The toxicity of naringenin to human corneal epithelial cells

doi:10.3877/cma.j.issn.2095-2007.2021.01.007

Abstract

Abstract:

Objective

The aim of this study was to investigate the toxicity of naringenin (Nar) to human corneal epithelial cells (HCECs).

Methods

HCECs purchased from Qingqi Shanghai Biotechnology Development Co., Ltd. were used for cytotoxicity test and scratch test. The tetrazolium salt colorimetric assay was used to detect the cell survival of Nar and DMSO on HCECs. Graphpad Prism8.0 software was used to map the cells survival curve and calculate the half inhibitory concentration (IC₅₀). The scratch experiment was used to observe the effect of different concentrations of Nar on the migration of HCECs. ImageJ was used to calculate the cell healing rate (HR). OD value, survival rate (SR) and HR were in accordance with normal distribution, and expressed by ±s.The independent sample t-testing method was used to compare SR of HCECs with different concentrations of DMSO solvent and Nar solution for 24 h, 48 h and 72 h; and the wound HR of cells at 6 h, 12 h and 24 h with different concentrations of Nar.

Results

The SR of HCECs in the DMSO 0.04%, 0.08% and 0.16% groups for 24 h was (94.57±0.83)%, (93.59±1.17)% and (91.43±1.47)%, respectively. After t testing, compared with the DMEM group, the difference in the SR of HCECs was statistically significant (t=3.436, 3.882, 4.960; P<0.05). The SR of HCECs was (94.43±0.38)% and (93.16±0.66)% in the DMSO group with a volume percentage of 0.08% and 0.16% for 48 h. After t testing, compared with the DMEM group, the difference in the SR of HCECs was statistically significant (t=2.953, 3.573; P<0.05). In the DMSO group with the volume percentage of 0.02%, 0.04%, 0.08% and 0.16% for 72 h, the SRs of HCECs were (96.28±0.29)%, (95.30±0.25)%, (94.65±0.82)% and (93.73±1.18)%. After t testing, the difference in the SR of HCECs between different DMSO volume percentage and the DMEM group was statistically significant (t=3.756, 4.763, 4.929, 5.263; P<0.05). The SRs of HCECs treated with 200 μM, 400 μM and 800 μM Nar solutions for 48 h were (60.17±0.46)%, (47.55±0.91)% and (29.19±1.25)%, respectively; the SRs at 72 h were ( 60.02±6.20)%, (31.20±1.96)% and (22.96±2.69)%. After t testing, the difference in SR of HCECs for 48 h and 72 h compared with that for 24 h, was statistically significant (t=12.903, 6.675, 11.215, 5.377, 12.696, 11.972; P<0.05). The SRs of HCECs treated with 25 μM, 400 μM and 800 μM Nar solutions for 72 h were (89.41±1.38)%, (31.20±1.96)% and (22.96±2.69)%, respectively. After t testing, the SR of HCECs for 72 h compared with that for 48 h was significantly different (t=4.442, 13.119, 3.631; P<0.05). The IC₅₀ of Nar solution for HCECs at 24 h, 48 h and 72 h was 690.8 μM, 343.4 μM and 237.7 μM, respectively, and the 95% confidence intervals of IC₅₀ were (574.1 to 845.5), (259.2 to 734.3) and (231.1 to 266.7). The migration area coverage rate of HCECs for 6 h in 0, 100 μM, 200 μM and 400 μM Nar solutions were (18.04±1.61)%, (16.91±1.56)%, (16.63±2.12)% and (12.20±2.06) %, respectively. The migration area coverage rate of HCECs gradually increased with time. Compared with the DMEM group, there was no significant difference in the migration area coverage rate of cells added with 100 μM Nar solution in per time period (t=0.872, 1.074, 1.620; P>0.05); the difference in that of cells added with 400 μM Nar was statistically significant (t=3.863, 9.869, 27.194; P<0.05); the difference in the scar HR of HCECs added with 200 μM Nar after scratching for 12 h and 24 h was significantly different (t=5.714, 9.119; P<0.05).

Conclusions

Nar could reduce the SR of HCECs and inhibit the migration and HR of HCECs, which is toxic to HCECs.

Key words: Human corneal epithelial cells, Naringenin, Dimethyl sulfoxide, Toxicity

Haoliang Li, Ran Zhu, Xixi Zhang, Shu Liang. The toxicity of naringenin to human corneal epithelial cells[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2021, 11(01): 41-47.

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Figures/Tables 3

References 34

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项目	0	0.0025%DMSO	0.005%DMSO	0.01%DMSO	0.02%DMSO	0.04%DMSO	0.08%DMSO	0.16%DMSO
24 h OD值	0.415±0.011	0.412±0.108	0.409±0.011	0.406±0.012	0.403±0.012	0.398±0.013	0.395±0.014	0.389±0.012
存活率(%)	100.00±2.61	98.92±0.48	98.05±0.55	97.18±0.53	95.98±0.61	94.57±0.83^*	93.59±1.17^*	91.43±1.47^*
t值		0.704	1.266	1.836	2.598	3.436	3.882	4.960
P值		>0.05	>0.05	>0.05	>0.05	<0.05	<0.05	<0.05
48 h OD值	0.477±0.016	0.474±0.015	0.470±0.016	0.467±0.015	0.464±0.015	0.461±0.014	0.456±0.014	0.452±0.013
存活率(%)	100.00±3.25	99.10±0.12	98.11±0.07	97.30±0.20	96.50±0.45	95.60±0.54	94.43±0.38^*	93.16±0.66^*
t值		0.479	1.009	1.436	1.851	2.315	2.953	3.573
P值		>0.05	>0.05	>0.05	>0.05	>0.05	<0.05	<0.05
72 h OD值	0.615±0.010	0.610±0.010	0.605±0.010	0.601±0.015	0.596±0.010	0.591±0.009	0.588±0.010	0.583±0.012
存活率(%)	100.00±1.69	98.96±0.08	98.04±0.06	97.19±0.19	96.28±0.29^*	95.30±0.25^*	94.65±0.82^*	93.73±1.18^*
t值		1.068	2.010	2.860	3.756	4.763	4.929	5.263
P值		>0.05	>0.05	>0.05	<0.05	<0.05	<0.05	<0.05

项目	0	0.0025%DMSO	0.005%DMSO	0.01%DMSO	0.02%DMSO	0.04%DMSO	0.08%DMSO	0.16%DMSO
24 h OD值	0.415±0.011	0.412±0.108	0.409±0.011	0.406±0.012	0.403±0.012	0.398±0.013	0.395±0.014	0.389±0.012
存活率(%)	100.00±2.61	98.92±0.48	98.05±0.55	97.18±0.53	95.98±0.61	94.57±0.83^*	93.59±1.17^*	91.43±1.47^*
t值		0.704	1.266	1.836	2.598	3.436	3.882	4.960
P值		>0.05	>0.05	>0.05	>0.05	<0.05	<0.05	<0.05
48 h OD值	0.477±0.016	0.474±0.015	0.470±0.016	0.467±0.015	0.464±0.015	0.461±0.014	0.456±0.014	0.452±0.013
存活率(%)	100.00±3.25	99.10±0.12	98.11±0.07	97.30±0.20	96.50±0.45	95.60±0.54	94.43±0.38^*	93.16±0.66^*
t值		0.479	1.009	1.436	1.851	2.315	2.953	3.573
P值		>0.05	>0.05	>0.05	>0.05	>0.05	<0.05	<0.05
72 h OD值	0.615±0.010	0.610±0.010	0.605±0.010	0.601±0.015	0.596±0.010	0.591±0.009	0.588±0.010	0.583±0.012
存活率(%)	100.00±1.69	98.96±0.08	98.04±0.06	97.19±0.19	96.28±0.29^*	95.30±0.25^*	94.65±0.82^*	93.73±1.18^*
t值		1.068	2.010	2.860	3.756	4.763	4.929	5.263
P值		>0.05	>0.05	>0.05	<0.05	<0.05	<0.05	<0.05

项目	0	25 μM Nar	50 μM Nar	100 μM Nar	200 μM Nar	400 μM Nar	800 μM Nar	1600 μM Nar
24 h OD值	0.416±0.006	0.393±0.011	0.378±0.016	0.358±0.011	0.355±0.013	0.297±0.020	0.276±0.013	0.164±0.016
存活率(%)	100.00±1.47	92.81±2.27^a	88.02±3.22^a	82.03±2.03^a	81.11±2.77^a	62.69±3.82^a	56.03±3.95^a	20.87±1.30^a
t值		4.596	5.86	12.421	10.421	15.775	18.058	69.858
P值		<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05
48 h OD值	0.461±0.008	0.439±0.005	0.404±0.007	0.401±0.006	0.320±0.007	0.275±0.006	0.210±0.005	0.167±0.005
存活率(%)	100.00±0.49	93.78±1.01^a	83.99±3.31^a	83.14±3.07^a	60.17±0.46^ab	47.55±0.91^ab	29.19±1.25^ab	17.04±2.00^a
t值		9.628	8.28	9.397	102.698	88.208	91.503	69.768
P值		<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05
72 h OD值	0.638±0.030	0.584±0.029	0.568±0.023	0.540±0.030	0.434±0.006	0.287±0.042	0.245±0.019	0.201±0.012
存活率(%)	100.00±1.90	89.41±1.38^ac	86.35±1.53^a	80.81±0.27^a	60.02±6.20^ab	31.20±1.96^abc	22.96±2.69^abc	14.40±4.02^a
t值		7.813	9.765	17.307	10.673	43.648	40.463	33.348
P值		<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05

项目	0	25 μM Nar	50 μM Nar	100 μM Nar	200 μM Nar	400 μM Nar	800 μM Nar	1600 μM Nar
24 h OD值	0.416±0.006	0.393±0.011	0.378±0.016	0.358±0.011	0.355±0.013	0.297±0.020	0.276±0.013	0.164±0.016
存活率(%)	100.00±1.47	92.81±2.27^a	88.02±3.22^a	82.03±2.03^a	81.11±2.77^a	62.69±3.82^a	56.03±3.95^a	20.87±1.30^a
t值		4.596	5.86	12.421	10.421	15.775	18.058	69.858
P值		<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05
48 h OD值	0.461±0.008	0.439±0.005	0.404±0.007	0.401±0.006	0.320±0.007	0.275±0.006	0.210±0.005	0.167±0.005
存活率(%)	100.00±0.49	93.78±1.01^a	83.99±3.31^a	83.14±3.07^a	60.17±0.46^ab	47.55±0.91^ab	29.19±1.25^ab	17.04±2.00^a
t值		9.628	8.28	9.397	102.698	88.208	91.503	69.768
P值		<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05
72 h OD值	0.638±0.030	0.584±0.029	0.568±0.023	0.540±0.030	0.434±0.006	0.287±0.042	0.245±0.019	0.201±0.012
存活率(%)	100.00±1.90	89.41±1.38^ac	86.35±1.53^a	80.81±0.27^a	60.02±6.20^ab	31.20±1.96^abc	22.96±2.69^abc	14.40±4.02^a
t值		7.813	9.765	17.307	10.673	43.648	40.463	33.348
P值		<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05

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