Evaluation of melatonin, galectin-3, TGF-β1, and NF-κB in hypertrophic scar patients

ABSTRACT

A hypertrophic scar (HTS) is a fibrotic proliferative tissue that develops following extensive skin trauma. HTS is defined by aberrant fibroblast proliferation and excessive collagen deposition. Nowadays, the most common therapies for HTS are pressure therapy, surgical excision, and corticosteroid injection, yet these approaches have limitations or side effects. Thus, developing novel approaches to treat hypertrophic scars has emerged as a focal point for wound healing research in recent years. This case-control study included 80 participants, divided into two groups. Group 1 included 40 HTS patients, and Group 2 had 40 age-matched healthy volunteers as controls. We are the first to assess blood levels of melatonin (MLT) and galectin-3 (Gal-3), as well as transforming growth factor beta (TGF-β1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), in HTS patients to understand the scarring mechanism and identify new therapeutic techniques. The study’s main finding is that HTS patients had significantly higher Gal-3 levels, TGF-β1, NF-kB expression, and substantially lower MLT levels than the control group. These findings collectively suggested that MLT may considerably reduce Gal-3, TGF-β1, and NF-κB levels in patients with hypertrophic scars. Therefore, it could be a practical therapeutic approach for hypertrophic scarring.

KEYWORDS:

• Melatonin
• galectin-3
• hypertrophic scar
• TGF-β1
• NF-κB

Introduction

Fibrotic disorders are characterized by aberrant collagen deposition in the extracellular matrix (ECM) [1,2]. This aberrant collagen deposition impairs regular structure formation. It impairs the function of numerous organs and tissues, imposing a heavy burden on individuals and the global healthcare system [3–5]. These fibrotic conditions include hypertrophic scars, which are abnormal skin scars. Wound healing and scar formation are two reactions to tissue damage that are intimately related. However, there are some significant distinctions between the two procedures. Wound healing is the process by which the body heals damaged tissue after an injury. It entails several overlapping stages, such as hemostasis, inflammation, proliferation, and remodeling [6]. On the other hand, excessive buildup of extracellular matrix components, mainly collagen, which substitutes the standard tissue architecture throughout the wound healing process, results in scar development. Scarring can vary based on the size and depth of the wound, the wound’s location, and the patient’s age and heredity [7,8]. Burns, wounds, and surgical operations frequently result in hypertrophic scarring; the incidence rate of hypertrophic scars is 70% [9,10]. It can be uncomfortable and itchy, and a variety of hypertrophic scars cause muscular rigidity and restricted movement that results in cosmetic disfigurement. These conditions cause psychological discomfort and lower patients’ quality of life [11]. Laser therapy, silicone sheeting, and pressure on the scar are the most common nonpharmacological therapies for hypertrophic scars. Even though chemotherapeutic drugs such as steroids and 5-fluorouracil have been utilized, their efficacy has been demonstrated to be limited and often causes discomfort and recurrence. Therefore, developing novel approaches to treat hypertrophic scars has recently emerged as a focal point for wound healing research [12,13].

Melatonin (MLT) is a simple biological substance produced by various organs, including the pineal gland [14]. Although other organs may occasionally emit a tiny amount of melatonin, most of the melatonin in the blood comes from the pineal gland [15,16]. MLT was initially explored for its ability to control seasonal reproduction but is now recognized as a crucial chemical in cellular physiology because of its wide range of functions [17]. MLT plays critical roles in regulating circadian rhythm, cardiovascular function, immune system regulation, retinal function, and pancreatic function. It is well-known for its antioxidant action and neuroprotective properties [18]. MLT has strong antioxidant properties and contributes to the detoxification of free radical intermediates such as hydroxyl, peroxyl, singlet oxygen, nitric oxide, and peroxynitrite anion [19,20]. Additionally, melatonin affects the expression of antioxidant enzymes, including glutathione reductase, glutathione peroxidase, catalase, and superoxide dismutase. Due to its cell-protective properties, numerous studies have examined its potential roles in the regeneration of various damaged tissues, the reduction of chronic wound severity, and the improvement of wound contraction [21]. Research has revealed a significant association between melatonin and several fibrotic disorders. For example, it was found that, in contrast to normal skin, the fibroblasts of human hypertrophic scar tissue overexpressed the melatonin receptor [22]. However, the fundamental mechanism of melatonin in fibrotic disorders is still poorly understood.

Galectin-3 (Gal-3) is a 29-35kDa carbohydrate-binding lectin found on the surface of various cell types, such as endothelial cells, fibroblasts, and inflammatory cells. The primary source of Gal-3 is activated macrophages, which are triggered by oligosaccharides. Other ligands that activate Gal-3 include glycosylated matrix proteins like fibronectin, collagen, elastin, laminin, and integrin. Upon activation, Gal-3 promotes cell proliferation, adhesion, and fibrosis. It has been demonstrated that Gal-3 is necessary for tissue fibrosis and remodeling [23]. Gal-3 could be a sign of oxidative stress since it is linked to disruptions in mitochondrial homeostasis, which may result in the depletion of glutathione (GSH) and the generation of reactive oxygen species (ROS) [24–26]. We aimed to understand the scarring mechanism and identify new therapeutic techniques. So, we hypothesized that melatonin may be involved in scarring. To our knowledge, we are the first to assess blood levels of MLT and Gal-3, as well as tissue TGF-β1 and NF-κB, in patients with hypertrophic scars.

Patients and methods

This case-control study included 40 patients with hypertrophic scars who attended the Dermatology Outpatient Clinic at Sohag University Hospital (group 1) and 40 age- and sex-matched scar-free volunteers with no history of HTS as controls (group 2). This control group was drawn from individuals undergoing abdominoplasty at Sohag University Hospital’s plastic surgery department. The Research and Ethical Committees of the Sohag Faculty of Medicine approved this study with the approval number (Soh-Med-21-09-51). Each participant in the study gave their informed written consent after receiving a thorough explanation of the study’s methodology. We included individuals with hypertrophic scars who had not received therapy within the previous six months. We excluded patients with systemic or cutaneous inflammatory illnesses, diabetes, asthma, autoimmune diseases, and cancer, and who had previously received treatment for HTS, to avoid interference with our measurements.

Methods

This study was conducted in the Biochemistry Department, Plastic and Reconstructive Surgery Department, and Dermatology, Venereology, and Andrology Department at Sohag University. The experiments were done in the central laboratory of Sohag University. All individuals in the study underwent a complete medical history and clinical evaluation.

Blood samples

We collected 5 ml of venous blood from the anti-cubital vein under strictly aseptic conditions. All samples were collected into a gel-filled vacutainer tube. The serum was separated by centrifugation and kept at −80°C till the time of measurement of MLT and Gal-3.

Tissue samples

Skin punch biopsies (5 mm) were collected from the lesions of patients with hypertrophic scars after they had received a 2% lidocaine local anesthesia. The control group was obtained from the surplus skin of patients having abdominoplasty. Every skin sample was preserved as frozen material at −80°C to evaluate the tissue expression of TGF-β1 and NF-κB levels using Western blotting.

Laboratory measurements

Serum MLT and Gal-3 concentrations were measured using the Human Melatonin and Galectin 3 ELISA Kits (Catalogue No EEL056 & BMS279–4). Kits were provided by ThermoFisher and followed the procedures advised by the manufacturer’s instructions. A microplate incubator (Stat fax 220,012 VAC2A), a microplate washer (Stat fax 2600), and a microplate reader (Stat fax 2100) were used.

Western blot analysis

The skin tissues were homogenized in Tris lysis buffer (400 mM NaCl, 0.5% Triton X-100, 50 mM Tris pH 7.4) and protease inhibitor cocktail (Biospes, China) at 4°C for 30 minutes. Centrifugation at 12,000 rpm for 15 minutes at 4°C was used to remove the remaining tissue. Total protein concentrations in each sample were determined using the Biuret technique. Using semidry transfer equipment (SD20, Cleaver Scientific, UK), the protein (40 μg per lane) was transferred to a PVDF membrane (Millipore, Merck, USA) after being resolved by 10% SDS-polyacrylamide gel electrophoresis [27]. After blocking the membranes with 5% nonfat milk in Tris-buffered saline with 0.1% 20 detergent (TBST) buffer for an hour at ambient temperature, primary antibodies were incubated on the membranes for an entire night at 4°C. β-actin was examined as an internal control of protein loading. The antibodies used were anti-NF-κB-p65 (YPA1020, Biospes, China, dilution 1:1000), anti-TGF-β (YPA1196, China, dilution 1:500), and anti-β-actin (E-AB-20031, Elabscience, China, dilution 1:3000). Afterward, the membranes were treated with an appropriate dilution of a secondary antibody conjugated with alkaline phosphatase (Biospes, China) for one hour. The BCIP/NBT substrate detection kit (Biospes, China) was used to visualize the bands. The generated bands were analyzed using Image J® software (National Institutes of Health, Bethesda, USA).

Statistical analysis

The data was analyzed using SPSS (Statistical Package for the Social Sciences) version 25. The distribution of different variables was determined using the Shapiro-Wilk normality test. We reported the mean, median, standard deviation, and interquartile range for quantitative data. The student t-test was used to compare the means of two groups with a normal distribution, while the Mann-Whitney test was used to compare two groups when the data was not normally distributed. We used Pearson correlation for correlations between serum factors. To create the graphs, Excel or SPSS software was utilized. The p-value was taken into consideration. We used G*power software version 9.3.1.7 to estimate the sample size [28].

Results

Power of the sample size

To achieve a statistical power of 95% (1-β) and an effect size of d = 0.9, we used G*Power and the Mann-Whitney U test to determine the appropriate sample size. Our secondary outcome was TGF-β1. To account for a potential dropout rate of 15%, we enrolled 40 hypertrophic scar patients in Group 1 and 40 age- and sex-matched scar-free volunteers with no history of hypertrophic scar (HTS) as controls in Group 2.

The two groups showed no statistical difference as regards the age with their mean age ± SD (24.2 ± 13.9 and 24.2 ± 11.0 years old, respectively). Moreover, there was no remarkable difference in sex, residence, smoking status, or occupation between the two studied groups. The comparison of socio-demographic characteristics of patients with HTS and controls is shown in Table 1.

Table 1. The sociodemographic characteristics of the two studied groups n = 80.

Parameter		Group 1 Hypertrophic scar group (n = 40)	Group 2 Control group (n = 40)	P value
Age (Years)	Mean ± SD	24.2 ± 13.9	24.2 ± 11.0	*0.6
	Median (IQR)	20.5 (12–33.5)	21.5 (16–32)
Sex	Male	17(42.5%)	20(50.0%)	#0.5
	Female	23(57.5%)	20(50.0%)
Residence	Rural	19(47.5%)	20(50.0%)	#0.8
	Urban	21(52.5%)	20(50.0%)
Occupation	Employee	7(17.5%)	11(27.5%)	#0.4
	Housewife	13(32.5%)	8(20.0%)
	Worker	5(12.5%)	8(20.0%)
	Student	15(37.5%)	13(32.5%)
Smoking	No	39(97.5%)	38(95.0%)	#0.5
	Smokers	1(2.5%)	2(5.0%)

SD: Standard deviation IQR: Interquartile range.

*By Mann-Whitney test.

#By Chi-square test.

We found that the mean duration ± SD of HTS was 12 ± 14.6 months. The Van Couver scar score evaluates the surface, thickness, height of the border, and color variations between a scar and the surrounding normal skin, ranging from 0 to 13 according to scar severity [29]. Our study showed that the mean ± SD of the Van Couver scar score was 8.2 ± 1.9, as shown in Table 2. In the HTS group, the scars were mainly located on the trunk (42.5%), upper limb (22.5%), and lower limb (22.5%), as shown in Table 2.

Table 2. Clinical characteristics of hypertrophic scare group, n = 40.

Duration (Months)	Mean ± SD	12.0 ± 14.6
	Median (IQR)	7.0 (3–9.5)
Vancouver Scar Scale	Mean ± SD	8.2 ± 1.9
Family history	Frequency (%)	5 (12.5%)
Site	Trunk	17 (42.5%)
	Upper limb	9 (22.5%)
	Lower limb	9 (22.5%)
	Face	5 (12.5%)

SD: Standard deviation IQR: Interquartile range.

To investigate Gal-3’s role in HTS, we measured the serum level of Gal-3 in HTS patients. The findings demonstrated a substantial rise in Gal-3 in the HTS group vs. the control group (mean ± SD; 3.7 ng/ml ±0.7, 1.8 ng/ml ±0.6, p < 0.001), respectively, as shown in Table 3. Also, we measured the expression of TGF-β1 with western blot analysis, and the findings revealed a very considerable difference between the two groups (p < 0.003) with a marked increase in TGF-β1 expression in the HTS group (Table 3, Figure 1a,c).

Figure 1. (a) western blot analysis of TGF-β1, (b) western blot analysis of NF-kB, (c) western blot analysis of TGF-β1 and NF-kB normalized to β-actin.

Table 3. Comparison between the two studied groups as regards (serum melatonin and serum Gal-3, n = 80.

Parameter		Hypertrophic scar group (n = 40)	Control group (n = 40)	*p value
Serum Melatonin (pg/ml)	Mean ± SD	35.6 ± 8.1	82.6 ± 9.1	<0.001
Serum Galectin-3 (ng/ml)	Mean ± SD	3.7 ± 0.7	1.8 ± 0.6	<0.001

SD: Standard deviation *p value was calculated by Mann-Whitney test.

Next, we tried to find out if MLT could have an anti-fibrotic impact by controlling oxidative stress levels, so we investigated the serum melatonin concentration in HTS patients and compared it to the control group. Interestingly, the two groups differed significantly from each other (p < 0.001), with a much-reduced level of MLT in the HTS group compared to controls (mean ± SD 35.6 pg/ml ±8.1 and 82.6 pg/ml ±9.2, respectively) (Table 3).

Numerous investigations have demonstrated that MLT regulates the NF-kB pathway during inflammation and that this regulation affects the expression of the genes responsible for the inflammatory response. To further elucidate the roles of MLT in HTS, we investigated the NF-kB expression by western blot analysis, and the results showed markedly increased levels in the HTS group (mean ± SD 2.1 ± 0.3) compared to controls (mean ± SD 0.3 ± 0.1) (Table 3, Figure 1b,c).

We also found significant negative correlations between serum MLT and the Vancouver scar scale (R = −0.646, p < 0.001; Figure 2) and between serum MLT and serum Gal-3 (R = −0.842, p < 0.001; Figure 3). There was also a considerable positive correlation between serum Gal-3 and the Vancouver scar scale (R = 0.669, p < 0.001; Figure 4).

Figure 2. Pearson correlation between serum melatonin and Vancouver scar scale.

Figure 3. Pearson correlation between serum melatonin and serum galectin-3.

Figure 4. Pearson correlation between serum galectin-3 and Vancouver scar scale.

Discussion

The study’s main finding is that HTS patients had significantly higher Gal-3 levels and substantially lower MLT levels than the control group. This study is the first to report the relationship between MLT, Gal-3, and HTS. Research has demonstrated that hypertrophic scarring develops through complex pathways [30]. We postulated that melatonin deficiency may be involved in scarring. Nevertheless, little is known about the underlying mechanism of MLT in fibrotic diseases. MLT is beneficial in numerous pathological circumstances. It exhibits strong antioxidant properties in vivo and in vitro [31]. Furthermore, it protects multiple tissues from the continual production of free radicals [32] and exhibits immunomodulatory, solid, and anti-inflammatory properties [33,34]. In this context, melatonin protects cells by regulating redox-sensitive transcription factors, including NF-κB [35]. It was discovered that external stimuli affected the expression of MLT receptors [36]. Both normal skin and hypertrophic scars in humans have a positive expression of melatonin receptors; however, the expression is higher in scars [22]. This finding supports our postulation that melatonin may be involved in scaring and encourages the possibility of using MLT to treat HTSs. Interestingly, Dong et al., 2024 revealed that melatonin therapy reduced the production of collagen and α-SMA and prevented the creation of HTS in a rabbit ear model [37].

Our study discovered that HTS patients had a discernible rise in NF-κB expression and substantially low serum melatonin levels. This observation may be explained by melatonin’s inhibition of NF-κB transduction through blocking its translocation into the nucleus [38]. Consequently, this inhibitory action serves as a crucial defense mechanism for cells against inflammation and the generation of reactive oxygen species (ROS) brought on by inflammation. The significant negative correlation between serum MLT and the Vancouver scar scale may be explained by this. In agreement with our findings, multiple studies have demonstrated that MLT regulates the NF-kB pathway during inflammation and that this regulation affects the expression of the genes that cause inflammation [39]. Also, Bona et al. 2018, discovered that using MLT decreased the expression of NF-kB, which reduced the inflammatory cascade [40].

Gal-3 exists on the surface of several cell types, including fibroblasts, endothelial cells, and inflammatory cells. It plays a role in the pathological breakdown of tissue structure, the creation of scars, and fibroblast activation in many tissues, such as the kidney, liver, and heart [41]. In this context, we showed for the first time that serum Gal-3 levels were greater in HTS patients than in controls. We postulated that this increase could be due to a decrease in serum melatonin levels, so MLT could mediate anti-fibrotic effects by reducing the expression level of Gal-3. This could account for the noteworthy negative correlation between serum MLT and Gal-3. Concurrent with our results, Fenton-Navarro et al., 2021 found that in male rats with acute global cerebral ischemia, MLT reduced the levels of Gal-3 in the blood. However, the exact mechanisms were still unclear [42]. Moreover, Lan et al., 2023 showed that bleomycin treatment caused a considerable increase in Gal-3 in the lungs of mice and showed that MLT reduces pulmonary fibrosis by blocking Gal-3 expression in vivo [43]. The exact mechanisms by which galectin-3 affects fibrosis and ECM remodeling are still unknown. At the same time, it has been proposed that oxidative stress and inflammation, as well as the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and protein kinase C (PKC) pathways, are involved. Furthermore, galectin-3 may directly promote the synthesis of ECM proteins [44,45]. This may explain the study’s notable finding that the Vancouver scar scale and serum Gal-3 were positively correlated.

To provide more insight into Gal-3’s functions in HTS, we examined TGF-β1 expression. TGF-β is distinguished by its participation in multiple wound healing processes, such as ECM production, expression of α-SMA, and fibroblast activity [46]. There are around 40 members of the TGF-β superfamily that have similar structural motifs and sequence components. Among the three isoforms of TGF (TGF-β 1, TGF-β 2, and TGF-β 3), TGF- β 1 is the most elevated in ECM remodeling and fibrosis and is therefore considered a key regulator of the ECM [47]. The production of ECM proteins, including fibronectin, collagen, and plasminogen activator inhibitor-1 (PAI-1), is enhanced by vascular TGF-β1 activation and its downstream signaling effector, SMAD [23]. According to our research, TGF-β1 was highly expressed in HTS patients. This result may be explained by an increase in Gal-3 or a decrease in melatonin. Supporting our conclusion, Lan et al., 2023 demonstrated that blocking Gal-3 expression decreased the amount of ROS and α-SMA in HFL1 cells that TGF-β1 triggered. They also stated that MLT inhibited ROS buildup, α-SMA expression, and Gal-3 expression in HFL1 cells induced by TGF-β1 [43]. Moreover, Bona et al., 2018 revealed that MLT effectively reduced liver fibrosis and markedly lowered the expression of TGF-β1 and α-SMA, indicating that MLT has an inhibitory effect on the deposition of ECM [40].

These findings collectively suggested that MLT may considerably reduce Gal-3, TGF-β1, and NF-κB levels in patients with hypertrophic scars.

Conclusion

In conclusion, our research revealed that people with hypertrophic scars had reduced melatonin levels. This reduction could be the reason behind the elevation of TGF-β1, NF-κB, and Gal-3, which puts these patients at risk for underlying fibrosis and scarring. As a result, MLT might be a useful therapeutic strategy for hypertrophic scarring. However, because of the small patient sample size in our study, we recommend conducting bigger multicenter trials with a greater number of HTS patients to confirm the promising findings before using them in clinical settings. Additionally, we recommend that randomized controlled trials (RCTs) be initiated.

Authors’ contributions

AA and MS: conceptualization and methodology.

MA, MS, and NO: writing – original draft preparation.

AA and MS: Review and edit the final manuscript.

MA and NO: taking the skin biopsy.

MS and ZM analyzed and interpreted the laboratory data.

All authors have read and agreed on the final manuscript.

Availability of data and materials

Data and materials are available on demand through the corresponding author.

Ethics approval and participation consent

Every patient provided informed consent to the procedures.

With permission from the Scientific Research Ethical Committee of the Faculty of Medicine at Sohag University, the study methodology was carried out following the Declaration of Helsinki (approved number: Soh-Med-21-09-51).

List of abbreviations

ECM	=	extracellular matrix
Gal-3	=	Galectin-3
ROS	=	Reactive oxygen species
MLT	=	Melatonin
HTS	=	Hypertrophic scar
TGF-β1	=	Transforming growth factor beta
NF-κB	=	Nuclear factor kappa-light-chain-enhancer of activated B cells
GSH	=	Glutathione
JAK	=	Janus kinase
STAT	=	Signal transducer and activator of transcription
PKC	=	Protein kinase C
α-SMA	=	Smooth muscle actin
PAI-1	=	Plasminogen activator inhibitor-1
HFL1	=	Fetal lung fibroblast
RCTs	=	Randomized controlled trials
TBST	=	Tris-buffered saline with 0.1% 20 detergent

Disclosure statement

No potential conflict of interest was reported by the author(s)

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.