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ORIGINAL ARTICLE
Year : 2021  |  Volume : 11  |  Issue : 1  |  Page : 50-56

A Novel Herbal Combination Decreased Lipid Droplets Accumulation and Cytokines Levels During Adipogenesis by Regulating Leptin, PPARγ and SREBP1c Genes Expression in 3T3L1 Cells


1 Department of anatomical sciences, School of Medicine, St. George’s University Grenada, West Indies
2 Department of biomedical sciences, School of Medicine, Nazarbayev University Nur-Sultan, Kazakhstan
3 Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Mysuru, India

Date of Submission08-Sep-2020
Date of Decision30-Nov-2020
Date of Acceptance08-Dec-2020
Date of Web Publication12-Feb-2021

Correspondence Address:
Arehally M Mahalakshmi
School of Medicine, St. George’s University Grenada, West Indies. & Arehally M. Mahalakshmi, Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Mysuru
India
Vasavi Rakesh Gorantla
Department of Anatomical Sciences, School of Medicine, St. George’s University Grenada, West Indies

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijnpnd.ijnpnd_88_20

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   Abstract 


The present study aims at demonstrating the anti-adipogenesis and anti-inflammatory of combination of extracts of commiphora wightti, curcuma longa, and tinospora cordifolia using mouse 3T3-L1 cells. Herbal formula (CCT) significantly suppressed the mRNA of PPARγ, leptin, and SREBP1c expression, which is reveals its significant anti-adipogenesis activity. In addition, CCT down-regulated TNFα and iNOS expression, this is an added advantage and helps to alleviate the inflammatory reactions during adipogenesis. These findings encourage for further studies using in vivo models of obesity and associated complications.

Keywords: Commiphora wightti, curcuma longa, tinospora cordifolia, anti-adipogenesis


How to cite this article:
Gorantla VR, Bolla SR, Tuladhar S, Bishir M, Mahalakshmi AM. A Novel Herbal Combination Decreased Lipid Droplets Accumulation and Cytokines Levels During Adipogenesis by Regulating Leptin, PPARγ and SREBP1c Genes Expression in 3T3L1 Cells. Int J Nutr Pharmacol Neurol Dis 2021;11:50-6

How to cite this URL:
Gorantla VR, Bolla SR, Tuladhar S, Bishir M, Mahalakshmi AM. A Novel Herbal Combination Decreased Lipid Droplets Accumulation and Cytokines Levels During Adipogenesis by Regulating Leptin, PPARγ and SREBP1c Genes Expression in 3T3L1 Cells. Int J Nutr Pharmacol Neurol Dis [serial online] 2021 [cited 2021 Oct 24];11:50-6. Available from: https://www.ijnpnd.com/text.asp?2021/11/1/50/309284




   Introduction Top


Obesity is a metabolic disorder and a major global medical concern irrespective of ethnicity, age, gender, and socioeconomic status.[1]. It is one of the major risk factors for cardiovascular diseases (CVD), type II diabetes, cancer, and nonalcoholic fatty liver disease (NAFLD).[2],[3],[4]. Approximately, 2 billion adults are obese and 650 million have BMI ≥ 30 kg/m2. By 2025, it is estimated that 177 million adults will suffer from obesity. In normal physiological state, adipocytes are dynamic component in the body, which regulated various cellular activities like insulin signalling, immune energy homeostasis, and hormonal functions.[5] In hyperlipidemic condition, cellular characterization of adipose tissues revealed increased lipids both in terms of numbers and size and accumulation as well. Various factors like lifestyle, decreased physical activity, stress, smoking, genetics, and epigenetics are indicated to be involved in obesity pathogenesis.[6],[7].

Basically, adipocytes are of two types, viz, white adipocytes, which stores triglycerides and serve as energy reservoir. Whereas, the brown adipocytes help to regulate the whole-body thermogenesis. Reports indicate that in obesity white adipose tissues become dysfunctional and do not inflate to store energy, which in turn leads to ectopic accumulation of lipids in body, a condition called lipotoxicity.[8] This condition disturbs the glucose regulatory mechanism, hormonal functions, and increased releases of adipokines and cytokines, which results in CVD.[9] Thus, a proinflammatory status produces a strong association between insulin resistance and endothelial dysfunction leading to atherosclerotic lesions in the vascular beds in obesity. Endothelial dysfunction decreases the production of nitric oxide and endothelial-derived hyperpolarizing factors (EDHF), which aggravates vascular elasticity. In obesity, preadipocytes, and adipocytes serve as sources of cytokines (IL-1, IL-6, and TNFα), and these cytokines trigger reactive oxygen and nitrogen species generation via monocytes and macrophages.[10] This information indicates the key role of inflammation and oxidative stress during adipogenesis.

Conversion of preadipocytes to adipocytes involves complex mechanisms and various transcription factors like proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer protein (C/EBPα), leptin, SREBP1C participate in adipogenesis.[11]. Pharmacological agents target these key genes for decreasing adipogenesis; however, they pose severe side effects like myopathy, gastric disturbances, liver toxicity.[12] Several herbal formulas are shown to possess anti-obesity activities in in vitro and in vivo models.[13],[14],[15] Many of herbal formula are proven efficacious and safe in clinical trials. The advantage of herbal combination is that the presence of multiple bioactives alleviates inflammation and oxidative stress in adipocytes. In the present study, we prepared a unique herbal combination with commiphora wightti, curcuma longa, and tinospora cordifolia (CCT) and screened for its anti-adipogenesis using 3T3-L1 cell line. In addition, the herbal combination is also evaluated for its anti-inflammatory and anti-oxidant potential during adipogenesis.


   Materials and Methods Top


Preparation of herbal formulations

Dried exudates of Commiphora wightti, dried rhizomes of Curcuma longa and dried stems and leaves of Tinospora cordifolia were obtained from local traditional medicine vendor and were authenticated by the botanist. Hot continuous Soxhlet’s extraction was carried out using ethyl acetate solvent for Commiphora wightti, 70% ethanol for curcuma longa and tinospora cordifolia. The individual extracts were concentrated under vacuum and dried to powder state. The extracts were then mixed in the ratio 1:1:1 to prepare the herbal formula CCT. The ratio of the individual herbs was fixed based on their cytotoxicity potential.

Determination of cytotoxicity of herbal formulation

3T3-L1 mouse fibroblasts (preadipocyte) were procured from National Centre for Cell Science (Pune, India). To establish the working concentrations for efficacy screening, cytotoxicity assay using MTT was performed. In brief, 3T3-L1 cells were seeded at density of 8 × 103 (±5%) cells/well in a 96 well plate. Cells were allowed to attach to the plate at 37°C in 5% CO2 incubator. After 24 hours, the cells were treated with various concentrations (1 ng/mL to 10 μg/mL) of the CCT for 20 hours. MTT was dissolved in PBS at 5 mg/mL and filtered and 10 µL of MTT solution was added to each well and incubated for 4 hours. After incubation period, the medium was aspired carefully from each well and 100 µL of DMSO was added to the wells. Color intensity was read at a test wavelength of 570 nm and a reference wavelength of 630 nm using PerkinElmer, USA. The experiments were performed in triplicates.[16]

Anti-adipogenesis screening of CCT

Experiments were performed in 60 mm petri plates and the cells were seeded at a density of 2 × 105 (±5%) cells/plate and allowed to attach overnight. 3T3-L1 mouse fibroblasts (preadipocyte) were procured from National Centre for Cell Science (Pune, India). Cells were grown in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% FBS and penicillin/streptomycin (100 IU, 100 mg/mL, respectively) at 37°C in 5% CO2 incubator. To induce differentiation, 2-day postconfluent 3T3-L1 preadipocytes (day 0) were stimulated for 24 hours by adding 51.8 mM 3-isobutyl-1-methylxanthine, 0.25 µM dexamethasone, and 0.1% insulin (MDI) to the DMEM/10% FBS medium. On day 3, the MDI medium was replaced with DMEM/10% FBS containing 1µM insulin. On day 4, the MDI medium was replaced with DMEM/10% FBS and refreshed for 2 days until analysis was performed on days 6 to 7. The experiments were performed in triplicates. Normal control cells (NCC) were represented by undifferentiated cells. Positive control cells (PCC) were represented by differentiated adipocytes treated with vehicle (0.1% DMSO). CCT was dissolved in 0.1%DMSO in culture medium. Cells received extracts of doses 10 and 100pg concentration/DMSO premixed with culture medium from day 0 to day 5, a period of time which covered the entire induction and post-induction stages.[17]

Oil red O staining

The formation of oil droplets in treated and untreated differentiated cells were analyzed by oil red O staining method. After differentiation, the media was removed, the cells were washed once with PBS, and then fixed for 1h with prechilled 10% formaldehyde prepared in PBS. Cells were stained with Oil Red O solution (a mixture of three parts of 0.5% (w/v) oil red O in isopropanol and two parts of water) for 2 hours at room temperature followed by washing with PBS twice, ethanol once and water twice. Cells were kept in water and photographed using inverted fluorescence microscope (Olympus, China) .[18]

Isolation of total RNA for mRNA expression studies by RT-PCR

Reverse transcriptase (RT) − PCR was performed to determine effect of the CCT on the mRNA expression of leptin, PPAR-γ, and SREBP1C, the key regulators in adipogenesis. Briefly, on day 6 after differentiation, total RNA was extracted from cultured cells using TRIzol Reagent (Sigma, USA). After homogenization, the tubes were incubated for 10min and centrifuged at 1000 rpm for 5 minutes 200 μL of chloroform was added to the supernatant, allowed to incubate for 5min at room temperature and centrifuged at 12,000 rcf for 20 minutes. Then 500 μL of isopropyl alcohol was added to the supernatant to precipitate the total RNA and centrifuged at 12,000 rcf for 15 minutes followed by 10 minutes incubation period. The supernatant was decanted carefully; the pellet was washed three times with 75% ethanol, centrifuged at 12,000 rcf for 15 minutes and the pellet was allowed to air dry. The pellet was resuspended in 20 μL of RNase free water and stored in −80°C until use .[19]

Determination of anti-inflammatory potential of the CCT

Degree of anti-inflammatory potency of the CCT was investigated by measuring the gene expression of proinflammatory markers such as TNFα and iNOS in the cytosolic lysates of treated and untreated 3T3 cells.

Data Analysis

Data were expressed as mean±SEM. The cytotoxicity assay was analyzed regression analysis. The mean differences between the groups were analyzed by one-way ANOVA followed by Dunnett multiple comparison as posthoc test. P ≤ 0.05 is considered as statistically significant. GraphPad Prism 7.0 is used for biostatics analysis.


   Results Top


Effect of CCT of 3T3-L1 cell viability

Exposure of 3T3-L1 cells to CCT produced a dose-dependent cell death with at IC50 of 139.60 μg/mL [Figure 1]. Further, experiments were carried out at 10 and 30 μg/mL.
Figure 1 Cytotoxicity of CCT: Exposure of 3T3-L1 to CCT has produced a dose-dependant cell death. The IC50 value was found to be 139.60µg/mL.

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Anti-adipogenesis effects of CCT–oil red O staining

Seven days after the initiation of adipogenesis experiments, oil red O stain was added to the treated and untreated cells. Vehicle (0.1%DMSO) treated differentiated cells showed highly incidences of lipid droplets and were found to be bigger in size when compared to the undifferentiated cells. Treatment with CCT produced a concentration dependent decrease in the size and number of lipid droplets as observed with oil red O staining when compared to the vehicle treated differentiated cells. This indicates that the CCT has the ability to decrease lipids/triglycerides formation [Figure 2].
Figure 2 Effect of CCT on lipid droplets formation – stained using Oil red O: Vehicle-treated differentiated cells have shown high incidence of lipid droplets and were found larger when compared to undifferentiated cells, whereas treatment with CCT has shown a dose-dependent decrease in the size and number of lipid droplets.

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Effects of CCT on key regulators of adipogenesis gene expression

mRNA expression of key regulators in the adipogenesis and lipid formation such as leptin, PPARγ and SREBP1C were measured in 3T3 −L1 cells treated with herbal formula. Treatment of 3T3-L1 preadipocyte with MDI, an adipogenic inducer, resulted in conversion of preadipocytes to matured adipocytes. Vehicle treated differentiated cells showed significant increase leptin (P < 0.01), and PPARγ (P < 0.01) and decreased SREBP1C (P < 0.01) mRNA expression when compared to vehicle undifferentiated cells. Treatment with herbal formulation, produced a dose-dependent decrease on leptin (13.05 and 58.92% ↓, respectively) and PPARγ (22.25% and 53.76% ↓, respectively) and upregulation of SREBP1C (8.21% and 71.86% ↑, respectively) gene expression [Figure 3].
Figure 3 Effect of CCT on the gene expression of PPARγ, leptin and SREBP1c in 3T3-Li differentiated cells: 3T3-L1 differentiated cells treated with MDI have resulted in the conversion of preadipocytes to matured adipocytes. Vehicle-treated differentiated cells showed significant increase leptin (P < 0.01), and PPARγ (P < 0.01) and decreased SREBP1C (P < 0.01) mRNA expression when compared to vehicle undifferentiated cells. Treatment with herbal formulation, produced a dose- dependent decrease on leptin (13.05% and 58.92% ↓, respectively) and PPARγ (22.25% and 53.76%↓, respectively) and up-regulation of SREBP1C (8.21% and 71.86%↑, respectively).

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Effect of CCT on TNFα and iNOS on mRNA expression

Differentiation of preadipocytes to adipocytes with MDI, produced a significant up-regulation of TNFα and iNOS mRNA expression when compared to the undifferentiated cells. This is indicating the formation of the increased inflammatory markers during adipogenesis. Treatment with CCT produced a dose-dependent (10 and 30 μg/mL) suppression of TNFα (38.97% and 68.19%, ↓, respectively) and iNOS (32.61 and 75.54% ↓, respectively) mRNA expression when compared to the vehicle treated differentiated cells [Figure 4] and [Figure 5].
Figure 4 Effect of CCT on the gene expression of TNFα and iNOS in 3T3-L1 differentiated cells: Treatment with MDI has resulted in up-regulation of TNFα and iNOS mRNA expression compared to undifferentiated cells. Treatment with CCT produced a dose-dependant (10 and 30 µg/mL) suppression of TNFα (38.97% and 68.19% ↓, respectively) and iNOS (32.61% and 75.54% ↓, respectively) mRNA expression compared to vehicle-treated differentiated cells.

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Figure 5 mRNA expression of β-actin, PPAγ, Leptin and SREBP1c.

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   Discussion Top


Slow deposition of lipids in the walls of the blood vessels causes vascular stiffness leading to plaque formation.[20] Hence, suppression of inflammation is one of the key important factors in treatment of hyperlipidemia and obesity. The present study demonstrates the dual actions viz the anti-adipogenesis and anti-inflammatory activities of a novel herbal combination containing commiphora wightti, curcuma longa and tinospora cordifolia. Cell viability assay indicate the relative safety of the herbal combination in 3T3-L1 cells with an IC50 value of 139.60 μg/mL.

Pathologically obesity is characterized by the presence of excessive adipose tissues and uncontrolled differentiation of preadipocytes to adipocytes, hyperplasia, and hypertrophy.[21], Hence, the initial prevention of adipocyte formation is crucial in the treatment of obesity.[22] The process of differentiation is very complex and involves various transcription factors. 3T3-L1 model of adipocyte differentiation is one of the gold standard model to screen the antiadipogenic activity of the compounds.[17],[23] Treatment of 3T3 −L1 preadipocyte with 3-isobutyl-1-methylxanthine, dexamethasone, and insulin (MDI), and then the continuous exposure insulin triggered differentiation. There is a significant increase in the adipocytes number, cell size, lipid accumulation.[24] Pretreatment with the CCT significantly decreased the lipid droplets formation, which reveals its potential interaction on the two stages of adipogenesis viz commitment and differentiation.

Peroxisome PPARγ is one of the crucial proadipogenic transcription factors and master regulator of adipocytes differentiation.[25] It exists as a heterodimer with retinoid receptor and binds to DNA and thus involved in adipogenesis.[26] In the present study, the CCT suppressed PPARγ mRNA expression which could be the possible reason for the decreased lipid droplets formation as observed in the oil red O staining. Leptin, a hormone secreted by adipose tissues, regulates energy intake and expenditure. Circulating leptin levels directly influence the fat mass or adipose tissue formation. Hyperleptinemia and body fat mass are direct indicators of leptin resistance, which as a causative factor of obesity.[27] CCT decreased leptin mRNA expression which reveals its ability to decrease leptin release and fat cells. PPARγ is a target gene for SREBP1c and reports indicate that SREBP1c increases the PPARγ activity by increasing the endogenous ligands for PAARγ and in turn increases adipogenesis.[27] Interestingly, the CCT suppressed the SREBP1c mRNA expression in the differentiated 3T3-L1 cells, indicating a potent anti-adipogenesis effect.

Chronic obesity is mostly associated with type II diabetes, hypertension, certain cancers, nonalcoholic fatty liver diseases and it impose major economic burden in healthcare system.[28] In fact, obesity is otherwise called as state of chronic low-grade systemic inflammation. Reports indicate that the circulating levels of TNFα, MCP-1, IL-1, etc., are elevated in obesity,[29],[30]and this is a predisposing factor in the development of insulin resistance.[31] The present CCT exerted significant anti-adipogenesis and anti-inflammatory activity in 3T3-L1 cell lines.Guggulsterone (GS), the major chemical constituent of Commiphora wightii which was reported to exert potential anti- adipogenesis effect by inhibiting maturation of preadipocytes and by initiating apoptosis and lipolysis of the matured adipocytes in 3T3-L1 cells.[32] GS also reported to exert prominent anti-inflammatory activity, treatment with GS has been found to inhibit IL-1β mediated chemokine production and epithelial neutrophil activating peptide-78 (ENA-78), MMP-1,-3 by suppressing NF-κB, nuclear p50, and p65.[33],[34],[35],[36] Curcuminoids like curcumin, demethoxycurcumin, and bisdemethoxycurcumin have shown promising anti-adipogenic and anti-inflammatory activity.[37] Curcumin blocks the adipocyte differentiation by inhibiting the adipogenic transcription factors like C/EBPα and PPARγ, also it reduces the phosphorylation of MAPK. In addition, curcumin activates Wnt/β-catenin signaling, which is a crucial regulator of adipocytes and plays a vital role in adipogenesis.[38] The anti-inflammatory properties of curcuminoids are associated with activation of PPAR-γ receptors, thereby reducing the levels of ALT and AST, lactate, and TNF-α.[39] In addition, curcuminoids also reduces the levels of C-reactive protein. Tinospora cordifolia is rich in phenolic and flavanoid contents. They inhibit TNF-α and thereby prevent the release of other inflammatory markers like IL-6, IL-8, and MCP-1. In addition, Tinospora cordifolia attenuates the lipopolysachride induced TNF-α transcription and secretion.[40] These evidences rationalize the combination formulation of commiphora wightii, curcuma longa and tinospora cardifolia is useful attempt and also potential anti-adipogenetic agent. It also helps to combat the inflammation associated with obesity.


   Conclusion Top


The present study demonstrates the potential anti-adipogenesis and anti-inflammatory activity of the unique combination of commiphora wightii, curcuma longa and tinospora cardifolia (1:1:1). CCT was found to regulate key anti-adipogenesis mRNA such as PPARγ, leptin, and SREBP1c and also anti-inflammatory genes TNFα and iNOS. Further, safety and efficacy studies using in vivo models are in progress in our laboratory.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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