Elsevier

Life Sciences

Volume 225, 15 May 2019, Pages 107-116
Life Sciences

1,25-(OH)2D3 protects Schwann cells against advanced glycation end products-induced apoptosis through PKA-NF-κB pathway

https://doi.org/10.1016/j.lfs.2019.03.068Get rights and content

Abstract

Aims

To explore the effect and mechanism of 1, 25-(OH)2D3 on Schwann cell apoptosis induced by advanced glycation end products.

Main methods

Schwann cells, isolated from rodent sciatic nerve were incubated with AGE-modified bovine serum albumin(AGE) to mimic diabetic conditions and 1,25-(OH)2D3 was used as protector. Cell apoptosis was detected by PI/Annexin-V staining, caspase 3 activity assay and western blotting for caspase 3 and PARP. The activation of protein kinase A (PKA) and nuclear factor kappa-B (NF-κB) was evaluated by western blot. Immunofluorescent staining was used for intercellular location of NF-κB. Cytokine secretion was evaluated by enzyme-linked immunosorbent assay.

Key findings

Schwann cell apoptosis accelerated after incubating with AGE. However, if combining 1,25-(OH)2D3 with AGE, apoptosis decreased significantly. 1,25-(OH)2D3 enhanced PKA activity, but inhibited AGE-induced nuclear translocation of NF-κB. Furthermore, PKA activator (8-bromoadenoside cyclic adenoside monophosphate, 8-Br-cAMP) or NF-κB inhibitor (caffeic acid phenethyl ester, CAPE) could reduce the apoptosis, decreased cleaved caspase 3 and cleaved PARP, suggesting the involvement of PKA and NF-κB pathways in the protection of 1,25-(OH)2D3 on Schwann cells. Moreover, 8-Br-cAMP and CAPE could inhibit AGE-induced secretion of interleukin(IL)-1β, prostaglandin E2(PEG2) and cyclooxygenase 2(COX2). Interestingly, 8-Br-cAMP decreased phospho-NF-κB and inhibited nucleus translocation of NF-κB. It hinted at the regulation of PKA to NF-κB. Finally, a pre-treatment of H-89 (an inhibitor of PKA) could block the protection of 1,25-(OH)2D3 on cell apoptosis. In conclusion, 1,25-(OH)2D3 could protect Schwann cell against AGE-induced apoptosis through PKA/NF-κB pathway.

Significance

These findings provide experimental rationales for using vitamin D for diabetic neuropathy.

Introduction

As one of the most common chronic complications of diabetes mellitus, diabetic peripheral neuropathy (DPN) is also a major cause of worsening quality of life in individuals with diabetes. Hyperglycemia results in an accumulation of advanced glycation end products (AGEs) and becomes an important causative factor of diabetic complications. However, the molecular mechanism of DPN is so complex that multiple factors and many types of cells are involved. Both injuries of peripheral nerve and microvessels supplying peripheral nerve might contribute to DPN and result in demyelination and axonal degeneration followed by nerve dysfunction and ultimately somatic and visceral denervation [1].

Myelin sheath is generated by Schwann cells in the peripheral nervous system. Thus demyelination in the peripheral nervous system is caused mostly by insults of Schwann cells [2,3]. In addition, Schwann cell are sensitive to hyperglycemic toxicity because of its insulin-independent glucose transporter [4,5]. Pathological abnormalities of nerve fiber are generally restricted to myelin sheath and Schwann cell in rat models of diabetes [6]. Apoptosis of Schwann cell has been detected in models of diabetes [[7], [8], [9], [10], [11]]. It indicated that protecting Schwann cell might be a potential strategy for preventing and treating DPN.

In recent year, AGEs are demonstrated as a key factor leading to diabetic complications. AGEs were produced from sugars reacting non-enzymatically with the amino groups of proteins to form reversible Schiff bases. Then the early glycation products undergo further complex reactions such as rearrangement, dehydration and condensation to become irreversibly cross-linked, heterogeneous fluorescent derivatives termed AGE [12]. The formation and accumulation of AGE are positively correlated with the progress of diabetes. Especially, the pathological role of AGE has been reported in various diabetic complications [[12], [13], [14], [15], [16]]. AGE via binding to their receptor (RAGE) could lead to dysfunction and death of various types of cells [[17], [18], [19]]. It is also shown that in vitro incubation of Schwann cells with AGE induces cell death [20].

The data obtained from patients and animals with diabetes showed that the levels of AGEs were increased not only in the serum but also in the peripheral nerves [21]. AGEs were found in the peripheral nerve of rat with diabetes, and the expression of RAGE was found in endothelial and Schwann cells, which may contribute to the impairment of nerve function [22].

Some recent studies have demonstrated that a low serum level of vitamin D was associated with diabetes complications [23,24] and vitamin D deficiency might be correlated with DPN [25,26]. Many international studies have reaffirmed that vitamin D deficiency was an independent risk factor for DPN [[27], [28], [29], [30]]. It is expected that supplementation of vitamin D could prevent or delay the onset of DPN. In another study, treatment of vitamin D improved the emotional distress of patients with painful diabetic neuropathy. Yet there was no significant effect on other painful symptoms [31]. Vitamin D was correlated positively with IL-13 and nerve growth factor [22] and negatively with IL-17 [32]. These effects of vitamin D indicated anti-inflammation and nerve protection. Chabas JF et al. examined the efficacy of vitamin D on neuropathy in a rat model of transected peripheral nerve. Vitamin D2 could increase axon diameter and potentiate nerve regeneration [33]. Both vitamins D2 and D3 improved myelination and recovery after nerve injury. However, vitamin D3 was more efficient than vitamin D2. Vitamin D3 not only increased the number of preserved or newly formed axons but also improved neurite myelination in both distal and proximal ends [34].

Despite an efficacy of vitamin D for DPN, the mechanism of vitamin D on peripheral nerve system required further investigation, especially under disease condition. Under diabetic conditions, AGE binding to RAGE activates a variety of signaling pathways leading to increased oxidative stress and synthesis of local growth factors, cytokines and adhesion molecules [35]. Activation of nuclear factor κB (NF-κB) is a key step for inflammatory response and following injury [36,37]. Moreover, in our previous studies, we found that PKA mediated protection in diabetic nephropathy [38]. Activating PKA was also reported to contribute a beneficial effect of cilostazol on DPN [39].

Considering improved myelination of vitamin D, it was speculated that vitamin D might have a beneficial effect on the survival or function of Schwann cells and PKA or NF-κB pathway may be involved. Therefore, the current study was intended for elucidating the effect and mechanism of 1,25(OH)2D3 on the AGE-induced apoptosis of Schwann cells.

Section snippets

Isolation and culture of Schwann cells

Schwann cells were isolated from rodent sciatic nerve as described previously with modification [40]. The experimental protocol was approved by our institutional Animal Ethics Committee. Six-week-old Wistar rats (200–250 g) (Beijing Vital River Laboratory Animal Technology, Beijing, China) were euthanized and sciatic nerves dissected. Sciatic nerves were cut into approximately 1 mm pieces and subsequently digested for 15 min at 37 °C in 0.2% type I collagenase solution. The mixture was agitated

Identification of Schwann cells

Cultured Schwann cells exhibited a monolayer with fibroblast-like morphology (Fig. 1a). All cells expressed S-100 by immunofluorescent staining (Fig. 1b). Purity of cultured cells was as high as 99% by flow cytometry for S-100 (Fig. 1c).

Effects of 1, 25(OH)2D3 on AGE-induced apoptosis of Schwann cell

For detecting the protection of 1, 25(OH)2D3 on Schwann cells, AGE were used for simulating diabetic condition. As shown in Fig. 2, Annexin-V positive cells were scarce under normal culture with BSA (Fig. 2a). However, exposure to apoptosis inducer (positive

Discussion

Vitamin D deficiency has been widely reported in patients with diabetic neuropathy [23,26,28,29]. However, the efficacy of vitamin D for diabetic neuropathy is not clear. Herein it was shown that 1,25-(OH)2D3 could protect Schwann cells against AGE-induced apoptosis via PKA/NF-κB pathway.

Schwann cells consist of myelin sheath in the peripheral nerve system. Such a role is vital for nerve conduction and neuron survival. During the development of diabetic neuropathy, Schwann cells became injured

Conclusion

Mounting evidence has confirmed the protective role of PKA for microvascular complications related to diabetes [38,53,54]. 1, 25(OH)2D3 decreases Schwann cell apoptosis through an activation of PKA followed by NF-κB suppression. These data hint at a potential application of vitamin D for clinical DPN.

Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (81670758 to L.P.), Nature Science Foundation of Jilin Province (20180101113JC to H.C.), Beijing Municipal Natural Science Foundation of China (7182147 to L.P.), Young Medical Talents Award Program of Chinese Academy of Medicine (2018RC310023 to L.P.) and Capital's Funds for Health Improvement and Research (2018-2-4062 to L.P.).

Conflicts of interest

The authors declare there are no conflicts of interest.

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