Label-free based quantitative proteomics analysis to explore the molecular mechanism of gynecological cold coagulation and blood stasis syndrome.

Cold coagulation and blood stasis (CCBS) syndrome is one of the common traditional Chinese medicine (TCM) syndromes of gynecological diseases. However, the molecular mechanism of CCBS syndrome is still unclear. Thus, there is a need to reveal the occurrence and regulation mechanism of CCBS syndrome, in order to provide a theoretical basis for the treatment of CCBS syndrome in gynecological diseases. The plasma proteins in primary dysmenorrhea (PD) patients with CCBS syndrome, endometriosis (EMS) patients with CCBS syndrome, and healthy women were screened using Label-free quantitative proteomics. Based on the TCM theory of "same TCM syndrome in different diseases," the differentially expressed proteins (DEPs) identified in each group were subjected to intersection mapping to obtain common DEPs in CCBS syndrome. The DEPs of gynecological CCBS syndrome in the intersection part were again cross-mapped with the DEPs of gynecological CCBS syndrome obtained by the research group according to the TCM theory of "different TCM syndromes in same disease" theory in the early stage, so as to obtain the DEPs of gynecological CCBS syndrome that were shared by the two parts. The common DEPs were subjected to bioinformatics analysis, and were verified by enzyme-linked immunosorbent assay (ELISA). A total of 67 common DEPs were identified in CCBS syndrome, of which 33 DEPs were upregulated and 34 DEPs were downregulated. The functional classification of DEPs involved in metabolic process, energy production and conversion, immune system process, antioxidant activity, response to stimulus, and biological adhesion. The subcellular location mainly located in the cytoplasm, nucleus, and extracellular. Gene ontology (GO) enrichment analysis showed that the upregulated DEPs mainly concentrated in lipid transport, cell migration, and inflammatory reaction, and the downregulated DEPs mostly related to cell junction, metabolism, and energy response. Protein domain enrichment analysis and clustering analysis revealed that the DEPs mainly related to cell proliferation and differentiation, cell morphology, metabolism, and immunity. The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis clustering analysis showed that the upregulated DEPs were involved in inflammation and oxidative damage, while the downregulated DEPs were involved in inflammation, cell adhesion, cell apoptosis, and metabolism. The results of ELISA showed significantly increased levels of Cell surface glycoprotein MUC18 (MCAM) and Apolipoprotein C1 (APOC1), and significantly decreased levels of Vasodilator-stimulated phosphoprotein (VASP), Fatty acid-binding protein 5 (FABP5), and Vinculin (VCL) in patients with CCBS syndrome compared with healthy women. We speculated that cold evil may affect the immune process, inflammatory response, metabolic process, energy production and conversion, oxidative damage, endothelial cell dysfunction, and other differential proteins expression to cause CCBS syndrome in gynecological diseases.

Effects of Bushen-Tiaojing-Fang on the pregnancy outcomes of infertile patients with repeated controlled ovarian stimulation.

Bushen-Tiaojing-Fang (BSTJF) is commonly used to treat infertility. This study investigated the effects of BSTJF on the pregnancy outcomes of patients with repeated controlled ovarian stimulation (COS), on mitochondrial function, and on oxidative stress in ovarian granulosa cells (GCs) and follicular fluid (FF). The samples and clinical data of 97 patients, including 35 in the control group, 29 in the placebo group and 33 in the BSTJF group, were collected for this study. The mitochondrial ultrastructure, ATP content, mitochondrial DNA (mtDNA) number, 8-hydroxy-2-deoxyguanosine (8-OHdG), Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase (GSH-Px) activity levels, and mRNA expression levels of Mn-SOD, GSH-Px, and nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) were analyzed. The high-grade embryo (P < 0.001), implantation (P = 0.033), and clinical pregnancy (P = 0.031) rates, as well as the ATP content (P = 0.014), mtDNA number (P = 0.035), GSH-Px activity (P = 0.004 in GCs and P = 0.008 in FF) and mRNA expression levels (P = 0.019), were significantly lower in the placebo group than in the control group, whereas the 8-OHdG content was significantly (P = 0.006 in FF) higher in the placebo group than in the control group. Compared with those in the placebo group, the high-grade embryo rate (P = 0.007), antioxidant enzyme activity (P = 0.037 and 0.036 in Mn-SOD; P = 0.047 and 0.030 in GSH-Px) and mRNA level (P < 0.001 in Nrf2, P = 0.039 in Mn-SOD and P = 0.002 in GSH-Px) were significantly higher in the BSTJF group, as were changes in mitochondrial ultrastructure, ATP (P = 0.040) and mtDNA number (P = 0.013). In conclusion, BSTJF can improve oxidative stress in patients with repeated COS and pregnancy outcomes.

[Effects of encircled needling plus electroacupuncture on hyperplastic mammary glands and serum sex hormones in the rat].

OBJECTIVE: To observe the effect of encircled needling plus electroacupuncture (EA) of the second pair of nipples on hyperplastic mammary glands, serum estradiol (E2), progesterone (P), prolactin (PRL) and testosterone (T) contents, estrogen receptor (ER) expression of the mammary glands in mammary hyperplasia rats. METHODS: Forty Wistar rats were equally randomized into normal control, model, acupuncture and medication groups. Mammary gland hyperplasia model was established by intramuscular injection of diethylstilbestrol (0.5 mg/kg, once daily for 25 days) and progesterone (4 mg/kg, once daily for 5 days). For rats of the acupuncture group, 4 filiform needles were inserted into the surrounding tissues of the second pair of breasts, respectively, followed by giving the animal with EA stimulation for 30 min. Another one acupuncture needle was inserted into "Danzhong" (CV 17) and retained for 30 min. For rats of the medication group, tamoxifen solution (1.8 mg/kg) was given by gavage. Both medication and EA intervention were given once daily for 30 days. The diameter and height of the second pair of nipples were measured before and on the 10th, 20th and 30th days of the treatment. Serum E2, P, PRL and T levels were assayed by radioimmunoassay, and the tissues of the second pair of nipples were stained with H. E. method. Estrogen receptor immunoactivity of the breast tissue was detected by immunohistochemistry. RESULTS: The diameter and height of the second pair of nipples, serum E2, PRL and T contents, and breast ER protein expression level were significantly higher in the model group than in the normal control group (P<0.05), while serum P content was remarkably lower in the model group than in the normal control group (P<0.05). In comparison with the model group, the diameter and height of the second pair of nipples, serum E2, PRL and T contents, and breast ER protein expression level were considerably lower in both acupuncture and medication groups (P<0.05), and serum P contents were significantly higher in the latter two groups (P<0.05). In addition, H.E. staining showed that the numbers of the lobule, alveolus and ducts of mammary glands were significantly increased, and the expansion of the acinar lumina and ducts was found in the model group. These pathological changes were relatively milder in both acupuncture and medication groups. CONCLUSION: Encircled needling plus EA stimulation of the surrounding tissues of the nipples can improve the hyperplasic nipples and pathological changes of the breast tissue in mammary gland hyperplasia rats, which is closely associated with its functions in lowering serum estradiol, prolactin, testosterone contents and estrogen receptor protein expression of the breast, and in increasing serum progesterone level.