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Advanced oxidation protein products sensitized the transient receptor potential vanilloid 1 via NADPH oxidase 1 and 4 to cause mechanical hyperalgesia

View Article: PubMed Central - PubMed

ABSTRACT

Oxidative stress is a possible pathogenesis of hyperalgesia. Advanced oxidation protein products (AOPPs), a new family of oxidized protein compounds, have been considered as a novel marker of oxidative stress. However, the role of AOPPs in the mechanism of hyperalgesia remains unknown. Our study aims to investigate whether AOPPs have an effect on hyperalgesia and the possible underlying mechanisms. To identify the AOPPs involved, we induced hyperalgesia in rats by injecting complete Freund’s adjuvant (CFA) in hindpaw. The level of plasma AOPPs in CFA-induced rats was 1.6-fold in comparison with what in normal rats (P<0.05). After intravenous injection of AOPPs-modified rat serum albumin (AOPPs-RSA) in Sprague-Dawley rats, the paw mechanical thresholds, measured by the electronic von Frey system, significantly declined. Immunofluorescence staining indicated that AOPPs increased expressions of NADPH oxidase 1 (Nox1), NADPH oxidase 4 (Nox4), transient receptor potential vanilloid 1 (TRPV1) and calcitonin gene-related peptide (CGRP) in the dorsal root ganglia (DRG) tissues. In-vitro studies were performed on primary DRG neurons which were obtained from both thoracic and lumbar DRG of rats. Results indicated that AOPPs triggered reactive oxygen species (ROS) production in DRG neurons, which were significantly abolished by ROS scavenger N-acetyl-l-cysteine (NAC) and small-interfering RNA (siRNA) silencing of Nox1 or Nox4. The expressions of Nox1, Nox4, TRPV1 and CGRP were significantly increased in AOPPs-induced DRG neurons. And relevant siRNA or inhibitors notably suppressed the expressions of these proteins and the calcium influxes in AOPPs-induced DRG neurons. In conclusion, AOPPs increased significantly in CFA-induced hyperalgesia rats and they activated Nox1/Nox4-ROS to sensitize TRPV1-dependent Ca2+ influx and CGRP release which led to inducing mechanical hyperalgesia.

No MeSH data available.


The expression of related proteins in DRG neurons. (A–D) The levels of TRPV1 and CGRP were up-regulated significantly in DRG neurons incubated with AOPPs-RSA in indicated concentrations and time durations. (E) DRG neurons were incubated with AOPPs-RSA (200 μg/mL) with or without pretreatment with Nox1 siRNA, Nox4 siRNA, NAC (2 mM), or SB366791 (1 μM) for 6 h. The expression of TRPV1 and CGRP could be trigged by AOPPs, meanwhile, inhibited by these siRNAs or inhibitors. Data represent mean±SEM of at least 3 independent experiments. *P<0.05 versus Control (0) group. # P<0.05 versus AOPPs-RSA group.
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f0030: The expression of related proteins in DRG neurons. (A–D) The levels of TRPV1 and CGRP were up-regulated significantly in DRG neurons incubated with AOPPs-RSA in indicated concentrations and time durations. (E) DRG neurons were incubated with AOPPs-RSA (200 μg/mL) with or without pretreatment with Nox1 siRNA, Nox4 siRNA, NAC (2 mM), or SB366791 (1 μM) for 6 h. The expression of TRPV1 and CGRP could be trigged by AOPPs, meanwhile, inhibited by these siRNAs or inhibitors. Data represent mean±SEM of at least 3 independent experiments. *P<0.05 versus Control (0) group. # P<0.05 versus AOPPs-RSA group.

Mentions: Western blot assay was used to verify the AOPPs-Nox1/Nox4-ROS-TRPV1-CGRP pathway. We stimulated DRG neurons with different concentrations of AOPPs-RSA (50, 100 and 200 μg/mL) for 6 h. The results showed that 50 μg/mL AOPPs-RSA could significantly activate TRPV1, and the expression level of TRPV1 was elevated with the increasing concentration of AOPPs-RSA (Fig. 6A). However, the expression of CGRP did not increase until the concentration of AOPPs-RSA reached 100 μg/mL (Fig. 6C). In addition, DRG neurons were stimulated by 200 μg/mL AOPPs-RSA for different time durations (0, 15, 30, 60, 120 and 360 min). The expression of TRPV1 was up-regulated at least ~3-fold at 30 min in comparison with that at 0 min (Fig. 6B). The level of CGRP was increased close to 1.4-fold after 60 min (Fig. 6D)..


Advanced oxidation protein products sensitized the transient receptor potential vanilloid 1 via NADPH oxidase 1 and 4 to cause mechanical hyperalgesia
The expression of related proteins in DRG neurons. (A–D) The levels of TRPV1 and CGRP were up-regulated significantly in DRG neurons incubated with AOPPs-RSA in indicated concentrations and time durations. (E) DRG neurons were incubated with AOPPs-RSA (200 μg/mL) with or without pretreatment with Nox1 siRNA, Nox4 siRNA, NAC (2 mM), or SB366791 (1 μM) for 6 h. The expression of TRPV1 and CGRP could be trigged by AOPPs, meanwhile, inhibited by these siRNAs or inhibitors. Data represent mean±SEM of at least 3 independent experiments. *P<0.05 versus Control (0) group. # P<0.05 versus AOPPs-RSA group.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037245&req=5

f0030: The expression of related proteins in DRG neurons. (A–D) The levels of TRPV1 and CGRP were up-regulated significantly in DRG neurons incubated with AOPPs-RSA in indicated concentrations and time durations. (E) DRG neurons were incubated with AOPPs-RSA (200 μg/mL) with or without pretreatment with Nox1 siRNA, Nox4 siRNA, NAC (2 mM), or SB366791 (1 μM) for 6 h. The expression of TRPV1 and CGRP could be trigged by AOPPs, meanwhile, inhibited by these siRNAs or inhibitors. Data represent mean±SEM of at least 3 independent experiments. *P<0.05 versus Control (0) group. # P<0.05 versus AOPPs-RSA group.
Mentions: Western blot assay was used to verify the AOPPs-Nox1/Nox4-ROS-TRPV1-CGRP pathway. We stimulated DRG neurons with different concentrations of AOPPs-RSA (50, 100 and 200 μg/mL) for 6 h. The results showed that 50 μg/mL AOPPs-RSA could significantly activate TRPV1, and the expression level of TRPV1 was elevated with the increasing concentration of AOPPs-RSA (Fig. 6A). However, the expression of CGRP did not increase until the concentration of AOPPs-RSA reached 100 μg/mL (Fig. 6C). In addition, DRG neurons were stimulated by 200 μg/mL AOPPs-RSA for different time durations (0, 15, 30, 60, 120 and 360 min). The expression of TRPV1 was up-regulated at least ~3-fold at 30 min in comparison with that at 0 min (Fig. 6B). The level of CGRP was increased close to 1.4-fold after 60 min (Fig. 6D)..

View Article: PubMed Central - PubMed

ABSTRACT

Oxidative stress is a possible pathogenesis of hyperalgesia. Advanced oxidation protein products (AOPPs), a new family of oxidized protein compounds, have been considered as a novel marker of oxidative stress. However, the role of AOPPs in the mechanism of hyperalgesia remains unknown. Our study aims to investigate whether AOPPs have an effect on hyperalgesia and the possible underlying mechanisms. To identify the AOPPs involved, we induced hyperalgesia in rats by injecting complete Freund&rsquo;s adjuvant (CFA) in hindpaw. The level of plasma AOPPs in CFA-induced rats was 1.6-fold in comparison with what in normal rats (P&lt;0.05). After intravenous injection of AOPPs-modified rat serum albumin (AOPPs-RSA) in Sprague-Dawley rats, the paw mechanical thresholds, measured by the electronic von Frey system, significantly declined. Immunofluorescence staining indicated that AOPPs increased expressions of NADPH oxidase 1 (Nox1), NADPH oxidase 4 (Nox4), transient receptor potential vanilloid 1 (TRPV1) and calcitonin gene-related peptide (CGRP) in the dorsal root ganglia (DRG) tissues. In-vitro studies were performed on primary DRG neurons which were obtained from both thoracic and lumbar DRG of rats. Results indicated that AOPPs triggered reactive oxygen species (ROS) production in DRG neurons, which were significantly abolished by ROS scavenger N-acetyl-l-cysteine (NAC) and small-interfering RNA (siRNA) silencing of Nox1 or Nox4. The expressions of Nox1, Nox4, TRPV1 and CGRP were significantly increased in AOPPs-induced DRG neurons. And relevant siRNA or inhibitors notably suppressed the expressions of these proteins and the calcium influxes in AOPPs-induced DRG neurons. In conclusion, AOPPs increased significantly in CFA-induced hyperalgesia rats and they activated Nox1/Nox4-ROS to sensitize TRPV1-dependent Ca2+ influx and CGRP release which led to inducing mechanical hyperalgesia.

No MeSH data available.