(W1030-01-02) Oral Nano-curcumin Alone or In Combination with Insulin Alleviates Diabetic Neuropathy in Rats

Purpose: Diabetes mellitus (DM) is a multifaceted metabolic disorder that, if not appropriately managed, leads to secondary complications which have limited approved medications for treatment [1]. Diabetic peripheral neuropathy (DPN) is one such complication caused by nerve damage that cannot be reversed, but can be delayed, if not prevented. Curcumin (CUR), the active component of an Indian spice turmeric, with natural anti-inflammatory property can help prevent underlying inflammation in diabetes but it is plagued with poor clinical bioavailability [2]. To better understand the role of bioavailability in clinical outcomes, we have tested previously [3] developed double-headed nanosystems containing curcumin (nCUR) on DPN. Because CUR does not influence glucose levels, we have combined nCUR with subcutaneous insulin (INS) and compared it to nCUR or INS-only groups.

Methods: nCUR was prepared by the emulsion-diffusion-evaporation method [3]. Streptozotocin (55 mg/kg, intraperitoneal) induced diabetic (glucose ≥150 mg/dl) rats were divided into five groups: diabetic control (D), D+CUR, daily doses (by oral gavage) of plain curcumin (40mg/kg, D+nCUR, daily doses nCUR (20mg/kg); D+nCUR+Insulin (daily nCUR by oral gavage and subcutaneous insulin) and Insulin (2IU/rat) for 10 weeks. Healthy SD rats formed the control group (C). Animals were euthanized and sciatic nerve and hind paw was harvested. Histological analyses were performed for peripheral nerves in hind paw and neurofilament in sciatic nerves.

Results: nCUR with or without INS alleviates DPN at two times the lower dose than CUR, as indicated by the qualitative and quantitative analysis of the hind paw, sciatic nerve, spleen, and L4-6 spinal cord. nCUR and nCUR+INS preserve hind paw peripheral nerve axons as evident by the Bielschowsky silver stain and intraepidermal nerve fibers (IENF) density measured by immunofluorescence. The mechanistic studies further corroborated the results, where nCUR or nCUR+INS showed a significant decrease in TUNEL positive cells, macrophage infiltration while preserving the NF200 expression in the sciatic nerve.

Conclusion: Together, these data confirms that CUR bioavailability is proportional to clinical outcomes and INS alone may not be one of the solutions for DM. This study highlights the potential of nCUR with or without INS in alleviating DPN and warrants further investigation of this combination.

References: [1] S. Yagihashi, H. Mizukami, K. Sugimoto, Mechanism of diabetic neuropathy: Where are we now and where to go?, J Diabetes Investig, 2 (2011) 18-32.
[2] R. Ganugula, M. Arora, P. Jaisamut, R. Wiwattanapatapee, H.G. Jorgensen, V.P. Venkatpurwar, B. Zhou, A. Rodrigues Hoffmann, R. Basu, S. Guo, N. Majeti, Nano-curcumin safely prevents streptozotocin-induced inflammation and apoptosis in pancreatic beta cells for effective management of Type 1 diabetes mellitus, Br J Pharmacol, 174 (2017) 2074-2084.
[3] G. Kaur, M. Arora, R. Ganugula, M. Kumar, Double-headed nanosystems for oral drug delivery, Chem Commun (Camb), 55 (2019) 4761-4764.

Acknowledgements: This work was supported by the NIH grant R01EY028169.


Fig.1. (A) Representative images (upper at a scale of 500 µm) with the Bielschowsky staining of a transverse section of the whole right hind paw and the globulus hind paw skin, magnified images (scale bar, 20 µm) of selected red box areas further analyzed for quantification (all images in the higher magnification-oriented uniformly). (B) Quantitative (n=8 images) analysis of the Bielschowsky staining. Statistical analysis was carried out using one-way ANOVA and Tukey post hoc test. Significant values were compared with the diabetic group, **** P < 0.001.


Fig. 2. Representative images of IENF and TUNEL stains in peripheral nerve of the right hind paw of all studied groups. (A) Cross-section of the peripheral nerve of the hind paw stained with an IENF antibody (red) at 20x magnification. (B) Quantitative representation of IENF expression. (C) TUNEL staining (green) of the selected area (white box from A images) in the peripheral nerve of the hind paw used for Quantification (n=8 images) representing TUNEL-positive cells. Statistical analysis was carried out using one-way ANOVA and Tukey post-hoc test for TUNEL and student t test for IENF staining. Significant values were compared with the diabetic group, * P < 0.05 ** P < 0.01, *** P < 0.001 & ****P < 0.001.


Fig. 3. NF200, F4/80+, and CD3+ expression in the sciatic nerve (A) cross section of sciatic nerve at 10x magnification, magnified image of selected area (white box) at 20x magnification showing NF200 antibody (red), F4/80+ (green), and CD3+ (turquoise) staining; Quantitative representation of (B) NF 200 expression, (C) F4/80 positive cells, and (D) CD3+ cells. Statistical analysis was carried out using one-way ANOVA and Tukey post-hoc except student t test for CD3+. Significant values were compared against the diabetic group, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.