骨桥蛋白通过PI3K/Akt信号通路激活胰腺星形细胞
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摘要
目的探讨骨桥蛋白(OPN)对胰腺星形细胞(PSC)的影响及其机制。方法构建OPN慢病毒过表达载体(OPN-O/E)并转染PSC,设置空载体转染细胞作为对照组。分别采用CCK-8实验和Transwell小室检测OPN-O/E转染及OPN-O/E转染联合Akt抑制剂LY294002(10μmol/L、50μmol/L)处理后PSC增殖活性和趋化活性,采用蛋白质印迹法检测α-平滑肌肌动蛋白(α-SMA)及PI3K/Akt信号通路相关蛋白的表达。结果 OPN-O/E转染上调OPN表达后,PSC增殖活性增高、趋化活性增高,细胞中磷酸化PI3K(p-PI3K)、磷酸化Akt(p-Akt)和α-SMA表达水平均增高,与对照组比较差异均有统计学意义(P<0.05或P<0.01);细胞中PI3K和Akt表达水平与对照组相比差异均无统计学意义(P均>0.05)。使用10μmol/L或50μmol/L Akt抑制剂LY294002干预后,细胞中α-SMA及p-Akt的表达被抑制,与OPN-O/E组相比差异均有统计学意义(P均<0.01),Akt的表达无明显变化。结论 OPN通过PI3K/Akt信号通路介导PSC活化,活化后PSC的增殖及趋化活性也增强。
Objective To explore the effect of osteopontin(OPN) on pancreatic stellate cell(PSC) and its mechanisms. Methods We transfected PSC with OPN lentiviral overexpression vector(OPN-O/E) and constructed empty vector control cells(control group). After PSCs were treated with OPN-O/E or OPN-O/E in combination with Akt inhibitor LY294002(10 μmol/L and 50 μmol/L), the proliferation ability and chemotactic activity were detected by CCK-8 and Transwell assays, respectively. The expression levels of α-smooth muscle actin(α-SMA) and related proteins of PI3 K/Akt signal pathway were determined by Western blotting. Results Compared with the control group, proliferation ability and chemotactic activity of PCS were signi?cantly increased in the OPN-O/E group, and the expression levels of phosphorylatedPI3 K(p-PI3 K), phosphorylated-Akt(p-Akt) and α-SMA were also signi?cantly increased(P<0.05 or P<0.01). There were no signi?cant differences in the expression levels of PI3 K or Akt between the OPN-O/E and control groups(both P>0.05).Compared with the OPN-O/E group, the expression levels of α-SMA and p-Akt were signi?cantly inhibited in the PCS treated with 10 μmol/L or 50 μmol/L LY294002(all P<0.01); however, there was no signi?cant difference in the Akt expression.Conclusion OPN can activate PSC through the PI3 K/Akt signaling pathway, and the proliferation ability and chemotactic activity of activated PSC are also increased.
Objective To explore the effect of osteopontin(OPN) on pancreatic stellate cell(PSC) and its mechanisms. Methods We transfected PSC with OPN lentiviral overexpression vector(OPN-O/E) and constructed empty vector control cells(control group). After PSCs were treated with OPN-O/E or OPN-O/E in combination with Akt inhibitor LY294002(10 μmol/L and 50 μmol/L), the proliferation ability and chemotactic activity were detected by CCK-8 and Transwell assays, respectively. The expression levels of α-smooth muscle actin(α-SMA) and related proteins of PI3 K/Akt signal pathway were determined by Western blotting. Results Compared with the control group, proliferation ability and chemotactic activity of PCS were signi?cantly increased in the OPN-O/E group, and the expression levels of phosphorylatedPI3 K(p-PI3 K), phosphorylated-Akt(p-Akt) and α-SMA were also signi?cantly increased(P<0.05 or P<0.01). There were no signi?cant differences in the expression levels of PI3 K or Akt between the OPN-O/E and control groups(both P>0.05).Compared with the OPN-O/E group, the expression levels of α-SMA and p-Akt were signi?cantly inhibited in the PCS treated with 10 μmol/L or 50 μmol/L LY294002(all P<0.01); however, there was no signi?cant difference in the Akt expression.Conclusion OPN can activate PSC through the PI3 K/Akt signaling pathway, and the proliferation ability and chemotactic activity of activated PSC are also increased.
引文
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[14]E R K A N M,W E I S N,PA N Z,S C H WA G E R C,S A M K H A R A D Z E T,J I A N G X,e t a l.O rg a n-,inflammation-and cancer specific transcriptional fingerprints of pancreatic and hepatic stellate cells[J/OL].Mol Cancer,2010,9:88.doi:10.1186/1476-4598-9-88.
[15]COOMBES J D,CHOI S S,SWIDERSKA-SYN M,MANKA P,REID D T,PALMA E,et al.Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis(NASH)fibrosis[J].Biochim Biophys Acta,2016,1862:135-144.
[16]XU J,YI Y,LI L,ZHANG W,WANG J.Osteopontin induces vascular endothelial growth factor expression in articular cartilage through PI3K/AKT and ERK1/2signaling[J].Mol Med Rep,2015,12:4708-4712.
[17]DAI J,PENG L,FAN K,WANG H,WEI R,JI G,et al.Osteopontin induces angiogenesis through activation of PI3K/AKT and ERK1/2 in endothelial cells[J].Oncogene,2009,28:3412-3422.
[2]B A C H E M M G,S C H N E I D E R E,G R O S S H,WEIDENBACH H,SCHMID R M,MENKE A,et al.Identification,culture,and characterization of pancreatic stellate cells in rats and humans[J].Gastroenterology,1998,115:421-432.
[3]UCHIBORI T,MATSUDA K,SHIMODAIRA T,SUGANO M,UEHARA T,HONDA T.IL-6 transsignaling is another pathway to upregulate osteopontin[J].Cytokine,2017,90:88-95.
[4]ZHANG H,GUO M,CHEN J H,WANG Z,DU X F,LIUP X,et al.Osteopontin knockdown inhibitsαv,β3 integrininduced cell migration and invasion and promotes apoptosis of breast cancer cells by inducing autophagy and inactivating the PI3K/Akt/mTOR pathway[J].Cell Physiol Biochem,2014,33:991-1002.
[5]DIANZANI C,BELLAVISTA E,LIEPE J,VERDERIOC,MARTUCCI M,SANTORO A,et al.Extracellular proteasome-osteopontin circuit regulates cell migration with implications in multiple sclerosis[J/OL].Sci Rep,2017,7:43718.doi:10.1038/srep43718.
[6]DONG B,PANG T T.LncRNA H19 contributes to Rh2-mediated MC3T3-E1 cell proliferation by regulation of osteopontin[J].Cell Mol Biol(Noisy-le-grand),2017,63:1-6.
[7]LOK S I,NOUS F M,VAN KUIK J,VAN DER WEIDEP,WINKENS B,KEMPERMAN H,et al.Myocardial fibrosis and pro-fibrotic markers in end-stage heart failure patients during continuous-flow left ventricular assist device support[J].Eur J Cardiothorac Surg,2015,48:407-415.
[8]SAKURAYA K,ENDO A,SOMEYA T,HIRANO D,MURANO Y,FUJINAGA S,et al.The synergistic effect of mizoribine and a direct renin inhibitor,aliskiren,on unilateral ureteral obstruction induced renal fibrosis in rats[J].J Urol,2014,191:1139-1146.
[9]CHEN Y,OU Y,DONG J,YANG G,ZENG Z,LIU Y,et al.Osteopontin promotes collagenⅠsynthesis in hepatic stellate cells by miRNA-129-5p inhibition[J].Exp Cell Res,2018,362:343-348.
[10]NAKAMURA M,OKA M,IIZUKA N,KAWAUCHI S,GONDO T,UENO T,et al.Osteopontin expression in chronic pancreatitis[J].Pancreas,2002,25:182-187.
[11]王域玲,安薇,李桂香,朱建伟,蒋斐.骨桥蛋白在大鼠慢性胰腺炎中的表达及意义[J].中华胰腺病杂志,2018,18:251-255.
[12]KHALIULLIN T O,KISIN E R,MURRAY A R,YANAMALA N,SHURIN M R,GUTKIN D W,et al.Mediation of the single-walled carbon nanotubes induced pulmonary fibrogenic response by osteopontin and TGF-β1[J].Exp Lung Res,2017,43:311-326.
[13]RY C H LíK O VáJ,V E C K A M,JáC H Y M O VáM,MACá?EK J,HRABáK P,ZEMAN M,et al.Osteopontin as a discriminating marker for pancreatic cancer and chronic pancreatitis[J].Cancer Biomark,2016,17:55-65.
[14]E R K A N M,W E I S N,PA N Z,S C H WA G E R C,S A M K H A R A D Z E T,J I A N G X,e t a l.O rg a n-,inflammation-and cancer specific transcriptional fingerprints of pancreatic and hepatic stellate cells[J/OL].Mol Cancer,2010,9:88.doi:10.1186/1476-4598-9-88.
[15]COOMBES J D,CHOI S S,SWIDERSKA-SYN M,MANKA P,REID D T,PALMA E,et al.Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis(NASH)fibrosis[J].Biochim Biophys Acta,2016,1862:135-144.
[16]XU J,YI Y,LI L,ZHANG W,WANG J.Osteopontin induces vascular endothelial growth factor expression in articular cartilage through PI3K/AKT and ERK1/2signaling[J].Mol Med Rep,2015,12:4708-4712.
[17]DAI J,PENG L,FAN K,WANG H,WEI R,JI G,et al.Osteopontin induces angiogenesis through activation of PI3K/AKT and ERK1/2 in endothelial cells[J].Oncogene,2009,28:3412-3422.