[Zuogui Pills and Yougui Pills in differential treatment of diminished ovarian reserve based on metabolomics].

Based on the metabolomics, this paper systematically analyzed the metabolic substance basis of Zuogui Pills and Yougui Pills in syndrome differentiation and treatment of diminished ovarian reserve(DOR), so as to provide a scientific basis for the traditional Chinese medicine(TCM) syndrome differentiation and treatment of DOR. Patients with DOR of kidney-Yin deficiency syndrome were collected from outpatient department of hospitals and treated with Zuogui Pills for 12 weeks. And kidney-Yang deficiency syndrome were treated with Yougui Pills for 12 weeks. Based on the non-targeted metabolomic research techniques, the potential biomarkers of Zuogui Pills and Yougui Pills in the treatment of DOR with kidney-Yin deficiency and kidney-Yang deficiency, respectively, were screened out, and metabolic pathways of biomarkers were analyzed. The pregnancy rate, basic serum hormone levels [basal follicle-stimulating hormone(bFSH), basal-luteinizing hormone(bLH), basal-estradiol(bE_2), and anti-Müllerian hormone(AMH)], TCM syndrome type score, and Kupperman score were recorded and statistically analyzed after treatment. The results showed that 23 patients with DOR of kidney-Yin deficiency syndrome and 25 patients of kidney-Yang deficiency syndrome were collected. Twenty-six differential metabolites, including L-carnitine, acetyl-CoA, coenzyme A, and coenzyme Q_(10)(CoQ10), were mapped to 12 metabolic pathways in patients with kidney-Yin deficiency treated with Zuogui Pills. Twenty-two differential metabolites, such as adipoyl-CoA, L-lysine, lysine arginine, and α-tocopherol, were mapped to 11 metabolic pathways in patients with kidney-Yang deficiency. After treatment, bFSH and bLH of patients with DOR were significantly lower than those before treatment(P<0.05). Although the comparison of bE_2 and AMH had no significant differences, there was a improvement trend. The TCM syndrome type score and Kupperman score of patients with DOR after TCM treatment were significantly lower than those before treatment(P<0.05).

Network pharmacology and experimental validation to explore the molecular mechanisms of Bushen Huoxue for the treatment of premature ovarian insufficiency.

Bushen Huoxue (BSHX) has been applied in clinical traditional Chinese medicine treatment, and has definitive clinical efficacy in the treatment of Premature Ovarian Insufficiency (POI) in China. However, little is known of the underlying mechanism of BSHX. The purpose of this paper is to study the pharmacological mechanisms of BSHX acting on POI based on a pharmacology and experimental validation. The pharmacological database of chinese medicine system and analysis platform (TCMSP) were used to search the effective active ingredients and potential action targets of BSHX. Drugbank, Online Mendelian Inheritance in Man (OMIM), Genecards, and Disgenet databases were used to obtain relevant targets of POI. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and the visual network of protein-protein interaction network were constructed by FunRich3.1. Pymol software, and Auto Dock tools 1.5.6 were used for molecular docking. Murine model of POI was used to further investigate the mechanism of BSHX against on POI. Finally, 127 active compounds were collected from TCMSP database, and 215 active targets were identified. There were 1366 targets related to POI and 99 targets of BSHX for the treatment of POI. Quercetin, kaempferol, and stigmasterol were recognized as the most effective compounds corresponding to targets. The top three genes according to degree value are TP53, Akt1, and VEGFA. Further, the results of GO and KEGG enrichment analysis revealed that those core targets were mainly enriched on TRAIL and TGF-ß receptor signaling. The results of molecular docking showed that stigmasterol had good binding ability to Akt1. Moreover, experimental validation suggests that BSHX significantly Increased the expression of TGF-ß1 and Smad2/3, regulating the release of serum sex hormones, which include Follicular stimulating hormone (FSH), Estradiol (E2), and Antimullerin hormone (AMH).

HIGHLIGHTSBSHX treats POI by regulating TGF-ß1 and Smad2/3 signaling pathways; Quercetin, kaempferol, and stigmasterol were the most effective compounds in BSHX.

The effect of Bu Shen Huo Xue Tang on autoimmune premature ovarian insufficiency via Modulation of the Nrf2/Keap1 signaling pathway in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu Shen Huo Xue Tang (BSHXT) is a traditional Chinese medicine formula that is clinically used in the treatment of premature ovarian insufficiency (POI). However, its therapeutic mechanism remains unclear. AIM OF THE STUDY: This study aimed to investigate the underlying molecular mechanism of pharmacological activity of BSHXT, via the Nrf2/Keap1 signaling pathway, in the treatment of autoimmune POI. MATERIALS AND METHODS: The chemical composition of BSHXT was analyzed using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry. The autoimmune POI mouse model was induced by immunizing mice twice, with zona pellucida (ZP) glycoprotein 3 antigen. The autoimmune POI mice were continuously administered BSHXT for 28 days. Body weight and organ indices were recorded. The pathological morphology of the ovaries was observed. The estrous cycle of each mouse was recorded. Immunofluorescence assay was used to detect the levels of ZP antibodies in the mouse ovaries. The levels of ZP antibodies, follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), estradiol (E2), luteinizing hormone (LH), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured. The expression of genes and proteins involved in the Nrf2/Keap1 signaling pathway were measured by Q-PCR and IHC, respectively. RESULTS: Twenty-one compounds were identified in the BSHXT water extract. BSHXT was found to increase the body weight and ovarian index, improve ovarian function, and reduce disorders in the estrus cycle. It also reduced the expression of ZP antibodies in the ovaries and serum of POI mice. BSHXT significantly increased E2 and AMH levels and decreased FSH and LH levels. It also increased the levels of SOD, and reduced MDA levels. The levels of Nrf2 and Keap1 were also increased, and the expression of Nrf2, HO-1 and NQO1 genes was upregulated. CONCLUSIONS: BSHXT has a therapeutic effect on autoimmune POI in mice, which may be a result of the enhanced antioxidant capacity and activation of the Nrf2/Keap1 signaling pathway. BSHXT is a good drug candidate for use as a protective agent for POI and may be used in clinical practice.

Tandem mass tag-based proteomic analysis reveals the treatment mechanism of Bushen Huoxue Formula on psychological stress-induced premature ovarian insufficiency.

ETHNOPHARMACOLOGICAL RELEVANCE: Bushen Huoxue formula (BSHXF) is a Chinese herbal prescription composed of eleven herbs widely used to treat psychological stress-induced premature ovarian insufficiency (POI) in clinical. However, the underlying mechanism is still unclear. AIM OF THE STUDY: The purpose of this study was to clarify the underlying molecular mechanisms of BSHXF in the treatment of psychological stress-induced POI. MATERIALS AND METHODS: The rat model was induced by corticosterone (CORT, 40mg/kg). Drugs were administered to rats once daily for 21 days. The serum E2, FSH and AMH levels were examined by enzyme-linked immunosorbent assays. Tandem mass tag-based proteomic analysis was used to identify differentially expressed proteins. Western blot was used to verify the results of proteomic. RESULTS: Our results indicate that BSHXF can improve ovarian disfunction. The levels of serum FSH were signally enhanced in model group compared to control group. As respected, BSHXF treatment for 3 weeks led to the decreased FSH levels than the model group. The concentrations of AMH showed an obvious decrease in the model group and were increased by BSHXF treatment. Moreover, the size and number of follicles in the BSHXF groups were similar to those in the control group. The proteomic screened out that Np4 and Angptl4 were simultaneously enriched by GO and KEGG, thus these two proteins were chosen for further study. CONCLUSIONS: These findings revealed that BSHXF might regulate the expression of Np4 and Angptl4 to improve psychological stress-induced POI.

Total C-21 steroidal glycosides, isolated from the root tuber of Cynanchum auriculatum Royle ex Wight, attenuate hydrogen peroxide-induced oxidative injury and inflammation in L02 cells.

Oxidative stress plays an important role in the pathology of liver disorders. Total C­21 steroidal glycosides (TCSGs), isolated from the root tuber of Cynanchum auriculatum Royle ex Wight, have been reported to exert numerous effects, including liver protective and antioxidant effects. In order to investigate the potential mechanisms underlying the protective effects of TCSGs on liver function, the present study used the human normal liver cell line, L02, to evaluate the effects of TCSGs on hydrogen peroxide (H2O2)­induced oxidative injury and inflammatory responses. The L02 cells were pretreated with various concentrations of TCSGs, followed by exposure to 1.5 mM H2O2. Cell viability was determined by a 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assay. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and nitric oxide (NO) were measured using colorimetric assays. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH­Px) and the production of malondialdehyde (MDA) were also determined. Intracellular reactive oxygen species (ROS) levels were detected using a fluorescent probe. H2O2­induced oxidative toxicity was attenuated following treatment with TCSGs, as indicated by the increase in cell viability, the decreased levels of ALT, AST, LDH, NO, MDA and ROS, and the increased activities of SOD, CAT and GSH­Px. To further explore the possible mechanisms of action of TCSGs, the nuclear factor erythroid 2­related factor 2 (Nrf2) and nuclear factor­κB (NF)­κB pathways were examined. The results revealed that treatment with TCSGs markedly induced Nrf2 nuclear translocation and upregulated the expression of heme oxygenase­1 (HO­1) in the L02 cells damaged by H2O2. In addition, pretreatment with TCSGs inhibited the NF­κB signaling pathway by blocking the degradation of the inhibitor of nuclear factor κBα (IκBα), thereby reducing the expression and nuclear translocation of NF­κB, as well as reducing the expression of tumor necrosis factor­α (TNF­α), interleukin-6 (IL­6), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX­2). On the whole, the findings of this study demonstrate that TCSGs can protect L02 cells against H2O2­induced oxidative toxicity and inflammatory injury by increasing the expression of Nrf2 and HO­1, mediated by the NF­κB signaling pathway.

Protective function of Bu Shen Huo Xue formula on the immunity of B6AF1 mice with experimental autoimmune premature ovarian failure.

Autoimmune abnormality is one of the main causes of premature ovarian failure (POF). Bu Shen Huo Xue formula (BSHXF), a traditional Chinese medicine formula, has been clinically used for the treatment of patients with POF in China. Regulatory T cells (Tregs) are important in the pathogenesis of autoimmune POF. The aim of the present study was to evaluate the immunoprotective effects of BSHXF on POF and the underlying mechanisms. An experimental autoimmune POF model was induced in B6AF1 mice with zona pellucida 3 (ZP3) fragments. Following modeling, BSHXF (31.53 g/kg/day) was orally administered for 4 weeks. CD4+ T lymphocytes, Tregs, anti-zona pellucida (anti-ZP) antibodies and cytokines were detected using flow cytometry, enzyme-linked immunosorbent assays, reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. The results revealed that BSHXF exhibited an immunoprotective function and reduced inflammatory cell infiltration and damage to the ovary. BSHXF upregulated the percentage of CD4+ CD25+ forkhead box P3+ T cells in the spleen, effectively inhibiting the activation of CD4+ T lymphocytes. The proliferation of Tregs was increased in serum obtained from mice in the BSHXF group in vitro. Anti-ZP antibodies and interleukin-10 and interferon-γ levels were decreased in the serum in the BSHXF-treated group. The present study demonstrated that BSHXF had an immunoprotective effect on POF model mice. Additionally, it indicated that the protective mechanisms of BSHXF may be associated with an increase in Treg cells.

Metabolomic study of biochemical changes in the plasma and urine of primary dysmenorrhea patients using UPLC-MS coupled with a pattern recognition approach.

Primary dysmenorrhea (PD) is characterized by painful menstrual cramps without any organic pathology and has a prevalence of up to 90% in adolescents. Recent advances in its etiology and pathogenesis are providing more speculative hypotheses focused on integral systems. Using a targeted tandem mass spectrometry (MS/MS)-based metabolomic platform, we explored the changes of metabolic profiling in plasma/urine simultaneously between PD patients and healthy controls before and after a 3-month herbal medicine (namely Shaofu Zhuyu formula concentrated-granule, SFZYFG) therapy. To detect and identify potential biomarkers associated with PD and SFZYFG treatment, we also performed a combined UPLC-QTOF-MS/MS-based metabolomic profiling of the plasma/urine samples, indicating a further deviation of the patients' global metabolic profile from that of controls. The total thirty-five metabolites (nineteen in plasma and sixteen in urine), up-regulated or down-regulated (p < 0.05 or 0.01), were identified and contributed to PD progress. These promising identified biomarkers underpinning the metabolic pathway including sphingolipid metabolism, steroid hormone biosynthesis, and glycerophospholipid metabolism are disturbed in PD patients, which were identified by using pathway analysis with MetPA. Twenty-four altered metabolites and fourteen biochemical indicators were restored back to the control-like level after the treatment of SFZYFG and could be potential biomarkers for monitoring therapeutic efficacy. These findings may be promising to yield a valuable insight into the pathophysiology of PD and to advance the approaches of treatment, diagnosis, and prevention of PD and related syndromes.

Biomarkers of primary dysmenorrhea and herbal formula intervention: an exploratory metabonomics study of blood plasma and urine.

Primary dysmenorrhea (PDM), a common clinical endocrine disorder affecting young women, is associated with endocrinopathy and metabolic abnormalities. Although some physiological and pathological function parameters have been investigated, little information about the changes of small metabolites in biofluids has been reported, which may cause poor diagnosis and treatment for PDM. The Xiang-Fu-Si-Wu Formula (XFSWF) is a Chinese herbal formula used to treat PDM for hundreds of years. The aim of this study was to establish the metabolic profile of PDM and investigate the action mechanism of XFSWF effect. In this cross-sectional study of 25 patients with PDM and 12 healthy controls, contents of small molecular endogenous metabolites in blood plasma and urine samples were measured by ultra performance liquid chromatography (UPLC) coupled with quadrupole-time-of-flight mass spectrometry (QTOF/MS) and triple quadrupole mass spectrometry (QqQ/MS) based techniques and analyzed by multivariate statistical methods. The levels of LPCs including lypso (16 : 1), lysoPC(20 : 4), lysoPC(18 : 2), lysoPC(16 : 0), lysoPC(18 : 1), lysoPC(10 : 1), estrone, 17-hydroxyprogesterone, myristoylglycine and palmitoylglycine increased significantly (p < 0.05) in PDM, while the levels of phytosphingosine, dihydrocortisol and sphingosine decreased significantly (p < 0.05) compared with the healthy controls. These significant perturbations are involved in glycerophospholipid metabolism and sphingolipid metabolism, as well as steroid hormone biosynthesis. The metabolic deviations recovered to the normal level after XFSWF intervention. The results demonstrated that biofluids metabonomics was a powerful tool in clinical diagnosis and treatment of PDM for providing information on changes in metabolites and neural, endocrinal and immune pathways. XFSWF can be used for the treatment of PDM cases, especially for those adolescents who do not desire a contraceptive method, to reduce the risk of secondary dysmenorrhea.

[Magnetic resonance characteristics of endometriosis rat model].

OBJECTIVE: To establish a non-invasive, repeatable and dynamic study method in endometriosis rat model using magnetic resonance imaging (MRI), in order to explore the magnetic resonance characteristics of the model. METHOD: Endometrium tissues were transplanted into left abdominal walls of unmated adult female SD rats. After surgery, pathological changes were observed and MRI scanning was made for the ectopic lesions. RESULT: The endometriosis rat model was successfully established and the ectopic lesions imaged strong hyperintense on DWI, hypointense on T1WI, hyperintense on T2WI with a clear border, without enhancement on CE-T1 WI. CONCLUSION: The lesions can be clearly observed in the MRI images on the endometriosis rat model established by this method, which facilitates repeat experiments and continuous observation studies.

[Effects of bushen huoxue decoction on neurobiochemical markers in the hippocampus of female rats with repeated immobilization stress].

OBJECTIVE: To study the effect o f Bushen Huoxue Decoction (BHD) on neurobiochemical markers in the hippocampus of female rats with repeated immobilization stress. METHODS: Sixty female rats were randomly divided into the normal group, the model group, the positive control group (treated with Liuwei Dihuang Pill at the dose of 3.3 g crude drug/kg), and the high, middle, and low BHD treated groups (at the dose of 8, 4, 2 g crude drug/kg), ten in each group. Chronic psychological stress was induced using repeated immobilization stress in rats. Medication was conducted by gastrogavage while modeling once a day for twenty successive days. The hippocampal neurohumoral levels were detected with high-performance liquid chromatography. The expression levels of BDNF and its receptor in the hippocampus were detected by Westem blot. Effect of BHD on neurobiochemical markers in the hippocampus of rats with repeated immobilization stress was observed. RESULTS: The levels of Glu, GABA, and BDNF in the hippocampus of the normal group were 1280.0 +/- 258.3 ng/mg, 588.3 +/- 115.1 ng/mg, and 13.26 +/- 2.57 gray value, respectively. But the hippocampal neurohumoral levels and the expression of BDNF in the model group obviously decreased when compared with the normal group, being 1016.9 +/- 215.9 ng/mg, 485.1 +/- 71.0 ng/mg, and 7.23 +/- 0.61 gray value, respectively. The levels of Glu (ng/mg) in hippocampus of the three BHD treated groups were 1459.1 +/- 413.5, 1894.7 +/- 542.8, and 1373.3 +/- 345.7, respectively. GABA levels (ng/mg) inthe hippocampus were 631.6 +/- 161.4, 899.1 +/- 262.1, and 656.4 +/- 140.8, respectively. BDNF levels (gray value) were 16.57 +/- 1.52, 29.85 +/- 1.37, and 24.44 +/- 3.81, respectively, significantly higher than that of the model group (P<0.05, P<0.01). The level of Glu in the positive control group (1216.5 +/- 193.8 ng/mg) was significantly higher than that of model group (P<0.05). CONCLUSION: BHD showed significant accommodation on the hippocampal neurohumoral levels and the expression of BDNF in the female rats with repeated immobilization stress.