*2.2. Both Short-Term and Long-Term 100 Hz EA Stimulation Reversed P2X3 Elevation in L4-6 DRG and SCDH after CFA Injection*

To investigate the effects of the short-term and long-term 100 Hz EA stimulation on the expression of P2X3 in the DRG and SCDH following CFA injection, we used immunofluorescence and western blotting to measure the P2X3 protein levels in the DRG and SCDH in the control group, CFA group, 100 Hz EA group and sham EA group, 3 or 14 days after 100 Hz EA stimulation. As expected, CFA injection significantly increased the mean intensity of P2X3-ir in L4-6 DRG (Figure 2A,B, *P* < 0.05; Figure 4A,B, *P* < 0.05) and SCDH (Figure 3A,B, *P* < 0.05; Figure 5A,B, *P* < 0.05). Short-term and long-term 100 Hz EA stimulation significantly reduced the mean intensity of P2X3-ir in L4-6 DRG (Figure 2A,B, *P* < 0.05; Figure 4A,B, *P* < 0.05) and SCDH (Figure 3A,B, *P* < 0.05; Figure 5A,B, *P* < 0.05) when compared with the control group. In contrast, sham EA had no observable effects. As shown in Figures 2C and 4C, P2X3 was mainly expressed in the small- and medium- diameter DRG neurons (diameter less than 35 μm). Neither short-term nor long-term 100 Hz EA changed the distribution of P2X3.

We also used western blotting to measure the P2X3 protein expression. CFA injection increased P2X3 protein expression in L4-6 DRG (Figure 2D,E, *P* < 0.05; Figure 4D,E, *P* < 0.05) and SCDH (Figure 3C,D, *P* < 0.05; Figure 5C,D, *P* < 0.05). Consistent with the immunofluorescence staining results, short-term and long-term 100 Hz EA stimulation significantly reversed paw inflammation induced P2X3 up-regulation in L4-6 DRG (Figure 2D,E, *P* < 0.05; Figure 4D,E, *P* < 0.05) and SCDH (Figure 3C,D, *P* < 0.05; Figure 5C,D, *P* < 0.05). Sham EA had no effect on P2X3 expression either in L4-6 DRG or SCDH.

**Figure 2.** The 100 Hz EA stimulation for 3 days repressed the up-regulation of P2X3 in the L4-6 dorsal root ganglions (DRG) of inflamed rats. (**A**) Representative images of L4-6 DRG immunofluorescence staining from the control, CFA, CFA+100 Hz EA and CFA + sham EA groups. Scale bars = 100 μm. (**B**) Mean intensity analysis of P2X3-ir in L4-6 DRG in each group. Data are presented as the mean ± SEM, *n* = 3. (**C**) Size distribution of P2X3 in L4-6 DRG in different groups. (**D**) Representative western blotting images of L4-6 DRG in each group. E. Relative protein level of P2X3 in rat L4-6 DRG from different groups. Data are presented as the mean ± SEM, *n* = 6. \* *P* < 0.05, compared with the control group; # *P* < 0.05, compared with the CFA group; -*P* < 0.05, compared with the CFA + sham EA group.

**Figure 3.** The 100Hz EA stimulation for 3 days inhibited the up-regulation of P2X3 in (spinal cord dorsal horn) SCDH of inflamed rats. (**A**). Representative images of SCDH immunofluorescence staining from the control, CFA, CFA + 100 Hz EA and CFA + sham EA groups. Scale bars = 100 μm. (**B**) Mean intensity analysis of P2X3-ir in SCDH in each group. Data are presented as the mean ± SEM, *n* = 3. (**C**) Representative western blotting images of SCDH in each group. (**D**) Relative protein level of P2X3 in SCDH from different groups. Data are presented as the mean ± SEM, *n* = 6. \* *P* <0.05, compared with the control group; # *P* <0.05, compared with the CFA group; - *P* <0.05, compared with the CFA + sham EA group.

**Figure 4.** The 100Hz EA stimulation for 14 days repressed the up-regulation of P2X3 in the L4-6 DRG of inflamed rats. (**A**) Representative images of L4-6 DRG immunofluorescence staining from the control, CFA, CFA + 100 Hz EA and CFA + sham EA groups. Scale bars = 100 μm. (**B**) Mean intensity analysis of P2X3-ir in L4-6 DRG in each group. Data are presented as the mean ± SEM, *n* = 3. (**C**) Size distribution of P2X3 in L4-6 DRG in different groups. (**D**) Representative western blotting images of L4-6 DRG in each group. (**E**) Relative protein level of P2X3 in rat L4-6 DRG from different groups. Data are presented as the mean ± SEM, *n* = 6. \* *P* < 0.05, compared with the control group; # *P* <0.05, compared with the CFA group; -*P* <0.05, compared with the CFA + sham EA group.

**Figure 5.** The 100 Hz EA stimulation for 14 days inhibited the up-regulation of P2X3 in the SCDH of inflamed rats. (**A**) Representative images of SCDH immunofluorescence staining from the control, CFA, CFA + 100 Hz EA and CFA + sham EA groups. Scale bars = 100 μm. (**B**) Mean intensity analysis of P2X3-ir in the SCDH in each group. Data are presented as the mean ± SEM, *n* = 3. (**C**) Representative western blotting images of SCDH in each group. (**D**) Relative protein level of P2X3 in the SCDH from different groups. Data are presented as the mean ± SEM, *n* = 6. \* *P* <0.05, compared with the control group; # *P* < 0.05, compared with the CFA group; -*P* < 0.05, compared with the CFA + sham EA group.
