Analysis of connexin Cx43 phosphorylation site and its relation with S368 site in rabbits after cerebral vasospasm / 中华神经医学杂志

ABSTRACT

Objective To investigate the phosphorylation site ofconnexin Cx43 and the changes ofS368 site phosphorylation levels in rabbits after cerebral vasospasm (CVS).Methods Seventy-eight New Zealand rabbits were randomly assigned into four groups:healthy control group (n=6),vehicle group (n=24),carbenoxolone (CBX) treatment group (n=24) and menstruum treatment group (n=24).Models of CVS after subara chnoid hemorrhage in the later three groups were established via double blood injection into the cistema; medications were given,respectively,to each group,and according to the different observation times (1,3,7 and 14 d after the success of model making),they were each divided into 4 subgroups (n=6).The phosphorylated Cx43 protein in the basilar arteries was gathered with PhosphoProtein Purification Kit,and mass spectrometric technique was employed to screen out its phosphorylated sites; Westem blotting was used to analyze the changes of Cx43 phosphorylation levels with its S368 antibody.And then,digital subtraction angiography (DSA) was performed to observe the diameter changes of the basilar arteries.Results Four phosphorylation sites (Y265,S364,S365 and S368) in Cx43 were detected by mass spectrometry.As compared with that in the healthy control group,the S368 site phosphorylation level in the vehicle group and menstruum treatment group was significantly increased at each observation time point (P＜0.05),reaching their peak level on the 7th d of observation,and decreasing from the 14th d of observation; the S368 site phosphorylation level in the vehicle group and menstruum treatment group was significantly higher than that in the CBX treatment group (P＜0.05).Meanwhile,DSA showed that the CBX treatment group had significantly reduced CVS areas as compared with the vehicle group (P＜0.05).Conclusion The phosphorylation of Cx43 S368 site may be associated with CVS,which might be one of the mechanisms of CBX releasing CVS.