Inhibitory Effect of Water Extract of Broussonetiae Fructus on Hepatocellular Carcinoma Induced by Diethylnitrosamine in Mice and Effect of PTEN/PI3K/Akt Signaling Pathway

HUANG Zihan; KONG Liang; YAN Aiwen; SONG Yuewen; JIANG Pan; JI Yu; YAN Yuhui

doi:10.13422/j.cnki.syfjx.20231826

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Inhibitory Effect of Water Extract of Broussonetiae Fructus on Hepatocellular Carcinoma Induced by Diethylnitrosamine in Mice and Effect of PTEN/PI3K/Akt Signaling Pathway

更新时间：2023-10-20

- Inhibitory Effect of Water Extract of Broussonetiae Fructus on Hepatocellular Carcinoma Induced by Diethylnitrosamine in Mice and Effect of PTEN/PI3K/Akt Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 22, Pages: 29-36(2023)
- 作者机构：
  
  1.江苏海洋大学海洋食品与生物工程学院，江苏连云港 222005
  2.江苏食品药品职业技术学院药学院，江苏淮安 223003
  3.辽宁中医药大学药学院，辽宁大连 116600
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20231826
  CLC： R2-0;R22;R242;R2-031;R285.5
- Published：20 November 2023，
  
  Published Online：28 July 2023，
  
  Received：29 May 2023，
- 稿件说明：
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黄梓涵,孔亮,严艾文等.楮实子水提物对二乙基亚硝胺诱发小鼠肝细胞癌的抑制作用及PTEN/PI3K/Akt信号通路的影响[J].中国实验方剂学杂志,2023,29(22):29-36.

HUANG Zihan,KONG Liang,YAN Aiwen,et al.Inhibitory Effect of Water Extract of Broussonetiae Fructus on Hepatocellular Carcinoma Induced by Diethylnitrosamine in Mice and Effect of PTEN/PI3K/Akt Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(22):29-36.
黄梓涵,孔亮,严艾文等.楮实子水提物对二乙基亚硝胺诱发小鼠肝细胞癌的抑制作用及PTEN/PI3K/Akt信号通路的影响[J].中国实验方剂学杂志,2023,29(22):29-36. DOI： 10.13422/j.cnki.syfjx.20231826.

HUANG Zihan,KONG Liang,YAN Aiwen,et al.Inhibitory Effect of Water Extract of Broussonetiae Fructus on Hepatocellular Carcinoma Induced by Diethylnitrosamine in Mice and Effect of PTEN/PI3K/Akt Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(22):29-36. DOI： 10.13422/j.cnki.syfjx.20231826.

摘要

目的

基于同源性磷酸酶-张力蛋白（PTEN）/磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）信号通路探讨楮实子水提物对二乙基亚硝胺（DEN）诱发小鼠肝细胞癌（HCC）的抑制作用。

方法

采用腹腔注射DEN的方法构建原发性HCC小鼠模型，将HCC小鼠随机分为模型组、索拉非尼组（0.01 g·kg

-1

·d

-1

）、楮实子水提物低、中、高剂量组（0.9、1.8、3.6 g·kg

-1

·d

-1

），每组10只；另取10只C57BL/6小鼠作为空白组，腹腔注射等体积生理盐水。在小鼠肝脏出现肝癌样白色结节后灌胃给予不同浓度的楮实子水提物；索拉非尼组给予索拉非尼灌胃；空白组和模型组小鼠给予生理盐水灌胃。生化分析仪检测小鼠血清碱性磷酸酶（ALP）、天门冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）及

-谷氨酰转移酶（

-GT）活性；酶联免疫吸附测定法（ELISA）检测肿瘤标志物甲胎蛋白（AFP）、癌胚抗原（CEA）表达水平；苏木素-伊红（HE）染色及马松（Masson）染色观察肝细胞癌变程度及肝细胞损伤情况；免疫组织化学染色观察肝癌细胞增殖情况；脱氧核苷酸末端转移介导的缺口末端标记法（TUNEL）染色观察小鼠肝癌细胞凋亡情况；蛋白免疫印迹法（Western blot）检测PTEN/PI3K/Akt信号通路相关蛋白PTEN、PI3K、磷酸化-Akt（p-Akt）蛋白表达水平变化。

结果

与空白组比较，模型组小鼠ALP、AST、ALT、

-GT活性及AFP、CEA表达水平显著升高（

0.01）；小鼠肝组织癌变和炎症细胞浸润现象明显，可见大量蓝色胶原纤维增生；肿瘤增殖抗原（Ki67）阳性的增殖细胞数量显著增加（

0.01）；PTEN蛋白表达被抑制（

0.01），并促进PI3K、p-Akt蛋白表达（

0.01）；与模型组比较，楮实子水提物中、高剂量组小鼠血清中ALP、AST、ALT、

-GT活性及AFP、CEA表达水平明显降低（

0.05，

0.01）；且小鼠肝组织癌变程度和炎症细胞浸润明显减轻，胶原纤维增生明显减少；楮实子水提物中、高剂量组Ki67阳性的增殖细胞数量明显减少（

0.05，

0.01），楮实子水提物高剂量组TUNEL阳性的凋亡细胞数量明显增加（

0.05，

0.01）；楮实子水提物楮实子水提物中、高剂量组促进PTEN蛋白表达（

0.05，

0.01），抑制p-Akt蛋白表达（

0.05，

0.01）。

结论

楮实子水提物对DEN诱发的小鼠原发性HCC具有显著的抑制作用，其作用机制可能与调控PTEN/PI3K/Akt信号通路中关键蛋白表达有关。

Abstract

Objective

To explore the inhibitory effect of water extract of Broussonetiae Fructus on hepatocellular carcinoma （HCC） induced by diethyl nitrosamine （DEN） in mice based on homologous phosphatase and tensin homolog/phosphatidylinositol 3-kinase/protein kinase B （PTEN/PI3K/Akt） signaling pathway.

Method

The primary HCC mouse model was constructed by intraperitoneal injection of DEN solution， and the HCC mice were randomly divided into model group， sorafenib group （0.01 g·kg

-1

·d

-1

）， low-dose Broussonetiae Fructus water extract group （0.9 g·kg

-1

·d

-1

）， medium-dose Broussonetiae Fructus water extract group （1.8 g·kg

-1

·d

-1

）， and high-dose Broussonetiae Fructus water extract group （3.6 g·kg

-1

·d

-1

）， with 10 mice in each group. Another 10 C57BL/6 mice were selected as a control group and intraperitoneally injected with an equal volume of normal saline. Mice were treated with different concentrations of Broussonetiae Fructus water extract when liver cancer-like white nodules appeared. sorafenib group was treated with sorafenib. The control group and model group were intraperitoneally injected with normal saline. The activities of alkaline phosphatase （ALP）， aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， and

-glutamyl transferase （

-GT） in the serum of mice were detected by the biochemical analyzer. The expression levels of alpha-fetoprotein （AFP） and carcinoembryonic antigen （CEA） were detected by enzyme-linked immunosorbent assay （ELISA）. The degree of hepatocyte canceration and hepatocyte injury were observed by Hematoxylin-eosin （HE） and Masson staining. The proliferation of HCC cells was observed by immunohistochemical staining. The apoptosis of HCC cells in mice was observed by erminal-deoxynucleotidyl transferase mediated nick end labelling （TUNEL） staining. The expression levels of PTEN， PI3K， Akt， and p-Akt proteins related to the PTEN/PI3K/Akt signaling pathway were detected by Western blot.

Result

Compared with the control group， the activities of ALP， AST， ALT， and

-GT， as well as the expression levels of AFP and CEA in the model group were significantly increased （

0.01）. Carcinogenesis and inflammatory cell infiltration were obvious in liver tissue of mice， and a large number of blue collagen fiber hyperplasia was found. The number of Ki67 positive cells was significantly increased （

0.01）， and the expression level of PTEN protein was significantly decreased， while PI3K and p-Akt protein expression was increased （

0.01）. Compared with the model group， the activities of ALP， AST， ALT， and

-GT， as well as the expression levels of AFP and CEA in the medium-dose and high-dose Broussonetiae Fructus water extract groups were significantly decreased （

0.05，

0.01）. The degree of carcinogenesis and inflammatory cell infiltration in liver tissue were reduced， and the collagen fiber hyperplasia was significantly reduced. The number of Ki67 positive cells was significantly decreased， and the number of TUNEL positive apoptotic cells was significantly increased （

0.05，

0.01）. PTEN protein expression was increased， while p-Akt protein expression was significantly decreased （

0.05，

0.01）.

Conclusion

The water extract of Broussonetiae Fructus has a significant inhibitory effect on DEN-induced primary HCC in mice， and its mechanism may be related to the regulation of key protein expressions in the PTEN/PI3K/Akt signaling pathway.

关键词

楮实子肝细胞癌增殖二乙基亚硝胺同源性磷酸酶-张力蛋白/磷脂酰肌醇3-激酶/蛋白激酶B（PTEN/PI3K/Akt）信号通路

Keywords

Broussonetiae Fructushepatocellular carcinomaproliferationdiethyl nitrosaminephosphatase and tensin homolog/phosphatidylinositol 3-kinase/protein kinase B （PTEN/PI3K/Akt） signaling pathway

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