TNF-alpha expression, evaluation of collagen, and TUNEL of <i>Matricaria recutita L.</i> extract and triamcinolone on oral ulcer in diabetic rats

OLIVEIRA, Bruna Vasconcelos; BARROS SILVA, Paulo Goberlânio; NOJOSA, Jacqueline de Santiago; BRIZENO, Luiz André Cavalcante; FERREIRA, Jamile Magalhães; SOUSA, Fabrício Bitú; MOTA, Mário Rogério Lima; ALVES, Ana Paula Negreiros Nunes

doi:10.1590/1678-775720150481

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Journal of Applied Oral Science

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ORIGINAL ARTICLES • J. Appl. Oral Sci. 24 (3) • May-Jun 2016 • https://doi.org/10.1590/1678-775720150481 copy

TNF-alpha expression, evaluation of collagen, and TUNEL of Matricaria recutita L. extract and triamcinolone on oral ulcer in diabetic rats

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

Diabetes mellitus (DM) is a disease associated with delayed wound healing of oral ulcers by increased expression of proinflammatory cytokines and cellular apoptosis.

Objective

to evaluate the influence of Tumor Necrosis Factor alpha (TNF-α) and apoptosis in rats with DM treated with chamomile extract or triamcinolone.

Material and Methods

Wistar male rats (210.0±4.2 g) were divided into five groups: negative control group (NCG) without diabetes; positive control group (PCG) with DM (alloxan, 45 mg/kg); and groups treated with chamomile extract (normoglycemic= NCG group and diabetic= DCG group) and with triamcinolone (TG). Traumatic ulcers were performed on all animals that received topical triamcinolone, chamomile extract or saline 12/12 hours for ten days.

Results

On days five and ten the animals were euthanized and the ulcers were analyzed by light microscopy, TUNEL assay, and immunohistochemically (TNF-α). The NCG (p=0.0062), PCG (p=0.0285), NCG (p=0.0041), and DCG (p<0.0001) groups were completely healed on the 10th day, however, there was no healing on the TG (p=0.5127) group. The TNF-α expression showed a significant reduction from the 5^th to the 10^th day in NCG (p=0.0266) and DCG (p=0.0062). In connective tissue, the TUNEL assay showed a significant reduction in the number of positive cells in NCG (p=0.0273) and CNG (p=0.0469) and in the epithelium only in CDG (p=0.0320).

Conclusions

Chamomile extract can optimize the healing of traumatic oral ulcers in diabetic rats through the reduction of apoptosis in the epithelium and TNF-α expression.

Oral ulcer; Diabetes mellitus; Chamomile; Matricaria; Triamcinolone

Groups	Clinical Evaluation
	Area of Ulcer (mm²)			Weight Loss (g) [Final weight – Initial weight]			Blood Glucose Changes (mg/dL) [Final Glucose – Initial Glucose]
	5^th	10^th	p1	5^th	10^th	p1	5^th	10^th	p1
Saline Treatment
Negative Control (n=13)	14.2±2.2	0.0±0.0	0.003	-25.0±5.4§	-8.3±2.1§	0.0105	-15.4±9.6	7.2±4.5	0.051
Positive Control (n=12)	18.4±2.2	5.5±0.9	0.0005	-19.0±5.1§	-15.0±4.2§	0.561	-134.2±46.0*§	-147.5±22.4*§	0.8183
Chamomile Treatment
Chamomile Normoglycemic (n=14)	18.9±2.2	0.4±0.2	0.0002	6.2±3.5	19.7±2.9	<0.0001	-16.9±12.1	5.2±5.0	0.1201
Chamomile Diabetic (n=17)	18.5±2.0	4.0±1.3	0.0004	-25.6±1.3§	-21.0±4.6§	0.2477	119.7±13.3†§	10.0±12.6†§	0.0002
Triamcinolone Treatment
Triamcinolone Diabetic (n=17)	18.8±0.5	10.5±3.3†	0.082	-47.8±3.0†§	-52.1±4.5*§	0.4165	55.8±70.5†§	32.5±24.7†§	0.5206

Groups	Microscopic Evaluation
	Histological Scores			Percentage of Collagen Deposition Area (%)			TNF-α Immunostaining			TUNEL connective tissue Staining			TUNEL epithelium Staining
	5^th	10^th	p1	5^th	10^th	p2	5^th	10^th	p1	5^th	10^th	p1	5^th	10^th	p1
Saline Treatment
Negative Control (n=13)	4	1	0.0062	39.2±4.9	57.8±1.8	0.0264	3	1	0.0266	3	1	0.0273	3	3	0.9222
	(2,4)	(0,3)					(2,3)	(0,3)		(3,3)	(0,3)		(1,3)	(1,3)
Positive Control (n=12)	4	2	0.0285	25.8±2.8*	38.7±4.2*	0.0452	3	3	0.3367	3	3	1.0000	3	3	1.0000
	(4,4)	(2,2)*§					(3,3)	(2,3)		(3,3)	(3,3)*§		(2,3)	(3,3)
Chamomile Treatment
Chamomile Normoglycemic (n=14)	3	0	0.0074	30.9±4.3	41.0±3.6	0.0297	1	1	0.1683	3	1	0.0469	3	2	0.1683
	(1,3)	(0,1)					(1,1)	(0,1)		(2,3)	(1,3)		(3,3)	(2,2)
Chamomile Diabetic (n=17)	3	1	0.0041	45.1±1.2†	51.5±6.2	0.1934	3	2	0.0062	3	3	0.3545	3	2	0.032
	(2,4)	(1,3)					(2,3)	(1,3)		(3,3)	(2,3)		(3,3)	(1,3)
Triamcinolone Treatment
Triamcinolone (n=17)	4	4	0.5127	38.2±1.9	30.6±4.4‡	0.3251	3	3	1.0000	3	3	1.0000	3	3	0.3545
	(4,4)	(3,4)†§					(3,3)	(3,3)*‡§		(3,3)	(3,3)*§		(2,3)	(3,3)‡§

	Correlation	Negative Control (n=13)	Positive Control (n=12)	Chamomile (n=17)	Triamcinolone (n=17)
Pearson Correlation	Area of Ulcer x Weight Loss	p=0.002* r=–0.766	p=0.010* r=–0.763	p=0.994 r=0.002	p=0.120 r=–0.392
	Area of Ulcer x Blood Glucose Changes	p=0.204 r=0.503	p=0.066 r=–0.573	p=0.089 r=0.471	p=0.999 r=0.000
	Blood Glucose Changes x Weight Loss	p=0.270 r=0.372	p=0.011* r=0.728	p=0.641 r=0.126	p=0.305 r=0.234
Spearman Correlation	Histologic Scores x Area of Ulcer	p<0.001* r=0.836	p=0.046* r=0.640	p=0.011* r=0.601	p=0.020* r=0.573
	Histologic Scores x Weight Loss	p=0.335 r=–0.291	p=0.192 r=–0.425	p=0.266 r=–0.286	p=0.540 r=–0.160
	Histologic Scores x Blood Glucose Changes	p=0.970 r=0.011	p=1.000 r = 0.000	p=0.070 r=0.790	p=0.270 r=–0.284

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[1] Corresponding address: Paulo Goberlânio de Barros Silva - Departamento de Odontologia Clínica - Setor de Patologia Oral - Faculdade de Odontologia - Universidade Federal do Ceará - Rua Alexandre Barauna, 949 - Rodolfo Teofilo - 60430-160 - Fortaleza - CE - Brazil - Phone/Fax +55 85 3366 8421 - Phone 2: +55 85 8705 7151 - e-mail: paulo_goberlanio@yahoo.com.br