Journal: Molecular Pain

Article Title: Electroacupuncture downregulates P2X3 receptor expression in dorsal root ganglia of the spinal nerve-ligated rat

doi: 10.1177/1744806919847810

Figure Lengend Snippet: SNL-induced changes in P2X3R expression in ipsilateral L4 and L5 DRG were altered by EA stimulation. (a) Examples of P2X3R expressions in different rat groups. (b) EA-induced changes in P2X3R protein expression in L4 DRGs. Following SNL+ s-EA stimulation, P2X3R expression was 1.4 times (= 1.1/0.77) higher than that in control rats. The expression was retuned close to control level (0.7/0.77 = 0.9) after EA stimulation. (c) EA-induced changes P2X3R protein expressions in L5 DRGs. The expression of P2X3R was much reduced after SNL+s-EA treatment (0.2/0.97 = 0.21) or after SNL+EA treatment (0.16/0.97 = 0.17). (n = 6 for each rat group, * P < 0.01). DRG: dorsal root ganglion; EA: electroacupuncture; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The samples were then electro-transferred to a polyvinylidene difluoride membrane, which was subsequently incubated in a Tris-buffered saline blocking buffer containing 5% w/v fat-free dry milk at room temperature (RT) for 1 h. The membrane was then immunoblotted with rabbit anti-P2X3R primary antibody (1:1000, Alomone Labs) at 4°C, overnight followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (1:10000, Abcam) at RT for 1 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), probed with rabbit anti-GAPDH antibody (1:1000, Cell Signaling Technilogy), was used as loading control.

Techniques: Expressing

Journal: Molecular Pain

Article Title: Electroacupuncture downregulates P2X3 receptor expression in dorsal root ganglia of the spinal nerve-ligated rat

doi: 10.1177/1744806919847810

Figure Lengend Snippet: The effect of immunohistochemical study of P2X3R-ir and ATF3-ir expression in L4 DRG neurons. (a) P2X3R-ir (red) + ATF3-ir (green) double labeled DRG neurons in Control (Con), SNL+ s-EA (s-EA) and SNL+EA (EA) rats. Bar =100 µm. (b) Percentages of P2X3R+ expressing neurons in Control and SNL+ s-EA rats were similar (28.24 ± 1.69% in Con vs. 26.83 ± 0.9% in s-EA rats), The percentage of P2X3R+ neurons in SNL+ EA rats was significantly reduced (15.37 ± 1.75%). (c) Integrated optical density measured in SNL+s-EA rats ((9.21 ± 1.08) × 10 5 ) was higher than that in control rats ((6.64 ± 0.75) ×10 5 ) and much reduced ((2.81 ± 0.39) × 10 5 ) in SNL+EA rats. (d) The percentage of ATF+ neurons in SNL+ s-EA and in SNL+EA rats were not significantly different (6.7 ± 1.7 vs. 6.1 ± 0.6%, P > 0.05). Both were higher than control rats (0%). (e) Almost no P2X3+/ATF3+ neurons were expressed in the three rat groups. Thus, all of the P2X3R+ were expressed in un-injured neurons (Con: n = 7, SNL+ s-EA: n = 3, SNL+EA: n = 3). * P < 0.01. EA: electroacupuncture; ATF3: activating transcription factor 3; IOD: integrated optical density.

Article Snippet: The samples were then electro-transferred to a polyvinylidene difluoride membrane, which was subsequently incubated in a Tris-buffered saline blocking buffer containing 5% w/v fat-free dry milk at room temperature (RT) for 1 h. The membrane was then immunoblotted with rabbit anti-P2X3R primary antibody (1:1000, Alomone Labs) at 4°C, overnight followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (1:10000, Abcam) at RT for 1 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), probed with rabbit anti-GAPDH antibody (1:1000, Cell Signaling Technilogy), was used as loading control.

Techniques: Immunohistochemical staining, Expressing, Labeling

Journal: Molecular Pain

Article Title: Electroacupuncture downregulates P2X3 receptor expression in dorsal root ganglia of the spinal nerve-ligated rat

doi: 10.1177/1744806919847810

Figure Lengend Snippet: EA stimulation did not affect P2X3R or ATF3 expression in injured neurons ipsilateral to L5 DRGs. (a) Examples of P2X3R-ir + ATF3-ir double labeled neurons in L5 DRGs of three rat groups, Bar = 100 µm. (b) Compared to control rats, L5 nerve ligation induced a substantial reduction in P2X3R+ expression in DRGs of SNL+ s-EA or SNL+EA rats (from 29.0 ± 2.1% to 13.7 ± 4.9% and to 11.0 ± 3.3% respectively). (c) Unlike the minimal expression of ATF3+ cells (0.4 ± 0.2%) in L5 DRG of control rats, a majority of L5 DRG neurons in SNL+ s-EA rat group (85.7 ± 1.6%) and in SNL+EA group (81.7 ± 3.1%) were ATF3+. (d) Most P2X3Rs in injured L5 DRGs were expressed in ATF3+ neurons (P2X3R/ATF3+ =10.6% in SNL+ s-EA rats and =8.8% in SNL+EA rats). (e) The cell count of P2X3R+/ATF3− cells was reduced from 30.0 ± 2.0% in control L5 DRGs to 2.7 ± 0.8% in SNL+ s-EA and 1.8 ± 0.5% in SNL+EA rats. EA stimulation did not reverse the reduction in P2X3R expression induced by SNL (Con: n = 7, SNL+ s-EA: n = 3, SNL+EA: n = 3). * P < 0.01. EA: electroacupuncture; ATF3: activating transcription factor 3.

Article Snippet: The samples were then electro-transferred to a polyvinylidene difluoride membrane, which was subsequently incubated in a Tris-buffered saline blocking buffer containing 5% w/v fat-free dry milk at room temperature (RT) for 1 h. The membrane was then immunoblotted with rabbit anti-P2X3R primary antibody (1:1000, Alomone Labs) at 4°C, overnight followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (1:10000, Abcam) at RT for 1 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), probed with rabbit anti-GAPDH antibody (1:1000, Cell Signaling Technilogy), was used as loading control.

Techniques: Expressing, Labeling, Ligation, Cell Counting

Journal: Molecular Pain

Article Title: F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

doi: 10.1177/1744806916660557

Figure Lengend Snippet: CPT increases the membrane expression of P2X3Rs in cultured DRGs. The P2X3R levels in total protein (Total), membrane protein (Mem), and cytoplasmic protein samples (Cytosol) before (Con) and after CPT (1 µM, 10 min) treatment were determined. Total, membrane, and cytosol protein samples were loaded onto gels. The expression of the ubiquitous membrane protein Na+/K+ ATPase was probed to indicate the membrane expressed protein levels in our samples. The expressions of β-tubulin used as sample loading controls were also indicated. CPT significantly increased P2X3Rs expressed in the cell membrane (total protein: CPT/Con = 1.19; membrane protein: CPT/Con = 1.93 and cytoplasmic protein: CPT/Con = 0.75). N = 3. * P < 0.05.

Article Snippet: Antibodies used were rabbit anti-pPKC ɛ (1:1000, Santa Cruz, Dallas, TX) and rabbit anti-P2X3R (1:2000, Alomone Labs).

Techniques: Expressing, Cell Culture

Journal: Molecular Pain

Article Title: F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

doi: 10.1177/1744806916660557

Figure Lengend Snippet: Epac control of the membrane expression of P2X3Rs in DRG neurons is F-actin mediated. (a) The membrane expression of P2X3Rs was examined in cultured DRG neurons using anti-ext-P2X3R antibody. The enlarged views of neurons (indicated by red arrows) are shown on the right side of each panel. Bars = 25 µm. (b) In the presence of CPT (1 µM), the percentage of cells expressing ext-P2X3R increased by 1.80 fold and the intensity of ext-P2X3R labels increased by 5.57 fold. The CPT-induced increase in membrane expressed P2X3Rs was significantly reduced by pre-incubating cells with the F-actin disrupter, LaA (1 µM, 60 min). A total of 1192 cells obtained from three experiments were used for the analyses. * P < 0.05, # P < 0.01. NS: not significant.

Article Snippet: Antibodies used were rabbit anti-pPKC ɛ (1:1000, Santa Cruz, Dallas, TX) and rabbit anti-P2X3R (1:2000, Alomone Labs).

Techniques: Expressing, Cell Culture