|Year : 2021 | Volume
| Issue : 4 | Page : 286-293
Desmodium gyrans dc modulates lipid trafficking in cultured macrophages and improves functional high-density lipoprotein in male wistar rats
MS Indu, Arunaksharan Narayanankutty, Smitha K Ramavarma, Jeksy Jose Manalil, Jose Padikkala, Achuthan Chathrattil Raghavamenon
Amala Cancer Research Centre, Thrissur, Kerala, India
|Date of Submission||22-Jul-2017|
|Date of Decision||13-Jun-2019|
|Date of Acceptance||23-Jun-2021|
|Date of Web Publication||18-Aug-2021|
Dr. Achuthan Chathrattil Raghavamenon
Department of Biochemistry, Amala Cancer Research Centre, Amala Nagar, Thrissur - 680 555, Kerala
Source of Support: None, Conflict of Interest: None
OBJECTIVE: High-density lipoprotein (HDL) cholesterol-mediated atherosclerotic plaque regression has gained wide therapeutic attention. The whole plant methanolic extract of the medicinal plant Desmodium gyrans Methanolic Extract (DGM) has shown to mitigate hyperlipidemia in high fat- and-cholesterol fed rats and rabbits with significant HDL enhancing property. The study aimed to assess the functionality and mechanistic basis of HDL promoting effect of DGM.
MATERIALS AND METHODS: Macrophage cholesterol efflux and foam cell formation assays were performed in THP-1 macrophages. Male Wistar rats were given DGM extract over 1 month and assessed the serum HDL, Apolipoprotein A1 (Apo-A1), and paraoxonase activity. Quantitative Polymerase chain reaction was carried out to assess the expression level of Apo-A1, SR-B1 (Scavenger receptor B1), and Cholesteryl ester transfer protein (CETP) on cDNA of HepG2 cells exposed to DGM.
RESULTS: Pretreatment of DGM inhibited uptake of oxidized lipids and enhanced the lipid efflux by THP-1-derived macrophages. Oral administration of DGM (100 and 250 mg/kg) progressively enhanced the serum HDL, Apo-A1 level, and associated paraoxonase activity in normal male Wistar rats. In support to this, DGM exposed HepG2 cells documented dose-dependent increase in the expression of SR-B1 and Apo-A1 mRNA, while reduced the CETP expression.
CONCLUSION: Overall the results indicated that DGM modulates lipid trafficking and possesses functional HDL enhancing potential through increased Apo-A1 levels and paraoxonase activity. Further, reduced CETP expression and increased expression of SR-B1 suggest the reverse cholesterol transport promoting role of DGM.
Keywords: Atheroprotection, cholesterol efflux, foam cells, high-density lipoprotein, reverse cholesterol transport
|How to cite this article:|
Indu M S, Narayanankutty A, Ramavarma SK, Manalil JJ, Padikkala J, Raghavamenon AC. Desmodium gyrans dc modulates lipid trafficking in cultured macrophages and improves functional high-density lipoprotein in male wistar rats. Indian J Pharmacol 2021;53:286-93
|How to cite this URL:|
Indu M S, Narayanankutty A, Ramavarma SK, Manalil JJ, Padikkala J, Raghavamenon AC. Desmodium gyrans dc modulates lipid trafficking in cultured macrophages and improves functional high-density lipoprotein in male wistar rats. Indian J Pharmacol [serial online] 2021 [cited 2023 Sep 27];53:286-93. Available from: https://www.ijp-online.com/text.asp?2021/53/4/286/324042
| » Introduction|| |
Atherosclerosis is a chronic degenerative disease manifested by the deposition of excessive cholesterol and oxidized lipids in the intima of large and medium-sized arteries that subsequently progresses to a vulnerable calcified plaque and eventually breaks, forming an intravascular clot. Reverse cholesterol transport (RCT) involving cholesterol and phospholipid efflux from lipid-laden macrophages and transport of these lipids to the liver for metabolism is expected to reduce plaque burden and thereby cardiac incidents. High-density lipoprotein cholesterol (HDL) plays a crucial role in RCT. Plasma membrane transporter proteins such as ATP-binding cassette transporter A1 (ABCA1), ATP-Binding Cassette Subfamily G Member 1 (ABCG1), and Scavenger receptor B1 (SR-B1) promote cholesterol efflux and these effluxed cholesterol and phospholipids are accepted by Apolipoprotein A1 forming nascent HDL. The nascent HDL, in course, matures and gets transported to the liver for metabolism and excretion. The functional HDL molecule has an esterase enzyme associated with it called paraoxonase, which protects low-density lipoprotein (LDL) from oxidation and acts as antiatherogenic molecule.
A drug that regulates cholesterol ingress to macrophage and thereby prevents its accumulation may be a promising candidate drug for the treatment of atherogenesis. HDL mimetic molecules have been used to enhance RCT and cholesteryl ester transfer protein (CETP) inhibitors are known to increase functional HDL. Number of studies have been reported that niacin is a known enhancer of functional HDL and a promoter of the RCT pathway. However, the clinical benefits from these molecules are nearly 25%–30% plaque regression. Hence, the quest for newer molecules with better activity is still under progress.
Desmodium gyrans (Thozhukanni) is a tropical shrub belonging to the Fabaceae family. The plant enjoys wide distribution in India and Sri Lanka, as well as regions of East-Southern Asia. Chinese medicine uses this plant for the cure of malaria, rheumatism, hepatitis cough, and dysentery. The plant is reported to have antimicrobial and wound healing properties with no toxic effects on the human body. Our studies have shown that D. gyrans increases HDL levels and lowers various lipid parameters in rats and rabbits fed high-cholesterol and-fat diet. The Safety of DGM extract in Wistar rats has been documented by our lab recently. The present study attempts to ensure the functional HDL enhancing effect as well as regulation of lipid trafficking in macrophages by DGM extract using cell culture and animal models.
| » Materials and Methods|| |
Plant authentication and preparation of extract
Plants collected from Marottichal (Thrissur District) were authenticated by Dr. C.N. Sunil (Department of Botany, SNM College Moothakunnam). Voucher specimen (SNMH-7012) was kept in the herbarium of SNM College, Moothakunnam. The collected aerial parts of the plant were cleaned, dried under shade, and powdered. The extract prepared using 70% methanol in the soxhlet apparatus was filtered and dried by evaporation. For cell culture studies, DGM was dissolved and diluted using dimethyl sulfoxide (DMSO) (10 μL/mL). For animal experimentations, DGM was dissolved in 1% propylene glycol.
THP-1 (Human monocytic leukemia) and HepG2 (hepatocellular carcinoma) cells were procured from the National Centre for Cell Science (NCCS) Pune, and cultured in DMEM containing fetal bovine serum (10% v/v), streptomycin (100 U/mL), penicillin (100 U/mL), and mercaptoethanol (0.05 mm) in a humidified incubator with 5% CO2 saturation set at 37°C.
For conversion of THP-1 monocyte into macrophages, THP-1 cells (1 × 105 cells/mL) were plated in 6 well plates. At a growth stage of 80%–90% confluence, the cells were overlaid with fresh DMEM containing 100 nm PMA (phorbol-12-myristate-13-acetate) and allowed to grow for 48 h. The cells which adhered to the growth surface were considered THP-1 derived macrophages.
Maintenance of experimental animal
Wistar rats with average weight of 150–180 g (gender male) were brought from SABS (Small Animal Breeding Station), College of Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala. They were maintained under standard conditions (22°C–28°C, 60%–79% relative humidity, and 12 h dark/light cycle) and had given food (Sai Durga Feeds and Foods, India) and water ad libitum. Previous studies conducted in our lab have shown the hypolipidemic properties of D. gyrans in high-cholesterol fed rats and rabbits in which significant increase in HDL level was observed. Further to this end, functional HDL promoting effect of DGM in normal rats fed normal laboratory diet was sought. The male Wistar rats were selected mainly to comply with our previous animal studies. The advantage of using rats over mice is that sufficient serum and liver tissue samples can be collected from rats for complete evaluation of lipid profiles and various enzyme activities. All the animal's experiments had prior permission (No. ACRC/IAEC/16-06-12) from IAEC (Institutional Animal Ethics Committee) keeping the standards of the Committee for the purpose of Control and Supervision of Experiments on Animals, Government of India.
MTT assay was performed on THP-1-derived macrophages and HepG2 cells to detect the toxicity of DGM extract toward cells. The cell lines were plated (1 × 105 cells) on different 12 well plates containing 1 mL complete Rosewell Park Memorial Institute (RPMI) medium and incubated at 37°C for 24 h. In the case of THP-I monocytes, the cells were converted to macrophages according to the method previously described. The attached macrophages and HepG2 cells were exposed to DGM at various concentrations (10–100 μg/mL) for 24 h. DMSO (100 μg/mL) was used as vehicle control and niacin (100 μg/mL) as standard. At the end of incubation, the cells were overlaid with fresh DMEM containing 0.5 mg/ mL MTT and incubated for another 3 h. The dark blue formazan crystals formed were dissolved in 1 mL solubilization solution (isopropanol-50 mL, 0.12N HCl-0.43 mL and triton ×100 [10%]-5 mL). The absorbance was measured at 570 nm. The optical density of untreated wells was set as 100% and the percentage cell viability was determined in the test well by comparing control optical density.
Oxidative modification of lipids
LDL was subjected to oxidation by 5 μM CuSO4. The oxidation reaction was carried out at 37°C and monitored over 24 h by measuring thiobarbituric acid-reactive substances generated in the reaction system.
Foam cell formation assay
The cells (THP-1 macrophages) were exposed to DGM (20, 60, and 100 μg/mL) for 4 h in a 12 well plate. About 50 μg/mL ox-lipids in fresh medium were added to these macrophages and incubated for 24 h. Following a brief wash with Phosphate-buffered saline (PBS), the cells were fixed using paraformaldehyde (4% in water), and neutral intracellular lipids stained with Oil Red O (60% in isopropanol). Stained cells were examined by an inverted microscope and images (×200 magnification) were taken by Magnus image capture system 5.20 [Figure 1].
Quantification of lipid accumulation
To each well, 1 mL isopropanol was added and kept for 15 min for complete extraction of stain, and absorbance was measured at 500 nm against isopropanol as blank. Absorption of untreated control samples was taken as 100% and percentage change in absorbance in the treated wells was then calculated.
Macrophage cholesterol efflux assay
The THP-1 macrophages (seed density 1 × 105 cells/mL) in 6 well plates at approximately 80%–90% growth stage were loaded with oxidized lipid (50 μg/mL) containing DMEM and incubated for 24 h. Following this, the spent medium was replaced with DGM extracts (20, 60, and 100 μg/mL) containing fresh medium and further incubated for 4 h washed with PBS and. At the end of the incubation period, the treatment medium was removed and cells were briefly washed and subjected to Oil Red O staining as described above. For quantitative measurements of the lipid retained, the stain was extracted using 60% isopropanol and absorbance read at 500 nm [Figure 2].
Screening high-density lipoprotein enhancing effect of DGM in vivo
Thirty male Wistar rats weighing approximately 160 g were grouped into five [Table 1]. Group I was the control without drug treatment and Group II and III animals were orally administered with 100 and 250 mg/kgb. wt DGM, respectively. Group IV animals were treated with clinically known drug niacin (10 mg/kgb. wt) and considered positive control. Those animals in Group V were treated with vehicle control (1% propylene glycol) over a period of 1-month. The level of HDL monitered at weekly intervals using commercially available kits. At the end, the animals were sacrificed following overnight fasting and serum HDL level and paraoxonase activity were measured. The liver tissues were excised and paraoxonase (PON1) activity was also assessed spectrophotometrically.
Gene expression of Apo-protein A1, Scavenger receptor B1 and cholesteryl ester transfer protein in HePG2 cells
cDNA synthesis and quantitative polymerase chain reaction
HepG2 cells were seeded at a concentration of one million cells per mL in 75 mL tissue culture flasks. The cells were allowed to grow till sub-confluency and incubated with DGM at varying concentrations (20, 60, and 100 μg/mL) for a day. Entire RNA from the cells was extracted and purified by the TRIzol reagent method. The yield of RNA was determined based on the absorbance at 260 nm and calculated using the formula A260 X dilution X 40.
For cDNA synthesis, total RNA (1ŋg) in a 20 μL reaction volume was reverse transcribed using Anchored Oligo (dT) primers (Verso cDNA synthesis kit). The thermal conditions for the reaction were 42°C for 55 min and an inactivation step done at 95°C for 2 min.
Real-time quantitative polymerase chain reaction (PCR) was run using GoTaq Master Mix added with 2 μL of template cDNA and 0.1 μM each of gene-specific primers (forward and reverse primers of respective genes) in a final volume of 20 μL. [Table 2] shows the primer sequences (Sigma-Aldrich Inc., St Louis, USA) used for the analysis. β-actin was the endogenous control gene used The PCR products (10 μL) were resolved on 1.2% agarose gel with ethidium bromide using 1X TAE buffer and visualized under UV light on a gel documentation system.
|Table 2: The primer sequences used for the real time-polymerase chain reaction analysis|
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Quantitative polymerase chain reaction analysis
Real-time quantitative PCR (qPCR) was performed on Eco™ Real-Time PCR system V126.96.36.199. Reaction mix (final volume10 μL) contained Power SYBR Green PCR Master Mix (Life technologies, UK) 1 μL of cDNA and gene-specific primers. Primers for β-actin were used (housekeeping gene) for normalization of gene expression. Based on the comparative computed tomography method the qPCR data were analyzed.
Data are presented as Mean ± standard deviation of three different experiments in duplicate. Significance difference among was analyzed by one-way analysis of variance and confirmed by wed by Dunnett's test using graphpad inStat 3 software. P ≤ 0.05 is considered statistically significant.
| » Results|| |
MTT cytotoxicity assay
Toxicity assessment of DGM extract toward THP-1-derived macrophages and HepG2 cells are depicted in [Figure 3]. The study showed that within the range of 10–100 μg/mL concentration, DGM did not show toxicity in the THP-1-derived macrophages [Figure 3]a and HepG2 cells [Figure 3]b.
|Figure 3: Toxicity analysis of DGM on THP-1 macrophages (a) and HepG2 cells (b). Both Cells (1 × 105 cells) in 24 well plates were treated with DGM at different concentrations (10–100 μg/mL) for 24 h in RPMI containing 10% fetal bovine serum. The cell viability was measured using MTT assay. Values were expressed as mean ± standard deviation of triplicate assays|
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Foam cell formation assay
[Figure 4] shows the Oil Red O-stained THP-1 macrophages at various experimental conditions. The numbers of lipid-stained cells were high in control wells [Figure 4]a and DMSO treated cells [Figure 4]b. Cells pretreatment with DGM extracts at 20, 60, and 100 μg/ml [Figure 4]d, [Figure 4]e, [Figure 4]f had comparatively fewer numbers of lipid-stained cells. The number of stained cells was comparatively less than the niacin-treated cells [Figure 4]c. The intracellular stains when extracted and quantified [Figure 6]a, showed that pretreatment with DGM reduced lipid uptake by the cells.
|Figure 4: Foam cell formation assay by DGM exposed THP-1 macrophages. Cells (1 × 105 cells) were exposed to (a) no additive, (b) Vehicle control (c) Niacin (100 μg/mL) (d) 20 (e) 60 and (f) 100 μg/mL of DGM extract for 24 h and 50 μg/mL ox-low-density lipoprotein was added to these macrophages and incubated for 24 h. Then Oil Red “O” staining was performed. Images were taken using Magnus INVI microscope at ×400 magnification. Vehicle control-dimethyl sulfoxide (10 μL/mL), standard - niacin (100 μg/mL)|
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|Figure 6: The percentage inhibition of foam cell formation by DGM in THP-1 macrophages induced by ox-low-density lipoprotein (a). The percentage of efflux of lipids by DGM in low-density lipoprotein in THP-1 macrophages induced by ox-low-density lipoprotein (b). For quantitative measurements of the lipid retained, the stains were extracted using 60% isopropanol for 15 min and absorbance was read at 500 nm. Values were expressed as mean ± standard deviation of triplicate experiments|
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Macrophage cholesterol efflux assay
[Figure 5] shows the Oil red O-stained macrophages at various experimental conditions. The numbers of lipid-stained cells were high in control cells [Figure 5]a or vehicle-treated cells [Figure 5]b. Here, niacin was used as standard [Figure 5]c. The DGM of three different concentrations [Figure 5]d, [Figure 5]e, [Figure 5]f showed maximum efflux of cholesterol. When the intracellular stains were extracted and quantified [Figure 6]b the untreated control and vehicle control cells had more quantity of lipids than the treated wells.
|Figure 5: Macrophage cholesterol efflux assay by DGM exposed THP-1 macrophages. The macrophages (1 × 105) were loaded with oxidized low-density lipoprotein (50 μg/mL) for 24 h, further incubated with (a) no additive, (b) Vehicle control (c) Standard (d) 20 (e) 60 and (f) 100 μg/mL of DGM for 4h and stained with Oil Red “O” stain. Images were taken using Magnus INVI microscope at ×400 magnification. Vehicle control- dimethyl sulfoxide (10 μL/mL), standard - niacin (100 μg/mL)|
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In vivo functional high-density lipoprotein enhancing effect of Desmodium gyrans
The normal male Wistar rats given DGM extract orally had significantly increased level of HDL. The increase was gradual, at the end of the 4th week, there was 71 ± 9.1 and 88 ± 1.6 mg/dL HDL in the 100 mg and 250 mg/kgb. wt. DGM ingested animals. The normal group of animals had 38 ± 4.1 mg/dL HDL [Figure 7].
|Figure 7: The level of high-density lipoprotein in normal Wistar rats ingested with DGM extracts orally over a period of one month. Values were expressed as Mean ± standard deviation of six animals per group; a = P < 0.01 compared to untreated group|
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The HDL-associated paraoxonase activity was also found increased in the DGM-treated animals. The serum paraoxonase activity in the normal animals was 0.0225 ± 0.0065 per mg protein. The activity was elevated to 0.053 ± 0.001 and 0.065 ± 0.005 per mg protein in 100 and 250 mg/kgb. wt ingested animals, respectively [Table 3]. The paraoxonase activity in the liver tissue of normal animals was 3.39 ± 0.81per mg proteins. The enzyme activity was increased to 4.91 ± 0.73 and 9.65 ± 0.38 per mg protein in 100 and 250 mg/kgb. wt ingested animals, respectively. In these animals, Apolipoprotein A1 (Apo-A1) was found to be increased with respect to the normal group of animals. The increase of Apo-A1 in groups treated with 100 and 250 mg/kgb. wt was found to be 221 and 291 mg/dL, respectively [Figure 8].
|Table 3: Serum and liver paraoxonase activity (.OD/min/mg protein) in wistar rats|
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|Figure 8: The level of Apo-protein A1 in normal Wistar rats ingested both DGM extract orally over a period of one month. Values were expressed as Mean ± standard deviation of six animals per group; a = P < 0.01 compared to untreated group|
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Effect of the crude DGM extract on the gene expression of Scavenger receptor B1, Apo-protein A1, and cholesteryl ester transfer protein
DGM crude extract significantly increased (4-fold) the mRNA expression of SR-B1 in HepG2 cells in a dose-dependent manner compared to normal untreated cells. The qPCR results also revealed a higher expression of Apo-A1 (4-fold) in DGM-treated cells. On the other hand, animals ingested with DGM crude extract at lower doses did not show significant change at low doses in the hepatic mRNA expression of CETP [Figure 7]. However, higher dose DGM (100 μg/mL treated) and niacin-treated animals hepatic CETP expression was found to be decreased [Figure 9].
|Figure 9: (a) Expressions of Scavenger receptor B1, Apo-protein A1 and cholesteryl ester transfer protein in HepG2 cells exposed to DGM extract. The gene expressions were compared by relative quantification using ΔΔCT method and the fold change was calculated as 2–ΔΔCT. Values were expressed as mean ± standard deviation. a = P < 0.01, ns = P > 0.01 compared to control group. (b) Electrophoresis profile of amplified genes. C-control N-Niacin|
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| » Discussion|| |
In the present study, DGM dose-dependently prevents the uptake of oxidized lipids by THP-1-derived macrophages and substantially enhances cellular lipid efflux. This observation has great significance in atherogenesis as macrophages are the primary cells involved in lipid uptake and foam cell formation. Previous studies from our lab have clearly indicated the hypolipidemic and HDL enhancing efficacy of DGM. Together, present observation that DGM modulates lipid trafficking in macrophage cells indicate its anti-atherogenic potential [Figure 10].
The oral administration of DGM is found to progressively enhance serum HDL, Apo-A1 levels, and associated paraoxonase activity in Wistar rats. The observed effect is found higher with the higher dose of DGM and comparatively higher than niacin. The enhanced paraoxonase activity in the serum and liver tissue and increased Apo-A1 levels of normal rats received DGM suggests that the HDL formed is functional. It is expected that increase in HDL may be due to the increase in the synthesis of Apo-A1.
This assumption is further ascertained by the observation that an upregulation occurs in the Apo-A1, SR-B1, and CETP genes in DGM exposed HepG2 cells. Therefore, observed increase in the Apo-A1 and paraoxonase protein expression in DGM exposed HepG2 cells, supports the in vivo observation that DGM increases functional HDL.
Moreover, the present study observes increase in SR-B1 and decrease in CETP expressions in HepG2 cells exposed to DGM extract. SR-B1 mediates lipid efflux from macrophage foam cells and also promotes hepatic uptake and biliary secretion of HDL cholesterol.,
The CETP inhibitors are reported to increase HDL-C in humans and animals. However, the notion that CETP deficiency prevents atherogenesis is a subject of debate. In this study, decreased CETP expression in DGM exposed cells could be supporting the enhanced HDL level by DGM ingested animals. The same could be the reason behind the increased HDL observed in high-fat diet fed rat model study reported by our lab recently.
Chemical analysis of the DGM extract revealed higher amounts of polyphenols. Recent studies show that polyphenols have a role in enhancing SR-B1 expression. Polyphenols of olive oil and grape increase functional HDL., Possibly the phenolics in DGM account for its functional HDL enhancing efficacy which needs to be further ascertained
| » Conclusion|| |
The study suggests that DGM possess functional HDL enhancing efficacy partially due to enhanced synthesis of Apo-AI and paraoxonase and also due to inhibition of CETP synthesis. Together with SR-B1 expression, DGM is likely to have RCT enhancing potential, and the active component responsible for these needs to be characterized to become a potential candidate for atheroprotection.
The study was financially supported by the Science Engineering Research Board, Department of Science and Technology, India (File no. SR/SO/HS-0240/2012).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
[Table 1], [Table 2], [Table 3]