Objective To evaluate the role of Sonic hedgehog (Shh) signaling pathway in the spinal cord in neuropathic pain (NP) in the rats.Methods Seventy-two male Sprague-Dawley rats,aged 8 weeks,weighing 250-300 g,were randomly divided into 2 groups (n=36 each) using a random number table:sham operation group (S group) and NP group.Spared nerve injury was produced by exposing the sciatic nerve and its branches and ligation and transection of tibial nerve and common fibular nerve in anesthetized rats.The mechanical paw withdrawal threshold (MWT) was measured before operation and at 1,4,7,14 and 21 days after operation.After measurement of the pain threshold at 1,4,7,14 and 21 days after operation,the animals were then sacrificed,and the lumbar segment (L46) of the spinal cord was obtained for determination of Shh,Patched (Ptch),Gli1 and glial fibrillary acidic protein (GFAP)expression (by Western blot),Shh,Ptch and Gli1 mRNA expression (by fluorescent quantitative real-time reverse transcriptase-polymerase chain reaction),and Shh and GFAP expression (by immunohistochemistry).Results Compared with group S,the MWT was significantly decreased,and the expression of Shh,Ptch and Gli1 protein and mRNA and GFAP in spinal cord tissues was up-regulated in group NP (P＜ 0.05).Shh was mainly expressed in the cytoplasm of spinal dorsal horn neurons and in the gap around glial cells.Conclusion Shh signaling pathway in spinal cord is involved in the development and maintenance of NP in the rats.

Objective:To understand the drug purchase behaviors and prescription flows of outpatients and their influencing factors. Methods: After selecting and investigating tertiary hospitals, secondary hospitals, community health service centers ( one per each level) in Beijing, Zhengzhou, Guangzhou, this paper uses structured question-naire method to analyze 3,155 valid questionnaires. A logistic regression analysis is performed to understand the fac-tors influencing the drug purchase behaviors. Results:The factors influencing the drug purchase behaviors of outpa-tients mainly include the region where the patient is, level of the hospital, type of health insurance that the patient has, frequency of drug purchase, etc. Conclusions: This paper suggests hospital administrators to take measures to improve prescription flows, such as reforming the health care insurance to make payment system more suitable for pharmacies standardizing supply channels in order to make varieties consistent between hospitals and pharmacies, strengthening education and publicity to raise the outpatients’ awareness for drug purchase outside of hospitals, etc.

Objective To investigate the correlation between vertebral artery dominance (VAD) and posterior circulation ischemic stroke (PCIS).Methods The consecutive patients with acute ischemic stroke hospitalized from November 2013 to October 2015 were collected.All patients underwent MRI and magnetic resonance angiography (MRA).The dominant vertebral artery was defined as having the larger diameter if left and right diameter difference ≥0.3 mm or the vertebral artery connected to the basilar artery in a more straight fashion if the diameter difference ＜ 0.3 mm.They were divided into either an anterior circulation ischemia stroke (ACIS) group or a PCIS group according to the classification criteria of Oxfordshire Community Stroke Project (OCSP).A multivariate logistic regression analysis was performed to investigate the correlation between VAD and PCIS.Results A total of 226 patients withacute ischemic stroke were enrolled,including 172 patients (76.1％) in the ACIS group and 54 (23.9％) in the PCIS group.Fifty-four patients (23.9％) had VAD,including 38 (70.3％) on the left and 16 (29.7％) on the right.The proportion of the patients with VAD in the PCIS group was significantly higher than that in the ACIS group (55.6％ vs.14.0％;x2 =39.115,P ＜ 0.001).Multivariate logistic regression analysis showed that VAD was an independent risk factor for patients occurring PCIS (odds ratio,13.60,95％ confidence interval 6.90-27.01;P＜0.001).Conclusions VAD is closely associated with the occurrence of PCIS,and it is an independent risk factor for PCIS.

AIM To study the therapeutic effects of Neiyi Kangfu Tablets (Trionycis Carapax,Ostreae concha,Rhei Radix et Rhizoma,etc.) on adenomyosis and its possible mechanism of action.METHODS The mouse model for adenomyosis was set up by the pituitary allograft,and received the gavage of Neiyi Kangfu Tablets,Dan'e Fukang Decoction (Salviae miltiorrhizae Radix et Rhizoma,Curcumae Rhizoma,Paeoniae Radix rubra,etc.) and gestrinone,respectively,for three months.Western blot,real-time PCR,IHC were used to measure the expression of GPER-Ras-STAT3 in ectopic endometrium and entopic endometrium.RESULTS High doses of Neiyi Kangfu Tablets could significantly lower GPER protein and gene expression;down-regulate the Ras and STAT3 protein expression;reduce the lesion severity score of ectopic tissue morphology.Its curative effect was better than Dan'e Fukang Decoction and gestrinone.CONCLUSION Neiyi Kangfu Tablets may control the development of ectopic lesions by inhibiting the GPER-Ras-STAT3 pathway activity and weakening the production of estrogen.

Objective To investigate the effects of sex hormone-binding globulin (SHBG)gene in the apoptosis of human trophoblastic cells.Methods The siRNA specific-targeting SHBG gene was transfected into human trophoblastic cells and they were divided into six groups:trophoblasts without transfection in normal control groups(group Ⅰ);transfect liposome in blank control groups(group Ⅱ);transfect nonspecific siRNA in negative control groups(group Ⅲ);transfect SHBG siRNA-Ⅰ,SHBG siRNA-Ⅱ,SHBG siRNA-Ⅲ respectively in trans-fection group(group Ⅳ,Ⅴ,Ⅵ).Hoechst 33258 dying method was used to detect cell apoptosis.SHBG and Caspase-3 mRNA profiling and the level of SHBG and caspase-3 protein were detected by real-time PCR and Western blot.Results There was no statistical significant difference in the gene expression and protein level of SHBG and caspase-3 in group Ⅰ,Ⅱ and Ⅲ (P >0.05).In Ⅳ,Ⅴ and Ⅵ group,there was no statistical significant difference in the expression level of SHBG and caspase 3 (P >0.05).Compared with group Ⅰ,Ⅱ and Ⅲ,the a-mount of SHBG gene expression decreased obviously,the caspase-3 mRNA and protein level increased obviously and the trophoblast cell ap-optosis increased markedly (P <0.05).Conclusion Through siRNA interference technology can reduce SHBG gene expression in human trophoblastic cells,and it can lead to excessive apoptosis of human trophoblasts cells.

Objective: To investigate the effect of chrysotile exposure on ribosomal DNA (rDNA) copy number and DNA damage response, so as to provide insights into the mechanism of asbestos-induced carcinogenesis. Methods: Human pleural mesothelial MeT-5A cells were treated with chrysotile suspensions at doses of 1.25, 2.5 and 5 μg/cm2 (low-, medium-, high-dose group), while PBS served as controls. MeT-5A cells were harvested 6, 24, 48 and 72 h post-treatment, and the rDNA copy numbers and the BIRC5, HRAS, GINS4 and RRM2 mRNA expression were determined using a quantitative real-time PCR (qPCR) assay. The apoptosis of MeT-5A cells and DNA damage were detected using Muse cell analyzer. The rDNA copy numbers, DNA damage responses and BIRC5, HRAS, GINS4 and RRM2 mRNA expression were compared in MeT-5A cells treated with different doses of chrysotile suspensions. Results: There were significant differences in 45S rDNA copy numbers among low-, medium-, high-dose groups and the control groups 6, 48 and 72 h post-treatment with chrysotile suspensions, and significantly lower 45S rDNA copy numbers were measured in low-, medium- and high-dose groups than in the control group 6 h post-treatment, while significantly higher 45S rDNA copy numbers were found in the high-dose group than in low- and medium-dose groups 48 and 72 h post-treatment (all P<0.05). There were significant differences in 5S rDNA copy numbers among low-, medium-, high-dose groups and the control groups 24, 48 and 72 h post-treatment with chrysotile suspensions, and significantly lower 5S rDNA copy numbers were measured in medium- and high-dose groups than in the control group 24 and 48 h post-treatment, while significantly lower 5S rDNA copy numbers were found in medium- and high-dose groups than in the low-dose group 24, 72 h post-treatment (all P<0.05). There were significant differences in the overall apoptotic rate of MeT-5A cells among groups at different time points, and the overall apoptotic rate of MeT-5A cells were significantly higher in medium- and high-dose groups than in the control group (all P<0.05), with late-stage apoptosis predominantly detected. There were significant differences in the rates of ATM activation and DNA double-strand break in MeT-5A cells among groups 72 h post-treatment, and higher rates of ATM activation and DNA double-strand break were measured in medium- and high-dose groups than in the control group (all P<0.05). In addition, there were significant differences in the relative mRNA expression of BIRC5, HRAS, GINS4 and RRM2 genes among groups 24 and 48 h post-treatment, and significantly lower BIRC5, HRAS, GINS4 and RRM2 mRNA expression was quantified in medium- and high-dose groups than in the control group (all P<0.05). Conclusion Exposure to chrysotile may induce rDNA copy number variations and altered expression of nucleolar proteins in human pleural mesothelial cells, which may be involved in the regulation of DNA damage responses.

Objective: To investigate the effect of ribosomal DNA (rDNA) copy number variation caused by hexavalent chromium exposure on DNA damage response in different cell lines, so as to provide insights into the involvement of hexavalent chromium-induced rDNA copy number variation in DNA damage responses. : Methods Human lung epithelial BEAS-2B cells and human embryonic lung MRC-5 cells were treated with 2 μmol/L potassium dichromate for 24 hours, and then cells were transferred to fresh media for further incubation, while cells treated with the same volume of phosphate buffer solution served as controls. Cells treated with potassium dichromate for 24 hours, and 3 and 7 days post-detoxification, were harvested, and rDNA copy number was quantified in cells using a quantitative fluorescent real-time PCR assay. Cell cycle, apoptosis and DNA damage were detected using a Muse cell analyzer, and the DNA damage was evaluated with the proportion of ataxia telangiectasia-mutated (ATM) gene activation, proportion of double-strand DNA breaks and the percentage of the H2A.X variant histone phosphorylatio. : Results The 45S and 5S rDNA copy numbers of were significantly higher in MRC-5 cells than in BEAS-2B cells [(1.54±0.26) vs. (1.02±0.18), P<0.05; (6.97±1.07) vs. (3.00±0.15), P<0.05]. The 45S rDNA copy number was lower in MRC-5 cells 3 days post-detoxification (0.80±0.04) than in controls (P<0.05), and was higher in BEAS-2B cells 3 days post-detoxification (1.43±0.07) than in controls (P<0.05) . G0/G1 phase arrest was found in MRC-5 cells 24 hours post-treatment, and the apoptotic rates were significantly higher in MRC-5 cells 3 and 7 days post-detoxification than in controls [(11.53±1.53)%, (18.33±0.70)% vs. (3.53±0.93)%, P<0.05]. The overall apoptotic rates 24 hours post-treatment and 3 days post-detoxification [(2.80±0.17)%, (3.33±0.57)% vs. (1.53±0.61)%, P<0.05], proportion of ATM gene activation 3 days post-detoxification [(3.37±0.67%) vs. (1.18±0.22)%, P<0.05], proportion of double-strand DNA breaks 3 days post-detoxification [(4.45±0.85)% vs. (0.97±0.21)%, P<0.05] and percentage of the H2A.X variant histone phosphorylation 3 days post-detoxification [(1.68±0.56)% vs. (0.29±0.06)%, P<0.05] in BEAS-2B cells were higher than in controls. Conclusions Hexavalent chromium-induced rDNA copy number variation affects DNA damage response in different cell lines. A stronger DNA damage response is found in BEAS-2B cells with a low rDNA copy number, and a relative stable response is observed in MRC-5 cells with a high rDNA copy number.

Objective To study the effect of mitogen-activated protein kinas/extracellular signalregulated kinase (MAPK/ERK) signaling pathway on cell proliferation modulated by Sonic Hedgehog (Shh) signaling in fibroblast-like synoviocytes (FLS) isolated from patients with active rheumatoid arthritis (RA).Methods The synovial tissue were collected by the synovial arthroscopic debridement or arthroscopic synovectomy of RA patients with active disease activity [disease activity score(DAS)28 ≥3.2].The RA-FLS were primarily cultured by the explanted culture,and then were treated with Shh agonist purmorphamine,inhibitor cyclopamine or MAPK/ERK signaling pathway inhibitor U0126,respectively.Western blotting was used to examine the phosphorylation level of ERK 1/2 (p-ERK1/2),which was the critical protein of MAPK/ERK signaling.The cell proliferation activity was detected using cell proliferation and cytotoxicity kit-8 (CCK8),and the cell proliferation rate was detected using a flow cytometry.Analysis of variance and Kruskal-Wallis H(K) test were used for statistical analysis.Results Compared with the control group,purmorphamine transiently increased p-ERK1/2 protein at the concentration of 1 μmol/L,and the peak activations of p-ERK1/2 took place at 15 min (P＜0.01).Cyclopamine and U0126 decreased the expression ofp-ERK1/2 protein (P＜0.01).After the RA-FLS treated with purmorphmine(1 μmol/L)for 48 hours,the cell proliferation activity was (114±4)％ and the percentage of S phase cells was (8.39±0.60)％,which was significantly higher than those of the control group (100±0)％ (P＜0.01) and (3.29±0.69)％ (P＜0.01).After treated with cyclopamine (10 μmol/L) for 48 hours,the cell proliferation activity of RA-FLS was (89±1)％ (P＜0.05) and the percentage of S phase cells was (1.53±0.22)％ (P＜0.05).When co-treated with purmorphamine (1 μmol/L) and U0126 (10 μmol/L),the cell proliferative activity was (89±2)％ (P＜0.05) and the percentage of S phase cells was(1.07±0.25)％(P＜ 0.05).Conclusion Shh may promote proliferation of RA-FLS via modulating MAPK/ERK signaling,which in turn contributes to hyperplasia of synovium and ultimately leading to RA.

Objective To evaluate the relationship between spinal Sonic hedgehog (Shh) signaling pathway and inflammatory responses in rats with neuropathic pain (NP).Methods Forty-eight cleangrade healthy male Sprague-Dawley rats,aged 8 weeks,weighing 250-300 g,were divided into 4 groups (n=12 each) using a random number table method:sham operation group (group S),group NP,dimethyl sulfoxide (DMSO) group (group D) and specific Shh signaling pathway inhibitor cyclopamine group (group CP).Spared nerve injury was produced by exposing the sciatic nerve and branches followed by ligation and transection of tibial and common fibular nerves in anesthetized rats.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before establishing the model and 1 and 7 days after establishing the model.,The animals were sacrificed after measurement of pain threshold at 7 days after operation,and the lumbar segment L4-6 of the spinal cord was obtained for determination of the expression of Shh,Patched homolog (Ptch),Smoothened (Smo) and zinc finger-containing transcription factors 1 (Gli1) (by Western blot) and contents of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) (by enzyme-linked immunosorbent assay).Results Compared with group S,the MWT was significantly decreased,the expression of Shh,Patched,Smo and Gli1 was up-regulated,and the contents of TNF-α and IL-1β were increased in NP,D and CP groups (P＜0.05).Compared with group NP,the MWT was significantly increased,the expression of Shh,Patched,Smo and Gli1 was down-regulated,and the contents of TNF-α and IL-1β were decreased in group CP (P＜0.05),and no significant change was found in the parameters mentioned above in group D (P＜0.05).Conclusion The mechanism by which Shh signaling pathway is involved in the development and mainterance of inflammatory responses is related to inhibiting inflammatory responses of the spinal cord in rats with NP.

Objective: To explore the changes in ribosomal DNA copy number in peripheral blood among patients with pneumoconiosis and its influencing factors, so as to provide insights into prevention and treatment of pneumoconiosis. Methods: Eighty-eight patients with pneumoconiosis who visited a designated hospital and 71 community residents with no history of pneumoconiosis or dust exposure were selected as the pneumoconiosis group and control group, and age, smoking history, drinking history and cumulative years of exposure to dust were collected through questionnaire surveys. The copy number of 45S rDNA and 5S rDNA was detected using real-time fluorescence quantitative PCR, and the differences between the two groups were compared. Factors affecting the copy number of 45S rDNA and 5S rDNA were identified by a multiple linear regression model. Results: The pneumoconiosis group had a median age of 56.00 (interquartile range, 15.25) and a mean cumulative dust exposure duration of (12.40±8.08) years, with 56.82% smoking and 62.50% drinking. The control group had a median age of 64.00 (interquartile range, 37.00) years, with 32.39% smoking and 26.76% drinking. The median copy number of 45S rDNA in the pneumoconiosis group was 1.29 (interquartile range, 0.59), which was lower than 2.10 (interquartile range, 1.88) in the control group; the median copy number of 5S rDNA in the pneumoconiosis group was 5.33 (interquartile range, 0.85), which was higher than 4.66 (1.34) in the control group (both P<0.05). Multiple linear regression analysis identified age (β=-0.034) and pneumoconiosis (β=-1.595) as factors affecting 45S rDNA copy number, age (β=-0.013) as a factor affecting 5S rDNA copy number, and age (β=0.018) as a factor affecting 5S rDNA copy number in the pneumoconiosis group (all P<0.05). Conclusions Compared with community residents with no history of pneumoconiosis or dust exposure, the copy number of 45S rDNA in peripheral blood among patients with pneumoconiosis is reduced and the copy number of 5S rDNA is increased.