温和灸和电针对TET2基因敲除溃疡性结肠炎小鼠结肠DNA甲基转移酶的调节作用

Modulation of colonic DNA methyltransferase by mild moxibustion and electroacupuncture in ulcerative colitis TET2 knockout mice

    摘要
  • 摘要:
    目的 探讨温和炙和电针通过调节DNA甲基转移酶 （DNMT）及DNA羟甲基化酶减轻10-11易位（TET）蛋白2 基因敲除（TET2-/-）溃疡性结肠炎（UC）小鼠结肠黏膜炎症的机制。
    方法 使用雄性无特定病原体（SPF）C57BL/6J野生型（WT）小鼠（共8只）和TET2-/-小鼠（共20只），通过自由饮用3%葡聚糖硫酸钠溶液7天建立UC模型。每种小鼠随机选取2只通过组织病理学检查进行模型验证后，剩余小鼠随机分为四组（每组6只）：WT模型组（WT + UC）、TET2-/-模型组（TET2-/- + UC）、TET2-/-温和灸组（TET2-/- + MM）和TET2-/-电针组（TET2-/- + EA）。TET2-/- + MM组小鼠每天在天枢（ST25）和气海（CV6）穴位接受温和艾灸10分钟，连续7天。TET2-/- + EA组小鼠每天在相同穴位接受电针（1 mA，频率2/100 Hz）10分钟，连续7天。每天评估各组小鼠疾病活动指数（DAI）评分，干预后测量结肠长度。通过苏木素-伊红（HE）染色观察结肠组织病理学改变。采用酶联免疫吸附试验（ELISA）检测血清中的白介素（IL）- 6、C-C 基序趋化因子17（CCL17）、C-X-C 基序趋化因子配体10（CXCL10）的浓度。免疫组化法检测小鼠结肠组织中DNMT蛋白（DNMT1、DNMT3A和DNMT3B）的表达。免疫荧光法检测5-甲基胞嘧啶（5-mC）、5- 羟甲基胞嘧啶（5-hmC）、组蛋白去乙酰化酶2（HDAC2）、DNA羟甲基化酶家族蛋白（ TET1、TET3）表达，以及观察TET1 与 IL-6 蛋白共定位情况。
    结果 与WT + UC组相比，TET2-/- + UC组的DAI评分显著升高，结肠长度显著缩短（P < 0.01）。温和灸和电针均显著降低了TET2-/-小鼠的DAI评分，改善了结肠缩短（P < 0.001）。TET2-/- + UC小鼠的组织病理学评分显著高于WT + UC组（P < 0.001），温和灸和电针干预后显著降低（P < 0.001）。与WT + UC组相比，TET2-/- + UC组的血清IL-6、CCL17和CXCL10水平显著升高（P < 0.001）。温和灸显著降低了TET2-/-小鼠IL-6、CCL17和CXCL10的水平（分别为P < 0.001、P < 0.001和P < 0.01），而电针同样显著降低了IL-6、CCL17和CXCL10的表达水平（分别为P < 0.05、P < 0.01和P < 0.01）。TET2-/- + UC小鼠DNMT1、DNMT3A、DNMT3B和5-mC的表达增加（分别为P < 0.01、P < 0.05、P < 0.01和P < 0.001），TET1、TET3、5-hmC和HDAC2的表达减少（P < 0.001）。温和灸显著降低了DNMT1、DNMT3B和5-mC水平（分别为P < 0.05、P < 0.01和P < 0.001），同时增加了TET1、TET3、5-hmC和HDAC2的表达（分别为P < 0.001、P < 0.001、P < 0.05和P < 0.001）。电针显著降低5-mC和DNMT3B水平（分别为P < 0.001和P < 0.01），增加5-hmC和HDAC2水平（分别为P < 0.05和P < 0.001），但对TET1和TET3表达没有显著影响（P > 0.05）。与TET2-/- + MM组相比，TET2-/- + EA组5-mC表达明显升高（P < 0.001）。TET2-/- + UC组小鼠IL-6表达明显增加，TET1和IL-6在黏膜上皮中的共定位较高，而其他组IL-6表达最低。
    结论 温和灸和电针通过表观遗传调节显著改善因TET2缺失而加重的UC小鼠结肠炎症。两种干预之间存在不同的机制：温和灸调节DNA甲基转移酶和DNA羟甲基化酶，而电针主要影响DNA甲基转移酶。

     

    Abstract:
    Objective To investigate the mechanism of in alleviating colonic mucosal inflammation in ten-eleven translocation (TET) protein 2 gene knockout (TET2-/-) mice with ulcerative colitis (UC) by regulating DNA methyltransferase (DNMT) and DNA hydroxymethylase.
    Methods Male specific pathogen-free (SPF) grade C57BL/6J wild-type (WT) mice (n = 8) and TET2-/- mice (n = 20) were used to establish UC models by freely drinking 3% dextran sulfate sodium solution for 7 d. After UC model validation through histopathological examination in two mice from each type, the remaining mice were divided into four groups (n = 6 in each group): WT model (WT + UC), TET2-/- model (TET2-/- + UC), TET2-/- mild moxibustion (TET2-/- + MM), and TET2-/- electroacupuncture (TET2-/- + EA) groups. TET2-/- + MM group received mild moxibustion on Tianshu (ST25) and Qihai (CV6) for 10 min daily for 7 d. The TET2-/- + EA group also applied electroacupuncture (1 mA, 2/100 Hz) at the same acupoints for 10 min daily for 7 d. The disease activity index (DAI) scores of each group of mice were accessed daily. The colon lengths of mice in groups were measured following intervention. The pathological changes in the colon tissues were observed with hematoxylin and eosin (HE) staining. The concentrations of interleukin (IL)-6, C-C motif chemokine 17 (CCL17), and C-X-C motif chemokine ligand 10 (CXCL10) in serum were detected by enzyme-linked immunosorbent assay (ELISA). The expression of DNMT proteins (DNMT1, DNMT3A, and DNMT3B) in the colon tissues was detected by immunohistochemistry. The expression of 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), histone deacetylase 2 (HDAC2), and DNA hydroxymethylase family proteins (TET 1 and TET3) was detected using immunofluorescence, which also determined the co-localization of TET1 and IL-6 protein.
    Results Compared with WT + UC group, TET2-/- + UC group exhibited significantly higher DAI scores and shorter colon lengths (P < 0.01). Both mild moxibustion and electroacupuncture significantly decreased DAI scores and ameliorated colon shortening in TET2-/- mice (P < 0.001). Histopathological scores of TET2-/- + UC mice were significantly higher than those of WT + UC group (P < 0.001) and were significantly reduced after both mild moxibustion and electroacupuncture interventions (P < 0.001). Serum levels of IL-6, CCL17, and CXCL10 were significantly elevated in TET2-/- + UC group compared with WT + UC group (P < 0.001). Mild moxibustion significantly reduced IL-6, CCL17, and CXCL10 levels (P < 0.001, P < 0.001, and P < 0.01, respectively), while electroacupuncture also significantly reduced IL-6, CCL17, and CXCL10 levels (P < 0.05, P < 0.01, and P < 0.01, respectively). TET2-/- + UC mice showed increased expression levels of DNMT1, DNMT3A , DNMT3B, and 5-mC (P < 0.05, P < 0.01 and P < 0.001, respectively), with decreased expression levels of TET1, TET3, 5-hmC, and HDAC2 (P < 0.001). Mild moxibustion significantly reduced DNMT1, DNMT3B, and 5-mC levels (P < 0.05, P < 0.01, and P < 0.001, respectively), while increasing expression levels of TET1, TET3, 5-hmC, and HDAC2 (P < 0.001, P < 0.001, P < 0.05, and P < 0.001, respectively). Electroacupuncture significantly decreased 5-mC and DNMT3B levels (P < 0.001 and P < 0.01, respectively) and increased 5-hmC and HDAC2 levels (P < 0.05 and P < 0.001, respectively), but did not significantly affect TET1 and TET3 expression (P > 0.05). Compared with TET2-/- + MM group, TET2-/- + EA group showed significantly higher 5-mC expression (P < 0.001). TET2-/- + UC group exhibited markedly increased IL-6 expression and higher co-localization of TET1 and IL-6 in mucosal epithelium, whereas minimal IL-6 expression was observed in the other groups.
    Conclusion Mild moxibustion and electroacupuncture significantly ameliorate colonic inflammation exacerbated by TET2 deficiency in UC mice via epigenetic modulation. Distinct mechanisms exist between the two interventions: mild moxibustion regulates both DNMT and hydroxymethylase, whereas electroacupuncture primarily affects DNMT.

     

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