Ziwuliuzhu acupuncture modulates Glu/GABA‑Gln metabolic loop abnormalities in insomniac rats

doi:10.12122/j.issn.1673-4254.2025.08.06

Abstract

Abstract:

Objective To investigate the therapeutic effect of Ziwuliuzhu acupuncture in a rat model of insomnia and its regulatory effect on the glutamic acid (Glu)/γ-aminobutyric acid (GABA)-glutamine (Gln) metabolic loop. Methods Forty male SD rats were randomly assigned to control group, model group, Najia group and Nazi group (n=10). In the latter 3 groups, rat models of insomnia were established by intraperitoneal injections of p-chlorophenylalanine and verified using a sodium pentobarbital-induced sleep test. After modeling, the rats in Najia and Nazi groups received acupuncture for 7 days at specifically chosen sets of acupoints based on the Ziwuliuzhu rationale in traditional Chinese medicine. Pathological changes in the hypothalamic tissue of the rats were examined with HE staining, and the levels of Glu and GABA in the hypothalamus were determined with high-performance liquid chromatography (HPLC)-mass spectrometry (MS)/MS. Immunohistochemistry was used to detect the expressions of GABAA receptors (GABAARs) in the hypothalamus, and the expression levels of glutamate decarboxylase (GAD65/67) and glutamine synthetase (GS) were determined with Western blotting. Results Compared with the model group, the rats in Najia and Nazi groups exhibited decreased Glu levels and GABAA receptor expression and increased GABA levels with a decreased Glu/GABA ratio in the hypothalamus. Ziwuliuzhu acupuncture significantly increased the protein expressions of GAD65 and GAD67 and lowered the expression of GS in the hypothalamus in the rat models of insomnia. Conclusion Ziwuliuzhu acupuncture produces sedative and hypnotic effects in rat models of insomnia possibly by regulating Glu and GABA-Gln metabolism to restore the excitatory/inhibitory balance between Glu and GABA.

Key words: acupuncture, glutamic acid, γ-aminobutyric acid, glutamic acid/γ-aminobutyric acid-glutamine metabolism loop, insomnia

Jiarong XU, Ao HUANG, Zhikai DING, Yu BAO, Canghuan ZHAO, Wenzhi CAI. Ziwuliuzhu acupuncture modulates Glu/GABA‑Gln metabolic loop abnormalities in insomniac rats[J]. Journal of Southern Medical University, 2025, 45(8): 1616-1624.

Figures/Tables 8

Fig.1 Illustration of the locations of the chosen acupoints in rats in the prone and supine positions. Rats were stimulated using specific acupuncture points with anatomical correspondence to human acupoints. The SP1 (Yinbai) is located at the medial aspect of the big toe, approximately 0.1 cun posterior to the corner of the nail. LR3 (Taichong) is in the depression distal to the junction of the 1st and 2nd metatarsal bones. HT7 (Shenmen) is in the transverse crease of the wrist of the forepaw, radial to the flexor carpi ulnaris tendon. PC7 (Daling) is on the midpoint of the transverse crease of the wrist between the tendons of the palmaris longus and flexor carpi radialis. LU8 (Jingqu) is in the depression between the styloid process of the radius and the radial side, 1 cun above the transverse crease of the wrist. KI2 (Rangu) is located on the medial border of the foot, inferior to the tuberosity of the navicular bone, at the junction of the pink and pale skin. KI10 (Yingu) is located on the medial side of the popliteal fossa between the tendons of the semitendinosus and semimembranosus.

Fig.2 Results of pentobarbital sodium-induced sleep test (sleep latency and sleep duration) in the 4 groups (Mean±SD, n=10). A: Sleep latency. B: Sleep duration. ****P<0.0001, ***P<0.001 vs the control group; ※P<0.05, ※※P<0.01, ※※※※P<0.0001 vs model group; €P<0.05 vsNajia group.

Fig. 3 Pathological examination of rat hypothalamus in each group (HE staining, scale bar=50 μm). A: Control group. B: Model group. C: Najia group. D: Nazi group.

Fig.4 Effect of Ziwuliuzhu acupuncture on the expression of GABA and Glu in the hypothalamus. The expression levels of Glu (A) and GABA (B) in the hypothalamus were assessed via HPLC-MS/MS. The values are the Means±SD (n=6). Post hoc LSD test: ***P<0.001 vs the control group; ※※※※P<0.0001, ※※※P<0.001, ※P<0.05 vs the model group.

Fig.5 Expressions of GABAARs in the hypo-thalamus of the rats in the 4 groups. A: Immunohistochemistry for GABAAR in the hypothalamus in each group (Original magnification: ×100 or ×400). B: Comparison of mean OD values among the groups (Mean±SD, n=6). Post hoc LSD test: **P<0.01 vs control group; ※※P<0.01 vs model group.

Fig.6 Effect of Ziwuliuzhu acupuncture on expressions of protein expressions in the hypothalamus of the rats. A: Western blotting for GAD65, GAD67, and GS in the hypothalamic tissues. B: Quantitative analysis of the protein expression levels (Mean±SD). Post hoc LSD test: ****P<0.0001, ***P<0.001 vs the control group; ※※※※P<0.0001, ※※※P<0.001, ※※P<0.01, ※P<0.05 vs model group; €€P<0.01 vsNajia group.

Fig.7 Ziwuliuzhu acupuncture corrects imbalance in the Glu/GABA-Gln metabolic loop.

Fig.8 Schematic diagram of the tricarboxylic acid (TCA) cycle.

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