Nanoparticle–microRNA-146a-5p polyplexes ameliorate diabetic peripheral neuropathy by modulating inflammation and apoptosis

BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 17 (2019) 188 – 197

Original Article

nanomedjournal.com

Nanoparticle–microRNA-146a-5p polyplexes ameliorate diabetic peripheral neuropathy by modulating inflammation and apoptosis Qiong Luo, MS a, b, 1 , Yonghao Feng, MS a, 1 , Yangmei Xie, MD candidate b , Yiye Shao, MD b , Men Wu, MS candidate a , Xiaolin Deng, MS candidate a , Wei-En Yuan, MD c,⁎, Yinghui Chen, MD d,⁎, Xiaohong Shi, MS a,⁎ a

Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China b Department of Neurology, Jinshan Hospital, Fudan University, Shanghai, China c Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China d Department of Neurology, Huashan Hospital North, Fudan University, Shanghai, China Revised 19 December 2018

Abstract Nontoxic and nonimmunogenic nanoparticles play an increasingly important role in the application of pharmaceutical nanocarriers. The pathogenesis of diabetic peripheral neuropathy (DPN) has been extensively studied. However, the role of microRNAs in DPN remains to be clarified. We verified in vitro that miR-146a-5p mimics inhibited the expression of proinflammatory cytokines and apoptosis. Then, we explored the protective effect of nanoparticle–miRNA-146a-5p polyplexes (nano-miR-146a-5p) on DPN rats. We demonstrated that nano-miR-146a-5p improved nerve conduction velocity and alleviated the morphological damage and demyelination of the sciatic nerve of DPN rats. The expression of the inflammatory cytokines, caspase-3, and cleaved caspase-3 in the sciatic nerve was inhibited by nano-miR-146a-5p. Additionally, nano-miR-146a-5p increased the expression of myelin basic protein. These results all indicated that nano-miR-146a-5p had a protective effect on peripheral nerves in the DPN rat model, which may occur through the regulation of the inflammatory response and apoptosis. © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/). Key words: Diabetic peripheral neuropathy; microRNA-146a; Nanoparticle; Inflammation; Apoptosis

Diabetic peripheral neuropathy (DPN), one of the most common chronic complications of type 2 diabetes mellitus (T2DM), is estimated to occur in 50% of patients with T2DM. 1 The most common clinical presentation of DPN is chronic distal symmetric sensory and motor multiple neuropathy, which cause morbidity and have a significant impact on the quality of life in people with diabetes. 2 The inflammatory response has been demonstrated to participate in the pathogenesis of DPN. 3,4 However, the application of exogenous anti-inflammatory drugs is limited in clinical use due to the prevalence of side effects and lack of tissue specificity. Therefore, finding endogenous molecules that efficiently regulate the inflammatory response

Acknowledgments: This work was supported by grants from the Shanghai Municipal Commission of Health and Family Planning (201540075). ⁎Corresponding authors. E-mail addresses: [email protected] (X. Shi), [email protected] (Y. Chen), [email protected] (W.-E. Yuan). 1 These two authors contributed equally to this work.

may help solve this problem. MicroRNAs (miRNAs) are highly conserved non-protein-coding endogenous small RNAs that have a length of approximately 18 to 25 nucleotides and can modulate important biological processes. 5,6 Some miRNAs have been shown to regulate the inflammatory response, 7 which might have a positive effect on DPN. We compared the miRNAs expression profiles in the sciatic nerve between T2DM rats and DPN rats. Three miRNAs were differentially expressed (≥ 2-fold), one of which, microRNA146a-5p (miR-146a-5p), has exhibited an anti-inflammatory property in the process of many diseases. 8,9 Our previous study also demonstrated that miR-146a-5p is involved in the pathophysiological process of DPN by regulating the inflammatory response. 10 This finding suggests that miR-146a-5p may be a target for the treatment of DPN. Therefore, delivering microR146a-5p into DPN rats might have a therapeutic effect on their peripheral neuropathy. The typical virus vectors of miRNAs have limited in vivo applications because of their immunogenicity, early degradation, etc. Appropriate carriers for miRNA delivery will be helpful for the in vivo application of miRNAs.

https://doi.org/10.1016/j.nano.2019.01.007 1549-9634/© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Luo Q, et al, Nanoparticle–microRNA-146a-5p polyplexes ameliorate diabetic peripheral neuropathy by modulating inflammation and apoptosis. Nanomedicine: NBM 2019;17:188-197, https://doi.org/10.1016/j.nano.2019.01.007

Q. Luo et al / Nanomedicine: Nanotechnology, Biology, and Medicine 17 (2019) 188–197

With the rapid development of nanotechnology, researchers have begun to turn their attention to low-toxicity and nonimmunogenic nanocarriers. In a previous study, our team constructed a cationic nanocarrier named imine backbone-based polymer (TPSP) for the delivery of siRNAs, where imine was used as the biodegradable bonds, spermine was used as the unit, and 1,4-phthalaldehyde was used as the linkers. 11 In addition, PEG-PCL-maltotriose-COO − assembled on the surface of the polymer as a shell, which could prevent leakage and nonspecific adsorption of siRNAs before the nanocarrier reached the target cell. In this study, we explored the protective effect of nanoparticle–miRNA-146a-5p polyplexes (nano-miR-146a-5p) on DPN. First, we explored the regulatory effect of miR-146a-5p mimics on the inflammatory response, apoptosis, and proliferation in Schwann cells induced by hyperglycemia in vitro. Then, we observed whether nano-miR-146a-5p polyplexes alleviated the peripheral neuropathy of DPN rats in vivo.

Methods Reagent miR-146a-5p mimics 5′-UGAGAACUGAAUUCCAUGG GUU-3′ and Lipofectamine RNA iMAX were purchased from Thermo Fisher technology. Streptozocin (STZ) was purchased from Sigma. TPSP was synthesized by Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University. Selection of target miRNA T2DM and DPN rats were induced by the combination of STZ and a high-fat, high-sugar diet (see supplementary files and Figure S1). Total RNA in the sciatic nerve of T2DM and DPN rats was extracted using the TRIzol method (Takara Technology, Japan) according to the manufacturer's instructions. Large-scale profiling of miRNA expression was achieved by a GeneChip miRNA 4.0 array (Affymetrix Inc., Santa Clara, CA, USA). All miRNA microarrays were performed at the Beijing CapitalBio Corporation. The experimental procedures were conducted as described in detail on the CapitalBio website (http://www. capitalbio.com). Raw data were normalized to the mean array intensity for inter-array comparison and analyzed using Significant Analysis of Microarray software (SAM, version 3.02) to identify differentially expressed genes between the T2DM and DPN groups. The differences are considered significant when log2-fold change ≥2 and adjusted P value b0.001. Then, we utilized R software to perform clustering analysis. According to the published literature, the appropriate miRNAs regulating the inflammatory response were selected. Cell culture and treatment RSC96 cells (rat Schwann cells) were obtained from the Cell Resource Center of the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, and cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO, USA) containing 25 mM glucose supplemented with 10% bovine serum (GIBCO-16000-044) in a humidified incubator under5%

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CO2 and 95% air at 37 °C. To obtain RSC96 cells that are similar to cells under hyperglycemic condition, we cultured RSC96 cells at glucose concentrations of 25 mM, 100 mM, and 200 mM. We measured the relative level of miR-146a-5p isolated from RSC96 cells induced by different concentrations of glucose (25 mM, 100 mM, 200 mM) at different times (24 h, 48 h, 12 d, 30 d) (see supplementary files). Then we determined the level of proliferation and apoptosis in RSC96 cells (see supplementary files). Cell transfection Lipofectamine RNA iMAX was used as a miR-146a-5p carrier. We measured the transfection efficiency and cytotoxicity of Lipofectamine (see supplementary files). Then, we conducted the transfection according to the instruction manual. The experimental cells were divided into the normal group, hyperglycemia group, negative control (NC) group, and miR146a-5p group. miR-146a-5p mimics or a negative control was transfected in each well. After incubation for 6 h, the old medium was discarded, and fresh normal culture medium was added and incubated for another 30 h. After that, the medium was substituted with the hyperglycemic culture medium and incubated for another 48 h. Preparation and characterization of nano-146a-5p polyplexes First, the cytotoxicity of TPSP nanoparticles and TPSP/miR146a-5p polyplexes (nano-miR-146a-5p) was determined (see supplemental file). Then, nano-miR-146a-5p were prepared by adding TPSP solution to miR-146a-5p solution at polymer-tomiRNA ratios (mass ratio) of 4.49, 44.90, 67.36, 89.80, 134.70 and 224.50. The polyplexes were incubated at room temperature for 30 min. The formed nano-miR-146a-5p were loaded on a 3.0% agarose gel containing 0.5 μg/mL ethidium bromide and subjected to electrophoresis at 110 V for 45 min. The retardation of miR-146a-5p was visualized by a UV illuminator to identify the condensing efficiency. The particle size and distribution of the polyplexes at different mass ratios were measured by using a Brookhaven particle size analyzer (90 Plus). The zeta potential of the nanoparticles was determined by using the same instrument. Particle size and zeta potential values were calculated from four individual experiments. A transmission electron microscope (TEM, JEM 2010 system, JEOL, Japan) was used to observe the image of the nano-miR-146a-5p. Then we measured the transfection efficiency of nano-miR-146a-5p at different mass ratios (see supplementary files). Experimental rats grouping and measurement of nerve conduction velocity and morphology All rats were randomly divided into six groups with fifteen rats in each group: a normal group, a T2DM group, a DPN group, an empty nanoparticle (NANO) group, a nano-miR-146a-5p group, and a VitB12 group. Treatment of each group rats and determination of nerve conduction velocity (NCV) are referred to the supplemental file. Then we observed the morphological changes of each group rats (the supplemental file).

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Hyperglycemia inhibited RSC96 cell viability and miR-146a-5p expression In the initial stage of the experiment, we verified RSC96 cell viability was obviously inhibited by 200 mM glucose (Figure S2, A). Then we demonstrated the miR-146a-5p expression was evidently inhibited in RSC96 cells induced by 200 mM at 30 d (Figure S2, B). miR-146a-5p increased the viability of RSC96 cells

Figure 1. Heat map showing the differentially expressed miRNAs (fold change ≥2) in the sciatic nerve between DPN (n = 3) and T2DM (n = 3) rats.

Western blot assay and Luminex® 200™ Assays The expression of COX-2, TNF-α, caspase-3, cleaved caspase-3 and myelin basic protein (MBP) in sciatic nerve tissue or RSC96 cells was determined by western blot assay (see supplemental file). The levels of interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and interleukin 10 (IL-10) in the plasma or supernatant were measured using Luminex® 200™ assays (see supplemental file). Statistical analysis Data are expressed as the mean ± SEM. Statistical analyses were performed using SPSS 25.0 (IBM Corp., Armonk, NY, USA). One-way ANOVA was conducted for multiple sample analyses. The paired t test was used for two paired samples. All tests were considered significant when P b 0.05.

Results Screening of differential miRNAs in the sciatic nerve by miRNA microarray A miRNA microarray was used to compare the miRNA expression levels in sciatic nerves between T2DM and DPN rats. Among the 232 rno-miRNAs detected by the microarray, miR146a-5p, miR-146b-5p and miR-30a-3p were found to be downregulated by at least 2.0-fold in the DPN group compared with the T2DM group (P b 0.001). The heat map vividly reflects the differential expression of the 3 miRNAs (Figure 1). After a comprehensive review of the literature, we found that miR-146a5p was obviously involved in the inflammatory response.

We verified that Lipofectamine could serve as the carrier of miR-146a-5p in vitro (Figure S3). To evaluate the effect of miR146a-5p on the viability of RSC96 cells, we measured the cell viability of each group using the CCK-8 method. There was a significant difference between each group (P b 0.01). Compared with that of the normal (25 mM) group, the cell viability of the 200 mM group decreased, while it was consistent with that of the NC group. The cell viability of the miR-146a-5p group was lower than that of the normal group; however, it was obviously higher than that of the 200 mM and NC groups (P b 0.05) (Figure 2, A). These results indicated that miR-146a-5p increased the viability of RSC96 cells. miR-146a-5p inhibited the expression of proinflammatory cytokines in RSC96 cells To verify whether miR-146a-5p could inhibit the inflammatory response in RSC96 cells induced by hyperglycemia, we examined the expression levels of COX-2 and TNF-α by western blot (Figure 2, B). The levels of COX-2 and TNF-α were significantly different between groups (P b 0.05). Compared with those in the normal group, the COX-2 and TNF-α levels in the 200 mM group were increased, while they were consistent with those in the NC group. The COX-2 and TNF-α levels in the miR-146a-5p group were higher than those in the normal group; however, in comparison to those in the 200 mM and NC groups, the COX-2 and TNF-α levels in the miR-146a-5p group were obviously decreased (P b 0.05) (Figure 2, C). At the same time, the level of IL-1β in the supernatant was measured by Luminex® 200™ assays. As shown in Figure 2, D, compared with those in the normal group the levels of IL-1β in the 200 mM and NC groups were remarkably upregulated (P b 0.01). The level of IL-1β was downregulated in the miR-146a-5p group in comparison to the 200 mM and NC groups (P b 0.01). These results implied that miR-146a-5p inhibited the inflammatory response. miR-146a-5p inhibited the apoptosis of RSC96 cells As shown in Figure 2, E and F, the apoptosis rate of RSC96 cells in each group was different (P b 0.05). The apoptosis rate in the 200 mM group was higher than that in the normal group, while it was consistent with the apoptosis rate in the NC group (P N 0.05). The apoptosis rate in the miR-146a-5p group was higher than that of the normal group; however, in comparison to the apoptosis rates in the 200 mM and NC groups, it was obviously decreased (P b 0.01). We also measured the expression of cleaved caspase-3 (Figure 2, B and C). The expression of cleaved caspase-3 in the 200 mM group was higher than that in the normal group, while it was consistent with that in the NC group. The expression of cleaved caspase-3 in the miR-146a-5p group was higher than that in the normal group;

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Figure 2. The effect of miR-146a-5p on cell viability, inflammation, and apoptosis in RSC96 cells induced by hyperglycemia. (A) The effect of miR-146a-5p on cell viability. (B) Western blot analysis showing the expression of COX-2, TNF-α, and cleaved caspase-3. (C) The relative expression of COX-2, TNF-α, and cleaved caspase-3 normalized to α-tubulin. (D) The effect of miR-146a-5p on the level of IL-1β. (E) Percentages of apoptotic Schwann cells analyzed by flow cytometry, where a represents the 25 mM group, b represents the 200 Mm group, c represents the NC group, and d represents the miR-146a-5p group. (F) Apoptosis rate of Schwann cells induced by a high concentration of glucose. ⁎P b 0.05, ⁎ ⁎P b 0.01.

however, in comparison to the cleaved caspase-3 expression in the 200 mM and NC groups, it was significantly decreased (P b 0.01). These results indicated that miR-146a-5p inhibited the apoptosis of RSC96 cells induced by hyperglycemia. Cytotoxicity of TPSP and nano-miR-146a-5p polyplexes In the initial stage, we measured cell viability in the presence of different concentrations of TPSP and polyethyleneimine (PEI) using the CCK-8 method. The results showed that when the concentration of TPSP and PEI was greater than 22.45 ng/μL, the cell viability significantly declined (P b 0.05). When the concentration of nanoparticles was greater than 44.90 ng/L, the cytotoxicity of PEI was greater than that of TPSP (P b 0.01) (Figure 3, A). We also determined cell viability nano-miR-146a5p and PEI-146a-5p polyplexes (Figure S4). Selecting the appropriate mass ratio of nano-miR-146a-5p polyplexes To screen the appropriate mass ratio of TPSP and miR-146a5p polyplexes, we observed the polymerization of nano-miR146a-5p polyplexes at different mass ratios by agarose gel electrophoresis. When the mass ratio of nano-miR-146a-5p polyplexes was more than 67.35, the nanoparticles could condense the miR-146a-5p mimics completely (Figure 3, B). As shown in Figure 3, C, when the mass ratio of nano-miR146a-5p polyplexes was greater than 67.35, the particle size stabilized between 200 nm and 400 nm. When the mass ratio of the nano-miR-146a-5p polyplexes was more than 44.9:1, the zeta potential of the polyplexes ranged from 20 to 35 mV (Figure 3, D). We also detected the miR-146a-5p level after polyplexes were transfected into the cytoplasm at different mass ratios. Compared with that in the control group, the relative expression levels of miR-146a-5p in the 89.80:1, 112.25:1, and 134.7:1

group were significantly increased (P b 0.01). However, the relative expression levels of miR-146a-5p in these three groups were not statistically different (Figure 3, E). We also used transmission electron microscopy to observe the morphology of nano-miR-146a-5p polyplexes at a mass ratio of 89.80:1. As shown in Figure 3, F, nano-miR-146a-5p polyplexes were uniform and formed spherical particles. This result indicated that nano-miR-146a-5p polyplexes prepared at a mass ratio of 89.80:1met the transport carrier requirements. Transfection efficiency of nano-miR-146a-5p polyplexes in Schwann cells A confocal microscope was used to observe the location of nano-miR-146a-5p polyplexes in Schwann cells. As shown in Figure 3, G, nano-miR-146a-5p polyplexes were mainly distributed in the cytoplasm. The experimental results showed that the transfection efficiency of TPSP met our experimental requirements (P b 0.01). Nano-miR-146a-5p has no noticeable effect on the blood glucose levels of DPN rats We conducted a small animal fluorescence imaging experiment, which indicated that the intramuscular administration method was more appropriate than the intraperitoneal administration method (Figure S5). After the administration method was chosen, we measured the blood glucose levels at different time points. At the 6th week, there was no difference in the blood glucose levels among the DPN group, the NANO group, the nano-miR-146a-5p group, and the VitB12 group (P N 0.05). The blood glucose levels in the DPN group, the NANO group, the nano-miR-146a-5p group, and the VitB12 group were slightly decreased at the 12th week compared with the 6th week

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Nano-miR-146a-5p increased the NCV of DPN rats attenuated the morphological damage to the sciatic nerve

Figure 3. The characterization and transfection efficiency of nano-miR-146a5p polyplexes. (A) The cytotoxicity of TPSP and PEI at the different concentrations. (B) Agarose gel electrophoresis showed the polymerization between TPSP and miR-146a-5p at different ratios, where number one represents miR-146a-5p alone, number two represents a mass ratio of 22.45:1, number three represents a mass ratio of 44.9, number four represents a mass ratio of 67.35:1, number five represents a mass ratio of 89.8:1, and number six represents a mass ratio of 112.25. (C) Particle size distribution of the nano-miR-146a-5p polyplexes prepared at mass ratios of 4.49, 44.90, 67.35, 89.80, 134.70 and 224.50. (D) Zeta potential of the TPSP-miRNA polyplexes prepared at ratios of 4.49, 44.90, 67.35, 89.80, 134.70 and 224.50. (E) The relative expression levels of nano-miR-146a-5p polyplexes prepared at mass ratios of 89.8:1, 112.25:1, and 134.7:1 after transfection for 48 h. (F) Transmission electron microscopic image of the nano-miR-146a-5p nanoparticles prepared at a ratio of 89.8. (G) The transfection efficiency of nano-miR-146a-5p polyplexes in RSC96 cells; a shows the cell background; b shows Cy3 with red fluorescence, c shows the merging of a and b. ⁎ P b 0.05, ⁎ ⁎ P b 0.01.

(P b 0.05). However, there was still no significant difference in the blood glucose levels among the DPN group, the NANO group, the nano-miR-146a-5p group, and the VitB12 group at the 12th week (P N 0.05) (Table 1).

To estimate the effect of miR-146a-5p on the function of the peripheral nerve, the NCV of each group was examined. The result indicated that nano-miR-146a-5p increased the MNCV and SNCV of DPN rats (Figure S6). H&E staining and Luxol fast blue staining were performed to observe the histopathological changes in the rat sciatic nerves from each group. In the normal group rats, the myelinated nerve fibers were similar in size. The myelin appeared dense, round, and uniform and had ordered lamellar structures presenting neither axonal shrinkage nor swelling. In the DPN and NANO groups, the myelin sheath of the myelinated nerve fibers was thin, loose, and disorganized and exhibited vacuolar-like defects. Some nerve fibers in the sciatic nerve appeared demyelinated. Lamellar spaces were expanded and separated from each other, and visible signs of axonal atrophy were evident. The endoneurial capillary displayed thick walls and irregular lumen. Furthermore, compared to those in the normal group, the nerve fibers decreased significantly. Morphological damage was alleviated in the nano-miR-146a-5p group compared with the DPN and NANO groups. There was no obvious difference in the histopathological changes between the nano-miR-146a-5p and ViB12 groups. Although the morphological damage in the nano-miR-146a-5p group was greatly alleviated, the improvement was not as large as that in the nanao-miR-146a-5p group (Figure 4, A and B). The result of toluidine blue staining was the same as that of H&E staining and Luxol fast blue staining (Figure S7). To observe the ultrastructure of myelin in the sciatic nerve, transmission electron microscopy was used. The cross-sectional structure of the sciatic nerve in the normal group was uniform, dense, and integrated. Lamellar structures also presented dark and concentric light circles and axonal shrinkage and swelling. The myelin structure in the DPN and NANO groups was disorganized and expanded toward the axonal or stromal side. A visible degree of lamellar fracture, acute demyelination, and myelin sheath separation was presented. The disruption and disorganization of axonal microtubules and microfilaments were observed, demonstrating signs of atrophy. The ultrastructure of Schwann cells showed nuclear irregularity, endoplasmic reticulum expansion, mitochondrial vacuolization, and basement membrane rupture. The amount and degree of axonal shrinkage and myelin degeneration of the nerve fibers in the miR-146a-5p group were lower and less severe than those in the DPN and NANO group. Although some stratification still existed, the overall signs of pathology were alleviated. There was no obvious difference in the ultrastructure changes of myelin between the nano-miR-146a-5p and ViB12 groups (Figure 5). We also found that nano-miR-146a-5p alleviated sciatic nerve damage at the molecular level (Figure S8). Nano-miR-146a-5p decreased the levels of inflammatory response- and apoptosis-related proteins in DPN rats As shown in Figure 6, A, IL-1β and IL-6 expression levels were remarkably upregulated in the DPN and NANO groups compared with the normal group (P b 0.01). Compared with those in the DPN and NANO groups, the IL-6 and IL-1β expression levels in in the nano-miR-146a-5p group were

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Figure 4. Neuromorphology images of H&E staining, Luxol fast blue staining, and toluidine blue staining of the sciatic nerve from rats in each group. (A) H&Estained neuromorphology images; (B) Luxol fast blue-stained neuromorphology images.

significantly downregulated (P b 0.01). There was no significant difference in the expression of IL-6 and IL-1β between the nanomiR-146a-5p and ViB12 groups. Additionally, no significant difference in the expression of IL-10 between each group (P N 0.05) was observed. Compared with those in the normal group, the COX-2, TNF-α, caspase-3 and cleaved caspase-3 expression levels in the DPN and NANO groups were significantly higher (P N 0.05). The expression of these cytokines was significantly decreased in the miR-146a-5p group in comparison to the DPN and NANO groups (P b 0.01). There was no significant difference in the expression

of COX-2 between the nano-miR-146a-5p and ViB12 groups. The expression of TNF-α, caspase-3, and cleaved caspase-3 in the VitB12 group was lower than that in the nano-miR-146a-5p group (P b 0.01) (Figure 6, B and C).

Discussion Recent studies have confirmed that the inflammatory response plays an important role in the pathogenesis of DPN. 12 A long period of hyperglycemia can activate

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Figure 5. The ultrastructure of myelin in the sciatic nerve of rats from each group. (A, B) Normal group; (C, D) DPN group; (E, F) NANO group; (G, H) nano-miR-146a-5p group.

macrophages, neutrophils and glial cells. 13 Activated immune cells can also promote tumor necrosis factor-α (TNF-α), IL-1β, and IL-6 expression. 14 Many studies have indicated that TNF-α is an essential proinflammatory cytokine in DPN and can activate both the nuclear factor-kappa B (NF-κB) and apoptotic pathways, causing cell viability inhibition and promoting nerve demyelination. 15,16 In previous studies, we found that the NCV of DPN rats treated with rhTNFR:Fc, an inhibitor of TNF-α, was significantly higher than that of untreated DPN rats. 17 COX (cyclooxygenase) is a key enzyme in the formation of prostaglandins (PGs), which are often highly expressed in various inflammation diseases and tumors. 18,19 COX-2 has been demonstrated to be involved in the development and progression of DPN. 20 The overexpression of COX-2 can lead to an

imbalance of TXA2/PGI2, which would further induce tissue hypoxia and damage the structural integrity and function of nerve tissue. 21 However, anti-inflammatory drugs are limited in clinical applications due to their obvious side effects and tissue nonspecificity. Therefore, endogenous small molecules regulating inflammation might be beneficial for the treatment of DPN. miRNAs are endogenous small molecules with lengths of 18 to 25 nucleotides. They can bind specific RNA by complementary base pairing of the 3’UTR and can negatively regulate the transcription or translation of gene expression. 22 Numerous studies have confirmed that miRNAs are involved in the regulation of inflammation. 6,7 Due to the important role of the inflammatory response in DPN, some miRNAs might participate in its pathogenesis. We performed miRNA microarray analysis in the sciatic nerve to compare between 3 T2DM and 3 DPN rats. We found 3 miRNAs to be differentially expressed (≥ 2-fold). One of the three, miR-146a-5p, has been demonstrated to be a negative regulator of inflammation. 9,10 Schwann cells, which are glial cells in the peripheral nervous system, can secrete and express a variety of neurotrophic factors. 23 Thus, they play a role in preventing neuronal cell death and promoting axonal myelination. 24 Under physiological conditions in vivo, Schwann cells are used as axons to encapsulate cells and exert protective effects on axons, nutrition support, injury repair, and repair of peripheral nerve damage caused by metabolic abnormalities. 25 Therefore, Schwann cells play an important role in preservation of the function of the peripheral nervous system and in the pathogenesis of peripheral neuropathy. Studies have demonstrated that in damaged nerve tissue, the proliferation and migration of Schwann cells are inhibited. 23 Therefore, RSC96 cells induced by hyperglycemia were used in vitro. We examined the effect of different concentrations of glucose at different times on the viability of Schwann cells and found that the RSC96 cells induced by 200 mM glucose at the 30th day were similar to cells under clinical hyperglycemic conditions. After miR-146a-5p was overexpressed in the RSC96 cells induced by 200 mM glucose, the inflammatory response and apoptosis were obviously inhibited. These results indicated that miR-146a-5p could inhibit the inflammatory response and apoptosis in RSC96 cells induced by hyperglycemia. A feasible nucleic acid vector is expected to accomplish interand intracellular trafficking for miRNA delivery. At present, the transfected vectors of miRNAs for animals mainly consist of retroviruses, adenoviruses or adeno-associated viruses. Their transfection efficiency might be high; however, the clinical use of viral vectors has been hindered by their immunogenicity, lack of tissue specificity and potential risk of inducing tumorigenic mutations. With the advancement of nanotechnology, we focused our attention on nanocarriers. Due to the nonimmunogenicity of nanoparticles, many nanocarrier drugs are already applied in clinical practice. 26,27 At present, nanodrug carriers can be mainly divided into organic nanocarriers and inorganic nanocarriers. The ideal nanocarrier for miRNAs should meet the requirements in the following aspects. First, it should remain stable after direct contact with miRNAs and transfer miRNAs with a high loading rate. Second, the nanocarrier should

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Table 1 Effect of nano-miR-146a-5p on the blood glucose levels of DPN rats (unit: mmol/L). Time

Normal

T2DM

DPN

NANO

nano-miR-146a-5p

VitB12

6th week 12th week

2.41 ± 1.80 3.01 ± 1.75

2.34 ± 1.83 18.98 ± 3.92

18.83 ± 3.65 17.39 ± 3.30

19.23 ± 3.75 16.44 ± 3.17⁎⁎

20.29 ± 4.29 15.88 ± 2.86

19.90 ± 4.23 16.08 ± 2.88

Figure 6. The expression levels of inflammatory response- and apoptosis-related proteins in each group. (A) The expression levels of IL-1β, IL-6 and IL-10 in plasma were detected by Luminex ® 200TM™ assays. (B) Western blot analysis showed the expression of COX-2, TNF-α, caspase-3, and cleaved caspase-3 in the rat sciatic nerve of each group. (C) The relative density of COX-2, TNF-α, caspase-3, and cleaved caspase-3.

selectively absorb on cell membranes and encapsulate miRNAs to protect miRNAs from degradation mediated by intracellular RNA-degrading enzymes. Third, after being phagocytosed, the polyplex should be able to withstand the destruction of endosomes and release the carried miRNAs inside the target cells. Finally, the carrier itself should be metabolized to endogenous molecules or other nontoxic small molecules. In our previous study, our team constructed a cationic nanocarrier meeting the basic conditions for the use of nanoparticles as pharmaceutical carriers; the main component of the carrier was TPSP. 11 Numerous cationic polymers for siRNA delivery have been reported, such as PEI. 28,29 We demonstrated that TPSP had low toxicity. Furthermore, we found that nano-miR-146a-5p polyplexes could be effectively transfected into RSC96 cells, implying that they could serve as a transport carrier. Neuroelectrophysiological examination is considered to provide direct evidence for the diagnosis of DPN. In this study, we tested the SNCV and MNCV. We demonstrated that the SNCV and MNCV of DPN rats were significantly decreased and that nano-miR-146a-5p elevated the NCV and reduced

morphological damage of DPN rats. This finding suggests that nano-miR-146a-5p plays a protective role in sciatic nerve injury in DPN rats. Moreover, the inflammatory responses and apoptosis of DPN rats were inhibited after treatment with nano-miR-146a-5p. This result implied that nano-miR-146a-5p might protect the sciatic nerve by inhibiting the inflammatory response and apoptosis. Based on our previous studies, we chose 16.7 mmol/L as our blood glucose standard in the DPN model. 10 Compared with the blood glucose levels of 11.11 mmol/L and 13.8 mmol/L in other studies, 30,31 16.7 mmol/L was higher and more readily induced injury in rats. Compared with 19.4 mmol/L, 32 our standard is slightly lower. However, the decreased level can prevent death induced by high glucose in rats and maintain the survival rate. We tested the blood glucose of the rats in each group at the 6th week and 12th week. The blood glucose levels in the DPN, NANO, nano-miR-146a-5p, and VitB12 groups were slightly decreased at the 12th week compared with the 6th week. However, the blood glucose levels in the four groups were not different from each other. This result indicated that nano-miR-

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inflammatory pathway, causing the expression of inflammationrelated genes. 36 Overexpression of IRAK1 and TRAF6 can activate NF-κB, resulting in the expression of related inflammatory genes regulated by NF-κB. 37 NF-κB is a nuclear protein factor with multidirectional transcriptional regulation. It widely exists in a variety of cells and plays a role in physiological and pathological processes such as inflammation and apoptosis. 38,39 As a proinflammatory transcription factor, NF-κB can promote the expression of inflammation-related genes and promote the occurrence of inflammatory responses. At the same time, NF-κB has been proven to have a promoting effect on apoptosis. 40 It has been confirmed that NF-κB can cause cell apoptosis by inhibiting the expression of antiapoptotic genes. 41 Downregulation of NF-κB in the process of UV-induced apoptosis of human melanoma cells can inhibit apoptosis. 42 Based on the current findings, we speculated that miR-146a-5p may inhibit the inflammatory response and apoptosis to reduce DPN by regulating the NF-κB signaling pathway (Figure 7). In contrast to previous studies using traditional treatments with chemical drugs and miR-146a-5p mimics, 17,43 our study adopted nano-miR-146a-5p as a therapeutic target. This research is mainly focused on inflammation and apoptosis in DPN. Because miR-146a-5p has multiple targeted sites, whether there are other potential mechanisms still needs to be explored. However, the present study provides experimental evidence to merit further exploration of the possible use of miR146a-5p and nanoparticles as a therapeutic approach in the treatment of DPN.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.nano.2019.01.007.

References Figure 7. The mode chart of nano-miR-146a-5p transfection and the potential pathway of miR-146a-5p inhibition of the inflammatory response and apoptosis.

146a-5p had no significant hypoglycemic effect on DPN rats. We considered that blood glucose fluctuations and the compensatory recovery of pancreatic β cells might be involved. Although the glucose concentrations were lower than those in the previous treatment, they were still higher than 11.11 mmol/L and 13.8 mmol/L. Compared with that in the normal group, the blood glucose concentration was still maintained at high levels. Since nano-miR-146a had no hypoglycemic effect on DPN rats, some other biological processes might be involved in the inhibition of the inflammatory response and apoptosis regulated by nano-miR-146a. Studies have confirmed that miR-146a-5p can be involved in the inflammatory response by direct binding on interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6). 33,34 IRAK1 and TRAF6 are widely expressed in various cells and are receptor proteins that localize to the cytoplasm and nucleus. 35 They mainly serve as receptor proteins participating in the Toll-like receptor-mediated

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