Effect of Topical H₁-antihistamine on the level of Transforming growth factor beta (TGF-β) and Collagen of Acute wound in animal model

 

Magda Rosalina Hutagalung, David S. Perdanakusuma, Pratidina Wulandari*

Department of Plastic Reconstructive and Aesthetic Surgery, Faculty of Medicine, Universitas Airlangga,

Dr. Soetomo General Hospital, Surabaya, 60115, Indonesia.

 

ABSTRACT:

Imbalance of collagen synthesis and degradation causes formation of abnormal scarring leading to hypertrophic scar or keloid. Previous studies have shown that applying antihistamines could reduce the level of dermal collagen. However, no data whether topical antihistamine could be used as preventative therapy for abnormal scar formation. The aim of this study was to assess whether the application of topical antihistamine in acute wounds could affect the level of transforming growth factor beta (TGF-β), a regulator of wound healing process, and collagen in wound healing phase in order to suppress the keloid formation. A randomized post-test only control group design study was conducted. Twenty-four acute wounds on Rattus novergicus rats were randomized divided into control and treatment groups. The animals of treatment group received daily topical antihistamine consisting 1% diphenhydramine hydrochloride and 0.1% zinc acetate. The full-thickness skin tissue from each rat were taken on day 5 and 21 and were subjected to ELISA test to measure the levels of TGF-β and collagen. Our data found that in the treatment group, the level of collagen decreased significantly over time (1623.61ng/mL on day 5 to 755.15ng/mL on day 21, p=0.001). Similarly, the level of TGF-b also decreased although was not statistically significant (p=0.766). In contrast, the levels of TGF-β level and collagen in the control group increased significantly from day 5 to day 21, p=0.003 and p=0.001, respectively. The level of TGF-b in treatment group was significantly higher compared to the control group p=0.001 on day 21 while the level of collagen had no different between treatment and control group (p=0.124). In conclusion, our data suggest that topical antihistamine reduced the levels of TGF-β and collagen at the end of the proliferation phase or the beginning of the remodeling phase. Further study is warrant to elucidate the effect of antihistamine on other would healing markers or parameters.

 

 

 

INTRODUCTION:

Following an injury, a hemostasis system will be initiated by human body by releasing and activating the cytokines such as epidermal growth factor (EGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-β) which all are critical for neutrophil chemotaxis, macrophages, mast cells, endothelial cells, and fibroblasts.^1,2 TGF-β has a major role in wound healing by influencing the inflammatory response, angiogenesis, granulation tissue formation, re-epithelialization, extracellular matrix disposition, and remodeling and contribute in scar formation.^3-6 In wound healing process, collagen will be synthesized  with involves the role of proline hydroxylase.⁷ This process is balanced with collagen degradation by the collagenase enzyme to prevent collagen accumulation. Imbalance of this collagen synthesis and degradation will cause hypertrophic scars or keloid.^8-11

 

Although there are many options of therapeutic, there is not satisfactory results in the management of keloids since suboptimal response often occurs with high recurrence leading to physiological issues and high expenditure. The surgical therapy provides a recurrence rate of 45-100% and this recurrence rate will decrease when in combination with nonsurgical therapy.¹² Currently, there are 25 non-surgical modalities available, one of which is systemic antihistamine. Some alternative medicines also have been explored.^13-31 A study conducted by Jailani showed that injection of histamine into the wounds of animal model increased the dermal collagen, while antihistamine injection reduced the level of dermal collagen.³² Another study found that oral antihistamine drug (pheniramin maleate) could decrease the collagen synthesis.³³

 

Some studies have used systematic antihistamine to prevent the keloid formation; however, it caused several side effects such as drowsiness, headache, psychomotor disturbances, and antimuscarinic effects such as urinary retention, dry mouth, blurred vision, and gastrointestinal disorders.^34,35 Since many possible side effects associated with systemic antihistamine, we sought to assess the effect of topical antihistamine in preventing keloids formation by measuring the levels of TGF-β and collagen.

 

METHODS:

This study used a randomized post-test only control group design. A full-thickness skin incision 1 cm long was made on each of the back of 24male rats, Rattus novergicus Wistar strain, aged three months, weighing 250-300grams. Rats were then randomly divided into two groups (i.e., control and treatment group). Each control and treatment group consisted two sub-groups (one sub-group was used for measurement on day 5 while another sub-group for measurement on day 21). All rats within the control group received the same procedures and all rats within the treatment group received the same treatment. Animals of control group received no treatment while animals of treatment group received topical antihistamine (Benadryl®) - a product from a Johnson and Johnson, with a composition of 1% diphenhydramine hydrochloride and 0.1% zinc acetate as a skin protectant.

 

The specimens of the full-thickness skin (5x5 mm2) in the middle of the would were taken on day 5 and day 21-post-incession. The skin tissues were sent to the Department of Anatomical Pathology and subjected for the enzyme-linked immunosorbent assay (ELISA) to measure the level of TGF-β and collagen.

 

The normality of the data of TGF-β and collagen level were measured using Kolmogorov-Smirnov test. Our data suggested that the data were normally distributed with p=0.748 and p=0.868 for the level of TGF-β and collagen, respectively. The data were analysed using parametric statistical tests. ANOVA test was used to compare the level of TGF-β and collagen from each group on day 5 and day 21. The Student’s t-test was employed to compare the level of TGF-β and collagen either between day 5 and day 21 within the same group or between control and treatment group on day 5 and    day ²¹.

 

RESULTS:

The mean levels of TGF-β and collagen from each group on day 5 and day 21 are presented in Table 1. The normality test using the Kolmogorov-Smirnov test suggested that both TGF-b and collagen levels had normal distribution. Data analysis using ANOVA suggested that there were significant differences of the TGF-b and collagen levels among groups on day 5 and day 21 with p<0.001 for both comparisons (Table 1).

 

Table 1. The mean level of the TGF-β and collagen from each group on day 5 and day 21

Day post-incision	Group	TGF-b level (pg/mL)	Collagen level (ng/mL)
Day 5	Control	44.65±28.37	315.07±137.40
	Treatment	94.72±14.55	1064.38±363.02
Day 21	Control	132.10±28.32	1623.61±368.17
	Treatment	128.39±9.09	755.15±268.64
p-value*		<0.001	<0.001

* Calculated using ANOVA test

 

We compared the levels of TGF-b and collagen within control and treatment group to see the changes of TGF-b and collagen production between day 5 and day 21 post-injury. The TGF-b level in control group was significantly higher on day 21 compared to day 5 (94.72 pg/mL vs. 44.65pg/mL, p=0.003) suggesting there was an increase in TGF-b production over time (Table 2). In treatment group, however, there was a reduction of TGF-b between day 5 and day 21, although this is not significantly different (p=0.766).

 

We also compared the level of collagen and found that collagen level increased significantly in control group over time from 315.07ng/mL in day 5 to 1064.38ng/mL on day 21(p=0.001) (Table 2). In contrast, the mean level of collagen significantly decreased on day 21 compared to day 5(755.15ng/mL vs. 1623.61ng/mL, p=0.001). These findings suggest that antihistamine decreased the level of collagen during the wound healing process.

 

Table 2. Comparison of the TGF-b and collagen levels between day 5 and day 21 in control and treatment groups

Marker	Group	Day post-incision	Mean (±SD)	p-value
TGF-b level (pg/mL)	Control	Day 5	44.65±28.37	0.003
		Day 21	94.72±14.55
	Treatment	Day 5	132.10±28.32	0.766
		Day 21	128.39±9.09
Collagen level (ng/mL)	Control	Day 5	315.07±137.40	0.001
		Day 21	1064.38±363.02
	Treatment	Day 5	1623.61±368.17	0.001
		Day 21	755.15±268.64

 

Next, we compared the levels of TGF-b and collagen between control and treatment group on day 5 and day 21. On day 5, the mean level of TGF-b in treatment group was significantly higher compared to control group (132.10pg/mL vs. 44.65pg/mL, p<0.001) (Table 3). The level of collagen on treatment group was also significantly higher compared to control group, 1623.61 ng/mL vs. 315.07ng/mL, p<0.001). On day 21, the level of TGF-b in treatment group was significantly higher compared to control group (128.39±9.09 vs 94.72±14.55 pg/mL, p=0.001) (Table 3). However, the level of collagen had no different between treatment and control group (p=0.124).

 

Table 3. Comparison of the TGF-b and collagen levels in the control and treatment group on day 5 and day 21

Marker	Day post-incision	Group	Mean ±SD	p-value
TGF-b level (pg/mL)	Day 5	Control	44.65±28.37	<0.001
		Treatment	132.10±28.32
	Day 21	Control	94.72±14.55	0.001
		Treatment	128.39±9.09
Collagen level (ng/mL)	Day 5	Control	315.07±137.40	<0.001
		Treatment	1623.61±368.17
	Day 21	Control	1064.38±363.02	0.124
		Treatment	755.15±268.64

 

DISCUSSION:

This study was conducted to assess the effect of topical antihistamine on the level of wound healing regulator and marker, TGF-β and collagen. TGF-β and collagen were chosen as the parameters because they are considered the best markers to describe the wound healing process and the formation of scar tissue. Topical antihistamine was chosen since antihistamine is one of the non-surgical therapeutic modalities currently available and has been shown to have a beneficial effect on wound healing and has minimal side effect compared to systemic antihistamine.^34,35

 

In wounds treated with antihistamine, our data suggest that the TGF-b level decreased from the 5^th day to the 21^st day and had a decrease trend in collagen level. In contrast, the TGF-β level in the control group increased from the 5^th day to the 21^st day and had an increase trend in collagen level. This was consistent with the hypothesis of this study that application of topical antihistamine to acute wounds could reduce the TGF-β and collagen level in the wound healing phase. Interestingly, our data indicated that the level of TGF-β in wound treated with antihistamine were significantly higher compared to control group on day 21. The literature suggests that TGF-β will decrease during the remodeling phase. Day 21 is the initial phase of remodeling and therefore TGF-β level was expected to be low. Further assessment is required to elucidate for this. One of the reasons probably because the TGF-b level baseline was quite different between control and treatment group. Increased TGF-b level in the control group on the day 21, led to activation of proliferative fibroblasts to synthesize collagen. This is what causes collagen buildup which later yields hypertrophic scars and keloid.^8,9

 

There are some limitations of our study. The number of animals used in this study was relatively small for each group and therefore study with bigger sample size is suggested. The levels of TGF-β and collagen were measured using ELISA in the present study, other methods might need to be employed to give more precise data.

 

CONCLUSION:

We attempted to elucidate one possible mechanism in the development of hypertrophic scars and keloid, in which antihistamine was used to inhibit the action of histamine in the wound healing phase. Our data suggest that topical application of antihistamines could reduce TGF-β and collagen levels in acute wounds in animal model at the end of the proliferation phase or the beginning of the remodeling phase. Further research is needed regarding the effect of topical antihistamine on the management of hypertrophic and keloid scars, such as application in humans.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors would like to thank Department of Anatomical Pathology for their kind support during ELISA testing.

 

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Received on 24.06.2021           Modified on 08.12.2021

Accepted on 11.04.2022         © RJPT All right reserved