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. 2024 May 21;20:17448069241252654. doi: 10.1177/17448069241252654

Mitochondria-derived peptide is an effective target for treating streptozotocin induced painful diabetic neuropathy through induction of activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator 1alpha -mediated mitochondrial biogenesis

Lingfei Xu 1,*, Xihui Tang 2,*, Long Yang 1, Min Chang 3, Yuqing Xu 2, Qingsong Chen 2, Chen Lu 1, Su Liu 1,2,✉, Jinhong Jiang 1,✉
PMCID: PMC11113074  PMID: 38658141

Abstract

Painful Diabetic Neuropathy (PDN) is a common diabetes complication that frequently causes severe hyperalgesia and allodynia and presents treatment challenges. Mitochondrial-derived peptide (MOTS-c), a novel mitochondrial-derived peptide, has been shown to regulate glucose metabolism, insulin sensitivity, and inflammatory responses. This study aimed to evaluate the effects of MOTS-c in streptozocin (STZ)-induced PDN model and investigate the putative underlying mechanisms. We found that endogenous MOTS-c levels in plasma and spinal dorsal horn were significantly lower in STZ-treated mice than in control animals. Accordingly, MOTS-c treatment significantly improves STZ-induced weight loss, elevation of blood glucose, mechanical allodynia, and thermal hyperalgesia; however, these effects were blocked by dorsomorphin, an adenosine monophosphate-activated protein kinase (AMPK) inhibitor. In addition, MOTS-c treatment significantly enhanced AMPKα1/2 phosphorylation and PGC-1α expression in the lumbar spinal cord of PDN mice. Mechanistic studies indicated that MOTS-c significantly restored mitochondrial biogenesis, inhibited microglia activation, and decreased the production of pro-inflammatory factors, which contributed to the alleviation of pain. Moreover, MOTS-c decreased STZ-induced pain hypersensitivity in PDN mice by activating AMPK/PGC-1α signaling pathway. This provides the pharmacological and biological evidence for developing mitochondrial peptide-based therapeutic agents for PDN.

Keywords: Adenosine monophosphate-activated protein kinase/PGC-1α, mitochondrial-derived peptide, mitochondrial function, painful diabetic neuropathy

Introduction

Painful Diabetic Neuropathy (PDN) is the common complication of type-I or type-II diabetes, characterized by severe hyperalgesia and allodynia. Approximately half of all diabetic patients develop neuropathy, however, 30%–40% of cases accompanied by PDN.1,2 Painful Diabetic Neuropathy is difficult to treat because the underlying mechanisms remain unclear. Increasing evidences suggest that increased glucose levels lead to glucose metabolism, resulting in increased oxidative free radicals (ROS) and inflammatory responses, leads to mitochondrial dysfunction.3,4 In chronic pain, oxidative stress and mitochondrial dysfunction have received widespread interest.5,6 Especially in PDN, mitochondria may mediate, drive, or even initiate pathologic molecular cascades, representing it a promising therapeutic target.7,8

Recently, Lee et al. reported a novel mitochondrial-derived peptide (MOTS-c), encoded by the open reading frame (sORF) in mitochondrial 12S rRNA. 9 Mitochondrial-derived peptide is a 16-amino-acid peptide, of which is extremely conserved among different species. 9 Recent studies have shown that MOTS-c regulates obesity, insulin sensitivity, metabolic homeostasis and inflammatory disease via targeting adenosine monophosphate-activated protein kinase (AMPK).10–13 Several studies confirmed the significance of AMPK, an energetic stress-sensing protein with neuroprotective properties, as a therapeutic target for the treatment of chronic pain.14–16 Activation of AMPK reduces the excitability of dorsal root ganglion neurons and reverses neuropathic allodynia in chronic neuropathic pain. 15 Notably, metformin, a well-known drug for type II diabetes, is also an indirect AMPK activator reported to attenuate hyperalgesia and allodynia in rats with PDN. 17 However, MOTS-c was reported as an indirect AMPK activator involved in regulating insulin resistance and obesity, osteoporosis and other diseases.9,11,18 Additionally, MOTS-c has gained increasing interest as a promising therapeutic or prevention strategy for obesity and diabetes mellitus.9,11 Moreover, the association of both MOTS-c and type 2 diabetic patients was evaluated in the clinical trial (NCT04027712). However, However, the interaction of MOTS-c with PDN remains unclear.

Several studies demonstrated that the pathological activation of microglia, intensive microgliosis, and peripheral and central sensitization are key contributors to chronic pain.19–22 Notably, Lee et al. found that MOTS-c inhibited cytokine expression, and chemokine activity, the innate immune response, interferon type1 biosynthesis, and inflammatory responses. 9 Our previous report showed that MOTS-c single treatment inhibited microglia activation in the acute spared nerve injury-induced neuropathic pain. 23 PDN is characterized as chronic peripheral neuralgia that presents therapeutic challenges and required long-term treatment, however, the analgesic effect of MOTS-c in PDN remains unknown. In this study, we evaluated the potential effects of MOTS-c in chronic PDN and attempted to identify the underlying regulatory mechanisms. We found that mice with streptozocin (STZ)-induced PDN had significantly lower serum MOTS-c levels than control animals. In addition, MOTS-c treatment showed strong and dose-dependent antinociceptive effects in PDN mice. These findings suggested that MOTS-c regulates PDN, thus understanding the underlying regulatory mechanism may provide novel and effective therapeutic targets for PDN treatment.

Materials and methods

Experimental mice

Male C57BL/6 mice (weighing 20–22 g, 8–10 weeks-old) were purchased from the Experimental Animal Center of Xuzhou Medical University (Xuzhou, China). The mice were housed in cages (5 animals/cage) with a standardized 12-h light (8:00 a.m.)/dark (8:00 p.m.) cycle. All mice were maintained in specific pathogen-free conditions (22 ± 2°C) with free access to tap water and food. All animal protocols in this study were approved by the Office of Laboratory Animal Research and the Institute of Animal Care and Use Committee of Xuzhou Medical University (approval number, 202207S104).

Drugs

Mitochondrial-derived peptide was synthesized by a standard Fmoc-based solid phase synthesis methodology as our previous report. 23 All amino acids and coupling reagents were purchased from GL Biochem Ltd (Shanghai, China). Dorsomorphin was purchased from MedChemExpress (Shanghai, China) and dissolved in 10% DMSO, kept at −20°C, and diluted in corn oil immediately before i.p. injection (10 mg/kg). STZ was purchased from Sigma Aldrich (St Louis, MO, USA) and dissolved in sterile cold citrate buffer pH 4.5. Lipopolysaccharide (LPS) was purchased from Sigma and dissolved in saline (1 μg/μL). Glucose was purchased from Sigma.

Animal model of painful diabetic neuropathy

To develop a PDN mice model, we followed the previously reported method. 24 Briefly, diabetes was induced in C57BL/6 mice aged 8–10 weeks. Streptozotocin (STZ, 60 mg/kg, Sigma-Aldrich) in sterile cold citrate buffer pH 4.5 was administered i.p. over 3 days after fasting overnight. The PDN model was considered successful as the mice’s blood glucose levels exceeded 200 mg/dL by developing hyperalgesia and allodynia. Experiments were performed 2–7 weeks post-onset of hyperglycemia in male mice.

Treatment

To investigate the effects of MOTS-c on STZ-induced diabetic neuropathic pain, mice were given intrathecal (i.t.), intraperitoneal (i.p.), or intraplantar (i.pl.) injection of MOTS-c or saline 2 weeks after STZ treatment. For the i.t. injection, mice were anesthetized with isoflurane, and i.t. administration of MOTS-c was carried out by using a 32-gauge needle. This needle was connected via a PE tube to a 25 μL Hamilton syringe and directly inserted between the L5 and L6 segments in mice. The total volume of intrathecal injection was 5 μL. When the reflex tail flicks sideways or the tail forms an “S” shape, the drug injection is considered successful. Dorsomorphin (10 mg/kg, 150 μL) was i.p. injected 30 min before the injection of MOTS-c. The behavior experiments were carried out immediately after the MOTS-c application. To minimize non-specific stress responses during injecting the drug, the mice were handled daily for 2–3 min before the start of the experiment.

Behavioral test

Von Frey filament test

The mechanical pain threshold of mice was measured by von Frey monofilaments (Bio-VF-M, Bioseb, USA) with the up-down method. 25 Firstly, the mice were placed on a metal mesh floor and covered by a transparent plastic box. After at least 30 min adjustment, mice were given MOTS-c, and the paw withdrawal threshold was recorded by grade-strength von Frey monofilaments (2.36, 2.44, 2.83, 3.22, 3.61, 3.84, 4.08, 4.17 and 4.31) at 30, 60, 90, and 120 min. In detail, the first von Frey monofilament (2.44) was applied to the plantar surface of the hindpaw. If a withdrawal response was observed within 2 s, the filament (2.36) was used. Conversely, if the monofilament failed to evoke a withdrawal response, the next filament (2.83) was applied. A total of six responses were recorded, and the 50% paw withdrawal threshold (PWT) was statistically analyzed based on the up-down method.26,27

Thermal pain test

Similar to the von Frey monofilament test, mice were placed on the glass surface and covered by a transparent plastic box. After at least 30–60 min adjustment, mice were given MOTS-c, and the paw withdrawal threshold was recorded by a plantar anesthesia tester (Boerni, Tianjin, China). A radiant heat source was focused through the glass under the plantar side of the hindpaw until the withdrawal response, paw withdrawal latency (PWL) was then recorded by an automatic timer. The cutoff time of heating was set to 20 s, the heat stimulation would be stopped to prevent thermal injury.

Immunofluorescence

Mice were sacrificed in a CO2 chamber, and transcardially perfused with 20 mL PBS followed by 40 mL 4% paraformaldehyde (PFA). The lumbar spinal cord was removed, postfixed in 4% paraformaldehyde overnight, and cut into consecutive frozen sections (20 μm). The sections were blocked in 5% donkey serum for incubated with primary antibodies overnight at 4°C. Subsequently, the secondary antibody was applied to the sections for 2 h at room temperature. The antibodies information is presented in Table S1. The sections were imaged with a confocal microscope (FluoView1000; Olympus, Japan). The images were processed and analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA). For quantification of the number of immunopositive cells, we randomly selected four sections from each mouse. Cell counts were averaged to reflect the number of positive cells in the entire spinal dorsal horn. Fiji Image J software was used to provide a quantitative measurement of the co-localization of 8-OHdG with NeuN, GFAP, or iba1 by Pearson’s correlation coefficient, which estimated the degree of overlap between fluorescence signals obtained in two channels. The degree of co-localization from the Pearson’s coefficient values was categorized as very strong (0.85 to 1.0), strong (0.49 to 0.84), moderate (0.1 to 0.48), weak (−0.26 to 0.09) based on previously reports.28,29

Immunohistochemistry

Similar to the immunofluorescence experiment, mice were sacrificed in a CO2 chamber, and transcardially perfused with 20 mL PBS followed by 40 mL 4% paraformaldehyde (PFA). The lumbar spinal cord was removed and cut into coronal paraffin sections (6 μm). Then sections were incubated with the primary antibodies at 4°C overnight. Subsequently, the sections were incubated with a biotinylated goat anti-rabbit secondary antibody (1:3000, Servicebio) for 2 h at room temperature. Then, sections were incubated for 40 min in vidin-biotin-horseradish peroxidase complex (1:200; Vector Laboratories, Burlingame, California) diluted with 0.1 M TBS at 37°C. The sections were treated with 0.05% 3, 3-diaminobenzidine in 0.1 M TBS containing 0.004% H2O2 for a chromogen reaction. Finally, Sections were washed with water, mounted, dehydrated, cleared in xylene, coverslipped, and observed under the microscope. The images were processed and analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA).

Quantitative polymerase chain reaction

Briefly, total RNA was extracted using RNA extraction reagent (Vazyme, R401-01), and 1 µg of RNA in each sample was reversely transcribed into a single-stranded complementary DNA with the 5X PrimeScript RT Master Mix (TaKaRa). qPCR reactions (TaKaRa) were carried out in a 25 µL reaction mixture consisting of 12.5 µL of 2X SYBR Premis Ex TaqTM II, 2 µL of cDNA, 1 µL of forward primer, 1 µL of reverse primer, and 8.5 µL of ddH2O; and was run under the following conditions: 95°C for 5 min, 40 cycles of 95°C for 5 s, 58°C for 30 s, and 72°C for 30 s. The primer pair P1/P2 was presented in Table S2. The GAPDH was used as an endogenous control in each sample. Relative levels were quantified with the 2-ΔΔCT method that was normalized to the GAPDH. 30

Western blotting

WB was carried out as described previously. 23 Briefly, the protein was extracted with RIPA buffer containing protease inhibitor (Servicebio, Wuhan). The protein concentration was determined using a BCA protein assay kit (Thermo Scientific, 23,225). The protein samples (40 μg per lane) isolated from the lumbar spinal cord were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, IPV H00010). PVDF membranes were blocked with 5% fat-free milk for 2 h and incubated with specific primary antibodies at 4°C overnight, followed by incubation with peroxidase-conjugated secondary antibodies (Servicebio, Wuhan). GAPDH, β-actin, and tubulin were used as loading control. The membrane was detected with ECL substrate (Thermo Scientific, 32,106), and exposed using a ChemiDoc XRS imaging system (Bio-Rad, USA). The intensity of blots was quantified by the Image J software (National Institutes of Health, Bethesda, MD, USA).

Enzyme-linked immunoassay

Total proteins in plasma were obtained from experimental mice and MOTS-c in plasma was quantified using commercially available ELISA kits (Cloud-Clone Crop, CEX132Mu), according to the manufacturer’s instructions.

In vitro experiments

SH-SY5Y cells were cultured in Dulbecco modified eagle medium (DMEM) (Gibco, 2193059) supplemental with 15% fetal bovine serum (FBS) (Gibco, A3160902) and 1% penicillin (Invitrogen, 10378016s) at 37°C in a humidified atmosphere with 5% CO2. Similar to previous studies, 3 45 mmol/L high glucose evokes the elevation of intracellular ROS. Therefore, we established high glucose group (HG, with a glucose concentration of 45 mmol/L), vehicle group (with a glucose concentration of 25 mmol/L), and HG + MOTS-c group treated with MOTS-c (10−5, 10−6, 10−7, 10−8, and 10−9 mol/L) at different concentrations. After 24 h of HG treatment, an equal volume of MOTS-c and PBS was added dropwise to HG-treated cells, and vehicle respectively, following 6 h of incubation, all cells were harvested. All cell lysates were prepared for evaluation of mitochondrial function. According to the manufacturer’s instruction, the intracellular ROS level was measured using the dye DCFH-DA (Sigma-Aldrich, USA), and mitochondrial membrane potential was investigated by JC-1 kit (Beyotime, C2003S).

BV2 microglial cells were cultured in DMEM (HyClone, SH30022.01) medium, which contained 10% fetal bovine serum (FBS) (Gibco, A3160902) and 1% penicillin and streptomycin (Beyotime, C0222), in a bacterial incubator at 37°C with 5% CO2. First, BV2 was treated with lipopolysaccharide (LPS, Sigma-Aldrich, L2880) at concentrations of 100 ng/mL. Simultaneously, different concentrations of MOTS-c (10−5, 10−6, 10−7, 10−8, and 10−9 mol/L) were added to the experimental group. An equal volume of PBS was added to the control group. Cells were harvested after 6 h of incubation. Following RNA extraction, the expression levels of related inflammatory factors were analyzed by qPCR.

Statistical analysis

All data are presented as the Mean ± SEM. Statistical analysis was evaluated using the SPSS 19.0 (IBM Corp., Armonk, NY, USA). The data of the two groups were statistically analyzed using the student’s t-tests. The data between groups was evaluated by one-way ANOVA, followed by bonferroni’s post-hoc test. Comparisons of time-series data were analyzed with repeated measurement two-way ANOVA. The p values < .05 was considered statistically significant.

Results

MOTS-c levels were decreased in plasma and spinal dorsal horn of STZ-induced diabetic painful mice

To understand the potential role of MOTS-c in STZ-induced diabetic painful mice, we examined whether MOTS-c is expressed in the spinal cord of STZ-induced diabetic mice and age-matched sham mice (STZ-untreated). As shown in Figure 1(b)–(e), 1 week following STZ treatment, mice’s blood glucose level increased dramatically, compared to the sham group (p < .0001, Figure 1(b)). Two weeks following STZ treatment, STZ-treated mice had considerably lower body weight than the sham group (p < .01, Figure 1(e)). Two weeks following STZ injection, the mechanical and thermal paw withdrawal thresholds were significantly lower than in the sham group (p < .05 for Figure 1(c) and (d)). Consistent with previous report, 31 the number of c-Fos positive neurons was significantly up-regulated in the spinal cord dorsal horn of STZ-treated mice compared to sham group (p < .01 for 14 days, p < .001 for 35 days, Figure 1(f) and (g)), suggesting the establishment of PDN in mice.

Figure 1.

Figure 1.

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MOTS-c is decreased in plasma and spinal dorsal horn after PDN. (a) Experimental design. (b)–(e) Body weight, mechanical allodynia, thermal hyperalgesia, and blood glucose were investigated before surgery and on days 7, 14, 21, 28, and 35 after STZ treatment (n = 8). (f), (g) Representative images of c-fos immunofluorescence (green) on the dorsal horn (scale bar 100 μm, n = 5). (h) Immunohistochemistry shows the levels of MOTS-c in the lumbar spinal dorsal horn (n = 5). (i) The levels of MOTS-c in plasma are examined using ELISA (n = 8). Data are expressed as the Mean ± SEM. *p < .05, **p < .01 and ***p < .001 compared with sham group.

Thirty-five days following the STZ injection, we measured MOTS-c expression in plasma and the lumbar spinal cord using ELISA and IHC (Figure 1(h) and (i)). The results showed that the level of endogenous MOTS-c in the plasma of STZ-treated mice was significantly lower than that of the sham group, especially in 35 days (Figure 1(i), p < .05 for 14 days, p < .01 for 35 days). IHC experiments revealed that the expression level of MOTS distributed in the spinal dorsal horn was significantly down-regulated in STZ-treated mice, compared with that in mice of the sham group (Figure 1(h)).

MOTS-c treatment produced an antinociceptive effect in PDN mice

Exogenous MOTS-c injections were used to determine their influence on mechanical pain thresholds in PDN mice using Von Frey monofilaments. Behavioral tests showed that STZ treatment evoked significant mechanical hyperalgesia, 2 weeks after STZ injection (Figure 1(c) and (d)). As shown in Figure 2(b), (c) and (i) t. injection of MOTS-c (0.5 and 1 μg) significantly reverse mechanical allodynia (p < .01 for 0.5 μg; p < .001 for 1 μg) (Figure 2(b) and (c)). Additionally, the AUCs were calculated from the MOTS-c dose-response curve using trapezoidal rules between 0 and 120 min and analyzed using one-way ANOVA with bonferroni’s post-hoc test. The extent and duration of analgesia were estimated according to the AUC values (0–120 min) presented in Figure 2(c) (p < .05 for 0.5 μg, p < .001 for 1 μg). Figure 2(d) showed that MOTS-c exhibited a notable dose-response relationship at 30, 60, and 90 min after injection.

Figure 2.

Figure 2.

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MOTS-c treatment attenuates STZ-induced mechanical pain hypersensitivity. (a) Experimental design. (b)–(d) Antinociceptive dose- and time-response curve for i.t. administration of MOTS-c (0.1, 0.5, and 1 μg) in STZ induced PDN mice. (e)–(g) Antinociceptive dose- and time-response curve for i.pl. administration of MOTS-c (1, 5, and 10 μg) in STZ-induced PDN mice. (h)–(j) Time- and dose-response curves of the antiallodynic effects of i.p. injection of MOTS-c (0.1, 0.5, 1, and 5 mg/kg) in STZ-induced PDN mice. Data are expressed as the Mean ± SEM, n = 8 for each group. The extent and duration of analgesia are estimated by the area under the curve (AUC (g ⋅ min)) of PWT versus time (0–120 min). *p < .05, **p < .01 and ***p < .001 compared with STZ group.

Meanwhile, we investigated the analgesic effect of MOTS-c administered peripherally to STZ-treatment mice. As shown in Figure 2(e)–(g), i.pl. administration of MOTS-c increased the withdrawal threshold (p < .05 for 5 μg, p < .001 for 10 μg), whereas MOTS-c (1 μg, i.pl.) did not affect on mechanical sensation. Similarly, as shown in Figure 2(h)–(j), i.p. administration of MOTS-c (0.5, 1, and 5 mg/kg) also showed analgesic effects in the mechanical hyperalgesia in PDN mice (Figure 2(h)–(j)). Taken together, these results indicate that MOTS-c treatment exhibited dose-dependent antinociceptive effects in PDN mice.

Chronic administration of MOTS-c produced an antinociceptive effect in PDN mice

Two weeks following the STZ injection, PDN mice were treated with a single dosage of MOTS-c (1 mg/kg, i.p.) for 21 consecutive days as indicated in Figure 3(a). The results indicated that compared to the STZ-treated group, i.p. injections of MOTS-c (1 mg/kg, daily) significantly improve STZ-induced weight loss (p < .01 for STZ group vs STZ + MOTS-c group, Figure 3(c)), elevation of blood glucose (p < .01 for STZ group vs STZ + MOTS-c group, Figure 3(b)), mechanical allodynia (p < .01 for STZ group vs STZ + MOTS-c group, Figure 3(d)) and thermal hyperalgesia (p < .01 for STZ group vs STZ + MOTS-c group, Figure 3(e)). We found that MOTS-c (i.p., 1 mg/kg, daily) significantly down-regulated the expression of c-Fos positive neurons induced by STZ (p < .01 for STZ group vs STZ + MOTS-c group) (Figure 3(f) and (g)). Taken together, these findings show that prolonged MOTS-c treatment improves STZ-induced weight loss, elevated blood glucose, mechanical allodynia and thermal hyperalgesia in PDN mice.

Figure 3.

Figure 3.

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Chronic administration of MOTS-c produces an antinociceptive effect in STZ-treated mice. (a) Experimental design. (b)–(e) Two weeks after STZ injection, STZ-treated mice were daily treated with MOTS-c (1 mg/kg, i.p.) for 21 consecutive days, and body weight, mechanical allodynia, thermal hyperalgesia, and blood glucose were investigated on days 7, 14, 21, 28 and 35 (n = 8). (f) Representative images of c-fos immunofluorescence (green) on the dorsal horn (scale bar 100 μm, n = 5). (g) Qualitative data show the number of c-fos positive neurons in the dorsal horn. **p < .01 and ***p < .001 compared with sham group. #p < .05, ##p < .01 and ###p < .001 compared with STZ group.

Effect of MOTS-c on mitochondrial dysfunction

Given that MOTS-c is a recently discovered mitochondrial derivative peptide, 9 we investigated whether it could regulate mitochondrial function following PDN. We found that MOTS-c treatment significantly reduced the up-regulation of mitochondrial dysfunction genes (CYP2E1 and Bax) in PDN mice (p < .01 for CYP2E1, p < .05 for Bax, Figure 4(a) and (b)). The expression of superoxide dismutase (SOD1) was considerably lowered in STZ-induced PDN mice, while MOTS-c-treated PDN mice showed elevated SOD1 gene expression (p < .05, Figure 4(d)).

Figure 4.

Figure 4.

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MOTS-c rescues STZ-induced mitochondrial dysfunction and DNA oxidative damage in spinal dorsal horn. (a)–(f) mRNA transcription of the genes related to mitochondrial function in the spinal dorsal horn samples of mice treated with sham, STZ, or STZ plus MOTS-c therapy. (g)–(h) Immunofluorescent images and quantification of 8-OHdG-immunolabelled activated cells in the spinal dorsal horn (n = 5).

Additionally, Nrros was reported to negatively regulate reactive oxygen species (ROS).32,33 The expression of Nrros has significantly decreased in STZ-induced PDN mice (p < .01, Figure 4(c)). With exposure to MOTS-c, the expression of Nrros was remarkably elevated by about 20% (p < .05 for STZ group vs STZ + MOTS-c group, Figure 4(c)). It’s been extensively shown that 8-hydroxydeoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, is the most frequently reflected ROS level studied in nuclear DNA and mitochondrial DNA damage.34,35 As shown in Figure 4(g) and (h), compared with sham group, STZ treatment caused a significant increase in 8-OHdG of the spinal dorsal horn (p < .01 for sham group vs STZ group). i.p. injection of MOTS-c (1 mg/kg, daily) significantly down-regulated the number of spinal 8-OHdG (p < .05 for STZ group vs STZ + MOTS-c group), indicating that MOTS-c treatment could down-regulate ROS level in spinal cord in PDN mice.

As previously stated, MOTS-c could inhibit the expression of c-Fos while functioned as a signal of neuronal activity. 36 Subsequently, SH-SY5Y cells were selected as in vitro models to evaluate the effect of MOTS-c on mitochondrial function (ROS, JC-1, and mitochondrial biogenesis-related genes). As shown in Figure 5(a)–(f), MOTS-c treatment (10−5 mol/L) significantly improved HG-induced expression of mitochondrial function-related genes such as CYP2E1, Bax, NQO1, SOD1, PGC-1α, and Nrros (p < .05 for CYP2E1, Bax, NQO1, and Nrros, p < .01 for SOD1 and PGC-1α). Next, we examined the effect of MOTS-c on the production of ROS in SH-SY5Y cells using the molecular probe DCFH-DA. The results indicated that MOTS-c treatment (10−5 mol/L) significantly inhibited HG-induced ROS production in SH-SY5Y cells (p < .01 for HG group vs HG + MOTS-c group, Figure 5(g) and (h)). Besides, JC-1 assay kit was used to evaluate the changes in mitochondrial membrane potential in SH-SY5Y cells. As shown in Figure 5(i)–(j), we found that HG treatment had a significant enhancement effect on JC-1 in SH-SY5Y cells (p < .001 for vehicle group vs HG group), whereas MOTS-c treatment (10−5 mol/L) markedly decreased the level of JC-1 (p < .05 for HG group vs HG + MOTS-c group). Taking together, these results indicated that MOTS-c treatment attenuated mitochondrial dysfunction in STZ-induced PDN mice.

Figure 5.

Figure 5.

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MOTS-c inhibited HG-induced mitochondrial dysfunction in SH-SY5Y cells. (a)–(f) MOTS-c treatment (10−5 mol/L) significantly reduced the expression of mitochondrial function-related genes in HG-treat SH-SY5Y cells (n = 5). (g), (h) The level of intracellular ROS in SH-SY5Y cells was determined using the fluorescent dye DCFH-DA and quantified by flow cytometry (n = 5). (i), (j) MOTS-c treatment (10−5 mol/L) markedly decreased the level of JC-1 (n = 5). *p < .05 and **p < .01 and ***p < .001 compared with vehicle group. #p < .05 and ##p < .01 compared with HG group.

MOTS-c inhibited oxidative damage of neuron, microglia activation and pro-inflammatory cytokines production in the spinal cord of PDN mice

Previous experiments showed that MOTS-c treatment down-regulated the expression of spinal 8-OHdG, widely used as a biomarker of endogenous oxidative DNA damage. To explore whether MOTS-c inhibited oxidative damage in microglia, astrocytes or neurons, we performed double immunofluorescence staining for 8-OHdG with NeuN (neuron), iba1 (microglia), or GFAP (astrocyte) in the spinal dorsal horn. Fiji image J software was used for the quantification of co-localization according to pearson’s correlation coefficient. As shown in Figure 6(a)–(d), 8-OHdG co-localized primarily with the neuronal marker NeuN, although some overlap with the microglial marker iba1 was also detected, to a lesser extent, with GFAP (Figure 6(a)–(d)). According to these findings, MOTS-c primarily protects spinal dorsal horn neurons from oxidative damage.

Figure 6.

Figure 6.

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MOTS-c inhibited STZ-induced oxidative damage of neuron and microglia activation. (a)–(d) Double immunofluorescence and quantification showed subcellular colocalization of 8-OHdG in the spinal dorsal horn (scale bar 100 μm, n = 5). 8-OHdG (red) is primarily co-staining with neurons (NeuN, green), rarely with microglial cells (iba1, green) and astrocytes (GFAP, green). (e), (f) Representative images showed the effects of MOTS-c on the activation of microglia (iba1, green) in the spinal dorsal horn (scale bar 100 μm). (g), (h) Representative images of GFAP immunofluorescence (green) on the spinal dorsal horn (scale bar 100 μm). n = 5. **p < .01 and ***p < .001 compared with sham group. ##p < .01 compared with the STZ group.

It is well documented that activated microglia secrete a variety of proinflammatory factors that activate neighboring neurons.19,37 Given the crucial roles of glia activation in PDN,19–22 we further investigated whether microglia and astrocyte activation was necessary for the antiallodynic effect of MOTS-c in PDN mice. As shown in Figure 6(e)–(h), immunofluorescent staining showed that STZ treatment caused a significant increase in iba1-positive microglia (p < .001 for sham group vs STZ group) and GFAP-positive astrocyte (p < .001 for sham group vs STZ group), compared with sham control. Then, i.p. injection of MOTS-c (1 mg/kg, daily) significantly reduced the number of iba1-positive microglia (p < .01 for STZ group vs STZ + MOTS-c group), whereas had no effects on GFAP-positive astrocyte. These result suggests that MOTS-c inhibits microglia activation in the spinal dorsal horn of PDN mice.

Numerous studies have shown that activated microglia in the spinal cord are considered to be the main source of pro-inflammatory cytokines.38–40 To examine the expression of pro-inflammatory cytokines in spinal cord, we investigated the effect of MOTS-c on the pro-inflammatory factors by qPCR and WB. As shown in Figure 7(a)–(h), qPCR analysis demonstrated that the pro-inflammatory genes (TNF-α, IL-1β, IL-6, and iNOS) were remarkably elevated in spinal cord of STZ-treat mice (p < .05 for TNF-α, IL-1β, p < .01 for IL-6 and iNOS, p < .001 for iba1). The anti-inflammatory genes (Arg-1, CD206, and CD68) were significantly decreased in spinal cord of STZ-treat mice (p < .05 for CD206 and CD68, p < .001 for CD206). However, these changes were significantly improved after MOTS-c application (p < .05 for TNF-α, IL-6, IL-1β, and Arg-1, p < .01 for iNOS and iba1, Figure 7(a)–(h)). Meanwhile, western blotting showed a significant reduction in the pro-inflammatory marker (iNOS) and elevation in the anti-inflammatory marker (Arg-1) after MOTS-c treatment (Figure 7(i)–(k)). In addition, we used lipopolysaccharide (LPS) treatment to quantify the expression of pro-inflammatory factors in BV2 microglial cells in vitro. We observed that following LPS treatment, there was a significant up-regulation of TNF-α, IL-1β, and IL-6 expression, which persisted for at least 24 h (data not shown). In contrast to LPS-treated BV2 cells, the expression of these factors was significantly down-regulated by MOTS-c (10−5 mol/L) at 6 h of LPS stimulation (p < .05 for TNF-α, IL-1β and IL-6, Figure 7(l)–(n). These results suggested that MOTS-c inhibits pro-inflammatory cytokines production in the spinal dorsal horn of PDN mice.

Figure 7.

Figure 7.

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MOTS-c reverses STZ-induced production of pro-inflammatory cytokines in the lumbar spinal dorsal horn. (a)–(h) The expression of TNF-α, IL-1β, IL-6, iNOS, iba1, CD68, CD206, and Arg-1 were measured in the spinal dorsal horn samples of mice treated with sham, STZ or STZ plus MOTS-c therapy by qPCR. (i)–(k) The expression of iNOS and Arg-1 was measured in the spinal dorsal horn by WB (n = 5). (l)–(n) MOTS-c (10−5 mol/L) significantly down-regulated LPS induced the expression of TNF-α, IL-1β and IL-6 in BV-2 cells. Data are expressed as the Mean ± SEM, n = 6 for each group. *p < .05, **p < .01 and ***p < .001 compared with sham group. #p < .05 and ##p < .01 compared with STZ group.

AMPK/PGC-1α signaling pathway could be involved in antiallodynic effects of MOTS-c in PDN mice

We investigated the activation of the AMPK/PGC-1α signaling pathway, which is crucial for mitochondrial biogenesis, to evaluate the molecular mechanism of MOTS-c on the regulation of diabetic painful neuropathy. As shown in Figure 8(a)–(d), western blotting analysis demonstrated that the phosphorylation of AMPK was remarkably reduced in spinal cord of STZ-treat mice (p < .05 for sham group vs STZ group), whereas MOTS-c treatment significantly elevated the expression of p-AMPK (p < .05 for STZ group vs STZ + MOTS-c, Figure 8(a)–(d)). Furthermore, immunofluorescent staining showed that STZ caused a significant decrease in the expression of p-AMPK of spinal cord, compared with sham group (p < .01 for sham group vs STZ group, Figure 8(e)). However, MOTS-c treatment exhibited more increase in the phosphorylation of AMPK, as compared to STZ group (p < .05 for STZ group vs STZ + MOTS-c, Figure 8(e)).

Figure 8.

Figure 8.

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MOTS-c attenuates STZ-induced pain hypersensitivity in PDN mice through activation of AMPK/PGC-1α signaling pathway. (a)–(d) Quantitative western blot analysis showing activating effects of MOTS-c on STZ-induced decreased phosphorylation of AMPK (n = 5). (e), (f) Immunofluorescent images and quantification of p-AMPK-immunolabelled activated cells in the spinal dorsal horn (n = 5). (g)–(j) Effect of pretreatment with dorsomorphin (10 mg/kg, i.p.) on the spinal antinociception of MOTS-c. n = 8. AUC was calculated during 0–120 min. (k) The expression of PGC-1α was measured in the spinal dorsal horn samples of mice treated with sham, STZ, or STZ plus MOTS-c therapy by qPCR. (l)–(m) Quantitative western blot analysis showed the expression of PGC-1α and β-actin in each group of spinal cord. Data are expressed as the Mean ± SEM, *p < .05, **p < .01 and ***p < .001 compared with sham group. #p < .05, ##p < .01 and ###p < .001 compared with STZ group.

In addition, dorsomorphin, an AMPK pathway inhibitor, 41 was used to evaluate the effect of MOTS-c. As shown in Figure 8(f)–(i), MOTS-c treatment dramatically alleviated thermal hyperalgesia and mechanical allodynia, as evidenced by the restored PWL and PWT values (p < .0001 for STZ + saline group vs STZ + MOTS-c group, Figure 8(f)–(i)). Then, pretreatment with dorsomorphin (10 mg/kg, i.p.), 30 min before i.p. injection of MOTS-c (1 mg/kg), significantly blocked the antinociceptive effects of MOTS-c in PWT and PWL (p < .001 for STZ + MOTS-c group vs STZ + Dorsomorphin + MOTS-c group, Figure 8(f)–(i)). These results suggested that AMPK signaling pathway was at least partially involved in antiallodynic effects of MOTS-c in PDN mice.

Previous studies reported that PGC-1α is a downstream molecule of AMPK pathway involved in regulating mitochondrial gene biosynthesis.42,43 As shown in Figure 8(k)–(m), qPCR and western blotting analysis demonstrated that the expression of PGC-1α was remarkably reduced in spinal cord of STZ-treat mice, whereas MOTS-c treatment significantly elevated the expression of PGC-1α. Taken together, these results indicate that MOTS-c attenuates STZ-induced pain hypersensitivity in PDN mice at least partially through activation of AMPK/PGC-1α signaling pathway.

Discussion

Painful diabetic neuropathy, a typical complication of diabetes that frequently cause severe hyperalgesia and allodynia, is difficult to manage because the underlying mechanism is unclear.44–46 Numerous studies demonstrated that mitochondrial dysfunction causes an increase level of ROS and inflammation, which may contribute to the development of PDN.6,47 Recently, a novel bioactive peptide (MOTS-c) was discovered from the open reading frame (sORF) in mitochondrial 12S rRNA. 9 It has been reported to regulate insulin sensitivity, metabolic homeostasis and inflammatory response via AMPK.9,13,48 Therefore, this study aimed to evaluate the effects of MOTS-c on PDN and investigate the putative underlying mechanisms.

Numerous studies have addressed the critical functions of MOTS-c in regulating metabolic homeostasis and insulin resistance.9,12 Here, we observed that the level of endogenous MOTS-c in the plasma and spinal dorsal horn of STZ-treated mice was significantly lower than that of sham group by ELISA and IHC. According to previous reports, lower circulating endogenous MOTS-c levels in human subjects are associated with impaired coronary endothelial function. 49 Obese male children and adolescents showed decreased levels of circulating MOTS-c, a biomarker associated with both insulin resistance and obesity. 50 Thus, this phenomenon implied that MOTS-c may be involved in the regulation of PND. Subsequently, we found that STZ treatment evoked significant mechanical allodynia and thermal hyperalgesia, 2 weeks after STZ injection, whereas exogenous injection of MOTS-c was able to significantly improve STZ-induced weight loss, elevation of blood glucose, mechanical allodynia and thermal hyperalgesia. Further studies showed that STZ treatment evoked a significant increase in c-fos expression, whereas after being activated by MOTS-c, c-fos in the spinal dorsal horn was decreased. Collectively, these data indicate that MOTS-c might play an important role in PDN mice.

Meanwhile, we observed that STZ stimulation increased the expression of spinal 8-OHdG, whereas MOTS-c application had a negative effect on 8-OHdG expression. 8-OHdG is an oxidized nucleoside of DNA which is most commonly studied as reflected ROS level in nuclear DNA and mitochondrial DNA damage.34,35 Moreover, with exposure to MOTS-c, the expression of Nrros, a widely reported negative regulator of ROS, 33 was remarkably elevated by about 20%. Recent evidence has highlighted a causative link between mitochondrial dysfunction and painful diabetic neuropathy pathway. 6 Chronic or intermittent hyperglycemia triggered the production of ROS, oxidative stress, and the secretion of pro-inflammatory cytokines, which were reported to have a pivotal role in PDN . 3 In vitro experiments found that MOTS-c treatment significantly reduced hyperglycemia-induced ROS production in SH-SY5Y cells.

Subsequently, we further evaluated the effects of MOTS-c on mitochondrial function (mitochondrial membrane potential [JC-1] and mitochondrial biogenesis-related genes). The JC-1 assay results showed that hyperglycemia treatment had a significant enhancement effect on JC-1, whereas MOTS-c treatment markedly decreased the level of JC-1. Meanwhile, the expression of mitochondrial function-related markers (CYP2E1, Bax, SOD1, NQO1, and ACC1) was improved with MOTS-c treatment both in vivo and in vitro. Taking together, these results indicate that MOTS-c treatment attenuates mitochondrial dysfunction in STZ-induced PDN mice. To explore mitochondrial damage associated the cell type in the spinal dorsal horn, we investigated co-staining of 8-OHdG with neuronal (NeuN) or glial markers (iba1 and GFAP). Double immunofluorescent staining additionally revealed that 8-OHdG was predominately co-localized on spinal neurons rather than microglia and astrocyte. These results suggested that the effect of MOTS-c in spinal neurons may be more important for PDN.

In the last two decades, the contribution of spinal cord microglia to the development of neuronal hypersensitivity in PDN has been widely discussed.19,37 Thus, we determined whether glia was involved in the roles of MOTS-c. The results showed that STZ treatment caused significant activation of microglia and astrocyte, whereas treatment with MOTS-c inhibited the remarkable activation of microglia. No differences were observed in the levels of astrocytes after MOTS-c application. There is evidence that activation of spinal microglia has been observed in STZ-treated animals, which is the most commonly used model of PDN. 51 Previous studies reported that hyperglycemia evokes the production of ROS and the change in local microenvironment in the spinal cord, triggering the activation of microglia.3,52 It is well documented that activated microglia synthesize and release a variety of pro-inflammatory factors that activate neighboring neurons.19,37 Therewith, our results showed that the pro-inflammation factors expression in spinal cord was also reduced after MOTS-c treatment both in vivo and in vitro. These data supported that microglia are also important for inhibition of PDN progress in MOTS-c treated mice.

To explore the molecular mechanism of MOTS-c on the regulation of diabetic painful neuropathy, we examined the activation of AMPK/PGC-1α signaling pathway, which is essential for mitochondrial biogenesis. Previous studies also showed that the activation of AMPK mitigated chronic pain in a broad variety of preclinical pain models, and AMPK is emerging as a new target for novel intervention and modulation for chronic pain.9,48,53 In this study, immunofluorescent staining and western blotting analysis demonstrated that STZ caused a significant decrease in the phosphorylation of AMPK1/2 in spinal cord of STZ-treat mice, while MOTS-c treatment significantly elevated the expression of p-AMPK1/2. Meanwhile, dorsomorphin, an AMPK pathway inhibitor, was able to block the antinociceptive effects of MOTS-c in PWT and PWL. These data point toward that AMPK signaling pathways may be involved in the antinociceptive effects of MOTS-c in PDN mice.

PGC-1α is a downstream molecule of AMPK pathway involved in regulating mitochondrial gene biosynthesis and plays an important role in regulating glucose homeostasis.42,43 Our observation demonstrated that the expression of PGC-1α was remarkably reduced in spinal cord of STZ-treat mice, whereas MOTS-c treatment significantly elevated the expression of PGC-1α. Taken together, these results indicate that MOTS-c attenuates STZ-induced pain hypersensitivity in PDN mice at least partially through activation of AMPK/PGC-1α signaling pathway.

Conclusion

In summary, the present study reveals that endogenous MOTS-c levels were significantly lower in STZ-treated mice than in control animals. MOTS-c attenuates diabetes-induced mechanical allodynia and thermal hyperalgesia at least partially through activation of AMPK/PGC-1α signaling pathway. Mechanistic studies indicated that MOTS-c significantly restored mitochondrial biogenesis, inhibited microglia activation, and decreased the production of pro-inflammatory factors, contributing to pain alleviation. These findings suggested that the MOTS-c could be a promising candidate for developing effective therapeutics for treating painful diabetic neuropathy.

Supplemental Material

Supplemental Material - Mitochondria-derived peptide is an effective target for treating streptozotocin induced painful diabetic neuropathy through induction of activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator 1alpha -mediated mitochondrial biogenesis
sj-pdf-1-mpx-10.1177_17448069241252654.pdf (177.9KB, pdf)

Supplemental Material for Mitochondria-derived peptide is an effective target for treating streptozotocin induced painful diabetic neuropathy through induction of activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator 1alpha -mediated mitochondrial biogenesis by Lingfei Xu, Xihui Tang, Long Yang, Min Chang, Yuqing Xu, Qingsong Chen, Chen Lu, Su Liu and Jinhong Jiang in Molecular Pain

Acknowledgements

We would like to thank all participants and our university. We are grateful to Naeem Ullah for revising the manuscript and correcting typos and grammatical errors.

Author contributions: Jinhong Jiang and Su Liu as the primary and corresponding authors designed the research, supervised all experiments, wrote the first draft and funded the work. Lingfei Xu and Xihui Tang performed the experiments and analyzed data. Long Yang, Yuqing Xu, and Qingsong Chen facilitated the equipment and software to perform the behavior experiments. Min Chang synthesized peptide drugs. Chen Lu revised the manuscript and funded the work.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The study was supported by grants from the National Natural Science Foundation of China (82201385 and 82071225), the grants from Natural Science Research Fund of Higher Education Institutions in Jiangsu Province (22KJA320007 and 21KJA320002).

Supplemental Material: Supplemental material for this article is available online. In the supporting information, Table S1 shows information relating to the antibody used for IF and WB. Table S2 shows information relating to the primers used for qPCR.

Ethical statement

Ethics approval and consent to participate

All of the experiments were approved by the Animal Ethics Committees of Xuzhou Medical University (Xuzhou, China) following the “Institutional Guidelines and Animal Ordinance” (Ethics No. 202207S104).

ORCID iDs

Su Liu https://orcid.org/0000-0001-5036-2104

Jinhong Jiang https://orcid.org/0000-0001-7953-7270

Data availability statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.*

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Supplementary Materials

Supplemental Material - Mitochondria-derived peptide is an effective target for treating streptozotocin induced painful diabetic neuropathy through induction of activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator 1alpha -mediated mitochondrial biogenesis
sj-pdf-1-mpx-10.1177_17448069241252654.pdf (177.9KB, pdf)

Supplemental Material for Mitochondria-derived peptide is an effective target for treating streptozotocin induced painful diabetic neuropathy through induction of activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator 1alpha -mediated mitochondrial biogenesis by Lingfei Xu, Xihui Tang, Long Yang, Min Chang, Yuqing Xu, Qingsong Chen, Chen Lu, Su Liu and Jinhong Jiang in Molecular Pain

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.*

Articles from Molecular Pain are provided here courtesy of SAGE Publications

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