The Synergistic Effect of Glucagon-Like Peptide-1 and Chamomile
Oil on Differentiation of Mesenchymal Stem Cells into
Saghahazrati S, Ayatollahi SAM, Kobarfard F, Minaii Zang D. The synergistic effect of glucagon-like peptide-1 and chamomile oil on differentiation of mesenchymal stem cells into insulin-producing cells. Cell J. 2020; 21(4): 371-378. doi: 10.22074/cellj.2020.6325.
Glucagon-like peptide-1 (GLP-1) has attracted tremendous attention for treatment of diabetes. Likewise, it seems that active ingredients of chamomile oil might have anti-diabetic effects. This work was conducted to investigate the effects of the combination of GLP-1 and chamomile oil on differentiation of mesenchymal stem cells (MSCs) into functional insulin-producing cells (IPCs).
Materials and Methods
In this experimental study, adipose MSCs derived from the adult male New Zealand white
rabbits were assigned into four groups: control (without any treatment); GLP-1 (in which cells were treated with 10 nM
GLP-1 every other day for 5 days); chamomile oil (in which cells were treated with 100 ug/ml
Our results demonstrated that isolated cells highly expressed MSC markers and were able to differentiate
into osteocytes and adipocytes. Additionally, using GLP-1 in combination with chamomile oil exhibited higher levels
of IPCs gene markers including NK homeobox gene 2.2 (
Collectively, these findings establish a substantial foundation for using peptides in combination with natural products to obtain higher efficiency in regenerative medicine and peptide therapy.
Type 1 diabetes mellitus (Fig T1DM,) is an autoimmune disease that is responsible for about 5-10% of all cases of diabetes around the world (1). During T1DM, initiation of chronic inflammatory responses gives rise to apoptotic and necrotic death of pancreatic ß-cells, and absolute insulin deficiency which, in turn, results in serious short-term and long-term side effects (2). It is urgent to discover new therapeutic options for treatment of T1DM and other degenerative diseases considering their high rate of morbidity and mortality (3-6). In recent years, stem cell-based therapy has been regarded as a promising strategy to treat immune-mediated diseases such as T1DM (7). Unique properties of mesenchymal stem cells (MSCs) including modulation of immune response, differentiation plasticity, easy attainability, and ability for inhibition of key factors involved in initiation of autoimmune disorders, make them excellent candidates to treat T1DM (8, 9). Although MSCs have demonstrated safety and efficacy in treatment of immune-mediated diseases such as T1DM, several drawbacks such as differentiation into undesired cells and migration to other body organs might limit their clinical applications (10).
Glucagon-like peptide-1 (GLP-1) is an incretin
hormone and food intake acts as a potent stimulator of its
secretion by intestinal cells. GLP-1 plays an important
role in a large number of physiological processes such
as modulation of gastric emptying, blood glucose
level, insulin secretion, glucose metabolism and
appetite (11). Some previous studies have shown that
GLP-1 might promote the growth and differentiation
of ß-cells. For example, Abraham et al. (12) reported
that GLP-1 contributed to the differentiation of nestinpositive
islet-derived progenitor cells, present in the
ducts and islet of the pancreas, into insulin-producing
cells (IPCs). They concluded that GLP-1 exerted this
function through alterations of gene expression profile.
In fact, GLP-1 increased the expression of
Materials and Methods
GLP-1, Collagenase type I, and
In this experimental study, male New Zealand white rabbits with a mean weight of 2.5 kg, were obtained from Razi Institute, Iran. All procedures and experimental tests were approved by the Animal Ethics Committee of Shahid Beheshti University of Medical Sciences (reference No. 1392. 49270). Rabbits were maintained in a temperature-controlled chamber set at 25 ± 1°C, with 12/12-hour light/dark cycles. They were fed with standard pellet chow and water ad libitum. After surgery and isolation of cells, the animals were permitted to recover spontaneous breathing and placed in their cage with free access to food and water.
Isolation of adipose-derived mesenchymal stem cells
Rabbits were anesthetized intraperitoneally (IP) using ketamine (40 mg/kg) and xylazine (5 mg/kg). A midline incision was made in abdominal region. Approximately, 100 ml of adipose tissue was dissected from the perivisceral area. The adipose tissue was divided into small pieces in cold phosphate-buffered saline (PBS, Biochrom, Germany, pH=7.4). Then, small pieces of adipose tissue were homogenized and centrifuged at 175 g for 5 minutes. After removing supernatant, pellet was digested using 0.1% collagenase type I at 37°C under continuous shaking for 60 minutes. Then, the cell suspension was centrifuged at 175 g for 5 minutes. The supernatant was removed, and pellets were resuspended in an appropriate volume of the DMEM (Gibco, USA) supplemented with 10% FBS, and 1% penicillin-streptomycin and incubated at 37°C in a humid incubator with 5% CO2 to acquire enough cell density.
Identification of mesenchymal stem cells
To determine cell surface antigen profile of MSCs, fluorescence-activated cell sorting (FACS) was performed. In brief, after trypsinizing and washing with cold PBS containing 1% fetal calf serum (FCS), cells were incubated for 30 minutes with 10 µg/ ml antibodies in PBS per 1×106 cells at 25°C in the dark. Antibodies applied in this work included CD45FITC, CD34-FITC, CD105-PE and CD73-PE (Dako, Denmark). To determine nonspecific fluorescence, cells were incubated with the isotype-matched antibody. A flow cytometer (Partec Pas III, Germany) was used to quantify the results.
Evaluation of osteogenic and adipogenic differentiation
To evaluate adipogenic differentiation, Oil red O staining was performed. MSCs were incubated in a medium including 100 µg/ml 3-isobutyl1- methylxanthine, 10 µg/ml insulin, 10-6 M dexamethasone, 50 µM indomethacin in alpha- MEM medium supplemented with 10% FBS, for 3 weeks. To determine osteogenic differentiation, cells were incubated with a medium including 10 mM glycerophosphate disodium, 10-7 M dexamethasone, 50 µg/ml ascorbic acid in alpha-MEM medium supplemented with 10% FBS, for 4 weeks. Alizarin red S staining was used to observe calcium deposits.
MSCs were cultured at a density of 1.5×106 cells/
mL in alpha-MEM medium supplemented with 10%
FBS containing 20 ng/ml of basic fibroblast growth
factor (bFGF) and epidermal growth factor (EGF).
Cells were randomly divided into the following four
groups of 12 flasks in each. For control groups, cells
did not receive any treatment (control). GLP-1 group
only received 10 nM GLP-1 every other day for 5 days.
Chamomile oil group only was treated with 100 µg/ml
Reverse transcription polymerase chain reaction
Qiagen RNeasy kit (Qiagen Company, Valencia, CA, USA) was used to extract total RNA from 1×106 differentiated cells following the manufacturer’s instructions. RNA concentration was determined using NanoDrop Microvolume Spectrophotometer and stored at -80°C. Then, total RNAwas converted into cDNA following the manufacturer’s protocol using a Dart cDNA kit. Quantitative polymerase chain reaction (PCR) was carried out using SYBR® Premix Ex Taq ™ II on a Rotor-Gene Q 5plex System (30-40 cycles). ß-actin was used as the internal control. The expression levels of each target gene was normalized against the internal control expression using 2-ΔΔCt method. Reverse transcription- PCR (RT-PCR) primer pairs are shown in Table 1.
Assessment of insulin/C-peptide release
To evaluate C-peptide release, we used Rabbit C-Peptide ELISA Kit. Measurement of insulin levels in culture media was performed using rabbit insulin ELISA kit. First, cells were pre-incubated with Krebs- Ringer buffer at 37°C for 2 hours. Then, cells were incubated with Krebs-Ringer buffer containing different doses of glucose (0, 15, and 30 mM) at 37°C for 1 hour. Finally, culture media was collected and assessments were performed.
All the data were presented as mean ± SD. GraphPad Prism software version 5.0 (CA, USA) was employed to analyze data. Values were subjected to a one-way analysis of variance (ANOVA) followed by Tukey multiple comparison tests. P<0.05 was accepted to be statistically significant.
Characterization of mesenchymal stem cells
Three days after initial plating, we found that MSCs possess fibroblast-like morphology. Fourteen days after the initial plating, a confluent monolayer of MSCs was formed. Flowcytometric analysis demonstrated that CD105 (MSC marker) was expressed in 95.76% of cultured MSCs. Additionally, CD73 (MSC marker) was expressed in 96.86% of MSCs.
The hematopoietic progenitor marker CD34 (expressed in 0.04% of MSCs) and the pan-leukocyte marker CD45 (expressed in 0.02% of MSCs) did not indicate significant expression levels (Fig .1,).
Osteogenic and adipogenic differentiation
Oil red O staining demonstrated that isolated MSCs have the ability to differentiate into adipocytes (Fig .2A,). Alizarin red S staining showed the ability of the isolated MSCs for mineralization and formation of calcium deposits. These findings confirmed that isolated MSCs are able differentiate into osteocytes (Fig .2B,).
|Gene||Primer sequence (5ˊ-3ˊ)||Accession number||Sequence detected (bp)|
|- Immunophenotypic characterization of adipose-derived cells. The expressions of mesenchymal stem cell (MSC) markers such as CD73-PE and CD 105 PE, were higher than those of the hematopoietic progenitor marker CD34 and the pan-leukocyte marker CD45.|
The effects of GLP-1 and chamomile oil on morphology 1 and chamomile oil into IPCs, we measured mRNA of cultured MSCs
The cells treated with GLP-1 and chamomile oil exhibited changes in their morphology. These cells were more flattened compared with control after 5 days, suggesting their differentiation into IPCs (Fig .3A,).
The effects of GLP-1 and chamomile oil on differentiation of MSCs into IPCs
To confirm differentiation of cells treated with GLP-1 and chamomile oil into IPCs, we measured mRNA
The effects of GLP-1 and chamomile oil on the cleaved C-peptide levels in culture media
To evaluate the function of treated cells, we measured C-peptide secretion by cells in response to different concentrations of glucose. As shown in Figure 4A, no significant differences were found among different groups in the absence of glucose (0 mM). Significant differences were observed in response to 15 and 30 mM concentrations of glucose. GLP-1+ chamomile oil group exhibited higher C-peptide secretion than cells treated either with chamomile oil alone or GLP-1 alone.
The effects of GLP-1 and chamomile oil on insulin levels in culture media
There were no significant differences among different groups in the absence of glucose (0 mM). Compared with other groups, GLP-1+chamomile oil showed the highest insulin secretion in response to 15 and 30 mM concentrations of glucose (Fig .4B,).
In this work, we demonstrated that using peptide therapy and natural products together can produce synergistic effects on differentiation of MSCs into IPCs. In recent years, GLP-1, a peptide produced by dipeptidyl peptidase-4 (DPP4) cleavage of the gut incretin hormone, has attracted tremendous attention from scientific community for T1DM therapy because it can act as a growth factor to increase mass expansion of ß-cells and subsequently, insulin secretion. In fact, it is well known that this peptide promotes survival and proliferation of ß-cells (20). However, some recent studies have shown that GLP-1 facilitated the formation of new mature ß-cells (neogenesis) in the adult pancreases (21). Moreover, many previous reports have demonstrated that chamomile oil possesses many active ingredients that act as anti-diabetic, antioxidant, anti-inflammatory and antibacterial agents (22-24). For example, luteolin, a bioactive compound present in chamomile oil, increases insulin secretion and activates adipokines/cytokines in adipocytes through induction of the peroxisome proliferator-activated receptor-γ (PPARγ) pathway (25, 26).
In this study, we investigated the synergistic effect of GLP-1 and chamomile oil on differentiation of MSCs into insulin-secreting cells. The isolated MSCs exhibited an increased expression of MSCs markers, whereas they did not demonstrate a significant expression of the hematopoietic progenitor and pan-leukocyte markers. These findings confirmed a highly purified MSC population. In agreement with the results of the present study, Razavi Tousi et al. (27) reported that the isolated MSCs strongly expressed MSCs marker CD105, but not CD 45 and CD34. On the other hand, isolated cells were able to differentiate into osteocytes and adipocytes. In agreement with this study, a previous report showed that the isolated MSCs can be differentiated into osteocytes and adipocytes (28). Furthuremore, a previous study indicated that addition of GLP-1 to the culture media of mouse embryonic stem cells, contributed to differentiation into IPCs (29).
To examine the synergistic effects of GLP-1 and
chamomile oil, we measured mRNA levels of
Collectively, our finding demonstrated that chamomile oil in combination with GLP-1 more efficiently enhances the differentiation of adipose-derived MSCs into IPCs. These findings establish a substantial foundation for using peptides in combination with natural products to obtain higher efficiencies in regenerative medicine.
This study was financially supported by a research grant from Phytochemistry Research Center of Shahid Beheshti University of Medical Sciences. The authors declare no conflict of interest.
S.A.A., B.M.Z.; Contributed to conception and design, and were responsible for overall supervision. S.S., F.K.; Contributed to all experimental work, data and statistical analysis, and interpretation of data. S.A.A.; Drafted the manuscript, which was revised by S.S. All authors read and approved the final manuscript.