Electroacupuncture regulates apoptosis/proliferation of intramuscular interstitial cells of cajal and restores colonic motility in diabetic constipation rats

Abstract

Injury of interstitial cells of Cajal (ICC) is associated with gut dysmotility in diabetic rats. We have shown an acceleration of the colonic contractility by electroacupuncture stimulation (EAS). However, little is known about potential roles of EAS on colonic transit and ICC. In this study, we evaluate the effect of EAS on colonic transit and investigate whether apoptosis/proliferation of ICC was involved in regulative effect of EAS on colonic transit. Rats were randomly assigned to normal, diabetic, diabetic-plus-sham stimulation, diabetic-plus-low-frequency stimulation, and diabetic-plus-high-frequency stimulation groups. Bead expulsion test was used for measuring the distal colonic transit. The Kit (ICC marker) was detected by western blot. Apoptotic ICC was detected by terminal dUTP nucleotide end labeling. Proliferating ICC was identified by Kit/Ki67 double immunofluorescent staining on whole mount preparations. Ultrastructure changes of ICC were studied using electron microscopy. Results showed that high-frequency stimulation significantly promoted colonic transit. Low- and high-frequency stimulation markedly rescued intramuscular ICC from apoptosis. Abundant proliferating intramuscular ICC was found in low- and high-frequency stimulation groups. Our results indicate that high-frequency EAS has stimulatory effect on the distal colonic transit, which may be mediated by downregulation of the apoptosis and upregulation of the proliferation of intramuscular ICC.

Figure 1

Blood glucose (a) and body weight (b) in the 5 groups. (a) All the diabetic rats displayed markedly increased blood glucose. In the SEA, LEA, and HEA groups, blood glucose did not show any changes compared with the DM group (P > 0.05). (b) No differences in the baseline body weight among each group. The HEA group gained weight compared with the DM group at the end of 4 weeks (P < 0.01) and 8 weeks (P < 0.01). (*Significantly different to the DM group for 4 weeks, ^#significantly different to the DM group for 8 weeks.)

Figure 2

Effects of EAS at ST-36 on distal colonic transit. In contrast to the control, the DM group displayed delayed colonic transit, while EAS with high-frequency at ST-36 significantly increased colonic transit. The colonic transit time was not changed obviously in the SEA and LEA group. Data are given as means ± SEM; an asterisk indicates statistical significance at a level of P < 0.05. (*Significantly different to the DM group for 4 weeks, ^#significantly different to the DM group for 8 weeks.)

Figure 3

Western blot analysis of Kit in colon tissue which the submucosa removed. Compared with the control group, the expression of Kit was decreased in the DM group; there were no significant difference between the DM and SEA group (P > 0.05). However, they were increased markedly in the LEA and HEA group. (*Significantly different to the DM group, ^#significantly different to the LEA group.)

Figure 4

Confocal images of ICC-IM in each group showing the apoptosis of ICC-IM. (a)–(c) In the control group, nearly no cell was labeled by TUNEL method and displayed a perfect network. (d)–(f) In the DM group, a large number of Kit⁺/TUNEL⁺ cells (arrowheads) were observed, and the Kit⁺ cellular networks were severely damaged. (g)–(i) In the SEA group, a number of apoptotic Kit⁺ ICC-IM (arrowheads) were observed and the network of ICC-IM was not restored. (j)–(l) In the LEA group, the network of ICC-IM was partially restored and rare double labeled cells (arrowheads) were distributed within ICC-IM. (m)–(o) In the HEA group, the apoptotic ICC-IM nearly vanished and was accompanied by a more intact network. Scale bar = 50 μm and refers to all panels.

Figure 5

Confocal images of ICC-IM in each group showing the proliferation of ICC-IM. (a)–(c) In the control group, a small number of proliferative ICC-IM (arrowheads) were observed and displayed a perfect network. (d)–(f) In the DM group, Kit⁺/Ki67⁺ were almost absent, and the network was severely damaged. (g)–(i) In the SEA group, there was also no proliferative ICC-IM and the network of ICC-IM was not restored. (j)–(l) In the LEA group, the network of ICC-IM was partially restored and a small number of double labeled cells (arrowheads) were distributed within ICC-IM. Such cells were in high density in the HEA group (m)–(o), accompanied by a more intact network. Scale bar = 50 μm and refers to all panels.

Figure 6

Ultrastructure of ICC. Comparing with the control group, ICC was seriously injured in the DM and SEA group, while they showed nearly normal structure and minor injury in the LEA and HEA group. ICC: interstitial cells of Cajal; SMC: smooth muscle cell; NF: nerve fibre.