Simvastatin Attenuates Neuropathic Pain By Inhibiting The RhoA/LIMK/cofilin Pathway

Objective:Neuropathic pain occurs due to deleterious changes in the nervous system caused by a lesion or dysfunction. Currently, neuropathic pain management is unsatisfactory and remains a challenge in clinical practice. Studies have suggested that actin cytoskeleton remodeling may be associated with neural plasticity and may involve a nociceptive mechanism. Here, we found that the RhoA/LIM kinase (LIMK)/cofilin pathway, which regulates actin dynamics, was activated after chronic constriction injury (CCI) of the sciatic nerve. In this study, we found that over-activation of the cytoskeleton caused by RhoA/LIMK/cofilin pathway activation may produce a scaffold for the trafficking of nociceptive signaling factors, leading to chronic neuropathic pain. Intrathecal administration of simvastatin significantly decreased the ratio of membrane/cytosolic RhoA, which was significantly increased after CCI, by inhibiting the RhoA/LIMK/cofilin pathway. We conclude that simvastatin attenuated neuropathic pain in rats subjected to CCI by inhibiting actin-mediated intracellular trafficking to suppress RhoA/LIMK/cofilin pathway activity.Methods:To evaluate the expression and activation of RhoA/LIMK/cofilin pathway in CCI rats, seventy-two male SD rats weighing 180-200 g were randomly divided into the CCI group (n=30), the sham group (n=30) and the naive group (n=12). The rats in each group were subjected to CCI of the sciatic nerve. Behavioral tests were performed on days 1,3,7,14 and 21 after surgery. L4-L6 dorsal root ganglia (DRGs) were removed at each time point. Real-time quantitative polymerase chain reaction (RT-PCR) was used to test RhoA mRNA expression. Western blot was performed to evaluate the protein expression of RhoA, phosphorylated LIM kinases, phosphorylated cofilin and the ratio of membrane/cytosolic RhoA. To evaluate the effects of treatments that reduced RhoA/LIMK/cofilin pathway activity on CCI rats’pain behavior and its molecular mechanism, rats were randomly divided into the following groups:the simvastatin group and the vehicle group. Simvastatin (1 μg/μl,10 ul/d) or the same volume of vehicle was injected into the intrathecal tube once daily after CCI for 7 days; Y-27632 (4 mg/ml in sterile saline,48 μg/d), a Rho kinase inhibitor, or the same volume of vehicle, was administered intrathecally after CCI surgery; Synthetic peptide Tat-S3 (1 μg/μl,30 μg/d) was administered intrathecally for 7 days. An equal volume of the peptide Tat-S3A, in which Ser is mutated to Ala, was used as a control. Behavioral tests were then performed. L4-L6 DRGs were removed on postoperative day 14, followed by RT-PCR and Western blot analyses to detect the expression and the location of RhoA, as well as the phosphorylation of LIMK and coffilin.Results:CCI surgery induced mechanical allodynia and thermal hyperalgesia in rats. Compared with the sham group, a decrease in the PWTL was observed from day 3 after surgery and a decrease in the PWMT was observed from day 7 after surgery in the CCI group. Administration of simvastatin significantly attenuated heat hyperalgesia and mechanical allodynia 3 days after administration (P<0.05). Y-27632 relieved CCI-induced neuropathic pain detected by increased PWTL and PWMT 3 days after administration (P<0.05). Intrathecal administration of the Tat-S3 peptide attenuated CCI-induced mechanical and thermal pain-related behavior (P<0.05). CCI induced the activation of the RhoA/LIMK/cofilin pathway in L4-L6 DRGs. The level of RhoA mRNA in L4-L6 DRGs was significantly increased from postoperative day 3. At the protein level, CCI resulted in the overexpression of RhoA from postoperative day 7 to postoperative day 21 compared with sham or naive treatment. The expression of phosphorylated LIMK and the phosphorylated cofilin were increased from postoperative day 7 to day 21 in the CCI group compared with the sham and naive groups. Increased RhoA expression was detected via immunofluorescence in CCI group compared with the naive group. Intrathecal simvastatin treatment inhibited RhoA/LIMK/cofilin pathway activation. The protein expression levels of RhoA, phosphorylated LIMK and phosphorylated cofilin were significantly decreased after intrathecal simvastatin administration compared with vehicle administration. Intrathecal treatment with simvastatin significantly alleviated the CCI-induced increase in the ratio of membrane/cytosolic RhoA.Conclusion:The RhoA/LIM kinase (LIMK)/cofilin pathwaywas activated after chronic constriction injury (CCI) of the sciatic nerve. Simvastatin attenuated neuropathic pain in rats subjected to CCI by inhibiting RhoA/LIMK/cofilin pathway activity and depressing the intracellular trafficking of RhoA. Therefore, simvastatin may be a novel target for neuropathic pain.