Liu Dengrui, Kang Hongxia, Gao Mingtai, Pei Wei, Wang Shimo, Chen Zhou
Department of Pediatric Surgery, The First Hospital of Lanzhou University, No. 1 Donggang West Road, Lanzhou, 730000 Gansu China.
Department of Pain, Gansu Provincial People's Hospital, Lanzhou, 730000 Gansu China.
3 Biotech. 2023 Sep;13(9):312. doi: 10.1007/s13205-023-03721-w. Epub 2023 Aug 23.
The current study aimed to explore the effect and underlying mechanism of the purinergic receptor P2Y2 in regulating the loss of intestinal neurons and the intestinal neural crest in Hirschsprung's disease (HSCR). Western blotting was used to assess the expression levels of P2Y2 in colon tissues. An in vivo HSCR mouse model was established following treatment with benzalkonium chloride (BAC). We overexpressed or silenced P2Y2 in SH-SY5Y cells, and cell proliferation, migration, and invasion were subsequently investigated by CCK-8, wound healing, and transwell assays, respectively. Additionally, we implemented a xenograft model to assess the impact of P2Y2 on tumor growth as well as the expression of extracellular signal-regulated kinase (ERK). The results showed that the expression of P2Y2 protein in the colon tissues of patients with HSCR was lower than that in the normal colon tissues. P2Y2 expression is downregulated in the colon tissues of mice with HSCR. Additionally, P2Y2 silencing inhibited SH-SY5Y cell proliferation, invasion, and migration. Furthermore, adenosine 5'-triphosphate (ATP, a strong agonist of P2Y2)-induced P2Y2 overexpression enhanced the proliferation, invasion, and migration of SH-SY5Y cells. Immunofluorescence staining and western blot analysis revealed that P2Y2 silencing downregulated phosphorylated (p)-ERK in SH-SY5Y cells. In addition, treatment with PD98059, a p-ERK inhibitor, reversed the effects of ATP on SH-SY5Y cell proliferation, invasion, and migration. Finally, we demonstrated that P2Y2 silencing suppressed tumor growth and decreased p-ERK expression. Overall, the results of the present study suggest that P2Y2 plays an important role in HSCR pathogenesis. P2Y2 silencing inhibited the proliferation, invasion, and migration of nerve cells by suppressing the ERK signaling pathway. P2Y2 silencing could be considered an innovative and possible target for treating HSCR.
本研究旨在探讨嘌呤能受体P2Y2在调节先天性巨结肠（HSCR）中肠神经元丢失和肠神经嵴方面的作用及潜在机制。采用蛋白质免疫印迹法评估结肠组织中P2Y2的表达水平。用苯扎氯铵（BAC）处理后建立体内HSCR小鼠模型。我们在SH-SY5Y细胞中过表达或沉默P2Y2，随后分别通过CCK-8、伤口愈合和Transwell实验研究细胞增殖、迁移和侵袭情况。此外，我们建立了异种移植模型以评估P2Y2对肿瘤生长以及细胞外信号调节激酶（ERK）表达的影响。结果显示，HSCR患者结肠组织中P2Y2蛋白的表达低于正常结肠组织。HSCR小鼠的结肠组织中P2Y2表达下调。此外，沉默P2Y2可抑制SH-SY5Y细胞的增殖、侵袭和迁移。此外，5'-三磷酸腺苷（ATP，P2Y2的强效激动剂）诱导的P2Y2过表达增强了SH-SY5Y细胞的增殖、侵袭和迁移。免疫荧光染色和蛋白质免疫印迹分析显示，沉默P2Y2可下调SH-SY5Y细胞中磷酸化（p）-ERK的表达。此外，用p-ERK抑制剂PD98059处理可逆转ATP对SH-SY5Y细胞增殖、侵袭和迁移的影响。最后，我们证明沉默P2Y2可抑制肿瘤生长并降低p-ERK表达。总体而言，本研究结果表明P2Y2在HSCR发病机制中起重要作用。沉默P2Y2通过抑制ERK信号通路抑制神经细胞的增殖、侵袭和迁移。沉默P2Y2可被认为是治疗HSCR的一种创新且可能的靶点。
