Effects of moxibustion on miRNA-133b, Pitx3/TH, and neurotransmitters in the midbrain of rats with diarrhea-predominant irritable bowel syndrome
Authors: Chen Jinyu , Wang Jiaojiao , Zou Ling , Zhu Shanshan , Li Kuiwu , Liao Lumin , Ruan Jingru , Chu Haoran 
Published: December 2022

Objective

To investigate the mechanism of moxibustion in the treatment of diarrhea-predominant irritable bowel syndrome (IBS-D), by observing the effects of moxibustion at Tianshu (ST25) and Shangjuxu (ST37) on microRNA-133b (miRNA-133b), pituitary homeobox family factor 3 (Pitx3)/tyrosine hydroxylase (TH), and neurotransmitters in the brain tissue of IBS-D rats.

Methods

Healthy Sprague-Dawley rats were randomly divided into a normal group, a model group, a moxibustion group, and a Western medicine group, with 12 rats in each group. Except for the normal group, the IBS-D rat model was established by mother-offspring separation and acetic acid enema combined with restraint stress stimulation in all the other groups. No intervention was performed in the normal and model groups. Mild moxibustion was applied to both Tianshu (ST25) and Shangjuxu (ST37) in the moxibustion group. Rifaximin was given by gavage in the Western medicine group. The physical status of rats in each group was observed at different periods. After the intervention, hematoxylineosin staining was performed to observe the histopathological morphology of rat colon; enzyme-linked immunosorbent assay was used to measure the levels of dopamine (DA), noradrenaline (NE), and 5-hydroxytryptamine (5-HT) in plasma, colon, and midbrain tissue of rats; the relative expression levels of miRNA-133b, Pitx3 mRNA, and TH mRNA in the midbrain tissue were measured by real-time fluorescence quantitative polymerase chain reaction, and the relative expression levels of Pitx3 and TH proteins in the midbrain tissue were measured by Western blotting and immunofluorescence.

Results

The body weights of rats among groups and at different time points were statistically different (P<0.01). The body weight of the normal group was higher than that of the other groups over time (P<0.01). After modeling, the minimum volume threshold of abdominal withdrawal reflex (AWR) was significantly lower (P<0.01) and the loose stool rate was significantly higher (P<0.01) in the model, moxibustion, and Western medicine groups compared with the normal group; the miRNA-133b expression in the midbrain tissue was significantly lower (P<0.01), the expression levels of Pitx3 and TH in the midbrain tissue were significantly higher (P<0.01), and the levels of DA, NE, and 5-HT in plasma, colon and midbrain tissue were significantly higher (P<0.01). After the intervention, the minimum volume threshold of AWR was significantly higher (P<0.01), the loose stool rate was significantly lower (P<0.01), the miRNA-133b expression was significantly increased (P<0.01 orP<0.05) and the expression levels of Pitx3 and TH were significantly decreased (P<0.01) in the midbrain tissue, the levels of DA, NE, and 5-HT in plasma, colon, and midbrain tissue were significantly reduced (P<0.01) in the moxibustion and Western medicine groups compared with the model group; the levels of 5-HT in the colon and midbrain tissue of the moxibustion group were significantly lower than those in the Western medicine group (P<0.05), and there was no statistical difference compared with the remaining groups (P>0.05). Linear correlation analysis showed that miRNA-133b was negatively correlated with Pitx3 (r<0,P<0.01); Pitx3 with TH, TH with DA, and NE with 5-HT were positively correlated (r>0,P<0.01).

Conclusion

Moxibustion at Tianshu (ST25) and Shangjuxu (ST37) improves diarrhea symptoms and visceral hypersensitivity in IBS-D rats. The mechanism may be related to up-regulating miRNA-133b, inhibiting Pitx3/TH, and reducing neurotransmitter expression levels in the midbrain tissue.