Study on the Mechanism Underlying the Regulation of the NMDA Receptor Pathway in Spinal Dorsal Horns of Visceral Hypersensitivity Rats by Moxibustion

Abstract

Visceral hypersensitivity is enhanced in irritable bowel syndrome (IBS) patients. Treatment of IBS visceral pain by moxibustion methods has a long history and rich clinical experience. In the clinic, moxibustion on the Tianshu (ST25) and Shangjuxu (ST37) acupoints can effectively treat bowel disease with visceral pain and diarrhea symptoms. To investigate the regulatory function of moxibustion on the Tianshu (ST25) and Shangjuxu (ST37) acupoints on spinal cord NR1, NR2B, and PKCε protein and mRNA expression in irritable bowel syndrome (IBS) visceral hypersensitivity rats, we did some research. In the study, we found that moxibustion effectively relieved the IBS visceral hyperalgesia status of rats. Analgesic effect of moxibustion was similar to intrathecal injection of Ro 25-6981. The expression of NR1, NR2B, and PKCε in the spinal dorsal horns of IBS visceral hyperalgesia rats increased. Moxibustion on the Tianshu and Shangjuxu acupoints might inhibit the visceral hypersensitivity, simultaneously decreasing the expression of NR1, NR2B, and PKCε in spinal cord of IBS visceral hyperalgesia rats. Based on the above experimental results, we hypothesized NR1, NR2B, and PKCε of spinal cord could play an important role in moxibustion inhibiting the process of central sensitization and visceral hyperalgesia state.

Figure 1

Diagram of moxibustion in rats. Rats were immobilized on a fixing frame. A moxa stick was lighted, and the lighted end was placed perpendicularly to the acupoint, approximately 2 cm above it. This was performed for 10 min once every day for 7 days.

Figure 2

AWR scores of rats in each group after moxibustion intervention. Under the same intensity of CRD stimulation, compared to the normal group, ^▲ P < 0.001; compared to the model group, ^■ P < 0.001; compared to the Ro 25-6981 group, ^⧫ P < 0.05, ^◊ P > 0.05.

Figure 3

Pathology of colon tissues (HE stain ×200). The observation of colon tissues under a light microscope showed that the overall structure of colon tissues of rats in the model group was clear. There were no abnormal pathological changes such as hyperplasia, erosions, or ulcers. There was no significant inflammatory cell infiltration and no obvious interstitial edema. The mucosal epithelium was complete, the glands in the lamina propria were arranged orderly, and the distribution of submucosa layer and muscular layer was regular.

Figure 4

NR1 mRNA expression in the spinal cord was significantly different between all groups (P < 0.001) except the moxibustion versus the Ro 25-6981 group (P > 0.05) (a). NR2B (b) and PKCε (c) followed the same pattern. Pairwise comparisons by LSD t-test. Compared to the normal group, ^▲▲ P < 0.001 and ^▲ P < 0.01; compared to the model group, ^■■ P < 0.001; compared to the Ro 25-6981 group, ^◊ P > 0.05.

Figure 5

The differences in the expression of NR1 ((a) and (d)), NR2B ((f)–(i)), and PKCε ((k)–(n)) in spinal cord were statistically significant between all groups (P < 0.001) ((e), (j), and (o)). The least significant difference (LSD) t-test was performed for pairwise comparison, and the results showed that the expression of NR1, NR2B, and PKCε in the model group significantly increased compared to the normal group (P < 0.001) ((b), (g), and (l)). Compared to the model group, the expression of NR1, NR2B, and PKCε in the moxibustion group ((d), (i), and (n)) and the Ro 25-6981 group ((c), (h), and (m)) significantly decreased (P < 0.001) ((e), (j), and (o)). Compared to the normal group, ^▲ P < 0.001; compared to the model group, ^■ P < 0.001. The comparison between the moxibustion group and the Ro 25-6981 group did not show a significant difference (^◊ P > 0.05) (magnification ×200).

Figure 6

The levels of NR1 and NR2B proteins in spinal cord of rats were different between all groups (P < 0.001) ((a), (b), (c), and (d)). Further pairwise comparison using the LSD t-test showed that the expression of NR1 and NR2B proteins in the model groups significantly increased compared to the normal group (P < 0.001). Compared to the model group, NR1 and NR2B proteins in the moxibustion group and Ro 25-6981 group both significantly decreased (P < 0.05). PKCε showed a similar pattern. ((e) and (f)) compared to the normal group, ^▲▲ P < 0.001, ^▲ P < 0.01, and ^△ P < 0.05; compared to the model group, ^□ P < 0.05; compared to the Ro 25-6981 group, ^◊ P > 0.05.