创面siRNA敲降HO-1改善小鼠放创复合伤创面愈合的实验研究

吕晓凡; 汪国建; 赵娜; 龙爽; 王双; 冉新泽; 王军平; 王涛

doi:10.16016/j.2097-0927.202401026

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创面siRNA敲降HO-1改善小鼠放创复合伤创面愈合的实验研究

基础医学 | 更新时间：2026-01-09

- 创面siRNA敲降HO-1改善小鼠放创复合伤创面愈合的实验研究
- Topical knockdown of HO-1 through siRNA improves skin wound healing in mice with radiation-wound combined injury
- 陆军军医大学学报 2024年46卷第11期页码：1194-1205
- 作者机构：
  
  1.400038 重庆，陆军军医大学(第三军医大学)：军事预防医学系防原医学教研室，全军复合伤研究所，创伤与化学中毒全国重点实验室
  2.400038 重庆，陆军军医大学(第三军医大学)：军事预防医学系防原医学教研室，全军复合伤研究所，药学与检验医学系药剂学教研室
  3.400715 重庆，西南大学前沿交叉学科研究院生物学研究中心
  4.400038 重庆，陆军军医大学(第三军医大学)：军事预防医学系防原医学教研室，全军复合伤研究所，军事预防医学系
- 作者简介：
  
  [王军平, E-mail: wangjunping@tmmu.edu.cnWANG Junping, E-mail: wangjunping@tmmu.edu.cn
  [王涛, E-mail: wangtao@tmmu.edu.cnWANG Tao, E-mail: wangtao@tmmu.edu.cn
- 基金信息：
  
  国家自然科学基金面上项目(82172219)
- DOI：10.16016/j.2097-0927.202401026
  中图分类号： R394.6;R64;R818.74
- 收稿：2024-01-07，
  
  修回：2024-03-06，
  
  纸质出版：2024-06-15
- 稿件说明：
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吕晓凡, 汪国建, 赵娜, 等. 创面siRNA敲降HO-1改善小鼠放创复合伤创面愈合的实验研究[J]. 陆军军医大学学报, 2024,46(11):1194-1205.

Xiaofan LYU, Guojian WANG, Na ZHAO, et al. Topical knockdown of HO-1 through siRNA improves skin wound healing in mice with radiation-wound combined injury[J]. Journal of Army Medical University, 2024, 46(11): 1194-1205.
吕晓凡, 汪国建, 赵娜, 等. 创面siRNA敲降HO-1改善小鼠放创复合伤创面愈合的实验研究[J]. 陆军军医大学学报, 2024,46(11):1194-1205. DOI： 10.16016/j.2097-0927.202401026.

Xiaofan LYU, Guojian WANG, Na ZHAO, et al. Topical knockdown of HO-1 through siRNA improves skin wound healing in mice with radiation-wound combined injury[J]. Journal of Army Medical University, 2024, 46(11): 1194-1205. DOI： 10.16016/j.2097-0927.202401026.

摘要

目的

检测血红素加氧酶1(heme oxygenase-1，HO-1)在放创复合伤(radiation-wound combined injury，R-W-CI)创面修复中的表达情况，评价通过siRNA敲降HO-1对放创复合伤创面愈合的改善作用。

方法

将36只8周龄雄性C57BL/6J小鼠按随机数字表法分为单纯皮肤创伤组(W组，

=18)和合并全身辐射(6 Gy)损伤的皮肤创伤组(R-W-CI组，

=18)，建立单纯皮肤创伤与放创复合伤的小鼠模型。在创面愈合过程中，拍照记录创面愈合情况并通过Image J量化分析残留面积；取材创面组织进行HE染色及病理组织学观察；动态检测外周血象评估造血系统损伤情况。通过创面组织定量PCR及Western blot检测创面HO-1表达水平及变化情况。将26只8周龄雄性C57BL/6J小鼠随机分为siRNA敲降HO-1组(si-HO-1组，

=13)和siRNA阴性对照组(si-NC组，

=13)。放创复合伤致伤后，si-HO-1组在每个创面涂抹负载si-HO-1(5 μmol/L)的F127凝胶60 μL，si-NC组创面涂抹等量负载阴性对照si-NC的F127凝胶。通过创面组织Western blot检测HO-1的敲降情况，观察创面面积变化，对伤后第3天样本进行定量PCR检测

-1

、

-6、

TNF-α

等炎症因子的表达变化，组织切片进行Ki67免疫组织化学和HE染色；对第9天创面组织进行HE染色病理评估；综合评价敲降HO-1对放创复合伤创面愈合的改善作用。

结果

与W组相比，创面残留面积的半定量分析表明R-W-CI组愈合在伤后第7、10天显著延迟(

0.01)；第7天的HE病理显示R-W-CI组再上皮化延缓，肉芽组织生长不良；同时R-W-CI组外周血白细胞及其分类计数显示在损伤后早期即显著下降(

0.05)。检测发现，R-W-CI组创面HO-1蛋白在伤后第3、7天表达略高于W组，但无显著差异(

＞0.05)，而在第10天显著升高(

0.05)，同时伴有全长与截短形式的分布改变；定量PCR显示R-W-CI组在伤后第7天、10天的创面组织

-1的表达显著高于W组(

0.05)。放创复合伤创面siRNA干预实验显示：与si-NC组比较，si-HO-1组能有效敲降创面HO-1蛋白含量(

0.05)，促进伤口收缩(

0.05)，减小创面宽度(

＜0.01)，上调致伤第3天创面

-6、

TNF-α

等炎症因子表达，促进创缘组织细胞增殖，改善肉芽组织生长情况。

结论

放创复合伤创面修复过程中存在HO-1蛋白的持续高表达，创面siRNA敲降HO-1可以改善R-W-CI组小鼠创面乏炎状态，促进创面愈合。

Abstract

Objective

To detect the expression profile of heme oxygenase-1 (HO-1) during the process of wound repair in radiation-wound combined injury (R-W-CI)

and evaluate its wound healing improving effects of R-W-CI by HO-1 knockdown with siRNA.

Methods

A total of 36 male C57BL/6J mice (8 weeks old) were randomly and equally divided into a simple skin wound group (W group) and a skin wound group combined with whole-body radiation (6 Gy) injury (R-W-CI group). During the wound healing process

the wounds were photographed and recorded

and the residual areas were quantified by Image J. Wound tissues were sampled and stained with HE staining for pathological and histological observation

and the damage to the hematopoietic system was assessed by dynamic examination of the peripheral blood. The expression and

changes of HO-1 in wound tissues were detected by q-PCR and Western blotting. Then

26 male C57BL/6J mice (8 weeks old) were randomly and equally divided into siRNA knockdown HO-1 group (si-HO-1 group) and siRNA negative control group (si-NC group). After radiation combined injury was inflicted

60 μL of F127 gel loaded with si-HO-1 (5 μm/L) was applied to each wound in the si-HO-1 group

and an equal amount of F127 gel loaded with negative control si-NC was applied to the wound in the si-NC group. The knockdown of HO-1 in wound tissues was detected by Western blotting

and the changes in wound area were observed. In the wound tissues harvested in 3 d after wounding

the expression of cytokines

-1

-6 and

TNF-α

was examined by q-PCR and the proliferation of granulation tissues was evaluated by Ki67 immunohistochemical staining. HE staining was performed on wound tissues on day 3 and day 9 post-injury to assess the improvement effect of knockdown of HO-1 on wound healing of radiation combined injuries.

Results

Compared with the W group

semi-quantitative analysis of the residual wound area showed that healing was significantly delayed in the R-W-CI group on days 7 and 10 post-injury (

0.01). HE staining on day 7 showed that in the R-W-CI group

the re-epithelialization was delayed

and the growth of granulation tissues was poor; and at the same time

peripheral blood leukocytes and their classified counts showed a significant decrease in the early period after injury (

0.05). Further tests indicated that the expression of HO-1 protein was slightly higher in the wound of the R-W-CI group than that of the W group in 3 and 7 d after injury

though no significant difference (

0.05)

whereas statistical difference was seen in 10 d (

0.05)

accompanied by the distribution of the full-length and truncated forms of HO-1 protein. Quantit

ative PCR obtained similar results in the mRNA expression of HO-1 in wounds in both 7 and 10 d after injury (

0.05). siRNA intervention could effectively knock down the HO-1 protein level of the wounds (

0.05)

promote wound contraction (

0.05)

reduce the width of the wound (

0.01)

up-regulate the inflammatory cytokines IL-6 and TNF-α in 3 d

enhance the proliferation of repair cells in wound margin

and improve the growth of the granulation tissue in the R-W-CI model when compared with the conditions after si-NC intervention.

Conclusion

There exists a sustained high expression level of HO-1 during wound repair

and wound knockdown of HO-1 by siRNA can improve the lack of inflammation status and promote wound healing in R-W-CI mice.

关键词

Keywords

references

程天民, 邹仲敏. 放射复合伤的研究进展[J]. 中华放射医学与防护杂志, 1998, 18(5): 299-304. DOI: 10.3760/cma.j.issn.0254-5098.1998.05.105.

CHENG T M, ZOU Z M. Research progress of radiation combined injury[J]. Chin J Radiol Med Prot, 1998, 18(5): 299-304. DOI: 10.3760/cma.j.issn.0254-5098.1998.05.105.

ZOU Z, SUN H, SU Y, et al . Progress in research on radiation combined injury in China[J ] . Radiat Res, 2008, 169(6): 722-729. DOI: 10.1667/RR1284.1.

RAN X Z, SHI C M, ZHENG H E, et al . Experimental research on the management of combined radiation-burn injury in China[J ] . Radiat Res, 2011, 175(3): 382-389. DOI: 10.1667/RR2198.1.

史春梦, 程天民. 放射损伤对创面愈合的影响[J]. 国外医学(放射医学核医学分册), 2005, 29(1): 30-32. DOI: 10.3760/cma.j.issn.1673-4114.2005.01.008.

SHI C M, CHENG T M. Effects of radiation injury on wound healing[J]. Foreign Med Sci Sect Radiat Med Nucl Med, 2005, 29(1): 30-32. DOI: 10.3760/cma.j.issn.1673-4114.2005.01.008.

王涛, 冉新泽, 王军平. 放射复合伤的研究进展与展望[J]. 中华损伤与修复杂志(电子版), 2023, 18(4): 353-357. DOI: 10.3877/cma.j.issn.1673-9450.2023.04.015.

WANG T, RAN X Z, WANG J P. Advances and prospect in research of radiation combined injuries[J]. Chin J Inj Repair Wound Heal Electron Ed, 2023, 18(4): 353-357. DOI: 10.3877/cma.j.issn.1673-9450.2023.04.015.

谷庆阳, 王德文, 崔彩彬, 等. 不同剂量照射对大鼠伤口愈合影响规律的分子病理学研究[J]. 中华放射医学与防护杂志, 1998, 18(3): 166-169. DOI: 10.3760/cma.j.issn.0254-5098.1998.03.010.

GU Q Y, WANG D W, CUI C B, et al . Molecular pathological study on the effect of different doses of radiation on wound healing in rats[J ] . Chin J Radiol Med Prot, 1998, 18(3): 166-169. DOI: 10.3760/cma.j.issn.0254-5098.1998.03.010.

刘霞, 刘建忠, 周元国, 等. 局部辐射对大鼠伤口愈合过程中细胞增殖和凋亡的影响规律[J]. 中华放射医学与防护杂志, 2002, 22(3): 187-188. DOI: 10.3760/cma.j.issn.0254-5098.2002.03.016.

LIU X, LIU J Z, ZHOU Y G, et al . Effect of local radiation on cell proliferation and apoptosis during wound healing in rats[J ] . Chin J Radiol Med Prot, 2002, 18(3): 187-188. DOI: 10.3760/cma.j.issn.0254-5098.2002.03.016.

WAGENER F A D T G, VAN BEURDEN H E, VON DEN HOFF J W, et al . The heme-heme oxygenase system: a molecular switch in wound healing[J ] . Blood, 2003, 102(2): 521-528. DOI: 10.1182/blood-2002-07-2248.

NAITO Y, TAKAGI T, HIGASHIMURA Y. Heme oxygenase-1 and anti-inflammatory M2 macrophages[J]. Arch Biochem Biophys, 2014, 564: 83-88. DOI: 10.1016/j.abb.2014.09.005.

LEAL E C, CARVALHO E. Heme oxygenase-1 as therapeutic target for diabetic foot ulcers[J]. Int J Mol Sci, 2022, 23(19): 12043. DOI: 10.3390/ijms231912043.

COSTA D L, AMARAL E P, ANDRADE B B, et al . Modulation of inflammation and immune responses by heme oxygenase-1: implications for infection with intracellular pathogens[J ] . Antioxidants, 2020, 9(12): 1205. DOI: 10.3390/antiox9121205.

汪国建, 谭雨龙, 龙爽, 等. 高温高湿环境暴露对重度放创复合伤小鼠损伤恢复的影响[J]. 中华损伤与修复杂志(电子版), 2023, 18(4): 285-292. DOI: 10.3877/cma.j.issn.1673-9450.2023.04.003.

WANG G J, TAN Y L, LONG S, et al . Effects of hot and humid environments exposure on recovery of mice subjected to severe radiation injury combined with skin-wound trauma[J ] . Chin J Inj Repair Wound Heal Electron Ed, 2023, 18(4): 285-292. DOI: 10.3877/cma.j.issn.1673-9450.2023.04.003.

LIN X L, LV J J, LV J, et al . Heme oxygenase-1 directly binds STAT3 to control the generation of pathogenic Th17 cells during neutrophilic airway inflammation[J ] . Allergy, 2017, 72(12): 1972-1987. DOI: 10.1111/all.13216.

ZHAO N, WANG G J, LONG S, et al . MicroRNA-34a deficiency leads to impaired wound closure by augmented inflammation in mice[J ] . Ann Transl Med, 2020, 8(7): 447. DOI: 10.21037/atm.2020.03.161.

CHEN L, MIRZA R, KWON Y, et al . The murine excisional wound model: contraction revisited[J ] . Wound Repair Regen, 2015, 23(6): 874-877. DOI: 10.1111/wrr.12338.

赵娜, 刘登群, 汪国建, 等. 自噬在小鼠皮肤创面修复中作用的初步研究[J]. 第三军医大学学报, 2019, 41(1): 25-32. DOI: 10.16016/j.1000-5404.201808194.

ZHAO N, LIU D Q, WANG G J, et al . Role of autophagy during cutaneous wound healing in mice[J ] . J Third Mil Med Univ, 2019, 41(1): 25-32. DOI: 10.16016/j.1000-5404.201808194.

陈晓红, 程天民, 冉新泽. 电离辐射对大鼠创面愈合早期阶段的影响[J]. 第三军医大学学报, 2001, 23(1): 24-26. DOI: 10.3321/j.issn:1000-5404.2001.01.008.

CHEN X H, CHENG T M, RAN X Z. Effects of irradiation on early stage wound healing in traumatic rats[J]. J Third Mil Med Univ, 2001, 23(1): 24-26. DOI: 10.3321/j.issn:1000-5404.2001.01.008.

HOLLEY T R, VAN EPPS D E, HARVEY R L, et al . Effect of high doses of radiation on human neutrophil chemotaxis, phagocytosis and morphology[J ] . Am J Pathol, 1974, 75(1): 61-72.

陈晓红, 程天民, 冉新泽. 电离辐射对大鼠伤口中性粒细胞肌动蛋白的影响[J]. 中华放射医学与防护杂志, 2000, 20(6): 388-390. DOI: 10.3760/cma.j.issn.0254-5098.2000.06.004.

CHEN X H, CHENG T M, RAN X Z. Effects of irradiation on actin in wound neutrophils of rats[J]. Chin J Radiol Med Prot, 2000, 20(6): 388-390. DOI: 10.3760/cma.j.issn.0254-5098.2000.06.004.

赵娜. 丝氨酸蛋白酶抑制因子Spink7促进炎症消退的作用与机制探索: 基于小鼠创面愈合与结肠炎模型的实验研究[D ] . 重庆: 陆军军医大学, 2021. DOI: 10.27001/d.cnki.gtjyu.2021.000054 http://dx.doi.org/10.27001/d.cnki.gtjyu.2021.000054 .

ZHAO N. Effect and mechanism of serine protease inhibitor Spink7 in promoting inflammation fading—an experimental study based on mouse wound healing and colitis model[D ] . Chongqing: Army Medical University, 2021. DOI: 10.27001/d.cnki.gtjyu.2021.000054 http://dx.doi.org/10.27001/d.cnki.gtjyu.2021.000054 .

GROCHOT-PRZECZEK A, LACH R, MIS J, et al . Heme oxygenase-1 accelerates cutaneous wound healing in mice[J ] . PLoS One, 2009, 4(6): e5803. DOI: 10.1371/journal.pone.0005803.

LIN Q, WEIS S, YANG G, et al . Heme oxygenase-1 protein localizes to the nucleus and activates transcription factors important in oxidative stress[J ] . J Biol Chem, 2007, 282(28): 20621-20633. DOI: 10.1074/jbc.M607954200.

YANG Q, WANG W Q. The nuclear translocation of heme oxygenase-1 in human diseases[J]. Front Cell Dev Biol, 2022, 10: 890186. DOI: 10.3389/fcell.2022.890186.

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