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J Zhejiang Univ (Med Sci)  2018, Vol. 47 Issue (5): 499-506    DOI: 10.3785/j.issn.1008-9292.2018.10.09
    
Anti-inflammatory effect of interleukin-35 in mice with colitis and its mechanism
LU Zhanjun1,2,3(),HU Yangyang2,3,LI Sisi2,3,ZANG Lijuan4,JIANG Weiliang2,3,WU Jianjiong2,WU Xiening2,ZENG Yue2,3,WANG Xingpeng1,2,3,*()
1. Department of Gastroenterology, Shanghai General Hospital of Nanjing Medical University, Shanghai 200080, China
2. Department of Gastroenterology, Shanghai General Hospital, Shanghai 200080, China
3. Shanghai Key Laboratory of Pancreatic Disease, Shanghai 200080, China
4. Pathology Center, Shanghai General Hospital, Shanghai 200080, China
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Abstract  

Objective: To investigate the anti-inflammatory effect and mechanisms of interleukin-35 (IL-35) in inflammatory bowel disease. Methods: BALB/c mice were divided into three groups with 10 mice in each group:control group, model group (oral administration of 4% glucan sodium sulfate for 7 d) and IL-35-treated group (oral administration of 4% glucan sodium sulfate for 7 d, intraperitoneal injection of 2 μg IL-35 at d2-5). Disease activity index (DAI) was scored every day. After 7 d, the mice were sacrificed, and the serum and intestinal tissue samples were collected. The gross morphology of the colon was observed; HE staining was used to observe the pathological changes of colon tissue; flow cytometry was employed to detect the change of macrophage polarization ratio in colon tissue; the mRNA expression levels of cytokines IL-6, TNF-α, IFN-γ, IL-10 and SHIP1 in colon tissue were determined by real-time quantitative RT-PCR; the expression and distribution of SHIP1 in colon tissue was measured by immunohistochemistry; Western blotting was adopted to detect the expression level of SHIP1 protein in colonic intestinal tissues of each group. Results: The DAI scores of the mice in the model group were higher than those in the control group, while the DAI scores in the IL-35-treated group were lower than those in the model group (all P < 0.01). Compared with the control group, the colon length was significantly shortened in the model group (P < 0.05), while the colon length of the IL-35-treated group had an increasing trend compared with the model group, but the difference was not statistically significant (P > 0.05). Compared with the model group, microscopic inflammatory infiltration score was decreased and microscopic crypt destruction and score was significantly lower in IL-35-treated group (all P < 0.05). The relative expression of proinflammatory cytokines IL-6, TNF-α and IFN-γ in the colon tissue of IL-35-treated group was decreased compared with the model group, while the relative expression of IL-10 mRNA was higher than that of the model group (all P < 0.05). Compared with the control group, the proportion of M1 macrophages in the model group increased (P < 0.05), while the proportion of M1 macrophages in the IL-35-treated group was lower than that in the model group (P < 0.05). The relative expression of SHIP1 mRNA and protein in the colon tissue of IL-35-treated group was higher than that in the model group (all P < 0.05). Conclusion: IL-35 can inhibit the polarization of M1 macrophages and regulate inflammatory cytokines to promote anti-inflammatory effect on mice with colitis.



Key wordsInflammatory bowel diseases/physiopathology      Interleukins/metabolism      Immunohistochemistry      Blotting, western      Randomized controlled trial      Disease models, animal     
Received: 20 July 2018      Published: 23 January 2019
CLC:  R574  
Corresponding Authors: WANG Xingpeng     E-mail: lzjdoctor@126.com;richardwangxp@163.com
Cite this article:

LU Zhanjun,HU Yangyang,LI Sisi,ZANG Lijuan,JIANG Weiliang,WU Jianjiong,WU Xiening,ZENG Yue,WANG Xingpeng. Anti-inflammatory effect of interleukin-35 in mice with colitis and its mechanism. J Zhejiang Univ (Med Sci), 2018, 47(5): 499-506.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2018.10.09     OR     http://www.zjujournals.com/med/Y2018/V47/I5/499


白介素35在炎性肠疾病中的抗炎作用机制

目的: 研究IL-35在炎性肠疾病中的抗炎作用及相关机制。方法: BALB/c雌性小鼠共30只,随机分为对照组、模型组(口服4%葡聚糖硫酸钠7 d)、IL-35组(口服4%葡聚糖硫酸钠7 d,第2~5天腹腔注射IL-35 2 μg/d),每组10只。每天对小鼠进行疾病活动指数(DAI)评分;7 d后处死小鼠,留取血清和肠道组织,观察结肠大体形态;HE染色观察各组结肠组织病理形态变化;流式细胞术检测各组结肠组织中巨噬细胞极化情况;实时定量RT-PCR检测各组结肠组织中细胞因子IL-6、TNF-α、γ干扰素(IFN-γ)、IL-10和Src同源系列2结构域的肌醇5-磷酸酶1(SHIP1)的mRNA表达量;免疫组织化学法检测各组结肠组织中SHIP1的表达及分布情况;蛋白质印迹法检测各组结肠组织中SHIP1蛋白的表达。结果: 模型组在实验过程中DAI评分较对照组增加,而IL-35组自第4天起DAI评分较模型组减少(均P < 0.01);与对照组比较,模型组结肠明显缩短(P < 0.05),而IL-35组的结肠长度长于模型组,但差异无统计学意义(P > 0.05);与模型组比较,IL-35组炎症细胞浸润减少、黏膜组织炎症评分和腺窝破坏组织评分较模型组减少(均P < 0.05);IL-35组结肠组织中促炎因子IL-6、TNF-α和IFN-γ的mRNA相对表达量较模型组减少,而抑炎因子IL-10的mRNA相对表达量较模型组增加(均P < 0.05);与对照组比较,模型组M1型巨噬细胞比例增加(P < 0.05),而IL-35组M1型巨噬细胞的比例较模型组减少(P < 0.05);IL-35组小鼠结肠组织中SHIP1 mRNA和蛋白相对表达量均较模型组增加(均P < 0.05)。结论: 在炎性肠疾病中,IL-35可以通过调控SHIP1表达抑制M1型巨噬细胞极化,以及调节炎症因子的表达发挥抗炎作用。


关键词: 炎性肠疾病/病理生理学,  白细胞介素类/代谢,  免疫组织化学,  印迹法, 蛋白质,  随机对照试验,  疾病模型, 动物 
引物名称 引物序列(5'→3')
IL-6 正向:ACAAAGCCAGAGTCCTTCAGAG
反向:GCCACTCCTTCTGTGACTCC
TNF-α 正向:CCACCACGCTCTTCTGTCTA
反向:GGTTTGCTACGACGTGGGG
IFN-γ 正向:AGACAATCAGGCCATCAGCA
反向:TGGACCTGTGGGTTGTTGAC
IL-10 正向:CAGTACAGCCGGGAAGACAAT
反向:TTGGCAACCCAAGTAACCCT
SHIP1 正向:GCGTGCTGTATCGGAATTGG
反向:TGGTGAAGAACCTCATGGAGAC
β-actin 正向:TGGACTTCGAGCAAGAGATG
反向:GAAGGAAGGCTGGAAGAGTA
GAPDH 正向:ACCACAGTCCATGCCATCACT
反向:TCCACCACCCTGTTGCTGTA
Tab 1 Primer sequences for real-time RT-PCR
Fig 1 Disease activity index scores in three groups
Fig 2 Morphology of colonic tissues in three groups
Fig 3 HE staining of colonic tissues in three groups
($\bar x \pm s$)
组别 n 黏膜组织炎症评分 腺窝破坏组织评分
与对照组比较,*P<0.05;与模型组比较,#P<0.05.IL:白介素.
对照组 10 1.02±0.43 0
模型组 10 16.12±1.05* 8.96±1.21*
IL-35组 10 8.12±0.78*# 5.93±0.56#
Tab 2 Microscopic inflammatory infiltration score and microscopic crypt destruction score of colonic tissues in three groups
Fig 4 mRNA expression levels of IL-6, TNF-α, IFN-γ and IL-10 in colonic tissues of three groups
Fig 5 Polarization of macrophage in colonic tissues of three groups
($\bar x \pm s$,%)
组别 n M1型巨噬细胞 M2型巨噬细胞
与对照组比较,*P<0.05;与模型组比较,#P<0.05.IL:白介素.
对照组 10 7.1±1.2 8.2±1.1
模型组 10 20.0±1.8* 13.1±2.8*
IL-35组 10 11.5±1.3# 9.9±4.2
Tab 3 M1 and M2 macrophage ratios in colonic tissues of three groups
Fig 6 Immunohistochemistry staining of SHIP-1 in colon tissues of three groups
Fig 7 Expression of SHIP1 in colonic tissues of three groups
($\bar x \pm s$)
组别 n SHIP1 mRNA SHIP1蛋白
与对照组比较,*P<0.05;与模型组比较,#P<0.05.SHIP1:含有Scr同源系列2结构域的肌醇5-磷酸酶1;IL:白介素.
对照组 10 1.02±0.32 0.37±0.26
模型组 10 4.71±0.32 0.42±0.07
IL-35组 10 18.1±4.18*# 1.03±0.24*#
Tab 4 mRNA and protein expression of SHIP1 in colonic tissues of three groups
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