Synaptotagmin 1 Is Involved in Neuropathic Pain and Electroacupuncture-Mediated Analgesic Effect

Abstract

Numerous studies have verified that electroacupuncture (EA) can relieve neuropathic pain through a variety of mechanisms. Synaptotagmin 1 (Syt-1), a synaptic vesicle protein for regulating exocytosis of neurotransmitters, was found to be affected by EA stimulation. However, the roles of Syt-1 in neuropathic pain and EA-induced analgesic effect remain unclear. Here, the effect of Syt-1 on nociception was assessed through an antibody blockade, siRNA silencing, and lentivirus-mediated overexpression of spinal Syt-1 in rats with spared nerve injury (SNI). EA was used for stimulating bilateral "Sanjinjiao" and "Zusanli" acupoints of the SNI rats to evaluate its effect on nociceptive thresholds and spinal Syt-1 expression. The mechanically and thermally nociceptive behaviors were assessed with paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) at different temperatures, respectively, at day 0, 7, 8, 14, and 20. Syt-1 mRNA and protein levels were determined with qRT-PCR and Western blot, respectively, and its distribution was observed with the immunohistochemistry method. The results demonstrated Syt-1 antibody blockade and siRNA silencing increased ipsilateral PWTs and PWLs of SNI rats, while Syt-1 overexpression decreased ipsilateral PWTs and PWLs of rats. EA significantly attenuated nociceptive behaviors and down-regulated spinal Syt-1 protein levels (especially in laminae I-II), which were reversed by Syt-1 overexpression. Our findings firstly indicate that Syt-1 is involved in the development of neuropathic pain and that EA attenuates neuropathic pain, probably through suppressing Syt-1 protein expression in the spinal cord.

Keywords: Synaptotagmin 1; allodynia; electroacupuncture; hyperalgesia; neuropathic pain.

Figure 1

The distribution of Syt-1 immunoreactive cells in the spinal cord dorsal horn (SCDH). (A,B) The sketches and tissues (in laminae I-II) showed that SNI resulted in increased Syt-1 immunoreactive (Syt-1-IR), mainly in the laminae I-II of the SCDH. B shows the representative of immunohistochemistry from 3 animals. Red arrows show Syt-1 positive cells. Scale bars, 100 µm.

Figure 2

The effect of Syt-1 antibody on paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) (Mean ± SD, n = 6). Rats in SNI+ different doses of Syt-1 antibody were intrathecally injected with 1, 4, or 8 µg antibody at day 8 and once every two days. SNI induced a decrease in PWLs (0, 43, 46, and 49 °C) and ipsilateral PWTs at day 7 to 20. Syt-1 antibody treatment caused increased PWLs (0, 43, 46, and 49 °C) and ipsilateral PWTs in a dose-dependent manner at day 8 to 20. Values with different letters (a, b and c) at the same day show different (p < 0.05); two-way ANOVA followed by Bonferroni test.

Figure 3

The effect of Syt-1 siRNA on PWTs and PWLs (Mean ± SD, n = 6). Rats in SNI-Lip, SNI-Con-si and SNI-Syt1-si groups were intrathecally injected with 15 µL lipofectamine, 10 µL lipofectamine + 5 µL control siRNA and 10 µL lipofectamine + 5 µL Syt-1 siRNA, respectively. Syt-1 siRNA induced increased ipsilateral PWTs and PWLs (0, 43, and 46 °C) at day 14 and 20 compared with SNI group. Values with different letters (a, b and c) at the same day show different (p < 0.05); two-way ANOVA followed by Bonferroni test.

Figure 4

The effect of Syt-1 knockdown, Syt-1 overexpression or EA stimulations on Syt-1 expression (Mean ± SD, n = 6). (A,B) Syt-1 mRNA and protein levels were detected at day 20. Intrathecal injection of Syt-1 siRNA after SNI treatment decreased the protein of Syt-1, but unchanged its mRNA. (C,D) Intrathecal injection of Syt-1 activation particles shows the similar tendency of Syt-1 protein as SNI surgery. The mRNA and protein levels of Syt-1 in Sham-Syt1-Lev rats were higher than those in Sham group. (E,F) Rats in SNI-EA group showed decreased Syt-1 protein levels compared with rats in SNI group. Intrathecal injection of Syt-1 lentiviral activation particles induced an increase in Syt-1 protein and mRNA compared with EA treatment. No change was observed in Syt-1 mRNA among Sham, SNI, SNI-EA and SNI-Con-Lev groups. A, C, and D shows Syt-1 protein; B, E and F shows Syt-1 mRNA. Values with different letters (a, b and c) at the same day show different (p < 0.05); one-way ANOVA followed by Bonferroni test.

Figure 5

The effect of Syt-1 lentiviral activation particles on PWTs and PWLs (Mean ± SD, n = 6). Rats in Sham-Con-Lev and Sham-Syt1-Lev groups were intrathecally injected with 15 µL control lentiviral particles and Syt-1 lentiviral activation particles, respectively. Bilateral PWTs and PWLs (0, 43, 46 and 49 °C) in Sham-Syt1-Lev rats were less than those in Sham-Con-Lev at day 14 and 20. Ipsilateral PWTs and PWLs (0, 43, and 46 °C) of Sham-Syt1-Lev rats were decreased during the experiment, but not different from those of SNI rats at day 20. Values with different letters (a, b, and c) at the same day show different (p < 0.05); two-way ANOVA followed by Bonferroni test.

Figure 6

The effect of EA on PWTs and PWLs (Mean ± SD, n = 6). Rats in SNI-EA, SNI-EA-Con-Lev, or SNI-EA-Syt1-Lev groups were treated with EA at day 8, 10, 12, 14, 16, 18, and 20, for 7 times in total. Rats in the SNI-EA-Syt1-Lev or SNI-EA-Con-Lev groups were intrathecally injected with 15 µL of Syt-1 lentiviral activation particles or lentiviral control particles. The SNI-EA group showed increased ipsilateral PWTs, PWLs (0, 43, and 46 °C) at day 8 to 20 and PWL (49 °C) at day 20 compared with the SNI group. EA induced an increase in ipsilateral PWTs and PWLs at (0, 43, and 46 °C) at day 14 and 20 and in PWL (49 °C) at day 20, which was reversed by the intrathecal administration of Syt-1 lentiviral activation particles. Values with different letters (a, b, and c) on the same day show different (p < 0.05); two-way ANOVA followed by a Bonferroni test.

Figure 7

Effect of EA on Syt-1 immunoreactive cells in the laminae I-II of the SCDH (Mean ± SD, n = 3). (A) Decreased Syt-1-IR, which EA induced were apparently reversed by Syt-1 overexpression. Red arrows show Syt-1 positive cells. Scale bars, 100 µm. (B) The optical density values of the Syt-1-IR cells in the laminae I-II. The values in SNI and SNI-EA-Syt1-Lev were higher than those in SNI-EA and SNI-EA-Con-Lev. ** p < 0.01, vs. the SNI group; one-way ANOVA followed by Bonferroni test.

Figure 8

Colocalization of Syt-1 with the marker of neurons, astrocytes, or microglial cells in the SCDH. Co-labeling with RBFOX3 (a marker for neurons) and GFAP (a marker for astrocytes) antibodies showed that Syt-1-IR (in green) mainly exists in neurons and astrocytes (in red). Weak Syt-1-IR signal was observed in few microglial cells stained with Iba1 (a marker for microglial cells) antibodies. White arrows show the colocalization of Syt-1 with neurons, astrocytes, or microglial cells. Scale bars, 100 µm.

Figure 9

The schematic diagram for the experimental design. (A) The diagram for Syt-1 antibody intrathecal injection. (B) The diagram for Syt-1 siRNA intrathecal injection. (C) The diagram for Syt-1 lentiviral activation particles intrathecal injection. (D) The diagram for electroacupuncture (EA) stimulation after spared nerve injury (SNI) surgery. (E) The methods for the SNI model: A ligation of the common peroneal and tibial nerves was conducted with a 5.0 silk, leaving the sural nerve intact. Then, the two ligated nerves were sectioned distal to the ligation with 2–4 mm of the distal nerve stump left. (F) EA acupoints: Bilateral acupoints of “Zusanli” and “Sanyinjiao”. i.t. means intrathecal injection.

Figure 10

Exploration of the optimal conditions for Syt-1 interference and overexpression (Mean ± SD, n = 3). (A,B) Five microliters of Syt-1 siRNA in mixture with 10 µL lipofectamine (1:2) caused less Syt-1 mRNA and protein expression compared with the other ratios; * p < 0.05, ** p < 0.01, vs. control group. (C,D) After a bolus administration of the optimal Syt-1 siRNA vs. lipofectamine ratio (1:2), decreased Syt-1 expression lasted for 5 days at least; * p < 0.05, ** p < 0.01, vs. 0 d group. (E,F) Intrathecal injection of 15 µL Syt-1 lentiviral activation particles caused increased Syt-1 mRNA and protein compared with control lentiviral particles; * p < 0.05, ** p < 0.01, vs. Con-Lev group. A, C, and E show Syt-1 mRNA; B, D, and F show Syt-1 protein. In the control (A and B), phosphate buffer saline (PBS) instead of the Syt-1 siRNA mixture was intrathecally administered. The significance of differences was calculated with a one-way ANOVA followed by a Bonferroni test.