|Year : 2023 | Volume
| Issue : 1 | Page : 23-30
Potential anti-adipogenic activity of Calligonum comosum cuminaldehyde on mouse 3T3-pre-adipocytes
Mohammad Ghaleb Mohammad1, Ahmed El-Serafi2, Mohamed Ibrahim Madkour1, Abeer Alhabshi3, Ansar Wadea3, Rola Abu Jabal3, Divyasree Sandeep3, Sameh S M. Soliman4
1 Research Institute for Medical and Health Sciences; Department of Medical Laboratory Sciences, College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates
2 Research Institute for Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates; Department of Biomedical and Clinical Sciences (BKV), Linköping University, Linköping, Sweden; Medical Biochemistry Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
3 Research Institute for Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates
4 Research Institute for Medical and Health Sciences; College of Pharmacy, University of Sharjah, Sharjah, United Arab Emirates
|Date of Submission||20-Jun-2022|
|Date of Decision||05-Oct-2022|
|Date of Acceptance||07-Oct-2022|
|Date of Web Publication||11-Nov-2022|
Dr. Mohammad Ghaleb Mohammad
Department of Medical laboratory Sciences, College of Health Sciences, University of Sharjah, P. O. Box 27272, Sharjah
United Arab Emirates
Source of Support: None, Conflict of Interest: None
Background: Obesity is a medical condition characterized by augmented body fat mass that can adversely affect human health. Several regimens were suggested to counteract obesity and fat accumulation with limited success. As plants are well-known source of medicinal products, we studied the potential anti-adipogenic activity of the essential oil extracted from Calligonum comosum plants growing in the desert of the United Arab Emirates.
Methods: C. comosum essential oil was extracted and fractionated on thin layer chromatography. The effect of total oil extract, the major compound-rich fraction, and the pure compound (cuminaldehyde) were tested on the viability, lipid content, and glucose uptake of 3T3-L1 cells. The capability of cuminaldehyde to reduce the formation of 3D-adipocyte pellets and expression of related transcripts was also tested.
Results: The results showed that C. comosum essential oil, particularly its major component cuminaldehyde, caused a significant reduction in the viability of 3T3-L1 cells when compared with fibroblasts, employed as controls. Furthermore, cuminaldehyde caused a significant reduction in the lipid content of 3T3 cells, as determined by Nile red stain, reduction in the glucose uptake, and reduction in the levels of both triglycerides and cholesterol. Moreover, cuminaldehyde significantly reduced the formation of 3D-adipocyte pellets and the expression of adipocyte-specific transcripts, CAAT-enhancer binding protein-alpha, and peroxisome proliferator-activated receptor-gamma.
Conclusion: Taken together, these results demonstrated a potential inhibition of lipid accumulation in 3T3 adipocytes after treatment with cuminaldehyde extracted from C. comosum oil. Thus, cuminaldehyde can be considered as a new potential anti-adipogenic agent for the prevention and treatment of obesity.
Keywords: 3T3 cells, adipocyte differentiation, anti-adipogenic, anti-obesity, cuminaldehyde, plant essential oil
|How to cite this article:|
Mohammad MG, El-Serafi A, Madkour MI, Alhabshi A, Wadea A, Jabal RA, Sandeep D, M. Soliman SS. Potential anti-adipogenic activity of Calligonum comosum cuminaldehyde on mouse 3T3-pre-adipocytes. Adv Biomed Health Sci 2023;2:23-30
|How to cite this URL:|
Mohammad MG, El-Serafi A, Madkour MI, Alhabshi A, Wadea A, Jabal RA, Sandeep D, M. Soliman SS. Potential anti-adipogenic activity of Calligonum comosum cuminaldehyde on mouse 3T3-pre-adipocytes. Adv Biomed Health Sci [serial online] 2023 [cited 2023 Jun 9];2:23-30. Available from: http://www.abhsjournal.net/text.asp?2023/2/1/23/361000
| Background|| |
Obesity is a medical condition in which the body fat mass is increased to a level that can adversely affect human health. According to the World Health Organization (WHO), obesity has tripled since 1975 and 39% of adults aged 18 years and above were overweight in 2020 and 13% were obese . Obesity is considered as a risk factor for life-threatening conditions, including cardiovascular diseases , musculoskeletal disorders , and some types of cancer . A fundamental cause of obesity is an energy imbalance between calories consumption and exhaustion . Obesity is largely preventable and can be managed by applying several strategies including the use of therapeutic compounds . A key strategy in reducing obesity is dampening adipocyte differentiation, reducing adipocyte hypertrophy and hyperplasia, as well as antagonizing fat accumulation . Several therapeutic compounds were developed in order to control general body obesity, as well as localized fat accumulation . In contrast, local communities accumulated considerable experience for the use of native growing plants as a treatment of adiposity. For example, compounds such as scoparone isolated from Chinese herb Artemisia scoparia , quinoa saponins , myricetin , 4-hydroxyderricin, and xanthoangelol from Japanese Ashitaba (Angelica keiskei)  have shown significant interference with adipocyte functions. Calligonum comosum L. has been employed by local communities in the United Arab Emirates (UAE) for decades in their foods for the treatment of several gastric and skin illnesses [13,14]. Various pharmacological activities of the plant were also reported, including anti-ulcer, anti-inflammatory, and anticancer activities [15,16]. Chemical analysis of C. comosum indicated the presence of major polar compounds with anticancer activity including (+)-catechin, dehydrodicatechin A, kaempferol-3-O-rhamnopyranoside, quercitrin (quercetin-3-O-rhamnopyranoside), beta-sitosterol-3-O-glucoside, isoquercitrin (quercetin-3-O-glucopyranoside), kaempferol-3-O-glucuronide, and mequilianin (quercetin-3-O-glucuronide) [15,17]. Besides, C. comosum is known as a source of volatile essential oil [15,17]; gas chromatography–mass spectrometry (GC–MS) analysis of the plant growing in the UAE indicated the presence of cuminaldehyde (50%), carene-10-al (~11%), and curcumene (~10%) as the major volatile compounds . Cuminaldehyde is known to mediate several physiological actions such as astringent, carminative, diuretic, stomachic, and stimulant . Its reported therapeutic activities included antidiarrheal, antidysenteric, antimicrobial, and anticancer [18,19]. Despite all of these reported activities, the mode of action of cuminaldehyde at the molecular level is still lacking. The purpose of this study was to investigate the anti-adipogenic activity of C. comosum oil and to determine a new lead compound that might interfere with adipocytes differentiation and metabolism.
| Materials and Methods|| |
C. comosum L. (Polygonaceae) plant materials (stems and roots) were collected during December 2016 from the desert of Sharjah Emirate (coordinates 25.3284° N, 55.5123° E), UAE . The plants were collected and identified according to the international standard of the plant taxonomy and by Prof. Ali El-Keblawy (Department of Biology, College of Sciences, Sharjah). There is no specific permission required for collecting plant samples from the open desert of UAE for scientific purposes.
Plant essential oil preparation and analysis
Air-dried plant stems were grinded, and the essential oil was extracted by the steam distillation method according to Soliman et al. [18,21]. Briefly, 200 g plant stem powder was extracted with water steam at 100oC for 3 h. The condensed oil was collected and dried over anhydrous sodium sulfate to yield 250 μL. The oil was diluted in chloroform in a ratio of 1:100 prior to the GC–MS analysis. The composition of essential oil was analyzed and identified according to Soliman et al.  using Agilent GC–MS. The identified compounds including the target compound, cuminaldehyde, were described in Soliman et al.  in both tabular and chromatogram forms.
Fractionation-based bioassay and partial purification of cuminaldehyde
Identification of the major anti-adipogenic compound in the plant essential oil extract was performed according to Soliman et al. . Briefly, the plant essential oil was separated on analytical thin layer chromatography (TLC) plates (Sigma-Aldrich, Germany) and developed by petroleum ether: acetone (5:1) as solvent system. The plate was visualized under UV lamp using short wavelength (250 nm). The major bright bands were scrapped off the plate and each fraction was tested for its anti-adipogenic activity using 3T3 cells. The fraction (# 3) containing the active compound, cuminaldehyde (CAS 122-03-2), was confirmed by GC–MS. The percentage of cuminaldehyde in the fraction was >50%. The fractionation using TLC and the corresponding GC–MS chromatogram were described in Soliman et al. .
The 3T3-L1 mouse pre-adipocyte cell line was originally developed by clonal expansion from murine Swiss 3T3 cells (ATCC, Virginia, USA). Normal human dermal fibroblast cell line (HDF, 106-05A, Sigma, EU) was employed as control. Both cell lines were cultured in the Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, Germany) supplemented with 25 mM glucose, 10% fetal bovine serum (FBS; Sigma-Aldrich), and 1% penicillin–streptomycin (Sigma-Aldrich) and maintained at 37oC in a humidified atmosphere of 5% CO2.
MTT cell viability assay and determination of LD50
The reduction of yellow tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) was used to measure cellular metabolic activity as a proxy for cell viability [18,23]. To measure the lethal dose that is required to induce 50% death of the cells (LD50), differentiated 3T3 and HDF cell lines were cultured until they reached 80% confluence. A 96-well plate was seeded with 4000 cells per 100 μL media and incubated at 37oC for 24 h. A serial dilution of C. comosum oil extract, its separated fractions, or cuminaldehyde-containing fraction was added onto the cells and incubated for a further 24 h. Freshly prepared MTT solution (5 mg/mL) was added to each well (20 μL per well) and followed by incubation for 2 h at 37oC. The supernatant was then removed and 100 μL dimethyl sulfoxide was added and incubated until formazan violet crystals were developed and the OD540 was measured.
Pre-adipocyte cell differentiation
Differentiation of pre-adipocytic 3T3 cells followed a protocol published by Vishwanath et al. . Briefly, 2 × 104 pre-3T3 cells were seeded in a 12-well plate until they reached confluence. The medium was then supplemented with a final concentration of 10 μM dexamethasone (Sigma-Aldrich, Germany), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Calbiochem, La Jolla, CA, USA), 100 μM indomethacin (Sigma-Aldrich, St Louis, MO, USA), and insulin-transferrin-sodium selenite (ITS) supplement to provide 10 μg/mL of insulin (Sigma-Aldrich). After 3 days, the medium was replaced with fresh media supplemented with ITS (10 μg/mL of insulin) only. The last step was repeated once again after 3 days. Cultures were maintained in a humidified incubator containing 5% (v/v) CO2 at 37°C. The accumulation of lipid droplets and adipocytes growth were monitored over the cultivation time.
Cholesterol, triglyceride, and glucose-intake measurements
Post-differentiated adipocytes were incubated for 24 h with C. comosum cuminaldehyde fraction in a 12-well plate at its LD50. Wells that received no oil were employed as negative controls. Cell lysates and culture supernatants were then obtained and stored at -80°C prior to testing. Enzymatic colorimetric assays were used to measure cholesterol, triglycerides, and glucose (Diasys Colorimetric Kits, Diagnostic Systems) in comparison to known standards (DIASYS) [25,26].
Nile red stain and flow cytometry
To quantify the changes in intracellular lipid droplets accumulation in differentiated adipocytes following the treatment for 24 h, Nile red stain was employed. Nile red stock solution (Sigma-Aldrich) was prepared in acetone and diluted in phosphate-buffered saline at a concentration of 0.5 μg/mL. The cells were then fixed with 4% paraformaldehyde. Fixed cells were stained with Nile red working solution (diluted 100× in 50 mM Tris/maleate) for 5 min at room temperature. Stained cells were further examined under an Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan) at excitation/emission wavelengths of 552/636 nm. Differential interference contrast images were taken to produce contrast by visually displaying the refractive index gradients of different areas of 3T3 cells. Other sets treated similarly were gently scrapped after staining, and positively stained cells were quantified using a BD FACS Area III flow cytometer.
RNA extraction, reverse transcription, and RT-PCR
To evaluate the gene expression related to the adipocyte differentiation, RNA was extracted from treated and untreated cells by the column-based RNeasy Mini Kit (Qiagen), according to manufacturer's instructions. Extracted RNA was quantified and reverse transcribed to cDNA using TruScript™ kit (Norgen, Thorold, Canada). For quantitative RT-PCR, cDNA was added as 100 ng/reaction to SYBR Green-based GoTaq® qPCR Master Mix (Promega, WI, USA). The primer sequence for CEBPa was F: 5′-TTACAACAGGCCAGGTTTCC-3′ and R: 5′-GGCTGGCGACATACAGATCA-3′, PPARγ was
F: 5′-TTTTCAAGGGTGCCAGTTTC-3′ and R: 5′-AATCCTTGGCCCTCTGAGAT-3′ [27,28]. The expression of Beta actin was employed as housekeeping gene for normalization. The primer sequence was F: 5′-GACAACGGCTCCGGCATGTGCAAAG-3′ and R: 5′-TTCACGGTTGGCCTTAGGGTTCAG-3′ . RT-PCR was carried out using Rotor Gene Q (Qiagen, Hilden, Germany) with initial incubation at 95°C for 15 min, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s, and 70°C for 30 s. Gene expression was calculated after normalization with β-actin following the 2−ΔΔCT method.
Pre-adipocyte pellet cultures
To evaluate the effects of cuminaldehyde on in-vitro morphological formation of 3T3 cells into tissue-like structures, a three-dimensional tissue culture approach was used. 3T3 pre-adipocytes were cultured as pellets, which provide a more physiological environment for cell-to-cell interaction, differentiation, and tissue-like formation. The procedure of pellet cultures preparation was performed according to the previously published protocols [29,30]. Briefly, 1 × 106 3T3 cells/pellet were suspended in 1 mL media and centrifuged for 10 min at 400g, followed by media change for three times a week. The pellets were then treated with cuminaldehyde at LD50 for 24 h. The weight and dimensions of the pellets were then measured, followed by fixation and standard processing for histological staining and examination.
Values were presented as means ± standard error of the mean (SEM) from three sets of experiments with three to six replicates per experiment. Data were analyzed by using t-test using GraphPad Prism software. Statistical significance was set at P < 0.05.
| Results|| |
C. comosum essential oil showed potential and safe
The effect of C. comosum essential oil on the viability of differentiated pre-adipocyte mouse cell line (3T3-L1) and normal fibroblast cell line (HDF) was determined using the MTT assay. The results showed that C. comosum oil caused selective inhibitory activities on 3T3 when compared with normal HDF cells. The oil at concentrations 0.4–0.8 μg/mL caused a significant reduction (~50%, P < 0.05) in the viability of differentiated 3T3 cells when compared with HDF fibroblasts, which showed a non-significant change in viability in response to both concentrations [Figure 1]. The results obtained indicated that C. comosum essential oil can be employed as a potential anti-adipogenic and 0.4–0.8 μg/mL was the concentration of choice for selective inhibition of 3T3 cells.
|Figure 1: Effect on viability of cuminaldehyde-rich oil separated from C. comosum against 3T3 and fibroblast. The viability of cuminaldehyde-treated and -untreated cells was measured using the MTT assay. Shown is the average of three separate experiments with six replicas per experiment ± SEM. *P < 0.05|
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Cuminaldehyde was the oil's major component responsible for the anti-adipogenic activity
The plant oil was subjected to fractionation-based 3T3 cell viability bioassay using the MTT assay. The oil was fractionated on TLC into five fractions and each fraction was tested on 3T3 cells. Fraction number 3 that contained >50% cuminaldehyde showed 90–100% killing activity when compared with other fractions, whereas its LD50 was determined as 0.4 μg/mL [Figure 1].
Cuminaldehyde was further tested on the intracellular lipid accumulation of 8-day post-differentiated 3T3 adipocytes using Nile red staining. The results indicated that cuminaldehyde at a concentration of 0.4 μg/mL caused a significant reduction (25%, P=0.038) in the intracellular lipid content of 3T3 cells [Figure 2]A, [Figure 2]B, [Figure 2]C, [Figure 2]D. The concentration of both triglycerides and cholesterol from cell lysates and the level of glucose in media supernatants were measured. The results showed a non-significant reduction in the triglyceride content (~10% reduction, P=0.495), whereas a 50% reduction in the cholesterol level content at a concentration of 0.4 μg/mL [P=0.009; [Figure 3]A was observed. In contrast, the glucose level after oil treatments was reduced by 40% [P=0.0012; [Figure 3]B].
|Figure 2: Reduction in the number of cells and the content of fat droplets within cuminaldehyde-treated 3T3 L1 cell line. Cuminaldehyde demonstrated significant reduction in the amount of fat droplet content within 3T3 L1 cell line after 24 h (A and B). 3T3 cells cultured in six-well plates were stained with Nile red and the images taken by a fluorescent microscope (FL-1 fluorescence and merged with DIC) where (A) represented control treatment and (B) represented treated cells with cuminaldehyde. (C) 3T3 cells were scraped, stained with Nile red, filtered, and measured by a BD FACS Area II flow cytometer. The histogram shows reduced fluorescence of treated cells (dark gray) compared with untreated cells (light gray). (D) Nile red fluorescence intensity was significantly decreased in comparison to the untreated control. Fluorescence intensity averages ± SEM are shown. Scale bar=100 μm, *P < 0.05.|
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|Figure 3: Effect of cuminaldehyde on the lipid and glucose metabolism of 3T3 cells. (A) Evaluation of triglycerides and cholesterol content in the cell lysate which is in response to treatment with cuminaldehyde showed a decrease in the cholesterol content, in comparison with untreated control. (B) Similarly, the glucose content in the media was less in response to treatment. *P < 0.05.|
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Expression of the genes CEBPa and PPARg coding for two adipogenesis transcription factors CEBPa and PPARg is crucial for the adipocyte maturation . Adding cuminaldehyde at a concentration of 0.4 μg/mL caused a significant reduction in the expression of CEBPα and PPARγ by ~50% and 70% [P=0.01 and 0.0001, respectively; [Figure 4]A].
|Figure 4: Cuminaldehyde significantly reduced the transcription of adipogenesis transcription factors and the weight of formed adipocyte pellets. (A) The effects of cuminaldehyde on the gene expression of adipogenesis transcription factors CEBPa and PPARg. The number of mRNA transcripts of both genes is significantly reduced as a result of treatment. β-actin gene expression was employed for normalization. (B) Three-week-old 3T3 cells were induced for the formation of 3D-culture pellets followed by treatment with cuminaldehyde compared with untreated control. Weight measurements of pellets indicated a significant reduction in the weight of treated pellets. (C) Representative photomicrographs showing hematoxylin/eosin-stained sections of treated and untreated pellets indicated interrupted core formation of the treated pellets. Scale bar= 200 μm. The results indicated as the mean ± SEM. *P < 0.05.|
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To investigate the effect of cuminaldehyde on the formation of tissue structures as another measure of safety of the compound, 3D tissue-like pellets with cuminaldehyde treatments were morphologically evaluated. Cuminaldehyde caused reduction by 30% in the weight of the 3D adipogenic pellets [P=0.044; [Figure 4]B]. Testing the histological structure of the formed pre-adipocytes pellets indicated an absence of intact inner center when compared with the untreated controls [Figure 4]C.
| Discussion|| |
Obesity is one of the world's health problems that affect approximately a third of the population with alarming projection of affecting half of the population by 2030 in many countries over the globe . Accumulation of adipocytes is obesity hallmark that involves the dysregulation of adipokines and metabolites that predispose and contribute to severity of many chronic diseases such as cancers, diabetes, and cardiovascular disease . An estimate of 20% of all cancers is attributed to obesity . Susceptibility genetic and environmental factors interact with each other to exacerbate the complexity of obesity. However, not all obesity is attributed to genetic-related factors. Obesity has been extensively investigated at the molecular level, and the impact of epigenetic modulation of gene expression in obesity has been recently highlighted . Several strategies and regimens have been established to control and reduce lipid accumulation, including those dependent on the inhibition of adipogenicity . Strategies used to reduce adiposity can include controlling calories intake, induction of lipolysis, enhancement of adipocyte apoptosis, and suppression of adipocyte differentiation . Here in this study, we demonstrated the ability of pure cuminaldehyde and cuminaldehyde-rich oil isolated from C. comosum as a natural product to enhance adipocyte metabolism and hence induction of lipolysis.
General or local obesity can be a result of increase in the tissue fat mass, which primarily consists of mature differentiated adipocytes . Thus, both lipolysis and inhibition of adipocyte differentiation can be considered as effective strategies in body weight loss or dissolution of local fat deposits. Consistently, our results indicated that cuminaldehyde selectively reduced the viability of differentiated 3T3 cells but not fibroblasts at 0.4 μg/mL. Inhibition of 3T3 is a known model associated with reduction in the fat mass of tissues . On the contrary, the cytotoxic effects of cuminaldehyde on 3T3 were accompanied by enhanced glucose intake and lipid turnover, which could be considered as an indicator of enhanced metabolism and/or cells metabolic stress. Enhanced metabolism can maintain the body energy stores and hence regulate the storage and mobilization of fat stores . In this regard, cuminaldehyde can be considered as a hypoglycemic inducing factor. The significant reduction in fat globules (stained with Nile red) due to the effect of cuminaldehyde could suggest that the compound interfered with fat formation by holding the adipogenesis or reflecting the dissolution of fat in adipocytes. A dampened adipogenesis would result in reduced fat production and formation of intact adipose tissue. This observation was supported by the significant reduction in the expression of transcripts known to control adipocyte maturation, as a result of treatment with the compound . These results can indicate that the anti-adipogenic potential effects of cuminaldehyde can be, at least partially, through antagonizing the expression of adipogenesis-related genes. The suppression effects of cuminaldehyde on adipocyte maturation are similar to inhibition effects of cocoa tea . Recently, cuminaldehyde has been reported as an agonist of the transient receptor potential Ankyrin 1 (TRPA1) channel , which influences obesity and metabolic syndrome through multiple actions . Possible mode of action is through the TRPA1-mediated cold sensing and thermal sensing generation, resulting in increased oxidation energy expenditure .
The process of adipogenesis is highly regulated by complex network including transcription factors, calories intake, fat utilization, and adipocyte maturation. C. comosum essential oil extract and its major component cuminaldehyde showed potential anti-adipogenic activities through reducing the expression of adipocytes differentiation-specific genes CEBPα and PPARγ. A similar inhibitory effect on these transcription factors was observed in previous studies [44-46]. Cuminaldehyde selectively reduced 3T3 cells' viability and increased the metabolic activities of adipocytes and hence the consumption of stored lipids for energy use along with suppression of pre-adipocytes differentiation. Additionally, cuminaldehyde interfered with size and formation of intact tissue-like structures in 3D cultured pellets. Further mechanistic studies addressing the fat metabolism and fat packaging are still lacking. Further, the anti-adipogenic effects of C. comosum essential oil have not been studied in laboratory animals. The aforementioned double activities of cuminaldehyde by reducing lipid content can be employed as a potential anti-adipogenic lead compound.
| Conclusion|| |
As concluding remarks, the results obtained demonstrated that cuminaldehyde and cuminaldehyde-rich essential oil extracted from C. comosum exhibited both suppression in pre-adipocytes maturation and an increase in lipid turnover, indicative of a potential anti-adipogenic activity. This study can be considered, up to our best of knowledge, as the first report for the anti-adipogenic activities of C. comosum essential oil extract and cuminaldehyde. The extract from C. comosum and/or its major component cuminaldehyde can be further investigated for the use as a potential therapeutic agent for the prevention and treatment of obesity.
This research was fulfilled with the competitive grant (1801050130-P) and targeted grant (1801050232-P) from University of Sharjah.
MGM, AE, and SSMS designed and developed the study. MGM and SSMS conceived the study and wrote the manuscript. SSMS was responsible for oil extraction, identification, and analysis of fractions and compound. AA, AE, AW, DS, RAJ, and MIM were responsible for laboratory work including cell culture, treatment, cell viability assay, Nile red staining, glucose, cholesterol, and triglyceride quantification. AA, AE, AW, and DS carried out the RNA extraction and gene expression experiments. MGM was responsible for data analysis and statistical calculations. MGM and SSMS interpreted the results and wrote the manuscript. All authors read and approved the final manuscript.
Conflict of interests
No conflict of interests declared.
Data availability statement
Data will be made available, if requested.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]