Herbal cake-partitioned moxibustion inhibits colonic autophagy in Crohn's disease via signaling involving distinct classes of phosphatidylinositol 3-kinases.
Herbal cake-partitioned moxibustion inhibits colonic autophagy in Crohn's disease via signaling involving distinct classes of phosphatidylinositol 3-kinases.
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药饼隔灸通过涉及不同类别磷脂酰肌醇 3-激酶的信号传导抑制克罗恩病的结肠自噬
DOI：
10.3748/wjg.v26.i39.5997
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发表时间：
2020-10-21
影响因子：
4.3
通讯作者：
Liu HR
中科院分区：
文献类型：
--
作者：
Wang SY;Zhao JM;Zhou CL;Zheng HD;Huang Y;Zhao M;Zhang ZY;Wu LY;Wu HG;Liu HR
自噬是真核细胞中进化上保守的生物学过程，其涉及溶酶体介导的相关细胞组分的降解和再循环。近年来的研究表明，自噬在克罗恩病（CD）的发病机制中起着重要作用。隔药饼灸（HM）在历史上一直被用于治疗CD。然而，HM调节CD中结肠自噬的机制仍不清楚。目的观察HM是否可以通过调节结肠自噬来缓解CD，并阐明其作用机制。方法大鼠随机分为正常对照组（NC）、CD组、HM组、胰岛素+ CD（I + CD）组、胰岛素+ HM（I + HM）组、雷帕霉素+ CD（RA + CD）组和雷帕霉素+ HM（RA + HM）组。给药2，4，6-三硝基苯磺酸建立CD模型。苏木精-伊红染色观察结肠黏膜形态，电镜观察自噬体的形成。免疫荧光染色观察自噬标志物微管相关蛋白1轻链3 β（LC 3B）的表达。胰岛素和雷帕霉素分别用于抑制和激活结肠自噬。通过RT-qPCR评估I类磷脂酰肌醇3-激酶（PI 3 KC 1）、Akt 1、LC 3B、隔离体1（p62）和哺乳动物雷帕霉素靶蛋白（mTOR）的mRNA表达水平。通过Western blot分析评估白细胞介素18（IL-18）、肿瘤坏死因子-α（TNF-α）、核因子κB/p65（NF-κB p65）、LC 3 B、p62、卷曲螺旋肌球蛋白样BCL 2相互作用蛋白（Beclin-1）、p-mTOR、PI 3 KC 1、III类磷脂酰肌醇3-激酶（PI 3 KC 3/Vps 34）和p-Akt的蛋白表达水平。结果与NC组相比，CD组结肠组织损伤严重，IL-18和NF-κB p65表达水平升高（均P < 0.01）。与CD组相比，HM组显示这些蛋白质的水平显著降低（PIL-18 < 0.01和Pp 65 < 0.05）。各组大鼠结肠组织TNF-α蛋白表达无明显差异。CD组和HM组均可见典型的自噬囊泡。CD组大鼠结肠组织中自噬蛋白LC 3B和Beclin-1的表达较NC组上调（均P < 0.01），而p62和p-mTOR的蛋白表达较NC组下调（均P < 0.01）。然而，与CD组相比，这些表达趋势在HM组中显著逆转（PLC 3B < 0.01，PBeclin-1 < 0.05，Pp 62 < 0.05，和Pm-TOR < 0.05）。RA + HM组PI 3 KC 1、Akt 1、mTOR和p62 mRNA表达水平显著高于RA + CD组（PPI 3 KC 1 < 0.01，PAkt 1、mTOR和p62 < 0.05），而LC 3B mRNA表达水平显著低于RA + CD组（P < 0.05）。与RA + CD组相比，RA + HM组结肠组织中PI 3 KC 1、p-Akt 1和p-mTOR蛋白水平显著升高（PPI 3 KC 1 < 0.01，Pp-Akt 1 < 0.05，Pp-mTOR < 0.01），p62蛋白水平升高（P = 0.057），LC 3B和Vps 34蛋白水平显著降低（均P < 0.01）。结论HM可激活CD大鼠结肠组织中PI 3 KC 1/Akt 1/mTOR信号通路，抑制PI 3 KC 3（Vps 34）-Beclin-1蛋白复合物，从而抑制过度激活的自噬，发挥治疗作用。
BACKGROUND Autophagy is an evolutionarily conserved biological process in eukaryotic cells that involves lysosomal-mediated degradation and recycling of related cellular components. Recent studies have shown that autophagy plays an important role in the pathogenesis of Crohn’s disease (CD). Herbal cake-partitioned moxibustion (HM) has been historically practiced to treat CD. However, the mechanism by which HM regulates colonic autophagy in CD remains unclear. AIM To observe whether HM can alleviate CD by regulating colonic autophagy and to elucidate the underlying mechanism. METHODS Rats were randomly divided into a normal control (NC) group, a CD group, an HM group, an insulin + CD (I + CD) group, an insulin + HM (I + HM) group, a rapamycin + CD (RA + CD) group, and a rapamycin + HM (RA + HM) group. 2,4,6-trinitrobenzenesulfonic acid was administered to establish a CD model. The morphology of the colonic mucosa was observed by hematoxylin-eosin staining, and the formation of autophagosomes was observed by electron microscopy. The expression of autophagy marker microtubule-associated protein 1 light chain 3 beta (LC3B) was observed by immunofluorescence staining. Insulin and rapamycin were used to inhibit and activate colonic autophagy, respectively. The mRNA expression levels of phosphatidylinositol 3-kinase class I (PI3KC1), Akt1, LC3B, sequestosome 1 (p62), and mammalian target of rapamycin (mTOR) were evaluated by RT-qPCR. The protein expression levels of interleukin 18 (IL-18), tumor necrosis factor-α (TNF-α), nuclear factor κB/p65 (NF-κB p65), LC3B, p62, coiled-coil myosin-like BCL2-interacting protein (Beclin-1), p-mTOR, PI3KC1, class III phosphatidylinositol 3-kinase (PI3KC3/Vps34), and p-Akt were evaluated by Western blot analysis. RESULTS Compared with the NC group, the CD group showed severe damage to colon tissues and higher expression levels of IL-18 and NF-κB p65 in colon tissues (P < 0.01 for both). Compared with the CD group, the HM group showed significantly lower levels of these proteins (PIL-18 < 0.01 and Pp65 < 0.05). There were no significant differences in the expression of TNF-α protein in colon tissue among the rat groups. Typical autophagic vesicles were found in both the CD and HM groups. The expression of the autophagy proteins LC3B and Beclin-1 was upregulated (P < 0.01 for both) in the colon tissues of rats in the CD group compared with the NC group, while the protein expression of p62 and p-mTOR was downregulated (P < 0.01 for both). However, these expression trends were significantly reversed in the HM group compared with the CD group (PLC3B < 0.01, PBeclin-1 < 0.05, Pp62 < 0.05, and Pm-TOR < 0.05). Compared with those in the RA + CD group, the mRNA expression levels of PI3KC1, Akt1, mTOR, and p62 in the RA + HM group were significantly higher (PPI3KC1 < 0.01 and PAkt1, mTOR, and p62 < 0.05), while those of LC3B were significantly lower (P < 0.05). Compared with the RA + CD group, the RA + HM group exhibited significantly higher PI3KC1, p-Akt1, and p-mTOR protein levels (PPI3KC1 < 0.01, Pp-Akt1 < 0.05, and Pp-mTOR < 0.01), a higher p62 protein level (P = 0.057), and significantly lower LC3B and Vps34 protein levels (P < 0.01 for both) in colon tissue. CONCLUSION HM can activate PI3KC1/Akt1/mTOR signaling while inhibiting the PI3KC3 (Vps34)-Beclin-1 protein complex in the colon tissues of CD rats, thereby inhibiting overactivated autophagy and thus exerting a therapeutic effect.
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影响因子：
19
作者：
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通讯作者：
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影响因子：
29.4
作者：
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通讯作者：
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影响因子：
3.9
作者：
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影响因子：
16.6
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通讯作者：
Vanhaesebroeck B
影响因子：
3.4
作者：
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通讯作者：
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