Objective:To observe effects of wrist-ankle acupuncture (WAA) onβ-endorphin (EP), nitric oxide (NO) in uterus tissue and prostaglandin F2α (PGF2α), substance P (SP) in serum of rats with primary dysmenorrhea. Methods:A total of 45 non-pregnant Wistar rats were randomly divided into a control group, a model group and a WAA group, 15 rats in each group. Rats in the model group and the WAA group received continuous abdominal subcutaneous injection of Diethylstilbestrol to establish dysmenorrhea rat models. On the first day after modeling, rats in the WAA group began to receive acupuncture on Point Lower 1 and Point Lower 2, once a day for 10 d. The control group and the model group didn’t receive any treatment. Writhing latencies and frequencies were recorded.β-EP and NO in uterus tissue homogenates and PGF2α, SP in serum were detected. Results:In the model group,β-EP and NO levels were the lowest among the groups, the serum PGF2α level was the highest, and serum SP level was the lowest. These measurements showed significantly difference between the model group and the control group (P<0.05). PGF2α in the WAA group was lower than that in the model group;β-EP, NO and SP levels were higher than those in the model group, with inter-group statistically significant differences (P<0.05). Conclusion: WAA may achieve analgesic effect through decreasing PGF2α, increasingβ-EP, NO and SP to relieve uterine cramps, increase blood flow and promote functional improvement.

Objective To observe the antidepressant effect of the volatile oil of Myristica in mice and investigate its mechanism.Methods Healthy male Kunming mice were divided into control group,fluoxetine hydrochloride group,high,medium and low dose group,and all of the mice were gavaged for 7 days.The role of antidepressant on the mice were observed with tail suspension tests and forced swimming tests.Then serotonin(5-HT),dopamine(DA) and norepinephrine (NE) in the brain of the mice were determined by enzyme-linked immunosorbent assay.Results Compared with the control group((82.60 ± 24.70)s),the medium dose group could shorten the immobility time of the tail suspension tests ((54.40± 15.87) s),and showed statistical significance (P＜ 0.05).The content of 5-HT,DA,NE in the brain of the medium group were (19.35±2.79) ng/ml,(12.16±0.71)pg/ml,(0.27±0.12) ng/ml,and control group were (14.95±4.83) ng/ml,(11.32±0.95) pg/ml,(0.20±0.11) ng/ml.Compared with the control group,the content of 5-HT of the medium dose group was increased and showed statistical significance(P＜0.05).Conclusion The volatile oil of Myristica fragrans Houtt has antidepressant effect,and may be related to raising the content of 5-HT,DA,NE in the brain of the mice,especially 5-HT.

Objective To investigate the value and diagnostic efficiency of the quantitative dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI) and intravoxel incoherent motion (IVIM) parameters using three dimention (3D)-histogram analysis for discriminating the Gleason score (GS) of prostate cancer. Methods A total of 53 patients pathologically confirmed as prostate cancer by systemic prostate biopsy who had routine , DCE and DWI-MRI scans were retrospectively analyzed. There were 15 cases for low-risk and 38 cases for intermediate/high-risk prostate cancer. The 3D ROI of all lesions based on T2WI was achieved by image registration to get the quantitative parameters of DCE-MRI and DWI-IVIM. The parameters of DCE-MRI contains: transfer constant (Ktrans), rate constant (Kep) and extracellular-extravascular volume fraction (Ve).The DWI-IVIM related quantitative parameters were ADC, diffusion coefficient (D), diffusion coefficient related to perfusion (D*) and perfusion fraction (f). Then the histogram analysis of these quantitative parameters was performed to get the mean, median, 25th percentile, 75th percentile, Skewness and Kurtosis. Using the Spearman rank correlation analysis to evaluate the correlation of these parameters and GS of prostate cancer. The diagnostic performance of these quantitative histogram parameters related to the GS in identifying low-risk and intermediate/high-risk of prostate cancer was carried by ROC. Results The Kep and Ktrans (mean, median, 25th, 75th) of DCE-MRI were positively correlated with GS (r value was 0.346 to 0.696, P<0.05). The ADC (mean, median, 25th, 75th), D (mean, median, 25th, 75th, Skewness, Kurtosis) and D*(25th) of DWI-IVIM were correlated with GS (r value was-0.544 to 0.428, P<0.05). The DCE-MRI quantitative parameters Kep (25th) had the highest area under curve (AUC, 0.961); The ADC (median) and D (25th) had higher AUC( 0.832, 0.888) in the quantitative parameters of DWI-IVIM, the difference between Kep(25th) and ADC (median) was statistically significant (Z value was 2.212, P value was 0.027). The difference of AUC between Kep (25th) and D (25th), D (25th) and ADC (median) was not statistically significant (Z values were 1.027 and 1.398, P values were 0.162 and 0.304, respectively).Conclusion DCE and IVIM quantitative parameters (Kep, Ktrans, ADC, D) histogram analysis results are correlated with GS, and can be used for distinguishing low-risk from intermediate/high-risk prostate cancer.

Objective:To screen the neutralizing epitope of enterovirus 71 (EV71) and determine the specific minimum amino acid sequence that triggers immunity for providing a theoretical basis for the development of synthetic peptide vaccines.Methods:EV71 neutralizing antibody-specific binding clones were panned and sequenced using a phage display random 12-peptide library to obtain the key sequences of neutralizing epitopes. A series of peptides containing the key sequences with N-terminal acetylation (AC) and C-terminal linking to Keyhole limpet hemocyanin (KLH) were synthesized. Serum samples were collected after immunizing mice with the modified peptides. Then the immunogenicity of the peptides and the neutralizing activity of serum samples were analyzed by Western blot, ELISA and neutralization test.Results:After three rounds of panning, cloning and sequencing, KQEKDL was identified as the key motif. The serum samples collected from the mice immunized with the modified series of peptides containing key motifs had different degrees of binding ability to EV71 and VP1 protein. The serum samples of mice immunized the synthetic peptide containing only the minimum key motif (AC-KQEKDL-KLH) had the strongest response to the other three peptides and EV71 and the highest neutralizing titer.Conclusions:The EV71 neutralizing epitope was successfully screened using the phage display random peptide library. The key motif of KQEKDL might be the specific minimum amino acid sequence that triggered the immune system. This study provides a theoretical basis for better understanding the immune response mechanism, evaluating the immunogenicity of the antigens and further research and development of polypeptide vaccines.

Objective:To evaluate anatomical locking plate combined with bone cement intramedullary support in the treatment of proximal humeral fractures in the elderly.Methods:From May 2016 to July 2018, 19 geriatric proximal humeral fractures were treated with anatomical locking plate combined with bone cement intramedullary support at Department of Orthopaedics, General Hospital of Shenyang Northern Theater. There were 8 females and 5 males, with an average age of 71 years (from 60 to 84 years). All the fractures were closed. According to the Neer classification, there were 4 two-part, 5 three-part and 4 four-part fractures. At 6, 12, 18 and 36 weeks and one year postoperatively, anteroposterior and lateral X-ray films of the scapula were taken. At the last follow-up, the Constant-Murley shoulder score and the Disabilities of the Arm, Shoulder and Hand (DASH) score for the upper limb dysfunction were recorded.Results:The 13 patients were followed up for 3 to 12 months (6 months on average). Bony union was achieved in all the 13 patients. Local necrosis and deformation of the humeral head and partial screw perforation were observed one year after operation in 2 cases, but other patients reported no such complications or loosening of internal fixation. At the last follow-up, their mean Constant-Murley score was 82.6 (from 71 to 95) and their mean ADSH score 19.2 (from 9.2 to 48.1).Conclusion:Anatomical locking plate combined with bone cement intramedullary support may lead to fine early efficacy for the treatment of proximal humeral fractures in the elderly, since it can facilitate fracture reduction, promote rigid fixation and prevent loss of reduction.

Objective To investigate the prevalence of DUOX2 mutations in Chinese patients with congenital hypothyroidism (CH) and to discuss the inheritance pattern of DUOX2 gene.Methods Blood samples were collected from 91 CH children and their genomic DNA was extracted from peripheral blood leukocytes.All exons and exon-intron boundaries of DUOX2 were analyzed by target next-generation sequencing and family trios was established to study the inheritance pattern of DUOX2 gene.Results Fifty-four out of 91 children with CH carried DUOX2 mutation, with a prevalence of 59.34%.Of the 54 CH children, 36 carried DUOX2 biallelic mutations.In all 12 family trios with probands carrying biallelic DUOX2 mutations, the parents carried heterozygous DUOX2 mutations while still showing normal thyroid function, suggesting that CH caused by DUOX2 mutations is inherited in an autosomal recessive manner.Conclusion DUOX2 gene is one of the most frequently mutated genes in Chinese CH patients and its inheritance pattern is an autosomal recessive one.

Objective: To evaluate whether simultaneous vaccination with live attenuated polio vaccine affects the immunogenicity of live attenuated rotavirus (RV) vaccine. Methods: Rotarix produced by GlaxoSmithKline was used as the research object. Two doses of Rotarix were orally administered on day 0 and month 1, and oral live attenuated polio vaccine (OPV) was administered on day 0, month 1 and month 2 according to the national vaccination plan. Healthy infants aged 6 to 16 weeks were randomly divided into two groups: interval vaccination group (Rotarix and OPV were vaccinated on different days) and simultaneous vaccination group (Rotarix and OPV were vaccinated on the same day). Serum samples were collected on day 0, month 2 and month 12, and serum RV-IgA was measured by enzyme linked immunosorbent assay. Statistical analysis was performed to evaluate whether there were statistical differences in the seroconversion rate and level distribution of RV-IgA between the two groups. Results: The seroconversion rate of serum RV-IgA in month 2 was 73.84% in the interval vaccination and 63.95% in the simultaneous vaccination group, and the difference between them was statistically significant (P<0.05). The geometric mean concentrations (GMC) of RV-IgA were 97 EU/ml and 90 EU/ml, respectively (P>0.05). Compared with the simultaneous vaccination group, the seroconversion rate and GMC of serum RV-IgA in month 12 were higher in the interval vaccination group, and the differences were statistically significant (P<0.05). Conclusions Simultaneous vaccination with live attenuated polio vaccine would affect the immune response of live attenuated rotavirus vaccine, especially the maintenance of RV-IgA antibody level.

Objective:To investigate the role of long non-coding RNA nuclear paraspeckle assembly transcript 1 (Neat1) in pyroptosis of ultraviolet B (UVB)-induced human lens epithelial cells (LECs) and to explore the possible mechanism.Methods:The human lens epithelial cell line HLE-B3 was cultured in vitro, and cells at log phase were exposed to ultraviolet B for 0, 2, 4 and 8 hours, respectively.The expression of cysteine aspartic acid-specific protease-1 (caspase-1), a protein related to pyroptosis, was detected by Western blot.The relative expression level of Neat1 in cells after different irradiation durations was determined by real-time quantitative PCR.Cell viability was determined by the cell counting kit-8 (CCK-8) method to screen the optimal irradiation duration for UVB-induced LECs pyroptosis, which was finally determined to be 4 hours.HLE-B3 cells were divided into negative siRNA transfection group, siRNA Neat1 transfection group, negative siRNA transfection+ irradiation group and siRNA Neat1 transfection+ irradiation group, and were transfected with corresponding reagents for 24 hours.The negative siRNA transfection+ irradiation group and siRNA Neat1 transfection+ irradiation group were irradiated with UVB for 4 hours after transfection.The cell viability was detected by the CCK-8 method.The pyroptosis rate was detected by flow cytometry.The expression levels of caspase-1, gasdermin D (GSDMD) and nod-like receptor protein 3 (NLRP3) proteins were detected by Western blot.The concentration of interleukin (IL)-1β was detected by enzyme-linked immunosorbent assay (ELISA). Ultrastructural changes in HLE-B3 cells were observed under a transmission electron microscope. Results:The grayscale of caspase-1 protein bands increased with the extension of irradiation duration.The relative expression levels of caspase-1 protein at 0, 2, 4 and 8 hours of irradiation were 0.05±0.01, 0.25±0.07, 0.51±0.04 and 0.74±0.02, respectively, with a statistically significant overall difference ( F=168.223, P<0.001), and significant differences were found in paired comparisons (all at P<0.05). With prolonged irradiation, the relative expression level of Neat1 mRNA increased and the cell viability decreased, with statistically significant differences in paired comparisons (all at P<0.05). Compared with negative siRNA transfection group, the cell viability was increased in siRNA Neat1 transfection group and decreased in negative siRNA transfection+ irradiation group, with statistically significant differences (both at P<0.01). Compared with negative siRNA transfection+ irradiation group, the cell viability was increased in siRNA Neat1 transfection+ irradiation group, showing a statistically significant difference ( P<0.05). The pyroptosis rate was significantly lower in negative siRNA transfection group and siRNA Neat1 transfection+ irradiation group than in negative siRNA transfection+ irradiation group, and the differences were statistically significant (both at P<0.01). The relative expression levels of caspase-1, NLRP3 and GSDMD proteins in negative siRNA transfection+ irradiation group were higher than those in negative siRNA transfection group and siRNA Neat1 transfection+ irradiation group and the differences were statistically significant (all at P<0.01). The concentration of IL-1β was significantly higher in negative siRNA transfection+ irradiation group than in negative siRNA transfection group and siRNA Neat1 transfection+ irradiation group, and the differences were statistically significant (all at P<0.05). Cell swelling, formed cell membrane pores, vacuolated cells and fuzzy mitochondrial cristae were seen in negative siRNA transfection+ irradiation group and siRNA Neat1 transfection+ irradiation group by transmission electron microscopy.Compared with negative siRNA transfection+ irradiation group, slighter cell swelling, fewer cell membrane pores and lighter mitochondrial swelling were seen in siRNA Neat1 transfection+ irradiation group. Conclusions:Neat1 is involved in human LECs pyroptosis induced by UVB through the classic pyroptosis pathway mediated by caspase-1.Knockdown of Neat1 can inhibit the pyroptosis of human LECs.

Objective To investigate the cell origin of interleukin (IL)-22-secreting cell of mice infected with Trichinella spiralis (T.spiralis) at the early encapsulated stage.Methods Twelve Balb/c mice were divided into the infected group and the control group according to body weight by random number table.The infected mice were intragastrically administrated with 300 muscle larvae of T.spiralis,and the control mice were given the same amount of normal saline.The IL-22-secreting cell subsets in mouse splenic lymphocytes were detected by flow cytometry at the fourth week after infection.Results The proportion of IL-22-secreting cells in splenic lymphocytes of T.spiralis infected mice was increased when compared with control group [(0.88 ± 0.25)％ vs (0.28 ±0.17)％,t =-4.899,P ＜ 0.05].There was no significant difference between the proportion of CD3+IL-22+ cells and CD3-IL-22+ cells in the splenic lymphocytes of the infected group [(0.29 ± 0.17)％ vs (0.51 ± 0.17)％,t =-2.195,P ＞ 0.05],and the percentage of CD3-IL-22+ cells were similar between the infected group and the control group [(0.51 ± 0.17)％ vs (0.44 ± 0.22)％,t =-0.600,P ＞ 0.05].The proportion of CD3+IL-22+ cells in the infected group was significantly higher than that in the control group [(0.29 ± 0.17)％ vs (0.07 ± 0.06)％,t =-3.068,P ＜ 0.05],and the percentage of CD4+IL-22+ T cells and γδTCR+IL-22+ T cells were obviously increased in CD3+ lymphocytes [(1.28 ± 0.54)％ vs (0.16 ± 0.07)％,(0.33 ± 0.22)％ vs (0.02 ± 0.00)％,t =-4.997,-3.342,P ＜ 0.05].Conclusions The proportion of IL-22-secreting splenic lymphocytes is increased in mice infected with T.spiralis at the early encapsulated stage.The rise is caused by increased numbers of IL-22-secreting CD3 + lymphocytes,especially CD4+ T cells and γδT cells.

Objective To validate a cell infection-based quantitative RT-PCR for evaluating the potency of rotavirus vaccine. Methods According to the ICH ( the International Council for Harmonization) Harmonised Tripartite Guideline, the method was validated for its specificity, accuracy, precision, linearity and robustness. Results The method had good specificity as it could only amplify and detect the corre-sponding type of rotavirus strain. The recovery rates for determining the potency against rotaviruses of G2, G3 and G4 types were 97％ to 108％. The percent coefficient of variation ( CV) of both intra-plate and in-ter-plate precision was≤2. 62％, while the intraday and interday CV was≤1. 76％ and≤2. 27％, respec-tively. The CV between the two experimenters was≤7. 68％. The linearity range of the method was 4. 4-6. 5 UI for G2 type rotavirus, 3. 9-8. 3 UI for G3 type and 3. 5-8. 1 UI for G4 type. Good robustness was observed using the cells of 140 to 160 generations. Conclusions The cell infection-based quantitative RT-PCR was shown to have satisfactory specificity, accuracy, precision, linearity and robustness, suggesting that it was a suitable method for evaluating the potency of multivalent rotavirus live vaccines.