β-Nicotinamide Mononucleotide (NMN) Administrated by Intraperitoneal Injection Mediates Protection Against UVB-Induced Skin Damage in Mice

Figures & data

Table 1 Sequences of the Primers Used for the Mice Liver and Skin Tissues

Gene	Sequences	Gene ID
NF-κBp65	F:5ʹ- GAGGCACGAGGCTCCTTTTCT −3’	XM_006531695.3
	R:5ʹ- GTAGCTGCATGGAGACTCGAACA −3’
IκB-α	F:5ʹ-TGAAGGACGAGGAGTACGAGC-3’	XM_021179000.1
	R:5ʹ-TGCAGGAACGAGTCTCCGT-3’
TNF-α	F:5ʹ-CAGGCGGTGCCTATGTCTC-3’	NM_013693.3
	R:5ʹ-GCTGCAACAGGGGGTAACAT-3’
IL-6	F:5ʹ-CTGCAAGAGACTTCCATCCAG-3’	NM_031168.2
	R:5ʹ-AGTGGTATAGACAGGTCTGTTGG-3’
IL-10	F:5ʹ-CTTACTGACTGGCATGAGGATCA-3’	NM_010548.2
	R:5ʹ-GCAGCTCTAGGAGCATGTGG-3’
SOD1	F:5ʹ-AACCAGTTGTGTTGTCAGGAC-3’	NM_011434.1
	R:5ʹ-CCACCATGTTTCTTAGAGTGAGG-3’
SOD2	F:5ʹ-CAGACCTGCCTTACGACTATGG-3’	NM_013671.3
	R:5ʹ-CTCGGTGGCGTTGAGATTGTT-3’
CAT	F:5ʹ-GGAGGCGGGAACCCAATAG-3’	NM_009804.2
	R:5ʹ-GTGTGCCATCTCGTCAGTGAA-3’
GSH	F:5ʹ-CCACCGTGTATGCCTTCTCC-3’	NM_008160.6
	R:5ʹ-AGAGAGACGCGACATTCTCAAT-3’
AMPK	F:5ʹ-GTCAAAGCCGACCCAATGATA-3’	NM_001013367.3
	R:5ʹ-CGTACACGCAAATAATAGGGGTT-3’
LKB1	F:5ʹ-CTGGACTCCGAGACCTTATGC-3’	NM_011492.5
	R:5ʹ-CAAGCTGGATCACATTCCGAT-3’
SIRT1	F:5ʹ-TGATTGGCACCGATCCTCG-3’	NM_001159589.2
	R:5ʹ-CCACAGCGTCATATCATCCAG-3’
mTOR	F:5ʹ-CAGTTCGCCAGTGGACTGAAG-3’	NM_020009.2
	R:5ʹ-GCTGGTCATAGAAGCGAGTAGAC-3’
PGC-1α	F:5ʹ-TATGGAGTGACATAGAGTGTGCT-3’	NM_008904.2
	R:5ʹ-GTCGCTACACCACTTCAATCC-3’
APPL1	F:5ʹ-AGCCAGTGACCCTTTATATCTGC-3’	NM_145221.2
	R:5ʹ-AGGTATCCAGCCTTTCGGGTT-3’
FOXO	F:5ʹ-CCCAGGCCGGAGTTTAACC-3’	NM_019739.3
	R:5ʹ-GTTGCTCATAAAGTCGGTGCT-3’
β-actin	F:5ʹ-CATGTACGTTGCTATCCAGGC-3’	NC_000071.7
	R:5ʹ-CTCCTTAATGTCACGCACGAT-3’

Figure 1 Photomicrograph of liver paraffin sections stain with (H&E). The black bars indicate 200 μm. The black arrows indicate necrotic cells.

Abbreviations: VC+UVB, mice treated with vitamin C(300mg/kg) and UVB irradiation; NMN+UVB, mice treated with nicotinamide mononucleotide (300mg/kg) and UVB irradiation.

Figure 2 Photomicrograph of skin paraffin sections. (A) H&E staining of the skin; (B) Masson’s staining of the skin; (C) toluidine blue staining; (D) epidermal thickness of skin; (E) dermal thickness of skin; (F) number of mast cells of skin. *p < 0.05 compared to the UVB group; **p < 0.01 compared to UVB group; ***p < 0.001 compared to the UVB group. → ← in (A) indicate the thickness of dermis; ↑ in (B) indicate collagen fiber; ↑ in (C) indicate mast cell.

Abbreviations: VC+UVB, mice treated with vitamin C(300mg/kg) and UVB irradiation; NMN+UVB, mice treated with nicotinamide mononucleotide (300mg/kg) and UVB irradiation.

Table 2 Levels of Oxidative Stress Indicators in Serum of Mice^A

Group	T-SOD (U/mL)	CAT (U/mL)	H2O2 (mmol/L)	AGEs (pg/mL)
Control	99.05±16.07^b	40.73±4.62^a	15.52±3.47^d	32.44±6.19^c
UVB	48.64±7.87^c	18.08±4.28^d	61.57±10.07^a	131.62±9.89^a
VC+UVB	81.87±11.86^b	23.46±4.25^c	39.15±7.60^b	113.53±12.34^b
NMN+UVB	198.92±21.26^a	34.15±5.58^b	27.35±4.79^c	29.69±8.11^c

Notes: ^AValues are mean ± standard deviation (N = 10/group). The difference in variance between the two groups was significant (p < 0.05). ^a–dMean values with different letters in the same column are significantly different (p < 0.05) according to Duncan’s honestly significantly different test.

Abbreviations: UVB, mice treated with UV irradiation; VC+UVB, mice treated with vitamin C (300 mg/kg) and UV irradiation; NMN+UVB, mice treated with nicotinamide mononucleotide (300 mg/kg) and UV irradiation; T-SOD, total superoxide dismutase; CAT, catalase; H₂O₂, hydrogen peroxide; AGEs, advanced glycation end products.

Table 3 Levels of Oxidative Stress in Skin of Mice^B

Group	T-SOD (U/mgprot)	CAT (U/mgprot)	H2O2 (mmol/gprot)	AGEs (pg/mL)	Na^+-K^+-ATPase (U/mgprot)	NADK (nmol/min/mgprot)
Control	27.69±6.32^a	24.24±3.11^a	10.99±1.19^b	366.76±7.24^bc	0.89±0.13^a	17.47±2.66^a
UVB	7.51±1.19^c	14.58±3.54^b	14.09±1.66^a	451.02±55.35^a	0.20±0.04^d	12.97±0.69^c
VC+UVB	17.01±3.93^b	21.84±4.53^a	13.50±2.44^a	405.36±26.62^b	0.49±0.14^c	14.58±1.47^b
NMN+UVB	20.06±3.07^b	22.66±4.59^a	8.46±1.99^b	339.61±43.18^c	0.66±0.12^b	16.48±1.57^a

Notes: ^BValues are mean ± standard deviation (N = 10/group). The difference in variance between the two groups was significant (p < 0.05). ^a–dMean values with different letters in the same column are significantly different (p < 0.05) according to Duncan’s honestly significantly different test.

Abbreviations: UVB, mice treated with UV irradiation; VC+UVB, mice treated with vitamin C (300 mg/kg) and UV irradiation; NMN+UVB, mice treated with nicotinamide mononucleotide (300 mg/kg) and UV irradiation; T-SOD, total superoxide dismutase; CAT, catalase; H₂O₂, hydrogen peroxide; AGEs, advanced glycation end products; NADK, nicotinamide adenine dinucleotide kinase.

Table 4 Levels of Inflammatory Indexes in Serum and Skin of Mice^C

Serum Levels
Group	TNF-α (ng/L)	IL-1β (ng/L)	IL-4 (pg/L)	IL-6 (pg/mL)	IL-10 (pg/mL)
Control	238.87±67.66^b	17.83±1.17^c	102.14±15.80^a	16.04±4.69^c	320.38±47.29^a
UVB	550.60±83.35^a	21.87±1.42^a	59.11±8.41^c	75.78±8.46^a	128.73±21.54^c
VC+UVB	317.87±60.20^b	20.12±1.18^b	57.88±9.30^c	50.12±8.02^b	135.54±10.48^c
NMN+UVB	250.27±51.28^b	17.95±1.11^c	71.40±10.87^b	17.33±6.75^c	179.35±50.09^b
Skin Levels
Control	99.34±14.92^b	6.34±0.69^c	134.45±23.71^a	34.67±4.65^b	653.74±68.16^a
UVB	158.79±14.03^a	15.20±1.16^a	41.06±6.94^d	50.79±7.30^a	95.71±19.20^c
VC+UVB	151.18±26.87^a	9.43±2.30^b	69.80±14.44^c	47.43±5.20^a	141.91±35.56^c
NMN+UVB	95.63±18.98^b	5.28±0.63^c	89.97±6.08^b	31.22±8.03^b	271.39±35.41^b

Notes: ^CValues are mean ± standard deviation (N = 10/group). The difference in variance between the two groups was significant (p < 0.05). ^a–dMean values with different letters in the same column are significantly different (p < 0.05) according to Duncan’s honestly significantly different test.

Abbreviations: UVB, mice treated with UV irradiation; VC+UVB, mice treated with vitamin C (300 mg/kg) and UV irradiation; NMN+UVB, mice treated with nicotinamide mononucleotide (300 mg/kg) and UV irradiation; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-4, interleukin-4; IL-6, interleukin-6; IL-10, interleukin-10.

Figure 3 Gene expression of SOD1, SOD2, CAT and GSH in skin and liver tissues. *p < 0.05 compared to the UVB group; **p < 0.01 compared to UVB group; ***p < 0.001 compared to the UVB group.

Abbreviations: VC+UVB, mice treated with vitamin C(300mg/kg) and UVB irradiation; NMN+UVB, mice treated with nicotinamide mononucleotide (300mg/kg) and UVB irradiation.

Figure 4 Gene expression of NF-κBp65, IκB-α, TNF-α, IL-6 and IL-10 in skin and liver tissues. *p < 0.05 compared to the UVB group; **p < 0.01 compared to UVB group; ***p < 0.001 compared to the UVB group.

Abbreviations: VC+UVB, mice treated with vitamin C(300mg/kg) and UVB irradiation; NMN+UVB, mice treated with nicotinamide mononucleotide (300mg/kg) and UVB irradiation.

Figure 5 Gene expression of AMPK, SIRT1, LKB1, PGC-1α, APPL1, mTOR and FOXO in skin and liver tissues. *p < 0.05 compared to the UVB group; **p < 0.01 compared to UVB group; ***p < 0.001 compared to the UVB group.

Abbreviations: VC+UVB, mice treated with vitamin C(300mg/kg) and UVB irradiation; NMN+UVB, mice treated with nicotinamide mononucleotide (300mg/kg) and UVB irradiation.

Figure 6 Protein expression of AMPK, NFκB-p65, IκB-α, SOD1 and CAT in skin tissues. (A) relative expression levels of proteins; (B) protein banding map. *p < 0.05 compared to the UVB group; **p < 0.01 compared to the UVB group; ***p < 0.001 compared to the UVB group.

Abbreviations: VC+UVB, mice treated with vitamin C(300mg/kg) and UVB irradiation; NMN+UVB, mice treated with nicotinamide mononucleotide (300mg/kg) and UVB irradiation.

Figure 7 The possible mechanism of this study. The black arrows “↑” indicated those indicators were increased after intraperitoneal injection treatment with NMN, but the red arrows “↓” indicated those indicators were decreased after intraperitoneal injection treatment with NMN.