›› 2017, Vol. 37 ›› Issue (11): 1459-.doi: 10.3969/j.issn.1674-8115.2017.11.002

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Bilirubin-induced calcium overload in synaptosomes isolated from brainstem neurons of rats#br#

LI Dan-ping, LAI Ke, WANG Ji-ping, SHI Hai-bo   

  1. Department of Otorhinolaryngology, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, China
  • Online:2017-11-28 Published:2018-01-10
  • Supported by:
     National Natural Science Foundation of China, 81371092,81570909; Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support, 20152233; Clinical Research Promotion Plan of Shanghai Shen Kang Hospital Development Center, 16CR3041A

Abstract: Objective · To observe real-time changes of calcium concentration ([Ca2+]i) exposure to bilirubin in synaptosomes isolated from brainstem nucleus of rats.  Methods · Forty P7-14 SD rats were randomly assigned to three groups: control group, bilirubin group (with levels of 0.1, 1 and 10 μmol/L) and bulirubin plus glycoursodeoxycholic acid (GUDCA) group. The synaptosomes were purified from brainstem nucleus by sucrose density gradient centrifugation. After loading OG-BAPTA in synaptosomes, two dimensional image of intracellular calcium and analysis of fluorescence intensity were achieved by Confocal laser scanning microscopy.  Results · Synaptosomes with well biological activity were obtained from brainstem of the SD rats. In the control group, a progressive increase in fluorescent intensity of [Ca2+]i was detected. In the bilirubin group, acuter increases in fluorescent intensity were observed in all levels of bilirubin, with a manner of both concentration and time-dependent (P<0.05). Fluorescent intensity of [Ca2+]i was reduced  in the present of GUDCA, which was not significant compared with the control group (P=0.656). However, GUDCA could abate the increase of fluorescent intensity of [Ca2+]i induced by bilirubin exposure, of which showing significant decrease in 10 μmol/L bilirubin exposure (P=0.000).  Conclusion · Bilirubin could induce calcium overload in synaptosomes. GUDCA could abate bilirubin-induced calcium overload in synaptosomes, possibly explaining its protection effect of neurons from bilirubin neurotoxicity.

Key words:  bilirubin, synaptosome, intracellular calcium, glycoursodeoxycholic acid, fluorescent probe