Role of LRRK2 in spinal dorsal horn in DNP and relationship with NF-κB signaling pathway in rats
10.3760/cma.j.cn131073-20240726-00315
- VernacularTitle:脊髓背角LRRK2在大鼠DNP中的作用及其与NF-κB信号通路的关系
- Author:
Yanyan XU
1
;
Jun ZHOU
;
Quanshui HAO
Author Information
1. 武汉市第一医院麻醉科，武汉 430022
- Publication Type:Journal Article
- Keywords:
Leucine-rich repeat serine-threonine protein kinase-2;
Diabetic neuropathies;
Microglia;
Inflammation;
NF-kappa B;
Spinal cord dorsal horn
- From:
Chinese Journal of Anesthesiology
2025;45(3):341-347
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To evaluate the role of leucine-rich repeat kinase 2 (LRRK2) in the spinal dorsal horn in diabetic neuropathic pain (DNP) and to determine whether the mechanism involved nuclear factor kappa B (NF-κB) signaling pathway in rats.Methods:SPF healthy male Sprague-Dawley rats, aged 8 weeks, weighing 205-238 g, were fed a high-sugar and high-fat diet and received intraperitoneal injection of streptozotocin to induce a type 2 diabetes mellitus model. Successful DNP model was defined as the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) being below 85% of the baseline in type 2 diabetes mellitus rats. Thirty-six rats with DNP were allocated into 3 groups ( n=12 each) using a random number table method: DNP group, MLi-2 (LRRK2 inhibitor) group, and MLi-2+ phorbol 12-myristate 13-acetate (PMA) (NF-κB activator) group. MLi-2 1 mg/kg was intrathecally injected at L 5-6 in MLi-2 group. In MLi-2+ PMA group, MLi-2 1 mg/kg was intrathecally injected, and 1 h later PMA 20 μg/10 μl was intrathecally injected. In group DNP, the equal volume of 5% dimethyl sulfoxide was intrathecally injected. Twelve healthy rats were randomly selected and served as control group (C group). The animals were fed a standard diet, normal saline 2 ml was intraperitoneally injected, and the equal volume of 5% dimethyl sulfoxide was intrathecally injected in group C. Intrathecal injection was performed once a day for 7 consecutive days. The MWT and TWL were measured at 1, 3, 5 and 7 days after intrathecal injection, and the motor nerve conduction velocity (MNCV) was measured using neuroelectrophysiological techniques. The L 4-6 segments of the spinal cord were collected, and histopathological changes were observed through HE staining. The co-localization of leucine-rich repeat kinase 2 (LRRK2) in the spinal dorsal horn with the microglial activation marker ionized calcium-binding adaptor molecule-1 (Iba-1) was observed by immunofluorescence staining. The L 4-6 segments of the spinal dorsal horn were harvested for determination of the level of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 (using enzyme-linked immunosorbent assay) and expression of LRRK2, Iba-1, nuclear factor kappa B (NF-κB) p65 and phosphorylated NF-κB p65 (p-NF-κB p65) (by Western blot). Results:Compared to C group, the TWL was significantly shortened, the MNCV was decreased, the percentage of cells co-expressing LRRK2 and Iba-1 in the spinal dorsal horn was increased, the contents of TNF-α, IL-1β and IL-6 were increased, the expression of LRRK2 and Iba-1 was up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was increased ( P<0.05), and histological examination revealed nuclear shrinkage and inflammatory cell infiltration in the spinal dorsal horn tissue in DNP group. Compared with DNP group, the MWT was significantly increased, the TWL was prolonged, the MNCV was elevated, the percentage of cells co-expressing LRRK2 and Iba-1 was decreased, the contents of TNF-α, IL-1β and IL-6 were decreased, the expression of LRRK2 and Iba-1 was down-regulated, the ratio of p-NF-κB p65/NF-κB p65 was decreased ( P<0.05), and pathological changes of the spinal dorsal horn tissue were significantly attenuated in MLi-2 group. Compared to MLi-2 group, the MWT was significantly decreased, the TWL was shortened, the MNCV was decreased, the percentage of cells co-expressing LRRK2 and Iba-1 was increased, the contents of TNF-α, IL-1β and IL-6 were increased, the expression of Iba-1 was up-regulated, and the ratio of p-NF-κB p65/NF-κB p65 was increased in MLi-2+ PMA group ( P<0.05). Conclusions:LRRK2 in spinal dorsal horn may be involved in the maintenance of DNP by promoting the inflammatory response mediated by microglial activation, and the mechanism may be related to the activation of NF-κB signaling pathway in rats.
