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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 NADPH oxidase in DRG neurons. (A, C) The levels of Nox1 and Nox4 were up-regulated significantly in DRG neurons incubated with AOPPs-RSA in indicated concentrations. (B) Indicated concentrations of AOPPs-RSA could not significantly up-regulate the expression of Nox2. (D, F) The levels of Nox1 and Nox4 were up-regulated significantly in DRG neurons incubated with 200 μg/mL AOPPs-RSA in different time durations. (E) The expression of Nox2 had no significant difference between indicated time durations in 200 μg/mL AOPPs-RSA-induced DRG neurons. (G-I) After Nox1 siRNA, Nox2 siRNA or Nox4 siRNA pretreatment, cells were stimulated with or without AOPPs-RSA (200 μg/mL) for 6 h, bar graph showed quantitative analysis of Nox1, Nox2 and Nox4. 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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f0020: The expression of NADPH oxidase in DRG neurons. (A, C) The levels of Nox1 and Nox4 were up-regulated significantly in DRG neurons incubated with AOPPs-RSA in indicated concentrations. (B) Indicated concentrations of AOPPs-RSA could not significantly up-regulate the expression of Nox2. (D, F) The levels of Nox1 and Nox4 were up-regulated significantly in DRG neurons incubated with 200 μg/mL AOPPs-RSA in different time durations. (E) The expression of Nox2 had no significant difference between indicated time durations in 200 μg/mL AOPPs-RSA-induced DRG neurons. (G-I) After Nox1 siRNA, Nox2 siRNA or Nox4 siRNA pretreatment, cells were stimulated with or without AOPPs-RSA (200 μg/mL) for 6 h, bar graph showed quantitative analysis of Nox1, Nox2 and Nox4. 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: To investigate the effect of AOPPs-RSA on NADPH oxidase expression in rat DRG neurons, the cells were stimulated with increasing concentrations of AOPPs-RSA (0, 50, 100 and 200 μg/mL) for different time (0, 15, 30, 60, 120 and 360 min). The protein levels of Nox1, Nox2 and Nox4 were analyzed by western blot from AOPPs-stimulated rat DRG neurons. The Nox1 and Nox4 protein level gradually increased after AOPPs stimulation at 0–200 μg/mL (Fig. 4A and C). AOPPs-RSA stimulation also time-dependently induced Nox1 and Nox4 protein expression (Fig. 4D and F). As for protein expression of Nox2 (Fig. 4B and E), the results showed no significant difference at indicated concentrations or time durations as analyzed by Western blot. Next, we knocked down Nox1, Nox2 and Nox4 gene in DRG neurons, and Nox1, Nox2 or Nox4 siRNA transfection dramatically reduced AOPPs-induced Nox1, Nox2 or Nox4 expression in rat DRG neurons (Fig. 4G-I). These results indicate that up-regulation of Nox1 and Nox4 may be critical for AOPPs-mediated ROS production in rat DRG neurons..


Advanced oxidation protein products sensitized the transient receptor potential vanilloid 1 via NADPH oxidase 1 and 4 to cause mechanical hyperalgesia
The expression of NADPH oxidase in DRG neurons. (A, C) The levels of Nox1 and Nox4 were up-regulated significantly in DRG neurons incubated with AOPPs-RSA in indicated concentrations. (B) Indicated concentrations of AOPPs-RSA could not significantly up-regulate the expression of Nox2. (D, F) The levels of Nox1 and Nox4 were up-regulated significantly in DRG neurons incubated with 200 μg/mL AOPPs-RSA in different time durations. (E) The expression of Nox2 had no significant difference between indicated time durations in 200 μg/mL AOPPs-RSA-induced DRG neurons. (G-I) After Nox1 siRNA, Nox2 siRNA or Nox4 siRNA pretreatment, cells were stimulated with or without AOPPs-RSA (200 μg/mL) for 6 h, bar graph showed quantitative analysis of Nox1, Nox2 and Nox4. 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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f0020: The expression of NADPH oxidase in DRG neurons. (A, C) The levels of Nox1 and Nox4 were up-regulated significantly in DRG neurons incubated with AOPPs-RSA in indicated concentrations. (B) Indicated concentrations of AOPPs-RSA could not significantly up-regulate the expression of Nox2. (D, F) The levels of Nox1 and Nox4 were up-regulated significantly in DRG neurons incubated with 200 μg/mL AOPPs-RSA in different time durations. (E) The expression of Nox2 had no significant difference between indicated time durations in 200 μg/mL AOPPs-RSA-induced DRG neurons. (G-I) After Nox1 siRNA, Nox2 siRNA or Nox4 siRNA pretreatment, cells were stimulated with or without AOPPs-RSA (200 μg/mL) for 6 h, bar graph showed quantitative analysis of Nox1, Nox2 and Nox4. 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: To investigate the effect of AOPPs-RSA on NADPH oxidase expression in rat DRG neurons, the cells were stimulated with increasing concentrations of AOPPs-RSA (0, 50, 100 and 200 μg/mL) for different time (0, 15, 30, 60, 120 and 360 min). The protein levels of Nox1, Nox2 and Nox4 were analyzed by western blot from AOPPs-stimulated rat DRG neurons. The Nox1 and Nox4 protein level gradually increased after AOPPs stimulation at 0–200 μg/mL (Fig. 4A and C). AOPPs-RSA stimulation also time-dependently induced Nox1 and Nox4 protein expression (Fig. 4D and F). As for protein expression of Nox2 (Fig. 4B and E), the results showed no significant difference at indicated concentrations or time durations as analyzed by Western blot. Next, we knocked down Nox1, Nox2 and Nox4 gene in DRG neurons, and Nox1, Nox2 or Nox4 siRNA transfection dramatically reduced AOPPs-induced Nox1, Nox2 or Nox4 expression in rat DRG neurons (Fig. 4G-I). These results indicate that up-regulation of Nox1 and Nox4 may be critical for AOPPs-mediated ROS production in rat DRG neurons..

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.