Abstract Depression is a common comorbidity of chronic pain. The comorbidity of pain and depression causes longer symptoms and poorer patient prognosis. Periaqueductal gray (PAG) is the key region for the regulation of pain and depression. Puerarin (Pue) is a natural isoflavone compound that has a neuroprotective effect, but the mechanisms on the comorbidity of chronic pain and depression remain unclear. In this study, the spared nerve injury (SNI) produced mechanical allodynia and depressive-like behaviors and elevated the neurological damage in ventrolateral (vl) PAG. Meanwhile, at the 8 weeks following injury, mitochondrial dysfunctions including the dysregulated protein levels, the decreased Mn-SOD activity and the reduced ATP contents were observed in vlPAG of SNI model mice. Pue administration improved mechanical pain, motor coordination, and depression-like behaviors, decreased the neuronal activity and neuroinflammation, and elevated the mitochondrial function in vlPAG. Database analysis and experimental assay showed that Pue bound with Bax at the affinity of 2.4 ± 0.1 μM via D102 residue, and decreased Bax level in vlPAG of mice and in primary astrocytic cells. In addition, Pue also recovered levels of mitochondrial membrane potential and reactive oxygen species, and decreased inflammation in primary astrocytic cells. These results suggest that Pue improves the comorbidity of chronic pain and depression by targeting Bax and reducing mitochondrial dysfunction in vlPAG. This study may provide a theoretical basis for Pue application in improving the comorbidity of chronic pain and depression. Keywords: Comorbidity of chronic pain and depression, vlPAG, mitochondria, puerarin, Bax Introduction Chronic pain is pain that is ongoing and persists for over 3 months. It affects approximately one-third of people globally and is considered to be a major public problem.^ [39]1 Depression is a common comorbidity of chronic pain and up to 60% of chronic pain patients display depression symptoms.^ [40]2 Patients who are suffering from pain and depression have a reduction in physical, mental, and social functioning, and exhibit longer physical and psychological symptoms and poorer prognosis than those who are suffering from either condition alone.^ [41]3 Understanding the mechanisms underlying the comorbidity of chronic pain and depression is helpful for the development of a effective treatment for pain and depression management. Periaqueductal gray (PAG) is an integration center for neuronal signals that is located at the interface between the forebrain and brainstem, which works as the key region for descending pain modulation and the center for controlling depression-like behavior.^ [42]4 Deep brain stimulation of PAG is used for the treatment of severe, refractory neuropathic pain.^ [43]5 Neuroplasticity and neuroinflammation have been proposed as common mechanisms for the comorbidity of chronic pain and depression.^ [44]6 Inhibition of dopaminergic or glutamatergic neuronal activity in ventrolateral (vl) PAG serves to exacerbate pain hypersensitivity and depression-like behaviors.^[45]7,[46]8 Modulation of the activity of vlPAG astrocytes influences the behaviors of anxiety and neuropathic pain.^ [47]9 Reducing the neuroinflammation in vlPAG improves chronic pain, including inflammatory pain.^ [48]10 and opioid analgesics.^ [49]11 Mitochondria are essential for the structure and function of neurons and glial cells via adenosine 5′-triphosphate (ATP) production, Ca^2+ regulation and reactive oxygen species (ROS) signaling.^ [50]12 Mitochondrial dysfunction is associated with chronic pain and depression and mitochondrial disease is often accompanied by chronic pain^ [51]13 and depression.^ [52]14 At the same time, mitochondrial morphology and functional damage have been observed in chronic pain and depression animal models.^ [53]15 Therefore, targeting the improvement on mitochondrial function in PAG could effectively alleviate the comorbidity of chronic pain and depression. Puerarin (Pue) is a natural isoflavone compound that is isolated from the root of Pueraria lobata and works as a potential natural neuroprotective agent in various neurological disorders.^ [54]16 Pue has been reported to protect dopaminergic neurons in Parkinson’s disease and hippocampus neurons in Alzheimer’s disease.^ [55]17 It has also been reported to inhibit neuroinflammation^ [56]18 and further relieve neuropathic pain^ [57]19 and depression.^ [58]20 Pue also improves the function and performance of mitochondria.^ [59]21 However, the mechanisms of Pue on the comorbidity of chronic pain and depression remain unclear. Therefore, a spared nerve injury (SNI) mouse model was constructed in this study and Pue was administered intraperitoneally for 7 days. Pain and depressive related behavior changes were both tested and neuronal and neuroinflammatory changes in the PAG region were detected. The aim of this study is to examine the effect Pue has on the comorbidity of chronic pain and depression and to clarify the related molecular mechanism. Materials and methods Antibodies and reagents Anti-β-actin (AF7018), anti-GFAP (BF0345), anti-IL-1β (AF5103), anti-Bax (AF0120), anti-c-Fos (AF5354) were purchased from Affinity Biosciences (Jiangsu, China). Anti-NeuN (66836-1-Ig) was purchased from Proteintech (Wuhan, China). Anti-GFAP (A19058), Anti-Iba1 (A19776), HRP goat anti-rabbit IgG (H+L) (AS014) were purchased from ABclonal (Wuhan, China). Goat anti-mouse IgG H&L (FITC) (ab6785) and goat anti-rabbit IgG H&L (FITC) (ab6717) were purchased from Abcam (Cambridge, UK). MitoSOX Red (S0061S), Mito-Tracker Red CMXRos (C1049B), antigen retrieval solution (P0083), blocking solution (P0102), RIPA lysis buffer (P0013B), DAB Horseradish Peroxidase Color Development Kit (P0202), H&E staining solution (C0105M), Nissl Staining kit (C0117), ATP Assay Kit (S0026), Mn-SOD Assay Kit (S0103) were obtained from Beyotime (Shanghai, China). Pue (A10081) was purchased from Shanghai yuanye (Shanghai, China). Animals Male C57BL/6J mice (6–8 weeks old, 18–20 g, n = 40) were performed from Hubei Province Experimental Animal Centre (Wuhan, China), kept under 12 h light/dark environment and unlimited access to food and water. The experiment was approved by the Laboratory Animal Ethics Committee of Hubei University of Science and Technology (2023-03-104). Model construction and drug administration Mice were divided into four groups at random (n = 10/each): Sham, Sham + Pue, SNI, and SNI + Pue groups. To construct the mouse model of sciatic nerve injury (SNI), mice in the SNI group were euthanized with 50 mg/kg pentobarbital sodium (i.e.). the skin of the left thigh was cut, the muscles were separated, the sciatic nerve trunk was exposed, the common peroneal and tibial were cut, and then the muscles and skin were fixed. Mice in the sham group were similarly operated without injuring the sciatic nerve. Following 7 weeks of surgery, mice in the Sham + Pue and SNI + Pue groups were intraperitoneally injected with Pue (10 mg/kg) for seven consecutive days. Pue was dissolved in DMSO and diluted with 0.9% NaCl (v/v = 1:1) before use. While, mice in the Sham and SNI groups were injected with the same volume (0.2 ml) of the vehicle (DMSO and 0.9% NaCl). Behaviors tests Mechanical threshold test Paw withdrawal threshold (PWT) values were tested to assay the mechanical pain of mice using the von Frey filaments (Stoelting, Wood Dale, USA) to stimulate the left plantar. Mice were placed in a 30 × 30 × 30 cm plexiglass chamber for 30 min to acclimatize before the behavioral tests. The filaments were vertically pressed onto the plantar surfaces of the paw until it bends and maintains for 3–5 s. Recorded a positive response when the paw is lifted or withdrawn, while no response was considered to be negative. Once a positive response is observed, a neighboring filament with lower force was applied. Recorded six times. The pattern of positive and negative responses was then converted to a PWT value (g). Motor coordination test The latency to fall on a rotating rod was tested to evaluate the motor coordination. The mice were trained for 3 days at a fixed pace for 10 min prior to the tests. In the experiments, the test was initially set at a constant speed of 10 revolutions per minute (r/min) for 10 sec, before being increased to 20 r/min for 30 sec. The latency (s) was then recorded. Sucrose preference test Sucrose preference test was performed to evaluate the depressive like behavior. The mice were housed for 2 days in a single cage for adaptive training. On day 1, two bottles of 1% sucrose solution were placed in the cage. On day 2, one bottle was changed to drinking water and the bottle positions were switched after 12 h. On day 3, the mice were deprived for 12 h and given the 1% sucrose solution and water. The consumption of water and 1% sucrose solution was recorded and a sucrose preference percentage (%) was then calculated. Tail suspension test The tail suspension test (TST) was performed to evaluate the depression-like behavior. The test apparatus consisted of white acrylic walls (20 × 40 × 60 cm). Mice were suspended by their tails and the head was 15 cm away from the bottom. The behaviors were recorded by a video camera for 6 min. The immobility time of mice in the last 4 min (3–6 min) was counted. Open field test An open field test was performed to evaluate the ability of locomotion by using apparatus XR-XZ301 (Shanghai Xinruan Co., Shanghai, China). The mice were initially placed in the central zone and their movements were recorded for 5 min. The total distances (cm) and center distances (cm) were calculated. Data collections The database [60]GSE91396 for the comorbidity of chronic pain and depression were downloaded from the website ([61]https://www.ncbi.nlm.nih.gov/gds/?“term=GSE91396). The differentially expressed genes (DEGs) in PAG between SNI and sham groups (n = 6) was analyzed. Mitochondrial gene database MitoCarta 3.0 was downloaded from the website ([62]http://www.broadinstitute.org/mitocarta). The structure information about Pue was downloaded from the Pubchem (CID 5281807, [63]https://pubchem.ncbi.nlm.nih.gov/compound/5281807). Pue related targets were predicted from the website of the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), Swiss Target Prediction and TargetNet. Data information was performed using venn diagram, GO, and KEGG pathway enrichment analysis on Metascape platform. The structure of Bax was downloaded from PDB database (ID 5W62, [64]https://www.rcsb.org/structure/5W62). The binding between Bax and Pue were docked using Autodock, and visualized using Pymol. Histomorphological analysis Following euthanization with 100 mg/kg pentobarbital sodium (i.p.) and transcardially perfusion using 4% PFA, the mice brain tissues were collected, dehydrated, embedded, and cut into 4 μm sections. For H&E staining, the sections were dyed with hematoxylin solution for 3 min, washed for 10 s, stained with eosin for 3 min, washed for 10 s. The dehydration and transparent procedure were conducted as follows: 80% ethanol (5 min), 90% ethanol (5 min), 95% ethanol (5 min), 100% ethanol (5 min, two times), xylene (5 min, times), and finally sealed with neutral gum. The degrees of inflammatory infiltrate were categorized as 0 (normal); 1 (meningeal and perivascular lymphocytic infiltration); 2 (1–10 lymphocytes present); 3 (11–100 lymphocytes); and 4 (>100 lymphocytes). For Nissl staining, the sections were dyed with toluidine blue stain for 30 min and washed for 10 s. Gradient dehydration is performed after differentiation and finally sealed with neutral gum. For immunohistochemical (IHC) staining, after deparaffinization, the sections were immersed in an antigen retrieval solution for antigen retrieval, heated at 100°C for 15 min, incubated with 3% hydrogen peroxide for 10 min, blocked at 25°C for 1 h, incubated with the primary antibodies overnight and secondary antibodies for 1 h, stained with diaminobenzidine (DAB), counterstained with hematoxylin, sealed with neutral gum, and finally observation under microscope (Olympus IX73, Olympus, Japan). Protein purification Plasmids of Bax and Bax-D102A in pET.3C vector (Beijing Tsingke Biotech Co., Ltd. Beijing, China) were then transfected into E. coli. Cells were harvested, lysed, and centrifuged. The supernatants were collected and purified by Ni^2+-sepharose affinity chromatography following by size-exclusion chromatography (Hiload 26/60 superdex 200, GE Healthcare, USA). The purified proteins were analyzed by 15% SDS-PAGE, and the concentrations were detected using the Lambert-Beer law. Isothermal titration calorimetry (ITC) assay The binding affinity for proteins and Pue was detected using iTC200 titration calorimetry (MicroCal, Northampton, MA). Bax or Bax-D102A in Tris-HCl buffer (pH 7.4) were loaded into the sample cell and Pue was loaded in the injection syringe. The data was analyzed using the MicroCal ORIGIN software. Primary astrocytic cells culture One day old mice were anesthetized, the brains were collected, the cerebral cortex tissues were isolated, cut into 1 mm^3 pieces digested with DNase I and trypsin at 37°C for 20 min, and centrifuged at 150 g for 6 min. The pellet was resuspended, filtered, seeded in T25 bottles, and cultured in DMEM/F-12 medium (10% FBS, 50 U/ml penicillin and 50 µg/ml streptomycin) at 37°C with 5% CO[2]. Transient transfection and Pue treatment Bax siRNA (sc-29213) and control siRNA (sc-44230) were purchased from Santa Cruz Biotechnology (CA, USA) and transfected with primary astrocyte cells in Control siRNA and Bax siRNA groups according to the siRNA Transfection Protocol. Cells were treated with 0 and 1 μM Pue for 24 h in DMSO and Pue groups. Cells were inoculated in a 24-well plate with poly-L-lysine-coated coverslips, stimulated with IL-1β (5 ng/ml) for 1 h and incubated with 0 and 1 μM Pue for 24 h in the IL-1β and IL-1β + Pue groups. Cells in the control group were untreated. Organotypic PAG slice cultures Organotypic PAG slices were prepared according to the method of Xie et al.^ [65]22 with some modifications. Briefly, mice in SNI model were decapitated, brains were rapidly dissected and placed in a Petri dish in ice-cold serum-free DMEM medium. The PAG were isolated and sectioned into 400-μm-thick transverse slices with McIlwain tissue chopper (Mickle Laboratory Engineering Co. Ltd., Goose Green, UK). The slices were then carefully separated and transferred on to Millicell cell culture inserts (Millipore, Billerica, MA, USA) and the inserts were transferred to a 6-well culture plate. Each well contained 1.5 ml of DMEM/F-12 medium (10% heat inactivated horse serum, 100 U/ml penicillin and 100 µg/ml streptomycin) at 37°C with 5% CO[2]. The day after preparation, the culture medium was replaced with fresh medium, and the siRNA and Bax siRNA were administered into the medium. After 24 h treatment, the slices were harvested for Western blot. Western blotting assay The cells or tissues were collected, homogenized in RIPA lysis buffer containing 1% protease inhibitor on ice for 15 min and centrifugated for 20 min at 12,000 g and 4°C. The supernatants were collected and quantified for protein concentration. After separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with blocking buffer for Western Blot for 90 min and then incubated with primary antibodies overnight at 4°C and secondary antibodies at 25°C for 1 h, visualized using ECL solution, and observed by iBright 1500 (Invitrogen, Carlsbad, USA). Immunofluorescence (IF) assay For tissues, the slices were dewaxed with xylene, conducted to antigen repair, incubated with 3% H[2]O[2] for 10 min, blocked with blocking solution for 1 h, subsequently incubated with primary antibodies for 24 h at 4°C, incubated with the fluorescent secondary antibody for 1 h at 25°C, mounted with antifade mounting medium. For cell experiments, the cells were rinsed with phosphate buffer solution, fixed with 4% PFA, treated with 0.5% tritonX-100, blocked with immunofluorescence blocking solution for 1 h, incubated with primary antibodies at 4°C for 24 h, fluorescent secondary antibodies at 25°C for 60 min and Hoechst 33342 (1 µg/ml) for 15 min at 25°C, mounted with antifade mounting medium. Th images were acquired by fluorescence microscope (Olympus IX73; Olympus Corporation) for cell research.^ [66]23 The fluorescence intensity was analyzed using ImageJ v1.48 software (National Institutes of Health). Mitochondrial membrane potential (MMP) and ROS assays MMP is essential for ATP generation and widely used for assessing mitochondrial function. In this study, it was evaluated using the fluorescent probe Mito-Tracker Red CMXRos. While, as a mitochondrially targeted fluorescent dye, MitoSOX can be oxidized by superoxide and produce the red fluorescence, and it was used for detecting the mitochondrial (mito) ROS. Briefly, cells were inoculated on a 24-cell plate, induced with IL-1β for 1 h, treated with Pue for 24 h, cultured with Mito-Tracker Red CMXRos or MitoSOX in the dark for 20 min and observed by fluorescence microscope. Statistical analysis All statistical analyses were performed by the SPSS 27 software. The differences of PWT values between groups with time course were analyzed using two-way ANOVA (group vs time) followed by Tukey’s test. The differences of data for other behavioral tests, H&E staining, IHC staining, IF staining, and western blotting were analyzed using one-way ANOVA followed by Tukey’s post-hoc test. Analytical correlations were analyzed using Spearman’s analysis. p < 0.05 was set to present statistical significance. Results Puerarin improves behaviors of pain and depression in the SNI model mice The experimental processes were shown in [67]Figure 1(a). A SNI mouse model was constructed by lesioning the common peroneal and tibial nerve branches. Behaviors of pain, depression, and locomotion were detected and data were shown in [68]Figure 1(b) to ([69]n). In comparison to mice in the sham groups, the SNI model mice showed the decreased PWT values on 2, 5, 7, and 8 weeks following surgery (p < 0.05, [70]Figure 1(b)–([71]d)). While, on the 8 weeks after surgery, the SNI model mice showed the declined latency to fall (p < 0.05, [72]Figure 1(e) and ([73]f)), the reduced sucrose preference percentage (p < 0.05; [74]Figure 1(g) and ([75]h)), the increased immobility time (p < 0.05; [76]Figure 1(i) and ([77]j)), and the shortened total or center distance (p < 0.05; [78]Figure 1(k)–([79]n)). However, the mice in the sham + Pue group were no statistical changes. Following 1 week of Pue treatment, the PWT value of the SNI + Pue group mice was increased (p < 0.05 vs SNI group), the latency to fall was increased (p < 0.05 vs SNI group), the sucrose preference percentage was raised (p < 0.05 vs SNI group), the immobility time was decreased (p < 0.05 vs SNI group), and the total or center distance both were elongated (p < 0.05 vs SNI group). In addition, correlation analysis showed the PWT values were correlated with latency to fall ([80]Figure 1(o)), sucrose preference percentage ([81]Figure 1(p)), immobility time ([82]Figure 1(q)), and the total or center distance ([83]Figure 1(r) and ([84]s)). These results suggested that Pue improved mechanical pain, motor coordination, and depression-like behaviors in the SNI model mice. Figure 1. [85]Figure 1. [86]Open in a new tab Effect of Pue on behaviors of pain and depression in mice. (a) Schematic diagram of the experimental procedures. (b–j) Schematic diagram and changes of PWT values, latency, sucrose preference, and immobility time of mice. (k and l) Schematic diagram and representative motion trajectories in the OFT. (m and n) Changes in total or center distance in mice. (o–s) Correlations of PWT values with latency, sucrose preference, immobility time, and total or center distance. Data were expressed as the mean ± SEM. *p < 0.05 versus sham group. #p < 0.05 versus SNI group. Puerarin decreases the neuronal activity and neuroinflammation in vlPAG of SNI model mice We analyzed the database [87]GSE61396, and found that 324 DEGs in the PAG for the comorbidity of chronic pain and depression were collected ([88]Figure 4(a)). GO enrichment analysis showed that the biological processes (BP) relating with synapse signaling such as the modulation of chemical synaptic transmission (GO:0050804) regulation of trans-synaptic signaling (GO:0099177), vesicle-mediated transport in synapse (GO:0099003), synapse organization (GO:0050808) were enriched. The cellular components (CC) relating with synapse signaling such as post synapse (GO:0098794), glutamatergic synapse (GO:0098978), presynapse (GO:0098793) were enriched. The molecular functions (MF) relating with synapse signaling such as active monoatomic ion transmembrane transporter activity (GO:0022853) and proton transmembrane transporter activity (GO:0015078) are enriched ([89]Table 1). Here, we found that in comparison to the mice in sham group, the numbers of Nissl bodies and the positive cells of neuronal activity marker c-Fos in the ventrolateral (vl) subregion of PAG in the SNI model mice were both increased (p < 0.05, [90]Figure 2(a) and ([91]b)). Meanwhile, the degrees of leukocytes infiltrate in H&E staining (p < 0.05, [92]Figure 3(a)) and the positive cells of astrocytic marker GFAP (p < 0.05, [93]Figure 3(b)) and microglial marker Iba1 (p < 0.05, [94]Figure 3(c)) in vlPAG of the SNI model group were all increased. However, the mice of sham + Pue group were no statistical changes (p > 0.05, [95]Figures (2) and ([96]3)). Following 1 week of Pue treatment, the SNI + Pue group mice displayed a decreased numbers of Nissl bodies, degrees of leukocytes infiltrate and positive cells of c-Fos, GFAP, and Iba1 in vlPAG (p < 0.05 vs SNI group). While, there were no statistical changes in dorsomedial (dm) and lateral (l) subregion PAG between groups. Figure 4. [97]Figure 4. [98]Open in a new tab Effect of Pue on the mitochondrial proteins. (a) Volcanic map of differentially expressed genes in [99]GSE91396. (b) Venn diagram for the comorbidity of chronic pain and depression, mitochondria and Pue-potential targets. (c and d) GO and KEGG enrichment analysis of overlapped genes. (e) Representative IHC staining images and quantitative analysis for UQCRB in PAG, dmPAG, lPAG, and vlPAG of mice. PAG Scale bar: 200 µm. dmPAG, lPAG, and vlPAG Scale bar: 50 µm. Data were expressed as the mean ± SD. *p < 0.05 versus sham group. #p < 0.05 versus SNI group. Table 1. GO enrichment analysis of the DEPs in PAG of database [100]GSE61396. Category Term Description LogP BP GO:0002181 Cytoplasmic translation −18.18 BP GO:0031175 Neuron projection development −11.78 BP GO:0006091 Generation of precursor metabolites and energy −11.45 BP GO:0006412 Translation −11.10 BP GO:0050804 Modulation of chemical synaptic transmission −10.93 BP GO:0099177 Regulation of trans-synaptic signaling −10.91 BP GO:0042176 Regulation of protein catabolic process −9.97 BP GO:0099003 Vesicle-mediated transport in synapse −9.38 BP GO:0050808 Synapse organization −8.54 BP GO:0006163 Purine nucleotide metabolic process −8.48 CC GO:0030424 Axon −22.65 CC GO:0022626 Cytosolic ribosome −20.59 CC GO:0044297 Cell body −20.58 CC GO:0043025 Neuronal cell body −17.90 CC GO:0005840 Ribosome −15.74 CC GO:0098794 Postsynapse −15.72 CC GO:0098978 Glutamatergic synapse −15.11 CC GO:0044391 Ribosomal subunit −15.05 CC GO:0098793 Presynapse −14.68 CC GO:0150034 Distal axon −14.49 MF GO:0003735 Structural constituent of ribosome −15.59 MF GO:0033218 Amide binding −8.01 MF GO:0001540 Amyloid-beta binding −7.74 MF GO:0015631 Tubulin binding −7.47 MF GO:0008047 Enzyme activator activity −7.19 MF GO:0022853 Active monoatomic ion transmembrane transporter activity −6.35 MF GO:0015078 Proton transmembrane transporter activity −6.16 MF GO:0070325 Lipoprotein particle receptor binding −6.11 MF GO:0050750 Low-density lipoprotein particle receptor binding −5.16 MF GO:0008017 Microtubule binding −5.07 [101]Open in a new tab Figure 2. [102]Figure 2. [103]Open in a new tab Effect of Pue on the neuronal activity in PAG of mice. (a) Representative images and quantitative analysis of Nissl bodies in PAG. (b) Representative IHC staining images and quantitative analysis for c-Fos in PAG. PAG Scale bar: 200 µm. dmPAG, lPAG, and vlPAG Scale bar: 50 µm. Data were expressed as the mean ± SD. *p < 0.05 versus sham group. #p < 0.05 versus SNI group. Figure 3. [104]Figure 3. [105]Open in a new tab Effect of Pue on the neuroinflammation in PAG of mice. (a) Representative images and quantitative analysis of H&E staining in PAG. (b and c) Representative IHC staining images and quantitative analysis for GFAP and Iba1 in PAG. PAG Scale bar: 200 µm. dmPAG, lPAG, and vlPAG Scale bar: 50 µm. Data were expressed as the mean ± SD. *p < 0.05 versus sham group. #p < 0.05 versus SNI group. Puerarin elevates the mitochondrial function in vlPAG of SNI model mice Venn analysis showed that there were 32 overlapping genes for the comorbidity of chronic pain and depression and mitochondria ([106]Figure 4(b) and [107]Tables 1 and [108]2). GO and KEGG pathway enrichment analysis for 32 overlapping genes were performed ([109]Figure 4(c) and ([110]d)). GO terms showed that cellular respiration (GO:0045333), oxidative phosphorylation (GO:0006119), and energy derivation by oxidation of organic compounds (GO:0015980) were enriched in BP category. Mitochondrial membrane (GO:0031966), mitochondrial inner membrane (GO:0005743), and mitochondrial protein-containing complex (GO:0098798) were enriched in CC category. Oxidoreductase activity (GO:0016491), electron transfer activity (GO:0009055), and oxidoreduction-driven active transmembrane transporter activity (GO:0015453) were enriched in MF category. KEGG terms showed that 12 genes (Bckdha, Cox6A1, Fasn, Gpx1, Ndufa2, Ndufs8, Uqcrc1, Grhpr, Uqcr11, Prdx5, Uqcr10, Ndufs7) were enriched in oxidative phosphorylation pathway (hsa00190), 11 genes (Atp5F1D, Atp5Mc2, Cox6A1, Ndufa2, Ndufs8, Uqcrc1, Uqcr11, Uqcr10, Ndufa13, Ndufa11, Ndufs7) were enriched in chemical carcinogenesis – reactive oxygen species pathway (hsa05208) and 11 genes (Atp5F1D, Atp5Mc2, Cox6A1, Ndufa2, Ndufs8, Uqcrc1, Uqcr11, Uqcr10, Ndufa13, Ndufa11, Ndufs7) were enriched in thermogenesis pathway (hsa04714). UQCRB (Ubiquinol cytochrome c reductase binding protein, a subunit of the mitochondrial complex III) protein level was verified. The IHC data showed that comparing with the sham group, the number of UQCRB positive cells in vlPAG of the SNI model group was increased (p < 0.05, [111]Figure 4(e)). To investigate the cellular localization of UQCRB in vlPAG, the IF co-staining of NeuN and GFAP along with UQCRB were performed. The results showed that comparing with the sham group, the intensity of UQCRB in vlPAG of the SNI model group was enhanced (p < 0.05, [112]Figure 3(f)–([113]m)). Moreover, the colocalization rates of GFAP with UQCRB and NeuN with UQCRB both were increased ([114]Figures 5 and [115]6(a)–([116]e)). In addition, antioxidative mitochondrial enzyme Mn-SOD activity was suppressed and ATP contents were found to be relatively reduced in the SNI model group (p < 0.05 vs sham group, [117]Figure 6(f) and ([118]g)). Following 1 week of Pue treatment, the number of UQCRB positive cells was increased, the Mn-SOD activity was increased, and the ATP content was up-regulated in vlPAG of SNI + Pue group (p < 0.05 vs SNI group, [119]Figure 6). However, there was no change between sham and sham + Pue groups (p > 0.05 vs SNI group, [120]Figures 4(e), [121]5, and [122]6). Table 2. Thirty-two overlapping genes for the comorbidity of chronic pain and depression and mitochondria. Gene name log2Fold change p Value Regulation Acot7 0.29 0.01 Up Antkmt 0.79 0.00 Up Arf5 0.49 0.02 Up Atp5f1d 0.49 0.02 Up Atp5mc2 0.54 0.01 Up Bax 0.61 0.02 Up Bckdha 0.69 0.03 Up Chchd10 0.47 0.02 Up Cox6a1 0.34 0.00 Up Fasn 0.52 0.00 Up Fkbp8 0.70 0.00 Up Ghitm −0.17 0.04 Down Gpx1 0.48 0.03 Up Grhpr 0.63 0.02 Up Isoc2a 0.41 0.00 Up Mtch1 0.21 0.03 Up Nat8l 0.33 0.02 Up Ndufa11 0.77 0.01 Up Ndufa13 0.50 0.01 Up Ndufa2 0.56 0.00 Up Ndufs7 1.16 0.00 Up Ndufs8 0.45 0.01 Up Pink1 0.39 0.00 Up Pnkd 0.28 0.03 Up Prdx5 0.49 0.00 Up Prepl −0.19 0.02 Down Slc25a22 0.48 0.04 Up Slc25a39 0.51 0.04 Up Timm13 1.05 0.00 Up Uqcr10 0.74 0.00 Up Uqcr11 0.56 0.01 Up Uqcrc1 0.26 0.01 Up [123]Open in a new tab Figure 5. [124]Figure 5. [125]Open in a new tab Effect of Pue on the UQCRB level in PAG of mice. (a–e) Representative fluorescence images (a–d) and quantitative analysis (e) of colocalized UQCRB with NeuN in PAG, dmPAG, lPAG, and vlPAG. PAG Scale bar: 200 µm. dmPAG, lPAG, and vlPAG Scale bar: 50 µm. Data were expressed as the mean ± SD. *p < 0.05 versus sham group. #p < 0.05 versus SNI group. Figure 6. [126]Figure 6. [127]Open in a new tab Effect of Pue on the UQCRB location and mitochondrial function in PAG of mice. (a–e) Representative fluorescence images (a–d) and quantitative analysis (e) of colocalized UQCRB with GFAP in PAG dmPAG, lPAG, and vlPAG. PAG scale bar: 200 μm. dmPAG, lPAG, and vlPAG scale bar: 50 μm. (f and g) Changes in Mn-SOD activity and ATP contents in PAG of mice. Data were expressed as the mean ± SD. *p < 0.05 versus sham group. #p < 0.05 versus SNI group. Puerarin reduces Bax level in vlPAG of SNI model mice via binding on the D102 residue To explore the mechanisms of Pue in the comorbidity of chronic pain and depression, 297 Pue predicted targets were obtained by TCMSP, Swiss Target Prediction, and TargetNet database. The Venn diagram showed that 6 genes (App, Bax, Calm3, Esrra, Mif, Ptprs) were overlapped for the comorbidity of chronic pain and depression and the Pue-potential targets, 17 genes (Acaca, Acacb, Aldh2, Bad, Bax, Bcl2, Casp3, Casp8, Casp9, Dhodh, Ephx2, Fdps, Maoa, Maob, Mcl1, Prkaca, Sod1) were overlapped for mitochondria and the Pue-potential targets, and only 1 gene (Bax) was overlapped for the comorbidity of chronic pain and depression, mitochondria, and the Pue-potential targets ([128]Figure 4(b)). Autodock was then used to predict the binding between Pue and Bax, and the docking results showed that Pue formed hydrogen bonds with Bax at residues M99, D102, and F176 with the binding energy at −6.7 kcal/mol ([129]Figure 7(a) and ([130]b)). The structure and conservation of Bax were analyzed to verify this prediction and the results showed that M99 residue was positioned on the helix structure of the DH1 domain, D102 residue was positioned on the turn structure of the DH1 domain, and F178 residue was positioned on the helix structure of the transmembrane (TM) motif ([131]Figure 7(c)). On the basis of these information, the mutation Bax-D102A was constructed ([132]Figure 7(d)) and the binding for Bax or Bax-D102A with Pue was detected using ITC. The results showed that the binding of Pue with Bax was an exothermic reaction with the binding affinity at 2.4 ± 0.1 μM ([133]Figure 7(e)). However, no reaction was observed between Pue and Bax-D102A ([134]Figure 7(f)). In addition, the IHC assay showed that the Bax positive cells in vlPAG of the SNI model mice increased comparing with the sham group (p < 0.05) and Pue treatment decreased the Bax-positive cells in vlPAG of the SNI + Pue group (p < 0.05 vs SNI group, [135]Figure 7(g) and ([136]h)). Figure 7. [137]Figure 7. [138]Open in a new tab Effect of Pue on Bax level in PAG of mice. (a) Chemical structure of puerarin. (b) Diagram of the binding between Pue and Bax. (c) Structural and conservative analysis of Bax. (d) Protein expression of Bax. (e and f) ITC data for the titration between Pue and protiens Bax or Bax-D102. (g and h) Representative immunohistochemistry staining images and quantitative analysis for Bax in PAG dmPAG, lPAG, and vlPAG. PAG scale bar: 200 μm. dmPAG, lPAG, and vlPAG scale bar: 50 μm. Mean ± SD. *p < 0.05 versus sham group. #p < 0.05 versus SNI group. Puerarin reduces the levels of Bax and IL-1β in primary astrocytic cells The primary astrocytic cells were cultured to verify the role of Pue on Bax level and inflammation. The Western blotting results showed that the levels of Bax and proinflammatory cytokine IL-1β were both reduced in the Pue treated group compared with the DMSO group (p < 0.05, [139]Figure 8(a) and ([140]b)). The Bax level was decreased using Bax siRNA and the IL-1β level was relevantly reduced (p < 0.05 vs siRNA group). No changes in the levels of Bax and IL-1β were observed in the siRNA treated group (p > 0.05 vs Control group, [141]Figure 8(a) and ([142]b)). Meanwhile, in organotypic PAG slice of the SNI model mice, the levels of Bax, IL-1β, and c-Fos were reduced in the Bax siRNA group compared with siRNA group detecting by Western blotting (p < 0.05, [143]Figure 8(c) and ([144]d)). The results revealed that Pue and Bax siRNA treatment both served to reduce the neuronal activity and inflammation. Furthermore, primary astrocytic cells were stimulated by IL-1β in order to mimic the activated inflammation. The results in [145]Figure 8(e) to ([146]h) showed that the fluorescence intensities and relative levels of GFAP and Bax in the IL-1β stimulated group were increased compared to the control group (p < 0.05) and Pue treatment weakened the GFAP and Bax intensities and levels in the IL-1β + Pue group (p < 0.05 vs IL-1β group). Figure 8. [147]Figure 8. [148]Open in a new tab Effect of Pue or Bax siRNA on Bax and IL-1β levels in primary astrocytic cells. (a and b) Representative Western blotting bands and quantitative analysis for Bax and IL-1β in primary astrocytic cells of Control, siRNA, Bax siRNA, DMSO, and Pue groups. *p < 0.05 versus siRNA or DMSO group. (c and d) Representative Western blotting bands and quantitative analysis for Bax, IL-1β, and c-Fos in organotypic PAG slice of the SNI model mice with Control, siRNA and Bax siRNA groups. *p < 0.05 versus siRNA group. (e and f) Representative immunofluorescence images and quantitative analysis for Bax and GFAP in primary astrocytic cells of Control, IL-1β and IL-1β + Pue groups. Scale bar: 50 μm. (g and h) Representative Western blotting bands and quantitative analysis for Bax and GFAP in primary astrocytic cells in Control, IL-1β and IL-1β + Pue groups. Data were presented as mean ± SD. *p < 0.05 versus Control group. #p < 0.05 versus IL-1β group. Puerarin recovers the levels of MMP and mito-ROS in primary astrocytic cells Bax protein is a crucial factor in MMP maintenance and mito-ROS release.^ [149]24 Mito-Tracker assay for MMP in [150]Figure 9a and MitoSOX assay for mito-ROS in [151]Figure 9b showed that compared to the control group, Mito-Tracker intensity was reduced while MitoSOX intensity was increased in IL-1β group (p < 0.05). Pue treatment recovered these intensities in the IL-1β + Pue group (p < 0.05 vs IL-1β group). Figure 9. [152]Figure 9. [153]Open in a new tab Effect of Pue on MMP and mito-ROS levels in primary astrocytes. (a) Representative fluorescence images and quantitative analysis of Mito-Tracker in primary astrocytes. (b) Representative fluorescence images and quantitative analysis of MitoSOX in primary astrocytes. Scale bar: 50 μm. Data were presented as mean ± SD. *p < 0.05 versus Control group. #p < 0.05 versus IL-1β group. Discussion Chronic pain is considered a critical factor for depression. In order to better explore the pathological mechanism of the comorbidity of chronic pain and depression, valid animal models are extremely important. Neuropathic pain constitutes a part of chronic pain, results from damage or abnormal function of the central or peripheral nervous system. It is relevantly linked to emotional disorders like anxiety, irritability, social isolation or depression.^ [154]25 The SNI model was reported as the increased mechanical allodynia on the ipsilateral hind paw lasting for 3–4 months,^[155]26–[156]28 and the inducement of depressive symptoms.^[157]29,[158]30 Meanwhile, the time factor is also critical. Some negative results are showed at 1–3 weeks following the inflammatory or neuropathy induction.^ [159]31 Here, the SNI mouse model was constructed to induce chronic neuropathic pain and depression and the behaviors tests were performed on 8 weeks following surgery. And we found that the SNI model mice showed as the increased mechanical pain, the impaired motor coordination, the induced anhedonia and despair. Then the SNI model was suitable for the research on the comorbidity of chronic pain and depression. In addition, we found that Pue administration improved the pain and depression behaviors in SNI model mice. Pue is reported to attenuate neuropathic pain in mice of SNI model,^ [160]32 chemotherapy induced model,^ [161]33 chronic constriction injury and diabetes-induced model.^ [162]34 Pue has been reported to alleviate depression-like behavior in a chronic unpredictable mild stress mouse model via repairing inflammatory damages and phospholipid metabolism disorders,^ [163]35 or remodeling their gut microbiota.^ [164]36 Pue is reported to effectively ameliorate depression and pain in SNI mice via activating ERK, CREB, and BDNF pathways.^ [165]37 Thus, we suggested that Pue may serve as a potential protective agent of novel therapeutics for the comorbidity of chronic pain and depression. Understanding the pathophysiology of the comorbidity of chronic pain and depression is offering the potential avenues for therapeutic strategies. Changes of neurobiology and neuroinflammation are considered to be the possible reason for the comorbidity.^ [166]38 It is reported that during the process of chronic pain and depression, the major neurotransmitter systems, such as serotonin (5-HT), gamma-aminobutyric acid (GABA), and glutamate are dys-functioned. The key neural circuits, including pain perception pathways, emotion regulation circuits, and the reward system are disrupted.^ [167]39 Meanwhile, peripheral nerve injury also triggers neuroinflammation in brain, such as the activation of glial cells, the releasing of cytokines and chemokines, and the enhanced infiltration of leukocytes.^ [168]40 In this study, we also found the neuronal activity and neuroinflammation were triggered in vlPAG of SNI model mice, and Pue treatment reduced these injuries. Then, we considered that improving the neurocircuitry and neuroinflammation may be responsible for the optimal modulation of the comorbidity of chronic pain and depression, and Pue can work as a neuroprotective agent. This study found that Pue bound with Bax and reduced its level in vlPAG of the SNI model mice. Bax (Bcl-2 associated X-protein) belongs to the Bcl-2 family, is widely expressed in the nervous system and works as a primary executor of mitochondrial membrane permeabilization and cell apoptosis.^ [169]41 The increased Bax level is accompanied by the apoptotic death of cortical neurons and blocking the Bax activity has a neuroprotective effect.^ [170]42 Bax level is up-regulated in animal models of inflammatory pain and neuropathic pain, while inhibiting Bax signal by Aju-I and daidzein relieves the pain behaviors.^[171]43,[172]44 Bax inhibitor-1 improves depression-like behavior by reducing the apoptotic and inflammatory signals.^ [173]45 It has also been reported that the numbers of motor neurons and cerebellar Purkinje cells are increased approximately 50% and 30% respectively in Bax-KO animals.^ [174]46 In addition, Pue has been reported to inhibit Bax expression in the models of several diseases.^[175]16,[176]47 By combining these information, we suggested that Bax could be a potential target for the treatment of comorbidity of chronic pain and depression. Mitochondrial functions on ATP and ROS generation were dysregulated in vlPAG of the SNI model mice in this study. Bax has been reported to negatively regulate the expression of mitochondrial respiratory complexes subunits and mitochondrial respiration.^ [177]48 At the same time, Bax functions on mitochondrial permeabilization by forming oligomeric pores in the mitochondrial outer membrane, which results in the release of mitochondrial contents.^ [178]49 Residues D102 and F176 are consisted for the pocket formation near residue S184 which is prone to phosphorylation and activates Bax.^ [179]50 In this study, the binding between Pue and Bax was found to be interrupted by the mutation on residue D102. We suggested that Pue inhibited Bax oligomerization and activation via binding on D102. In addition, Pue has been reported to exert antioxidant and anti-inflammatory effects by improving FUNDC1-mediated mitophagy^ [180]51 or mitochondrial dysfunction.^ [181]52 Pue also has a neuron-protective effect by increasing MMP, mitochondrial ATP generation, and electron leakage.^ [182]53 In this study, among 17 genes of mitochondrial and Pue-potential targets, 12 genes (Acaca, Bad, Bax, Bcl2, Casp3, Casp8, Casp9, Fdps, Maob, Mcl1, Sod1, Prkaca) were found to be enriched in the apoptosis pathway (hsa04210). Thus, we suggested that Pue could be applied for the treatment of mitochondrial related disease. Conclusion In SNI model mice, neuropathic pain and depressive related behaviors are evoked, neuronal activity and neuroinflammation in vlPAG are triggered, mitochondrial dysfunction in vlPAG is developed. Pue administration targets Bax, improves mitochondrial function, decreases the neuronal activity and inflammation in vlPAG, and reduces pain hypersensitivity and depression ([183]Figure 10). This study helps to reveal the pathogenesis of comorbidity of chronic pain and depression and may provide a theoretical basis for Pue application in improving the comorbidity of chronic pain and depression. Figure 10. [184]Figure 10. [185]Open in a new tab Schematic diagram of the potential mechanisms of Pue on the comorbidity of chronic pain and depression. Pue targets on bax, improves mitochondrial function, reduces inflammation and neuronal damage in vlPAG, ameliorates pain and depression. Footnotes Author contributions: HLZ conceptualized and designed the study. GGS, YW, and LXY carried out the experiment and collected data. HYL, PGZ, and CCS analyzed the data. HLZ and GLW wrote and edited the manuscript. All authors read and approved the final manuscript. Data availability statement: The data sets used and analyzed during the current study are available from the corresponding author on reasonable request. 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: This work was supported by the National Natural Science Foundation of Hubei Province (Nos. 2023AFB1087, 2023AFD111, 2023ZRKX102); the Hubei University of Science and Technology Program (Nos. 2022YKY02, 2023YKY06, 2023YKY10, 2023TNB06). ORCID iD: Haili Zhu Inline graphic [186]https://orcid.org/0000-0001-7802-676X References