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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.


Intracellular free calcium was detected by Fura-2/AM. (A) The levels of calcium were shown in F340/F380. (B) DRG neurons were incubated with AOPPs-RSA (200 μg/mL) with or without Nox1 siRNA, Nox4 siRNA, NAC (2 mM, 2 h), or SB366791 (1 μM, 2 h). F340/F380 was used to indicate Intracellular free calcium. Data represent mean±SEM of at least 3 independent experiments. *P<0.05 versus Control group. # P<0.05 versus AOPPs-RSA group.
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f0035: Intracellular free calcium was detected by Fura-2/AM. (A) The levels of calcium were shown in F340/F380. (B) DRG neurons were incubated with AOPPs-RSA (200 μg/mL) with or without Nox1 siRNA, Nox4 siRNA, NAC (2 mM, 2 h), or SB366791 (1 μM, 2 h). F340/F380 was used to indicate Intracellular free calcium. Data represent mean±SEM of at least 3 independent experiments. *P<0.05 versus Control group. # P<0.05 versus AOPPs-RSA group.

Mentions: ROS increase intracellular free calcium by acting specifically on TRPV1 [22]. AOPPs -treated DRG neurons (200 μg/mL, 2 h) showed more calcium influxes comparing to those in the control group (Fig. 7A). We further assessed calcium fluxes in DRG neurons which were pretreated with Nox1 siRNA, Nox4 siRNA, NAC (2 mM, 2 h), or SB366791 (1 μM, 2 h) and then incubated with 200 μg/mL AOPPs-RSA for 2 h. Fig. 7B showed that, comparing with the control group, despite of the fact that the concentration of intracellular calcium were higher in AOPPs+siNox1 group, AOPPs+siNox4 group and AOPPs+NAC group, these inhibitors could efficiently decrease the concentration of intracellular free calcium which was induced by AOPPs..


Advanced oxidation protein products sensitized the transient receptor potential vanilloid 1 via NADPH oxidase 1 and 4 to cause mechanical hyperalgesia
Intracellular free calcium was detected by Fura-2/AM. (A) The levels of calcium were shown in F340/F380. (B) DRG neurons were incubated with AOPPs-RSA (200 μg/mL) with or without Nox1 siRNA, Nox4 siRNA, NAC (2 mM, 2 h), or SB366791 (1 μM, 2 h). F340/F380 was used to indicate Intracellular free calcium. Data represent mean±SEM of at least 3 independent experiments. *P<0.05 versus Control 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

f0035: Intracellular free calcium was detected by Fura-2/AM. (A) The levels of calcium were shown in F340/F380. (B) DRG neurons were incubated with AOPPs-RSA (200 μg/mL) with or without Nox1 siRNA, Nox4 siRNA, NAC (2 mM, 2 h), or SB366791 (1 μM, 2 h). F340/F380 was used to indicate Intracellular free calcium. Data represent mean±SEM of at least 3 independent experiments. *P<0.05 versus Control group. # P<0.05 versus AOPPs-RSA group.
Mentions: ROS increase intracellular free calcium by acting specifically on TRPV1 [22]. AOPPs -treated DRG neurons (200 μg/mL, 2 h) showed more calcium influxes comparing to those in the control group (Fig. 7A). We further assessed calcium fluxes in DRG neurons which were pretreated with Nox1 siRNA, Nox4 siRNA, NAC (2 mM, 2 h), or SB366791 (1 μM, 2 h) and then incubated with 200 μg/mL AOPPs-RSA for 2 h. Fig. 7B showed that, comparing with the control group, despite of the fact that the concentration of intracellular calcium were higher in AOPPs+siNox1 group, AOPPs+siNox4 group and AOPPs+NAC group, these inhibitors could efficiently decrease the concentration of intracellular free calcium which was induced by AOPPs..

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.