Tmprss6基因对小鼠放射性肠损伤的影响

doi:10.3969/j.issn.1674-8115.2021.09.007

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (9): 1175-1182.doi: 10.3969/j.issn.1674-8115.2021.09.007

• 论著 · 基础研究 • 上一篇

Tmprss6基因对小鼠放射性肠损伤的影响

毛久昂¹(), 翁震²(), 钮晓音³, 何杨²^,⁴, 王振欣¹()

^1.苏州大学附属第一医院肿瘤科，苏州 215006
^2.苏州大学血液和血管疾病诊疗药物技术教育部工程研究中心，苏州 215123
^3.上海交通大学医学院，上海市免疫学研究所，上海 200025
^4.江苏省血液研究所，苏州大学附属第一医院，苏州 215006

收稿日期:2021-02-24 出版日期:2021-08-24 发布日期:2021-08-24
通讯作者: 翁震,王振欣 E-mail:maojiuang@163.com;zhweng@suda.edu.cn;zhenxw316@163.com
作者简介:毛久昂（1994—），女，硕士生；电子信箱：maojiuang@163.com。
基金资助:
国家自然科学基金(81773356);上海市卫生和计划生育委员会科研课题(201640137)

Role of Tmprss6 gene in radiation-induced intestinal injury of mice

Jiu-ang MAO¹(), Zhen WENG²(), Xiao-yin NIU³, Yang HE²^,⁴, Zhen-xin WANG¹()

^1.Department of Oncology, the First Affiliated Hospital of Soochow University, Suzhou 215006, China
^2.MOE Engineering Center of Hematological Disease, Soochow University, Suzhou 215123, China
^3.Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^4.Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, Suzhou 215006, China

Received:2021-02-24 Online:2021-08-24 Published:2021-08-24
Contact: Zhen WENG,Zhen-xin WANG E-mail:maojiuang@163.com;zhweng@suda.edu.cn;zhenxw316@163.com
Supported by:
National Natural Science Foundation of China(81773356);Research Program of Shanghai Municipal Commission of Health and Family Planning(201640137)

摘要/Abstract

摘要：

目的·观察Tmprss6基因对小鼠放射性肠损伤的影响。方法·将7~8周龄雄性野生型（WT）小鼠和Tmprss6基因敲除（Tmprss6^-/-）小鼠各自随机分成未辐照组以及辐照后6 h、24 h、3.5 d和7 d组。其中，辐照组采用11.5 Gy X射线全腹部照射，建立放射肠损伤模型。通过TUNEL染色观察辐照后6、24 h不同肠段隐窝细胞凋亡情况；通过苏木精-伊红染色观察辐照后3.5 d不同肠段病理损伤情况；测量不同肠段辐照前和辐照后3.5、7 d的铁含量，以及抗氧化指标包括超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽（glutathione，GSH）、总抗氧化能力（total antioxidant capacity，T-AOC）和丙二醛（malondialdehyde，MDA），并对铁含量与各项抗氧化指标进行相关性分析。结果·在辐照后3.5 d，Tmprss6^-/-和WT小鼠十二指肠绒毛长度均缩短，平均每毫米横截面隐窝计数均减少；Tmprss6^-/-小鼠的十二指肠绒毛长度和隐窝计数低于WT小鼠（均P<0.05）。辐照后6、24 h，Tmprss6^-/-小鼠十二指肠段每隐窝TUNEL染色阳性细胞数相较于WT小鼠均显著增多（均P<0.05）。而回肠段则相反，辐照后Tmprss6^-/-小鼠绒毛长度和隐窝计数显著增加，TUNEL染色阳性细胞数显著减少（均P<0.05）。WT和Tmprss6^-/-小鼠十二指肠部位铁含量在辐照后7 d，相较于辐照前均显著升高（均P<0.05）；并且Tmprss6^-/-小鼠辐照前后的十二指肠铁含量均显著高于同时间点的WT小鼠（均P<0.05）。WT和Tmprss6^-/-小鼠辐照后回肠段铁含量较辐照前均升高，其中WT小鼠辐照后3.5 d与辐照前的差异有统计学意义，Tmprss6^-/-小鼠辐照后7 d与辐照前的差异有统计学意义（均P<0.05）。辐照后，Tmprss6^-/-小鼠十二指肠部位SOD、GSH、T-AOC含量均较WT小鼠低，而MDA含量较高；但只有在辐照后3.5 d的差异具有统计学意义（均P<0.05）。与十二指肠段相反，辐照后Tmprss6^-/-小鼠回肠部位SOD、GSH、T-AOC含量较WT小鼠高，而MDA含量则较低；但T-AOC、GSH、MDA含量在辐照后3.5 d的差异才具有统计学意义（均P<0.05），SOD含量在辐照后7 d的差异具有统计学意义（P=0.031）。相关性分析结果显示，未辐照组的MDA、GSH、SOD和T-AOC水平与小肠铁含量均无相关性；辐照后WT小鼠十二指肠SOD和MDA水平与其铁含量相关（r=0.639，r=-0.710，均P<0.05），回肠T-AOC、MDA和GSH与其铁含量相关（r=-0.817，r=-0.658，r=-0.726，均P<0.05）；辐照后Tmprss6^-/-小鼠十二指肠段MDA、SOD和T-AOC水平与其铁含量相关（r=-0.606，r=0.771，r=-0.712，均P<0.05），回肠段各抗氧化指标与铁含量均无相关性。结论·小肠铁含量与辐照诱导的氧化应激具有相关性；Tmprss6基因敲除小鼠表现为十二指肠铁累积和辐照诱导的氧化应激加剧；Tmprss6基因缺失在十二指肠放射损伤中具有增敏作用。

关键词: Tmprss6, 放射性肠损伤, 铁代谢, 氧化应激

Abstract:

Objective·To explore the effect of Tmprss6 gene on radiation-induced intestinal injury in mice.

Methods·7?8 weeks old wild-type (WT) male mice and Tmprss6 knockout (Tmprss6^-/-) mice were randomly divided into non-irradiated (IR) group and groups of 6 h, 24 h, 3.5 d and 7 d post-IR. The IR group received total abdominal X-ray irradiation (TAI) at a dose of 11.5 Gy to establish radiation intestine injury models. H-E staining was performed in the 3.5 d post-IR group, and TUNEL immunofluorescence was performed in the 6 h and 24 h post-IR groups to observe the pathological damage and apoptosis of different intestinal segments, respectively.The levels of iron of different intestinal segments were measured 0, 3.5 and 7 d after TAI, as well as superoxide dismutase (SOD), glutathione (GSH), total antioxidant capacity (T-AOC) and malondialdehyde (MDA). The correlation analysis between iron and each antioxidant index was performed.

Results·The length of duodenal villi and the number of crypts decreased significantly in both Tmprss6^-/- mice and WT mice 3.5 d after TAI. Meanwhile, the duodenal villi length and crypt count of Tmprss6^-/- mice were lower than those of WT mice (both P<0.05). The number of TUNEL-positive apoptotic cells per crypt in the duodenal segment of Tmprss6^-/- mice was significantly increased compared with that of WT mice 6 and 24 h after TAI (both P<0.05). On the contrary, the length of ileum villi and the count of crypts increased significantly, while the number of TUNEL-positive apoptotic cells decreased significantly in Tmprss6^-/- mice compared with that of WT mice (all P<0.05). The iron content in the duodenum of WT and Tmprss6^-/-mice 7 d after TAI was significantly increased compared with that of non-irradiated mice (both P<0.05). And the iron content of the duodenum in the Tmprss6^-/- mice before and after TAI was significantly higher than that of WT mice at the same time points (all P<0.05). The iron content of the ileum in WT and Tmprss6^-/- mice also increased after TAI. And the increase was statistically significant at 3.5 d post-IR in the WT mice, so was at 7 d post-IR in the Tmprss6^-/- mice (both P<0.05). In addition, the SOD, GSH and T-AOC levels in the duodenum of Tmprss6^-/- mice were lower than those in the WT mice after TAI, while the content of MDA was higher. Among them, the difference of SOD, GSH, MDA and T-AOC levels were statistically significant 3.5 d after TAI (all P<0.05). Contrary to the duodenum, the contents of SOD, GSH, and T-AOC in the ileum of Tmprss6^-/- mice were higher than those in the WT mice, while the content of MDA was lower. Among them, the content of T-AOC, GSH and MDA had a statistically significant difference 3.5 d after TAI (all P<0.05), and the content of SOD had a statistically significant difference 7 d after TAI (P=0.031). The results of correlation analysis showed that there was no significant correlation between the MDA, GSH, SOD and T-AOC levels and the iron content of the small intestine in the non-IR group. The levels of SOD and MDA in the duodenum (r=0.639, r=-0.710, both P<0.05), and T-AOC, MDA and GSH in the ileum (r=-0.817, r=-0.658, r=-0.726, all P<0.05) of WT mice were significantly correlated with their respective iron content after TAI. The levels of MDA, SOD and T-AOC in the duodenum of Tmprss6^-/-mice were significantly correlated with the iron content after TAI (r=-0.606, r=0.771, r=-0.712, all P<0.05), and all the antioxidant indexes of the ileum were not significantly correlated with the iron content.

Conclusion·The iron level in the small intestine is significantly correlated with the oxidative stress response after irradiation. Tmprss6 gene-deficient mice show increased duodenal iron accumulation and irradiation-induced oxidative stress, and Tmprss6 gene deletion exacerbates duodenal radiation damage.

Key words: Tmprss6, radiation-induced intestinal injury, iron metabolism, oxidative stress

中图分类号:

R818

毛久昂, 翁震, 钮晓音, 何杨, 王振欣. Tmprss6基因对小鼠放射性肠损伤的影响[J]. 上海交通大学学报(医学版), 2021, 41(9): 1175-1182.

Jiu-ang MAO, Zhen WENG, Xiao-yin NIU, Yang HE, Zhen-xin WANG. Role of Tmprss6 gene in radiation-induced intestinal injury of mice[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(9): 1175-1182.

图/表 9

图1 Tmprss6-/-小鼠与WT小鼠行TAI后十二指肠损伤和细胞凋亡情况的比较Note： A. Duodenum sections of WT and Tmprss6-/- mice after TAI (H-E staining, scale bar=50 μm). B/C. Quantitative results of villus length (B) and crypts count (C). D. Immunofluorescence images for TUNEL in the duodenum tissues at the indicated time points after TAI (scale bar=75 μm). E. The number of TUNEL-positive cells per crypt at the indicated time points after TAI. ①P=0.000, ②P=0.001, compared with WT mice.

Fig 1 Comparison of duodenal injury and apoptosis between Tmprss6-/- mice and WT mice after TAI

图2 Tmprss6-/-小鼠与WT小鼠行TAI后回肠损伤和细胞凋亡情况的比较Note： A.Ileum sections of WT and Tmprss6-/- mice after TAI (H-E staining, scale bar=50 μm). B/C. Quantitative results of villus length (B) and crypts count (C). D. Immunofluorescence images for TUNEL in the ileum tissues at the indicated time points after TAI (scale bar=75 μm). E. The number of TUNEL-positive cells per crypt at the indicated time points after TAI. ①P=0.000, compared with WT mice.

Fig 2 Comparison of ileum injury and apoptosis between Tmprss6-/- mice and WT mice after TAI

图3 WT和Tmprss6-/-小鼠行TAI后不同肠段铁含量的变化Note： A. Duodenum section. B. Ileum section.

Fig 3 Iron concentration of different intestine segments in WT and Tmprss6-/- mice after TAI

图4 WT和Tmprss6-/-小鼠行TAI后十二指肠段抗氧化指标的变化Note： ①P=0.041, ②P=0.046, ③P=0.044, ④P=0.007, compared with WT mice.

Fig 4 Antioxidant index changes of duodenums in WT and Tmprss6-/- mice after TAI

图5 WT和Tmprss6-/-小鼠行TAI后回肠段抗氧化指标的变化Note： ①P=0.031, ②P=0.030, ③P=0.006, ④P=0.009, compared with WT mice.

Fig 5 Antioxidant index changes of ileums in WT and Tmprss6-/- mice after TAI

表1 辐照前后小肠T-AOC、MDA、SOD和GSH水平与铁含量的相关性分析

Tab 1 Correlation analysis of the levels of T-AOC, MDA, SOD and GSH and iron content in small intestines before and after irradiation

Item	Before irradiation		7 d after irradiation
Item	r value	P value	r value	P value
T-AOC	0.135	0.675	0.233	0.243
MDA	0.019	0.948	0.511	0.006
SOD	-0.377	0.205	-0.461	0.012
GSH	0.114	0.687	0.200	0.299

图6 小鼠小肠组织铁含量与抗氧化指标的相关性分析Note： A?D. Duodenum section. E?H. Ileum section.

Fig 6 Correlation analysis of iron content and antioxidant indexes of mice intestines

图7 WT小鼠小肠铁含量与抗氧化指标相关性分析Note： A?D. Duodenum section. E?H. Ileum section.

Fig 7 Correlation analysis of iron content and antioxidant indexes of intestines in WT mice

图8 Tmprss6-/-小鼠小肠铁含量与抗氧化指标相关性分析Note： A?D. Duodenum section. E?H. Ileum section.

Fig 8 Correlation analysis of iron content and antioxidant indexes of intestines in Tmprss6-/- mice

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Tmprss6基因对小鼠放射性肠损伤的影响

Role of Tmprss6 gene in radiation-induced intestinal injury of mice

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