Interplay between exosomes and autophagy machinery in pain management: State of the art.

Exo are nano-sized EVs with an endosomal compartment origin. It has been suggested that these biological nano-carriers are secreted from all cell types into biofluids in response to stimuli during physiological and pathological conditions ( Coughlan et al., 2020 , Datta et al., 2018 ). Exo enable the transfer of several signaling molecules from donor cells toward acceptor cells to regulate bioactivities and various biological phenomena in a paracrine manner ( Isola and Chen, 2017 , Henne et al., 2011 ). Within the host cells, Exo are generated via invagination into the lumen of early endosomes and multivesicular bodies (MVBs) where they are named intraluminal vesicles (ILVs). These compartments are destined for lysosomal degradation or secretion into the extracellular matrix (ECM). Upon fusion of MVBs with the plasma membrane, ILVs are released into the ECM, hereafter known as Exo ( Mathivanan and Simpson, 2009 ). To be specific, MVBs can be formed via early endosomes originating from endocytosis-based mechanisms or trans -Golgi network activity where specific cargos are sequestrated into releasing ILVs ( Ge et al., 2012 ). Several biomolecules such as proteins, lipids, miRNAs, and mRNAs are transferred into recipient cells after the uptake of Exo ( Colombo et al., 2013 ). Given the highly intricate underlying mechanisms involved in Exo biogenesis, the endosomal sorting complex required for transport (ESCRT) machinery has a considerable role in Exo biogenesis. Several studies have demonstrated that the endosomal ESCRT complex accounts for MVB biogenesis, plasma membrane reconstruction, abscission, and virus release from the host cells ( Rahbarghazi et al., 2021 ). ESCRT consisted of five functional subsets: ESCRTs −0, -I, -II, and -III, and AAA ATPase Vps4 complex ( Mathivanan and Simpson, 2009 ). Among these factors, ESCRT-0 functions to classify and sequestrate ubiquitinated proteins into generating ILVs. ESCRT-I, in collaboration with ESCRT–II, accelerates invagination of the MVB membrane, and the addition of ESCRT-III completes this mechanism by cleaving vesicles into the MVB lumen. Molecular investigations have suggested that the ESCRT-0 complex consists of two subunits: Hrs and STAM1/2. Hrs determines the ubiquitinated proteins with STAM, Eps15, and clathrin. Besides, Hrs can recruit TSG101 from the ESCRT-I complex, facilitating the addition of other ESCRT subsets such as ESCRT-II and -III and ALIX ( Stuffers et al., 2009 ). Beyond these mechanisms, some reports demonstrate the existence of the ESCRT-independent pathway in the biogenesis of MVBs, which were confirmed after the suppression of four main subunits of the ESCRT complex ( Juan and Fürthauer, 2018 ). In support of this notion, it has been shown that the release of Exo in mouse oligodendrocytes is an ESCRT-free mechanism via the activity of ceramide-generating enzyme neutral sphingomyelinases (nSMase) ( Heidarzadeh et al., 2021 ). Tetraspanins, as intracellular effectors, partake in the biogenesis of Exo and cargo sorting ( Chairoungdua et al., 2010 ). These factors are determined based on four transmembrane domains. Different members of Tetraspanins have been supposed to be involved in ILV formation. Among different Tetraspanins, the suppression of CD9 in murine cells can diminish the Exo secretion ( Nazarenko et al., 2010 ). Tetraspanin 8 can also alter the genomic and proteomic contents of Exo ( Ghossoub et al., 2020 ). The precise collaboration of other Tetraspanin members such as Tetraspanin 6 and syndecan can regulate the balance between exosomal secretion and lysosomal degradation, having a pivotal role in the intracellular orientation of Exo. Of note, the close interaction of syntenin-syndecan via ALIX, in turn, increases the exosomal release of CD63, while the recruitment of CD9 and CD81 is syntenin-syndecan independent ( Janas et al., 2015 ). In contrast to protein loading in Exo, sorting RNAs into the exosomal lumen is closely associated with lipid activity. For instance, certain genomic cargo can be sorted into Exo after interaction with lipid rafts and sphingosine activity ( Hessvik and Llorente, 2018 ). To promote the release of Exo from host cells, the functionality of small GTPases, cytoskeletal proteins, molecular motors such as dyneins and kinesins, and the membrane fusion apparatus soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex, including Vamp7, Vti1b, syntaxin 7, and 8 are mandatory ( Yue et al., 2020 ). Rab GTPase family consists of about 70 subtypes that can facilitate the fusion and budding of vesicles ( Hsu et al., 2010 ). Additionally, each Rab type has a critical role in Exo development and biogenesis. For instance, Rab35 regulates the docking of endocytic vesicles with the plasma membrane in a GTP-dependent manner ( Fukuda, 2013 ), whereas Rab27 can activate effectors involved in the transportation and fusion of secretory vesicles to the cell membrane ( Villarroya-Beltri et al., 2014 ). The molecular motors and microtubules support anchorage sites to dispatch MVBs to the plasma membrane ( Dingjan et al., 2018 ). The molecular investigation revealed that SNAREs are indispensable in membrane fusion ( Zheng et al., 2019 ). SNARE complex consists of three subsets in a coiled-coil shape, which interlinks two dissenting membranes in a zipper-like form ( Chairoungdua et al., 2010 , Hsu et al., 2010 ). Upon reaching released Exo to the host cells, general non-specific cell responses such as direct fusion, macropinocytosis, micropinocytosis, phagocytosis, clathrin-dependent endocytosis, lipid rafts, and clathrin-independent endocytosis can help Exo to internalize into the target cell cytosol ( van Dongen et al., 2016 , Fitzner et al., 2011 ). In addition, the close interaction of exosomal ligands with host cell receptors can accelerate Exo entry. Therefore, both specific and non-specific mechanisms can manage exosomal internalization. Whether and how these mechanisms dominate is the subject of debate ( Kamerkar et al., 2017 ). The presence of specific peptides on the Exo surface can inhibit the direct ligand-receptor interaction, leading to delayed Exo entrance into the acceptor cells. It has been postulated that exosomal CD47, an integrin-associated protein, blocks direct phagocytosis of reaching Exo and increases circulation time ( Mathieu et al., 2019 ). In contrast to the activity of CD47, it was suggested that the existence of distinct superficial proteins like integrins, lectins/proteoglycans, T cell immunoglobulin, and mucin domain-containing protein 4 (Tim4) support timely internalization of Exo ( Kanada et al., 2015 ). Soon after Exo’s arrival, Exo cargo can be directly targeted for lysosomal degradation or released into the specific sites in the cytosol. However, there is an inconsistent hypothesis about the exact place of delivery. It has been thought that the formation of endosomes is the putative leading site for delivery of content, and membrane assimilation in response to acidic pH has been considered a feasible system comparable to the process used in some viruses ( Kanada et al., 2015 , Rezaie et al., 2019 ). Previous studies have revealed several therapeutic aspects of Exo under pathological conditions ( Rezaie et al., 2018 ) ( Table 1 ; Fig. 3 ). As mentioned above, these nanoparticles can be released by several cell types, especially stem cells, with the potential to manage NP ( Ren et al., 2019 ). In the context of CNS and PNS, mesenchymal stem cell (MSC)-derived Exo can exert analgesic effects in chronic pain models via the transfer of certain miRNAs ( Hmadcha et al., 2020 ). It is noteworthy that MSCs are the most common stem cell types used to regenerate injured tissue in the different animal models and human counterparts ( Martins and Schratt, 2021 ). It is believed that Exo are eligible to transfer therapeutic factors to injured neurons. These Exo can enhance axonal growth and neuronal viability ( Zhang et al., 2021 ). Besides genetic materials, the existence of several neurotrophic factors, including GDNF, IGF-1, BDNF, NGF, and FGF-1 in Exo lumen, can intensify therapeutic effects ( Tofiño-Vian et al., 2017 ). The promotion of inflammation is touted as the leading cause of pain. Exo can also suppress the production of pro-inflammatory cytokines such as IL-1β, −6, TNF-α, and PGE 2 in injured areas and stimulate the release of IL-10, leading to anti-nociceptive effects ( Cata et al., 2021 ). Table 1 Role of Exo in the alleviation of pain in the experimental settings. Model Year Exo source Target cells or tissues Outcome Ref In vitro model of osteoarthritis 2020 Bone marrow MSCs IL-1β-treated chondrocytes Chondrocyte Migration↑, Proliferation↑, COL2A1↑ and ACAN↑, ADAMTS5↓, MMP13↓ ( Console et al., 2019 ) In vivo rat model of osteoarthritis induced by sodium iodoacetate 2020 Bone marrow MSCs Knee joint Reduced neuropathic pain via CGRP↓ and iNOS↓, improved PWL values ( Console et al., 2019 ) Plasma exchange in patients with complex regional pain syndrome 2019 Plasma Exo miRNAs Blood hsa-miR-338-5p↓, IL-6↓, Inflammation↓ ( Shiue et al., 2019 ) Administration of alginate-loaded Exo in rats with mechanical allodynia and thermal hyperalgesia 2020 Human umbilical MSCs Right L5/6 spinal nerve Withdrawal threshold and latency↑, Fos↓, GFAP↓, Iba1↓, TNF-α↓ and IL-1β↓, GDNF↑, Antinociceptive properties↑, Inflammation↓ ( Zhang et al., 2019 ) Intrathecal injection of Exo in rats with nerve injury-induced neuropathic pain 2019 Human umbilical MSCs L5/6 spinal nerve Mechanical and thermal hypersensitivities↓, Pain↓, c-Fos↓, CNPase↓, GFAP↓, and Iba1↓, TNF-α↓ and IL-1β↓, IL-10↑, ( Zhou et al., 2021 ) Application of Exo in rat model of osteoarthritis 2019 MSCs Temporomandibular joint Pain↓, Degeneration↓, Inflammation↓, Subchondral bone formation↑, ( Jean-Toussaint et al., 2021 ) In vitro exposure of Exo with chondrocytes 2019 MSCs Chondrocytes Akt/Erk/AMPK↑, s-GAG synthesis↑, IL-1β↓, iNOS↓, MMP13↓ ( Jean-Toussaint et al., 2021 ) Different doses of Exo in a rat model of osteoarthritis induced by monoiodoacetate-induced 2021 MSCs knee joint PWT and PWL values↑, GAP-43↑, ATF-3↓, Dose-dependent regeneration activity↑, Pain relief↑ ( Simeoli et al., 2017 ) Macrophages Exo 2021 mouse RAW 264.7 macrophages-induced by lipopolysaccharide Cortical neurons, Microglia, and Astrocytes Pro-inflammatory miRNA↓ ( Moen et al., 2017 ) Acute mouse model of acute inflammation induced by formalin 2021 mouse RAW 264.7 macrophages-induced by lipopolysaccharide A single intrathecal injection Mechanical hyperalgesia↓, prophylactic pain relief↑ ( Moen et al., 2017 ) Dorsal root ganglia sensory neurons treated with capsaicin 2020 Exposure of macrophages to miR-21-5p antagomir-loaded Exo Cell culture Macrophage NOS2↓, Spry2↓, ( Simeoli et al., 2017 ) Enhanced spinal cord nociceptive responses in rat model using placing nucleus pulposus onto dorsal nerve roots (Vertebraes Th13-L1, and L3–S1) 2017 Nucleus pulposus grafts Exo Dorsal nerve roots miR-223↑, Nociceptive spinal signaling↓, ( Moen et al., 2017 ) Mesenchymal stem cells: MSCs; Aggrecan: ACAN; ADAM Metallopeptidase With Thrombospondin Type 1 Motif 5: ADAMTS5; Type II collagen: COL2A1; Matrix metalloproteinases-13: MMP13; Calcitonin Gene-Related Peptide: CGRP; Inducible nitric oxide synthase: iNOS; Paw withdrawal latency: PWL; Glial Cell Derived Neurotrophic Factor: GDNF; Ionized calcium binding adaptor molecule 1: Iba1; Fos Proto-Oncogene, AP-1 Transcription Factor Subunit: FOS; Glial fibrillary acidic protein: GFAP; Sulfated glycosaminoglycan: s-GAG; Growth Associated Protein 43: GAP-43; Cyclic AMP-dependent transcription factor: ATF-3; Nitric Oxide Synthase 2: NOS2; Sprouty RTK Signaling Antagonist 2: Spry2. Fig. 2 A cross-talk between endosomal-derived Exo biogenesis and the autophagy process in favor of pain management. Exo are synthesized via entering into the lumen of early endosomes and MVBs. Rab GTPase family (e.g., Rab-11, −27, −35) and SNARE are mainly involved in Exo development and membrane fusions, respectively. Final fusion of autophagosomes with MVBs can lead to the formation of amphisomes encompassing Exo-autophagic cargos, exiting the cell through exocytosis. Abbreviations: (SNARE) Exosomes: Exo; Multivesicular Bodies: MVBs; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptors: SNARE. Fig. 3 Modulatory effect of Exo on pain via different mechanisms. Role of Exo in the alleviation of pain in the experimental settings. Mesenchymal stem cells: MSCs; Aggrecan: ACAN; ADAM Metallopeptidase With Thrombospondin Type 1 Motif 5: ADAMTS5; Type II collagen: COL2A1; Matrix metalloproteinases-13: MMP13; Calcitonin Gene-Related Peptide: CGRP; Inducible nitric oxide synthase: iNOS; Paw withdrawal latency: PWL; Glial Cell Derived Neurotrophic Factor: GDNF; Ionized calcium binding adaptor molecule 1: Iba1; Fos Proto-Oncogene, AP-1 Transcription Factor Subunit: FOS; Glial fibrillary acidic protein: GFAP; Sulfated glycosaminoglycan: s-GAG; Growth Associated Protein 43: GAP-43; Cyclic AMP-dependent transcription factor: ATF-3; Nitric Oxide Synthase 2: NOS2; Sprouty RTK Signaling Antagonist 2: Spry2. A cross-talk between endosomal-derived Exo biogenesis and the autophagy process in favor of pain management. Exo are synthesized via entering into the lumen of early endosomes and MVBs. Rab GTPase family (e.g., Rab-11, −27, −35) and SNARE are mainly involved in Exo development and membrane fusions, respectively. Final fusion of autophagosomes with MVBs can lead to the formation of amphisomes encompassing Exo-autophagic cargos, exiting the cell through exocytosis. Abbreviations: (SNARE) Exosomes: Exo; Multivesicular Bodies: MVBs; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptors: SNARE. Modulatory effect of Exo on pain via different mechanisms. Another study suggested that miRNA-29-enriched Exo originated from stem cells and can alleviate pro-inflammatory responses in osteoarthritis in a rat model ( Jin et al., 2020 ). Xenogenic injection of human MSC Exo enriched with miR-26a-5p can reduce pathological changes via the down-regulation of cyclooxygenase-2 (PTGS2) in rat synovial fibroblasts ( Wu and Shen, 2020 ). Along with these changes, free radical and nitrosative stress production is ipsilaterally diminished by the regulation of inducible nitric oxide synthase activity in dorsal root ganglia ( Console et al., 2019 ). Another analgesic facet of Exo is associated with tightly controlled immune cell response and antigen presentation ( Wang et al., 2021 ). For example, it has been shown that Schwann cell-derived Exo can reduce neuropathic injuries of dorsal root ganglia via the control of neuronal sensitization and macrophage recruitment under pathological conditions ( Jin et al., 2020 ). Likewise, intrathecal injection of Exo originating from hypoxic neurons can diminish NP in a rat model of spinal cord injury via the suppression of IL-1β, −6, TNF-α, and NF-kB, leading to M1 to M2 polarization of microglia ( Liu et al., 2020 , Hsu et al., 2020 ). Based on previous data, sustained release and therapeutic effects of Exo were also investigated in a mouse model of L5/6 spinal nerve ligation. To this end, Jong-Ming and co-workers isolated Exo from human umbilical cord MSCs, loaded them in gelfoam, and implanted them into the vicinity of the injury site. They claimed that this approach could blunt the increase of TNF-α, IL-1β, GFAP, Iba1, and c-Fos coincided with the restoration of myelin formation and up-regulation of IL-10 ( Shiue et al., 2019 ). In a similar work, human umbilical cord MSCs Exo administration led to therapeutic effects in a rat model of nerve ligation–induced pain ( Degli Esposti et al., 2021 ). It was suggested that the transplanted Exo could distribute ipsilaterally in the L5 spinal dorsal horn and dorsal root ganglion and reach IB41 + , CGRP + , and NF200 + sensory neurons. Along with these changes, the expression of GFAP, Iba1, c-Fos, and CNPase was diminished ( Degli Esposti et al., 2021 ). These data show that Exo can manage pain by reducing pro-inflammatory cytokines and free radicals and promoting neuronal proliferation and function. Emerging data have shown that Exo can be used for early-stage monitoring of pain following several pathologies ( Rezaie et al., 2018 ). Of note, the type of parent cells, epigenetic changes, and insulting conditions can change exosomal cargo ( D’Agnelli et al., 2020 ). Regarding synaptic activity in the nervous system, Exo can be interchanged between neurons and participate in inter-neuronal communication ( Ramanathan et al., 2019 ). It was suggested that the existence of pain-related factors in the Exo lumen is indicative of pathological conditions ( Ramanathan et al., 2019 ). These features help us to detect the type and intensity of pain via monitoring the exosomal cargo, indicating the diagnostic and prognostic values of Exo ( Orlova et al., 2011 ). To be specific, the molecular signature in Exo secreted during the injury of different tissues can be differed. For instance, in complex regional pain syndrome (CRPS) patients Exo carry large amounts of miR-338-5p, indicating pathological conditions ( Kowalski et al., 2022 ). It was suggested that exosomal miR-338-5p could modulate NP after spinal cord injury via the regulation of apoptosis and neuroinflammation ( Freger et al., 2021 ). Molecular analyses have revealed a close relationship between exosomal miRNA and the production of pro-inflammatory cytokines such as IL-6 in response to pathological conditions ( Orlova et al., 2011 ). The occurrence of pathological conditions can result in specific cargo sorting and enrichment with unique signaling molecules. For example, Freger and collaborators demonstrated that in females with endometriosis and pelvic pain, circulating Exo harbor specific long non-coding RNAs (lncRNAs) and proteins that can potentially regulate neurogenesis, and angiogenesis, immune system response, and histone modification ( D'Agnelli et al., 2020 ). These features show that Exo are the key regulators during physiological and pathological conditions with specific factors that give us valuable data about the status and intensity of pathologies. Because Exo can easily distribute in the systemic circulation, studying exosomal profiles in blood samples is helpful in the determination of pain-related factors ( Heidarzadeh et al., 2021 ). Despite the existence of a blood–brain barrier with selective permeability, Exo can engage several strategies to cross this natural barrier from the brain to the blood side and vice versa ( van Dijk et al., 2010 ). Besides, the production of several inflammatory cytokines can deteriorate endothelial barrier integrity and lead to the easier transfer of inflammatory Exo into the circulation system, resulting in the dissemination of Exo from original cells to remote sites ( van Dijk et al., 2010 ). Unlike blood, the collection of Exo from CSF during different CNS pathologies is touted as an invasive approach. Despite this disadvantage, the proximity of CSF to the brain and spinal cord enables us to directly monitor possible molecular alteration and signature ( Pegtel et al., 2014 ). Exo with distinct markers can be produced by a wide range of cells within the nervous system and released into the CSF ( Hornung et al., 2020 ). Therefore, molecular analysis of the Exo proteome and genome can give us valuable diagnostic and prognostic data about various neurodegenerative conditions ( Hercher et al., 2021 ). Like CNS, the occurrence of pain in PNS can contribute to the production of Exo, which are identical to the status of pain and inflammatory response ( Gurunathan et al., 2019 ). In animal models of neuritis, Schwann cells produce and release Exo carrying neural cell adhesion molecules 1 and P75, which can show degenerative conditions related to axonal demyelination ( Leoni et al., 2015 ). These factors are increased in human counterparts with several peripheral neuropathy types ( Leoni et al., 2015 ). In the circumstances associated with chronic pain conditions such as osteoarthritis and other diseases, Exo with large contents of Annexin A-1 can be detected in the blood ( Descalzi et al., 2015 ). It is suggested that Annexin A-1 acts as an anti-nociceptive agent with pro-inflammatory potential ( Descalzi et al., 2015 ). These features demonstrate that Exo are eligible biological nanovesicles that can reflect pathological changes and the pain intensity in different tissue types.

Epigenetic changes in the spinal cord and peripheral nerves have been introduced during chronic pain, which may guide substantial advances in novel therapeutic interventions. In this regard, autophagy can also be an aid for exploring the effects of experimental therapeutic options targeted at epigenetic mechanisms ( Jung and Lim, 2015 ). Moreover, previous literature proposed a correlation between the activation of microRNAs (miRNAs, potent nano-sized regulators of gene expression) and the autophagic flux in the terms of NP ( Shi et al., 2013 ). The underlying mechanisms involved in the modulation of autophagy via epigenetic changes following chronic pain in the brain and spinal cord are yet to be completely deciphered. However, miRNAs are involved in the autophagic regulation in the case of NP ( Shi et al., 2013 ). For instance, the miRNA-195 simultaneously stimulates neuroinflammation and NP via inhibiting autophagy flux following peripheral nerve injury ( Jung and Lim, 2015 , Xu et al., 2018 ). Emerging data have indicated varied intracellular effectors shared between Exo biogenesis and autophagy machinery ( Rahbarghazi et al., 2021 ). In Fig. 2 , a presumed cross-talk between autophagy and Exo biogenesis has been depicted to exert a therapeutic potential in the face of pain. As mentioned above, ATGs are the main signaling molecules regulating autophagic procedures. Of these ATGs, ATG5 promotes the fusion of MVBs with the plasma membrane and the release of ILVs ( Murrow et al., 2015 ). On the other hand, the activation of LC3, a well-known autophagy biomarker, can stimulate Alix from the ESCRT complex ( Villarroya-Beltri et al., 2016 ). Further fusion of autophagosomes with MVBs can also lead to the formation of amphisomes. These intracellular vesicles harbor the content of autophagy machinery and MVBs to ECM via engaging certain Rabs such as Rab8a and Rab27 ( Murrow et al., 2015 , Kuo et al., 2021 ). Alternatively, amphisomes join the lysosome for further digestion and degradation ( Murrow et al., 2015 ). By 2021, increasing evidence has designated a close link between Exo secretion and autophagy flux in the case of pain management, declaring a new field of research for advanced cell therapy in clinics. For example, following the sciatic nerve crush injury model, a novel combination therapy consisting of adipose-derived stem cells-derived Exo (ADSC-F-Exo) and immunosuppressive drug tacrolimus (FK506) was used ( Pan et al., 2021 ). The results showed that local administration of ADSC-F-Exo could alleviate the aberrant autophagy process in the DRG and spinal cord dorsal horn after nerve crush injury ( Pan et al., 2021 ). In addition, a proteome analysis revealed the presence of 22 exosomal proteins, heat shock protein family A member 8 (HSPA8), and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) in ADSC-derived Exo ( Pan et al., 2021 ). Pan et al. declared that the application of primary Schwann cell-derived Exo (SCDEs) promoted autophagy and inhibited apoptosis to retrieve spinal cord injury and axonal/motor functions by regulation of epidermal growth factor receptor (EGFR)/Akt/mTOR signaling pathway ( Luo et al., 2021 ). Intervertebral disc degeneration (IDD) is a challenging clinical condition causing back pain and subsequently affects a patient’s quality of life. In this regard, degeneration of the cartilage endplate plays a crucial role in IDD induction. Upon the promotion of IDD, certain factors such as matrix metalloproteinases (MMPs) and proinflammatory cytokines are up-regulated. These features coincide with decreased numbers of functional nucleus pulposus cells (NPCs) and prominent morphological changes. It has been indicated that the Exo-autophagy interface has therapeutic potential in the context of IDD ( Zhang et al., 2021 ). Recently, the potential impacts of normal and degenerated cartilage endplate stem cell-derived Exo (CESC-Exo) on autophagy activation were investigated. Luo et al. claimed that normal CESC-Exo is more effective in modulating autophagy flux and NPC apoptosis rate. Moreover, they highlighted that PI3K/AKT/autophagy axis has a pivotal role in retrieving CEP-induced IDD ( Zhang et al., 2021 ). In parallel with these findings, it has also been proved that autophagy can mitigate the MMPs activity and inflammatory responses to prevent NPCs matrix degradation ( Liu et al., 2021 ). In detail, autophagy-activated NPCs-derived Exo (NPCs-Exo) could recover IDD at least partly by exosomal miR-27 recruitment and targeting MMP-13 ( Liu et al., 2021 ). Long non-coding RNAs (lncRNAs) belong to regulatory ncRNAs that mainly participate in DPN development. Recent research conducted by Liu et al. reported a favorable role of lncRNA X-inactive specific transcript (XIST) in attenuating DPN in diabetic mice ( Ramanathan et al., 2019 ). Data showed that hyperglycemia triggered a significant suppression in the expression of XIST, sirtuin1 (SIRT1), LC3II, and Beclin-1, while the expression of microRNA-30d-5p (miR-30d-5p) was increased in the trigeminal sensory neurons which reversed by XIST administration ( Ramanathan et al., 2019 ).

Primary afferent nociceptors (PAN) are touted as the first structures involved in the perception of chemical, mechanical, and thermal stimuli. PAN are peripheral nerve fibers originating from pseudounipolar sensory neurons that locate in the trigeminal ganglion in the face and the dorsal root ganglion (DRG) in the body ( Wang et al., 2010 ) ( Fig. 1 ). Fig. 1 Simplified mechanism of pain perception via ascending and descending pathways in peripheral nerves and spinal cord. Simplified mechanism of pain perception via ascending and descending pathways in peripheral nerves and spinal cord. Molecular investigations indicated that large amino acid transporter 1 (LAT1) is highly expressed in the DRG region following the spinal cord injury. These data support the crucial role of LAT1/4F2hc/Wnt/frizzled/β-catenin signal transduction pathway in the pain process ( Alles et al., 2020 ). Of note, voltage-gated potassium channels (Kv1.1 and Kv1.2), the voltage-gated sodium channel (Nav1.7), and to a lesser extent, voltage-activated calcium channel auxiliary subunit α2δ-1 are also involved in the pain process. Besides, LAT1 has the potential to stimulate the mTOR signaling pathway, particularly in the inflammatory responses and NP ( Alles et al., 2020 ). In a recently published study, it was suggested that autophagy and apoptosis, as adaptive mechanisms, can interact through various proinflammatory cytokine regulations when peripheral neuropathy occurs. In this era, proinflammatory cytokines can encourage autophagic/apoptotic function and subsequent NP formation. Autophagy per se quenches the proinflammatory cytokine activities and further modulates pain behavior ( Liao et al., 2022 ). Mounting evidence highlighted the role of neurogenic inflammation and neuroinflammation in developing pain patterns ( Matsuda et al., 2019 ). Inflammatory mediators, including prostaglandins (PGs), pro-inflammatory cytokines, and chemokines are elevated in response to noxious stimuli and concurrently persuade pain perception via direct stimulation of primary sensory neurons, namely nociceptors ( Matsuda et al., 2019 ). The promotion of cytokines can per se stimulate cytokine receptors, resulting in modified nociceptive signaling and neuronal activity. Along with these changes, excitatory synaptic transmission is induced while the basal inhibitory synaptic transmission is prohibited ( Vanderwall and Milligan, 2019 ). During several pathological conditions, specific cytokines such as TNF-α and other interleukins are frequently expressed at the injury site. TNF-α could intensify spontaneous excitatory post-synaptic activity via the elevation of excitatory α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA. Along with these changes, IL-6 blunts spontaneous inhibitory post-synaptic action via the suppression of GABA and glycine ( Kawasaki et al., 2008 ). Interestingly, the increase of endogenous pyrogen namely IL-1 can bilaterally mediate these features ( Kawasaki et al., 2008 ). Besides the overactivity of neurons during the inflammatory conditions, the critical role of glial cells should not be neglected in the context of pain perception. Given the highly intricate interaction of glial cells such as microglia and astrocytes within the nervous system, it is logical to hypothesize that these cells actively partake in the modulation of painful conditions. In support of this notion, it has been shown that these cells are juxtaposed to neural synapses and eligible to express several types of neurotransmitter receptors as described in neurons ( Matejuk and Ransohoff, 2020 ). For instance, astrocytes and microglia can express neurokinin 1, glutamate, AMPA, and purinergic receptors in presynaptic terminals. The activation of astrocytes and microglia occurs in the shortest time after the exposure to chemokines, colony-stimulating factor- 1, ATP, and substance P. In response to these conditions, the production of specific factors such as IL-1, −6, TNF-α, NO, chemokines, ATP, and other excitatory factors are increased ( Zhang et al., 2017 ). Holmes and co-workers found that the modulation of primary motor and somatosensory areas, changes in nerve divisions, and overexpression of cytokines involved in pain perception of ankle injury in human ( Holmes et al., 2020 ). In this regard, neurogenic inflammation applies catastrophic effects by neuropeptide release with rapid plasma extravasation. On the other hand, neuroinflammation can activate the glial cells located in the DRG or spinal ganglion to propagate pro-inflammatory cytokines and chemokines in both the peripheral nervous system (PNS) and CNS ( Matsuda et al., 2019 ). Of course, extravasation is indicated by the unilateral leakage of plasma factors into the injured tissues, resulting in local pain and swelling. Therefore, NP is the main source of pain markers, but in the later steps, the involvement of supporting vessels can increase the intensity of the pain ( Kim et al., 2020 ).

Exo secretion from glial cells such as astrocytes and microglia seems to be a critical event in the pathogenesis of pain. As a correlate, these nanoparticles can exchange different biomolecule types between the cells and dictate specific behavior in recipient cells such as neurons. However, there is a long way to underpin the underlying mechanisms behind exosomal delivery of certain factors and pain modulation in several pathologies. It is suggested that autophagy can affect the exocytosis capacity and thus alter the distribution of certain biomolecules within the CNS and PNS via autophagic response. Whether and how autophagy can change exosomal cargo and biogenesis during acute and chronic pain is the subject of debate. It is suggested that different therapeutic agents and compounds can be loaded onto the Exo using different engineering strategies to alleviate the pain. Due to close cross-talk between autophagy and Exo biogenesis, the modulation of these pathways can be touted as an alternative approach after the onset of pain. Despite these availabilities, numerous studies are needed to establish an optimized approach to managing acute and chronic pain.

The occurrence of pain, especially chronic type with limited therapeutic options, is relatively common among individuals ( Hylands-White et al., 2017 , Golzari et al., 2014 ). Pain is generally classified into acute and chronic forms in terms of duration ( Soleimanpour et al., 2022 , Ghojazadeh et al., 2019 ). Acute pain can last for a short period and accompanies anxiety and distress while the latter type is associated with a long-term painful condition because of nerve injury ( Glare et al., 2020 , Ghojazadeh et al., 2019 , Dolati et al., 2020 ). It is believed that pain is a multidimensional phenomenon and occurs because of an injury to the nervous system (neuropathic pain; NP) or tissue damage (nociceptive pain) ( Hylands-White et al., 2017 ). Irrespective of the pain type, this phenomenon is regulated by engaging several cellular and molecular mechanisms ( Cho and Huh, 2020 ). Changes in the activity of glia and neuronal lineages, and delivery of several cytokines within the nervous system can contribute to neuropathic effects ( Oh et al., 2018 ). The increase of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1) stimulates phenotype shifting in astrocytes from A2 to A1 types, which per se causes chronic pain. By contrast, astrocyte phenotypic switching from A1 to A2 reduces chronic pain. In response to inflammatory conditions, the increase of glial fibrillary acidic protein (GFAP), a well-known astrocyte hallmark, leads to NP generation ( Li et al., 2019 ). Upon the stimulation of astrocytes, the activation of microglia results in the local release of several biomolecules with a protective potential for neurons and glial cells ( Salaffi et al., 2018 ). Exosomes (Exo) are a subclass of extracellular vesicles (EVs) released from several cell types and participate in paracrine interaction between the cells ( Amini et al., 2021 ). Regarding their unique size ranging from 40 to 200 nm, Exo can easily distribute in biofluids and transfer signaling biomolecules such as proteins, lipids, and nucleic acids from donor cells to the recipient cells. In line with these statements, it is thought that Exo are key mediators to modulate biochemical reactions and cellular activity during physiological and pathological conditions ( Rahbarghazi et al., 2021 ). Of note, the critical roles of Exo on specific pathological conditions like neurodegenerative disease, inflammatory responses, osteoarthritis, etc., have been shown previously ( Lin et al., 2020 ). The reciprocal Exo exchange between the cells within the central nervous system (CNS) and peripheral nervous system (PNS) makes these nanoparticles a novel therapeutic modality ( Bahlakeh et al., 2021 ). Regarding the ability of Exo to transfer specific cargo, these particles can be used to retrieve several pathological conditions ( Ni et al., 2020 ). From a biological viewpoint, Exo are originated from the endosomal system consisting of several regulatory mechanisms ( Rahbarghazi et al., 2021 ). Based on numerous studies, there is synergy between Exo biogenesis and other secretory pathways like autophagy machinery, as an early-stage mechanism activated in the host cells to turnover damaged materials ( Colletti et al., 2021 , Hassanpour et al., 2020 ). It is also suggested that the close crosstalk between the autophagy-lysosomal pathway and Exo biogenesis is an adaptive response to further preserve cellular hemostasis ( Rahbarghazi et al., 2021 ). Here, we aimed to highlight the synergy of Exo-autophagy in alleviating pathological conditions and reducing pain within the nervous system.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.