Korean J Pain.  2025 Jan;38(1):14-28. 10.3344/kjp.24244.

Temporal therapy utilizing exosomes derived from M2 macrophages demonstrates enhanced efficacy in alleviating neuropathic pain in diabetic rats

• Wei W
• Fang J
• Yang B
• Cui C
• Wei J
• Xue Y

• KMID: 2563868
• DOI: http://doi.org/10.3344/kjp.24244

Abstract

Background
Diabetic pain patients have increased pain at night. Exosomes can relieve neuropathic pain. This study aimed to investigate the efficacy of exosome administration at different time points in relieving diabetic neuropathic pain (DNP) in rats.
Methods
M2 macrophages from bone marrow were induced in mice and exosomes were extracted. A diabetic rat model was induced using streptozotocin, with the mechanical withdrawal threshold (MWT) of the rats being measured at ≤ 80% of the basal value after 14 days, indicating successful construction of the DNP rat model. Exosomes were administered on three consecutive days at ZT0 (zeitgeber time) and ZT12. Parameters including blood glucose levels, body weight, MWT, and thermal withdrawal latency (TWL) were assessed in the rats. The lumbar spinal cord of rats was examined on days 21 and 28 to measure inflammatory factors and observe the expression of M1 and M2 microglia. Furthermore, microglia were exposed to lipopolysaccharide (LPS) and LPS + exosomes in a controlled in vitro setting to assess alterations in microglia phenotype involving the NF-kB p65 and IKBα inflammatory signaling pathways.
Results
The findings revealed that administration of exosomes during the rat resting period at ZT12 resulted in increased MWT and TWL, as well as a shift in microglia polarization towards the M2 phenotype. In vitro analysis indicated that exosomes influenced microglia polarization and suppressed the phosphorylation of NF-kB p65 and IKBα. 
Conclusions
Temporal therapy with exosomes effectively reduces pain in DNP rats by polarizing microglia and affecting NF-kB p65 and IKBα signaling pathways.

Keyword

Chronotherapy; Circadian Rhythm; Diabetic Neuropathies; Exosomes; Injections, Spinal; Macrophages; Microglia; Neuralgia; Spinal Cord

Figure

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  Fig. 1 (A) Detection of rat macrophage marker CD206 by flow cytometry. (B, C) Morphology and particle size analysis of exosomes observed by transmission electron microscopy. (D) Detection of marker proteins of exosomes.

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  Fig. 2 Intrathecal injection of exosomes does not change body weight (A) or blood glucose levels (B), but reduces mechanical (C) and thermal pain in diabetic rats (D). DNP rats were given streptozotocin (STZ, intraperitoneal injection, 65 mg/kg), and blood glucose levels and body weights were measured before (baseline) and on days 3, 7, 14, 21, and 28 after STZ administration, respectively. Mechanical and thermal pain thresholds were measured before (baseline) and on days 14, 17, 19, 21, and 28 after STZ administration. Data are expressed as mean ± standard error of the mean (n = 10, day 28 n = 5). DNP: diabetic neuropathic pain, ZT: zeitgeber time, DNP + exo-ZT0: DNP rats in the ZT0 exosome-injected group, DNP + exo-ZT12: DNP rats in the ZT12 exosome-injected group, TWL: thermal withdrawal latency, MWT: mechanical withdrawal threshold. Comparison with control group, *P < 0.05, ***P < 0.001; with DNP (ZT8) group, #P < 0.05, ###P < 0.001; with DNP (ZT20) group, &&P < 0.01, &&&P < 0.001; with DNP+exo-ZT0 group, $P < 0.05, $$$P < 0.001.

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  Fig. 3 Exosome injection during the resting period of DNP rats can more effectively promote the polarisation of microglia from M1 to M2 phenotype. (A, B) Levels of pro-inflammatory factor TNF-α and anti-inflammatory factor IL-10 in the lumbar segment of the rat spinal cord were determined by ELISA after 7 days and 14 days of exosome injection (n = 3). (C, D) qRT-PCR to detect relative mRNA levels of M1-related iNOS, IL-1β, and M2-related Arg-1 and IL-10 in the lumbar segment of the rat spinal cord after 7 and 14 days of exosome injection (n = 3). Relative mRNA levels of M1-associated iNOS, IL-1β, and M2-associated Arg-1, IL-10 in the spinal cord (n = 3). (E, F) Western blot detection of iNOS, ARG-1 protein levels in the lumbar segment of rat spinal cords 7 days after exosome injection and 14 days after exosome injection (n = 4). Data are expressed as mean ± standard error of the mean. DNP: diabetic neuropathic pain, ZT: zeitgeber time, DNP + exo-ZT0: DNP rats in the ZT0 exosome-injected group, DNP + exo-ZT12: DNP rats in the ZT12 exosome-injected group, TNF-α: tumor necrosis factor-α, IL: interleukin, ELISA: enzyme-linked immunosorbent assay, qRT-PCR: quantitative reverse transcription polymerase chain reaction. Compared with control group, ns represents P > 0.05, **P < 0.01, ***P < 0.001; compared with DNP group, #P < 0.05, ##P < 0.01, ###P < 0.001; compared between the exosomal intervention groups, $P < 0.05, $$P < 0.01, $$$P < 0.001.

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  Fig. 4 Significant activation of microglia in the dorsal horn of the spinal cord of diabetic rats, exosome administration enhances the expression of the M2 microglia marker ARG-1 and attenuates the expression of the M1 microglia marker iNOS, and nighttime administration is more effective than daytime administration. (A) Representative images of immunofluorescence assay for protein expression of Iba1 and ARG-1 in the lumbar spinal cord of the rat. (B) Representative images of Iba1, iNOS protein expression immunofluorescence assay (DAPI is blue fluorescence, Iba1 is red fluorescence, ARG-1 is green fluorescence), Bar = 100 um, quantitative analysis of immunofluorescence intensity (n = 3). Data are expressed as mean ± standard error of the mean. DNP: diabetic neuropathic pain, ZT: zeitgeber time, DNP + exo-ZT0: DNP rats in the ZT0 exosome-injected group, DNP + exo-ZT12: DNP rats in the ZT12 exosome-injected group, DAPI: 4,6-diamidino2-phenyl-indole dihydrochloride. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control group; #P < 0.05, ###P < 0.001 compared with DNP group; $P < 0.05, $$P < 0.01 for comparison between exosome intervention groups.

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  Fig. 5 Exosomes regulate microglia M1/M2 polarisation through NF-kB p65, IKBα signaling pathway. (A, B) Western blot strip plots of IKBα, p-IKBα, p-NF-kB p65 proteins in rat spinal cord on the seventh and 14th day after exosome injection, respectively, and quantification of grey values of protein expression levels (n = 4). Data are expressed as mean ± standard error of the mean. DNP: diabetic neuropathic pain, ZT: zeitgeber time, DNP + exo-ZT0: DNP rats in the ZT0 exosome-injected group, DNP + exo-ZT12: DNP rats in the ZT12 exosome-injected group. ***P < 0.001 compared with control group; ##P < 0.01, ###P < 0.001 compared with DNP group; $$P < 0.01, $$$P < 0.001 compared between exosome intervention groups.

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  Fig. 6 Exosomes convert BV2 microglia from M1 phenotype to M2 phenotype through NF-kB p65, IKBα signaling pathway. (A, B) The concentration of pro- and anti-inflammatory cytokines in BV2 microglia in different groups. (C, D) mRNA expression levels of M1-related genes (CD86) and M2-related genes (CD206) detected by qRT-PCR. (E) The protein expression levels of M1- and M2-related genes and the expression of NF-kB p65 and IKBα signaling pathway in BV2 microglia in different groups were detected by Western blot. Data were expressed as mean ± standard error of the mean (n = 3). LPS: lipopolysaccharide, TNF-α: tumor necrosis factor-α, IL: interleukin, qRT-PCR: quantitative reverse transcription polymerase chain reaction, ns: no significance. Compared with control group, **P < 0.01, ***P < 0.001, ****P < 0.001; compared with LPS group, #P < 0.05, ###P < 0.001.

Reference

1. Saeedi P, Petersohn I, Salpea P, Malanda B, Karuranga S, Unwin N, et al. IDF Diabetes Atlas Committee. 2019; Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: results from the International Diabetes Federation Diabetes Atlas, 9th edition. Diabetes Res Clin Pract. 157:107843. DOI: 10.1016/j.diabres.2019.107843. PMID: 31518657.

2. Abbott CA, Malik RA, van Ross ER, Kulkarni J, Boulton AJ. 2011; Prevalence and characteristics of painful diabetic neuropathy in a large community-based diabetic population in the U.K. Diabetes Care. 34:2220–4. DOI: 10.2337/dc11-1108. PMID: 21852677. PMCID: PMC3177727.

3. Shillo P, Sloan G, Greig M, Hunt L, Selvarajah D, Elliott J, et al. 2019; Painful and painless diabetic neuropathies: what is the difference? Curr Diab Rep. 19:32. DOI: 10.1007/s11892-019-1150-5. PMID: 31065863. PMCID: PMC6505492.

4. Bhandari R, Sharma A, Kuhad A. 2022; Novel nanotechnological approaches for targeting dorsal root ganglion (DRG) in mitigating diabetic neuropathic pain (DNP). Front Endocrinol (Lausanne). 12:790747. DOI: 10.3389/fendo.2021.790747. PMID: 35211091. PMCID: PMC8862660.

5. Kalluri R, LeBleu VS. 2020; The biology, function, and biomedical applications of exosomes. Science. 367:eaau6977. DOI: 10.1126/science.aau6977. PMID: 32029601. PMCID: PMC7717626.

6. Kim H, Wang SY, Kwak G, Yang Y, Kwon IC, Kim SH. 2019; Exosome-guided phenotypic switch of M1 to M2 macrophages for cutaneous wound healing. Adv Sci (Weinh). 6:1900513. DOI: 10.1002/advs.201900513. PMID: 31637157. PMCID: PMC6794619.

7. Dibner C, Schibler U, Albrecht U. 2010; The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annu Rev Physiol. 72:517–49. DOI: 10.1146/annurev-physiol-021909-135821. PMID: 20148687.

8. Oliverio A, Castellano C, Puglisi-Allegra S. 1982; Opiate analgesia: evidence for circadian rhythms in mice. Brain Res. 249:265–70. DOI: 10.1016/0006-8993(82)90060-9. PMID: 7139304.

9. Gilron I, Ghasemlou N. 2014; Chronobiology of chronic pain: focus on diurnal rhythmicity of neuropathic pain. Curr Opin Support Palliat Care. 8:429–36. DOI: 10.1097/SPC.0000000000000085. PMID: 25111256.

10. Gilron I, Bailey JM, Tu D, Holden RR, Weaver DF, Houlden RL. 2005; Morphine, gabapentin, or their combination for neuropathic pain. N Engl J Med. 352:1324–34. DOI: 10.1056/NEJMoa042580. PMID: 15800228.

11. Walton JC, Walker WH 2nd, Bumgarner JR, Meléndez-Fernández OH, Liu JA, Hughes HL, et al. 2021; Circadian variation in efficacy of medications. Clin Pharmacol Ther. 109:1457–88. DOI: 10.1002/cpt.2073. PMID: 33025623. PMCID: PMC8268638.

12. Palada V, Gilron I, Canlon B, Svensson CI, Kalso E. 2020; The circadian clock at the intercept of sleep and pain. Pain. 161:894–900. DOI: 10.1097/j.pain.0000000000001786. PMID: 31895268.

13. Pathak R, Sachan N, Chandra P. 2022; Mechanistic approach towards diabetic neuropathy screening techniques and future challenges: a review. Biomed Pharmacother. 150:113025. DOI: 10.1016/j.biopha.2022.113025. PMID: 35658222.

14. Ying W, Cheruku PS, Bazer FW, Safe SH, Zhou B. 2013; Investigation of macrophage polarization using bone marrow derived macrophages. J Vis Exp. 76:50323. DOI: 10.3791/50323-v.

15. Tahara Y, Aoyama S, Shibata S. 2017; The mammalian circadian clock and its entrainment by stress and exercise. J Physiol Sci. 67:1–10. DOI: 10.1007/s12576-016-0450-7. PMID: 27084533. PMCID: PMC5138246.

16. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. 1994; Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 53:55–63. DOI: 10.1016/0165-0270(94)90144-9. PMID: 7990513.

17. Jiang Y, Wang J, Li H, Xia L. 2020; IL-35 promotes microglial M2 polarization in a rat model of diabetic neuropathic pain. Arch Biochem Biophys. 685:108330. DOI: 10.1016/j.abb.2020.108330. PMID: 32156533.

18. Kang J, Guo Y. 2022; Human umbilical cord mesenchymal stem cells derived exosomes promote neurological function recovery in a rat spinal cord injury model. Neurochem Res. 47:1532–40. DOI: 10.1007/s11064-022-03545-9. PMID: 35132478.

19. Tsuda M, Ueno H, Kataoka A, Tozaki-Saitoh H, Inoue K. 2008; Activation of dorsal horn microglia contributes to diabetes-induced tactile allodynia via extracellular signal-regulated protein kinase signaling. Glia. 56:378–86. DOI: 10.1002/glia.20623. PMID: 18186080.

20. Wu J, Ding DH, Li QQ, Wang XY, Sun YY, Li LJ. 2019; Lipoxin A4 regulates lipopolysaccharide-induced BV2 microglial activation and differentiation via the notch signaling pathway. Front Cell Neurosci. 13:19. DOI: 10.3389/fncel.2019.00019. PMID: 30778288. PMCID: PMC6369213.

21. Shu B, He SQ, Guan Y. 2020; Spinal cord stimulation enhances microglial activation in the spinal cord of nerve-injured rats. Neurosci Bull. 36:1441–53. DOI: 10.1007/s12264-020-00568-6. PMID: 32889636. PMCID: PMC7719153.

22. Yuan J, Fei Y. 2022; Lidocaine ameliorates chronic constriction injury-induced neuropathic pain through regulating M1/M2 microglia polarization. Open Med (Wars). 17:897–906. DOI: 10.1515/med-2022-0480. PMID: 35647302. PMCID: PMC9106111.

23. Zhang Y, Chen Q, Nai Y, Cao C. 2020; Suppression of miR-155 attenuates neuropathic pain by inducing an M1 to M2 switch in microglia. Folia Neuropathol. 58:70–82. DOI: 10.5114/fn.2020.94008. PMID: 32337959.

24. Afsari ZH, Renno WM, Abd-El-Basset E. 2008; Alteration of glial fibrillary acidic proteins immunoreactivity in astrocytes of the spinal cord diabetic rats. Anat Rec (Hoboken). 291:390–9. DOI: 10.1002/ar.20678. PMID: 18360886.

25. Liao YH, Zhang GH, Jia D, Wang P, Qian NS, He F, et al. 2011; Spinal astrocytic activation contributes to mechanical allodynia in a mouse model of type 2 diabetes. Brain Res. 1368:324–35. DOI: 10.1016/j.brainres.2010.10.044. PMID: 20971097.

26. Mohawk JA, Green CB, Takahashi JS. 2012; Central and peripheral circadian clocks in mammals. Annu Rev Neurosci. 35:445–62. DOI: 10.1146/annurev-neuro-060909-153128. PMID: 22483041. PMCID: PMC3710582.

27. Turk DC, Dworkin RH, Allen RR, Bellamy N, Brandenburg N, Carr DB, et al. 2003; Core outcome domains for chronic pain clinical trials: IMMPACT recommendations. Pain. 106:337–45. DOI: 10.1016/j.pain.2003.08.001. PMID: 14659516.

28. Xia T, Cui Y, Qian Y, Chu S, Song J, Gu X, et al. 2016; Regulation of the NR2B-CREB-CRTC1 signaling pathway contributes to circadian pain in murine model of chronic constriction injury. Anesth Analg. 122:542–52. DOI: 10.1213/ANE.0000000000000991. PMID: 26440419.

29. Fonken LK, Frank MG, Kitt MM, Barrientos RM, Watkins LR, Maier SF. 2015; Microglia inflammatory responses are controlled by an intrinsic circadian clock. Brain Behav Immun. 45:171–9. DOI: 10.1016/j.bbi.2014.11.009. PMID: 25433170. PMCID: PMC4386638.

30. Korszun A. 2000; Sleep and circadian rhythm disorders in fibromyalgia. Curr Rheumatol Rep. 2:124–30. DOI: 10.1007/s11926-000-0052-4. PMID: 11123049.

31. Gilron I, Bailey JM, Vandenkerkhof EG. 2013; Chronobiological characteristics of neuropathic pain: clinical predictors of diurnal pain rhythmicity. Clin J Pain. 29:755–9. DOI: 10.1097/AJP.0b013e318275f287. PMID: 23370066.

32. Vetter C. 2020; Circadian disruption: what do we actually mean? Eur J Neurosci. 51:531–50. DOI: 10.1111/ejn.14255. PMID: 30402904. PMCID: PMC6504624.

33. Li H, Yang J, Wang Y, Liu Q, Cheng J, Wang F. 2019; Neuroprotective effects of increasing levels of HSP70 against neuroinflammation in Parkinson's disease model by inhibition of NF-κB and STAT3. Life Sci. 234:116747. DOI: 10.1016/j.lfs.2019.116747. PMID: 31408661.

34. Shabab T, Khanabdali R, Moghadamtousi SZ, Kadir HA, Mohan G. 2017; Neuroinflammation pathways: a general review. Int J Neurosci. 127:624–33. DOI: 10.1080/00207454.2016.1212854. PMID: 27412492.

35. Zenker J, Ziegler D, Chrast R. 2013; Novel pathogenic pathways in diabetic neuropathy. Trends Neurosci. 36:439–49. DOI: 10.1016/j.tins.2013.04.008. PMID: 23725712.

36. Wang Q, Ye Y, Yang L, Xiao L, Liu J, Zhang W, et al. 2024; Painful diabetic neuropathy: the role of ion channels. Biomed Pharmacother. 173:116417. DOI: 10.1016/j.biopha.2024.116417. PMID: 38490158.

37. Shiue SJ, Rau RH, Shiue HS, Hung YW, Li ZX, Yang KD, et al. 2019; Mesenchymal stem cell exosomes as a cell-free therapy for nerve injury-induced pain in rats. Pain. 160:210–23. DOI: 10.1097/j.pain.0000000000001395. PMID: 30188455.

38. Ramanathan S, Ajit SK. 2016; MicroRNA-based biomarkers in pain. Adv Pharmacol. 75:35–62. DOI: 10.1016/bs.apha.2015.12.001. PMID: 26920008.

39. Sun G, Li G, Li D, Huang W, Zhang R, Zhang H, et al. 2018; hucMSC derived exosomes promote functional recovery in spinal cord injury mice via attenuating inflammation. Mater Sci Eng C Mater Biol Appl. 89:194–204. DOI: 10.1016/j.msec.2018.04.006. PMID: 29752089.

40. Guo S, Perets N, Betzer O, Ben-Shaul S, Sheinin A, Michaelevski I, et al. 2019; Intranasal delivery of mesenchymal stem cell derived exosomes loaded with phosphatase and tensin homolog siRNA repairs complete spinal cord injury. ACS Nano. 13:10015–28. DOI: 10.1021/acsnano.9b01892. PMID: 31454225.

41. Tran PH, Xiang D, Nguyen TN, Tran TT, Chen Q, Yin W, et al. 2020; Aptamer-guided extracellular vesicle theranostics in oncology. Theranostics. 10:3849–66. DOI: 10.7150/thno.39706. PMID: 32226524. PMCID: PMC7086349.