Preventive Electroacupuncture Alleviates Oxidative Stress and Inflammation via Keap1/Nrf2/HO-1 Pathway in Rats with Cyclophosphamide-Induced Premature Ovarian Insufficiency.

Electroacupuncture (EA) is a popular therapeutic therapy for premature ovarian insufficiency (POI). However, little has been known about the underlying processes of EA therapy. To investigate the benefit of EA and reveal the mechanism, thirty SD female rats were allocated into the control, model, sham, EA, and GnRHa groups at random. Vaginal smears were used to monitor the rats' estrous cycle. Serum liver and renal function (ALT, AST, BUN, and Cr), sex hormone (FSH, E2, and AMH), oxidative stress markers (SOD, GSH, and MDA), and inflammatory cytokine (IL6, IL1ß, and TNFα) levels were measured by enzyme-linked immunosorbent assay (ELISA). Their ovary morphology was observed by hematoxylin-eosin staining. Transmission electron microscope was used to remark the ultrastructure of the granulocytes. Protein and gene expressions of Keap1/Nrf2/HO-1 pathway were detected by western blot and RT-PCR. Compared with the model group, in the EA group, the levels of serum sex hormones recovered to normal levels. Moreover, it reduced oxidative stress in rats, as demonstrated by increased SOD and GSH levels and decreased MDA levels. Meanwhile, Keap1 mRNA and protein expression dropped considerably in the EA group, while the mRNA and protein expressions of Nrf2 and HO-1 increased. We found that preventive EA might rescue rats with CTX-induced ovarian dysfunction. The anti-inflammatory and antioxidative stress properties of EA, which elevated the Keap1/Nrf2/HO-1 signaling pathway, might be the underlying mechanism. Furthermore, as compared to GnRHa, electroacupuncture did not raise the burden of the liver (ALT and AST) or the kidney (BUN and Cr). Electroacupuncture has a meaningful impact and a sufficient level of safety, making it useful for therapeutic setting in POI.

[Simultaneous determination of baicalin and chlorogenic acid in human plasma by UPLC-MS/MS].

To develop and validate an ultra performance liquid chromatography tandem mass spectrometric (UPLC-MS/MS) method for the simultaneous quantification of baicalin and chlorogenic acid in human plasma after iv infusion of Yinhuang injection, the analytes were isolated from plasma by protein precipitation with methanol. Then they were chromatographied on an Acquity UPLC BEH C18 column (50 mm x 2.1 mm ID, 1.7 microm) at 40 degrees C. The mobile phase A consisted of water and 0.1% formic acid. The mobile phase B consisted of methanol and 0.1% formic acid. The analytes were eluted from the column with a linear gradient from 5% B to 80% B in 5 min, then hold for 0.5 min before returning to initial condition. The flow rate was 0.35 mL x min(-1). A tandem mass spectrometer equipped with electrospray ionization source was used as detector. Multiple reaction monitoring (MRM) using the precursor to product ion pairs of m/z 447-->271 (for baicalin), m/z 353-->191 (for chlorogenic acid) and m/z 287-->287 (for internal standard) were used to quantification. The linear concentration ranges of the calibration curves for baicalin and chlorogenic acid ranged from 9.6 to 1540 ng x mL(-1) and from 7.5 to 1200 ng x mL(-1), respectively. The intra- and inter-day relative standard deviation (RSD) across three validations run over the entire concentration range was less than 10.2% for both baicalin and chlorogenic acid. After iv infusion of Yinhuang injection to the volunteers, the concentration-time curves of baicalin and chlorogenic acid fitted the two-compartment and three-compartment model. T(1/2)alpha were (4.47 +/- 0.89) and (7.65 +/- 4.42) min, T(1/2)beta were (46.22 +/- 10.03) and (34.40 +/- 19.16) min, respectively. The method was proved to be highly sensitive, selective, and suitable for pharmacokinetic investigations of both baicalin and chlorogenic acid.