Objective:To investigate the effect of nuclear receptor subfamily 2 group F member 2(NR2F2)on the biological behaviors of human ovarian cancer SKOV3 cells,and to clarify its molecular mechauism and provide the new idea for treatment of ovarian cancer.Methods:Gene Expression Profiling Interactive Analysis(GEPIA)Database analyse the expression level of NR2F2 gene in ovarian tissue,and analyse its correlation with clinical prognosis of ovarian cancer patients.The human ovarian cancer SKOV3 cells were divided into control group and NR2F2 over-expression(NR2F2 OE)group,which were transfected with mCherry control virus and NR2F2 OE over-expression virus,respectively,when the cell deusity reached 70％,and the stable transfection SKOV3 cell lines were screened with puromycin(puro)48h lafter.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the transfection efficiencies of the cells;RT-qPCR method was used to detect the expression levels of NR2F2 and sex-determining region Y-box 2(SOX2)mRNA in the cells in two groups;Western blotting method was used to detect the expression levels of NR2F2,ATP-binding cassette superfamily G member 2(ABCG2),and programmed cell death 1-ligand 1(PD-L1)protcins in the cells in two groups.CCK-8 assay was used to detect the proliferation activities of the cells in two groups;Wound assay was used to detect the migration rates of the cells in two groups;Transwell chamber assay was used to detect the number of transmembrane cells;Spheroidization assay was used to detect the numbers of spheroids in the cells;peripheral blood mononuclear cells(PBMCs)-mediated tumor cell killing assay was used to detect the relative densities of surviving tumor cells;CCK-8 assay was used to detect the half maximal inhibitory concentration(IC50)of paclitaxel(PTX)and carboplatin(CBP).Results:Compared with normal ovarian tissue,the expression level of NR2F2 gene in ovarian tumor tissue was decreased(P＜0.05),and decreased with the improvement of clinical pathological grading of ovarian tumor.The patients with higher expression level of NR2F2 gene had better clincal prognosis.The SKOV3 cells with NR2F2 over-expresson were successfully constructed,and the expression levels of NR2F2 mRNA and protein in the cells in NR2F2 OE group were increased compared with control group(P＜0.001).The CCK-8 assay results showed that compared with control group,the proliferation activities of the cells in NR2F2 OE group were decreased at different time points(1,2,3,and 4 d)(P＜0.05 or P＜0.01).The cell wound assay results showed that compared with control group,the migration rate of the cells in NR2F2 OE group was decreased(P＜0.001).The Transwell assay results showed that compared with control group,the number of transmembrane cells in NR2F2 OE group was decreased(P＜0.01).Compared with control group,the number of the spheroids in NR2F2 OE group was decreased(P＜0.05),and the expression levels of SOX2 mRNA(P＜0.01)and protein(P＜0.001)were increased.Compared with control group,the relative density of surviving tumor cells in NR2F2 OE group was decreased,but the difference was not significant(P＜0.05),and the expression level of PD-L1 protein was decreased(P＜0.05).Compared with control group,the proliferation activities of cells in NR2F2 OE group were decreased(P＜0.05),and the drug sensitivities of the cells to PTX and CBP were enhanced(P＜0.05);the IC50 of PTX was significantly reduced,while the IC50of CBP could not be calculated due to excessively high drug concentration;the expression level of ABCG2 protein was decreased(P＜0.05).Conclusion:The over-expression of NR2F2 may inhibit the proliferation,migration,and invasion of the human ovarian cancer SKOV3 cells,decrease the expression levels of SOX2,PD-L1 and ABCG2 proteins,suppress the stemness and immune evasion ability of the SKOV3 cells,and enhance the sensitivities of the SKOV3 cells to PTX and CBP.

The complement system is an important part of the innate immune system,including more than 50 secretory proteins and membrane-bound proteins,and it contributes to the clearance of apoptotic cells and invading pathogens to limit inflammatory immune responses and maintaining brain homeostasis.Complement activity is strictly regulated to protect cells from random attacks or to prevent the deposition of complement proteins in physiological cases.However,overactivation or abnormal regulation of the complement cascade in the brain can lead to neuronal damage and brain dysfunction.Recent studies have pointed out that changes in complement molecules exist in patients with psychiatric diseases and play an important role in the occurrence and development of diseases by regulating the function of neurons and glial cells.Therefore,summarizing the latest research progress of complement system in psychiatric diseases such as schizophrenia,autism spectrum disorder,major depression,bipolar disorder and anxiety disorder can provide new ideas for preventing and controlling psychiatric diseases caused by abnormal activation of complement system.

Objective : To investigate the effect and mechanism of Urolithin B ( UB) on proliferation , migration , invasion , apoptosis and cell cycle of glioblastoma cells. Methods : The effects of UB on the proliferation , migration and invasion of glioblastoma U251 cells were detected by CCK⁃8 , clone formation assay , scratch healing assay , Transwell invasion assay respectively. The regulation effect of UB on cell cycle and apoptosis was detected by flow cytometry. Western blot was used to detect the effects of UB on the phosphorylation levels of downstream signaling pathway proteins ERK , AKT , p38 and JNK. Results : Compared with the control group , at 24 h and 48 h , the absorbance value of U251 cells was decreased by UB (P < 0. 01) in a dose dependent manner. UB reduced the percentage of cell clone formation (P < 0. 01) . The percentage of the scratch healing area was reduced by UB (P < 0. 05 or P < 0. 01) . The percentage of the number of invaded cells was reduced by UB (P < 0. 01) . UB could induce apoptosis (P < 0. 01) and cause the cells to stagnate in G2/M phase (P < 0. 01) . UB could significantly inhibit the phosphorylation of ERK (P < 0. 05 or P < 0. 01) and AKT (P < 0. 05 or P < 0. 01) . Conclusion UB can inhibit proliferation , migration , invasion , apoptosis and cell cycle of glioblastoma cells , and regulate the phosphorylation levels of ERK and AKT.

Objective:To investigate the correlation between serum calcitonin gene-related peptide (CGRP) and the culture outcome of in vitro fertilization embryo in male patients with infertility.Methods:In this study, the randomized samples from 25 male patients who received in vitro fertilization-embryo transfer (IVF-ET) in the 1st Affiliated Hospital of Fujian Medical University from June 2019 to October 2019 were analyzed by multiple linear stepwise regression, with some important clinical outcomes, such as the logarithmic conversion index of serum CGRP, fertilization method, masturbation difficulty, age, infertility duration, and prolactin, as independent variables, while total fertilization rate, normal fertilization rate, high quality embryo rate at day 3, blastocyst formation rate as dependent variables.Results:The Pearson correlation analysis showed that the D3 high-quality embryo rate was related to the normal sperm morphology rate in the primary infertility group ( r=0.537, P=0.048), the blastocyst formation rate was correlated with sperm density ( r=0.760, P=0.002), the CGRP logarithm was correlated with the total fertilization rate ( r=0.693, P=0.006). The logarithmic conversion index of serum CGRP was related to the total fertilization rate and normal fertilization rate in the secondary infertility group ( r=0.614, P=0.042 and r=0.611, P=0.046). In the secondary infertility group, there was a linear relationship between normal fertilization rate and total sperm count, serum CGRP log conversion, and sperm normal morphology rate, with standardized regression coefficients of 0.2, -0.729, and 6.8, respectively. Conclusion:Serum CGRP level, together with total sperm count and normal sperm morphology rate may affects normal fertilization rate in male patients with infertility.

Objective To observe the clinical therapeutic effect of Bushen Huoxue formula for treatment of diabetic nephropathy at G3a stage. Methods Sixty patients with stage G3a diabetic nephropathy were admitted to the Department of Nephrology of Affiliated Hospital of Tianjin Institute of Traditional Chinese Medicine from June 2017 to June 2018, and according to difference in treatment, they were divided into an integrated traditional Chinese and western medicine treatment group and a western medicine treatment group with 30 cases in each group. The two groups were treated with Huangkui capsule 2.5 g/time, 3 times a day; the integrated traditional Chinese and western medicine treatment group was additionally given Bushen Huoxue formula, one dose daily, twice a day taken orally; 2 months for 1 course. The changes of 24-hour urinary protein, serum creatinine (SCr) and urea nitrogen (BUN) were observed after 3 courses of treatment. Results After 3 courses of treatment, the levels of 24-hour urinary protein, SCr and BUN of the integrated traditional Chinese and western medicine treatment group were lower than those of the western medicine treatment group [24-hour urinary protein quantification (g): 1.45±0.26 vs. 2.11±0.35, SCr (μmol/L): 105.15±12.31 vs. 158.32±17.26, BUN (mmol/L): 7.26±2.41 vs. 12.87±3.24], the differences being statistically significant (all P < 0.05). The total effective rate of integrated traditional Chinese and western medicine treatment group was significantly higher than that of western medicine treatment group [86.67% (26/30) vs. 53.33% (16/30), P < 0.05]. Conclusion Bushen Huoxue formula in the treatment of patients with diabetic nephropathy at stage G3a can decrease their urinary protein and SCr, BUN significantly.

Objective:To investigate the anti-inflammation effects by activation of the cholinergic anti-inflammatory pathway and its mechanisms in non-alcoholic steatohepatitis (NASH)model mice.Me-thods:6-week-old male C57BL/6J (B6)mice were randomly divided into four groups:the first group was normal mice,injected with saline;the second group was normal mice,injected with nicotine;the third group was NASH model mice,injected with saline;the fourth group was NASH model mice,injec-ted with nicotine.The experimental mice were fed with either standard chow (SC)or high-fat and high-fructose (HFHF)for 17 weeks to generate an NASH model mice.The mice received injection once daily for 3 weeks [nicotine dose,400 μg/kg].Then,their pathological characteristics and function of the liver were assessed.The expressions of interleukin-6 (IL-6)and tumor necrosis factor-α(TNF-α)in se-rum were analyzed by enzyme linked immunosorbent assay (ELISA).The expressions of alpha 7 nicotinic acetylcholine receptors (α7nAChR),Toll-like receptors-4 (TLR-4)and nuclear factor κB of phosphory-lation (p-NF-κB)in Kupffer cells were determined by Western blot and immunofluorescence assays.Re-sults:We successfully generated NASH model mice by imitating the high-fat and high-fructose dietary style of NASH patients.The results of our investigation demonstrated that nicotine could reduce signifi-cantly the levels of IL-6,and TNF-αin serum (P <0.05).The expression of p-NF-κB protein in the group which was NASH model mice injected with nicotine declined significantly as compared with the group which was NASH model mice injected with saline (P <0.05).And the expression of α7nAChR protein elevated significantly conversely (P <0.05 ).Conclusion:Activation of the cholinergic anti-inflammatory pathway could inhibit the release of inflammatory factors as TNF-αand IL-6 in NASH model mice,and the mechanism for the inhibition of inflammatory was mediated by NF-κB pathway.

Objective To investigate the effect of nicotinic acetylcholine receptor αt7 (a7nAChR) subunit gene on liver inflammation in mice with nonalcoholic steatohepatitis (NASH) and related mechanisms.Methods C57BL/6J mice and α7nAChR gene knockout mice were fed for 24 weeks to establish the NASH model,and the mice were sacrificed to isolate and culture the primary liver macrophages.After the treatment with nicotine and endotoxin,ELISA was used to measure the levels of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in supematant;indirect immunofluorescence assay and Western blot were used to observe the effect on the NF-κB signaling pathway,and quantitative PCR was used to measure the mRNA expression of Toll-like receptor-4 (TLR-4) in macrophages.An analysis of variance was used for comparison of means between multiple groups.Results The results of ELISA showed that compared with the endotoxin+nicotine group of C57 NASH mice,the endotoxin+nicotine group of gene knockout NASH mice had significantly higher levels of IL-6 and TNF-α in supernatant (IL-6:1 599±65 pg/ml vs 1 465±45 pg/ml,P ＜ 0.05;TNF-α:1 567±66 pg/ml vs 1 433±50 pg/ml,P ＜ 0.05).The results of Western blot showed that compared with the endotoxin+nicotine group of C57 NASH mice,the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative protein expression of phosphorylated NF-κB and TLR-4 (NF-κB:69 425±600 vs 51 1334200,P ＜ 0.05;TLR-4:93 387±684 vs 64 198±630,P ＜ 0.05).The results of indirect immunofluorescence assay showed that the endotoxin+nicotine group of gene knockout NASH mice had a significantly higher fluorescence intensity of NF-κB than the endotoxin+nicotine group of C57 NASH mice.The results of PCR showed that the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative mRNA expression of TLR-4 than the endotoxin+nicotine group of C57 NASH mice (4.13±0.13 vs 2.93±0.14,P ＜ 0.05).Conclusion The α7nAChR gene knockout can aggravate the degree of inflammatory reaction in NASH,and its mechanism may be related to the fact that the NF-κB signaling pathway cannot be inhibited,which aggravates inflammatory reaction.

OBJECTIVETo investigate the effects and mechanisms of the inflammatory reaction related to nonalcoholic steatohepatitis (NASH) and induced by activation of the cholinergic anti-inflammatory pathway.

METHODSA mouse model of NASH was established by feeding a high-fat and high-sugar diet.Activation of the cholinergic anti-inflammatory pathway was achieved by nicotine administration to the NASH modeled mice and normal controls. Liver biopsies were taken and the concentrations of cytokines were measured. Isolated liver primary Kupffer cells and RAw264.7 cells were cultured, pre-treated or not with lipopolysaccharide (LPS) and exposed to nicotine, after which the supernatant concentrations of IL-6 and TNFa were determined by ELISA. The protein expression levels of phosphorylated (p)-NF-kB and I k B were detected in primary cultured Kupffer cells by western blotting.

RESULTSThe mouse model of NASH was successfully established, as evidenced by findings from liver biopsy and serum liver function tests. The degree of liver inflammation in the NASH mice decreased after nicotine administration, and the level of serum TNFa also significantly decreased. The levels of serum TNFa were 21.95+/-0.8 pg/mL in nicotine-treated mice and 38.07+/-1.7 pg/mL in the non-nicotine-treated NASH mice (P less than 0.05). The nicotine treatment also significantly reduced the concentration of TNFa in the culture supernatants of Kupffer cells after LPS stimulation; moreover, the supernatant level of TNFa decreased significantly after the nicotine treatment (Pless than 0.05). LPS stimulation of the RAw264.7 cells led to an increased level ofp-NF-kB and a reduced level ofI-kB, suggesting that the NF-kB pathway had been activated; different doses of nicotine pre-treatment led to down-regulation of the p-NF-kB level and up-regulation of the I-kB level, both in dose-dependent manners.

CONCLUSIONActivating the cholinergic anti-inflammatory pathway inhibits the NASH-related inflammatory reaction, and the mechanism for this inhibition involves the NF-kB signaling pathway.

Animals ; Cholinergic Agents ; Down-Regulation ; Inflammation ; Interleukin-6 ; Kupffer Cells ; Lipopolysaccharides ; Mice ; NF-kappa B ; Non-alcoholic Fatty Liver Disease ; Phosphorylation ; Up-Regulation

Objective To investigate the effect of rat macrophage α7-acetylcholine receptor (α7-AChR )-mediated cholinergic anti-inflammatory pathway on endotoxin-induced inflammation reaction . Methods Density gradient centrifugation method was used to isolate rat primary macrophages and flow cytometry was used to identify the cell purity .α7-AChR in macrophages was detected by fluorescence confocal microscopy and Western blot .After 1ipopolysaccharides ( LPS) was added to the culture media of primary culture of macrophages , the concentration of tumor necrosis factor ( TNF)-αin the supernatant was determined by enzyme linked immunosorbent assay (ELISA).The expression of p-NF-κB protein in primary cultured macrophages was detected by Western blot .ANOVA was used to analyze TNF-αlevels after adding different concentrations of nicotine .Results α7-AChR was observed by fluorescence confocal microscope in primary macrophages .Nicotine could significantly reduce the concentration of TNF-αin culture supernatants of macrophages after LPS stimulation .When the concentrations of nicotine were 0, 1, 10, 100μmol/L, the concentrations of TNF-αwere (2 123 ±86), (1 486 ±80), (1 316 ±83) and (1 090 ±77)pg/mL, respectively (t=16.33, 20.18 and 26.83, P<0.05).The level of p-NF-κB in macrophages was also reduced when nicotine added .Conclusion Activation of macrophage α7-AChR can inhibit the endotoxin-induced release of inflammatory factor TNF-α, which may be through NF-κB signal pathway .

Objective To explore the distribution of age,reasons for treatment,risk factors,and causes of vulvovaginitis in prepubertal girls.Methods A total of 4 214 prepubertal girls with vulvovaginitis who were admitted to the Girl's Sub-department,Zhongshan Boai Hospital from January 2010 to June 2013 was reviewed retrospectively.All clinical data were from medical records with files.Results Atotalof1 587 patients (37.7％) was0to1 years old,954 (22.6％) ＞ 1 to3 years old,1 289 (30.6％) ＞ 3 to 7 years old,and 384 (9.1％) ＞ 7 years old.Many risk factors were associated with vaginitis,including poor hygiene (2 924 girls; 69.4％),allergies and exposure to allergens (875 girls; 20.8％).Most common reason for treatment was a referral from physical examination accounting for 919 girls (21.8％),followed by vaginal secretions 812 girls (19.3％).The causes of vulvovaginitis of all patients were evaluated,1 771 of which (42.0％) were nonspecific vulvovaginitis,1 309 (31.1％) labial adhesions,375 (8.9％) bacterial vulvovaginitis,266 (6.3％) allergic vulvovaginitis,and 266 (6.3％) affective leg rubbing action.Conclusions Prepubertal vulvovaginitis occurred mainly in infancy and preschool.Floating population was common.They were caused by many risk factors including poor hygiene,allergies,poor urination habits,etc.The most common causes of vulvovaginitis were nonspecific vulvovaginitis and labia adhesion,yet allergies and affective leg rubbing action were the more common causes of recurrent vulvovaginitis.We propose that focusing on girls' reproductive health,timely treating allergic and crossing rub legs and other diseases would help reduce the prevalence of vulvovaginitis in the prepubertal girls.