Urolithin A alleviates neuropathic pain and activates mitophagy

Abstract

Neuropathic pain (NP) occurs frequently in the general population and has a negative impact on the quality of life. There is no effective therapy available yet owing to the complex pathophysiology of NP. In our previous study, we found that urolithin A (UA), a naturally occurring microflora-derived metabolite, could relieve NP in mice by inhibiting the activation of microglia and release of inflammation factors. Here in this study, we sought to investigate whether mitophagy would be activated when UA alleviated NP in mice. We showed that the autophagy flow was blocked in the spinal dorsal horn of the chronic constriction injury (CCI) mice when the most obvious pain behavior occurs. Intraperitoneal injection of UA markedly activated the mitophagy mediated by PTEN-induced kinase 1/Parkin, promoted mitobiogenesis in both neurons and microglia, and alleviated NP in the CCI mice. In summary, our data suggest that UA alleviates NP in mice and meanwhile induces mitophagy activation, which highlights a therapeutic potential of UA in the treatment of NP.

Neuropathic pain (NP) is a chronic pain caused by primary lesions or dysfunctions of the central or peripheral nervous system, characterized by spontaneous pain, hyperalgesia and allodynia. ¹ The incidence of NP in the general population is as high as 6.9%–10%. However, the complex pathophysiology of NP is not yet fully elucidated, which contributes to a lack of effective treatments. ² The development of drugs and methods that can effectively relieve NP is still an urgent problem to be solved at home and abroad. Therefore, it is necessary to conduct in-depth research on the mechanism of NP.

Autophagy is a “self-cleaning” process prevalent in eukaryotes, which degrades long-lived, dysfunctional or excessive proteins to maintain normal tissue homeostasis. ³ NP is often accompanied by injury and dysfunction of nerve cells. If not controlled, these neurons will eventually undergo cell death through a process that may involve autophagy. ⁴ Mitophagy is an autophagy process that selectively removes damaged mitochondria and is an important way to maintain normal mitochondrial function and quantity. ⁵ Mitophagy and mitobiogenesis may maintain cell survival and function in a coordinated manner. Generally, mitophagy removes damaged mitochondria, and then mitobiogenesis will happen in order to maintain mitochondrial homeostasis. ⁶ Currently, the proposed mitophagy mechanisms can be divided into two types: ubiquitin-dependent and non-ubiquitin-dependent. ⁷ Among them, the ubiquitin-dependent regulation pathway mediated by PTEN-induced kinase 1 (PINK1)/Parkin (an E3 ubiquitin ligase) is the focus of current research. In the damaged mitochondria, PINK1 gathers in the mitochondrial outer membrane to recruit Parkin for phosphorylation, which further mediates the binding of adaptor proteins to LC3, thus promoting the degradation of ubiquitination proteins through autophagy. ⁸ Studies have shown that PINK1 mediates mitophagy in the spinal cord in NP, and PINK1 knockout mice display reduced neuropathic hypersensitivity. ⁹ Therefore, the search for drugs that can target PINK1-activated mitophagy may become a potential treatment scheme for NP.

Urolithin A (UA), as a main product of ellagic acid metabolized by intestinal flora, has attracted more and more attention in recent years due to its broad range of biological activities and potentials to treat various diseases, such as anti-inflammatory, anti-cancer, heart protection, and neuroprotection.^10–12 Among them, it is worth noting that UA has been confirmed to have the effect of activating mitophagy. In a mouse model of hyperuricemic nephropathy, UA can reduce STING-NLRP3 axis-mediated inflammatory response by activating Parkin-dependent mitophagy. ¹³ At the same time, it has been reported that the mitophagy in the damaged endothelial cells is activated upon the treatment with UA, which can reduce heart microvascular injury by improving mitobiogenesis. ⁷ Besides, studies have shown that UA can activate mitophagy and thus play a neuroprotective role. ¹⁴ In our previous study, the potential of UA in analgesia was discovered, and it was confirmed that UA could relieve NP by inhibiting the activation of microglia and the release of inflammation factors. ¹⁵ Given that UA is an activator of mitophagy and its mechanism is involved in the PINK1/Parkin signaling pathway, we hypothesize that UA may protect neurons via PINK1/Parkin-mediated mitophagy activation when performing analgesic effect. In this study, we investigated whether mitophagy would be activated when UA alleviated NP in mice. We found that administration of UA activated the blocked autophagy flow and alleviated pain-related behaviors in the chronic constriction injury (CCI) mice. We further confirmed that UA activated PINK1/Parkin-mediated mitophagy and promoted mitobiogenesis in neurons and microglia in the spinal dorsal horn of the CCI mice. These findings deepen our understanding of the development of autophagy in NP and elucidate the changes of mitophagy when UA alleviates NP, and provide new ideas for clinical treatment of NP.

Materials and methods

Animals

Male C57BL/6J mice aged 8 weeks and weighting 30–40 g were obtained from the Experimental Animal Center of Xuzhou Medical University (ethics approval license No. 202208S114). All mice were kept in individual standard cages under a dark/light cycle of 12 h with controlled temperature and humidity and full access to food and water. All studies were approved by the Animal Care and Use Committee of Xuzhou Medical University and in accordance with the National Institutes of Health standards.

Administration of drugs

UA (Cat# GC15168, GlpBio) was dissolved in DMSO (Cat# VIC147, VICMED) to make a 40 mg/mL stock solution. Before being used for intraperitoneal injections, the stock solution was diluted to a prescribed concentration with 0.9% normal saline and 0.5% Tween-80. The first dose of UA (2.5 mg/kg) was injected immediately after CCI. Extra doses of UA were injected at the same concentration and volume every 24 h until sacrifice. The same volume of DMSO-saline mixed solvent was given to control group as vehicle.

CCI model preparation

Mice were anesthetized by 2% isoflurane. CCI model were prepared with reference to Bennett’s 16 method in which a posterolateral incision in the middle and back of the left thigh skin was made. We surrounded the left sciatic nerve with 4-0 sutures, made three light ligation loops with a spacing of 1 mm, and tied the knot with the proper strength, by which it would not affect the epineurium’s blood supply, and the thigh muscle twitches or kick reflex would be retained. In the Sham group, the sciatic nerve of mice was exposed but not ligated.

Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) testing

Pain-related behaviors were detected at the indicated time. MWT was tested using von Frey filament stimulation. Mice were placed in a plexiglass chamber on a wire net floor and allowed 10 to 15 min to habituate before experiment. A series of filaments 0.02, 0.07, 0.16, 0.4, 0.6, 1.0, 1.4, and 2.0 g were applied to the midplantar surface of the hind paw with sustaining pressure to observe the hind paw withdrawal response. TWL was measured to represent thermal hyperalgesia. Mice were placed in a plexiglass chamber on a glass plate and allowed 10 to 15 min to habituate before testing. A mobile radiant heat source located under the glass was focused onto the hind paw of each mouse. The duration from the onset of radiant heat stimulus to the withdrawal of the hind paw was defined as the latency (s), and a 20 s cut-off was set to avoid potential tissue damage.

Gait analysis

GA was measured to represent spontaneous pain. According to the literatures, we designed a simple apparatus composed of a runway (5 cm wide, 45 cm long, 7 cm height). Mice were habituated to the apparatus for 3 days before experiment. After being stained with the commercially available blue ink on the hind paws, the mice were forced to trot on a strip of paper (4.5 cm wide, 45 cm long) down the runway from one side to the other. Stride length (distance between left fore and hind paw) and print areas of the left hind paw were measured by Image J manually. The two successive longest stride length (stable velocity) were measured from each run. Pawprints made at the beginning (7 cm) and the end (7 cm) of the run were excluded because of velocity changes.

Western blot assay

The L4-5 spinal dorsal horn tissues were collected and homogenized in ice-cold radio immunoprecipitation assay lysis buffer (Cat# P0013B, Beyotime) containing 1% phenylmethylsulphonyl fluoride. After determination of the protein concentration, samples were separated by SDS-PAGE and subsequently transferred to PVDF membrane. Membranes were blocked by blocking buffer (Cat# P0252, Beyotime) for 30 min at room temperature, then incubated at 4°C overnight with the following primary antibodies: anti-LC3A/B (1: 1000, Cat# 12,741, CST), anti-p62 (1: 1000, Cat# ab109012, Abcam), anti-β-actin (1: 2000, Cat# 60,008-1, Proteintech), anti-Parkin (1: 1000, Cat# ab77924, Abcam), anti-PΙΝΚ1 (1: 1000, Cat# BC100-494, Novus Biologicals). After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1: 1000, Cat# A0216 and A0208, Beyotime) for 2 h at room temperature. Protein binds were visualized with a chemiluminescent imaging system and quantified densitometrically with ImageJ software.

Immunofluorescence and image analysis

Mice were anesthetized and perfused intracardially with saline and 4% paraformaldehyde in 0.1 M phosphate buffer. L4-5 spinal tissues were collected, fixed in 4% paraformaldehyde overnight at 4°C, then transferred to 30% sucrose in 0.1 M phosphate buffer at 4°C. After that, specimens were embedded by embedded medium (Cat# 4583, Sakura). Subsequently, 30-μm-thick serial sections were prepared using a freezing microtome and blocked with 1% bovine serum albumin (Cat# MB4219, Meilunbio) for 45 min. Spinal sections were then incubated overnight at 4°C with the following primary antibodies: antibody against translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20) (1: 250, Cat# ab186735, Abcam), antibody against neuronal nuclei (NeuN) (1: 500, Cat# ab104224, Abcam), anti-Iba-1 (1: 500, Cat# ab48004, Abcam). After washing, free-floating sections were incubated for 2 h at room temperature with the corresponding Alexa Fluor 488- or 594-conjugated secondary antibodies (1: 500, Cat# A-21,207 and A-21,202, Thermo Fisher Scientific). Fluorescence images were captured using a confocal scanning laser microscope (FV1000, Olympus), and images were shown as merged Z-stack projections consisting of approximately 10 optical slices (1 μm per slice). The number of Iba-1 positive cells and the count of TOMM20 positive neurons and microglia were measured using ImageJ software.

Quantification of mitochondrial DNA (mtDNA) copy number

DNA was isolated from the L4-L5 spinal segments using the Tissue Genomic DNA Extraction Kit (Cat# EP007, ELK Biotechnology) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qPCR) was performed with a kit for the mitochondrial NADH dehydrogenase subunit two gene (mt-ND2) (Cat# qMmuCEP0062243, Bio-Rad) and a kit for GAPDH gene (Cat# qqMmuCEP0039581, Bio-Rad). For estimation of relative mtDNA content, mt-ND2 was measured and normalized to the nuclear-encoded GAPDH gene.

Data analysis and statistics

GraphPad Prism 9.0 was used for data analysis and figure generation. Data are shown as the mean values ±SD. Results with homoscedastic datasets were compared by two-way ANOVA followed by Bonferroni’s multiple comparisons test and Tukey’s multiple comparisons test, and one-way ANOVA followed by Tukey’s multiple comparisons test. Sample sizes were indicated in the figure legends, and p < 0.05 was considered statistically significant.

Results

Altered expression of autophagy-related proteins in the spinal dorsal horn of CCI mice

It has been reported that the basal autophagy is impaired in NP. ¹ To confirm this, we used CCI mice as a model to detect the changes of autophagy-related proteins p62 and LC3 in the spinal dorsal horn during the occurrence and development of NP. Before CCI surgery, we performed MWT test to exclude the mice with significant differences in pain perception, and then performed sciatic nerve ligation in the left hind foot of the remaining mice. Next, we tested mice for pain-related behaviors, including MWT, TWL as well as the gait. The results showed that MWT and TWL of the CCI+Vehicle group were significantly lower than those of the Sham+Vehicle group on day 5, 7 and 14 after surgery (^*p < 0.05) (Figure 1(a) and (b)). Although TWL of the CCI+Vehicle group began to decrease significantly on the 3rd day after surgery, both the MWT and TWL of the CCI+Vehicle group showed the lowest values on the 7th day of all the test time points (^*p < 0.05). In addition, the results of GA suggested that the CCI+Vehicle group displayed abnormal gaits. Not only the stride length was significantly shortened on postoperative days 5 and 7 (Figure 1(c)) (^*p < 0.05), but also the footprint area was significantly reduced on postoperative day 7 (Figure 1(d)) (^*p < 0.05). These results indicated that the CCI model was successfully established. Subsequently, we collected the L4-L5 spinal cord tissues of mice on days 3, 5, 7, and 14 after surgery, and detected the expression levels of p62 and LC3 by western blot. p62 and LC3 are commonly used as the main markers of autophagy, among which the accumulation of p62 often indicates the inhibition of autophagy flow while the increased ratio of LC3-II/LC3-I may represent the increase of autophagosomes after the activation of autophagy in the early stage, or the accumulation of autophagosomes caused by the failure of autophagy lysosome clearance in the later stage. ¹ Our results showed that the expression of p62 increased gradually and reached the highest value on the 7th day after CCI and decreased on the 14th day (Figure 2(b) and (c)) (no statistical significance). On the contrary, the ratio of LC3-II/LC3-I gradually decreased and reached the lowest value on the 7th day, and then rose on the 14th day (Figure 2(b) and (d)) (no statistical significance). Compared with the Sham+Vehicle group, the expression level of p62 in the CCI+Vehicle group decreased on day 3 and increased on day 7 (Figure 2(c)) (^*p < 0.05), while the ratio of LC3-II/LC3-I increased on day 3 and 7 (Figure 2(d)) (^*p < 0.05). The data above indicated that autophagy flow was blocked in the spinal dorsal horn of the CCI mice when the most obvious pain-related behaviors occurred (day 7). Therefore, the spinal dorsal horn of the mice on the 7th day after CCI was taken for study during the subsequent experiments.

Figure 1.

Pain-related behavior tests of the mice after CCI surgery. MWT (a), TWL (b), stride length (c), and print area (d) of the left hind paw were collected prior to and at 3, 5, 7, and 14 days after CCI. The values are expressed as the means ± SD (n = 8). Statistical analysis was performed with two-way analysis of variance followed by Bonferroni's multiple comparisons test. ^*p < 0.05 compared with the Sham+Vehicle group.

Figure 2.

The expression of autophagy-related proteins p62 and LC3 in the spinal dorsal horn of the mice after CCI surgery. The L4-L5 spinal cord tissues were collected at 3, 5, 7, and 14 days after CCI and then analyzed by western blot. (a-b) Representative western blots of p62 and LC3. (c) Quantification of p62 expression (n = 4). (d) Quantification of the ration of LC3-Ⅱ/LC3-Ⅰ (n = 4). The values are expressed as the means ± SD. Statistical analysis was performed with two-way analysis of variance followed by Tukey's multiple comparisons test. ^*p < 0.05 compared with the Sham+Vehicle group.

UA activated PINK1/Parkin-mediated mitophagy when NP was alleviated

After finding out the relationship between autophagy flow and pain-related behaviors in the CCI mice, we speculated that administration of autophagy activator might increase autophagy in NP-alleviated mice. In our previous study, we have confirmed that UA could alleviate NP in mice by inhibiting the activation of microglia and its inflammatory response in the spinal dorsal horn. ¹⁵ Considering that UA is an autophagy activator, we deduced that autophagy activation might also be induced when NP was alleviated by UA. To verify this, we treated CCI mice with UA (2.5 mg/kg) through intraperitoneal injection to explore whether UA could alleviate NP caused by CCI surgery and activate autophagy. The administration of UA and the time points for behavioral tests are shown in Figure 3(a). Results of behavioral tests showed that compared with the CCI+Vehicle group, after UA administration, both MWT and TWL of the CCI+Vehicle group were significantly increased on day 7 (Figure 3(b) and (c)) (^#p < 0.05). In addition, we found that both stride length and footprint area of the mice in the CCI+UA group increased significantly on day 7 (Figure 3(d) and (e)) (^#p < 0.05), indicating that the limp gait was improved. These data suggested that UA could alleviate NP, which was consistent with our previous finding. ¹⁵ Next, we detected the expression levels of p62 and LC3 in spinal dorsal horn of the CCI mice treated with UA. Compared with the CCI+Vehicle group, the expression of p62 in the CCI+UA group decreased (Figure 4(a) and (b)) (^*p < 0.05), while the ratio of LC3-II/LC3-I increased (Figure 4(a) and (c)) (^*p < 0.05). These results suggested that the blockage of autophagy in the spinal dorsal horn caused by CCI was effectively activated by UA.

Figure 3.

UA alleviated the NP of CCI mice. (a) Chart of the time point for drug injection and behavioral test (BT). MWT (b), TWL (c), stride length (d), and print area (e) of the left hind paw were collected prior to and at 3, 5, and 7 days after CCI. The values are expressed as the means ± SD (n = 8). Statistical analysis was performed with two-way analysis of variance followed by Bonferroni's multiple comparisons test. ^*p < 0.05 Sham+Vehicle group versus CCI+Vehicle group. ^#p < 0.05 CCI+Vehicle group versus CCI+UA group.

Figure 4.

The expression of p62, LC3, PINK1, and Parkin in the spinal dorsal horn of the mice after CCI and drug injection. The L4-L5 spinal cord tissues were collected for the detection of p62, LC3, PINK1, and Parkin expression by western blot analysis at 7 days after CCI and drug injection. (a) Representative western blots of p62, LC3, PINK1, and Parkin. (b) Quantification of p62 expression (n = 4). (c) Quantification of the ration of LC3-Ⅱ/LC3-Ⅰ (n = 4). (d) Quantification of PINK1 expression (n = 4). (e) Quantification of Parkin expression (n = 4). The values are expressed as the means ± SD. Statistical analysis was performed with one-way analysis of variance followed by Tukey's multiple comparisons test. ^*p < 0.05, ^**p < 0.01.

Several lines of evidence suggest that UA enhances cellular health by increasing mitophagy ¹⁴ , ^16–19, and also show that UA could activated PINK1/Parkin-mediated mitophagy in hyperuricemia nephropathy model. ¹³ To make sure that UA ameliorated CCI-induced NP in mice and also induced PINK1/Parkin-mediated mitophagy activation, we examined the expression of PINK1 and Parkin by Western blot. As displayed in Figure 4, results showed that the expression of PINK1 (Figure 4(a) and (d)) (^*p < 0.05) and Parkin (Figure 4(a) and (e)) (^**p < 0.01) in the CCI+UA group increased after being treated with UA compared with the CCI+Vehicle group, indicating that UA activated mitophagy by regulating PINK1/Parkin signal pathway. In conclusion, based on the results above, we confirmed that UA could ameliorate NP and meanwhile activated PINK1/Parkin-mediated mitophagy.

UA promoted the mitobiogenesis in the neuron and microglia in the spinal dorsal horn of the CCI mice

Evidence has showed that NP could injury mitobiogenesis ²⁰ and many works have demonstrated that UA had ability to improve mitochondrial function by promote mitobiogenesis after mitophagy.^21,22 Therefore, we presumed that UA could increase mitobiogenesis in the CCI mice. To verify this, we used qPCR to measure the content of mtDNA in the spinal dorsal horn of CCI mice to evaluate the mitobiogenesis. As shown in Figure 5, compared with the Sham+Vehicle group, the content of mtDNA in the CCI+Vehicle group decreased significantly (^****p < 0.0001). After UA administration, the content of mtDNA in the CCI+UA group remarkably increased (^****p < 0.0001). These results indicated that UA could increase mitobiogenesis following the mitophagy wave.

Figure 5.

Relative mtDNA content in the spinal dorsal horn of the mice after CCI and drug injection. qPCR was used for analyzing the mtDNA copy number (n = 6 in each group). The values are expressed as the means ± SD. Statistical analysis was performed with one-way analysis of variance followed by Tukey’s multiple comparisons test. ^****p < 0.0001.

To find out the types of cell in which UA-promoted mitobiogenesis occurs, spinal dorsal horn sections were stained with antibodies specific to the neuron marker NeuN and the mitophagy marker TOMM20. As displayed in Figure 6, compared with the Sham+Vehicle group, the number of TOMM20 positive neurons in the CCI+Vehicle group was markedly increased on the 7th day after surgery (^***p < 0.001). After the injection of UA, the TOMM20 positive neurons increased even more (^*p < 0.05), indicating that neurons undergoing mitobiogenesis increased significantly. These results suggested that UA promoted neuronal mitobiogenesis in the spinal dorsal horn of CCI mice.

Figure 6.

UA promoted neuronal mitobiogenesis. (a) Double staining against NeuN (red, Alexa Fluor 594) and TOMM20 (green, Alexa Fluor 488) in dorsal horn of L4-L5 spinal cord showed that treatment with UA (2.5 mg/kg) on the CCI mice significantly increased the number of TOMM20 positive neurons. Representative spinal cord sections are shown. Scale bar = 250 µm. (b) Quantification of TOMM20 positive neurons for each group (n = 4). The values are expressed as the means ± SD. Statistical analysis was performed with one-way analysis of variance followed by Tukey’s multiple comparisons test. ^*p < 0.05, ^***p < 0.001.

As UA has been demonstrated to act on microglia in our previous study, ¹⁵ we speculated that UA might also promote the mitobiogenesis in microglia. To verify this, we detected the expression of Iba-1, a specific marker of microglia, and TOMM20 in the spinal cord horn of mice by immunofluorescence. As shown in Figure 7, compared with the Sham+Vehicle group, the expression of Iba-1 and the number of the cells co-labeled by Iba-1 and TOMM20 in the CCI+Vehicle group increased on day 7 after surgery (^****p < 0.0001), indicating that the number of microglia and the number of microglia undergoing mitobiogenesis increased significantly after CCI. After intraperitoneal injection of UA, the expression of Iba-1 in the CCI+UA group decreased (^****p < 0.0001), while the number of the cells co-labeled by Iba-1 and TOMM20 increased even more (^*p < 0.05), suggesting that UA inhibited the activation of microglia and promoted the mitobiogenesis in microglia. These results suggested that UA not only inhibited the activation of microglia, but also promoted the mitobiogenesis in microglia.

Figure 7.

UA promoted mitobiogenesis in microglia. (a) Double staining against Iba-1 (red, Alexa Fluor 594) and TOMM20 (green, Alexa Fluor 488) in dorsal horn of L4-L5 spinal cord showed that treatment with UA (2.5 mg/kg) on the CCI mice significantly decreased the numbers of Iba-1 positive cells and increase the TOMM20 positive microglia. Representative spinal cord sections are shown. Scale bar = 200 µm. (b) Quantification of Iba-1 positive cells for each group (n = 4). (c) Quantification of TOMM20 positive microglia for each group (n = 4). The values are expressed as the means ± SD. Statistical analysis was performed with one-way analysis of variance followed by Tukey’s multiple comparisons test. ^*p < 0.05, ^****p < 0.0001.

Discussion

Being a secondary metabolite of the natural polyphenol compound ellagtannin, UA has various biological activities such as anti-oxidation, anti-inflammation, anti-aging and induction of mitophagy. ¹² In our previous study, we found the analgesic effect of UA for the first time, and suggested that its possible mechanism was to inhibit the activation of microglia and the release of inflammatory factors in the spinal dorsal horn of the mice after CCI. ¹⁵ In this study, we found for the first time that mitophagy was activated when UA ameliorated NP in CCI mice at the spinal cord level. In short, after intraperitoneal injection of UA in CCI mice, NP was relieved and PINK1/Parkin-mediated mitophagy was activated in the spinal dorsal horn to promote mitobiogenesis in neurons and microglia (Figure 8). However, we can not deduce that activating mitophagy is one of the mechanisms for UA to alleviate NP since we did not study the analgesic effect of UA using specific inhibitors of mitophagy. Besides, we did not study the analgesic mechanism of UA in PINK1 knockout mice and therefore could not determine whether UA specifically activates PINK1/Parkin-mediated mitophagy to play the analgesic role.

Figure 8.

Schematic illustration of the UA’s effect on CCI mice. ①UA alleviates NP. ②UA activates PINK1/Parkin-mediated mitophagy in the spinal dorsal horn of the CCI mice, thus further facilitating the mitobiogenesis in neurons and microglia.

As innate immune cells in the central nervous system, microglia play an important role in the occurrence and development of NP.^23,24 When central or peripheral nerves are injured, microglia are activated and release a large number of proinflammatory factors and cytotoxic substances, resulting in neuronal disability and cell death, and exacerbating the development of NP. Our previous findings showed that UA could inhibit the activation of microglia and down-regulate the expression of IL-1β, IL-6, and TNF-α, thereby exerting remarkable anti-inflammation effects and alleviating NP in the CCI mice.^23,24 Interestingly, in this study, mitophagy was found to be activated in the spinal dorsal horn of CCI mice after the administration of UA, promoting microglia mitobiogenesis, suggesting that the anti-inflammation effect of UA may be related to the mitophagy and microglial mitobiogenesis. Microglia are highly dynamic cells that depend on mitochondria for their energy supply. When NP occurs, mitophagy of microglia are blocked, leading to the failure of damaged mitochondria to be cleared in time and the generation of excessive ROS, which further activates microglia and aggravates neuroinflammation.^25,26 On the contrary, the activation of mitophagy in microglia can inhibit the activation of microglia, reduce the release of inflammation factors, improve the microenvironment of neurons, and thus play a neuroprotective role. ²⁷ Similar to our results, some studies have shown that autophagy is activated in SNI rats after resveratrol treatment, which alleviated the activation of microglia and the release of downstream inflammation factors. ²⁸ Therefore, we speculate that UA may inhibit microglia-mediated neuroinflammation by activating mitophagy in order to promote mitobiogenesis in microglia, but the underlying mechanism needs to be further studied. Together, targeting mitophagy activation in microglia is a potential therapeutic strategy for NP.

UA is a drug with great potential for clinical use. ²⁹ The clinical safety of oral UA has been proven. ²⁹ In addition, the technology for industrial-scale synthesis of UA has become matured. Our data confirmed that UA could notably alleviate NP in CCI mice, implying that UA may become a potential novel drug for the treatment of NP. In spite of this, there are some limitations in our study. First, we selected male mice for research in order to avoid the influence of estrogen levels on the experiments. It has been shown that there are sex differences in pain, analgesia, and autophagy.^30–32 Whether UA plays the same analgesic effect in female mice remains unclear, which needs to be verified by experiments. Secondly, extensive data have shown that autophagy plays an important role in neurons, glial cells, vascular cells and extracellular matrix in the central nervous system.^33–35 Our study focused on mitobiogenesis that was promoted by mitophagy in neurons and microglia. However, whether mitophagy also occurs in other cells such as astrocytes and oligodendrocytes and whether they are involved in the regulation of pain needs further study.

Appendix.

Abbreviations

NP

Neuropathic pain

PINK1

PTEN-induced kinase 1

UA

Urolithin A

CCI

Chronic constriction injury

MWT

Mechanical withdrawal threshold

TWL

Thermal withdrawal latency

GA

Gait analysis

mtDNA

Mitochondrial DNA

mt-ND2

The mitochondrial NADH dehydrogenase subunit 2 gene

qPCR

Quantitative real-time polymerase chain reaction

NeuN

Neuronal nuclei

TOMM20

Translocase of the outer mitochondrial membrane complex subunit 20

OMM

Outer mitochondrial membrane

ORCID iD

Chenyi Wang https://orcid.org/0000-0002-6333-6439