure 2) but increased them in mBAT (Figure 3).

Quantification of Oil Red O staining confirmed these findings. In 3T3-L1, 2.00μM and 0.40μM hypericin reduced lipid content by 14.3% (P<0.01) and 10.2% (P<0.05), respectively (Figure 4A). In mBAT, 0.40μM hypericin increased lipid droplets by 9.5% (P<0.01), while the low dose had no significant effect (Figure 4B).

Figure 1: Viability of (A) 3T3-L1 and (B) mBAT cells after hypericin treatment, assessed by CCK-8 (x±s, n=6). *P<0.05, **P<0.01 vs. CON group.

Figure 2: Hypericin reduces lipid droplet accumulation in 3T3-L1 adipocytes.

Figure 3: Hypericin increases lipid droplet accumulation in mBAT adipocytes.

Figure 4: Quantification of lipid droplet accumulation in (A) 3T3-L1 and (B) mBAT cells via Oil Red O staining (x±s, n=6). *P<0.05, **P<0.01.

Figure 5: mRNA expression of adipogenic, lipolytic, and thermogenic genes in (A–C) 3T3-L1 and (D–F) mBAT cells after hypericin treatment (x±s, n=3). *P<0.05, **P<0.01.

Gene Expression Analysis

We examined the mRNA levels of key adipogenic (pparγ, ap2), lipolytic (atgl, hsl, lpl), and thermogenic (ucp1, prdm16) genes. In 3T3-L1 cells, 0.40 μM hypericin significantly downregulated ap2 (Figure 5A) (P<0.05). The 2.00μM dose downregulated both ap2 (P<0.01) and atgl (Figure 5B) (P<0.05). Notably, 2.00μM hypericin markedly upregulated ucp1 expression (Figure 5C) (P<0.01). Expression of pparγ, hsl, lpl, and prdm16 was unaffected. In mBAT cells, 0.08μM hypericin downregulated pparγ, ap2, atgl, and lpl (Figure 5D, 5E) (P<0.05). The 0.40μM dose downregulated atgl, hsl, and lpl (Figure 5E) (P<0.05). A non-significant increasing trend was observed for ucp1 and prdm16 after 0.40μM treatment (Figure 5F) (P>0.05).

Discussion

Adipose tissue, comprising various adipocyte types, serves essential roles in energy storage, thermal insulation, cushioning, thermogenesis, and endocrine signaling [6-9]. White adipocytes store energy, brown adipocytes generate heat, and beige adipocytes offer a recruitable thermogenic capacity in adults [10-14]. Obesity arises from a chronic positive energy balance, leading to excessive lipid droplet expansion in white adipose tissue.

Lipid droplets are dynamic organelles involved in lipid storage, fatty acid trafficking, mitochondrial cooperation, signaling, and immune regulation [15-18]. In white adipocytes, oversized lipid droplets contribute to metabolic dysfunction. In brown adipocytes, lipid droplets serve as fuel for thermogenesis; their abundance can indicate enhanced energy-burning capacity [19-23]. Thus, reducing white adipocyte lipid content while stimulating brown adipocyte function represents a promising anti-obesity strategy. Our findings demonstrate that hypericin reduces lipid storage in white 3T3- L1 adipocytes while increasing it in brown mBAT adipocytes. In 3T3-L1, hypericin downregulated the adipogenic marker ap2 and upregulated the thermogenic marker ucp1, suggesting a promotion of browning. In mBAT, hypericin downregulated lipolytic genes (atgl, hsl, lpl), potentially conserving lipid stores to sustain thermogenesis. Although not statistically significant, the upward trend in ucp1 and prdm16 expression hints at possible enhanced browning.

In summary, hypericin exhibits distinct, cell-type-specific effects: it attenuates lipid accumulation and induces browning in white adipocytes, while potentially augmenting the thermogenic fuel reserve in brown adipocytes. These combined actions may synergistically improve whole-body lipid metabolism. Future studies should identify the molecular targets of hypericin in adipocytes and validate these findings in vivo.

Acknowledgments

None.

Conflicting Interest

None.

References

1. Pietro Delcanale, Eleonora Uriati, Matteo Mariangeli, Andrea Mussini, Ana Moreno, et al. (2022) The Interaction of Hypericin with SARS-CoV-2 Reveals a Multimodal Antiviral Activity. ACS Appl Mater Interfaces 14(12): 14025-14032.
2. Ellen J Kim, Aaron R Mangold, Jennifer A DeSimone, Henry K Wong, Lucia Seminario-Vidal, et al. (2022) Efficacy and Safety of Topical Hypericin Photodynamic Therapy for Early-Stage Cutaneous T-Cell Lymphoma (Mycosis Fungoides): The FLASH Phase 3 Randomized Clinical Trial. JAMA Dermatol 158(9): 1031-1039.
3. RC Shelton, MB Keller, A Gelenberg, DL Dunner, R Hirschfeld, et al. (2001) Effectiveness of St John's wort in major depression: a randomized controlled trial. Jama 285(15): 1978-1986.
4. Chen Liang, Yan Li, Miao Bai, Yanxin Huang, Hang Yang et al. (2020) Hypericin attenuates nonalcoholic fatty liver disease and abnormal lipid metabolism via the PKA-mediated AMPK signaling pathway in vitro and in vivo. Pharmacol Res 153: 104657.
5. M Barathan, V Mariappan, EM Shankar, BJJ Abdullah, KL Goh, et al. (2013) Hypericin-photodynamic therapy leads to interleukin-6 secretion by HepG2 cells and their apoptosis via recruitment of BH3 interacting-domain death agonist and caspases. Cell Death Dis 4(6): e697.
6. Yugo Miyata, Michio Otsuki, Shunbun Kita, Iichiro Shimomura (2016) Identification of Mouse Mesenteric and Subcutaneous in vitro Adipogenic Cells. Sci Rep 6: 21041.
7. Matthias Rosenwald, Aliki Perdikari, Thomas Rülicke, Christian Wolfrum (2013) Bi-directional interconversion of brite and white adipocytes. Nat Cell Biol 15(6): 659-667.
8. G Barbatelli, I Murano, L Madsen, Q Hao, M Jimenez, et al. (2010) The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation. Am J Physiol Endocrinol Metab 298(6): E1244-253.
9. Qiong A Wang, Caroline Tao, Rana K Gupta, Philipp E Scherer (2013) Tracking adipogenesis during white adipose tissue development, expansion and regeneration. Nat Med 19(10): 1338-1344.
10. Badman MK, Flier JS (2007) The adipocyte as an active participant in energy balance and metabolism. Gastroenterology 132(6): 2103-2115.
11. Chouchani ET, Kajimura S (2019) Metabolic adaptation and maladaptation in adipose tissue. Nat Metab 1(2): 189-200.
12. Wang W, Seale P (2016) Control of brown and beige fat development. Nat Rev Mol Cell Biol 17(11): 691-702.
13. Kirsi A Virtanen, Martin E Lidell, Janne Orava, Mikael Heglind, Rickard Westergren, et al (2009) Functional brown adipose tissue in healthy adults. N Engl J Med 360(15): 1518-1525.
14. Scheja L, Heeren J (2016) Metabolic interplay between white, beige, brown adipocytes and the liver. J Hepatol 64(5): 1176-1186.
15. Barbosa AD, Siniossoglou S (2017) Function of lipid droplet-organelle interactions in lipid homeostasis. Biochim Biophys Acta Mol Cell Res 1864(9): 1459-1468.
16. Lei Han, Dayang Huang, Shiyong Wu, Sheng Liu, Cheng Wang, et al. (2023) Lipid droplet-associated lncRNA LIPTER preserves cardiac lipid metabolism. Nat Cell Biol 25(7): 1033-1046.
17. Kohlwein SD, Veenhuis M, van der Klei IJ (2013) Lipid Droplets and Peroxisomes: Key Players in Cellular Lipid Homeostasis or A Matter of Fat--Store ’em Up or Burn ’em Down. Genetics 193(1): 1-50.
18. Marta Bosch, Miguel Sanchez-Alvarez, Alba Fajardo, Ronan Kapetanovic, Bernhard Steiner, et al. (2020) Mammalian lipid droplets are innate immune hubs integrating cell metabolism and host defense. Science 370(6514).
19. Farese RV Jr, Walther TC (2009) Lipid droplets finally get a little R-E-S-P-E-C-T. Cell 139(5): 855-60.
20. Gao Q, Goodman JM (2015) The lipid droplet-a well-connected organelle. Front Cell Dev Biol 3: 49.
21. Anying Song, Wenting Dai, Min Jee Jang, Leonard Medrano, Zhuo Li, et al. (2020) Low- and high-thermogenic brown adipocyte subpopulations coexist in murine adipose tissue. J Clin Invest 130(1): 247-57.
22. Hyunsu Shin, Yinyan Ma, Tatyana Chanturiya, Qiang Cao, Youlin Wang, et al. (2017) Lipolysis in Brown Adipocytes Is Not Essential for Cold-Induced Thermogenesis in Mice. Cell Metab 26(5): 764-77.e5.
23. Ilan Y Benador, Michaela Veliova, Kiana Mahdaviani, Anton Petcherski, Jakob D Wikstrom, et al. (2018) Mitochondria Bound to Lipid Droplets Have Unique Bioenergetics, Composition, and Dynamics that Support Lipid Droplet Expansion. Cell Metab 27(4): 869-85.e6.