The administration of low-dose Curcuma longa extract induces M2 polarization in peritoneal macrophage culture

 

Afiat Berbudi1,2*, Nur Rahmi3*, Nur Atik4, Tenny Wikayani5, Nurul Qomarilla5,

Nurul Setia Rahayu6, Almahitta Cintami Putri7

1Department of Biomedical Sciences, Parasitology Division, Faculty of Medicine,

Universitas Padjadjaran, Bandung, Indonesia.

2Faculty of Medicine, Universitas Pasundan, Bandung, West Java, Indonesia.

3 Undergraduate Program, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, Indonesia.

4Department of Biomedical Sciences, Cell Biology Division, Faculty of Medicine,

Universitas Padjadjaran, Bandung, Indonesia.

5Cell Culture and Cytogenetic Laboratory, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia.

6Molecular Genetic Laboratory, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia.

7Department of Surgery, Plastic and Reconstructive Surgery Division, Faculty of Medicine, Universitas Padjadjaran/Dr. Hasan Sadikin General Hospital, Bandung, Indonesia.

*Corresponding Author E-mail: a.berbudi@unpad.ac.id.

 

ABSTRACT:

Curcuma longa (turmeric) has been widely used to accelerate wound healing, but the underlying mechanism remains unclear. Wound healing consists of four phases which are correlated and overlapping, i.e., coagulation, inflammation, proliferation and remodeling. Macrophages play an important role in most phases. Macrophage polarization to M2 initiates the proliferation phase, which is characterized by the production of several crucial growth factors. Since turmeric has been known to be an herb that accelerates wound closure, we investigated whether Curcuma longa extract administration in macrophage culture induces M2 macrophage switching. An in vitro study was performed using peritoneal macrophages from Swiss Webster strain mice. Peritoneal cells were collected and cultured in a 24-well plate. After 2 hours of incubation, macrophages (adherent cells) were treated with 0.5 ppm, 1.0 ppm and 5.0 ppm of ethanolic extract of Curcuma longa and incubated for 2 days. Quantitative real time PCR was performed to quantify M2 and M1 marker gene expression. The results revealed the upregulation of M2 marker (Arginase-1) expression upon administration of 0.5 ppm of Curcuma longa extract, but not of higher doses (1.0 and 5.0 ppm). In parallel, the ratio of Arg-1/Inos was high upon administration of 0. 5ppm of extract. In conclusion, Curcuma longa extract induces in vitro M2 polarization in low-dose administration.

 

KEYWORDS: Arginase-1; Turmeric; Curcuma longa extract; Inos; M2 macrophage.

 

 


INTRODUCTION:

A wound can be defined as a loss or disturbance in the continuity of the normal tissue structure. In many cases, a wound often contributes to the hospitalization of patients1,2. More than 1 billion USD was spent on wound care globally, becoming one of the highest health costs1,2.

 

In the USA, more than 11 million people have acute injuries and 300,000 people have to be hospitalized per year3. Moreover, approximately 6.5 million people have chronic wounds that are caused by pressure, venous stasis, and diabetes mellitus3. In developing countries, 1-2% of the population are estimated to be at risk of chronic wounds and the case incidence continues to grow1,4. The incidence of chronic wounds is worsened by diabetes mellitus and the lack of effective treatment1,4,5. Such wounds have a negative impact upon not only the patient but also his/her family economically, psychologically or socially4. The mechanisms of wound healing are focused in many studies in order to find a novel strategy with which to improve the healing process and help to prevent and reduce its negative effects2.

 

The wound-healing process is a dynamic process of restoring the structural integrity and the functional tissue that was damaged as much as possible to its original state6. This process consists of four phases which are correlated and overlapping, i.e. coagulation, inflammation, proliferation and remodelling3,6. These phases must occur at the right time and in sequence to produce the optimal results. A macrophage is one of the key components in the wound-healing process, since it plays an important role in almost all of those phases7. Based on its polarization, a macrophage is divided into two types, i.e. classically activated macrophage (CAM or M1), which plays a role in inflammation and in cleaning the wound debris through producing pro-inflammatory cytokines and phagocytosis activity, and alternatively activated macrophage (AAM or M2), which produces an anti-inflammatory effect by producing IL-108. Furthermore, M2 macrophages may induce cell proliferation, angiogenesis and remodelling through producing several growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-β)8. A disturbance in M2 macrophage polarization results in prolonged wound healing (chronic wound)5,8.

 

In general, pharmacological therapy of the wound is carried out through the administration of analgesic, antiseptic and antibiotic drugs6. However, such therapy is quite expensive and some drugs, such as nonsteroidal anti-inflammatory drugs (NSAID), can slow down the healing process6. Since various medicinal plants with an affordable cost are known to be potential agents to be used as an alternative treatment for accelerating the wound-healing process, further scientific identification and validation are required before the medicinal plants can be recommended to patients.

 

Curcuma longa (C. longa/ turmeric) is one of the medicinal plants that potential to be used to improve the wound-healing process. C. longa contains various substances such as vitamins, minerals, tannin, flavonoids, and curcumin (diferuloylmethane), which have anti-inflammatory, antioxidant and anti-infection effects and can facilitate a proper wound-healing process7,9,10. Since C. longa has been used empirically to improve wound healing, and because M2 macrophages play an important role in the wound-healing process, an in vitro experiment was conducted to investigate whether the administration of ethanolic extract of C. longa modulates the M2 macrophage polarization in macrophage culture.

 

MATERIAL AND METHODS:

Research design:

This study was a quantitative analytical study using primary data taken from in vitro laboratory experimental studies. The experiments were conducted in the Laboratory of Cell Culture and Cytogenetic Universitas Padjadjaran teaching hospital.

 

Animals and animal care:

Peritoneal macrophages were collected from three male wild-type Swiss Webster mice. Before peritoneal lavage was performed, the mice were bred and housed at the animal facilities of the Universitas Padjadjaran teaching hospital. They were maintained in an animal room with a 12-hour day/night cycle and food and water ad libitum. All the experiments using animal and cell culture had been approved by the ethical committee of Universitas Padjadjaran (No. 459/UN6. KEP/EC/2018) and carried out according to the International Guide for the Care and Use of Laboratory Animals.

 

Curcuma longa extract:

C. longa rhizomes were obtained from Lembang, West Java. After being cleaned, C. longa rhizomes were dried and ground to obtain powder. A total of 100 grams of C. longa rhizome powder was placed in a container and extracted using a macerator by adding 900 ml of 96% v/v ethanol for 3 days at 37˚C with occasional shaking and stirring. Thereafter, the macerate was transferred to a rotary evaporator at a temperature of 50˚C to evaporate the ethanol and produce a liquid extract. This liquid was then reheated in a water bath at 40˚C until a C. longa extract paste was obtained. C. longa extract was then dissolved using DMSO (Sigma-Aldrich, USA) to produce C. longa extract stock solution with 40,000 ppm. For administration, the C. longa stock solution was then diluted in supplemented medium to obtain final concentrations of 0.5 ppm, 1.0 ppm and 5.0 ppm.

 

Macrophage isolation:

To obtain peritoneal cells from mice, all the mice were anesthetised using inhaled isoflurane for ~5 minutes until they were dead. The surface of the abdomen skin of the mice was washed using 70% ethanol for aseptic treatment. A little cut was made on the abdomen skin using scissors and the skin was retracted to expose the mice’s peritoneum. Thereafter, sterile pre-cold phosphate buffer saline (PBS) (Gibco, USA) was injected into the mice’s peritoneal cavity and shaken gently11. The intraperitoneal washed PBS was aspirated back and was pooled in a 50 ml tube on ice. To obtain peritoneal cells, centrifugation was performed at 3000 rpm and at 4oC for 8 minutes. Afterwards, the supernatant was removed and replaced with supplemented RPMI (Gibco, USA) (containing 10% FBS (foetal bovine serum) (Gibco, USA) and 1% penicillin-streptomycin), and the cell number was then obtained using an improved Neubauer chamber.

 

Macrophage culture:

The peritoneal cells were placed in 24-well cell culture plates (Jet Biofil, China) with each well containing ~3 million cells. The plates were then incubated at 37oC and 5% CO2 for 2 hours. After incubation, the non-adherent (floating) cells were removed, while the adherent cells (macrophages) were washed three times using warmed PBS12,13. A new supplemented medium (RPMI, 10% FBS, 1% penicillin-streptomycin) without C. longa extract was transferred into each well of the control group. As for the treatment group, the supplemented medium containing C. longa extract with the final concentrations of 0.5 ppm, 1.0 ppm and 5.0 ppm were then added into each well. The macrophages in 24-well culture plates were then incubated at 37oC and 5% CO2 for 2 days.

 

RNA extraction:

Firstly, to detach the macrophages, scrapping was performed on the base of each well using a scrapper (Jet Biofil, China). Moreover, 1ml of Qiazol (Qiagen, Germany) was added into each well to induce lysis upon the cells, and then pipetting was carried out gently. The lysate was then transferred into 1.5 ml of micro-centrifuge tube and 100 μl of chloroform was added gently. The tube was shaken for 10 seconds and incubated for 10 minutes at room temperature. Centrifugation was then carried out at 12,000 xg for 12 minutes at a temperature of 4oC. After three phases were formed, 350 μl of the clear top layer consisting of the RNA was transferred into a new tube and washed with one volume of 70% ethanol. Thereafter, RNA precipitate was transferred into a mini-column tube, consisting of an RB column and a 2 ml collection tube, to be purified using the column method (Geneaid RNA extraction kit, Taiwan) and centrifuged for 1 minute at 14,000 xg and at a temperature of 4oC. The next step was performed according to the manufacturer’s (Geneaid) instructions. The extracted RNA step was then quantified using a spectrophotometer. The concentration of RNA was measured with absorbance at 260 nm and 280 nm.

 

Real-time PCR:

At least 4.2 µg/ml of the total RNA was reverse transcribed to be complementary DNA (cDNA) using ReverTra Ace® qPCR RT Master Mix (Toyobo, Japan) according to the manufacturer’s instructions. PCR reaction was then carried out using Thunderbird®SYBR®qPCR Mix (Toyobo, Japan) according to the manufacturer’s instructions. Real-time PCR was performed using a Rotorgene Q cycler (Qiagen, Germany). The performed gene expression analysis were comprised Arginase-1 (Arg-1) as a marker for the M2 macrophage and inducible nitric oxide synthase (Inos) as M1’s marker. The gene expressions were analyzed using the following formula: 2-ΔΔCt, in which

 

ΔΔCt = (Ctgene of interest- Ctreference gene) treatment group – (Ctgene of interest- Ctreference gene) control group.

 

The degree of gene expression was normalized with housekeeping gene β-actin as a reference gene and the result was presented as a fold change to the control group. The primer sequences (mouse) are presented in Table 1.

 

Statistical analysis:

The gene expression data were processed and analyzed using GraphPad Prism (Version 8) software. An ANOVA test was conducted for normal distributed data, while abnormal distributed data were analyzed using the Kruskal–Wallis test and a post hoc Mann–Whitney test. The difference between groups is classified as significant when p<0.05.

 

RESULTS AND DISCUSSION:

As presented in Figure 1A, upon administration of 0.5 ppm of C. longa extract, the Arg-1 (M2 marker) was significantly upregulated compared to the untreated group (control) (p=0.022). Interestingly, in the higher-dose administration of C. longa extract (1.0 ppm and 5.0 ppm), the Arg-1 expression was not upregulated any more compared to the control. Similarly, the Inos expression upon administration of 0.5 ppm of C. longa was also upregulated (p=0.044). Upon administration of 1.0 ppm and 5.0 ppm of C. longa extract, the Inos expression tended to be upregulated, but not significant (Figure 1B).


 

Table 1: Primer sequences used for quantitative real-time PCR

Gene

Forward (5’---3’)

Reverse (5’---3’)

Mouse Arginase-1

CCTATGTGTCATTTGGGTGGA

CAGGAGAAAGGACACAGGTTG

Mouse Inos

CCAAGCCCTCACCTACTTCC

CTCTGAGGGCTGACACAAGG

Mouse β-actin

AGAGGGAAATCGTGCGTGAC

CAATAGTGATGACCTGGCGGT

Quantitative real-time PCR was carried out at a temperature of 95oC for 10 minutes, followed by at least 40 cycles at 95oC for 20 seconds and at 60oC for 1 minute.

 


Since Inos (M1 marker) was also upregulated upon C. longa administration, we then analyzed the ratio of Arg-1 compared to Inos to determine the proportion of M2 macrophages to M1 macrophages after the administration of C. longa extract. The data show that the administration of 0.5 ppm of C. longa extract tended to produce more M2 macrophages than M1 macrophages compared to the untreated cells (p=0.133) and the cells treated with 5.0 ppm of C. longa extract (p=0.0023) (Figure 1C), suggesting that M2 polarisation occurred upon a low dose of C. longa extract administration, but not upon a high dose.


 

Figure 1: Administration of Curcuma longa extract at low dose induced M2 macrophage switching. A) Relative gene expression of Arg-1; B) Inos within peritoneal macrophage culture; C) The proportion of M2 macrophages to M1 macrophages, represented by ratio of Arg-1/Inos. Gene expression is given as fold change after normalized to β-actin. Statistical significance between comparisons of two groups was determined using Mann–Whitney U-test. Data are expressed as means + SEM. *p<0.05, **p<0.01.

 


Turmeric contains three main curcuminoids, i.e. curcumin, demethoxycurcumin and bisdemethoxycurcumin10,14. Curcumin (C21H20O5), which is known as diferuloylmethane or 1,7- bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-dione, is a curcuminoid that has been widely studied14. Based on previous studies, curcumin has an immunomodulatory effect; therefore, it has the potency to be used as an anti-inflammatory, anti-infection and anti-tumour15. Curcumin suppresses both pro-inflammatory cytokine production and the activity of reactive oxygen species (ROS) in macrophages. Furthermore, curcumin induces the production of IL-4 and IL-13 in macrophages16,17. These cytokines have been known to stimulate anti-inflammatory effects through several mechanisms, such as activating the signal transducer and activator of transcription 6 (STAT6), which is a transcription factor that plays a role in the induction of M2 macrophage polarisation16–19. In line with this, the upregulation of Arg-1 and the Arg-1/Inos ratio in our in vitro study may be the result of IL-4 and IL-13 induction by the curcumin compound in C. longa extract. Further study should be conducted to investigate this hypothesis.

 

As shown in our results, the administration of low-dose ethanolic extract of C. longa was able to upregulate Arg-1 (M2 marker) and the Arg-1/Inos ratio in macrophage culture, which indicates that low-dose administration of C. longa extract increases the proportion of M2 macrophages to M1 macrophages in the culture. Both M1 and M2 macrophages are known to be essential cells in the wound-healing process. The depletion of macrophages during wound healing reduces TGF-b and VEGF, leads to a signalling disturbance, and delays the formation and maturation of new tissue20. At the beginning of wound healing, M1 macrophages play a role in the clearance of pathogens and debris in wound tissue, while M2 macrophages predominate in the later stages of wound healing, repairing damaged tissue and promoting new tissue formation21. Since M2 macrophages play crucial roles in producing several growth factors in new tissue formation during wound healing, our findings in this study support the hypothesis that the revealed acceleration of wound healing upon C. longa treatment may derive from increased M2 macrophages in wound tissue.

 

In addition to several growth factors that are produced by M2 macrophages, Arginase-1, which is an intracellular enzyme produced by M2, was also predicted to be one of the proteins that improve wound healing. One of the functional differences between M1 macrophages and M2 macrophages is the metabolism of amino acid arginine, which is influenced by the environmental stimuli of macrophages22. L-arginine can act as the substrate of iNOS (inducible nitric oxide synthase), which is produced by M1 to produce L-citrulline and nitric oxide (NO), one of the effector molecules of cytotoxic activity of macrophages in a state of infection or inflammation5,22. On the other hand, L-arginine can also act as the substrate of Arginase-1, which is an intracellular enzyme of M2 macrophages that produces L-ornithine and urea5. L-ornithine plays a role in polyamine production for cell proliferation. In addition, L-ornithine increases L-proline for collagen synthesis22. The disturbance or imbalance in the metabolism of L-arginine that is mediated by Arginase-1 and iNOS may result in prolonged inflammation and abnormality in matrix deposition, which would delay the wound-healing process5. Accordingly, the upregulation of Arg-1 in macrophage culture upon the low dose of C. longa extract administration in our study may explain how C. longa (turmeric) has a positive impact in the form of accelerating wound healing, as shown by a previous study23. In their study, the excision of the mice’s skin which was treated with C. longa extract showed acceleration in the wound excision23. Similarly, our in vivo study regarding the impact of topical C. longa extract gel administration on skin wound healing in mice revealed the increase of Arg-1 expression in the wound’s tissues that were treated with C. longa extract gel (the data have not been published). Meanwhile, in our current in vitro study, the expression of Arg-1 was significantly higher upon administration of 0.5 ppm of C. longa extract and tended to be higher in the treatment groups upon administration of 1.0 ppm of C. longa compared to the control group (Figure 1A). The insignificant result in the administration of 1.0 ppm of Curcuma longa extract may be caused by the limited samples in this study. In addition, it could be caused by the high expression of Inos in all the treatment groups compared to the control group. This may be caused by contamination by a contaminant such as lipopolysaccharides (LPS) during the macrophage isolation procedure. LPS can both induce M1 polarisation and stimulate inflammation and increase the activity of iNOS24,25. On the other hand, it reduces the activity of Arginase-122. This could be one of the factors causing the expression of Arg-1 at high doses (1.0 and 5.0 ppm) to be not significant.

 

The limitation of this study is that it only focused on one marker from each macrophage type, i.e., Arg-1 and Inos. Further studies using different gene markers may be required. Another factor that could have influenced the significance of this research is the density of the macrophage cells that were cultured in each well, which might have been too narrow or too dense. This can cause the growth and cell differentiation to become less optimal26.

 

Few studies have stated that in order to stimulate the differentiation and the polarization of macrophages, culturing should be carried out for 6-14 days in a “condition of interest” before the isolation of RNA is undertaken26,27. In our study, the incubation was carried out for 2 days after treatment, which might have been too short or long to see the effects of the administration of C. longa extract. Another limitation in this study is the absence of a positive control with which to compare the effects of C. longa extract administration with an exogenous M2 classic inducer such as IL-4 or IL-13.

 

CONCLUSION:

Our experiment results suggest that the administration of C. longa extract at a low dose induced M2 polarization via upregulating the expression of Arg-1. Further studies should be conducted to confirm the result of this research by utilizing more samples and including a positive control group. In addition, confirmation with other macrophage gene markers is required to produce more accurate data. Our findings reveal that the upregulation of Arg-1 in macrophages could be one of the mechanisms of wound healing improvement upon administration of C. longa.

 

AUTHORS’ CONTRIBUTIONS:

AB NR and ACP: research design and manuscript preparation; performing peritoneal macrophage isolation and in vitro experiment. TW and NQ: performing in vitro experiments. NSR: performing quantitative real-time PCR. NA: reviewing the manuscript.

 

FUNDING/SUPPORT:

This work was supported and funded by the Internal research grant for fundamental research of Universitas Padjadjaran (No. 855/UN6.3.1/PL/2017).

 

ACKNOWLEDGEMENT:

We thank the Directorate for Research and Community Service, Universitas Padjadjaran, for their financial support through the Internal research grant for fundamental research.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 27.02.2020            Modified on 13.04.2020

Accepted on 29.05.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(2):1079-1084.

DOI: 10.5958/0974-360X.2021.00194.3