Korean J Pain 2021; 34(1): 19-26
Published online January 1, 2021 https://doi.org/10.3344/kjp.2021.34.1.19
Copyright © The Korean Pain Society.
1Department of Convergence Medical Science, Gyeongsang National University, Jinju, Korea
2Department of Anesthesiology and Pain Medicine, Gyeongsang National University Changwon Hospital, Changwon, Korea
3Department of Anesthesiology and Pain Medicine, Gyeongsang National University Hospital, Jinju, Korea
4Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju, Korea
5Department of Anesthesiology and Pain Medicine, Gyeongsang National University College of Medicine, Jinju, Korea
Correspondence to:Yeon A Kim
Department of Anesthesiology and Pain Medicine, Gyeongsang National University Changwon Hospital, 11 Samjeongja-ro, Seongsan-gu, Changwon 51472, Korea
Department of Anesthesiology and Pain Medicine, Gyeongsang National University Hospital, 79 Gangnam-ro, Jinju 52727, Korea
Handling Editor: Jeong-Il Choi
Received: June 19, 2020; Revised: September 18, 2020; Accepted: October 7, 2020
Background: Prolotherapy is a proliferation therapy as an alternative medicine. A combination of dextrose solution and lidocaine is usually used in prolotherapy. The concentrations of dextrose and lidocaine used in the clinical field are very high (dextrose 10%-25%, lidocaine 0.075%-1%). Several studies show about 1% dextrose and more than 0.2% lidocaine induced cell death in various cell types. We investigated the effects of low concentrations of dextrose and lidocaine in fibroblasts and suggest the optimal range of concentrations of dextrose and lidocaine in prolotherapy.
Methods: Various concentrations of dextrose and lidocaine were treated in NIH-3T3. Viability was examined with trypan blue exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Migration assay was performed for measuring the motile activity. Extracellular signal-regulated kinase (Erk) activation and protein expression of collagen I and α-smooth muscle actin (α-SMA) were determined with western blot analysis.
Results: The cell viability was decreased in concentrations of more than 5% dextrose and 0.1% lidocaine. However, in the concentrations 1% dextrose (D1) and 0.01% lidocaine (L0.01), fibroblasts proliferated mildly. The ability of migration in fibroblast was increased in the D1, L0.01, and D1 + L0.01 groups sequentially. D1 and L0.01 increased Erk activation and the expression of collagen I and α-SMA and D1 + L0.01 further increased. The inhibition of Erk activation suppressed fibroblast proliferation and the synthesis of collagen I.
Conclusions: D1, L0.01, and the combination of D1 and L0.01 induced fibroblast proliferation and increased collagen I synthesis via Erk activation.
Keywords: Actins, Cell Migration Assay, Cell Proliferation, Collagen Type 1, Extracellular Signal-Regulated MAP Kinases, Fibroblast, Glucose, Lidocaine, Muscle, Smooth, Prolotherapy.
Prolotherapy is a complementary and alternative treatment for strengthening the laxative tendon and ligament, and therefore relieves pain by using tissue irritant solutions. Solutions used in prolotherapy are considered to be inducing cellular osmotic stress or inflammation, thus stimulating the synthesis of growth factors, and beginning the healing process; however, its mechanism is not fully understood [1,2].
Dextrose solution is popularly used in prolotherapy as an irritant solution, and applied in a concentration from 10%-25%. Because it is painful during injection, local anesthetics, like lidocaine, are usually treated with dextrose solution. Considering the fact that normal blood glucose concentration is about 100 mg/dL (≈ 0.1%), 10%-25% is very high concentration. However, there is no
The first purpose of our study was to determine which range of concentrations of dextrose solution and lidocaine would be less cytotoxic in fibroblasts. Second, we investigated the molecular mechanism of fibroblast proliferation, which was induced by dextrose and lidocaine through evaluating the extracellular signal-regulated kinase (Erk) pathway activity. Finally, it was to provide information regarding which concentration would be proper for prolotherapy in the clinical field through an
The mouse embryonic fibroblast cell line, NIH-3T3, was purchased from the American Type Culture Collection (ATCC, Rockville, MD) and was maintained at 37°C with 5% CO2 in an air atmosphere; the cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (glucose 4,500 mg/L, 0.45%) supplemented with 10% bovine calf serum.
The following reagents were obtained commercially: Polyclonal rabbit anti-Erk 1/2, phospho-Erk 1/2, and RIPA buffer (10×) were purchased from cell Signaling Technology (Danvers, MA); the Polyclonal rabbit anti-collagen I was from Abcam (Cambridge, MA); the DMEM high glucose and bovine calf serum were from Gibco (Gaithersburg, MD); the Monoclonal mouse anti-β-actin, anti-α-smooth muscle actin (SMA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, thiazolyl blue (MTT), dimethyl sulfoxide (DMSO), trypan blue solutions, and lidocaine hydrochloride monohydrate (L5647-15g) were from Sigma (St. Louis, MO); the Erk inhibitor (PD98059) was from Merck millipore (Bedford, MA); 50% dextrose solution was purchased from Dai Han Pharm Co., Ltd. (Seoul, Korea). The enhanced chemiluminescence western blotting detection reagents SuperSignal West Pico Chemiluminescent Substrate were from Pierce (Rockford, IL).
Cell viability was determined by trypan blue exclusion assay at 24 hours after treating the lidocaine and dextrose solutions. Cells were collected using trypsin-EDTA, stained with 0.2% trypan blue solution, and counted in a Neubauer’s cell counting chamber. The result was related to the number of cells in an untreated control, which was considered 100%.
NIH-3T3 cells were plated on 96-well plates with 70,000 cells per well, and 6 parallel wells for each condition. On the next day, the medium was replaced with new medium (control medium, medium with 0.01%-0.2% [0.43-8.54 μM] of lidocaine, medium with 0.1%-10% [0.005-0.5 mM] of dextrose, and medium a with mixture of lidocaine and dextrose). The powder of lidocaine and dextrose were treated, and after that it was mixed with media at each weight together. The degrees of proliferation were measured after 24 hours of drug exposure with the MTT. MTT reagent was added to the wells at a final concentration 0.5 g/L. The cells were allowed to reduce MTT into formazan (4 hr at 37°C), the amount of which was measured spectrophotometrically at a wavelength of 560 nm against the background (650 nm) after lysing the cells in DMSO.
NIH-3T3 cells were seeded into the Culture-Insert 2 well (ibidi GmbH, Gräfelfing, Germany). Cells were incubated for 24 hours and separated from the Culture-Inserts. Closure of the resulting wound was monitored over the next 24 hours. Images of the wound were captured at different time points using a Nikon eclipse Ti-S inverted microscope (Nikon Instruments Inc., Tokyo, Japan). The cell-free area was measured using ImageJ densitometry software (National Institutes of Health, Bethesda, MD).
The cells (2 × 106) were washed twice with ice-cold phosphate buffered saline, suspended in 100 μL of ice-cold RIPA buffer (1×) (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride), and incubated at 4°C for 30 minutes. The lysates were centrifuged at 14,000 rpm for 30 minutes at 4°C. Protein concentrations of the cell lysates were determined using a Bradford protein assay reagent (Bio-Rad, Hercules, CA) and 20 μg of proteins were loaded onto 7.5%-15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The gels were transferred to a nitrocellulose membrane (Merk Millipore Ltd., Darmstadt, Germany) and reacted with the indicated antibodies. Immunostaining with antibodies was performed using the SuperSignal West Pico enhanced chemiluminescence substrate and detected using the ChemiDoc Touch Imaging System (Bio-Rad).
At least three independent experiments were conducted. The results are expressed as the means ± standard deviation. The statistical significance of the differences was primarily determined using the Student
To investigate the effects of the concentration of dextrose and lidocaine used in the clinical field, concentrations from 0.01% (0.043 mM) to 0.2% (0.86 mM) of lidocaine, and from 1% (5.56 mM) to 10% (55.6 mM) of dextrose, were tested in mouse fibroblasts, NIH-3T3, for 24 hours. More than 0.1% of lidocaine and 5% of dextrose decreased cell viability, and cells exposed to 0.2% of lidocaine and 10% of dextrose almost died (Fig. 1). Examining the cells with an inverted microscope, we found that 0.01%-0.05% of lidocaine and 1%-3% of dextrose appeared to mildly induce the proliferation of fibroblasts (Fig. 1B, D), therefore MTT assay was performed. The results were similar with microscopic observation. The concentrations in 0.01% and 0.05% of lidocaine and 1, 3, and 4% of dextrose increased the cell numbers (Fig. 2A, B). Because lidocaine and dextrose are usually combined in prolotherapy, we treated with them concurrently in the fibroblasts and there was no negative effect (Fig. 2C).
Motile activity, which is an important factor for wound healing, was also increased in 0.01% of lidocaine (L0.01) 1%, and of dextrose (D1), combined treatment of L0.01 and D1 was further increased the cell motility (Fig. 3).
L0.01, D1, and L0.01 + D1 increased Erk activation (Fig. 4A-C) and collagen I synthesis (Fig. 4D, E), and one of the markers of differentiation from fibroblast to myofibroblast, α-SMA expression, also increased (Fig. 4D, F). Fibroblast proliferation and collagen I synthesis were suppressed by Erk activation inhibition (Fig. 5).
This study demonstrated that relatively lower concentrations of lidocaine and dextrose, compared with concentrations used in clinical practice, induced fibroblast proliferation and increased collagen I synthesis
Since the prolotherapy technique has been applied, several irritant solutions have been used,
Lidocaine is commonly used in prolotherapy for reliving pain provoked due to the high osmolality of dextrose. It is known that lidocaine induces apoptotic cell death of various types of cells [17-21]. Sung et al.  reported there were few live human tenofibroblasts left in 1% lidocaine treatment. Onizuka et al.  also showed that the clinical dose of lidocaine,
The representative pathway of cell proliferation is the Erk pathway . Although the direct mechanism for glucose-induced cell proliferation is not understood, it is well known that glucose-stimulated cell proliferation takes place through the Erk pathway [24,25]. In order to confirm the proliferative effects of L0.01 and D1, we examined this pathway. Erk was activated distinctly at D1, however, L0.01 also increased phospho-Erk1/2 levels and L0.01 + D1 treatment further elevated it. The inhibition of Erk phosphorylation was also suppressed the proliferative effect of dextrose and lidocaine. It means lidocaine as well as dextrose multiplies the cell numbers through Erk phosphorylation.
It is not certain that increased fibroblast numbers would produce more collagen. In an inflamed condition, fibroblasts differentiate to myofibroblasts and have the increased ability of the extracellular matrix protein synthesis. Differentiated myofibroblasts contain α-SMA and it is a characteristic marker of myofibroblasts . It was increased at D1, notably, and it partially explains the increase of collagen I synthesis. In the study of Zhang et al. , the inhibition of Erk2 expression effectively prevented epidural fibrosis and collagen synthesis, and Tang et al.  reported collagen I and III production from cardiac fibroblasts was dependent on the Erk1/2 phosphorylation. In present study, Erk phosphorylation inhibition suppressed the collagen I synthesis, and high glucose stimulated collagen I synthesis in fibroblasts, also
The migration of fibroblasts or progenitor cells to the injured site is a key step in wound healing. In our study, L0.01 promoted the motile ability of fibroblast much more than D1. The lower concentration of lidocaine and dextrose were also effective in recruiting circumferential fibroblasts. Although there was more than 10% dextrose-induced cell death in the
We examined a simple pathway for fibroblast proliferation and collagen synthesis, however, it is only the first step for evaluating the mechanism of the treatable effects of prolotherapy. We expect prolotherapy to develop into orthodox and conventional medicine, from its current status as complementary and alternative medicine,
This article was presented at the 1st International Congress on Spinal Pain in Gwangju, Korea, 2016.
Min Seok Woo: Investigation; Jiyoung Park: Investigation; Seong-Ho Ok: Methodology; Miyeong Park: Investigation; Ju-Tae Sohn: Supervision; Man Seok Cho: Investigation; Il-Woo Shin: Supervision; Yeon A Kim: Study conception.
No potential conflict of interest relevant to this article was reported.
This study was funded by National Research Foundation of Korea (NRF-2011-0021216).