ObjectiveTo explore the regulation of hypothalamic-pituitary-gonadal （HPG） axis by modified Erxian decoction in rats with perimenopausal syndrome （PMS） and to further analyze the expression of proteins related to the silent information regulator 1 （SIRT1）/hypothalamic kisspeptin （Kisspeptin）/gonadotropin-releasing hormone （GnRH） signaling pathway in the arcuate nucleus region （ARC） of the hypothalamus， so as to reveal the potential target of action and molecular biological mechanism of modified Erxian decoction for the treatment of perimenopausal syndrome. MethodsAn animal model was established via the incomplete castration method， with successful modeling confirmed by the exfoliated cervical cell smear method. The 48 rats were divided into six groups based on the randomization principle after successful modeling， including a sham operation group， a model group， an estradiol valerate group （0.09 mg∙kg^-1∙d^-1）， high-， medium-， and low-dose modified Erxian decoction groups （7.614， 3.807，1.903 5 g∙kg^-1∙d^-1）， with 8 rats in each group. The estradiol valerate group and the high-， medium- and low-dose modified Erxian decoction groups were continuously administered by gavage for 28 days， and the indicators were detected 24 hours after the last administration. Body weights and uterine indices were measured. The pathological changes of the uterus were observed by hematoxylin-eosin （HE） staining. Enzyme-linked immunosorbent assay （ELISA） was performed to measure the levels of estradiol （E₂）， follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and gonadotropin-releasing hormone （GnRH）. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to determine the expression levels of SIRT1， Kisspeptin， kisspeptin receptor （GPR54）， and GnRH in the ARC region of the hypothalamus and gonadotropin-releasing hormone receptor （GnRH-R） in pituitary. ResultsCompared with the sham operation group， rats in the model group had a significantly increased body weight （P0.01）， reduced wet weight and index of uterus （P0.01）， endometrial thinning or atrophy， glandular atrophy， and a decreasing number of glands. Additionally， serum levels of E₂ and the expression of SIRT1 in the ARC region of the hypothalamus significantly decreased （P0.01）. Serum levels of FSH， LH， and GnRH， the expression of Kisspeptin， GPR54， and GnRH in the ARC region of the hypothalamus， and GnRH-R in pituitary significantly increased （P0.01）. Compared with the model group， the estradiol valerate group and the high-， medium-dose modified Erxian decoction groups had significantly reduced body weight， serum levels of FSH， LH， and GnRH， and expression of Kisspeptin， GPR54， and GnRH in the ARC region of the hypothalamus and GnRH-R in pituitary （P0.05， P0.01） and significantly increased wet weight and index of uterus， serum level of E₂， and expression of SIRT1 in the ARC region of the hypothalamus （P0.05， P0.01）. In addition， they showed thickened endometrium， increased number of endometrial glands， and improved glandular atrophy. ConclusionModified Erxian decoction regulates the function of the HPG axis through multi-targets， and its mechanism of action may be related to the up-regulation of the expression of SIRT1 in the ARC region of the hypothalamus， the inhibition of the over-activation of the Kisspeptin/GnRH signaling pathway， the regulation of the expression of GnRH-R in the pituitary， the restoration of secretion balance of gonadotropins， and the elevation of the estrogen level. This study provides an experimental basis for the interpretation of the scientific connotation of modified Erxian decoction in the treatment of perimenopausal syndrome and a theoretical reference for the development of a novel therapeutic strategy based on the SIRT1/Kisspeptin/GnRH pathway.

Based on the androgen receptor(AR)/mammalian target of rapamycin(mTOR)signaling pathway, the effects of Xihuang Pills-medicated serum on the proliferation and apoptosis of prostate cancer LNCaP cells were investigated. The drug-containing serum of SD rats was prepared by intragastric administration of Xihuang Pills suspension. The effects of low-, medium-, and high-dose Xihuang Pills-containing serum on the in vitro proliferation of LNCaP cells were detected by cell counting kit-8(CCK-8). Flow cytometry was used to detect the apoptosis level of LNCaP cells after intervention with different concentrations of Xihuang Pills. Protein expression of cleaved cysteinyl aspartate-specific proteinase caspase-3(cleaved caspase-3), B-cell lymphoma-2(Bcl-2), and AR as well as the phosphorylation level of mTOR protein were detected by Western blot. The results showed that compared with the blank serum, the drug-medicated serum could blunt the activity of LNCaP cells. Low-, medium-, and high-dose Xihuang Pills-containing serum could significantly increase the cell apoptosis rate, increase the expression of cleaved caspase-3 protein, decrease the expression of Bcl-2 protein, reduce the expression of AR protein, and down-regulate the level of phosphorylated mTOR(p-mTOR). To study the effect of Xihuang Pills on the growth of LNCaP cells in vivo, different doses of Xihuang Pills were used to intervene in the subcutaneous graft model in nude mice inoculated with LNCaP cells. The expression levels of AR, mTOR, p-mTOR, Bcl-2, and cleaved caspase-3 were detected by Western blot. The results showed that the volumes of subcutaneous graft tumor in the low-dose, medium-dose, and high-dose Xihuang Pills groups significantly decreased compared with that in the model group. The weight of subcutaneous transplanted tumor in each group with drug intervention was significantly lower than that in the model group. Compared with the model group, the low-dose, medium-dose, and high-dose Xihuang Pills groups showed increased cleaved caspase-3 protein expression, decreased Bcl-2 and AR protein expression, and reduced p-mTOR protein expression. Further experiments showed that AR agonist R1881 could block the anti-proliferation and pro-apoptotic effects of Xihuang Pills. The mechanism of Xihuang Pills against prostate cancer is related to the inhibition of the AR/mTOR signaling pathway, inhibition of LNCaP cell proliferation, and induction of apoptosis in cancer cells.
Humans ; Male ; Mice ; Rats ; Animals ; Caspase 3/metabolism* ; Mice, Nude ; Cell Line, Tumor ; Rats, Sprague-Dawley ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Prostatic Neoplasms/pathology* ; Cell Proliferation ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Mammals/metabolism*

OBJECTIVE：To establish UPLC method for the content determination of related substances in Terlipressin for injection. METHODS ：UPLC method was used to determine the contents of related substances in 5 batches of Terlipressin for injection. The separation was performed on Xtimate UPLC C 18 column with mobile phase A consisted of ammonium sulfate buffer （pH 2.3）-methanol（90 ∶ 10，V/V）and mobile phase B consisted of ammonium sulfate buffer （pH 2.3）-methanol（60 ∶ 40，V/V） （gradient elution ）at the flow rate of 0.2 mL/min. The detection wavelength was set at 210 nm，and sample size was 5 μL. RESULTS：The linear range of impurity A ，B，C，D，F，H，I，K，L and N were 0.43-3.86，0.44-3.95，0.44-3.97，0.45-4.08， 0.45-4.05，0.50-4.50，0.47-4.26，0.47-4.23，0.46-4.13，0.44-3.96 μg/mL（r≥0.999 7），respectively. The detection limits were 0.04， 0.04，0.05，0.04，0.05，0.05，0.05，0.05，0.04 μg/mL. The quantitation limits were 0.13，0.13，0.14，0.13，0.15，0.14，0.14， 0.14，0.13 μg/mL，respectively. RSDs of precision ，reproducibility and stability tests were all lower than 8％. The average recoveries were 94.95％，97.81％，101.88％，95.26％，93.40％，102.48％，104.26％，102.31％，96.42％，90.42％，with RSD s of 1.89％，1.86％，0.68％，1.30％，1.98％，3.36％，1.26％，1.30％，1.19％，1.40％（n＝9），respectively. Total contents of impurities in 5 batches of Terlipressin for injection were all lower than 4％. CONCLUSIONS ：Established method is rapid ，simple， accurate and specific ，which can be used for the quantitative analysis for related substances in Terlipressin for injection.

Objective To study the therapeutic effect of phages on extensively drug-resistant Acinetobacter baumannii-induced sepsis in mice.Methods (1) Sixty BALB/c mice were divided into blank control group,sepsis control group,antibiotics treatment group,phage treatment group,and phage control group according to the random number table,with 12 mice in each group.Mice in blank control group were intraperitoneally (the same injection position below) injected with 1 mL normal saline.Mice in sepsis control group,antibiotics treatment group,and phage treatment group were injected with 1 mL extensively drug-resistant Acinetobacter baumannii (the strain was isolated from the blood of a severely burned patient hospitalized in our unit) in the concentration of 5 × 107 colony-forming unit/mL to reproduce sepsis model.Two hours later,mice in sepsis control group,antibiotics treatment group,and phage treatment group were injected with 1 mL saline,1 mg/mL imipenem/cilastatin,and 1 × 108 plaque-forming unit (PFU)/mL phages screened based on above-mentioned Acinetobacter baumannii (the same phages below) respectively.Mice in phage control group were injected with 1 mL phages in the titer of 1 × 108 PFU/mL.The injection was performed continuously for 7 days in each living mouse,and the survival situation of mice was observed each day to calculate the survival ratio in one week.(2) Another 60 BALB/c mice were grouped and treated as in experiment (1),and the injection was performed continuously for 5 days in each living mouse.On experiment day 2,4,and 6,3 mice from each group were selected (if the number of survived mouse in any group was less than 3 at sample collecting,all the survived mice were selected),and blood was drawn to determine white blood cell count (WBC,with 3 samples at each time point in each group).On experiment day 2,blood was drawn from the mice that had their blood taken earlier for bacterial culture,and lung,liver,kidney,and spleen tissue was collected from the same mice.The tissue samples were added to the LB solid medium after being homogenized and diluted for bacterial culture.The content of bacteria was calculated after the bacterial colony number was counted.Data were processed Wilcoxon rank sum test,one-way analysis of variance,LSD test and Kruskal-Wallis rank sum test.Results (1) On experiment day 7,there were 12,8,10,and 12 mice survived in blank control group,antibiotics treatment group,phage treatment group,and phage control group respectively,while no mouse survived in sepsis control group.Compared with that in sepsis control group,the survival ratio of mice was significantly higher in the other four groups (with Z values from 55.635 to 106.593,P values below 0.05).The survival ratio of mice in phage treatment group was slightly higher than that in antibiotics treatment group,without statistically significant difference (Z =2.797,P ＞0.05).(2) On experiment day 2,WBC data of mice in blank control group,phage treatment group,and phage control group were close [respectively (5.60 ± 0.94) × 109/L,(5.16 ±0.36) × 109/L,and (5.26 ± 1.89) × 109/L],all significantly lower than the datum in sepsis control group [(8.64 ±0.64) × 109/L,P ＜0.05 orP ＜0.01],and the WBC data in the latter two groups were significantly lower than the datum in antibiotics treatment group [(7.80 ± 1.76) × 109/L,with P values below 0.05].On experiment day 4,WBC data of mice in antibiotics treatment group,phage treatment group,and phage control group were close,all significantly lower than the datum in blank control group (P ＜ 0.05 or P ＜ 0.01),and WBC data in the above-mentioned four groups were all lower than the datum in sepsis control group (with P values below 0.01).On experiment day 6,there was no statistically significant difference in WBC among blank control group,antibiotics treatment group,phage treatment group,and phage control group (x2 =4.128,P ＞0.05).On experiment day 2,respectively 12,7,and 2 mice were detected as blood bacterial culture-positive in sepsis control group,antibiotics treatment group,and phage treatment group,while no positive result was detected in the other two groups.Positive ratios of blood bacterial culture of mice in blank control group,phage treatment group,phage control group were significantly lower than the ratio in sepsis control group (with x 2 values from-30.000 to 30.000,P values below 0.01).Positive ratio of blood bacterial culture of mice in antibiotics treatment group was significantly higher than that in blank control group or phage control group (withx 2 values respectively 17.500 and-17.500,P values below 0.05).On experiment day 2,except for the kidney tissue of mice in phage treatment group,the bacteria load in each viscus of mice in blank control group,phage treatment group,and phage control group was significantly lower than that in sepsis control group (withx 2 values from-9.000 to 9.000,P values below 0.01).The bacteria load in kidney of mice in antibiotics treatment group was significantly higher than that in blank control group or phage control group (withx2 values respectively-7.500 and 7.500,P values below 0.05).Conclusions Phages can significantly improve survival ratio,control inflammation response,and effectively clean bacteria in lung,liver,spleen,and kidney in treating extensively drug-resistant Acinetobacter baumannii-induced sepsis in mice.