Mouse DRG neurons were purchased from Qincheng Biotechnology Co., Ltd. (cat. no. MIC-QC-242; Beijing Solarbio Science & Technology Co., Ltd.). DRG neurons were cultured in DMEM with 10% FBS, 100 µg/ml streptomycin and 100 U/ml penicillin (all Beijing Solarbio Science & Technology Co., Ltd.) at 37˚C with 5% CO₂ in a humid atmosphere. The neurons were cultured for a few passages prior to subsequent experiments.

To mimic diabetes and DN in vitro, DRG neurons were cultured in complete medium supplemented with 50 mM glucose (cat. no. G8150; Beijing Solarbio Science & Technology Co., Ltd.). DRG neurons were maintained under high-glucose conditions at 37˚C with 5% CO₂ for 48 h (D) or 72 h (DN). DRG neurons in the negative control (NC) group were cultured in complete medium containing 25 mM glucose conditions at 37˚C with 5% CO₂ for 48 h, which is optimal for DRG neuron survival and growth (37).

To determine the effect of CXB (cat. no. IC0230; Beijing Solarbio Science & Technology Co., Ltd.) on DN, DRG neurons in the DN group were incubated with CXB at various concentrations (0, 1, 5, 15, 30 or 50 µM) for 24 h prior to subsequent experiments.

miR-155 inhibitors (5'-ACCCCUAUCACAAUUAGCAUUAA-3'), NC inhibitors (5'-CAGUACUUUUGUGUAGUACAA-3'), miR-155 mimics (5'-UUAAUGCUAAUCGUGAUAGGGGU-3'), mimic NC (5'-UCACAACCUCCUAGAAAGAGUAGA-3') and predesigned vectors expressing siRNAs targeting COX-2 mRNAs (si-COX-2, 5'-AATGTCCGGGTACAATCGCACCCTGTCTC-3') were purchased from Cytiva. Scrambled siRNA (si-control, 5'-AATTCTCCGAACGTGTCACGT-3'; Qiagen GmbH) was used as the NC for si-COX-2. A total of 2x10⁵ DRG neurons of the DN group were cultured in 24-well plates and transfected using Lipofectamine™ 3000 (cat. no. L3000008; Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. miR-155 inhibitors, miR-155 mimics, NC inhibitors and mimic NC were used at a concentration of 100 nM and si-COX-2 and si-controls were used at a concentration of 50 nM. Cells were treated with CXB at 48 h post-transfection, after which cell suspensions were collected for further analysis.

Cell viability was measured using a Cell Counting Kit-8 (CCK-8; cat. no. C0038; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. DRG neurons were cultured into a 96-well microplate (5x10³ cells/well) and treated with glucose, CXB and/or transfected. A total of 10 µl of CCK-8 reagent was added to the microplates and incubated at 37˚C for 2 h. Absorbance at 450 nm (A450) was detected using a microplate reader (Bio-Rad Laboratories, Inc.). Cell growth curves were plotted based on the average A450 value from 3 replicate measurements.

Apoptosis was determined with an Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit (cat. no. E606336-0500; Sangon Biotech, Co. Ltd.) according to the manufacturer's protocol. DRG neurons under various treatment conditions were seeded into 6-well plates (5x10⁵ cells/well) and reacted with 5 µl of Annexin V-FITC and 10 µl of PI in the dark at a room temperature for 5 min. Flow cytometric analysis was performed using a flow cytometer (FACSCalibur; BD Biosciences) and FlowJo software version 7.6 (FlowJo, LLC). The results reflect the combination of early and late apoptotic cells.

ROS generation was detected using a ROS assay kit (cat. no. S0033S; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. DRG neurons under various treatment conditions were seeded into 6-well plates (5x10⁵ cells/well). Then, 10 µM 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) was added into each well and incubated at 37˚C for 30 min in the dark. The fluorescence intensity at 485 and 530 nm was examined with a flow cytometer (FACSCalibur; BD Biosciences) to evaluate ROS generation.

ELISAs were performed with ELISA kits for malondialdehyde (MDA; cat. no. SBJ-M0411; SenBeiJia Biological Technology Co., Ltd.) and superoxide dismutase (SOD; cat. no. ml037856; Mlbio). A total of 1x10⁵ cells under various treatment conditions (glucose, CXB and/or transfection) added to 96-well microplates and processed according to the manufacturer's protocol. Conditioned mediums were collected from the wells at 24 h after processing. Finally, the absorbance at A450 was detected with a microplate reader (Bio-Rad Laboratories, Inc.) and quantities were calculated with standard curves.

A total of 5x10⁶ DRG neurons/sample were harvested following treatments or transfections. Total cellular RNA was isolated using TRIzol™ Reagent (cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.). RNA concentrations were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). miR-155 expression was examined by TransScript^® Green miRNA Two-Step RT-qPCR SuperMix (cat. no. AQ202-01; Beijing Transgen Biotech Co., Ltd.). The specific primers used were as follows: miR-155 forward, 5'-CTGTATCAAAAGGCCAACTGAA-3' and reverse, 5'-GTGTCTATCCTTATGAATCGCCA-3'; U6 forward, 5'-AACGAGACGACGACAGAC-3' and reverse, 5'-GCAAATTCGTGAAGCGTTCCATA-3'. PCR amplification was then conducted using a LightCycler 480 instrument (Roche Diagnostics). The thermocycling conditions were as follows: 95˚C for 10 sec followed by 40 cycles of 95˚C for 10 sec and 60˚C for 30 sec. U6 was used as internal controls. Relative expression levels were calculated using the 2^-ΔΔCq method (38).

DRG neurons under various treatment conditions were seeded into 6-well plates (5x10⁵ cells/well). Crude cell lysates were harvested using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's protocol. The purity of the protein in extracts was examined by the BCA method. Proteins (100 µg/lane) were separated by 10% SDS-PAGE and transferred to a PVDF membranes. Nonspecific protein binding was prevented with the addition of blocking buffer [5% milk, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl and 0.1% Tween-20] for 40 min at a room temperature. The membranes were incubated with specific primary antibodies (1:2,000; Abcam) against nerve growth factor (NGF; cat. no. ab52918), brain-derived neurotrophic factor (BDNF; cat. no. ab226843), COX-2 (cat. no. ab188183), kelch-like ECH-associated protein 1 (Keap1; cat. no. ab227828), nuclear factor erythroid-2-related factor 2 (Nrf2; cat. no. ab137550), heme oxygenase-1 (HO-1; cat. no. ab189491), superoxide dismutase 1 (SOD1; cat. no. ab13498), SOD2 (cat. no. ab137037) or β-actin (cat. no. ab115777) were incubated in blocking buffer at 4˚C overnight. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin F (1:10,000; cat. no. ab205718; Abcam) for 60 min in the dark (at room temperature), and developed using an ECL reagent (cat. no. 32209; Thermo Fisher Scientific, Inc.). Membranes were exposed using chemiluminescence apparatus (Bio-Rad Laboratories, Inc.). ImageJ software (version 1.8.0; National Institutes of Health) was used to quantify the protein grayscale.

starBase database (http://starbase.sysu.edu.cn) provides a widely-used noncoding RNA interaction from crosslinking-immunoprecipitation and high-throughput sequencing (CLIP-seq) (39). The database was used to predict potential target sequences of miR-155 and COX-2.

The direct binding of COX-2 and miR-155 was verified by dual-luciferase reporter assays. Portions of the wild-type (WT) and mutant (MUT) 3'-untranslated regions (UTRs) of COX-2 mRNA containing the predicted miR-155-targeting regions were synthesized and inserted into the pGL3-report luciferase reporter vector (Sigma-Aldrich; Merck KGaA). miR-155 mimics and NC mimics were cotransfected into cells with pGL3-3'-UTR wild-type or mutant plasmid DNA using Lipofectamine^® 3000 (cat. no. L3000008; Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After 24 h, relative luciferase activity was analyzed using a dual-luciferase reporter gene analysis system (Promega Corporation) and Renilla luciferase reference plasmids were used for standardization.

Statistical analysis was performed using SPSS software (version 19.0; IBM Corp.). Unpaired Student's t-test was used to evaluate the differences between two groups. One-way ANOVA with Tukey's Multiple Range post hoc test was used to determine differences between multiple groups. Data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

To detect the effect of glucose on DRG neurons, DRG neurons were treated with glucose for 48 or 72 h. Cell viability was suppressed and apoptosis was induced by 50 mM glucose for 48 h compared with the NC group and cell survival was further inhibited by 50 mM glucose treatment for 72 h (P<0.05; Fig. 1A and B). These results indicated that prolonged exposure to a high glucose concentration damaged the DRG neurons. Thus, high-glucose treatment of DRG neurons for 48 or 72 h mimicked D or DN, respectively, in vitro.

CXB (various doses, 1-50 µM) was utilized to treat DRG neurons. Cell viability of DRG neurons was examined to evaluate the cytotoxicity of CXB, which indicated that cell viability was unchanged by treatment with CXB at different concentrations (Fig. 2A). To examine the effect of CXB on DRG neurons, DRG neurons were treated with CXB at various concentrations. CXB increased DRG neuron viability in the DN group to a certain extent (P<0.05; Fig. 2B). Considering that DN DRG neurons treated with 30 µM CXB exhibited the highest viability, 30 µM was used as the optimum CXB dose in subsequence experiments. Additionally, apoptosis in DN neurons was attenuated by CXB treatment (Fig. 2C). Furthermore, DN-induced neurotrophic factors NGF and BDNF suppression was attenuated with CXB treatment (Fig. 2D; P<0.05). These results indicated that CXB rescued DN-mediated inhibition of DRG neuron survival to a certain extent.

DRG neurons were treated with 30 µM CXB and the protective effect of CXB against oxidative stress in neurons was evaluated. The ROS and MDA levels in the DN group were decreased by CXB treatment (P<0.05; Fig. 3A and B). However, the SOD levels were enhanced by CXB treatment (P<0.05; 3C). Additionally, the levels of oxidative stress-related proteins in each group were assessed. The levels of COX-2 and Keap1 were reduced by CXB treatment, while the levels of Nrf2, HO-1, SOD1 and SOD2 were partially increased by CXB treatment (P<0.05; Fig. 3D). These data indicated that CXB effectively ameliorated oxidative stress in DRG neurons in the DN group.

The regulatory effect of miR-155 on CXB-alleviated DN was investigated. RT-qPCR data demonstrated that miR-155 expression was suppressed in the DN group (Fig. 4A). Furthermore, miR-155 expression was increased in a dose-dependent manner by CXB treatment (P<0.05). miR-155 expression in DN-treated DRG neurons was decreased by miR-155 inhibitor transfection (P<0.05; Fig. 4B). Following this, the importance of miR-155 in the protective function of CXB was determined. Compared to transfection with NC inhibitor, transfection with miR-155 inhibitor significantly decreased cell viability (Fig. 4C), promoted apoptosis (Fig. 4D), suppressed NGF and BDNF expression (Fig. 4E) and accelerated oxidative damage (Fig. 4F-I; all, P<0.05). Moreover, the protective functions of CXB against DN-induced damage in DRG neurons were partially abrogated by inhibition of miR-155 expression (Fig. 4C-I). These results indicated that miR-155 served an important role in CXB-mediated alleviation of DN.

The online bioinformatics tool starBase predicted COX-2 as a target of miR-155. The complementary binding sites of COX-2 and miRNA-155 are presented in Fig. 5A. To further confirm the interaction between miR-155 and COX-2, luciferase reporter gene vectors that expressed WT or MUT 3'UTR of the downstream target luciferase gene COX-2 were constructed. The results demonstrated a significant decrease in luciferase activity in the COX-2 3'UTR-WT/miR-155 mimic group compared with the COX-2 3'UTR-MUT/miR-155 mimic group (P<0.01; Fig. 5B). miR-155 mimic transfection efficiency was detected using RT-qPCR (P<0.05; Fig. 5C). The regulation of COX-2 by miR-155 in DRG neurons was then investigated. The protein expression level of COX-2 was significantly decreased after cells were treated with miR-155 mimics compared with NC mimics (P<0.05; Fig. 5D). These results demonstrated that miR-155 targeted COX-2.

The mechanism by which miR-155 and COX-2 are involved in CXB-mediated alleviation of DN was investigated. Vectors that expressed si-COX-2 or si-COX-2 plus miR-155 inhibitor were transfected into DRG neurons in the DN group. The protein expression of COX-2 was decreased by si-COX-2 transfection (P<0.05; Fig. 6A). Following this, the association between miR-155 and COX-2 with CXB-mediated alleviation of DN was investigated. Compared to the cell viability in the DN group and CXB treatment group, transfection with si-COX-2 plus CXB treatment increased cell viability (P<0.05; Fig. 6B), decreased apoptosis (Fig. 6C), upregulated NGF and BDNF expression (P<0.05; Fig. 6D) and reduced oxidative damage (P<0.05; Fig. 6E-H). Furthermore, the protective functions of CXB against DN-induced damage in DRG neurons were partially reduced by si-COX-2 and miR-155 inhibitor transfection plus CXB treatment (P<0.05; Fig. 6B-H). In summary, these results indicated that CXB attenuated DN-induced DRG neuron damage and oxidative stress by regulating the miR-155/COX-2 axis.