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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

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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.


AOPPs challenge increased reactive oxygen species (ROS) generation in DRG neurons via NADPH oxidase. (A) Viability of DRG neurons treated with AOPPs in different concentrations after cultured for 6 h. (B) DRG neurons were cultured as control cells, RSA group or with indicated concentration of AOPPs-RSA as AOPPs group for 2 h. (C) DRG neurons were incubated with 200 μg/mL AOPPs-RSA for indicated time. (D) DRG neurons were treated with AOPPs-RSA (200 μg/mL) with or without Nox1 siRNA, Nox2 siRNA, Nox4 siRNA or NAC (2 mM, 2 h). AOPPs-induced ROS production was significantly diminished by NAC and Nox1 siRNA, Nox4 siRNA. (E) DRG neurons were cultured with control medium, RSA, AOPPs-RSA (200 µg/mL), AOPPs+siNox1, AOPPs+siNox2, AOPPs+siNox4, AOPPs+NAC for 120 min. Confocal laser scanning microscope system was used to visualize ROS generation in DRG neurons with the use of DCFH-DA. Data represent mean±SEM of at least 3 independent experiments. Scale bar, 20 µm. *P<0.05 versus control (0) group. # P<0.05 versus AOPPs-RSA group.
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f0025: AOPPs challenge increased reactive oxygen species (ROS) generation in DRG neurons via NADPH oxidase. (A) Viability of DRG neurons treated with AOPPs in different concentrations after cultured for 6 h. (B) DRG neurons were cultured as control cells, RSA group or with indicated concentration of AOPPs-RSA as AOPPs group for 2 h. (C) DRG neurons were incubated with 200 μg/mL AOPPs-RSA for indicated time. (D) DRG neurons were treated with AOPPs-RSA (200 μg/mL) with or without Nox1 siRNA, Nox2 siRNA, Nox4 siRNA or NAC (2 mM, 2 h). AOPPs-induced ROS production was significantly diminished by NAC and Nox1 siRNA, Nox4 siRNA. (E) DRG neurons were cultured with control medium, RSA, AOPPs-RSA (200 µg/mL), AOPPs+siNox1, AOPPs+siNox2, AOPPs+siNox4, AOPPs+NAC for 120 min. Confocal laser scanning microscope system was used to visualize ROS generation in DRG neurons with the use of DCFH-DA. Data represent mean±SEM of at least 3 independent experiments. Scale bar, 20 µm. *P<0.05 versus control (0) group. # P<0.05 versus AOPPs-RSA group.

Mentions: Previous studies demonstrated that DRG neurons produced intracellular ROS under oxidative stress [34]. To determine whether AOPPs, as oxidative stress products, can cause redundant ROS accumulation inside DRG neurons, we examined intracellular ROS levels in AOPPs-treated DRG neurons. According to 6 h MTT test results (Fig. 5A), 400 μg/mL AOPPs-RSA caused significant DRG neurons death (cell viability was 61.80±4.20%), while incubation in 0 to 200 μg/mL AOPPs-RSA had little effects on cell fatality (cell viability was above 95%). Thus, we subjected DRG neurons cultures to increasing concentrations of AOPPs-RSA (0, 50, 100 and 200 μg/mL) for different time (0, 5, 15, 30, 45, 60 and 120 min). ROS production was increased in DRG neurons cultured with AOPPs-RSA in a dose- and time-dependent manner (Fig. 5B and C). For further confirmation, ROS generation in DRG neurons cultured with control medium, RSA, AOPPs-RSA (200 µg/mL), AOPPs+siNox1, AOPP+siNox2, AOPP+siNox4, AOPP+NAC (2 mM, 2 h) was observed under a confocal laser scanning microscope system using DCFH-DA probe (Fig. 5E)..


Advanced oxidation protein products sensitized the transient receptor potential vanilloid 1 via NADPH oxidase 1 and 4 to cause mechanical hyperalgesia
AOPPs challenge increased reactive oxygen species (ROS) generation in DRG neurons via NADPH oxidase. (A) Viability of DRG neurons treated with AOPPs in different concentrations after cultured for 6 h. (B) DRG neurons were cultured as control cells, RSA group or with indicated concentration of AOPPs-RSA as AOPPs group for 2 h. (C) DRG neurons were incubated with 200 μg/mL AOPPs-RSA for indicated time. (D) DRG neurons were treated with AOPPs-RSA (200 μg/mL) with or without Nox1 siRNA, Nox2 siRNA, Nox4 siRNA or NAC (2 mM, 2 h). AOPPs-induced ROS production was significantly diminished by NAC and Nox1 siRNA, Nox4 siRNA. (E) DRG neurons were cultured with control medium, RSA, AOPPs-RSA (200 µg/mL), AOPPs+siNox1, AOPPs+siNox2, AOPPs+siNox4, AOPPs+NAC for 120 min. Confocal laser scanning microscope system was used to visualize ROS generation in DRG neurons with the use of DCFH-DA. Data represent mean±SEM of at least 3 independent experiments. Scale bar, 20 µm. *P<0.05 versus control (0) group. # P<0.05 versus AOPPs-RSA group.
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f0025: AOPPs challenge increased reactive oxygen species (ROS) generation in DRG neurons via NADPH oxidase. (A) Viability of DRG neurons treated with AOPPs in different concentrations after cultured for 6 h. (B) DRG neurons were cultured as control cells, RSA group or with indicated concentration of AOPPs-RSA as AOPPs group for 2 h. (C) DRG neurons were incubated with 200 μg/mL AOPPs-RSA for indicated time. (D) DRG neurons were treated with AOPPs-RSA (200 μg/mL) with or without Nox1 siRNA, Nox2 siRNA, Nox4 siRNA or NAC (2 mM, 2 h). AOPPs-induced ROS production was significantly diminished by NAC and Nox1 siRNA, Nox4 siRNA. (E) DRG neurons were cultured with control medium, RSA, AOPPs-RSA (200 µg/mL), AOPPs+siNox1, AOPPs+siNox2, AOPPs+siNox4, AOPPs+NAC for 120 min. Confocal laser scanning microscope system was used to visualize ROS generation in DRG neurons with the use of DCFH-DA. Data represent mean±SEM of at least 3 independent experiments. Scale bar, 20 µm. *P<0.05 versus control (0) group. # P<0.05 versus AOPPs-RSA group.
Mentions: Previous studies demonstrated that DRG neurons produced intracellular ROS under oxidative stress [34]. To determine whether AOPPs, as oxidative stress products, can cause redundant ROS accumulation inside DRG neurons, we examined intracellular ROS levels in AOPPs-treated DRG neurons. According to 6 h MTT test results (Fig. 5A), 400 μg/mL AOPPs-RSA caused significant DRG neurons death (cell viability was 61.80±4.20%), while incubation in 0 to 200 μg/mL AOPPs-RSA had little effects on cell fatality (cell viability was above 95%). Thus, we subjected DRG neurons cultures to increasing concentrations of AOPPs-RSA (0, 50, 100 and 200 μg/mL) for different time (0, 5, 15, 30, 45, 60 and 120 min). ROS production was increased in DRG neurons cultured with AOPPs-RSA in a dose- and time-dependent manner (Fig. 5B and C). For further confirmation, ROS generation in DRG neurons cultured with control medium, RSA, AOPPs-RSA (200 µg/mL), AOPPs+siNox1, AOPP+siNox2, AOPP+siNox4, AOPP+NAC (2 mM, 2 h) was observed under a confocal laser scanning microscope system using DCFH-DA probe (Fig. 5E)..

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.