Protective Role of MerTK in Diabetic Peripheral Neuropathy via Inhibition of the NF-κB Signaling Pathway

Abstract

Introduction Diabetic peripheral neuropathy (DPN) impacts patient quality of life. In such patients, increased expression of mer tyrosine kinase (MerTK) has been demonstrated; however, its mechanism of action remains unclear. In this study, type 2 diabetes mellitus (T2DM) and DPN models were established in Sprague Dawley rats via low-dose streptozotocin and a high-fat diet and the mode of action of MerTK was examined.

Methods MerTK-specific inhibitors were administered by gavage once daily for 2 weeks. Sciatic nerve conduction velocity and nerve structure were measured. The levels of MerTK, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and relevant biochemical indexes were detected.

Results The study revealed upregulation of MerTK expression in T2DM and more so in DPN groups. Inhibiting MerTK led to reduced nerve conduction velocity and further deterioration of sciatic nerve structure, as evidenced by structural morphology. Concurrently, serum levels of total cholesterol, glycated hemoglobin, and triglyceride significantly increased. Moreover, levels of NF-κB increased in both serum and nerve tissue, alongside a significant rise in TNF-α and IL-1β expressions. MerTK could bind to the inhibitor of kappa B kinase beta (Ikbkb) in Schwann cells, establishing Ikbkb as a precursor to NF-κB activation.

Discussion Inhibition of MerTK exacerbates neuropathy, indicating its protective role in DPN by suppressing the NF-κB pathway, highlighting a potential new target for its diagnosis and treatment.

Keywords

diabetic peripheral neuropathy - MerTK - NF-κB signaling pathway - inflammatory factors - protective effect

Introduction

Diabetes mellitus (DM) is a chronic metabolic disorder with increasing incidence in developing countries, representing a global health concern. Its prevalence is estimated to reach 642 million by 2040 [1] [2]. Patients with diabetes usually present a series of macrovascular and microvascular complications, such as ischemic heart disease, nephropathy, retinopathy, and neuropathy [3] [4]. In addition to disease duration and glucose control, genetic background may influence diabetic complications [5]. Diabetic neuropathy (DN) is a syndrome characterized by lesions in the central and peripheral nervous systems caused by chronic hyperglycemia; notably, it is one of the most common diabetic target organ damage affecting both patients with type 1 DM and those with type 2 DM (T2DM) [6]. Diabetic peripheral neuropathy (DPN) is a major type of DN with an incidence rate of 58.5% in patients with diabetes, which severely impairs the quality of life of patients and is also associated with a high mortality rate [7] [8]. However, the clinical onset of DPN is often severe, and disease development is relatively long [9]. The early diagnosis of DPN is challenging. Thus, studying the pathogenesis and identifying early biomarkers of DPN will contribute to the effective prediction and intervention of DPN progression.

In our previous studies, we reported that mer tyrosine kinase (MerTK) levels were significantly increased both in the peripheral blood of patients with DPN and in the sciatic nerve of early DPN mice, suggesting that changes in the MerTK gene may be a potential biomarker for the early diagnosis of DPN [10]. Specifically, MerTK, a member of the MER/AXL/TYRO3 tyrosine receptor kinase family, is involved in phagocytosis and encodes a transmembrane protein that is distributed on the surface of various specific and non-specific phagocytic cells, such as retinal pigment epithelial cells, macrophages, and dendritic cells [11]. Recent studies have implicated the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the pathophysiological mechanism of DPN [12] [13]. NF-κB is an important precursor of proinflammatory factor and a heterodimer comprising p65 (RelA) and p50 [14]. The occurrence of DPN is commonly attributed to endothelial cell activation and inflammatory gene induction, regulated centrally by the transcription factor NF-κB [15] [16]. Previous studies have found the association of the MER/AXL/TYRO3 family with the NF-κB pathway [17], while MerTK can alleviate Staphylococcal lipophosphate-induced inflammatory response by blocking the activation of NF-κB [18]. Therefore, we speculate that MerTK may influence the development of DPN by regulating the NF-κB signaling pathway.

To simulate the disease characteristics of T2DM progressing to DPN, a T2DM animal model was established in Sprague Dawley (SD) rats by injecting a high-sugar and -fat diet combined with a low-dose streptozotocin (STZ). The DPN model was established by continuously feeding a high-sugar and high-fat diet for 8 weeks. We focused on the administration of MerTK-specific inhibitors MRX-2843, the conduction velocity and structural changes of the sciatic nerve, and the levels of MerTK, NF-κB, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and relevant biochemical indexes. Our study aimed to determine the regulatory role of MerTK in the NF-κB signaling pathway and its effect on the pathogenesis of DPN to provide a new biomarker for the early prediction of DPN as well as an effective therapeutic target for the treatment of DPN.

Materials and methods

Animal studies

All experiments were performed after obtaining approval from the Animal Experimentation Committee of Kunming Medical University and in accordance with the institutional regulations (No. kmmu20221595). Overall, 70 SD rats (8-week-old, 310–350 g) from the Animal Center of Kunming Medical University (No. 2019–0004) were divided into 5 groups, with 14 rats per group. The control (CON) group received an ordinary diet and 0.2 mL of 0.9% normal saline (NS) by gavage daily. The CON+MRX-2843 group was similarly fed but received a 0.2 mL 65 mg/kg MRX-2843 suspension daily for 2 weeks. The T2DM group was fed a high-fat and -sugar diet for 6 weeks, and then injected with a 1.5 mL solution of STZ (35 mg/kg) to induce diabetes. Blood glucose levels were measured on days 3, 7, and 14 post-STZ administration, with levels+≥+16.7 mmol/L indicating successful T2DM modeling. Rats then received an ordinary diet and 0.2 mL 0.9% NS gavage daily. For the DPN group, post-T2DM establishment, rats were switched to a high-fat and -sugar diet for 8 weeks, followed by an ordinary diet and daily 0.2 mL 0.9% NS gavage. In the DPN+MRX-2843 group, after DPN establishment, a 65-mg/kg MRX-2843 suspension was administered daily for 2 weeks.

Detection of sciatic nerve conduction velocity (NCV)

Based on the body weight and status of the rats, 0.9% pentobarbital sodium (30 mg/kg) was injected intraperitoneally, the sciatic nerve was isolated, and the NCV was detected. The double-stimulation needle electrode was placed at the left sciatic notch, and the recording electrode was placed between the second toe of the right plantar. The reference electrode was placed between the stimulation electrode and the recording electrode, approximately 1 cm away from the recording electrode. The intensity of the stimulation was gradually increased from a small value, and the measured electromyography was recorded over time. NCV was indicated according to the ratio of latency to conduction distance of compound action potential, calculated by dividing the distance between the stimulation electrode and the recording electrode by the difference obtained from the proximal latency minus the distal latency and expressed in meters per second. The Biological Function Experimental System was provided by the Biological Function Laboratory of the Kunming Medical University. Sciatic NCV measurement of<40 m/s indicated successful DPN modeling [19].

Detection of blood biochemical indexes

Blood was collected from the hearts of rats after execution, separated at 2795 G for 15 min at 4°C, and the upper plasma was obtained after stratification. Blood glucose, triglyceride (TG), and total cholesterol (TC) levels were measured using a biochemical analyzer (BS-240VET, Shenzhen Mairead Company). Insulin (INS) and glycated hemoglobin (GHB) levels were measured using enzyme-linked immunosorbent assay (ELISA) kits (INS: Elabscience Item, No. E-EL-R2466; GHB: Mlbio Item, No. ml059468).

Enzyme-linked immunosorbent assay

The levels of MerTK, NF-κB (P65), TNF-α, and IL-1β in the serum were determined by the double antibody sandwich method. Purified rat antibodies were coated onto each microwell plate to prepare solid-phase antibodies, and allowed to react with horseradish peroxidase (HRP)-labeled antibodies added to the corresponding coated microwell. The antibody-antigen-enzyme-labeled antibody complex was formed, and tetramethylbenzidine (TMB) substrate was added for color development after thorough washing. TMB was transformed into blue under the catalysis of the HRP enzyme and into yellow under the action of an acid. Absorbance (optical density value) was measured at 450 nm using a microplate reader to calculate the concentrations of MerTK, NF-κB (P65), TNF-α, and IL-1β in samples via standard curves from ELISA kits (MerTK: Thermo Fisher, EHMER; NF-κB(P65): Elabscience, E-EL-R0674; TNF-α: Elabscience, E-EL-R2856; IL-1β: Elabscience, E-EL-R0012).

Detection of sciatic nerve histopathology

The sciatic nerve was fixed in 10% buffered formaldehyde. The fixed tissues were soaked in alcohol for dehydration and placed in dimethylbenzene. The tissue was soaked and permeated in paraffin, and the paraffin-embedded tissue was sliced into about 5-µm-thick slices. The tissue was baked at 60°C in an incubator for 30 min. The tissues were then hydrated. Hematoxylin-eosin (HE) staining was performed using hematoxylin for 10–20 min. After rinsing with water, the solution was placed in a weakly alkaline aqueous solution until blue color appeared. Next, 85% alcohol was added for 3–5 min, followed by staining with eosin for 3–5 min. The toluidine blue-stained section was incubated in 1% toluidine blue aqueous solution at 50°C, then at 56°C for 20 min. After rinsing, samples were immersed in 70% alcohol for 1 min, dehydrated, sealed, and examined under a light microscope.

Observation by electron microscopy

Sciatic nerve tissue, approximately 1 mm thick, was treated with 4% glutaraldehyde for pre-fixation and 1% acid for post-fixation. After rinsing with phosphate buffered saline (PBS), the sections were dehydrated with acetone, embedded in epoxy resin, sliced using an ultrathin slicer, stained with uranium acetate and lead citrate solution in a box, and observed and photographed using a transmission electron microscope (JEM-1400).

Immunohistochemistry

The sciatic nerve was fixed in 10% buffered formaldehyde, dehydrated using an ethanol gradient, embedded in paraffin, and sliced. The sections were immersed in citrate buffer (PH 6.0) for repair and then cleaned with PBS. The slices were then treated with a buffer-blocking solution (5% goat serum). The blocking solution of goat serum was added, and the first antibody of MerTK (1:100, Affinity, USA) and NF-κB (1:100, Affinity, USA) was added. The glass slides were placed overnight in a 4°C refrigerator. The sections were rinsed with PBS and incubated with secondary antibodies, including goat anti-rabbit IgG (H+L) HRP (1:200, Affinity, USA) and goat anti-mouse IgG (H+L) HRP (1:200, Affinity, USA). The 3,3'-Diaminobenzidine (DAB) chromogen solution was added to terminate the staining. Finally, the experimental results were observed under an optical microscope; the brown or brown-yellow particles in the cell membrane or cytoplasm were considered protein expression. Four fields from each section were randomly selected under a microscope.

Immunofluorescence staining

The sciatic nerve was fixed in 10% buffered formaldehyde, dehydrated using an ethanol gradient, embedded in paraffin, and sliced. The sections were then immersed in a citrate buffer (PH 6.0) for repair. Goat serum was added to the blocking solution, and the primary antibody against MerTK (Affinity: DF4785) was added. The glass slides were put in a wet box and placed in a 4°C refrigerator overnight. After rinsing with PBS buffer, the slices were added with the second antibodies of goat anti-rabbit IgG (H+L) HRP (1:200, Affinity, USA) and treated at 37°C for 30 min. After washing thrice with PBS, DAPI was added and incubated at room temperature for 10 min. This was rinsed with PBS and the slices were sealed with anti-fluorescence attenuating tablets. Finally, images were captured and observed by fluorescence microscopy.

Western blot analysis

Total protein was extracted using RIPA buffer (Beyotime Biotechnology, China), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride membrane (Millipore ISEQ00010). Subsequently, the membranes were incubated with primary antibody (1:1000 for anti-MerTK, Abcam, ab52981; 1:1000 for anti-NF-κB, Proteintech, 80979–1-RR; 1:3000 for anti-TNF-α, Proteintech, 60291–1-Ig; 1:3000 for anti-β-actin, Proteintech, 81115–1-RR) and kept at 4°C overnight. Detection was performed using a horseradish peroxidase secondary antibody (goat anti-mouse IgG[H+L] HRP, 1:3000, Affinity, USA) after incubation at room temperature for 2 h. Subsequently, protein expression was visualized using an ECL kit and an X-ray film. Membranes displayed luminescence using a Bio-Rad luminescence imaging system.

Co-immunoprecipitation (co-ip) assays

The interaction of endogenous MerTK and the inhibitor of kappa B kinase beta (Ikbkb) was detected by the co-ip assays using RSC96 cells. RSC96 cells were lysed and centrifuged. The input protein was retained, and MerTK, Ikbkb, and IgG primary antibodies were added to the resulting product. After overnight incubation at 4°C, 5 µL Protein A and Protein G were added and incubated, washed, and centrifuged. Following incubation, washing, centrifugation, resuspension in SDS buffer, and denaturation, the final product was obtained, and finally, the interaction between MerTK and Ikbkb was detected by immunoblotting. An immunoprecipitation kit was purchased from Shanghai Ebixin Technology Co.

Statistical analysis

All experiments were performed at least three times. Data are expressed as the mean+±+standard deviation. A normality test was performed to determine normal distribution. Student t-test was used to compare the means between two groups, while the data among multiple groups were analyzed using one-way analysis of variance followed by the "Tukey or Dunnet post hoc" test. All statistical analyses were performed using SPSS 22.0 software. Statistical significance was defined as a P-value<0.05.

Results

Successful establishment of the diabetic peripheral neuropathy rat model, and a decrease in nerve conduction velocity after mer tyrosine kinase inhibition

Rats fed on a high-fat and -sugar diet combined with STZ injections exhibited typical symptoms of T2DM, such as hyperglycemia, polyuria, polydipsia, polyphagia, and weight loss. The DPN rat model was established by continuously feeding rats high-fat and -sugar diets and was confirmed by blood glucose detection and electrophysiological experiments. After 8 weeks of continued high-fat and -sugar feeding, diabetic rats exhibited significantly lower body weights and higher blood glucose levels than the non-diabetic rats. MRX-2843 had no significant influence on the body weight or blood glucose levels in the inhibitor group ([Fig. 1a], b). Moreover, sciatic NCV was significantly reduced in the DPN group, and NCV was significantly lower in the DPN+MRX-2843 group than in the DPN group ([Fig. 1c]).

Fig. 1 Basic parameters of rats in each group. (a) Changes in body weight in each group at weeks 2, 4, 6, 8, 10, 12, 14, and 16. No significant changes were observed in the body weight of the rats after MerTK inhibition. (b) Changes in blood glucose levels in each group. (c) Changes in sciatic NCV in each group at week 18. *p<0.05. Group counts were as follows: CON: 14; CON+MRX-2843: 14; T2DM: 10; DPN: 12; DPN+MRX-2843: 10. All rats were aged 96 weeks. MerTK, mer tyrosine kinase; NCV, nerve conduction velocity; CON, control group; MRX, MerTK-specific inhibitor; T2DM, type 2 diabetes mellitus; DPN, diabetic peripheral neuropathy.

Changes of mer tyrosine kinase expression in each group

MerTK expression was detected in the longitudinal section of the sciatic nerve using immunofluorescence ([Fig. 2]), immunohistochemistry ([Fig. 3]), and western blotting ([Fig. 4a]), and in the serum of rats using ELISA ([Fig. 4b,c]). The results showed that MerTK expression was upregulated in both the T2DM and DPN groups and increased significantly in the DPN group than in the CON and T2DM groups. After the MerTK inhibitor MRX-2843 was administered, MerTK expression decreased significantly in the CON+MRX-2843 and DPN+MRX-2843 groups, indicating that the MerTK inhibitor effectively inhibited MerTK.

Fig. 2 Immunofluorescence detection of MerTK expression in each group. Confocal images of MerTK (green) and DAPI (blue) staining of the nucleus. Scale bar=500 μm (first column). Scale bar=50 μm (columns 2, 3, and 4). Rat counts per group were: CON: 14, CON+MRX-2843: 14, T2DM: 10, DPN: 12, DPN+MRX-2843: 10, with a uniform age of 96 weeks. Sciatic nerves served as the study tissue. MerTK, mer tyrosine kinase; CON, control group; MRX, MerTK-specific inhibitor; T2DM, type 2 diabetes mellitus; DPN, diabetic peripheral neuropathy.

Fig. 3 Immunohistochemical detection of MerTK expression in each group. MerTK was expressed in all the groups and was significantly upregulated in the DPN group. Scale bars=50 μm. Rat counts per group were: CON: 14, CON+MRX-2843: 14, T2DM: 10, DPN: 12, DPN+MRX-2843: 10, with all rats aged 96 weeks. The sciatic nerve was the tissue of interest. MerTK, mer tyrosine kinase; CON, control group; MRX, MerTK-specific inhibitor; T2DM, type 2 diabetes mellitus; DPN, diabetic peripheral neuropathy.

Fig. 4 Detection of MerTK expression in each group by western blotting and ELISA. (a) Immunoblotting images of MerTK are representative of each group. MerTK protein levels were detected in different groups using western blotting. Gene expression was normalized to the β-actin levels in each sample. (b) Serum MerTK levels were measured by ELISA. (c) Phosphorylated MerTK (p-MerTK) serum levels were also determined by ELISA. *p<0.05; ns=no statistical difference. Rat numbers were: CON: 14, CON+MRX-2843: 14, T2DM: 10, DPN: 12, DPN+MRX-2843: 10; rats of all groups were 96 weeks old. ELISA, enzyme linked immunosorbent assay; MerTK, mer tyrosine kinase; CON, control group; MRX, MerTK-specific inhibitor; T2DM, type 2 diabetes mellitus; DPN, diabetic peripheral neuropathy.

Changes of insulin, total cholesterol, glycated hemoglobin, and triglyceride in each group

INS, TC, GHB, and TG levels were notably higher in the DPN group than in the CON group, indicating the involvement of glucose and lipid metabolism disturbances and insulin resistance in the development of DPN. Inhibiting MerTK expression significantly raised TC, GHB, and TG serum levels, exacerbating these metabolic disorders ([Fig. 5]).

Fig. 5 Levels of INS, TC, GHB, and TG in the serum. (a) INS levels in each group. (b) TC levels in each group. (c) GHB levels in each group. (d) TG levels in each group. *p<0.05; ns, no statistical difference. Rat group counts were: CON: 14, CON+MRX-2843: 14, T2DM: 10, DPN: 12, DPN+MRX-2843: 10. Rats in all groups were 96 weeks old. INS, insulin; TC, total cholesterol; GHB, glycated hemoglobin; TG, triglyceride; CON, control; MRX, MerTK-specific inhibitor; DPN, diabetic peripheral neuropathy; T2DM, type 2 diabetes mellitus.

Changes in structural morphology in the longitudinal sections of the sciatic nerve

After HE staining, the sciatic nerves in the CON group showed that the medullated nerve fibers were evenly distributed, continuous, complete, neat, and orderly, and the myelin sheaths were regular in shape and densely arranged. In the DPN group, the nerve fibers were arranged sparsely and chaotically, with irregular morphology and unclear borders. Some axons were shrunken or lost, which was accompanied by demyelination and myelinolysis. The structure of the medullated nerve fibers was disordered in cross-section, with axonal swelling accompanied by axon shrinkage and partial demyelination. Toluidine blue staining revealed that the nerve fibers in the CON group were dense, the individual fibers were full, and the thickness of the myelin sheath was uniform. In the DPN group, the nerve fibers were loose, the myelin sheath was thin, and the axons were small and centrally located. After MerTK inhibition, the structure of the nerve fibers was further disrupted, and the lesion worsened ([Fig. 6a]).

Fig. 6 Structural changes in the sciatic nerve were observed by light and electron microscopes. (a) The structure of the sciatic nerve was observed under light microscopes after the HE and toluidine blue staining, respectively (scale bar=50 μm). (b) The structure of the sciatic nerve was observed under an electron microscope at magnifications of 4000x,8000x and 20000x. The rat group counts were: CON: 14, CON+MRX-2843: 14, T2DM: 10, DPN: 12, DPN+MRX-2843: 10; the age of rats in each group was 96 weeks. HE, hematoxylin and eosin; CON, control; MRX, MerTK-specific inhibitor; DPN, diabetic peripheral neuropathy; T2DM, type 2 diabetes mellitus.

Under an electron microscope, the sciatic nerve in the CON group exhibited evenly arranged medullated nerve fibers, the structure of the myelin sheath was intact and dense, and the morphology of Shewanʼs cells showed no obvious abnormalities. In the DPN group, the shape of myelinated nerve fibers was irregular in cross-section, the myelin sheath was loose and foveolar, some of the myelin sheaths were deformed and vacuolated, and microtubules and microfilaments in axons were disturbed or had disappeared. The nuclei of Shewan cells had an irregular morphology with dilated perinuclear gaps, and the nuclear membranes were partially dissolved and ruptured. After MerTK inhibition, the looseness of the nerve fibers was aggravated, axons were reduced, and lesions worsened ([Fig. 6b]).

Expression of nuclear factor-κB and downstream inflammatory factors was upregulated upon the inhibition of mer tyrosine kinase expression

The expression of NF-κB (P65) was determined via immunohistochemistry ([Fig. 7]), western blotting ([Fig. 8]), and ELISA ([Fig. 9a]). The inflammatory factors TNF-α and IL-1β were detected by western blotting ([Fig. 8]) and ELISA ([Fig. 9 (b and c)], respectively). The results indicated upregulated expressions of P65, TNF-α, and IL-1β in the DPN group, with further significant increases following MerTK inhibition, suggesting the protective role of MerTK in DPN by inhibiting the NF-κB pathway.

Fig. 7 Immunohistochemical detection of NF-κB (P65) expression level in each group. P65 was expressed in all groups, and the expression of P65 was upregulated in the DPN group and further upregulated after MerTK inhibition. Scale bar=50 μm. Rat numbers per group were: CON: 14, CON+MRX-2843: 14, T2DM: 10, DPN: 12, and DPN+MRX-2843: 10, all rats aged 96 weeks. The sciatic nerve was selected as the study tissue. CON, control; MerTK, mer tyrosine kinase; MRX, MerTK-specific inhibitor; DPN, diabetic peripheral neuropathy; T2DM, type 2 diabetes mellitus.

Fig. 8 Detection of NF-κB (P65) and TNF-α expression levels in each group by western blotting. Immunoblotting images of P65 and TNF-α are representative of each group. The levels of P65 and TNF-α proteins were detected in different groups by western blotting. Gene expression was normalized to the β-actin levels in each sample. *p<0.05; ns, no statistical difference. Group rat numbers were: CON: 14, CON+MRX-2843: 14, T2DM: 10, DPN: 12, and DPN+MRX-2843: 10; rats in all groups were aged 96 weeks. The sciatic nerve was the chosen tissue. CON, control; MRX, MerTK-specific inhibitor; DPN, diabetic peripheral neuropathy; T2DM, type 2 diabetes mellitus; TNF-α, tumor necrosis factor α.

Fig. 9 Detection of P65, TNF-α, and IL-1β levels in the serum by ELISA. (a) P65 levels in each group. (b) TNF-α levels in each group. (c) IL-1β levels in each group. *p<0.05; ns, no statistical difference. Rat counts for each group were: CON: 14, CON+MRX-2843: 14, T2DM: 10, DPN: 12, and DPN+MRX-2843: 10. Rats in all groups were aged 96 weeks. ELISA, enzyme linked immunosorbent assay; CON, control; MRX, MerTK-specific inhibitor; DPN, diabetic peripheral neuropathy; T2DM, type 2 diabetes mellitus; TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β.

The binding of Mer tyrosine kinase to the inhibitor of kappa B kinase beta in Schwann cells

Immunoprecipitation assays demonstrated specific binding between MerTK and Ikbkb antibodies to their respective immunoprecipitates in RSC96 cells ([Fig. 10a], b). Western blot analysis revealed an increase in Ikbkb expression concurrent with a decrease in MerTK expression (P<0.05) ([Fig. 10c, d]). Immunofluorescence double staining indicated enhanced fluorescence of Ikbkb (red) and P65 (green) following MerTK-siRNA3 interference ([Fig. 10e]), highlighting the endogenous binding of MerTK to Ikbkb and its regulatory role in the NF-κB signaling pathway.

Fig. 10 MerTK and Ikbkb interaction in Schwann cells via immunoprecipitation assay. (a) Ikbkb immunoprecipitate was probed with MerTK antibody. (b) MerTK immunoprecipitate was probed with Ikbkb antibody. “Input” signifies the positive control, while “IgG” marks the negative control. (c) Ikbkb expression post-treatment with three MerTK siRNAs and a negative control agent (NC) was determined by western blotting. (d) Levels of Ikbkb protein across groups. (e) Ikbkb and P65 expression post-MerTK-siRNA3 treatment was observed by immunofluorescence (blue fluorescence: nuclei; red fluorescence: Ikbkb; green fluorescence: P65). MerTK, mer tyrosine kinase; Ikbkb, inhibitor of kappa B kinase beta.

Discussion

In this study, the T2DM model was established using high-sugar and -fat diets for 6 weeks combined with low-dose STZ injection in SD rats (35 mg/kg), and was considered a successful model when the blood glucose level was>16.7 mmol/L. Based on the T2DM model, the rats were fed with high-sugar and -fat diets continuously for 8 weeks, and the DPN model was confirmed by detecting the blood biochemical index, sciatic NCV<40 m/s, and the morphology of the nerve structure, which is easy to generate and widely available for exploring behavioral or pharmacological tests associated with long-term diabetic complications, as well as for studying the pathogenesis of diabetic complications.

MerTK is expressed in both immune and non-immune cells and is involved in inflammation, metabolism, and vascular homeostasis [20]. Previous studies have indicated that MerTK protects against liver fibrosis and cancer [21] [22] [23]. The expression of MerTK in the sciatic nerve and serum of rats was detected in our study by immunohistochemistry, immunofluorescence, western blotting, and ELISA, which showed that upregulation of MerTK in the T2DM and DPN groups, and the expression of MerTK was significantly increased in the DPN group than in the CON and T2DM groups, consistent with the results of our previous study [10]. After administering MerTK-specific inhibitors, sciatic nerve conduction velocity decreased, and structural disruption was observed, indicating DPN exacerbation upon MerTK inhibition and its protective role in DPN development. MerTK, a crucial tyrosine kinase, significantly influences PI3K/AKT and NF-κB pathway regulation and apoptotic cell-induced monocyte-derived dendritic cell inhibition [24] [25]. In addition, MerTK has been reported to affect organ function during inflammatory processes or any other injury, such as pathogen invasion [26].

In clinical studies, the levels of inflammatory markers have been shown to predict the onset and progression of diabetic complications [27]. NF-κB is a protein complex that controls transcribed DNA, cytokine production, and cell survival; it is present in almost all animal cell types and is involved in cellular responses to stimuli. It also plays a key role in regulating the immune response to infections [28] [29]. Previous studies have shown the central role of NF-κB pathway-mediated inflammatory response in the pathogenesis of diabetes-related complications, including peripheral neuropathy. NF-κB activation was involved in the insulin resistance and apoptosis of pancreatic β-cells [30]. TNF-α and IL-1β, the NF-κB-mediated downstream inflammatory factors, play a central role in the inflammatory response, including immediate, acute, and chronic inflammation [31]. Our studies showed NF-κB (P65), TNF-α, and IL-1β expressions were higher in the DPN group, further increasing with MerTK inhibition, indicating the protective effect of MerTK in DPN by inhibiting the NF-κB pathway. Additionally, MerTK influences metabolism-related indices by programming macrophages metabolically and epigenetically [24] [32]. We observed a significant increase in serum levels of TG, TC, and GHB in the serum after MerTK inhibition, indicating the aggravation of blood glucose and lipid metabolism disorders.

To elucidate the MerTK-NF-κB interaction, we identified Ikbkb (IKKβ, IKK2), a catalytic subunit of the IκB kinase complex and key NF-κB pathway regulator, from the National Centre for Biotechnology Information. This complex swiftly activates NF-κB, orchestrating target gene expression [33]. Diabetes induces IκB phosphorylation and degradation, allowing P50 and P65 to signal the nucleus and activate genes that regulate DPN-associated pathophysiological changes [34]. We verified the endogenous interaction of MerTK with Ikbkb in Ceibomb cells via immunoprecipitation. Interfering with MerTK increased Ikbkb expression, significantly enhancing Ikbkb and P65 immunofluorescence (P<0.05); this indicates that MerTK silencing upregulates Ikbkb, activating the NF-κB pathway and P65 expression, thus suggesting the role of MerTK in inflammation through NF-κB signaling.

In summary, this study found that MerTK increased significantly in the DPN rats, while upon MerTK inhibition, neuropathy was aggravated, and inflammatory factors were upregulated. MerTK played a protective role in the progression of DPN by inhibiting the NF-κB pathway. Our findings provide a valuable reference for studies on other diabetic complications owing to their similar pathogenesis and suggest that MerTK may be an early diagnostic marker and a new target for the treatment of DPN.

Authors contributions

Danfeng Lan, Qiuping Yang, and Yan Zhao defined the concept of the study. Wenting Chen and Yidan Fu performed the experiments and wrote the manuscript. Bian Wu and Fugang Mao analyzed the results. Danfeng Lan and Qiuping Yang revised the manuscript. All the authors have read and approved the publication of the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgment

We thank Prof. Di Lu and Prof. Lechun Lv for their valuable discussions and technical assistance. We thank the scientific and technological achievements of the incubation center of Kunming Medical University for providing the experimental platform.

• References

• 1 L'Heveder R, Nolan T. International diabetes federation. Diabetes Res Clin Pract 2013; 101: 349-351
• 2 Tesfaye S, Selvarajah D. Advances in the epidemiology, pathogenesis and management of diabetic peripheral neuropathy. Diabetes Metab Res Rev 2012; 28: 8-14
• 3 Lotfy M, Adeghate J, Kalasz H. et al. Chronic complications of diabetes mellitus: A mini review. Curr Diabetes Rev 2017; 13: 3-10
• 4 Tesfaye S, Selvarajah D, Gandhi R. et al. Diabetic peripheral neuropathy may not be as its name suggests: Evidence from magnetic resonance imaging. Pain 2016; 157: S72-S80
• 5 Elzinga SE, Eid SA, Mcgregor BA. et al. Transcriptomic analysis of diabetic kidney disease and neuropathy in mouse models of type 1 and type 2 diabetes. Dis Model Mech 2023; 16: dmm050080
• 6 Yu Y. Gold standard for diagnosis of DPN. Front Endocrinol (Lausanne) 2021; 12: 719356
• 7 Iqbal Z, Azmi S, Yadav R. et al. Diabetic peripheral neuropathy: Epidemiology, diagnosis, and pharmacotherapy. Clin Ther 2018; 40: 828-849
• 8 Tesfaye S, Boulton AJ, Dyck PJ. et al. Diabetic neuropathies: Update on definitions, diagnostic criteria, estimation of severity, and treatments. Diabetes Care 2010; 33: 2285-2293
• 9 Sloan G, Alam U, Selvarajah D. et al. The treatment of painful diabetic neuropathy. Curr Diabetes Rev 2022; 18: e2001717820
• 10 Lan D, Jiang HY, Su X. et al. Transcriptome-wide association study identifies genetically dysregulated genes in diabetic neuropathy. Comb Chem High Throughput Screen 2021; 24: 319-325
• 11 Audo I, Mohand-Said S, Boulanger-Scemama E. et al. Mertk mutation update in inherited retinal diseases. Hum Mutat 2018; 39: 887-913
• 12 Yang J, Wei Y, Zhao T. et al. Magnolol effectively ameliorates diabetic peripheral neuropathy in mice. Phytomedicine 2022; 107: 154434
• 13 Feng Y, Chen L, Luo Q. et al. Involvement of microrna-146a in diabetic peripheral neuropathy through the regulation of inflammation. Drug Des Devel Ther 2018; 12: 171-177
• 14 Ren C, Han X, Lu C. et al. Ubiquitination of NF-κB p65 by FBXW2 suppresses breast cancer stemness, tumorigenesis, and paclitaxel resistance. Cell Death Differ 2022; 29: 381-392
• 15 Perry BD, Caldow MK, Brennan-Speranza TC. et al. Muscle atrophy in patients with type 2 diabetes mellitus: Roles of inflammatory pathways, physical activity and exercise. Exerc Immunol Rev 2016; 22: 94-109
• 16 Li JS, Ji T, Su SL. et al. Mulberry leaves ameliorate diabetes via regulating metabolic profiling and AGES/RAGE and p38 MAPK/NF-κB pathway. J Ethnopharmacol 2022; 283: 114713
• 17 Korbecki J, Simińska D, Gąssowska-Dobrowolska M. et al. Chronic and cycling hypoxia: Drivers of cancer chronic inflammation through HIF-1 and NF-κB activation: A review of the molecular mechanisms. Int J Mol Sci 2021; 22: 10701
• 18 Shi X, Chen Y, Nadeem L. et al. Beneficial effect of TNF-α inhibition on diabetic peripheral neuropathy. J Neuroinflammation 2013; 10: 69
• 19 Zahoor A, Yang C, Yang Y. et al. Mertk negatively regulates Staphylococcus aureus induced inflammatory response via SOCS1/SOCS3 and Mal. Immunobiology 2020; 225: 151960
• 20 Musso G, Cassader M, De Michieli F. et al. Mertk rs4374383 variant predicts incident nonalcoholic fatty liver disease and diabetes: Role of mononuclear cell activation and adipokine response to dietary fat. Hum Mol Genet 2017; 26: 1747-1758
• 21 Cai B, Dongiovanni P, Corey KE. et al. Macrophage MerTK promotes liver fibrosis in nonalcoholic steatohepatitis. Cell Metab 2020; 31: 406-421
• 22 Huelse JM, Fridlyand DM, Earp S. et al. Mertk in cancer therapy: Targeting the receptor tyrosine kinase in tumor cells and the immune system. Pharmacol Ther 2020; 213: 107577
• 23 Yan D, Huelse JM, Kireev D. et al. Mertk activation drives osimertinib resistance in EGFR-mutant non-small cell lung cancer. J Clin Invest. 2022 132. e150517
• 24 Pipitone RM, Calvaruso V, Di Marco L. et al. Mer tyrosine kinase (MERTK) modulates liver fibrosis progression and hepatocellular carcinoma development. Front Immunol 2022; 13: 926236
• 25 Sen P, Wallet MA, Yi Z. et al. Apoptotic cells induce Mer tyrosine kinase-dependent blockade of NF-kappaB activation in dendritic cells. Blood 2007; 109: 653-660
• 26 Lin J, Xu A, Jin J. et al. Mertk-mediated efferocytosis promotes immune tolerance and tumor progression in osteosarcoma through enhancing M2 polarization and PD-L1 expression. Oncoimmunology 2022; 11: 2024941
• 27 Spranger J, Kroke A, Möhlig M. et al. Inflammatory cytokines and the risk to develop type 2 diabetes: Results of the prospective population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study. Diabetes 2003; 52: 812-817
• 28 Dolcet X, Llobet D, Pallares J. et al. NF-kB in development and progression of human cancer. Virchows Arch 2005; 446: 475-482
• 29 Schlein LJ, Thamm DH. Review: NF-kB activation in canine cancer. Vet Pathol 2022; 59: 724-732
• 30 He F, Huang Y, Song Z. et al. Mitophagy-mediated adipose inflammation contributes to type 2 diabetes with hepatic insulin resistance. J Exp Med. 2021 218. e20201416
• 31 Olefsky JM, Glass CK. Macrophages, inflammation, and insulin resistance. Annu Rev Physiol 2010; 72: 219-246
• 32 Thai LM, O'Reilly L, Reibe-Pal S. et al. Β-cell function is regulated by metabolic and epigenetic programming of islet-associated macrophages, involving Axl, Mertk, and TGFβ receptor signaling. iScience 2023; 26: 106477
• 33 Pai YW, Lin CH, Lin SY. et al. Reconfirmation of newly discovered risk factors of diabetic peripheral neuropathy in patients with type 2 diabetes: A case-control study. PLoS One 2019; 14: e220175
• 34 Mu ZP, Wang YG, Li CQ. et al. Association between tumor necrosis factor-α and diabetic peripheral neuropathy in patients with type 2 diabetes: A meta-analysis. Mol Neurobiol 2017; 54: 983-996

Publication History

Received: 28 November 2023
Received: 21 March 2024

Accepted: 26 March 2024

Accepted Manuscript online:
08 April 2024

Article published online:
10 May 2024