Elsevier

Brain Research

Volume 1707, 15 March 2019, Pages 198-207
Brain Research

Research report
miR-146a mediates thymosin β4 induced neurovascular remodeling of diabetic peripheral neuropathy in type-II diabetic mice

https://doi.org/10.1016/j.brainres.2018.11.039Get rights and content

Highlights

  • miR-146a mediates Tβ4-promoted endothelial cell function under hyperglycemia.

  • miR-146a regulates Tβ4-induced axonal outgrowth under hyperglycemia.

  • miR-146a contributes the therapeutic effect of Tβ4 on diabetic neuropathy.

Abstract

Diabetes induces neurovascular dysfunction leading to peripheral neuropathy. MicroRNAs (miRNAs) affect many biological processes and the development of diabetic peripheral neuropathy. In the present study, we investigated whether thymosin-β4 (Tβ4) ameliorates diabetic peripheral neuropathy and whether miR-146a mediates the effect of Tβ4 on improved neurovascular function. Male Type II diabetic BKS. Cg-m+/+Leprdb/J (db/db) mice at age 20 weeks were treated with Tβ4 for 8 consecutive weeks, and db/db mice treated with saline were used as a control group. Compared to non-diabetic mice, diabetic mice exhibited substantially reduced miR-146a expression, and increased IL-1R-associated kinase-1 (IRAK1), tumor necrosis factor (TNFR)-associated factor 6 (TRAF6) levels and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activity in sciatic nerve tissues. Treatment of diabetic mice with Tβ4 significantly elevated miR-146a levels and overcame the effect of diabetes on these proteins. Tβ4 treatment substantially improved motor and sensory conduction velocity of the sciatic nerve, which was associated with improvements in sensory function. Tβ4 treatment significantly increased intraepidermal nerve fiber density and augmented local blood flow and the density of fluorescein isothiocyanate (FITC)-dextran perfused vessels in the sciatic nerve tissue. In vitro, treatment of dorsal root ganglion (DRG) neurons and mouse dermal endothelial cells (MDEs) with Tβ4 significantly increased axonal outgrowth and capillary-like tube formation, whereas blocking miR-146a attenuated Tβ4-induced axonal outgrowth and capillary tube formation, respectively. Our data indicate that miR-146a may mediate Tβ4-induced neurovascular remodeling in diabetic mice, by suppressing pro-inflammatory signals.

Introduction

Diabetic peripheral neuropathy is a significant complication of diabetes. The vast majority of people with diabetes have type II diabetes. Type II diabetes is associated with subclinical inflammation and activation of the innate immune system (Pickup, 2004). Diabetic patients with peripheral neuropathy have increased serum levels of inflammatory cytokines (Bilir et al., 2016, Doupis et al., 2009). Hyperglycemia activates pro-inflammatory mediators, which induce vascular dysfunction and nerve damage. Inflammatory mediators participate in the pathogenesis of diabetic peripheral neuropathy (Kawamura et al., 2008, Wright, 2011).

Thymosin β4 (Tβ4), a small acidic polypeptide, has multiple biological functions including neurorestoration and anti-inflammation (Chopp and Zhang, 2015). Tβ4 promotes central nervous system plasticity and neurovascular remodeling leading to neurological recovery in a variety of neurological diseases (Morris et al., 2010, Xiong et al., 2011). Tβ4 attenuates inflammatory cell infiltration and promotes wound healing in diabetic animals (Evans et al., 2013). Our previous studies have found that Tβ4 ameliorates diabetes-induced neurovascular dysfunction, and thereby promotes neurological recovery of diabetic peripheral neuropathy (Wang et al., 2012, Wang et al., 2015a). However, the mechanisms by which Tβ4 improves neurological outcome in diabetic neuropathy have not been fully investigated.

MicroRNAs (miRNAs) are small non-coding RNAs that are key players in mediating physiological and pathogenesis functions of diabetes mellitus and diabetic complications (Kantharidis et al., 2011, Lorenzen et al., 2012). Dysregulation of miRNAs may affect inflammation and lead to development of diabetic neuropathy (Andersen et al., 2014, Gong et al., 2015). miRNAs are potential biomarkers and therapeutic targets in clinical pain disorders (Andersen et al., 2014). miR-146a modulates inflammatory and innate immunity responses by negatively regulating its target genes IRAK1, TRAF6 and NFkB activation, which subsequently down-regulate pro-inflammatory mediators such as MCP-1 and VCAM-1 (Landry et al., 1997, Taganov et al., 2006). The serum level of miR-146a is significantly decreased in type II diabetic patients (Balasubramanyam et al., 2011, Baldeon et al., 2014). miR-146a attenuates neuropathic pain by suppressing TRAF6 signaling in the spinal cord (Lu et al., 2015). Moreover, miR-146a over-expression represses the endothelial inflammatory response (Cheng et al., 2013). We recently demonstrated that treatment of diabetic db/db mice with miR-146a mimics reduces diabetic peripheral neuropathy (Liu et al., 2017). In addition, the miR-146a increases DRG neuronal survival and axonal outgrowth under hyperglycemia conditions (Jia et al., 2016, Wang et al., 2014). miR-146a upregulated by Tβ4 promotes oligodendrocyte differentiation by suppression of pro-inflammatory pathways (Santra et al., 2014). Furthermore, Tβ4 inhibits the expression and secretion of inflammatory mediators through miR-146a in hypoxia injured rats (Zhou et al., 2015). However, whether miR-146a mediates the therapeutic effect of Tβ4 on diabetic peripheral neuropathy has not been investigated.

In the present study, we elucidate the molecular mechanisms underlying the therapeutic effect of Tβ4 on diabetic peripheral neuropathy with a focus on miR-146a. We hypothesize that miR-146a is involved in Tβ4-improved neurovascular function in diabetic db/db mice.

Section snippets

Tβ4 reverses the effect of diabetes on miR-146a expression and its target proteins in sciatic nerve tissues

Our previous study showed that diabetes reduced miR-146a expression in DRG neurons (Wang et al., 2014). To extend our findings, we measured miR-146a levels in sciatic nerve tissues by means of quantitative real time RT-PCR and found that diabetic db/db mice exhibited significantly decreased miR-146a expression compared to that in non-diabetic mice, which was reversed by Tβ4 treatment (Fig. 1). These data indicate that Tβ4 treatment overcomes diabetes-reduced miR-146a expression in the sciatic

Discussion

The present study observed that miR-146a expression was considerably reduced in sciatic nerve tissues of diabetic db/db mice, and this reduction was associated with an increase in its target proteins IRAK1 and TRAF6, and NFkB activation, resulting in increased MCP-1 and VCAM-1 levels. Treatment of diabetic peripheral neuropathy with Tβ4 significantly upregulated miR-146a levels and suppressed expression of miR-146a target pro-inflammatory mediators, as well as improved neurovascular remodeling

Animals

All experimental procedures were approved by the Henry Ford Hospital Institutional Animal Care and Use Committee (IACUC#1486). Adult male BKS. Cg-m+/+Leprdb/J (db/db) mice were used as a model of type II diabetes, and heterozygote mice (db/m, a non-penetrant genotype) served as nondiabetic controls. Mice were purchased from Jackson Laboratories.

Tβ4 treatment

Diabetic db/db mice (age 20 weeks) were randomized to intraperitoneal (i.p.) injection with Tβ4 (30 mg/kg, RegeneRx, Inc. n = 10/group) daily for

Author contributions statement

All authors have read and approved submission of our manuscript. Conceived and designed the experiments: LW, MC, ZGZ. Performed the experiments: LW, XL, AS, LJ, XSL. Analyzed the data: LW AS KW ML. Wrote the paper: LW, MC, ZGZ.

Declaration of conflicting interests

None.

Acknowledgements

The authors thank Landschoot-Ward Julie, and Qing-e Lu for their technical assistance and Deborah Jewell for secretarial support.

Funding

This work was supported by the U.S. National Institutes of Health grants, RO1 NS075084 (LW), RO1 DK097519 (LW) and RO1 RDK102861A (XSL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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