Alterations in the expression level of visfatin in the lungs of piglets infected with PRRSV and its effect on PRRSV replication.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by PRRS virus (PRRSV), characterized by sow reproductive failure and respiratory symptoms in pigs of all ages. PRRSV mainly causes severe lung damage by invading alveolar macrophages. Visfatin is closely related to acute lung injury, immune response and inflammation along with virus invasion to the host. Therefore, the current study was performed to clarify the relationship between visfatin and PRRSV infection. We used ternary piglets to construct a piglet model to explore the expression of visfatin and tight junction protein in lung injury induced by PRRSV infection, and then further studied the inhibition effect of visfatin on PRRSV replication by PRRSV infection of Marc-145 cells. Our results indicated that both PRRSV attenuated and virulent infections could damage the lung tissues, which could not only lead to severe inflammatory reaction (such as increased expression of TNF-α, TGF-ß, IL-8 and IL-10) in lung tissues of piglets, but also brought about the sharp decrease of ZO-1 and Tricellulin expressions resulting in impaired alveolar epithelial barrier. Meanwhile, we found significantly up-regulated expression of visfatin in lungs and serum of pigs after PRRSV infection that were related to both the degree of lung injury and the virulence of PRRSV strain. Moreover, visfatin might inhibit the PRRSV infection to Marc-145 cells in time dependent fashion. Hence, the current investigation provides the novel information about the effect of visfatin and PRRSV co-culture on Marc-145 cells and the effect of visfatin on PRRSV proliferation at different time points.

Visfatin Regulates Inflammatory Mediators in Mouse Intestinal Mucosa Through Toll-Like Receptors Signaling Under Lipopolysaccharide Stress.

Visfatin is a multifunctional protein involved in inflammatory immune stress. The aim of current study was to explore the role of visfatin in lipopolysaccharide (LPS)-induced intestinal mucosal inflammation and to confirm its cellular effect in inflammatory immune response through silencing of Toll-like receptors (TLRs). We divided Kunming mice into three groups: Saline group, LPS group, and LPS + visfatin group and performed hematoxylin and eosin staining, immunohistochemistry, quantitative polymerase chain reaction, Western blot, enzyme linked immunosorbent assay and RNA-seq analysis. Pretreatment of visfatin improves LPS-stimulated reduction of tight junction protein 1 (ZO-1) and secretory immunoglobulin A, inhibits overexpression of Claudin-1 and vascular endothelial growth factor, and reduces intestinal mucosal damage and inflammation. RNA-seq analysis of cellular transcriptomes indicated that visfatin is involved in down-regulation of mRNA level of TLR4 as well as attenuation of protein levels of TLR8 and nucleotide-binding oligomerization domain-containing protein 2, revealing that visfatin could reduce intestinal mucosal inflammation through TLR signaling pathway in mice ileum. In RAW264.7 cells, the genes silencing of Toll/IL-1R family, such as TLR4, TLR2, and IL-1R1, was accompanied by decreased expressions of inflammatory factors (TNF-α, IL-1ß, IL-6 and MCP-1) along with lower cellular visfatin levels. Hence, visfatin maintains the intestinal mucosal barrier structure and attenuates the intestinal mucosal inflammation through the TLR signaling pathway. Likewise, the Toll/IL-1R family regulates the release of visfatin, which can participate in the inflammatory reaction through the regulation of inflammatory factors.

Optimization and bioactivity verification of porcine recombinant visfatin with high expression and low endotoxin content using pig liver as template.

In order to obtain the porcine recombinant visfatin protein with high expression and low endotoxin content, the current study aims to express and verify the biological activity of the purified porcine recombinant visfatin protein. Firstly, four different expression strains were successfully constructed. Then they were simultaneously induced at 37 °C for 4 h and 16 °C for 16 h. The results showed that Visfatin-pET28a-Transetta was the best strain with high protein expression and purity at 16 °C induction for 16 h. After that, endotoxin was reduced from the recombinant visfatin until the residual endotoxin was less than one endotoxin units per milliliter (EU/mL). Finally, the purified porcine recombinant visfatin protein was incubated with RAW264.7 cells. The results of cell counting kit-8 (CCK-8) showed the survival rate of the cells first increased and then decreased with the increase in visfatin concentration. When the concentration of visfatin was 700 ng/mL, the survival rate of the cells was the highest. Thereafter, control (PBS), Visfatin and Visfatin + PolymyxinB (Ploy.B) groups were incubated with the RAW264.7 cells for 6 h. Real-time quantitative polymerase chain reaction (RT-qPCR) and Enzyme Linked Immuno-Sorbent Assay (ELISA) results showed that, as compared to the control group, the expressions of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 in Visfatin group were significantly increased (P < 0.05). However, there was no significant difference between the Visfatin and Visfatin + Poly.B groups, indicating that porcine recombinant visfatin protein promoted the inflammatory activity of RAW264.7 cells while the residual endotoxin did not play a role, suggesting biological activity of porcine recombinant visfatin protein.

[A Study on the Risk Factors for Early Postpartum Urinary Incontinence in Chengdu].

OBJECTIVE: To investigate the incidence of early postpartum urinary incontinence in parturients from Chengdu, and to find out the high-risk factors for reference for clinical diagnosis and treatment. METHODS: A total of 9 918 parturient women who gave delivery at the West China Second University Hospital of Sichuan University from January 2014 to January 2018 were enrolled and reviewed 6 weeks after delivery. The prevalence of urinary incontinence at 6 weeks postpartum was investigated by questionnaire. χ2 test and multivariate logistic regression analysis were used to analyze the risk factors affecting the prevalence. RESULTS: 9 550 parturient women were actually investigated. The prevalence of urinary incontinence was 15.53% (1 483/9 550) at 6 weeks postpartum in Chengdu, among which stress urinary incontinence was the most common (73.03%, 1 083/1 483). Univariate analysis showed that age, pelvic surgery history, prenatal body mass index (BMI), urinary incontinence during pregnancy, neonatal body mass, the number of parturition, delivery mode, lateral perineal incision, perineal laceration and prolonged second stage of labor were all correlated with the occurrence of urinary incontinence at 6 weeks postpartum (P < 0.05). Multivariate logistic regression analysis showed that cesarean section can reduce the risk of urinary incontinence compared with vaginal delivery 〔odds ratio (OR)=0.373, P < 0.001〕. Age≥35 yr. (OR=1.803, P=0.001), pelvic surgery history (OR=1.260, P=0.003), BMI≥28 kg/m2 during pregnancy (OR=1.694, P=0.025), urinary incontinence during pregnancy (OR=2.605, P < 0.001), neonatal body mass ≥4 kg (OR=2.307, P=0.040), multipara (OR=1.284, P=0.023) and perineal laceration (OR=1.372, P=0.035) were independent risk factors for urinary incontinence at 6 weeks postpartum. CONCLUSIONS: Urinary incontinence at 6 weeks postpartum is not rare in Chengdu, and stress urinary incontinence is more frequent. Eutocia, elderly parturient, multipara, pelvic surgery history, prenatal obesity, urinary incontinence during pregnancy, large neonatal body mass and perineal laceration are the main risk factors for urinary incontinence at 6 weeks postpartum.

[Effects of Cellular Density on the Induction of Suspension Globe of Ovarian Cancer Stem Cells].

OBJECTIVE: To determine the effect of cellular density on the separation and identification of cancer stem cells from human ovarian clear cell carcinoma cell line ES-2 and adenocarcinoma cell line A2780. METHODS: ES-2 and A2780 cells were cultured with human recombinant epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and bovine serum albumin and insulin in serum free medium. The cancer stem cells were obtained through serial passages. Changes in cell morphology,expressions of surface marker CD133 and CD44,and soft AGAR clone forming in the stem cells were examined under different cell density,either in serum-supplemented medium (SSM group) or in serum free medium (SFM group). RESULTS: Under the density of 2×104 mL-1,ES-2 cells survived in SFM,but did not form stem cells. When the density increased to 5×104 mL-1 or 1×105 mL-1,ES-2 cells survived in SFM,proliferated and formed stem cells. Compared with adherent cells,the suspension globe of stem cells expressed high levels of CD133 and CD44 ( P<0.05),with proliferation and clone forming ability after serial passages. The stem cell balls under the density of 5×104 mL-1 had stronger ability of tumor formation. A2780 cells formed suspension globe under the density of 1×104 mL-1 and 3×104 mL-1,but larger and more transparent balls were observed under the density of 3×104 mL-1 density. No suspension globe was formed under the density of 5×104 mL-1. More CD133+/CD44+cells were detected by flow cytometry under the density of 3×104 mL-1,compared with that under the density of 1×104 mL-1 ( P<0.05). Tumor stem cells grew faster under the density of 3×104 mL-1. CONCLUSION: The optimal density for identifying stem cells from human ovarian cancer is 5×104 mL-1 for ES-2 and 3×104 mL-1 for A2780,respectively.

[Relationship of Human Papillomavirus Subtypes and Multiple Infection with Different Cervical Precancerous Diseases in Sichuan Province].

OBJECTIVE: To investigate the relationship of human papillomavirus (HPV) subtypes and multiple infections with different cervical precancerous diseases. METHODS: Retrospective study was done to review 1 226 patients with different cervical lesions who were pathologically diagnosed and scanned for HPV 23 subtypes with positive results from June 2006 to May 2012. These patients were divided into the following groups, chronic cervicitis, cervical condyloma, cervical intraepithelium neoplasia grade I (CIN I), grade II (CIN II), grade III (CINIII). RESULTS: There were significant differences in the proportion of HPV low risk types and high risk types between cervicitis, condyloma, CIN I group and CIN II + III groups (P<0. 05). HPV low risk types in condyloma group were mainly 6 and 11 subtype, while the other four groups were 42 and 43 subtype. The four most prevalence high risk types were 58, 16, 52,18 subtype. The infection rates of HPV16 were significant different in cervicitis (11. 0%), CIN II (20. 3%), and CIN III (20. 2%)(P<0. 01), and the infection rates of HPV58 was quite different between cervicitis (15. 9%) and CIN II (21. 4%) (P<0. 05). HPV multiple infection rate in condyloma (68. 8%) was significant different from that of cervicitis (23. 1%), CINI (26. 1%), CIN II (27. 8%) and CIN III (27. 1%) (P<0. 01); while the rest four groups were not significantly different (P>0. 05). CONCLUSION: There is a unique epidemiologic characteristic of HPV infection in Sichuan Province. The HPV low risk types were mainly 42 and 43, and high risk types were mainly 58, 16, 52, 18. It seems that HPV multiple infection is not the leading cause of progression of cervical disease.

[Early human papillomavirus testing predicts residual/recurrent disease after LEEP risk factors for predicting residual disease in high-grade squamous cervical intraepithelial neoplasia following LEEP conization].

OBJECTIVE: To explore the risk factors for residual/recurrent disease of cervical intraepithelial neoplasia (CIN) 2 or worse after loop electrosurgical excision procedure (LEEP) and the timing point for postoperative follow-up. METHODS: 428 patients with CIN 2 or CIN 3 who were treated with LEEP were retrospectively reviewed. Postoperative follow-up was performed by Pap smear and human papillomavirus (HPV) hybrid capture 2 (HC2) testing. The definition of persistent/recurrent disease was biopsy-proven CIN 2 or worse. RESULTS: 296 patients were CIN 2 and 132 were CIN 3 among 428 patients. The positive rate of HPV HC2 before LEEP was 86.7% (371/428). During follow-up, 26 patients (6.1%) had residual/recurrent disease, the positive LEEP margin, especially the cone top status, was a significant risk factor for persistent/recurrent disease. Other factors such as age, HPV viral load [> or =100 relative light units (RLU)], and HPV typing (type 16/18 vs. other types) did not predict recurrence. HPV HC2 test at 3 months after LEEP can find all the residual/recurrent disease, the sensitivity and negative predictive value of the HPV HC2 test for residual/recurrent disease were both 100% at 3 and 6 months. CONCLUSION: The positive margin of LEEP specimen especially the cone top status was a significant risk factor for residual/recurrent disease after LEEP. HPV test at 3 months during follow-up can offer timely information about residual/recurrent disease and help for the risk control in treatment selection.

Bioactive proteins in healthy pregnancies and preeclampsia: relevance to hypertension and proteinuria.

BACKGROUND: Bioactive proteins, such as cytokines and chemokines, have not been systematically evaluated in healthy and preeclamptic pregnancies. We aimed to investigate the difference of these proteins between healthy and preeclamptic pregnancies in order to help clarify their potential roles in the pathogenesis of hypertension and proteinuria in preeclampsia. METHODS: Samples of amniotic fluid and maternal/umbilical cord blood were collected from normal pregnancies and women with preeclampsia for examination of bioactive proteins. Fifty-three pregnant women were enrolled in this study. Of them, 30 pregnant women were recruited as healthy controls, and 23 pregnant women were diagnosed with preeclampsia. An antibody array was used to screen for higher levels of cytokines and related proteins in amniotic fluid than in the blood samples, and these proteins were then selected for quantification by immunoassay. RESULTS: Interleukin-1 receptor 4, hepatocyte growth factor, and urokinase plasminogen activator receptor were significantly elevated in the blood of preeclampsia patients. In particular, interleukin-1 receptor 4 was 8-fold higher in preeclampsia patients than in the healthy pregnancies. Moreover, in cord blood samples hepatocyte growth factor and interleukin-8 were significantly higher in preeclampsia patients. CONCLUSIONS: Because of the biologic activities, Interleukin-1 receptor 4, hepatocyte growth factor, urokinase plasminogen activator receptor and interleukin-8 in maternal and/or cord blood could play a role in the pathogenesis of hypertension and proteinuria in preeclampsia.

[Effect of siRNA inhibiting HPV16 E6 gene expression on proliferation and cell cycle of cervical cancer cell line CaSki].

OBJECTIVE: To observe whether human papillomavirus 16 (HPV16) E6-specific small interfering RNAs (siRNAs) can be employed to inhibit the growth of cervical cancer cell line, and to investigate the associated mechanism. METHODS: RNAi was performed using synthetic small interfering RNAs transferred into CaSki cell line by lipofectamine. The cell growth curves, live cell ratio and inhibition ratio of cells were measured by using cell counting. At various time points of post-transfection, the distributions of cell cycle, the expression levels of HPV16 E6, p53, p21 mRNA and proteins were detected by using flow cytometry (FCM) and real-time quantitative reverse transcription-polymerase chain reaction (real-time RT-PCR). RESULTS: The growth inhibition of E6 siRNA to CaSki cells was demonstrated after cells treated with E6 siRNA. No substantial G1 arrest was observed by FCM analysis. For 24 hours after cell transfection, the level of E6 mRNA was decreased by 20. 11 folds compared with control (P < 0.05). However, p53 and p21 mRNA levels appeared unaffected. 48 hours after cell transfection, the expression level of E6 protein was efficiently decreased, but the P53 and P21 protein levels increased in comparison. CONCLUSIONS: The inhibitory effect of HPV16 E6 siRNA to CaSki cell maybe due to specially and efficiently silence E6 mRNA expression, decrease the degradation of wild type P53 protein, and then recover the function activity of P53 protein.

[Experimental study on inhibition of cervical carcinoma siha cell proliferation by siRNA expression vector].

OBJECTIVE: To observe the effect of HPV16E7 specific expression vector on cell proliferation in cervical carcinoma SiHa cells. METHODS: The HPV16E7 siRNA expression vector and empty expression vector were transfected into SiHa cells by liposome. The effects on E7 mRNA and E7 protein expression, cell cycle phase and cell growth rate were examined respectively by real-time RT-PCR, FCM and MTT assay. RESULTS: The HPV16E7 siRNA expression vector significantly inhibited the expression levels of E7 mRNA and E7 protein, the inhibition rates being 92.15% and 84.30% respectively. It also inhibited the transition from G, phase to S phase and the growth of SiHa cell line. CONCLUSION: HPV16E7 specific siRNA expression vector could specifically and efficiently inhibit the expression of E7 gene and hence it could regulate cell cycle and inhibit cell proliferation in cervical carcinoma SiHa cells. siRNA expression vector

[Inhibiting E7 oncogene in CaSki cells of cervical cancer by vector-based RNA interference technique].

OBJECTIVE: To test of the effect of vector-based RNA interference (RNAi) technique on inhibiting HPV16E7 gene in CaSki cells of cervical cancer. METHODS: The HPV16E7-specific siRNA expression vectors P1, P2 and P3 were constructed and transfected into CaSki cells by liposome. The expression of HPV16E7 mRNA and protein were detected by real-time RT-PCR and Western blot. RESULTS: The expression of HPV16E7 mRNA and protein decreased with the transfection of P1, P2 and P3. Vector P1 had the strongest inhibition effect, with an inhibition rates of 92.86% and 81.0% for the expression of HPV16E7 mRNA and protein respectively three weeks after transfection. CONCLUSION: The expression of E7 gene in CaSki cells can be inhibited by HPV16E7-specific siRNA expression vector.

[Inhibiting effect of HPV16 E6 small interfering RNA on the growth of human cervical carcinoma xenotransplanted in nude mice].

OBJECTIVE: To explore the inhibiting effect of human papillomavirus 16 E6-specific small interfering RNA (siRNA) on cervical cancer transplanted in nude mice. METHODS: CaSki cells transfected with HPV16 E6 siRNA were transplanted into nude mice. HPV16 E6 siRNA was injected into the tumor, and the control group was treated with NS. Tumor growth was monitored once every other day. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) was performed to detect apoptosis. The expression of E6 and p53 protein was assessed by immunohistochemistry. The contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured and the liver and kidney were examined by histopathology. RESULTS: Inhibition of tumor growth was demounstrated after treatment with HPV16 E6 siRNA. The volume and weight of tumors were significantly decreased in comparison with those of control group (P < 0.05). Apoptosis occurred more frequently in the experiment group than in the control. The expression of E6 and p53 was down-regulated. The contents of ALT and AST underwent no significant changes, and the histopathology of liver and kidney showed no abnormal changes. CONCLUSION: The growth ability of human cervical cancer xenograft tumors in nude mice can be inhibited by HPV16 E6-specific siRNA, with no toxic side effect on the liver. It may provide an useful method of gene-therapy against cervical cancer.

[Inhibition of HPV16 E6 oncogene in cervical cancer by RNA interference].

OBJECTIVE: The efficiency of HPV16 E6 gene silenced by RNA interference in vitro and in vivo was assessed. METHODS: The specific siRNA of HPV16 E6 was designed and transfected into CaSki cells by liposome. Cell apoptotic rates and the changes in HPV16 E6 mRNA and protein before and after transfection were measured. Cervical cancer nude mice models were set up, siRNA was injected directly into subcutaneous tumor. The function of siRNA was evaluated by the changes in tumor volume, HPV16 E6 protein expression and apoptosis of tumor cells. RESULTS: In vitro research, the cell apoptotic rates were 7.7%, 11.8%, 37.4% and 12.6% respectively at 24 h, 48 h, 5th day and 9th day after transfection. The HPV16 E6 mRNA was reduced by 77%, 83%, 59% and 41% at 24 h, 48 h, 5th day and 9th day after transfection. The inhibition rates of E6 protein measured by Flow cytometry were 79.7%, 80.4%, 71.3% and 57.4% at 24 h, 48 h, 5th day and 9th day after transfection, which were confirmed by the results of Western blot. In vivo research, E6 siRNA administration groups had great power in inhibiting tumor growth, restraining E6 protein expression, increasing tumor necrosis and apoptosis. The result of repeated injections of siRNA was better than that of single injection. CONCLUSION: RNA interference with HPV16 E6 is specific and highly efficient in vitro and in vivo.

[The inhibitory effects of siRNA expression vector on the expression of human papillomavirus E6 gene].

OBJECTIVE: To investigate the inhibitory effects of RNAi(RNA interference)expression vector on the expression of human papilomavirus E6 gene. METHODS: siRNA expression vectors were constructed to be aimed directly at HPV16 E6 gene. The recombinants were transfected into cervical cancer cell line, caski, with liposomes. Expression of E6 was detected with fluorescence quantitative PCR (FQ-PCR) and flow cytometry. RESULTS: Three kinds of expression vectors could reduce the expression of E6 mRNA and protein all in caski-B cell. The expression of E6 mRNA reduced to 21.7% of control group, and the protein inhibition ratio reached 98.1%. CONCLUSION: The RNAi expression vector can effectively inhibit the expression of HPVE6 gene.

[Inhibitory effect of RNA interference on expression of HPV16 E6 oncogene in cervical cancer cell line CaSki].

BACKGROUND & OBJECTIVE: Tumorigenesis and progression of cervical cancer closely relate with human papilloma virus (HPV) E6 and E7 oncogenes. Ribozyme and antisense oligonucleotides had been used to inhibit the expression of HPV E6 or E7 oncogenes to treat cervical cancer, but problems, including low efficiency, short-period maintenance, hard work, and high costs, still exist. This study was to evaluate the specific inhibitory effect and time-efficiency of RNA interference (RNAi) on HPV16 E6 gene in cervical cancer cell line CaSki. METHODS: The specific small interfering RNA (siRNA) of HPV16 E6 modified by fluorescein was synthesized, and transfected into CaSki cells. The transfection efficiency of siRNA was evaluated by calculating the ratio of fluorescent cells to total cells. Cell apoptosis was evaluated by flow cytometry (FCM). The mRNA level of HPV16 E6 before and after siRNA transfection was measured by RT-PCR, and protein level of HPV16 E6 was measured by Western blot and FCM. RESULTS: The transfection efficiency of siRNA was 81%. Apoptosis rates of CaSki cells at 1, 2, 5, and 9 d after transfection were 7.7%, 11.8%, 37.4%, and 12.6%, respectively. The mRNA level of HPV16 E6 at 1, 2, 5, and 9 d after transfection reduced by 77%, 83%, 59%, and 41%, respectively, but the mRNA level of beta-actin, as internal control, had no change. The inhibition rates of HPV16 E6 protein at 1, 2, 5, and 9 d after transfection were 79.7%, 80.4%, 71.3%, and 57.4%, respectively, but the protein level of Lamin A/C, as internal control, had no change at each time point. CONCLUSION: RNAi exists in CaSki cells, and has specific high efficiency on HPV16 E6 gene.