Healthy male Sprague-Dawley rats (200–220 g) were maintained 4–5 per cage in specific-pathogen free conditions with (24 ± 1)°C, 40–50% relative humidity, 12/12 h light/dark cycles, and provided with food and water ad libitum. The animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Xuzhou Medical University (approval number: 202012A145).

The model of chronic neuropathic pain was induced via SNI as previously reported (Decosterd and Woolf, 2000). Briefly, a 1–2 cm incision was made along the lateral skin of the left thigh to expose the sciatic nerve and its three branches. The sural nerve was reserved, and the tibial nerve and the common peroneal nerve were carefully ligated with chromic gut ligatures (4.0) and transected. The incision was closed with 4.0 silk sutures. Rats in the sham groups underwent the same surgical procedure but without the ligation or transection.

Mechanical allodynia was assessed with von Frey filaments according to the up-down method (Chaplan et al., 1994). Briefly, after a 30-min accommodation period, a series of von Frey filaments (North Coast Medical, Morgan Hill, CA, United States) were applied sequentially to the lateral plantar area of the left hind paw of rats. Each filament was perpendicular to the paw and held for 5 s, and a sharp withdrawal of the hind paw was considered as a positive response. The von Frey filament force (g) that produced a 50% positive response was calculated and expressed as mechanical withdrawal threshold (MWT).

Cold allodynia was evaluated as previously described (Flatters and Bennett, 2004). After a 30-min accommodation period, a drop of acetone was applied to the lateral plantar surface of the ipsilateral hind paw. The responses of the rats were evaluated by the 4-point method: 0, no response; 1, quick withdrawal or flick of the paw; 2, prolonged withdrawal or repeated flicking of the paw; 3, repeated flicking of the paw with licking directed at the ventral side of the paw. The test was repeated for three times at an interval of 5 min, and the threshold of cold pain in rats was represented as the cumulative scores.

The forced swim test was performed as we previously described (Zhou et al., 2020). An animal was placed in a transparent plexiglass cylinder (65 cm in height, 30 cm in diameter) filled with water (25 cm in depth) at (25 ± 1)°C. After a 15 min pretest, 24 h later the animal was allowed to swim in the cylinder for 6 min and the immobility time during the last 5 min was recorded. Immobility was defined as the cessation of all active swimming and escaping activities.

The sucrose preference test was performed as we previously described (Zhou et al., 2020). Briefly, rats were initially trained to adapt to the sucrose solution before the formal test (Day 1, two bottles of 1% sucrose solution; Day 2, one bottle of 1% sucrose solution and one bottle of water). After the adaptation period, the rats were deprived of water for 23 h. The test was conducted when the rats were housed in individual cages and had free access to two bottles containing the sucrose solution and water, respectively. After 1 h, the weights of the consumed sucrose solution and water were recorded. Sucrose preference (%) was calculated as the consumption of sucrose water divided by the total liquid consumption.

Open field test was performed as we described previously (Zhou et al., 2020). The animal was placed in the central area of an open arena (100 × 100 × 50 cm) and allowed to move freely for 5 min. Locomotor activity of the animal was video-recorded and analyzed by an automated video-tracking system (ANY-maze, Stoelting, Kiel, WI, United States).

The elevated plus-maze apparatus was consisted of two open arms and two closed arms separated by a center square platform (10 × 10 cm). Each arm was 10 cm wide and 50 cm long. The maze was positioned 60 cm from the ground, and 40-cm-high black walls surrounded the closed arms. The apparatus was placed in a mildly lit room different from the room where the animals were maintained and handled. The rat was placed in the center platform and allowed to freely explore for 5 min. Between consecutive tests, the apparatus was cleaned with 75% ethanol solution. The total number of visits were automatically scored using a video tracking system (ANY-maze, Stoelting, Kiel, WI, United States).

The rAAV2/9-EF1a-lncRNA (LOC102552829-201)-bGH polyA-CMV-mCherry-hGH polyA (AAV-lnc) vector and control vector rAAV2/9-CMV-mCherry-WPRE-polyA (AAV-mCherry), the rAAV2/9-U6-shRNA (LOC102552829-201)-U6-shRNA (LOC102552829-201)-CMV-mCherry-SV40 polyA (AAV-lnc shRNA) vector and control vector rAAV2/9-U6-shRNA (scramble)-U6-shRNA (scramble)-CMV-mCherry-SV40 polyA (AAV-mCherry), and the rAAV2/9-hSyn-BFP-pre-Mir128-2-WPRE-hGH polyA (AAV-miR) vector and control vector rAAV2/9-hSyn-BFP-WPRE-hGH polyA (AAV-BFP) were obtained from BrainVTA Co., Ltd. (Wuhan, China). The pAAV2/9-U6-TuD (rno-miR-128-3p)-CMV-EBFP2-WPRE (AAV-miR TuD) vector and control vector pAAV2/9-U6-shRNA (NC2)-CMV-EBFP2-WPRE (AAV-EBFP) were obtained from OBiO Co., Ltd. (Shanghai, China). The AAV2/9-Hsyn-r-Sirt1-3xflag-ZsGreen (AAV-SIRT1) vector and control vector AAV2/9-Hsyn-ZsGreen (AAV-ZsGreen), and the AAV2/9-Hsyn-mir30-r-Sirt1-ZsGreen (AAV-SIRT1 shRNA) vector and control vector AAV2/9-Hsyn-ZsGreen (AAV-ZsGreen) were obtained from Hanbio Co., Ltd. (Shanghai, China). A volume of 0.3–0.6 μl virus (depending on the expression strength and viral titer) was delivered bilaterally into the CeA of rats (anteroposterior, −2.1 mm from the bregma; mediolateral, ±4.5 mm; dorsoventral, −8.6 mm from dura) at a rate of 0.1 μl/min. Viral injection sites were histologically verified by confirming the fluorescence signal in the CeA of brain slices with a fluorescence microscope.

The frozen CeA tissues were homogenized in ice-cold RIPA lysis buffer containing protease inhibitors and phenylmethylsulfonyl fluoride (Beyotime Biotech, Jiangsu, China). The supernatants were collected after centrifugation at 12,000 g for 15 min at 4°C, and the protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, United States). Equal amounts of protein samples were separated by SDS-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology, China) and transferred onto a nitrocellulose membrane. Then, the membrane was incubated with the following primary antibodies at 4°C overnight: anti-SIRT1 (Cell Signaling Technology, Beverly, MA, United States), and anti-β-actin (Bioworld, Louis Park, MN, United States), followed by incubation with the IRDye 800CW second antibody (Li-Cor, Lincoln, NE, United States). The immunoreactive bands were detected using an Infrared Imaging System (Gene Company Limited, Hong Kong, China) and analyzed with ImageJ software.

Total RNA was isolated from CeA tissues using a TRIzol reagent kit (Invitrogen, Carlsbad, CA, United States) and reversely transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, United States). Then the relative mRNA levels were measured by real-time PCR using the Roche 480 LightCycler detection system. Each sample was analyzed in triplicate. The primers used in the RT-qPCR assays are given in Supplementary Table 1. The relative mRNA levels of lncRNA-84277 and SIRT1 were normalized to β-actin. The relative mRNA level of miR-128-3p was normalized to U6.

The wild-type (wt) and mutant type (mut) luciferase reporter vectors for lncRNA-84277 or SIRT1 with miR-128-3p binding regions were constructed by Hanbio Co., Ltd. (Shanghai, China). The vectors were co-transfected with miR-128-3p mimics into 293T cells, and luciferase activities were measured 48 h later using a dual-luciferase reporter system (Promega, Madison, WI, United States).

Carboxyfluorescein (FAM)-labeled probe targeting to lncRNA-84277 was designed and synthesized by Wuhan Servicebio Technology Co. Ltd. (Wuhan, China). Probe sequence was shown in Supplementary Table 1. For FISH analysis, PC12 cells were fixed in 4% paraformaldehyde for 20 min. After prehybridization, the cells were hybridized with the specific probe at 37°C overnight. Then, cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). Images were photographed using a fluorescence microscope (Tokyo, Japan).

Nuclear and cytosolic fractions were separated using a Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s instruction. U6 was used as a nuclear control while β-actin was a cytoplasmic control. The expression levels of β-actin, U6, and lncRNA-84277 in the nuclear and cytoplasm were detected by RT-qPCR assays.

The data are presented as the mean ± SEM. Unpaired Student’s t-test was used to compare the differences between two groups. One-way analysis of variance (ANOVA) followed by Bonferroni test was applied to compare the differences among multiple groups. A repeated-measures ANOVA was used to compare the differences of mechanical pain. Mann-Whitney U-test was used to compare the differences of cold pain between two groups. Kruskal–Wallis test was used to compare the differences of cold pain among multiple groups. The difference was considered statistically significant at P < 0.05.

We used a rat neuropathic pain model of SNI, and the sensory pain was evaluated by Von Frey test and acetone test (Figure 1A). As shown in Figure 1B, compared with sham rats, SNI rats displayed mechanical allodynia with decreased MWT in the ipsilateral hind paw from day 1 to at least day 28 after the SNI surgery. SNI rats also developed cold allodynia, manifested by an increased acetone test score over the same time frame (Figure 1C).

We then determined the depression-like behaviors in SNI rats, using the forced swim test and the sucrose preference test (Figure 1A). We found that, compared with sham rats, SNI rats showed increased immobility time (P < 0.01; Figure 1D) and decreased sucrose preference rate (P < 0.05; Figure 1E) 30 days after the SNI surgery, suggesting that SNI rats developed depression-like behaviors.

Since motor impairment may influence pain perception and depression-like behaviors, the total distance traveled in the open field test and the total number of arm entries in the elevated plus-maze test were measured as the indicators of locomotor activity (Figure 1A). In the open field test, we found that there was no significant difference in the total distance traveled between sham rats and SNI rats (Figure 1F). In the elevated plus-maze test, the total number of arm entries was not altered in SNI rats when compared with sham rats (Figure 1G). These results indicate that the locomotor activity of the rats was not affected by SNI surgery.

In our previous study, we performed lncRNA microarray analysis to identify differentially expressed lncRNAs in the CeA tissues of SNI rats and sham rats, and total 50 novel lncRNAs, including 38 upregulated and 12 downregulated lncRNAs, were identified. To explore the upstream mechanism for SIRT1 to regulate the chronic pain-related depression-like behaviors, we analyzed the differentially expressed lncRNAs, and the novel lncRNA-84277 drew our attention. Further study showed that the expression of lncRNA-84277 in CeA was decreased in SNI rats 31 days after surgery when compared with sham rats (P < 0.01; Figure 1H).

Considering that lncRNA-84277 was downregulated in CeA of SNI-induced depression rats, we then investigated whether overexpression of lncRNA-84277 would alter the depression-like behaviors in SNI rats. An AAV vector expressing lncRNA-84277 and mCherry (AAV-lnc) was microinjected into the CeA of SNI rats, and control rats were injected with negative control AAV expressing mCherry only (AAV-mCherry). Then the sensory pain and depression-like behaviors of rats were evaluated 2 weeks after the AAV infusion (Figure 2A). The successful transfection of AAV was verified by the numerous mCherry-positive cells (Figure 2B) and the upregulation of lncRNA-84277 expression in the CeA of SNI rats (P < 0.01; Figure 2C). Moreover, lncRNA-84277 overexpression reversed the SNI-induced increase of immobility time in the forced swim test (P < 0.01; Figure 2D) and the decrease of sucrose preference rate in the sucrose preference test (P < 0.05; Figure 2E). However, the total distance traveled in the open field test (Figure 2F) and the total number of arm entries in the elevated plus-maze test (Figure 2G) did not alter by lncRNA-84277 overexpression. In addition, lncRNA-84277 overexpression had no influence on SNI-induced mechanical allodynia (Figure 2H) and cold allodynia (Figure 2I). These results suggest that the overexpression of lncRNA-84277 in CeA could improve SNI-induced depression-like behaviors.

Next, to further confirm the role of lncRNA-84277 in chronic pain-related depression-like behaviors, an AAV vector carrying lncRNA-84277-specific shRNA (AAV-lnc shRNA) was injected into the CeA of naïve rats, and control rats were injected with a negative control AAV containing scrambled shRNA (AAV-mCherry) (Figure 3A). The successful transfection of AAV-lnc shRNA was verified by the numerous mCherry-positive cells (Figure 3B) and the decreased expression of lncRNA-84277 in the CeA of naïve rats (P < 0.01; Figure 3C). Moreover, lncRNA-84277 knockdown induced depression-like behaviors in naïve rats, manifested by the increased immobility time in the forced swim test (P < 0.01; Figure 3D) and the decreased sucrose preference rate in the sucrose preference test (P < 0.05; Figure 3E). However, the total distance traveled in the open field test (Figure 3F), and the total number of arm entries in the elevated plus-maze test (Figure 3G) did not alter after the knockdown of lncRNA-84277. In addition, lncRNA-84277 knockdown had no influence on the baseline threshold of mechanical pain (Figure 3H) and cold pain (Figure 3I). These results suggest that the knockdown of lncRNA-84277 in CeA is sufficient to induce the depression-like behaviors in the absence of sensory pain in naïve rats.

To explore the mechanism for lncRNA-84277 to regulate SIRT1 expression, we observed the cellular localization of lncRNA-84277 using nuclear and cytoplasmic fractionation and FISH experiments. We found that lncRNA-84277 was mainly located in the cytoplasm (Figures 4A,B), suggesting that it may function as miRNA sponge to regulate SIRT1 expression. Therefore, we used the bioinformatic databases (miRDB, RNAhybrid, and TargetScan) to predict the potential miRNAs that interact with both lncRNA-84277 and SIRT1. We found that three of the top five miRNAs binding to lncRNA-84277 (miR-128-3p, miR-27a-3p, and miR-27b-3p, Figures 4C,D) could also bind to the 3’-UTR region of SIRT1 (Figure 4E). We then assessed the expression levels of the three miRNAs in the CeA of SNI-induced depression rats. The results showed that the expression of miR-128-3p increased (P < 0.01) while the expression of miR-27a-3p and miR-27b-3p had no significant change (Figure 4F).

Dual-luciferase reporter assay revealed that miR-128-3p mimics reduced the luciferase activity of the lncRNA-84277 wild type reporter (P < 0.01) but not the mutated reporter (Figures 5A,B). In addition, overexpression of lncRNA-84277 in CeA reversed the increase of miR-128-3p in SNI rats (P < 0.01; Figure 5C), and the improvement of lncRNA-84277 overexpression on the depression-like behaviors in SNI rats was also reversed by miR-128-3p overexpression (Figures 5D,E). Furthermore, knockdown of lncRNA-84277 in CeA induced the increase of miR-128-3p in naïve rats (P < 0.01; Figure 5F), and the depression-like behaviors in naïve rats induced by lncRNA-84277 knockdown were reversed by miR-128-3p knockdown (Figures 5G,H). However, the effects of lncRNA-84277 on the locomotor activity and sensory pain were not influenced by miR-128-3p (Supplementary Figures 1A–H). These findings suggest that lncRNA-84277 improves depression-like behaviors through inhibiting miR-128-3p.

As shown in Figure 4E, SIRT1 was predicted as a target gene of miR-128-3p. To confirm this finding, we constructed a luciferase reporter vector with the wild type or mutated SIRT1-3’UTR binding site for miR-128-3p (Figure 6A). The results showed that the reduced luciferase activity was observed in SIRT1 wild type reporter (P < 0.01) but not in SIRT1 mutated reporter (Figure 6B). Moreover, knockdown of miR-128-3p in CeA by infusion of AAV-miR-128-3p TuD (P < 0.01; Figure 6C) relieved the depression-like behaviors and upregulated SIRT1 expression in SNI rats, while these effects were abolished by SIRT1 knockdown (Figures 6D–G). Conversely, overexpression of miR-128-3p in CeA (P < 0.05; Figure 6H) induced the depression-like behaviors and decreased SIRT1 levels in naïve rats, which were partially rescued following SIRT1 overexpression (Figures 6I–L). In addition, the effects of miR-128-3p on the locomotor activity and sensory pain were not influenced by SIRT1 (Supplementary Figures 2A–H).

As shown in Figures 7A,B, Overexpression of lncRNA-84277 in CeA increased SIRT1 expression in SNI rats (P < 0.01, P < 0.01), which was abrogated by miR-128-3p overexpression (Figures 7C,D). In contrast, SIRT1 expression was inhibited by knockdown of lncRNA-84277 in CeA of naïve rats (P < 0.01, P < 0.01; Figures 7E,F), and reversed by miR-128-3p knockdown (Figures 7G,H). Similarly, the improvement of the depression-like behaviors and the increased SIRT1 expression in SNI rats caused by lncRNA-84277 overexpression were reversed by SIRT1 knockdown (Figures 8A–D). Conversely, the depression-like behaviors and the decreased SIRT1 expression in naïve rats caused by lncRNA-84277 knockdown were substantially rescued by SIRT1 overexpression (Figures 8E–H). In addition, the effects of lncRNA-84277 on the locomotor activity and sensory pain were not influenced by SIRT1 (Supplementary Figures 3A–H).

Taken together, these results reveal that lncRNA-84277 serves as a sponge for miR-128-3p to regulate SIRT1 expression and improves SNI-induced depression-like behaviors via a ceRNA mechanism (Figure 9).