Effect of targeting p38MAPK on scar hyperplasia after rabbit cleft lip repair in vivo

doi:10.3877/cma.j.issn.1674-1366.2023.01.005

Abstract

Abstract:

Objective

The recombinant adenovirus of p38 mitogen-activated protein kinases (p38MAPK) gene was used to knock down the expression of target genes. By detecting the influence of p38MAPK signal pathway obstruction on upper lip scar hyperplasia in different time periods, the optimal therapeutic time of gene therapy was determined.

Methods

A total of ninety New Zealand white rabbits with cleft upper lip of 2.0-2.5 kg were treated with cleft lip repair. Recombinant virus was injected into the scar center at week 0, 1 and 2 after surgery, and the scar tissue was made into specimens at week 3 after surgery. Western blot, Real-time fluorescence quantitative RT-PCR and immunohistochemical staining were used to quantitatively and qualitatively detect the relative expression levels of proteins and mRNA of p38MAPK, scar formation related factors and Smad. The results of Western blot and RT-PCR were analyzed with software (SPSS v. 24.0) .

Results

The results of Western blot, RT-PCR and immunohistochemical staining showed that the expressions of Col Ⅲ (1.373 ± 0.073, F = 8.027, P = 0.002) and MMP1 (1.715 ± 0.028, F = 9.262, P = 0.001) in scar tissue of the injection group at week 1 after surgery were significantly higher than those at week 0 and 2. The expressions of ColⅠ (0.424 ± 0.015, F = 7.794, P = 0.003) and TIMP1 (0.464 ± 0.025, F = 6.196, P = 0.007) significantly decreased. The expression level of Smad protein and mRNA in the injection group at 1 week after surgery decreased, and the results were statistically significant compared with other groups (F_p-smad2 = 14.123, P_p-smad2 = 0.029; F_p-smad3 = 3.796, P_p-smad3 = 0.037) .

Conclusions

Inhibition of p38MAPK expression may play a role in inhibiting scar hyperplasia through the Smad dependent signaling pathway. Targeted p38MAPK knockdown had the greatest effect on scar hyperplasia at one week after cleft-lip surgery in rabbit upper lip.

Key words: p38 mitogen-activated protein kinases (p38MAPK), Gene silencing, Adenovirus, Hypertrophic scar, Gene therapy

Yingying Ge, Jin Yue, Lingfa Xue, Yaoxiang Xu, Haoran Zhao, Mingxue Cui, Wenlin Xiao. Effect of targeting p38MAPK on scar hyperplasia after rabbit cleft lip repair in vivo[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2023, 17(01): 37-44.

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References 24

[1]	Kim S, Choi TH, Liu W，et al. Update on scar management：guidelines for treating Asian patients[J]. Plast Reconstr Surg，2013，132（6）：1580-1589. DOI：10.1097/PRS.0b013e3182a8070c.
[2]	Masuoka H. A new technique of unilateral cleft lip repair with scarless Cupid′s bow peaks[J]. Plast Reconstr Surg，2021，148（3）：597-604. DOI：10.1097/PRS.0000000000008254.
[3]	Pouzet L, Ramon A, Jayyosi L，et al. Use of the surgical glue in the cutaneous closure of cheiloplasties for cleft lip[J]. Ann Chir Plast Esthet，2019，64（1）：89-92. DOI：10.1016/j.anplas.2018.06.007.
[4]	Jahanbin A, Eslami N, Layegh P，et al. Fractional CO₂ laser treatment for post-surgical lip scars in cleft lip and palate patients[J]. Lasers Med Sci，2019，34（8）：1699-1703. DOI：10.1007/s10103-019-02819-z.
[5]	Coentro JQ, Pugliese E, Hanley G，et al. Current and upcoming therapies to modulate skin scarring and fibrosis[J]. Adv Drug Deliv Rev，2019，146：37-59. DOI：10.1016/j.addr.2018.08.009.
[6]	Du QC, Zhang DZ, Chen XJ，et al. The effect of p38MAPK on cyclic stretch in human facial hypertrophic scar fibroblast differentiation[J]. PLoS One，2013，8（10）：e75635. DOI：10.1371/journal.pone.0075635.
[7]	贾铠宁，许尧祥，于果，等.不同时间p38MAPK信号通路受阻对兔上唇瘢痕增生的影响[J].中华医学美学美容杂志，2019，25（3）：243-247. DOI：10.3760/cma.j.issn.1671-0290.2019.03.020.
[8]	Yamanaka O, Saika S, Ohnishi Y，et al. Inhibition of p38MAP kinase suppresses fibrogenic reaction in conjunctiva in mice[J]. Mol Vis，2007，13：1730-1739.
[9]	McArdle C, Abbah SA, Bhowmick S，et al. Localized temporal co-delivery of interleukin 10 and decorin genes using amediated by collagen-based biphasic scaffold modulates the expression of TGF-β1/β2 in a rabbit ear hypertrophic scarring model[J]. Biomater Sci，2021，9（8）：3136-3149. DOI：10.1039/d0bm01928c.
[10]	Aoki M, Matsumoto NM, Dohi T，et al. Direct delivery of apatite nanoparticle-encapsulated siRNA targeting TIMP-1 for intractable abnormal scars[J]. Mol Ther Nucleic Acids，2020，22：50-61. DOI：10.1016/j.omtn.2020.08.005.
[11]	Yue J, López JM. Understanding MAPK signaling pathways in apoptosis[J]. Int J Mol Sci，2020，21（7）：2346. DOI：10.3390/ijms21072346.
[12]	Zhang YE. Non-smad signaling pathways of the TGF-β family[J]. Cold Spring Harb Perspect Biol，2017，9（2）：a022129. DOI：10.1101/cshperspect.a022129.
[13]	Gabbiani G. The cellular derivation and the life span of the myofibroblast[J]. Pathol Res Pract，1996，192（7）：708-711.DOI：10.1016/S0344-0338(96)80092-6.
[14]	Arif S, Attiogbe E, Moulin VJ. Granulation tissue myofibroblasts during normal and pathological skin healing：The interaction between their secretome and the microenvironment[J]. Wound Repair Regen，2021，29（4）：563-572. DOI：10.1111/wrr.12919.
[15]	Serini G, Gabbianl G. Mechanisms of myofibroblast activity andphenotypic modulation[J]. Exp CelI Res，1999，250（2）：273-283. DOI：10.1006/excr.1999.4543.
[16]	Sabir N, Hussain T, Mangi MH，et al. Matrix metalloproteinases：Expression，regulation and role in the immunopathology of tuberculosis[J]. Cell Prolif，2019，52（4）：e12649. DOI：10.1111/cpr.12649.
[17]	Tian M, Chang X, Zhang Q，et al. TGF-β1 mediated MAPK signaling pathway promotes collagen formation induced by Nano NiO in A549 cells[J]. Environ Toxicol，2019，34（6）：719-727. DOI：10.1002/tox.22738.
[18]	Parekh A, Hebda PA. The contractile phenotype of dermal fetal fibroblasts in scarless wound healing[J]. Curr Pathobiol Rep，2017，5（3）：271-277. DOI：10.1007/s40139-017-0149-3.
[19]	Sato M, Shegogue D, Gore EA，et al. Role of p38 MAPK in transforming growth factor beta stimulation of collagen production by scleroderma and healthy dermal fibroblasts[J]. J Invest Dermatol，2002，118（4）：704-711. DOI：10.1046/j.1523-1747.2002.01719.x.
[20]	Murai M, Yamamura K, Hashimoto-Hachiya A，et al. Tryptophan photo-product FICZ upregulates AHR/MEK/ERK-mediated MMP1 expression：Implications in anti-fibrotic phototherapy[J]. J Dermatol Sci，2018，91（1）：97-103. DOI：10.1016/j.jdermsci.2018.04.010.
[21]	Walimbe T, Calve S, Panitch A，et al. Incorporation of types Ⅰ and Ⅲ collagen in tunable hyaluronan hydrogels for vocal fold tissue engineering[J]. Acta Biomater，2019，87：97-107. DOI：10.1016/j.actbio.2019.01.058.
[22]	Rajshankar D, Wang B, Worndl E，et al. Focal adhesion kinase regulates tractional collagen remodeling，matrix metalloproteinase expression，and collagen structure，which in turn affects matrix-induced signaling[J]. J Cell Physiol，2020，235（3）：3096-3111. DOI：10.1002/jcp.29215.
[23]	Ashcroft GS, Mills SJ, Flanders KC，et al. Role of Smad3 in the hormonal modulation of in vivo wound healing responses[J]. Wound Repair Regen，2003，11（6）：468-473. DOI：10.1046/j.1524-475x.2003.11614.x.
[24]	刘峰. p38MAPK基因沉默对唇裂术后瘢痕增生影响的实验研究[D].青岛：青岛大学，2017.

基因	引物序列	产物长度（bp）
GAPDH	正向：5′-AAGGTCGGAGTGAACGGATTTG-3′	148
	反向：5′-CGTGGGTGGAATCATACTGGAAC-3′
ColⅠ	正向：5′-CAAGGGAGAGAGTGGTAACAAG-3′	92
	反向：5′-TTCCCACTTTCACCCTTGAC-3′
ColⅢ	正向：5′-CACTCGGACTTGCAGGAATTA-3′	109
	反向：5′-TTCCCACTTTCACCCTTGAC-3′
MMP1	正向：5′-GATGACAGGTGGACCAAAGAT-3′	102
	反向：5′-CCCAATATCAGTAGAATGGGAGAG-3′
TIMP1	正向：5′-CTACCTTGTACCAGCGTTATGA-3′	140
	反向：5′-GTTCTGGGATTTGTGGGAGTA-3′

基因	引物序列	产物长度（bp）
GAPDH	正向：5′-AAGGTCGGAGTGAACGGATTTG-3′	148
	反向：5′-CGTGGGTGGAATCATACTGGAAC-3′
ColⅠ	正向：5′-CAAGGGAGAGAGTGGTAACAAG-3′	92
	反向：5′-TTCCCACTTTCACCCTTGAC-3′
ColⅢ	正向：5′-CACTCGGACTTGCAGGAATTA-3′	109
	反向：5′-TTCCCACTTTCACCCTTGAC-3′
MMP1	正向：5′-GATGACAGGTGGACCAAAGAT-3′	102
	反向：5′-CCCAATATCAGTAGAATGGGAGAG-3′
TIMP1	正向：5′-CTACCTTGTACCAGCGTTATGA-3′	140
	反向：5′-GTTCTGGGATTTGTGGGAGTA-3′

基因	引物序列	产物长度（bp）
GAPDH	正向：5′-AAGGTCGGAGTGAACGGATTTG-3′	148
	反向：5′-CGTGGGTGGAATCATACTGGAAC-3′
p38MAPK	正向：5′-CAAATCCTCCGAGGCCTAAA-3′	103
	反向：5′-GGATCTTCAGCTCACAGTCTTC-3′
Smad2	正向：5′-CCAGTTCTGCCTCCAGTATTAG-3′	91
	反向：5′-TTTCTGGAATGGAGTGGGTATAG-3′
Smad3	正向：5′-TACCTGAGCGAAGATGGAGAG-3′	119
	反向：5′-GGCTGCAGGTCCAAGTTATT-3′

基因	引物序列	产物长度（bp）
GAPDH	正向：5′-AAGGTCGGAGTGAACGGATTTG-3′	148
	反向：5′-CGTGGGTGGAATCATACTGGAAC-3′
p38MAPK	正向：5′-CAAATCCTCCGAGGCCTAAA-3′	103
	反向：5′-GGATCTTCAGCTCACAGTCTTC-3′
Smad2	正向：5′-CCAGTTCTGCCTCCAGTATTAG-3′	91
	反向：5′-TTTCTGGAATGGAGTGGGTATAG-3′
Smad3	正向：5′-TACCTGAGCGAAGATGGAGAG-3′	119
	反向：5′-GGCTGCAGGTCCAAGTTATT-3′

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