基于SNAIL信号通路及肠道菌群研究密点麻蜥抑制胃癌肝转移的作用机制

Mechanism of Eremias multiocellata on inhibiting gastric cancer with liver metastasis based on SNAIL signaling pathway and gut microbiota

目的基于SNAIL信号通路及肠道菌群研究密点麻蜥Eremias multiocellata对胃癌肝转移的抑制作用及机制。方法36只BALB/c裸鼠随机分为对照组和造模组,造模组脾脏注射HGC-27胃癌细胞,造模4周剖腹探查验证造模成功。随后将造模组随机分为模型组、5-氟尿嘧啶(5-fluorouracil,5-FU,0.025 g/kg)组和密点麻蜥(2.6 g/kg)组,每组8只。对照组和模型组ig生理盐水,密点麻蜥组ig密点麻蜥水煎剂,5-FU组ip 5-FU,连续干预4周后,采用苏木素-伊红(HE)染色观察各组裸鼠肝组织病理变化;采用Western blotting检测各组裸鼠肝组织SNAIL及上皮间质转化(epithelial-mesenchymal transition,EMT)相关蛋白表达;取末次给药2 h后各组裸鼠新鲜粪便进行16S rRNA高通量测序检测肠道菌群的变化。结果与对照组比较,模型组裸鼠肝组织出现肝小叶破坏、肝组织坏死,中性粒细胞浸润,癌细胞核大深染;肝组织SNAIL、N-cadherin蛋白表达水平显著升高(P<0.001),E-caherin蛋白水平显著降低(P<0.001)。与模型组比较,密点麻蜥组肝细胞排列趋于整齐,结构完整,癌组织灶变小;肝组织中SNAIL、N-cadherin蛋白表达水平显著降低(P<0.001),E-cadherin蛋白表达水平显著升高(P<0.001)。16S rRNA结果显示,与对照组比较,模型组裸鼠肠道菌群失调紊乱,厚壁菌门/拟杆菌门(Firmicutes/Bacteroidetes,F/B)值下降,经密点麻蜥干预后F/B值升高。门水平上,密点麻蜥主要对厚壁菌门进行调节,厚壁菌门在对照组中丰度占比40.9%,与对照组比较,模型组中厚壁菌门的丰度降低到26.4%。经密点麻蜥干预后厚壁菌门丰度提高到30.2%。属水平上,密点麻蜥主要对胃癌肝转移裸鼠肠道菌群中差异菌属(拟杆菌属Bacteroides、梭状芽孢杆菌属Clostridia_UCG-014、副拟杆菌属Parabacteroides、幽门螺旋杆菌Helicobacter)发挥调节作用。结论密点麻蜥可能通过降低SNAIL蛋白表达,增加E-cadherin蛋白表达,降低N-cadherin蛋白表达,来抑制EMT发展进程,发挥对胃癌肝转移的抑制作用。此外,密点麻蜥通过调节厚壁菌门丰度,增加作用于癌细胞表面G蛋白偶联受体的短链脂肪酸(short-chain fatty acids,SCFAs)含量,调节机体自身免疫相关菌属(拟杆菌属、梭状芽孢杆菌属、副拟杆菌属),降低致病菌幽门螺旋杆菌的丰度,发挥对胃癌肝转移的抑制作用。

Objective To study the effect and mechanism of Eremias multiocellata on inhibiting gastric cancer with liver metastasis based on SNAIL signaling pathway and intestinal flora.Methods Thirty-six BALB/c nude mice were randomly divided into control group and model group.The model group was injected HGC-27 gastric cancer cells into spleen,and model was successfully established by laparotomy for four weeks.Subsequently,model group was randomly divided into model group,5-fluorouracil(5-FU,0.025 g/kg)group and E.multiocellata(2.6 g/kg)group with eight rats in each group.Control group and model group were ig normal saline,E.multiocellata group was ig E.multiocellata water decoction,and 5-FU group was ip 5-FU.After four weeks of continuous intervention,pathological changes of liver tissues of nude mice in each group were observed by hematoxylin-eosin(HE)staining;Western blotting was used to detect the expressions of SNAIL and epithelial mesenchymal transition(EMT)related proteins in liver tissues of nude mice in each group;Two hours after the last administration,fresh feces of nude mice in each group were taken for 16S rRNA highthroughput sequencing to detect the changes of gut microbiota.Results Compared with control group,liver tissue of nude mice in model group showed destruction of liver lobule,necrosis of liver tissue,neutrophil infiltration,and large and deep staining of cancer cell nucleus;SNAIL and N-cadherin protein expression levels in liver tissue of model group were significantly increased(P<0.001),and E-cadherin protein expression level was significantly decreased(P<0.001).Compared with model group,liver cells tended to be arranged in order,with complete structure,and tumor focus became smaller in E.multiocellata group;SNAIL and N-cadherin protein expression levels of liver tissue in E.multiocellata group were significantly decreased(P<0.001),and E-cadherin protein level was significantly increased(P<0.001).16S rRNA results showed that compared with control group,gut microbiota of nude mice in model group was in disorder,Firmicutes/Bacteroides(F/B)was decreased,and F/B value was increased after the intervention of E.multiocellata.At the level of phyla,E.multiocellata mainly regulated Firmicutes,abundance of Firmicutes accounted for 40.9%in control group.Compared with control group,abundance of Firmicutes in model group was decreased to 26.4%.The abundance of Firmicutes was increased to 30.2%after the intervention of E.multiocellata.At the genus level,E.multiocellata mainly played a regulatory role in differential bacteria(Bacteroides,Clostridia_UCG-014,Parabacteroides,Helicobacter)in gut microbiota of nude mice with liver metastasis of gastric cancer.Conclusion E.multiocellata may inhibit the development of EMT and liver metastasis of gastric cancer by decreasing SNAIL protein expression,increasing E-cadherin protein expression and decreasing N-cadherin protein expression.In addition,by regulating the abundance of Firmicutes,E.multiocellata can increase the content of short chain fatty acids(SCFAs)acting on G protein coupled receptors on the surface of cancer cells,regulate the autoimmune related bacteria(Bacteroides,Clostridium,Parabacteroides),reduce the abundance of pathogenic bacteria Helicobacter pylori,and play an inhibitory role in liver metastasis of gastric cancer.