Study Of The Interaction Between Endoplasmic Reticulum Chaperone BIP And Nav1.8 Protein In Neuropathic Rats

Objective:In this study,the chronic constriction injury?CCI?model was established to identify the interaction between heavy-chain immunoglobulin binding protein?BIP?and Nav1.8 protein.In order to lay a foundation for further studying the interaction sites between BIP and Nav1.8 in neuropathic pain?NP?,and the mechanism of BIP in the expression and redistribution of Nav1.8 after nerve injury.Methods:Twenty four male Sprague-Dawley rats?220²50 g?were randomly divided into two groups?n=12?:CCI group and Sham group.The CCI model was built according to methods of Bennett.The Sham group only exposed the sciatic nerve and did not ligate the sciatic nerve.To evaluate the behavioral changes,the paw withdrawal mechanical threshold?PWMT?and Tail flick latency?TFL?were measured on 1 day before operation and 1 day,3 days,5days,7 days,10 days and 14days after operation,respectively.After finishing behavioral tests on 7 days and 14 days after operation,L₄^<sub>6 dorsal root ganglion?DRG?and sciatic nerve tissue of the injured side were taken for Western blot to detect the expression of BIP and Nav1.8 protein,immunofluorescence staining to observe the colocalization between BIP and Nav1.8 protein in the cells,and co-immunoprecipitation to identify the interaction between BIP and Nav1.8protein.Results:?1?There was no significant difference in PWMT between 1 day before operation and 1 day,3 days,5 days,7 days,10 days and 14days after surgery in Sham group?P>0.05?.There was no significant difference in PWMT between CCI group and Sham group on 1 day before surgery?P>0.05?.Compared with the baseline value of PWMT on 1 day before surgery,the PWMT at different times after surgery in the CCI group was significantly decreased?P<0.05?.Compared with Sham group,the PWMT at different times after surgery in the CCI group was significantly decreased?P<0.05?.?2?There was no significant difference in TFL between 1 day before operation and 1 day,3 days,5 days,and 7 days after surgery in Sham group?P>0.05?.There was no significant difference in TFL between CCI group and Sham group on 1 day before surgery?P>0.05?.Compared with the baseline value of TFL on 1 day before surgery,the TFL at different times after surgery in the CCI group was significantly decreased?P<0.05?.Compared with Sham group,the TFL at different times after surgery in the CCI group was significantly decreased?P<0.05?.?3?Compared with Sham group,the expression of BIP protein on DRG in CCI group was significantly increased?P<0.05?;Compared with Sham group,the expression of Nav1.8 protein on DRG in CCI group was significantly decreased?P<0.05?.Compared with Sham group,the expression of BIP protein in sciatic nerve in CCI group was significantly increased?P<0.05?.Compared with Sham group,the expression of Nav1.8 protein in sciatic nerve in CCI group was significantly increased?P<0.05?.?4?BIP and Nav1.8 were widely distributed in DRG.Compared with Sham group,the fluorescence density of BIP protein on DRG in CCI group was significantly increased?P<0.05?.There was no significant difference in the fluorescence density of Nav1.8 protein on DRG between CCI group and Sham group?P>0.05?.What's more,there was a significant co-localization between Bip and Nav1.8 on the DRG of CCI rats.BIP and Nav1.8 were widely distributed in the sciatic nerve.Compared with the Sham group,the fluorescence intensity of BIP protein on the sciatic nerve in the CCI group was significantly increased?P<0.05?.Compared with the Sham group,the fluorescence density of Nav1.8 protein on the sciatic nerve in the CCI group was significantly increased?P<0.05?.Moreover,there was a significant co-localization between BIP and Nav1.8 in the sciatic nerve of CCI rats.?5?BIP can co-precipitate Nav1.8 from DRG of CCI rats,while IgG in negative control group cannot precipitate Nav1.8;conversely,Nav1.8 also can co-precipitate BIP from DRG of CCI rats,but IgG cannot precipitate BIP.Conclutions:?1?The expression of Nav1.8 protein in CCI rats was redistributed and accumulated on the sciatic nerve near the injury site.?2?ERS occurred in the DRG and sciatic nerve of CCI rats,which increased the expression of BIP protein.?3?There is an interaction between BIP and Nav1.8 in the DRG of CCI rats.