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

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+/+Lepr^db/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.

Keywords: Diabetes; Mice; Peripheral neuropathy; Thymosin β4; miR-146a.

Fig.1:. Tβ4 treatment increased miR-146a level and decreased IRAK1, TRAF6 and p-NFkB expression, as well as MCP-1 and VCAM-1 levels in sciatic nerve tissues.

Panels A to C show real-time RT-PCR analysis of miR-146a expression (A) and Western blot analysis of IRAK1, TRAF6 and p-NFkB, as well as MCP-1 and VCAM-1 levels in the sciatic nerve tissues, β-actin was used as an internal control (B and C). Double immunofluorescent staining shows that IRAK1 (E, G red, arrows) and TRAF6 (I, K, red, arrows) immunoreactivity were localized to vWF positive vessels (D, G, H, K green, arrows). Panels F and J show DAPI nuclear staining (blue). *p<0.05 and #p<0.05 versus the db/m mouse and the saline treated db/db mouse, respectively. n=6/group. Bar=100 μm. dm=db/m mouse; db=db/db mouse. dbtb=diabetic mouse treated with Tβ4.

Fig.2:. miR-146a mediates Tβ4-improved vascular function in the diabetic sciatic nerve.

Panels A to F show that whole-mounted (A to C) and cross sections (D to F) of the sciatic nerve tissues revealed FITC-dextran perfused vessels from a representative dm mouse (A, D), db mouse treated with saline (B, E), and db mouse treated with Tβ4 (C,F). Panels L and M show quantitative data of the density of FITC-dextran perfused vessels in cross section (L, n=6/group) and percentage changes of sciatic nerve tissue blood flow with a reference of dm mice at 100% (M, n=6/group). *p<0.05 and #p<0.05 versus the db/m mouse and the saline treated db/db mouse, respectively. Bar=100μm. dm=db/m mouse; db=db/db mouse. dbtb=diabetic mouse treated with Tβ4. Panels G to K show representative microscopic images (G-K ) and quantitative data (N) of capillary-like tube formation in MDEs cultured in normal glucose (RG), high glucose (HG), high glucose with Tβ4 (HG+TB), high glucose with Tβ4 in the presence of miR-146a inhibitor (+si146) and scramble negative control (+NC). Bar in K=50μm. Panel O shows quantitative data of real-time RT-PCR analysis of miR-146a expression in MDEs. Panels P and Q show Western blot analysis of IRAK1 and TRAF6 levels in MDEs transfected with miR-146a mimics and scramble control, respectively. Panel R shows quantitative data of real-time RT-PCR analysis of miR-146a expression in MDEs. Panels S to U show quantitative data of real-time RT-PCR (S) and Western blot analysis (T and U) of IRAK1,TRAF6, p-NFkB, MCP-1 and VCAM-1 levels in MBEs under different conditions, and HSC70 and β-actin were used as internal controls, respectively. *p<0.05, #p<0.05 and $p<0.05 versus the normal glucose (RG), high glucose (HG) and scramble negative control (NC) groups, respectively. n=6/group.

Fig.3:. miR-146a mediates Tβ4-induced axonal remodeling in vivo and in vitro.

Panels A and B show PGP 9.5 immunoreactive intraepidermal nerve fibers (red, arrows) in the plantar skin from a representative non-diabetic mouse treated with saline (dm), diabetic mouse treated with saline (db) and diabetic mouse treated with Tβ4 (dbtb). Panel B shows quantitative data of intraepidermal nerve fiber density (IENFD). *p<0.05 and #p<0.05 versus the non-diabetic mouse (dm) and the diabetic mouse treated with saline (db), respectively. n=10/group. Bar=50μm. Panels C and D show representative microscopic images (C) and quantitative data (D) of axonal outgrowth in DRG neurons cultured in normal glucose (RG), high glucose (HG), high glucose with Tβ4 (+TB), high glucose with Tβ4 in the presence of miR-146a inhibitor (+si146) and scramble negative control (+NC). Panel E shows quantitative data of real-time RT-PCR analysis of miR-146a (E) and IRAK1 and TRAF6 (F) levels in DRG neurons under different conditions, and HSC70 was used as an internal reference gene (F). *p<0.05, #p<0.05 and $p<0.05 versus the normal glucose (RG), high glucose (HG) and scramble negative control (NC) groups, respectively. n=6/group. Bar in C =100μm

Fig.4:. Effect of Tβ4 on neurological function in diabetic db/db mice.

Treatment of db/db mice with Tβ4 improved neurological function measured by MCV (A), SCV (B) and Plantar Test (C). *p<0.05 and #p<0.05 versus the db/m mouse and the db/db mouse treated with saline, respectively. n=10/group. dm=db/m mouse; db=db/db mouse. dbtb=diabetic mouse treated with Tβ4.

Fig.5:

A schematic shows potential interaction of miR-146a increased by Tβ4 with its target genes and downstream pro-inflammatory proteins. Diabetic peripheral neuropathy down-regulates miR-146a, upregulates IRAK1 and TRAF6, and activates downstream NFkB. Tβ4-elevated miR-146a represses IRAK1 and TRAF6, which results in attenuation of NFkB activation and pro-inflammatory genes VCAM-1 and MCP-1.