Electroacupuncture Promoting Axonal Regeneration in Spinal Cord Injury Rats via Suppression of Nogo/NgR and Rho/ROCK Signaling Pathway

Abstract

Purpose: To observe the changes of Nogo/NgR and Rho/ROCK signaling pathway-related gene and protein expression in rats with spinal cord injury (SCI) treated with electroacupuncture (EA) and to further investigate the possible mechanism of EA for treating SCI.

Methods: Allen's method was used to create the SCI rat model. Sixty-four model rats were further subdivided into four subgroups, namely, the SCI model group (SCI), EA treatment group (EA), blocking agent Y27632 treatment group (Y27632) and EA+blocking agent Y27632 treatment group (EA+Y), according to the treatment received. The rats were subjected to EA and/or blocking agent Y27632 treatment. After 14 days, injured spinal cord tissue was extracted for analysis. The mRNA and protein expression levels were determined by real-time fluorescence quantitative PCR and Western blotting, respectively. Cell apoptosis changes in the spinal cord were evaluated by in situ hybridization. Hindlimb motor function in the rats was evaluated by Basso-Beattie-Bresnahan assessment methods.

Results: Except for RhoA protein expression, compared with the SCI model group, EA, blocking agent Y27632 and EA+blocking agent Y27632 treatment groups had significantly reduced mRNA and protein expression of Nogo-A, NgR, LINGO-1, RhoA and ROCK II in spinal cord tissues, increased mRNA and protein expression of MLCP, decreased p-MYPT1 protein expression and p-MYPT1/MYPT1 ratio, and caspase3 expression, and improved lower limb movement function after treatment for 14 days (P<0.01 or <0.05). The combination of EA and the blocking agent Y27632 was superior to EA or blocking agent Y27632 treatment alone (P < 0.01 or <0.05).

Conclusion: EA may have an obvious inhibitory effect on the Nogo/NgR and Rho/ROCK signaling pathway after SCI, thereby reducing the inhibition of axonal growth, which may be a key mechanism of EA treatment for SCI.

Keywords: MLCP; MYPT1; Nogo/NgR; Rho/ROCK; Y27632; electroacupuncture; spinal cord injury.

Figure 1

BBB score of each group before treatment and on the 14th days. ① P< 0.01, ② P<0.05 versus sham; ③ P<0.01, ④ P<0.05 versus SCI; ⑤ P<0.01; ⑥ P<0.05 versus EA; ⑦ P<0.01; ⑧P<0.05 versus Y27632. Data are expressed as the mean±SD (1-way analysis of variance and Student-Newman-Keuls post hoc test, n=8 rats/group). Abbreviations: sham, sham operation; SCI, spinal cord injury; EA, electroacupuncture; Y, blocking agent Y27632.

Figure 2

Nogo-A, NgR and LINGO-1 activation following SCI. (A) Western blotting bands for Nogo-A, NgR and LINGO-1. Compiled results in a bar graph for the relative protein expression (B) of Nogo-A. (C) of NgR. (D) of LINGO-1. ① P < 0.01, ② P<0.05 versus sham. ③ P<0.01, ④ P<0.05 versus SCI. Data are shown as the mean±standard error of the mean (1-way analysis of variance and Student-Newman-Keuls post hoc test, n=4 rats/group). Abbreviations: sham, sham operation; SCI, spinal cord injury; EA, electroacupuncture.

Figure 3

Comparison of the mRNA relative expression of Nogo-A, NgR and LINGO-1. Compiled results in a bar graph for the relative mRNA expression (A) of Nogo-A. (B) of NgR. (C) of LINGO-1. ① P < 0.01, ② P<0.05 versus sham. ③ P<0.01. Data are shown as the mean±standard error of the mean (1-way analysis of variance and Student-Newman-Keuls post hoc test, n=4 rats/group). Abbreviations: sham, sham operation; SCI, spinal cord injury; EA, electroacupuncture.

Figure 4

MLCP, ROCK II and RhoA activation following SCI. (A) Western blotting bands for MLCP, ROCK II, RhoA and GAPDH expression. Compiled results in a bar graph for the ratio (B) of MLCP/GAPDH expression. (C) of ROCK II/GAPDH expression. (D) of RhoA/GAPDH expression. ① P<0.01, ② P<0.05 versus sham. ③ P<0.01, ④ P<0.05 versus SCI. ⑥ P<0.05 versus EA. ⑦ P<0.01, ⑧P<0.05 versus Y27632. Data are shown as the mean±standard error of the mean (1-way analysis of variance and Student-Newman-Keuls post hoc test, n=4 rats/group). Abbreviations: sham, sham operation; SCI, spinal cord injury; EA, electroacupuncture; Y, blocking agent Y27632; MLCP, myosin light chain phosphatase; ROCK II, Rho-associated kinase II; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Effect of electroacupuncture on changes in mRNA expression of MLCP, ROCK II and RhoA in injured spinal cords. (A) Compiled results in a bar graph for MLCP expression. Compiled results in a bar graph (B) for ROCK II expression. (C) for RhoA expression.① P<0.01,. ③ P<0.01. ⑤ P<0.01; ⑥ P<0.05 versus EA. ⑦ P<0.01 versus Y27632. Data are expressed as the mean±SD (1-way analysis of variance and Student-Newman-Keuls post hoc test, n=4 rats/group). Abbreviations: sham, sham operation; SCI, spinal cord injury; EA, electroacupuncture; Y, blocking agent Y27632; ROCK II, Rho-associated kinase II; MLCP, myosin light chain phosphatase.

Figure 6

Myosin phosphatase target subunit 1 (MYPT1) activation following SCI. (A) Western blotting bands for phosphorylated MYPT1 (p-MYPT1), MYPT1, and GAPDH expression. Compiled results in a bar graph for the ratio (B) of p-MYPT1/GAPDH expression. (C) of MYPT1/GAPDH expression. (D) of p-MYPT1/MYPT1. ① p<0.01, ② P<0.05 versus sham. ③ P<0.01, ⑥ P<0.05 versus EA. Data are shown as the mean±standard error of the mean (1-way analysis of variance and Student-Newman-Keuls post hoc test, n=4 rats/group). sham: sham operation. Abbreviations: SCI, spinal cord injury; EA, electroacupuncture; Y, blocking agent Y27632; MYPT1, myosin phosphatase target subunit 1; p-MYPT1, phosphorylated MYPT1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 7

mRNA expression of caspase3 in injured spinal cords at 14 days following treatment (in situ hybridization, ×400). (Bar =25 µm). Abbreviations: sham, sham operation; SCI, spinal cord injury; EA, electroacupuncture; Y, blocking agent Y27632.

Figure 8

Compiled results in a bar graph for the number of caspase3 mRNA-positive cells. ① P<0.01. ③ P<0.01. ⑤ P<0.01 versus EA. Data are expressed as the mean±SD (1-way analysis of variance and Student-Newman-Keuls post hoc test, n=4 rats/group). Abbreviations: sham, sham operation; SCI, spinal cord injury; EA, electroacupuncture; Y, blocking agent Y27632.