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. 2023 Feb 15;29(1):22.
doi: 10.1186/s10020-023-00606-9.

AAV-glycine receptor α3 alleviates CFA-induced inflammatory pain by downregulating ERK phosphorylation and proinflammatory cytokine expression in SD rats

Hung-Chen Wang  1 , Kuang-I Cheng  2   3 , Kuang-Yi Tseng  2   3 , Aij-Lie Kwan  4 , Lin-Li Chang  5   6   7   8   9
Affiliations

AAV-glycine receptor α3 alleviates CFA-induced inflammatory pain by downregulating ERK phosphorylation and proinflammatory cytokine expression in SD rats

Hung-Chen Wang et al. Mol Med. .

Abstract

Background: Glycine receptors (GlyRs) play key roles in the processing of inflammatory pain. The use of adeno-associated virus (AAV) vectors for gene therapy in human clinical trials has shown promise, as AAV generally causes a very mild immune response and long-term gene transfer, and there have been no reports of disease. Therefore, we used AAV for GlyRα1/3 gene transfer in F11 neuron cells and into Sprague-Dawley (SD) rats to investigate the effects and roles of AAV-GlyRα1/3 on cell cytotoxicity and inflammatory response.

Methods: In vitro experiments were performed using plasmid adeno-associated virus (pAAV)-GlyRα1/3-transfected F11 neurons to investigate the effects of pAAV-GlyRα1/3 on cell cytotoxicity and the prostaglandin E2 (PGE2)-mediated inflammatory response. In vivo experiment, the association between GlyRα3 and inflammatory pain was analyzed in normal rats after AAV-GlyRα3 intrathecal injection and after complete Freund's adjuvant (CFA) intraplantar administration. Intrathecal AAV-GlyRα3 delivery into SD rats was evaluated in terms of its potential for alleviating CFA-induced inflammatory pain.

Results: The activation of mitogen-activated protein kinase (MAPK) inflammatory signaling and neuronal injury marker activating transcription factor 3 (ATF-3) were evaluated by western blotting and immunofluorescence; the level of cytokine expression was measured by ELISA. The results showed that pAAV/pAAV-GlyRα1/3 transfection into F11 cells did not significantly reduce cell viability or induce extracellular signal-regulated kinase (ERK) phosphorylation or ATF-3 activation. PGE2-induced ERK phosphorylation in F11 cells was repressed by the expression of pAAV-GlyRα3 and administration of an EP2 inhibitor, GlyRαs antagonist (strychnine), and a protein kinase C inhibitor. Additionally, intrathecal AAV-GlyRα3 administration to SD rats significantly decreased CFA-induced inflammatory pain and suppressed CFA-induced ERK phosphorylation, did not induce obvious histopathological injury but increased ATF-3 activation in dorsal root ganglion (DRGs).

Conclusions: Antagonists of the prostaglandin EP2 receptor, PKC, and glycine receptor can inhibit PGE2-induced ERK phosphorylation. Intrathecal AAV-GlyRα3 administration to SD rats significantly decreased CFA-induced inflammatory pain and suppressed CFA-induced ERK phosphorylation, did not significantly induce gross histopathological injury but elicited ATF-3 activation. We suggest that PGE2-induced ERK phosphorylation can be modulated by GlyRα3, and AAV-GlyRα3 significantly downregulated CFA-induced cytokine activation.

Keywords: Adeno-associated virus; Extracellular signal-regulated kinase (ERK) phosphorylation; Glycine receptors; Inflammatory pain; Prostaglandin E2.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Effect of PGE2 and pAAV/pAAV-GlyR1/3 on ERK phosphorylation and ATF-3 activation in F11 cells. A Time schedule of PGE2 (100 µM) administration and the time when cells were collected to measure ERK phosphorylation and ATF-3 activation. F11 cells were seeded into 6-well plates, and 24 h later, PGE2 was applied for 5, 15, 30 or 60 min. Finally, these treated cells were harvested for protein extraction. B Western blot images and quantitative evaluation of C p-ERK and D ATF-3 activation in PGE2-treated F11 cells. E Time schedule of F11 cells transfected with 2 μg pAAV, pAAV-GlyRα1 or pAAV-GlyRα3 for 48 h. Then, cell pellets were harvested for protein extraction. F Images of western blots and quantitative evaluation of G p-ERK and H ATF3 expression in pAAV-, pAAV-GlyR1 or pAAV-GlyRα3-transfected F11 cells are shown. I Time schedule to evaluate the effect of pAAV, pAAV-GlyRα1/3 transfection on PGE2 (100 µM)-induced ERK phosphorylation in F11 cells. F11 cells were transfected with 2 μg pAAV, pAAV-GlyRα1 or pAAV-GlyRα3 for 48 h. Serum-free medium replaced the initial medium and was incubated for another 24 h, and the cells were then treated with PGE2 for 60 min. The cells were harvested for J western blotting of phosphorylated ERK, and K quantification of phosphorylated ERK in F11 cells is shown. F11 cells without vector transfection or PGE2 treatment were used as controls. All the data are expressed as the fold change measured in from three to five independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA
Fig. 2
Fig. 2
Effects of prostaglandin EP2-receptor antagonist (PF-04418948), glycine-receptor antagonist (strychnine), and PKC inhibitor (G06983) on PGE2-induced ERK phosphorylation. A Time schedule in which F11 cells were seeded into a 6-well plate for 24 h; PF-04418948 (10 μM) or strychnine (10 μM) was applied and incubated for 24 h before PGE2 treatment. G06983 (3 μM) was applied 30 min before PGE2 treatment. One hour after PGE2 (100 μM) application, cells were harvested for protein extraction. B Western blotting of ERK phosphorylation and C quantification of ERK phosphorylation in F11 cells. F11 cells without inhibitors or PGE2 treatment were used as a control. The data are presented on the basis of from three to five independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA
Fig. 3
Fig. 3
Immunofluorescence images of AAV-GlyRα3, and ATF-3 expression in the DRG. A Expression of green fluorescent protein (GFP) in L5 DRGs was detected by immunofluorescence six to eight weeks after intrathecal AAV-GlyRα3 (2.5 × 1012 vg) injection. Double immunofluorescence staining showing GlyRα3 and NeuN co-localization in the L5 DRG. Positive for GlyRα3 are shown in green, positive for NeuN are shown in blue, merge images of GlyRα3 and neuron are indicated in peacock blue. A injury assessment by ATF-3 expression of DRG tissue sections from CFA (100 μl) subcutaneous hind paw injection. Group intrathecal NaCl plus CFA injection (GNF), group intrathecal AAV (2.5 × 1012 vg) plus CFA injection (GVF), and group intrathecal AAV-GlyRα3 (2.5 × 1012 vg) plus CFA injection (Gα3F) are compared. ATF-3 expression in the DRG was measured by B immunofluorescence and C western blotting. The data represent the means ± SE (n = 6–9 per group). One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. ATF-3 indicates activating transcription factor; scale bars: 250 μm
Fig. 4
Fig. 4
Behavioral responses to thermal and mechanical stimuli on hind paws. The hind paw withdrawal response to A thermal hyperalgesia and B mechanical allodynia was assessed at day 0 (baseline); weekly for two months after intrathecal injection of AAV-GlyRα3 (2.5 × 1012 vg), AAV (2.5 × 1012 vg), or NaCl; and daily for four days after CFA (100 μl) injection. Mann–Whitney U test, **p < 0.01, ***p < 0.001 (used in comparison between GNF and Gα3F groups). #p < 0.05, ##p < 0.01, ###p < 0.001 (used in comparison between GVF and Gα3F groups); D, days
Fig. 5
Fig. 5
Effects of AAV-GlyRα3 on ERK and p38 phosphorylation in CFA‑treated rats. Intrathecal NaCl, AAV (2.5 × 1012 vg), or AAV-GlyRα3 (2.5 × 1012 vg) injection 8 weeks later CFA (100 μg/100 μl) was given through hind paw injection. After 2 days of CFA treatment, L5 DRGs were collected to evaluate ERK and p38 phosphorylation. By A immunofluorescence, B western blotting and quantification, a magnification image showing the induction of ERK phosphorylation in the GNF and GVF groups. CFA induces sustained activation of ERK, which was repressed in the Gα3F group. Double immunofluorescence labeling of C p-ERK with NeuN and D p-ERK with GFAP indicated colocalization of p-ERK with NeuN in neurons and satellite glial cell in the DRG. E Immunofluorescence imaging revealed p38 phosphorylation in the GNF, GVF and Gα3F groups. Double immunofluorescence labeling of F p-p38 with NeuN and G p-p38 with NF200 revealed p-p38 expression in small neurons. The data represent the means ± SE (n = 6–9 per group). One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 50–100 μm
Fig. 6
Fig. 6
Effects of AAV-GlyRα3 on the expression level of cytokines in the L5 DRGs of CFA‑treated rats. For this experiment, CFA (100 μg/100 μl) hind paw injection was administered 8 weeks after the intrathecal injection of AAV-GlyRα3 (2.5 × 1012 vg). After 2 days of CFA treatment, L5 DRGs (25 μg/100 μl) were collected to evaluate cytokines expression. By ELISAs, the expression of A TNF-α, B IL-1β and C IL-6 was determined. The results are expressed as the means ± SE, and the data shown represent from three to five independent experiments. Mann–Whitney U test was used. *p < 0.05, **p < 0.01, ***p < 0.001
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References

    1. Ahmadi S, Lippross S, Neuhuber WL, Zeilhofer HU. PGE(2) selectively blocks inhibitory glycinergic neurotransmission onto rat superficial dorsal horn neurons. Nat Neurosci. 2002;5(1):34–40. doi: 10.1038/nn778. - DOI - PubMed
    1. Akhilesh, Baidya ATK, Uniyal A, Das B, Kumar R, Tiwari V. Structure-based virtual screening and molecular dynamics simulation for the identification of sphingosine kinase-2 inhibitors as potential analgesics. J Biomol Struct Dyn. 2022;40(23):12472–12490. doi: 10.1080/07391102.2021.1971559. - DOI - PubMed
    1. Akhilesh, Uniyal A, Gadepalli A, Tiwari V, Allani M, Chouhan D, et al. Unlocking the potential of TRPV1 based siRNA therapeutics for the treatment of chemotherapy-induced neuropathic pain. Life Sci. 2022;288:120187. doi: 10.1016/j.lfs.2021.120187. - DOI - PubMed
    1. Aley KO, Messing RO, Mochly-Rosen D, Levine JD. Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C. J Neurosci. 2000;20(12):4680–4685. doi: 10.1523/JNEUROSCI.20-12-04680.2000. - DOI - PMC - PubMed
    1. Aubrey KR, Supplisson S. Heterogeneous signaling at GABA and glycine co-releasing terminals. Front Synaptic Neurosci. 2018;10:40. doi: 10.3389/fnsyn.2018.00040. - DOI - PMC - PubMed

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