Objective To explore the therapeutic mechanism of kidney-tonifying and blood-activating herbal medicine for anovulatory dysfunctional uterine bleeding(ADUB).Methods Thirty-seven ADUB patients were randomized into two groups.Group A(N=25)was treated with Gongxue Yin(mainly composed of Fructus Psoraleae,Radix Dipsaci,Fructus Corni,Pollen Typhae,Radix Notoginseng,Radix Codonopsis,Os Draconis,Concha Ostreae,Herba Hedyotis Diffusae),and group B(N=17)received medroxyprogesterone.One menstrual cycle constituted one treatment course and the treatment lasted 3 continuous courses.Histostain-plus(HP)immunohistochemical assay was adopted to test the Bcl-2 and Bax protein expression in endometrium of the two groups.Ten healthy volunteers served as the control.Results Bcl-2 protein expression was higher in endometrium of ADUB patients at proliferative stage or with proliferative changes than that at secretory phase of the normal control group(P0.05).Conclusion Both progesterone and kidney-tonifying and blood-activating herbal medicine decrease endometrial Bcl-2 protein expression and increase Bax expression,by which inducing the endometrial cell apoptosis in patients with anovulatory dysfunctional uterine bleeding.But their detailed mechanism may be different.

Objective To observe the protective effect of Apelin-13 on the cerebral ischemia-reperfusion injury (CIRI), and to explore the possible mechanism in rat model. Methods Fifty male SD rats were randomly divided into five groups:sham group, CIRI model group and Apelin-13 (0.1, 1.0 and 10.0 μg/kg) treatment groups. The model of CIRI was established by filament. After 2 h ischemia, the focal middle cerebral artery was followed by 72 h reperfusion. Apelin-13 was administrated by intracerebroventricular injection 30 minutes before reperfusion. The score of neural function was estimated in different time points. The 2,3,5-triphenyl tetrazolium chloride (TTC) dye was used to calculate the volume and percentage of cerebral infarction. The endoplasmic reticulum stress (ERS) protein markers including glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) in cerebral cortex were measured by Western blot assay. Results Compared with the sham group, the score of neural function was significantly increased, the infarct rate was reached(47.63 ± 5.81)%and the protein expressions of GRP78 and CHOP were significantly up-regulated in CIRI model group (P＜0.05). There were no significant differences in these data between the CIRI model group and 0.1 μg/kg Apelin-13 treatment group (P＞0.05). Compared with the CIRI group, the neural function defect was significantly improved, the muscle strength was significantly enhanced and the infarct rate was significantly decreased, and the protein expressions of GRP78 and CHOP were significantly down-regulated in the 1.0 and 10.0 μg/kg Apelin-13 treatment groups (P＜0.05). Conclusion Apelin-13 protects the cerebral ischemia-reperfusion injury in rat model, which may be related with the inhibition of endoplasmic reticulum stress.

Objective:To investigate the site and characteristic ofp53 gene mutations in familial or early-onset breast cancer patients in part population of southern China.Methods:A total of 150 patients with familial and early-onset breast cancer in parts population of southern China were enrolled.Genomic DNA was isolated from each peripheral blood sample,and the entire coding sequence and exon and intron splicing region of p53 gene were amplificated by PCR in the 150 patients.The mutation analysis were detected by denaturing high performance liquid chromatography (DHPLC) and confirmed by DNA sequence analysis.Results:In the 150 patients with familial and early-onset breast cancer,6 mutations including one novel pathogenic mutation 869_888 ins20 (insert mutation) and 5 previously reported pathogenic mutations (deletion mutation 643_660de118 and 4 missense mutation 91G＞A,215C＞G,537T＞G,743G＞A) were identified in p53 gene encoding region in 9 patients of breast cancer.Moreover,one same sense mutation 141G＞A in exon 4,one 16 bases deletion in intron 3,and 9 single nucleotide polymorphisms in p53 gene introns were also identified.The total mutation frequency ofp53 gene in 150 patients with familial breast cancer and early-onset breast cancer from part population of southern China was 6.00％,and the mutation frequency of familial breast cancer and early-onset breast cancer was 6.81％ and 6.25％,respectively.Conclusion:The total mutation frequency ofp53 gene in 150 patients with familial breast cancer and early-onset breast cancer from partpopulation of southern China is higher than the frequency previously reported.The pathogenicity of the novel mutations (insert mutation) 869_888ins20 will be confirmed by function analysis in the future study.The deletion mutation 643_660de118 enriches the p53 gene mutation database among Chinese population,which is probably the specific mutation of breast cancer in Chinese population.

Objective To evaluate the role of ibuprofen and hydrocortisone in early treatment of patent ductus arteriosus ( PDA ) in premature infants with low cortisol level . Method A prospective randomized controlled trial on 144 very low birth weight infants in the Hospital within 24 hours after birth with gestational age of 28~32 weeks and birth weight of 1000~1499 grams,who had asymptomatic PDA diagnosed by echocardiography , introducing early administration of drugs including ibuprofen and /or hydrocortisone within the first 24 ~48 hours after birth.According to the baseline of serum cortisol level measured prior to the administration of drugs , the preterm were assigned into two groups .The low cortisol level group ( the cortisol level <150μg/L) were further subdivided into four groups each being allocated to hydrocortisone or ibuprofen or both of hydrocortisone and ibuprofen combined or placebo treatment .The high cortisol level group ( the cortisol level≥150μg/L) were allocated to either ibuprofen or placebo treatment in randomization.Diameter of ductus arteriosus and cortisol value were measured again after treatment , and the follow-ups were undertaken till discharge .All data was collected and analyzed by statistical software .Result A total of 91 cases were in low cortisol level group ( 22 cases of hydrocortisone , 23 cases of ibuprofen , 21 cases of both hydrocortisone and ibuprofen , and 25 cases of placebo ) and 53 cases in high cortisol level group (26 cases of placebo and 27 cases of Ibuprofen ).Low cortisol level group , combined therapy , closure of the ductus at a rate of 81.0%, was higher than other methods of therapy ( P<0.05);high cortisol level group, the ductus arteriosus closed in 20 patients of ibuprofen therapy ( 74.1%) and in 13 patients of placebo treatment (50.0%) (P<0.05).Early treatment did not significantly increase the drug adverse effects, including impaired renal function , gastrointestinal bleeding , hyperglycemia and others. After comparisons between laboratory changes in early targeted groups and non-early targeted groups after treatment, findings were as follows: decrease in the incidence of apnea , myocardial damage , feeding intolerance , intraventricular hemorrhage and reduce the duration of phototherapy .Conclusion This trial proved the efficacy and safety of early therapy with ibuprofen and hydrocortisone for closure of ductus arteriosus in premature infants with low cortisol level and the decreasing incidence of complications due to PDA without increasing the risk of adverse effects .

Objective: To investigate the Effects of entinostat on the expression of NKG2D ligands in the non-small cell lung cancer (NSCLC) cell lines, A549 and HCC-827, and to detect the effect of entinostat-mediated NK cell killing of A549 and HCC-827 cells. Meth-ods: The effect of entinostat on A549 and HCC-827 cell proliferation was measured by MTT assay. Flow cytometry was used to detect the expression of NKG2D ligands. mRNA levels of the ligands were detected by RT-PCR . The level of soluble MICA in cell culture super-natant was evaluated by ELISA. The cytotoxicity of NK cells against A549 and HCC-827 cell lines (treated with entinostat) was assessed using lactate dehydrogenase release assay. Results: Entinostat showed a time-and dose-dependent inhibition effect on the prolifera-tion of A549 and HCC-827 cell lines. The expression of NKG2D ligands and mRNA transcription levels of MICA and MICB were en-hanced after treatment with 0.5, 1μmol/L entinostat for 48 h. The soluble MICA level in A549 cell culture supernatant was increased by 1μmol/L entinostat. The sensitivity of HCC-827 cells to NK cells was enhanced upon treatment with 0.5, 1μmol/L entinostat. Con-clusions: entinostat enhanced the killing effect of NK cells on non-small cell lung cancer cells by up-regulating the expression of NKG2D ligands. This provides a new method and theory for the treatment of NSCLC.

Objective:To observe the efficacy of anti-sensitive moisturizing tolerance-extreme cream combined with isotretinoin in the treatment of severe acne.Methods:Fifty patients with severe acne were selected in the Dermatology Clinic of the Third People＇s Hospital of Hangzhou from November 2018 to July 2019. They were randomly divided into the experimental group of 25 cases and the control group of 25 cases. The experimental group was treated with anti-sensitive moisturizing tolerance-extreme cream combined with isotretinoin orally. The control group was treated with isotretinoin orally alone. Before and after treatment for 56 days, lactate score, skin cuticle hydration (SCH), transepidermal water loss (TEWL) and skin physiological indexes were measured.Results:After 56 days of treatment, the TEWL and SCH of the control group were 15.75±3.31 and 10.13±3.62, the TEWL and SCH of the experimental group were 12.17±3.61 and 28.07±3.17, respectively; the difference was statistically significant ( T was 3.610 and 12.398, P was 0.002 and 0.000, respectively). The volume and depth of cyst nodule, scar depression, skin roughness, absolute value and area of erythema in the experimental group were significantly lower than those in the control group ( T was 2.280, 1.676, 2.332, 1.508, 4.813 and 3.637; P was 0.031, 0.011, 0.027, 0.040, 0.000 and 0.001, respectively). Conclusions:Anti-sensitive moisturizing tolerance-extreme cream combined with isotretinoin has a good effect on severe acne and it can reduce the barrier damage and other adverse reactions.

Objective To explore the prevalence of mild cognitive impairment (MCI) among elderly veterans. Methods 2 674 veterans ( aged 60 years and over) from 26 military sanatorium in Shijiazhuang city were studied. The Mini-Mental State Examination, Global Deterioration Scale, Activity of Daily Living, Hachinski Ischemic Scale and Hamilton Depression Scale were served as screening tools. Results The prevalence of total MCI was 8 08% in elderly people. The standardized prevalence of MCI was 6 87% in male and 10 38% in female (P

Objective:To investigate the proliferation and apoptosis characteristics of polyploid non-small cell lung cancer (NSCLC) cell model induced by docetaxel (Doc), and to analyze the potential role of polyploid tumor cells in chemotherapy resistance and tumor recurrence.Methods:NSCLC A549 cells were treated with dimethyl sulfoxide (DMSO) or 1 μmol/L Doc for 24 h. After drug removal, the cells were cultured in complete medium until the third day or the 5th day, and then they were recorded as the control group, Doc 24 h group, Doc 24 h+ 3 d group, Doc 24 h + 5 d group, respectively. The cell morphology was detected by using immunofluorescence staining. Flow cytometry was used to determine cell ploidy and cell cycle. Dil labeling and CFSE labeling were applied to detect cell proliferation. Flow cytometry by Annexin-V/PI double labeling was used to detect apoptosis. The changes of cyclin and apoptotic protein were analyzed by using Western blot.Results:Immunofluorescence staining results showed that compared with the control group, the volume of a small number of surviving cells in Doc 24 h group was increased slightly and the cells showed multinuclear status; while the cell volume in Doc 24 h+ 3 d group and Doc 24 h+ 5 d group continued to increase, and the nucleus remained multinuclear. The results of cell ploidy analysis also showed that the percentage of polyploid cell subsets was (3.40±0.95)%, (20.80±2.87)% in Doc 24 h group, (55.67±3.85)% in Doc 24 h+3 d group and (76.20±2.51)% in Doc 24 h+5 d group. With the prolongation of withdrawal time, the percentage of polyploid cell subsets was increased, and the difference was statistically significant ( F= 478.054, P < 0.05). The percentage of G 1 and S phase cell subsets in Doc 24 h group was lower than that in the control group, and the percentage of G 2/M phase cell subsets was higher than that in the control group, and the difference was statistically significant (both P < 0.05). The protein expression level of cdc2, P-cdc2 (Thr14), P-cdc2 (Tyr15), P-cyclin B1 (Ser128), P-cyclin B1 (Ser147) in the cells of the control group, Doc 24 h group, Doc 24 h+ 3 d group and Doc 24 h+ 5 d group was down-regulated in sequence, while the expression level of cyclin B1 was up-regulated, and cdc25c was down-regulated in Doc 24 h + 3 d group and Doc 24 h+ 5 d group. Dil staining results showed that the fluorescence of cell-labeled Dil in Doc 24 h group, Doc 24 h+ 3 d group and Doc 24 h + 5 d group did not decrease significantly. CFSE staining showed that the fluorescence intensity of CFSE labeled by polyploid A549 cells did not change significantly with the prolonged withdrawal time. Annexin-V/PI double staining showed that the percentage of apoptotic cell subsets in Doc 24 h group was higher than that in the control group ( P < 0.05), but the percentage of apoptotic cell subsets in Doc 24 h + 3 d group and Doc 24 h + 5 d group was lower than that in Doc 24 h group, while there was no statistically significant difference when compared with the control group ( P > 0.05). Western blot results showed that the expression of bcl-xl and mcl-1 in the control group, Doc 24 h group, Doc 24 h + 3 d group and Doc 24 h + 5 d group was up-regulated in sequence, while the expression of bax and bak in Doc 24 h + 3 d group and Doc 24 h + 5 d group was up-regulated, but down-regulated in Doc 24 h+5 d group. Conclusions:Doc can induce polyploidy of A549 cells in vitro. The cell cycle is blocked in G 2/M phase. After doc treatment, the proliferation of A549 cells is significantly decreased, and the apoptosis of A549 cells is promoted. However, with the prolongation of withdrawal time, apoptosis resistance occurs, and the expression levels of corresponding pro-apoptosis and anti-apoptosis proteins show significant changes. This may be helpful for polyploid tumor cells to produce drug resistance and tumor recurrence after chemotherapy intervention.

Objective:To study the migration of polyploid tumor cells induced by docetaxel, the characteristics of epithelial-mesenchymal transition, and the killing effect of immune cells on them, in order to explore the potential role of polyploid tumor cells in tumor recurrence.Methods:The human non-small cell lung cancer A549 cells were treated with 1 μmol/L docetaxel for 24 h, and the cells were collected as Doc 1 d group. After drug removal, the cells were cultured in fresh and complete medium for 3 or 5 days, then the cells were collected as Doc 3 d group or Doc 5 d group respectively. The A549 cells were treated with DMSO for 24 h as control group. Immunofluorescence staining was used to detect cell morphology, flow cytometry was used to analyze cell ploidy, scratch test was used to detect cell migration, Western blotting was used to detect the expression of epithelial-mesenchymal transition related proteins, and lactate dehydrogenase release method was used to evaluate the killing activity of cytokine-induced killer (CIK) cells.Results:Compared with the control group, most of the cells in the Doc 1 d group, Doc 3 d group and Doc 5 d group were apoptotic, a few of the surviving cells were significantly larger, and the nucleus was polynuclear. The proportions of polyploid cell subset (DNA content > 4N) in the control group, Doc 1 d group, Doc 3 d group and Doc 5 d group were (1.93±0.55)%, (22.97±2.37)%, (51.30±12.51)% and (67.87±8.31)% respectively, and the difference among the four groups was statistically significant ( F=26.521, P<0.001). The proportion of polyploid cell subset in Doc 1 d group, Doc 3 d group and Doc 5 d group was significantly higher than that in the control group (all P<0.001). With the prolongation of withdrawal time, the proportion of polyploid cell subset in Doc 3 d group and Doc 5 d group was significantly higher than that in Doc 1 d group ( P=0.009; P=0.004). After 24 h and 48 h culture, the wound healing rates of the control group were both 100%, and the wound healing rates of the Doc 3 d group were (39.10±2.12)% and (46.13±5.32)% respectively, with no significant difference ( t=2.126, P=0.051). Compared with the control group at 24 h and 48 h, the cell migration abilities of Doc 3 d group were significantly lower ( t=49.756, P<0.001; t=30.825, P<0.001). Compared with the control group, the expression of E-cadherin protein decreased gradually in the Doc 1 d group, Doc 3 d group and Doc 5 d group, the expression of Vimentin protein increased gradually, and the expressions of Snail protein and N-cadherin protein did not change significantly. The killing efficiencies of CIK cells against the cells of the control group, Doc 3 d group and Doc 5 d group were (27.27±1.91)%, (17.87±2.35)%, (9.47±0.51)% respectively, and the difference was statistically significant ( F=11.294, P<0.001). The killing efficiency of Doc 3 d group and Doc 5 d group was significantly lower than that of the control group ( P=0.004; P<0.001). The killing efficiency of Doc 5 d group was significantly lower than that of Doc 3 d group ( P=0.003). Conclusion:The migration ability of polyploid tumor cells induced by docetaxel is weakened, but epithelial-mesenchymal transition is likely to occur, and the killing effect of immune cells on them is reduced.

Objective:To investigate the correlation between triglyceride-glucose (TyG) index and high on-treatment platelet reactivity (HTPR) during clopidogrel treatment in patients with ischemic stroke.Methods:Patients with ischemic stroke who received maintenance dose of clopidogrel (75 mg/d) in the Department of Neurology, Guangdong Provincial Hospital of Chinese Medicine from January 2017 to March 2021 were retrospectively included. The highest quartile (Q4) of the TyG index was defined as insulin resistance. Platelet reactivity was assessed by thromboelastogram and clopidogrel HTPR was defined as the clot strength induced by adenosine diphosphate (MA ADP) >47 mm. Multivariate regression model was used to analyze the independent correlation between TyG index and platelet reactivity. Results:A total of 83 patients were included. The TyG index showed a linear correlation with MA ADP. The patients were divided into 4 groups according to the quartile of TyG index. The incidence of clopidogrel HTPR increased significantly with the increase of the quartile of the TyG index ( Ptrend=0.017). Multivariate analysis showed that there was a significant independent correlation between insulin resistance and clopidogrel HTPR (odds ratio 4.597, 95% confidence interval 1.285-16.446; P=0.019). Conclusions:In patients with ischemic stroke treated with clopidogrel, the incidence of clopidogrel HTPR gradually increases with the increase of the quartile of the TyG index. The insulin resistance assessed by the TyG index is independently associated with clopidogrel HTPR.