The Mechanism Of Autism-related Gene Shank3 On The Neuropathic Pain

Objective: Neuropathic pain is the most common refractory pain in clinical practice and the specific mechanism remains unclear.The purpose of this study is to explore the role of Shank3 in neuropathic pain and its possible molecular mechanism.Methods: Sixty adult male Sprague Dawley(SD)rats of 180～250g were randomly divided into 4 groups: sham group(Sham),neuropathic pain group(SNI),neuropathic pain with negative control virus group(SNI plus NC-LV),and neuropathic pain with virus group(SNI plus Shank3-LV),with 15 rats per group.The Sham group was established by exposing the sciatic nerve of the left thigh and then suturing.The SNI model was performed by selective sciatic nerve injury on the left thigh.The lentiviruses of NC-LV and Shank3-LV were injected into the spinal cord dorsal horn of SNI rats in SNI plus NC-LV group and SNI plus Shank3-LV group.The paw paw mechanical threshold(PWMT)was measured on days 0,1,3,5,7,9,11,and 14,respectively.On the 5th day,part of the rats were sacrificed and collected.The Shank3 in the spinal cord of L4-L6 was examined by RT-PCR and Western blot.On the 14 th day,the remaining rats were sacrificed and the spinal cord and dorsal root ganglion(DRG)of L4-L6 were removed.The immunohistochemical staining was performed to detect the co-localization of Shank3 and HCN2,Shank3 and PSD95 in spinal cord dorsal horn.RT-PCR and Western blot was performed to detect the expression of Shank3 and PSD95 in the spinal cord and HCN2 in the DRG and spinal cord.Results: All rats had the same basic PWMT before surgery,but from the 3rd day,the PWMT began to decrease and the tendency was most significant on the5 th day(P<0.05)in the SNI group,and it lasted until the 14 th day when compared with Sham group.Compared with SNI group,the PWMT of SNI plus NC-LV group had no significant difference(P>0.05),while the SNI plus Shank3-LV group decreased significantly from day 5th day(P<0.05)and lasted until the 14 th day.The immunohistochemical staining showed that Shank3 andHCN2,Shank3 and PSD95 were co-located in the dorsal horn of the spinal cord and the expression levels were up-regulated in the SNI group.The quantitative analysis showed that the expression of Shank3 was increased(P<0.05)in the spinal dorsal horn of SNI group and was decreased in SNI plus Shank3-LV group when compared with SNI plus NC-LV group(P<0.05)on the 5th day.On the14 th day,compared with Sham group,Shank3 and PSD95 were increased in the spinal dorsal horn and HCN2 was increased in spinal dorsal horn and DRG of SNI group(P<0.05).Compared with SNI plus NC-LV group,Shank3 and PSD95 were decreased in the spinal dorsal horn in SNI plus Shank3-LV group(P<0.05),while HCN2 was decreased in the spinal dorsal horn and DRG in SNI plus Shank3-LV group(P<0.05).Conclusion: Our study found that Shank3 induces neuropathic pain by regulating the expression of HCN2 and PSD95 in the spinal dorsal horn and DRG.