Electro-Acupuncture Regulates Metabolic Disorders of the Liver and Kidney in Premature Ovarian Failure Mice

Abstract

As per the theory of traditional Chinese medicine (TCM), the liver and kidney dysfunction are important pathogenies for premature ovarian failure (POF). POF is a common gynecological disease that reduced the pregnancy rate. Electro-acupuncture (EA) is a useful non-pharmaceutical therapy that supposedly regulates the function of the liver and kidney in the treatment of POF with TCM. However, the underlying mechanism of EA in the treatment of POF has not been adequately studied through metabonomics with reference to the theory of TCM. Accordingly, we investigated the effect of EA on the liver and kidney metabolites in POF mice through metabolomics. POF mice were established via intraperitoneal injection of cisplatin. Both Sanyinjiao (SP6) and Guanyuan (CV4) were stimulated by EA for 3 weeks. The biological samples (including the serum and the ovary, liver, and kidney tissues) were evaluated by histopathology, molecular biology, and hydrogen-1 nuclear magnetic resonance (¹HNMR)-based metabolomics to assess the efficacy of EA. ¹HNMR data were analyzed by the orthogonal partial least squares discriminant analysis (OPLS-DA). The results revealed that EA was beneficial to ovarian function and the menstrual cycle of POF. Both the energy metabolism and neurotransmitter metabolism in the liver and kidney were regulated by EA. Notably, EA played an important role in regulating energy-related metabolism in the kidney, and the better effect of neurotransmitter-related metabolism in the liver was regulated by EA. These findings indicated that the ovarian functions could be improved and the metabolic disorder of the liver and kidney caused by POF could be regulated by EA. Our study results thus suggested that the EA therapy, based on the results for the liver and kidney, were related to POF in TCM, as preliminarily confirmed through metabolomics.

Keywords: 1HNMR; electro-acupuncture; energy metabolism; metabonomics; neurotransmitter metabolism; premature ovarian failure.

Figure 1

The location of acupoints (CV4 and SP6) and corresponding non-acupoints on mice (A); the EA instrument (B); stainless steel acupuncture needle of 0.25 mm × 13 mm (C).

Figure 2

The weight of four groups (A), and the typical morphology of vaginal exfoliated cells in each estrous cycle (B), proestrus; (C), estrus; (D), metestrus; (E), diestrus; (F), POF model; ×40 magnification) (* means P < 0.05; ** means P < 0.01).

Figure 3

Pathological morphology and granulosa cell apoptosis of ovaries in four groups (A), HE staining and TUNEL detection; (B), the positive rate of granulosa cell apoptosis in antral follicles) (×40 and ×200 magnification; * means P < 0.05; ** means P < 0.01).

Figure 4

The levels of sex hormones were detected by ELISA (A), FSH; (B), LH; (C), E2; (D), AMH.) and the relative expression of estrogen receptor (E), ER-α; (F), ER-β; (G), GRP30) (* means P < 0.05; ** means P < 0.01).

Figure 5

¹HNMR spectra of liver and kidney extracts (1, lactate; 2, alanine; 3, methionine; 4, glutamate; 5, pyruvate; 6, creatine; 7, phosphocholine; 8, betaine; 9, taurine; 10, glycine; 11, glycerol; 12, lysine; 13, serine; 14, aspartate; 15, cysteine; 16, threonine; and 17, glucose).

Figure 6

The OPLS-DA scores plots of four groups in liver [A, R2X(cum) = 0.632, R2Y(cum) = 0.748, Q2(cum) = 0.55] and kidney (B, R2X(cum) = 0.498, R2Y(cum) = 0.75, Q2(cum) = 0.378]. The OPLS-DA scores plots of the control group and the POF group in liver [C, R2X(cum) = 0.326, R2Y(cum) = 0.916, Q2(cum) = 0.621] and kidney [D, R2X(cum) = 0.535, R2Y(cum) = 0.947, Q2(cum) = 0.754].

Figure 7

OPLS-DA and S-plots of liver [A and a, R2X(cum) = 0.606, R2Y(cum) = 0.966, Q2(cum) = 0.689] and kidney extracts in POF group and EA group B and b, R2X(cum) = 0.499, R2Y(cum) = 0.913, Q2(cum) = 0.65].

Figure 8

The relative concentrations of characteristic metabolites in the liver (* means P < 0.05; ** means P < 0.01).

Figure 9

The relative concentrations of characteristic metabolites in the kidney (* means P < 0.05; ** means P < 0.01).

Figure 10

The metabolic pathways of characteristic metabolites in liver and kidney after EA (1, glycine, serine and threonine metabolism; 2, taurine and hypotaurine metabolism; 3, alanine, aspartate, and glutamate metabolism; 4, glycerolipid metabolism; 5, arginine and proline metabolism; 6, arginine biosynthesis; 7, glycolysis/gluconeogenesis; 8, pyruvate metabolism; 9, lysine degradation; 10, glycerophospholipid metabolism; green shows metabolites in liver; blue shows metabolites in kidney; red shows metabolites in liver and kidney).

Figure 11

The correlation analysis between POF and characteristic metabolites in the liver.

Figure 12

The correlation analysis between POF and characteristic metabolites in the kidney.