Effects of substance P on the secretory function and the expression of endogenous VEGF, bFGF and PDGF in radiation-injured skin fibroblasts

doi:10.11958/20220599

Abstract

Abstract:

Objective To explore the the effects of neuropeptide substance P (SP) on the secretory function and the expression of endogenous vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) in skin fibroblasts after radiation injured. Methods HFF-1 cells in logarithmic growth phase were divided into 10 experimental groups according to the irradiation dose: the 0 Gy, 2 Gy, 6 Gy, 12 Gy, 18 Gy, 0 Gy+SP, 2 Gy+SP, 6 Gy+SP, 12 Gy+SP and 18 Gy+SP. Exogenous SP intervention was performed before irradiation with 0 Gy+SP, 2 Gy+SP, 6 Gy+SP, 12 Gy+SP and 18 Gy+SP. The enzyme linked immunosorbent assay (ELISA) was used to detect the expression levels of typeⅠcollagen (Col-Ⅰ), type Ⅲ collagen (Col-Ⅲ), calcitonin gene related peptide (CGRP) in cell culture medium and Col-Ⅰ, Col-Ⅲ, CGRP, VEGF, bFGF and PDGF in cell lysate 24 h after irradiation. Quantificational real-time polymerase chain reaction (qPCR) was used to detect the expression levels of VEGF, bFGF and PDGF mRNA. Results Compared with the 0 Gy experimental group, Col-Ⅰ, Col-Ⅰ/Col-Ⅲ in culture medium and CGRP and VEGF proteins in cell lysate were increased in the irradiated groups, bFGF and PDGF mRNA were increased in the irradiated groups (6 Gy, 12 Gy and 18 Gy). The Col-Ⅰ was highly expressed in the culture medium and cell lysate of the 6 Gy group. The Col-Ⅰ, Col-Ⅲ, Col-Ⅰ/Col-Ⅲ and CGRP were highly expressed in the culture medium and cell lysate of the 18 Gy group. The bFGF was highly expressed at protein and mRNA levels in irradiated groups (12 Gy and 18 Gy). When SP intervention was given before irradiation, the CGRP expression was consistently high in the culture medium and lysate of the 0 Gy+SP group than that of the corresponding group without intervention. The Col-Ⅰ and Col-Ⅰ/Col-Ⅲ were consistently low expressed in the culture medium and lysate of the intervention groups (2 Gy+SP, 6 Gy+SP) and (2 Gy+SP, 6 Gy+SP and 18 Gy+SP), respectively. VEGF and PDGF were highly expressed at protein and mRNA levels in the intervention groups (2 Gy+SP, 12 Gy+SP and 18 Gy+SP) and (12 Gy+SP, 18 Gy+SP), respectively. Conclusion Exogenous SP could regulate the expression and secretion of Col-Ⅰ and Col-Ⅲ, decrease the ratio of Col-Ⅰ/Col-Ⅲ and promote the high expression of VEGF and PDGF in radiation injured skin fibroblasts, so as to coordinate the stress response of fibroblasts to electron beam irradiation.

Key words: radiation damage, skin diseases, fibroblasts, vascular endothelial growth factors, fibroblast growth factors, neuropeptide substance P

CLC Number:

Figures/Tables 5

References 13

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基因名称	引物序列（5'→3'）	产物大小（bp）
VEGF	上游：CGAGACCCTGGTGGACATCTT	180
VEGF	下游：TGGCCTTGGTGAGGTTTGATC	180
bFGF	上游：AGAAGAGCGACCCTCACATCA	226
bFGF	下游：CGTTTCAGTGCCACATACCAA	226
PDGF	上游：ACCAGGACGGTCATTTACGA	136
PDGF	下游：AGGGCTGGCACTTGACACT	136
GAPDH	上游：TGGGTGTGAACCATGAGAAGT	126
GAPDH	下游：TGAGTCCTTCCACGATACCAA	126

基因名称	引物序列（5'→3'）	产物大小（bp）
VEGF	上游：CGAGACCCTGGTGGACATCTT	180
VEGF	下游：TGGCCTTGGTGAGGTTTGATC	180
bFGF	上游：AGAAGAGCGACCCTCACATCA	226
bFGF	下游：CGTTTCAGTGCCACATACCAA	226
PDGF	上游：ACCAGGACGGTCATTTACGA	136
PDGF	下游：AGGGCTGGCACTTGACACT	136
GAPDH	上游：TGGGTGTGAACCATGAGAAGT	126
GAPDH	下游：TGAGTCCTTCCACGATACCAA	126

组别	Col-Ⅰ （μg/L）	Col-Ⅲ （μg/L）	Col-Ⅰ/ Col-Ⅲ	CGRP （ng/L）
0 Gy组	14.34±0.25	31.50±0.69	0.46±0.02	52.48±2.04
2 Gy组	20.50±0.19^a	30.14±0.35	0.68±0.01^a	53.07±3.50
6 Gy组	19.31±0.51^a	32.52±1.18	0.59±0.03^a	61.48±1.86^a
12 Gy组	21.07±0.84^a	33.37±0.74^a	0.63±0.02^a	99.91±4.94^a
18 Gy组	26.33±0.63^a	33.50±0.74^a	0.79±0.03^a	103.80±2.82^a
0 Gy+SP组	21.86±0.93^a	28.16±1.26^a	0.78±0.01^a	62.57±4.17^a
2 Gy+SP组	18.22±0.63^b	31.97±0.57^b	0.57±0.01^b	76.89±4.11^b
6 Gy+SP组	16.10±0.57^c	33.35±1.01	0.48±0.03^c	98.92±2.78^c
12 Gy+SP组	23.31±1.25^d	42.24±1.15^d	0.55±0.02^d	102.29±3.64
18 Gy+SP组	25.07±0.99	41.37±0.74^e	0.61±0.02^e	108.88±3.13
F	76.684^＊＊	77.852^＊＊	80.440^＊＊	135.039^＊＊

组别	Col-Ⅰ （μg/L）	Col-Ⅲ （μg/L）	Col-Ⅰ/ Col-Ⅲ	CGRP （ng/L）
0 Gy组	14.34±0.25	31.50±0.69	0.46±0.02	52.48±2.04
2 Gy组	20.50±0.19^a	30.14±0.35	0.68±0.01^a	53.07±3.50
6 Gy组	19.31±0.51^a	32.52±1.18	0.59±0.03^a	61.48±1.86^a
12 Gy组	21.07±0.84^a	33.37±0.74^a	0.63±0.02^a	99.91±4.94^a
18 Gy组	26.33±0.63^a	33.50±0.74^a	0.79±0.03^a	103.80±2.82^a
0 Gy+SP组	21.86±0.93^a	28.16±1.26^a	0.78±0.01^a	62.57±4.17^a
2 Gy+SP组	18.22±0.63^b	31.97±0.57^b	0.57±0.01^b	76.89±4.11^b
6 Gy+SP组	16.10±0.57^c	33.35±1.01	0.48±0.03^c	98.92±2.78^c
12 Gy+SP组	23.31±1.25^d	42.24±1.15^d	0.55±0.02^d	102.29±3.64
18 Gy+SP组	25.07±0.99	41.37±0.74^e	0.61±0.02^e	108.88±3.13
F	76.684^＊＊	77.852^＊＊	80.440^＊＊	135.039^＊＊

组别	Col-Ⅰ （μg/L）	Col-Ⅲ （μg/L）	Col-Ⅰ/ Col-Ⅲ	CGRP （ng/L）
0 Gy组	38.78±0.53	73.59±0.66	0.53±0.01	26.56±0.61
2 Gy组	39.10±0.62	72.29±0.90	0.54±0.01	33.53±0.71^a
6 Gy组	42.98±1.28^a	78.70±1.94^a	0.55±0.00	31.10±0.58^a
12 Gy组	39.69±1.12	72.84±0.62	0.54±0.02	32.55±0.73^a
18 Gy组	43.86±0.71^a	78.05±1.83^a	0.56±0.01^a	31.48±0.09^a
0 Gy+SP组	37.81±0.49	72.13±0.90	0.52±0.01	29.52±1.25^a
2 Gy+SP组	33.64±0.42^b	65.40±1.88^b	0.51±0.02^b	28.81±0.41^b
6 Gy+SP组	37.25±0.31^c	79.14±1.70	0.47±0.01^c	32.70±0.96^c
12 Gy+SP组	38.19±0.37^d	68.44±0.77^d	0.56±0.01	31.07±0.95^d
18 Gy+SP组	37.19±0.44^e	70.83±0.84^e	0.53±0.00^e	38.15±0.58^e
F	52.361^＊＊	34.876^＊＊	14.151^＊＊	51.309^＊＊