Effect of Sishenwan on PI3K/Akt/mTOR Signal Pathway in Colonic Tissue of Rats with Ulcerative Colitis Model of Spleen Kidney Yang Deficiency

Rong LIU; Yan WANG; Xiang-dong ZHU; Yan-kui GAO; Huan WANG; Xing-teng ZHONG

doi:10.13422/j.cnki.syfjx.20210437

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Effect of Sishenwan on PI3K/Akt/mTOR Signal Pathway in Colonic Tissue of Rats with Ulcerative Colitis Model of Spleen Kidney Yang Deficiency

更新时间：2021-12-03

- Effect of Sishenwan on PI3K/Akt/mTOR Signal Pathway in Colonic Tissue of Rats with Ulcerative Colitis Model of Spleen Kidney Yang Deficiency
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 4, Pages: 16-23(2021)
- 作者机构：
  
  甘肃中医药大学，兰州 730000
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20210437
  CLC： R2-0;R289;R574.4
- Received：30 September 2020，
  
  Published Online：05 January 2021，
  
  Published：20 February 2021
- 稿件说明：
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柳荣,王燕,朱向东等.四神丸对脾肾阳虚型溃疡性结肠炎模型大鼠结肠组织PI3K/Akt/mTOR信号通路的影响[J].中国实验方剂学杂志,2021,27(04):16-23.

LIU Rong,WANG Yan,ZHU Xiang-dong,et al.Effect of Sishenwan on PI3K/Akt/mTOR Signal Pathway in Colonic Tissue of Rats with Ulcerative Colitis Model of Spleen Kidney Yang Deficiency[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(04):16-23.
柳荣,王燕,朱向东等.四神丸对脾肾阳虚型溃疡性结肠炎模型大鼠结肠组织PI3K/Akt/mTOR信号通路的影响[J].中国实验方剂学杂志,2021,27(04):16-23. DOI： 10.13422/j.cnki.syfjx.20210437.

LIU Rong,WANG Yan,ZHU Xiang-dong,et al.Effect of Sishenwan on PI3K/Akt/mTOR Signal Pathway in Colonic Tissue of Rats with Ulcerative Colitis Model of Spleen Kidney Yang Deficiency[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(04):16-23. DOI： 10.13422/j.cnki.syfjx.20210437.

摘要

目的

探讨四神丸对脾肾阳虚型溃疡性结肠炎（UC）模型大鼠结肠组织磷脂酰肌醇-3激酶/蛋白激酶B/雷帕霉素靶蛋白（PI3K/Akt/mTOR）信号通路相关基因mRNA和蛋白以及血清中细胞因子白细胞介素-1

（IL-1

），白细胞介素-10（IL-10）表达水平的影响。

方法

将120只SPF级Wistar大鼠在SPF级实验室适应性喂养7 d后分为空白组和造模组，造模组大鼠采用二硝基苯磺酸盐（DNBS）/乙醇溶液灌肠+皮下注射氢化可的松+番泻叶灌胃法建立脾肾阳虚型UC大鼠模型。将成功建立模型的大鼠随机分为5组，分别为模型组，美沙拉嗪（0.36 g·kg

-1

）组，四神丸高、中、低剂量（3.2，1.6，0.8 g·kg

-1

）组，灌胃体积均为10 mL·kg

-1

，模型组和空白组灌服等体积蒸馏水，1次/d，连续21 d。每日观察大鼠一般情况，并记录小鼠体质量、粪便性状及隐血情况进行疾病活动指数（DAI）评分。取大鼠结肠组织，观察大体形态及结肠损伤情况，进行结肠黏膜损伤指数（CMDI）评分。采用苏木素-伊红（HE）染色观察各组大鼠结肠组织病理改变，实时荧光定量聚合酶链式反应（Real-time PCR）检测结肠组织PI3K，Akt，mTOR mRNA的表达水平，酶联免疫吸附测定（ELISA）检测血清IL-1

，IL-10的含量，蛋白免疫印迹法（Western blot）检测结肠组织PI3K，磷酸化（p-）PI3K，Akt，p-Akt，mTOR，p-mTOR蛋白的表达。

结果

与空白组比较，模型组大鼠一般生存状况相对较差，DAI评分，CMDI评分显著升高（

0.01）；大鼠病理切片见肠黏膜部分消失，腺体消失，有大量的炎性细胞浸润，聚集于黏膜层和基层；PI3K，Akt，mTOR mRNA表达水平显著升高（

0.01）；IL-1

含量显著升高，IL-10含量显著降低（

0.01）；p-PI3K，p-Akt，p-mTOR蛋白表达水平显著升高（

0.01）。与模型组比较，四神丸高、中剂量组及美沙拉嗪组大鼠DAI评分明显下降（

0.05）；四神丸高、中、低剂量组及美沙拉嗪组大鼠CMDI评分显著降低（

0.01）；大鼠病理切片显示各给药组炎性细胞减少，黏膜层结构不同程度的恢复正常，美沙拉嗪组和四神丸中剂量组效果最好，黏膜结构接近空白组；四神丸高、中剂量组及美沙拉嗪组大鼠PI3K，Akt，mTOR mRNA及四神丸低剂量组Akt mRNA表达水平明显降低（

0.05，

0.01），四神丸低剂量组PI3K，mTOR mRNA的表达水平虽有降低，但差异无统计学意义；四神丸高、中剂量组及美沙拉嗪组IL-1

含量明显降低，IL-10含量明显升高（

0.05，

0.01），四神丸低剂量组IL-1

含量降低，IL-10含量升高，但差异无统计学意义；四神丸高、中、低剂量组及美沙拉嗪组p-PI3K，p-Akt，p-mTOR蛋白表达水平均有不同程度下降（

0.05，

0.01）。

结论

四神丸具有改善脾肾阳虚型UC模型大鼠的一般情况和肠黏膜损伤的作用，其机制可能与抑制PI3K/Akt/mTOR信号通路有关。

Abstract

Objective

To discuss the effect of Sishenwan on phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin（PI3K/Akt/mTOR） signaling pathway related genes and proteins in colon tissue and interleukin-1

（IL-1

） and interleukin-10（IL-10） expression levels in serum of ulcerative colitis （UC） model rats with spleen kidney Yang deficiency.

Method

The 120 SPF Wistar rats were randomly divided into normal group and model group after 7 days of adaptive feeding in SPF laboratory. The model group were given dinitrobenzene sulfonate （DNBS）/ethanol solution enema+hydrocortisone subcutaneously injection+senna leaf gavage to establish UC model of spleen kidney yang deficiency. The rats who successfully established the model were randomly divided into five groups：model group， mesalazine （0.36 g·kg

-1

） group， and Sishenwan high， medium and low dose （3.2，1.6，0.8 g·kg

-1

） groups， the volume of which was 10 mL·kg

-1

. The model group and the blank group were given distilled water of the same volume. Once a day for 21 days. Observe the general conditions of the rats daily， and record the weight， fecal traits and occult blood of the mice for disease activity index （DAI） scoring.Take the rat colon tissue to observe the gross morphology and colon injury， and score the colon mucosal injury index （CMDI）. Hematoxylin-eosin （HE） staining was used to observe pathological changes. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）was used to detect the expression level of PI3K， Akt， mTOR mRNA in colon tissue. The levels of IL-1

and IL-10 in serum were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the expression level of PI3K， phosphorylation （p）-PI3K， Akt， p-Akt， mTOR， p-mTOR protein in colon tissue.

Result

Compared with blank group， the general survival status of the rats in model group was relatively poor， the DAI score and the CMDI index were significantly increased （

0.01）. The intestinal mucosa partially disappears， the glands disappear， and a large number of inflammatory cells infiltrate and gather in the mucosal layer and the base layer in the pathological sections of the model group. The expression levels of PI3K， Akt， and mTOR mRNA were significantly increased （

0.01）. The IL-1

content was significantly increased and the IL-10 content was significantly decreased （

0.01）. The expression levels of p-PI3K，p-Akt and p-mTOR protein were significantly increased （

0.01）. Compared with the model group， the DAI scores of Sishenwan high and medium dose groups and mesalazine group decreased （

0.05）. The CMDI index of mesalazine group and the high， middle and low dose groups of Sishenwan was significantly reduced （

0.01）. Pathological sections of rats showed that the inflammatory cells in the drug group decreased， and the mucosal layer structure returned to normal to varying degrees. The mesalazine group and the Sishenwan medium-dose group had the best effects， and the mucosal structure was close to the blank control group. The expression levels of PI3K， Akt， mTOR mRNA in the high and medium dose groups of Sishenwan， mesalazine group and Akt mRNA in low dose group of Sishenwan were significantly reduced （

0.05，

0.01）. The expression levels of PI3K and mTOR mRNA in low-dose group of Sishenwan decreased， but the difference was not statistically significant. The IL-1

content was significantly reduced and the IL-10 content was significantly increased in high， medium dose groups of Sishenwan and mesalazine groups （

0.05，

0.01）. The level of IL-1

decreased and the level of IL-10 increased in the low-dose group of Sishenwan， but the difference was not statistically significant. The expression levels of p-PI3K， p-Akt and p-mTOR protein in the high， medium， and low dose groups of Sishenwan and mesalazine group decreased to varying degrees， and the differences were statistically significant（

0.05，

0.01）.

Conclusion

Sishenwan has the effect of improving the general condition and intestinal mucosal damage of ulcerative colitis model rats with spleen and kidney Yang deficiency. The mechanism may be related to the inhibition of PI3K/Akt/mTOR signaling pathway.

关键词

Keywords

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