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Cong-Qi Du,1 Dong-Xue Zhang,1 Jing Chen,1,2 Qiu-Fen He,1,2 Wen-Qin Lin1 1Reproductive Medicine Center, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China; 2Department of Embryo Laboratory, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of ChinaCorrespondence: Wen-Qin Lin, Tel +86-0571-87235031, Email wzlwq70@126.comBackground: Poor sleep quality has been linked to lower semen quality, but it is unclear whether this result in decreased fertility. To address this question, we retrospectively evaluated the relationship between men’s sleep quality and treatment outcomes in subfertile couples receiving assisted reproductive technology (ART).Patient Enrollment and Methods: From September 2017 to November 2019, 282 subfertile couples referred to a Chinese fertility clinic and eligible for ART procedures were enrolled in our study. Sociodemographic characteristics, life habits, and sleep habits in the year prior to ART were recorded. Sleep quality was measured using the Pittsburgh Sleep Quality Index (PSQI). We first divided the patients into two groups based on sleep quality (good sleep: PSQI < 5 and poor sleep: PSQI ≥ 5). Then, the ART outcomes (fertilization rate, good quality embryo rate, implantation rate, positive pregnancy rate, clinical pregnancy rate, live birth rate, miscarriage rate, and birth weight) of each group were analyzed. Finally, multivariate linear and logistic regression analysis were used to examine the relationship between sleep quality (discrete variable or dichotomous variable) and ART outcomes.Results: The participants in the poor sleep group showed a lower fertilization rate of 60.13% (543/903) when compared with 67.36% for the good sleep group (902/1339), P < 0.001. The global PSQI score had a significant influence on birth weight (β, − 63.81; 95% CI, − 119.91- − 8.52; P = 0.047), and live birth rate (OR, 0.88; 95% CI, 0.78- 0.99; P = 0.047) after adjusting for the interfering factors. Men’s sleep quality was unrelated to good quality embryos rate, implantation rate, positive pregnancy rate, clinical pregnancy rate, or miscarriage rate.Conclusion: Men’s sleep quality was positively associated with fertilization rate, birth weight, and live birth rate among couples undergoing ART.Keywords: sleep quality, PSQI, fertility, male reproduction, in vitro fertilization, intracytoplasmic sperm injection

Cong-Qi Du,1 Yong-Yi Yang,2 Jing Chen,1 Lei Feng,3 Wen-Qin Lin1 1Reproductive Medicine Center, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China; 2Department of Gynaecology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China; 3Information Center, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of ChinaCorrespondence: Wen-Qin LinThe First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, People’s Republic of ChinaEmail wzlwq70@126.comBackground: The effects of sleep duration on semen quality have been documented in many epidemiological studies. However, the association between sleep quality and semen parameters and reproductive hormones is still unclear.Patients Enrollment and Methods: We conducted a cross-sectional study among 970 outpatients from the Reproductive Medicine Center in Zhejiang, China between October 2017 and July 2019. All participants delivered a semen sample, underwent a physical examination, and answered a questionnaire to provide the following information: demographics, life habits, and sleep habits. Sleep quality was measured using the Pittsburgh Sleep Quality Index (PSQI). We first divided the patients into two groups according to sleep quality (good sleep: PQSI < 5 and poor sleep: PSQI ≥ 5). Then, we analyzed routine sperm parameters (semen volume, sperm total motility, progressive motility, sperm concentration, total sperm number, and normal sperm morphology) and reproductive hormones (follicle-stimulating hormone, luteinizing hormone, estrogen, testosterone, and prolactin) of each group. Finally, we used multivariate linear regression analysis and Spearman correlation coefficients to examine the relationship between sleep quality (discrete variable or dichotomous variable) and sperm parameters, reproductive hormones.Results: A negative correlation was found between the general PSQI scores and several semen parameters: total motility (r= −0.187979, p< 0.001), progressive motility (r= −0.192902, p< 0.001), concentration (r= −0.167063, p< 0.001), total sperm number (r= −0.160008, p< 0.001), and normal sperm morphology (r= −0.124511, p< 0.001). However, there was no significant correlation between the semen volume, all reproductive hormones and the general PSQI scores. After adjusting for confounders, men with poor sleep had lower total motility (β= −9.287; 95% CI, −12.050, −6.523), progressive motility (β= −8.853; 95% CI, −11.526, −6.180), concentration (log scale, β= −0.131; 95% CI, −0.181, −0.082), total sperm number (log scale, β= −0.137; 95% CI, −0.189, −0.084), and normal sperm morphology (β= −1.195; 95% CI, −1.844, −0.547), but semen volume and all reproductive hormones were not markedly altered.Conclusion: Poor sleep quality might be related to impaired semen quality, but we found no evidence that poor sleep quality affects reproductive hormones.Keywords: sleep quality, fertility, male reproduction, sperm quality, PSQI

Purpose: Testicular sperm aspiration (TESA) is widely used to retrieve sperm from testis. Diagnostic testicular biopsy should not be routinely performed for azoospermia. Therefore, a good predictive model is needed before TESA., Methods: A total of 1972 azoospermia patients constituted the modelling set, and 260 azoospermia patients from two other centres constituted the validation set. An integrated predictive model was built using logistic regression. Receiver operating characteristic (ROC), calibration and decision curve analyses were performed to evaluate the performance of follicle-stimulating hormone (FSH), semen volume, testicular volume and the integrated model., Results: The FSH level was the best univariate predictor for successful sperm retrieval (SSR) and was better than semen volume and testicular volume alone (p<0.001, threshold 6.17 IU/L, modelling set area under receiver operating characteristic curve (AUC) 0.80, accuracy 0.79; validation set AUC 0.87, accuracy 0.78). The integrated predictive model had excellent accuracy for predicting SSR (modelling set: AUC 0.93, accuracy 0.89; validation set: AUC 0.96, accuracy: 0.89). Calibration curve analysis indicated that the integrated model calibration was good and better than that of FSH, semen volume and testicular volume alone. Decision curve analysis indicated with a threshold probability between 0.05 and 0.98, the integrated model added more benefit than treating either all or no patients., Conclusions: The integrated model has excellent discrimination and good calibration. It can help azoospermic men make better decisions before TESA. It should be noted that TESA is not the first-line treatment for non-obstructive azoospermia because of a low sperm retrieval rate., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Continuous glucose monitoring (CGM) were performed in children with hepatic glycogen storage disease (GSD) to accurately understand the situation of glucose levels during their treatment, and to provide support for optimizing their nutritional management. Methods: In this retrospective research, 42 patients with hepatic GSD who under went 72 h CGM were collected from Department of Pediatrics, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology from October 2019 to January 2020. According to the genetic test results, they were divided into 5 groups: type Ⅰa, type Ⅰb, type Ⅲa, type Ⅵ and type Ⅸa. After long-term follow up and regular treatment, the clinical data (induding course, age, height, weight and biochemical parameters, etc.) on the day of CGM were summarized, and 72 h CGM were performed to assess the occurrence of hypoglycemia and hyperglycemia.χ² test, Fisher exact probability method, t test, analysis of variance or nonparametric test were used for comparison between groups. Results: Forty-two cases of hepatic GSD patients included 25 males and 17 females (20 cases of type Ⅰa, 3 cases of type Ⅰb, 10 cases of type Ⅲa, 3 cases of type Ⅵ and 6 cases of type Ⅸa).The age was 9.5 (6.7, 12.9) years, and the course of disease was 6.8 (5.1, 11.3) years. The average levels of glucose of the patients were all normal. However, the levels of standard deviation of blood glucose (SDBG) and mean amplitude of glycemic excursion (MAGE) were significantly different ( F =2.747, 3.029,both P< 0.05). Among them, the SDBG of type Ⅰa and Ⅲa were significantly higher than those of type Ⅸa ((1.10±0.36), (0.98±0.30) vs. (0.62±0.26) mmol/L, t =3.010, 2.440, both P< 0.05), while the MAGE of type Ⅰ was higher than that of Ⅸa and Ⅲa ((2.3±0.9) mmol/L vs. (1.2±0.6) and (1.7±0.6) mmol/L, t =2.734, 2.302, both P <0.05, respectively). Conclusions: CGMS can accurately assess the fluctuations of blood glucose and effectively detect hidden hypoglycemia and hyperglycemia in hepatic GSD patients. For different types of hepatic GSD, individualized corn starch treatment doses should be given according to the different situation of blood glucose, so as to optimize the patient's treatment and improve their prognosis.

In the previous research, our laboratory established a mouse model combining disease with syndrome of human coronavi-rus pneumonia with pestilence attacking the lung syndrome, based on the national traditional Chinese medicine clinical classification of Novel Coronavirus Infected Pneumonia Diagnosis and Treatment Plan. In this study, a mouse model combining disease with syndrome of human coronavirus pneumonia with pestilence attacking the lung syndrome was used to evaluate the effectiveness of Reyanning Mixture to provide animal experimental support for clinical application. Mice were divided into normal group, 229 E infection group, cold-dampness group, cold-dampness+229 E infection group(the model group), Reyanning high and low dose groups. The cold-dampness group, cold-dampness+229 E infection group, two Reyanning groups were given cold and damp stimulation for 7 days. On the 5 th day, the 229 E infection group, cold-dampness+229 E infection group, and two Reyanning groups were infected with HCoV-229 E virus. Reyanning was administered for 3 days, starting from the day of infection. Blood was collected on the 4 th day and the lung tissue was dissected to calculate the lung index and inhibition rate; flow cytometry was used to detect the percentage of T and B lymphocytes in peripheral blood; RT-PCR was used to detect the nucleic acid virus load in lung tissue; ELISA was used to detect motilin and gastrin in serum, and inflammatory factors TNF-α, IFN-γ, IL-6, IL-10 in lung tissue proteins. Reyanning Mixture could reduce the lung index(P<0.01) of coronavirus pneumonia mice with pestilence attacking the lung; it could significantly increase the percentage of CD8~+ T lymphocytes and CD4~+ T lymphocytes in peripheral blood of model mice(P<0.05, P<0.01). The low dose of Reyanning could effectively increase the percentage of total B lymphocytes(P<0.05), reduce virus load in lung tissue of model mice(P<0.01), reduce the levels of TNF-α, IFN-γ, IL-6, IL-10 in the lung tissue of model mice(P<0.01), reduce the content of motilin in the serum of model mice(P<0.01). Reyanning Mixture convey a better effect in treating coronavirus pneumonia mice with pestilence attacking the lung. It manifested obvious effects in improving lung lesions, enhancing the gastrointestinal function of mice, improving the autoimmune function of mice, and reducing the expression of inflammatory factors in vivo, which could provide evidences for clinical research.

As glutamate dehydrogenases (GDHs) of microorganisms usually have higher affinity for NH 4 + than do those of higher plants, it is expected that ectopic expression of these GDHs can improve nitrogen assimilation in higher plants. Here, a novel NADP(H)-GDH gene ( TrGDH ) was isolated from the fungus Trichurus and introduced into rice ( Oryza sativa L.). Investigation of kinetic properties in vitro showed that, compared with the rice GDH ( Os GDH4), Tr GDH exhibited higher affinity for NH 4 + ( K m = 1.48 ± 0.11 mM). Measurements of the NH 4 + assimilation rate demonstrated that the NADP(H)-GDH activities of TrGDH transgenic lines were significantly higher than those of the controls. Hydroponic experiments revealed that the fresh weight, dry weight and nitrogen content significantly increased in the TrGDH transgenic lines. Field trials further demonstrated that the number of effective panicles, 1,000-grain weight and grain weight per plant of the transgenic lines were significantly higher than those of the controls, especially under low-nitrogen levels. Moreover, glutelin and prolamine were found to be markedly increased in seeds from the transgenic rice plants. These results sufficiently confirm that overexpression of TrGDH in rice can improve the growth status and grain weight per plant by enhancing nitrogen assimilation. Thus, TrGDH is a promising candidate gene for maintaining yields in crop plants via genetic engineering., (Copyright © 2019 by JAPANESE SOCIETY OF BREEDING.)

Background: This study assessed the effect of ibandronate (IBN), a farnesyl pyrophosphate synthase (FPPS) inhibitor, on vascular remodeling in diabetic rats., Methods: A rat model of diabetes was induced by a high-fat and high-sugar diet combined with a small dose of streptozotocin. The diabetic rats received 5 µg/kg of ibandronate solution or normal saline subcutaneously every morning for 16 weeks. The morphology of the thoracic aorta was assessed by hematoxylin and eosin and Masson's trichrome staining techniques. Gene expression levels of connective tissue growth factor (CTGF) and FPPS were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. CTGF and FPPS protein levels were determined by Western blotting analysis., Results: Rats with diabetes mellitus showed moderate hyperglycemia, insulin resistance, hyperlipidemia and thoracic aortic fibrosis. FPPS was significantly upregulated in the thoracic aorta from diabetic animals. Interestingly, IBN treatment for 16 weeks alleviated the diabetes-induced histopathologic changes in the thoracic aortic wall and reduced CTGF protein and mRNA levels., Conclusions: These findings provided evidence that FPPS is involved in thoracic aortic fibrosis in diabetic rats. Meanwhile, IBN could alleviate vascular remodeling in diabetic animals., (Copyright © 2019. Published by Elsevier Inc.)

Wnt1-inducible signaling pathway protein-1 (WISP1), a member of the CCN family, is increasingly being recognized as a potential target for obesity and type 2 diabetes mellitus. Recent studies have shown that WISP1 can regulate low-grade inflammation in obese mice, and circulating WISP1 levels are associated with obesity and type 2 diabetes mellitus in adults. Herein, we measured serum WISP1 levels in obese youth and explored its relationships with pro-inflammatory cytokine interleukin 18 (IL-18) and other metabolic indexes. Totally, 44 normal-weight and 44 obese children and adolescents were enrolled. Physical and laboratory data were recorded, and then serum levels of WISP1 and IL-18 were determined by enzyme-linked immunosorbent assays. Results showed that serum levels of WISP1 were significantly higher in obese children and adolescents than in normal-weight healthy controls (1735.44±15.29 vs. 1364.08±18.69 pg/mL). WISP1 levels were significantly positively correlated with body mass index (BMI) and BMI z-score (r=0.392, P=0.008; r=0.474, P=0.001, respectively) in obese group; circulating IL-18 was increased in obese individuals (1229.06±29.42 vs. 295.87±13.30 pg/mL). Circulating WISP1 levels were significantly correlated with IL-18 (r=0.542, P<0.001), adiponectin (r=0.585, P<0.001) and leptin (r=0.592, P<0.001). The multivariate stepwise regression analysis showed that higher IL-18 levels represented the main determinant of increased WISP1 levels after adjusting for BMI, waist circumference, fasting insulin, homeostatic model assessment of insulin resistance (HOMA-IR) and HbA1c in obese individuals (β=0.542, P=0.000). WISP1 can be involved in glucose/lipid metabolism in obese youth, which may be modulated by IL-18. Increased WISP1 levels may be a risk factor of obesity and insulin resistance, and WISP1 has a potential therapeutic effect on insulin resistance in obese children and adolescents.

To study how hydrogen-rich saline (HS) promotes the recovery of testicular biological function in a hemi-sectioned spinal cord injury (hSCI) rat model, a right hemisection was performed at the T11-T12 of the spinal cord in Wistar rats. Animals were divided into four groups: normal group; vehicle group: sham-operated rats administered saline; hSCI group: subjected to hSCI and administered saline; HRST group: subjected to hSCI and administered HS. Hind limb neurological function, testis index, testicular morphology, mean seminiferous tubular diameter (MSTD) and seminiferous epithelial thickness (MSET), the expression of heme oxygenase-1 (HO-1), mitofusin-2 (MFN-2), and high-mobility group box 1 (HMGB-1), cell ultrastructure, and apoptosis of spermatogenic cells were studied. The results indicated that hSCI significantly decreased the hind limb neurological function, testis index, MSTD, and MSET, and induced severe testicular morphological injury. The MFN-2 level was decreased, and HO-1 and HMGB-1 were overexpressed in testicular tissues. In addition, hSCI accelerated the apoptosis of spermatogenic cells and the ultrastructural damage of cells in the hypophysis and testis. After HS administration, all these parameters were considerably improved, and the characteristics of hSCI testes were similar to those of normal control testes. Taken together, HS administration can promote the recovery of testicular biological function by anti-oxidative, anti-inflammatory, and anti-apoptotic action. More importantly, HS can inhibit the hSCI-induced ultrastructural changes in gonadotrophs, ameliorate the abnormal regulation of the hypothalamic-pituitary-testis axis, and thereby promote the recovery of testicular injury. HS administration also inhibited the hSCI-induced ultrastructural changes in testicular spermatogenic cells, Sertoli cells and interstitial cells.

Reactive oxygen species (ROS), originating predominantly from vascular smooth muscle cells (VSMCs), lead to vascular damage and endothelial dysfunction in rats with hypertension. The downstream signaling pathways of farnesyl pyrophosphate (FPP) synthase, Ras-related C3 botulinum toxin substrate 1 (Rac1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mediate the generation of ROS. The present study investigated the effect of the FPP synthase inhibitor, ibandronate, on ROS production, the possible beneficial effect on endothelial dysfunction and the underlying mechanisms in spontaneously hypertensive rats (SHRs). The SHRs were treated with ibandronate for 30 days. Endothelium‑dependent and independent vasorelaxation were measured in isolated aortic rings. Additionally, VSMCs from the SHRs and Wistar‑Kyoto (WKY) rats were cultured. The production of ROS and activation of NADPH oxidase were determined using fluorescence and chemiluminescence, respectively, in vivo and in vitro. Angiotensin II (Ang II) increased ROS production in the cultured VSMCs from the WKY rats and SHRs, in a concentration‑dependent manner. The Ang II‑induced responses were more marked in the SHR VSMCs, compare with those in the WKY VSMCs, however, the response decreased significantly following ibandronate pretreatment. Treatment with ibandronate significantly decreased the production of ROS, translocation of NADPH oxidase subunit p47phox, and activities of NADPH oxidase and Rac1 in the aorta and VSMCs, and improved the impaired endothelium‑dependent vasodilation in the SHRs. Adding geranylgeraniol, but not farnesol or mevalonate, reversed the inhibitory effects of ibandronate. In addition, inhibiting geranylgeranyl-transferase mimicked the effect of ibandronate on the excess oxidative response. Ibandronate exerted cellular antioxidant effects through the Rac1/NADPH oxidase pathway. These effects may have contributed to the vasoprotective effects on the impaired endothelium in SHRs.

Pharmacological studies have shown that the active components in Dendranthema morifolium exhibit protective effects against ischemia/reperfusion injury; however, its pharmacological action on blood vessels has not yet been investigated. The purpose of the present study was to assess the effects of the total flavones extracted from D. morifolium (Ramat.) Tzvel. cv. Hangju (FDM) on the vasocontraction and proliferation of vascular smooth muscle cells (VSMCs). The tension of rat thoracic aortic rings was measured using a mechanical force transducer attached to a recording system. FDM induced a dose‑dependent relaxation of rings with endothelium pre‑contracted by either phenylephrine (PE; 10(‑6) mol/l) or a high concentration of potassium chloride (KCl; 60 mmol/l). FDM did not significantly affect the vasorelaxant effects on mechanically removed endothelium. In endothelium‑denuded aortic rings depolarized by 60 mmol/l KCl, FDM inhibited the contraction induced by Ca2+. FDM reduced the transient contraction caused by PE in a Ca2+‑free solution, but did not affect the contraction induced by phorbol ester. Furthermore, FDM inhibited the proliferation of VSMCs with or without growth stimulation by insulin. In conclusion, that the vasorelaxation induced by FDM in rat aortic rings is not dependent on the endothelium but is mediated via a reduction of the influx of extracellular Ca2+ through the voltage‑dependent and receptor‑operated channels and via the inhibition of the release of intracellular Ca2+ in VSMCs. The anti‑proliferative activity of FDM suggests that it may be beneficial in inhibiting atherosclerosis.

Through the regulation of the RhoA/Rho kinase (ROCK) pathway, angiotensin II (Ang II)-induced fibrotic responses contribute to vascular remodeling. Farnesyl pyrophosphate synthase (FPPS) plays an important role in cardiovascular remodeling through the modulation of the above-mentioned pathway. However, the role of FPPS in Ang II-induced fibrotic responses and the related molecular mechanisms have not yet been elucidated. In the present study, vascular smooth muscle cells (VSMCs) from Sprague-Dawley (SD) rats were stimulated with Ang II. Cell proliferation was measusred usin the cell counting kit-8 (CCK-8). The levels of connective tissue growth factor (CTGF), FPPS, and those of phosphorylated and total extracellular signal-regulated kinase (ERK)1/2, p38 and c-Jun N-terminal kinase (JNK) were determined by western blot analysis. RhoA activity was determined using a pull-down assay. The results revealed that stimulation with Ang II enhanced cell proliferation, and increased the protein expression levels of FPPS and CTGF in the VSMCs. The inhibition of FPPS with ibandronate sodium attenuated the Ang II-induced increase in cell proliferation, CTGF expresison and RhoA activity; these effects were partially reversed by treatment with geranylgeraniol and were mimicked by GGTI-286. Furthermore, both SB203580 (a specific inhibitor of p38) and SP600125 (JNK1, JNK2 and JNK3 inhibitor) diminished the Ang II-induced production of CTGF; however, the inhibition of FPPS reduced the Ang II-induced activation of p38 mitogen-activated protein kinase (MAPK) and JNK. In conclusion, our data indicate that FPPS may play an important role in Ang II-induced fibrotic responses in VSMCs, and the underlying mechanisms at least partly involve the modulation of RhoA activity, and the p38 and JNK pathways.

The proliferation of vascular smooth muscle cells (VSMCs) is one of the main features of atherosclerosis induced by high glucose. Mevalonate pathway is an important metabolic pathway that plays a key role in multiple cellular processes. The aim of this study was to define whether the enzyme expression in mevalonate pathway is changed in proliferated VSMCs during atherogenic process in diabetic mice. Diabetes was induced in BALB/c mice with streptozotocin (STZ, 50 mg/kg/day for 5 days). Induction of diabetes with STZ was associated with an increase of lesion area and media thickness after 8 and 16 weeks of diabetes. In aorta, there were overexpressions of some enzymes, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), farnesyl pyrophosphate synthase (FPPS), geranylgeranyl pyrophosphate synthase (GGPPS), farnesyltransferase (FNT), and geranylgeranyltransferase-1 (GGT-1), and unchanged expression of squalene synthase (SQS) and phosphor-3-hydroxy-3-methylglutaryl-coenzyme A reductase (P-HMGR) in 8 and 16 weeks of diabetes. In vitro, VSMCs were cultured and treated with different glucose concentrations for 48 h. High glucose (22.2 mM) induced VSMC proliferation and upregulation of HMGR, FPPS, GGPPS, FNT, and GGT-1 but did not change the expressions of SQS and P-HMGR. In conclusion, altered expression of several key enzymes in the mevalonate pathway may play a potential pathophysiological role in atherogenic process of diabetes macrovascular complication.

Both norepinephrine (NE) and connective tissue growth factor (CTGF) contribute to vascular fibrosis during hypertension. Recent studies indicate that farnesyl pyrophosphate synthase (FPPS) plays an important role in cardiac remodeling in hypertension. However, the role of FPPS in NE-induced fibrotic responses and related molecular mechanisms is unknown. Vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were stimulated with NE. The fibrotic responses were assessed by measuring CTGF, hydroxyproline (hyp), and α-1 procollagen I levels using Western blot, a hydroxyproline test kit, and real-time quantitative PCR assays, respectively. Ras activity was determined by a pull-down assay using a Ras activation assay kit and detected by Western blot. NE dose-dependently increased fibrosis in SHR-VSMCs, and this increase was significantly reduced by ibandronate, an inhibitor of FPPS. The addition of farnesol, but not geranylgeraniol, partially reversed the inhibitory effects of ibandronate. Furthermore, the anti-fibrotic effects of ibandronate could be mimicked by FTI-276 but not by GGTI-286. A pull-down assay showed that ibandronate reduced the NE-induced Ras activation. Moreover, ibandronate inhibited the NE-induced activation of p38, JNK, and ERK1/2. Only SB203580 (specific inhibitor of p38) diminished the NE-induced CTGF production. These results demonstrated that inhibiting FPPS prevents NE-induced fibrotic responses in SHR-VSMCs and that the Ras kinase and p38 pathways were the underlying mechanisms involved in this process.

Objective: To study the expressions of differential proteins in the expressed prostatic secretion (EPS) of patients with III A chronic prostatitis and healthy men., Methods: We collected EPS samples from 35 patients with III A chronic prostatitis and 18 age-matched healthy men, and detected the differentially expressed proteins in EPS by MALDI-TOF/MS. Based on the data obtained, we conducted a statistical analysis on the mass-to-charge (m/z) ratios of different proteins and a retrieval analysis on the relevant proteins using the protein database., Results: In the comparative studies of the III A chronic prostatitis patients and healthy men, 5 proteins were detected as at least 2-fold differentially expressed, which were probably brevinin-2Eg, big endothelin-1, alpha-defensin 15, beta-defensin 134 and prostatic steroid-binding protein C2. The m/z ratios were significantly up-regulated in 3 372, 3 487, 425 and 5 325 Da proteins (P < 0.01) and down-regulated in 10631Da (P < 0.01)., Conclusion: Proteins are differentially expressed in the EPS of III A chronic prostatitis patients and healthy men, and these proteins may be significantly correlated with the development and progression of III A chronic prostatitis.

Aims: Farnesyl pyrophosphate synthase (FPPS) is a key enzyme in the mevalonate pathway. In our previous study, we found that inhibition of FPPS attenuates cardiac hypertrophy in spontaneously hypertensive rats (SHRs) and prevents angiotensin (Ang) II-induced hypertrophy in cardiomyocytes. Here, we further investigate the role of FPPS in cardiac hypertrophy and heart failure (HF) using a transgenic (Tg) model, and its mechanisms., Methods and Results: Tg mice with cardiac-specific expression of FPPS were studied as an experimental model. The results showed that Tg mice with overexpression of FPPS exhibited cardiac hypertrophy, fibrosis, and HF, as well as increased synthesis of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate in heart tissue. These pathological changes were associated with the activation of RhoA and other known kinases in the hypertrophic signalling pathway, such as extracellular signal-related kinases 1/2 and p38. Adenoviral infection of FPPS in cultured neonatal cardiomyocytes induced a hypertrophic response characterized by an increased cell size and an increased extent of sarcomeric organization, as well as an increased activation profile of small GTPases and downstream protein kinases concordant with those seen in vivo. Further investigation showed a marked increase of FPPS protein levels in hypertrophic ventricles of patients with valvular heart disease., Conclusion: Taken together, these results suggest that FPPS may function as a potent regulator in myocardial remodelling. The FPPS-regulated signalling pathway is relevant to the pathological changes in cardiac hypertrophy and HF.

Objective: To evaluate the effect of Recession of both horizontal rectus muscles in Duane Retraction Syndrome with significant globe retraction., Methods: Sixteen cases with DRS were summarized retrospectively. All patients had undergone surgery with recession of both horizontal rectus muscles. All clinical records, including sex, age, types of DRS, clinical features, surgical methods and clinical outcomes were analyzed. All patients were followed up for 3 months to 1 year., Results: Fifteen cases had only monocular involvement while one had both eyes. The number of type I DRS was 3 cases, 1 case was esotropia while others were orthotropic in primary position. Type III DRS was observed 13 cases. Esotropia was seen in 6 cases (7 eyes), exotropia of 1 cases and orthotropic in primary position of 6 cases. 10 cases exhibited marked face turn. An upshoot or downshoot and variable severity of retraction of globe were found in all patients on attempt adduction of the affected eye. All patients had undergone surgery with recession of both horizontal rectus muscles. The medical rectus muscles were recessed from 5 mm to 7 mm and lateral rectus muscles 3 mm to 9 mm simultaneously, which was based on the amount of primary position deviation. Among these 2 cases were combined with Y-splitting of lateral rectus muscle. After surgery, all patients were orthotropic in primary position. Their symptom of unacceptable abnormal head position, significant globe retraction, noticeable narrowing of the palpebral fissure and significant upshoot or downshoot were ameliorated or disappeared. Especially the recession of lateral rectus muscle in addition to Y-splitting combining with the simultaneous medial rectus recession resulted in further amelioration of globe retraction in addition to upshoot and downshoot., Conclusion: Recession of both horizontal rectus muscles is effective in the treatment of significant globe retraction in Duane syndrome. Type III DRS with significant globe retraction but has no marked deviation and face turn can adopt this method to ameliorate their aspect. The method of lateral rectus muscle in addition to Y-splitting plays an important role in the treatment of upshoot and downshoot.

Interleukin-1β (IL-1β) is a multifunctional proinflammatory cytokine upregulated in acute phase of heart ischemic disease. Controversial effects of IL-1β have been demonstrated on endothelial progenitor cells (EPCs) functional activity. The aim of this study was to investigate the in vitro effect of IL-1β on activity of human origin EPCs and the possible mechanism involved. EPCs were isolated from peripheral blood of healthy volunteers without cardiovascular risk factors and characterized. After ex vivo cultivation, EPCs were stimulated with a series of final concentrations (0, 0.1, 1, and 10 ng/ml) of IL-1β for 24 h. In some other experiments, EPCs were pretreated with 10 μM LY294002 (Akt inhibitor) for 30 min and then stimulated with 1 ng/ml IL-1β for 24 h. Cell proliferation, apoptosis, adhesion, and migration were determined, respectively, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, annexin V/propidium iodide binding assay, adhesion assay, and transwell migration assay. In addition, the vascular endothelial vascular growth factor-A (VEGF-A) production has been examined using quantitative real-time RT-PCR and ELISA assay. Furthermore, the total and phosphorylation level of Akt was determined by Western blot. IL-1β significantly stimulated EPC proliferation, migration, and adhesion and upregulated the angiogenic growth factor VEGF-A at mRNA and protein level, while exerted no influence on cell apoptosis. However, pretreatment with LY294002 significantly diminished IL-1β-induced proliferation, migration, adhesion, and VEGF-A production. One nanogram per milliliter IL-1β for 15 min activated phosphorylation of Akt. These results suggest a potent role for IL-1β in upregulating EPCs functions. The phosphatidyl-inositol-3-kinase-Akt signaling pathway could be involved in the regulation of EPCs functions induced by IL-1β.

Overproduction of circulating S100A8/A9 occurs in patients following acute myocardial infarction (AMI). It remains unclear whether ischemia insult per se induces S100A8 and S100A9 expression in cardiac myocytes or even whether the cardiac myocytes participate as a source of these proteins. In this study, western blot analysis and quantitative real-time reverse transcription polymerase chain reaction were used to test samples obtained from isolated spontaneously hypertensive rat hearts and Wistar-Kyoto rat hearts subjected to global normothermic ischemia and from neonatal Wistar rat cardiac myocytes undergoing hypoxia. Ischemia did not increase the expression of S100A8 and S100A9 proteins and mRNA in the myocardium either from the spontaneously hypertensive rat hearts or the Wistar-Kyoto rat hearts. In addition, the levels of S100A8 and S100A9 proteins were unchanged in the neonatal rat cardiac myocytes undergoing hypoxia. However, both ischemia and hypoxia activated NF-kappaB in ischemic myocardium and in hypoxic cardiac cells in a time-dependent manner. The results suggest that the increased serum S100A8/A9 concentrations following AMI were not of cardiac myocyte origin.

Objective: To explore the clinical features and treatment of spermatic cord liposarcoma (SCL)., Methods: We retrospectively analyzed the clinical data of a case of SCL, reviewed the related literature and investigated the diagnosis and treatment of the disease., Results: The patient underwent tumor resection and left inguinal orchidectomy. Postoperative pathology confirmed the case to be s SCL. Neither recurrence nor metastasis was found during the five-month follow-up., Conclusion: SCL is a rare medical condition with no specific imaging and laboratory features. Radical orchidectomy with wide local excision of the mass is recommended for its treatment, and adjuvant radiotherapy can be considered in intermediately or highly differentiated tumors and recurrent liposarcomas, while the role of chemotherapy is not well-defined.

Background: The mevalonate pathway is an important metabolic pathway that plays a key role in multiple cellular processes. The aim of this study was to define whether the enzyme expression in mevalonate pathway changes during cardiovascular remodelling in spontaneously hypertensive rats (SHR)., Methods and Results: Hearts and thoracic aortas were removed for the study of cardiovascular remodeling in SHR and Wistar-Kyoto rats (WKY). The protein expression of the enzymes in hearts, aortas and livers was analyzed by western blot. The histological measurements showed that the mass and the size of cardiomyocytes, the media thickness and the media cross-sectional area (MCSA) of the thoracic aorta were all increased in SHR since 3 weeks of age. In the heart, there was overexpression of some enzymes, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), farnesyl diphosphate synthase (FDPS), and geranylgeranyltransferase type I (GGTase-I), and downregulation of squalene synthetase (SQS) in SHR since 3 weeks of age. In the aorta, besides similar expressions of HMGR, SQS, FDPS and GGTase-I as in the heart, there was upregulation of farnesyltransferase α at 16 and 25 weeks of age and of farnesyltransferase β in 25-weeks-old SHR. Western blot demonstrated overexpression of HMGR and downregulation of SQS in SHR livers at all ages tested., Conclusions: The cardiovascular remodeling of SHR preceded the development of hypertension, and altered expression of several key enzymes in the mevalonate pathway may play a potential pathophysiological role in cardiovascular remodeling.

The aim of this study was to determine whether proteasome inhibitor MG132 treatment has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation. EPCs were identified as adherent cells double positive for DiLDL-up-take and lectin binding by direct fluorescent demonstrated under a laser scanning confocal microscope. After 7 days in culture, EPCs were stimulated with proteasome inhibitor MG132 in series of final concentrations of 20, 50, 100, 200 nmol/l for 12, 24, 48 h. Cell proliferation and apoptosis were determined, respectively, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V/propidium iodide binding assay. Colony-forming capacity was performed by colony assay. EPCs adhesion and migration were assayed with adhesion assay and transwell migration assay, respectively. The expression of endothelial nitric oxide synthase (eNOS) was assayed by Western blot analysis, while nitric oxide (NO) generation was detected using the Griess method. It was found that proteasome inhibitor MG132 decreased the number of EPCs and EPC colonies, increased EPC apoptosis, decreased EPC proliferative, adhesive, migration capacity and eNOS/NO production in a concentration- and time-dependent manner. These data indicate that proteasome inhibitor MG132 suppresses the number and function of EPCs, and these actions may involve decreased eNOS/NO production in the EPCs.

Objectives: Thrombosis and inflammation are associated with the pathogenesis of pulmonary arterial hypertension (PAH). However, there are no solid data supporting the involvement of platelet and leukocyte activation and interaction in PAH. The present study thus investigated the activation and interaction of circulating platelets and leukocytes in a rat model of monocrotaline (MCT)-induced pulmonary hypertension., Methods: Mean pulmonary arterial pressure (mPAP) was monitored in rats (n = 24) before and 2, 3 and 7 weeks after MCT (60 mg/kg)injection. In parallel, activation of circulating platelets and leukocytes and platelet-leukocyte aggregates were measured by whole-blood flow cytometry., Results: Two weeks after MCT injection, mPAP had increased significantly, i.e. from 11.25 ± 0.92 mm Hg at baseline to 15.71 ± 1.66 mm Hg (p < 0.05), and it had increased even further at week 7 (26.83 ± 3.29 mm Hg; p < 0.01). Fibrinogen binding of circulating platelets had increased from the basal level of 1.45 ± 0.61 to 4.08 ± 1.59% 3 weeks after MCT injection (p < 0.01). Platelet responsiveness to ADP was also significantly enhanced. CD11b expression of circulating neutrophils was elevated; i.e. mean fluorescence intensity increased from 1.67 ± 0.38 before MCT injection to 2.37 ± 0.31 3 weeks after MCT injection (p < 0.01), and N-formyl-methionyl-leucyl-phenylalanine (1 × 10⁻⁷M) stimulation induced more marked elevation of neutrophil CD11b expression in MCT-treated animals. Circulating platelet-neutrophil aggregates were already increased 2 weeks after MCT treatment (14.93 ± 4.22%; p < 0.01) compared to baseline (6.01 ± 2.91%) and remained elevated at 3 weeks (15.19 ± 4.78%; p < 0.01)., Conclusions: MCT-induced PAH in rats is associated with increased platelet and leukocyte activation and platelet-leukocyte interaction in vivo, which may play an important role in the pathogenesis of PAH., (Copyright © 2010 S. Karger AG, Basel.)

This study was aimed to investigate the expressions of genes hoxb2 and hoxb4 after interference of the proliferation and differentiation of hematopoietic stem cells (HSC) to the erythroid progenitors (CFU-E) in vitro by using all-trans retinoic acid (ATRA). The cord blood was collected from 12 cases of fetal placenta umbilical vein and cultured by using culture technique of HSC in vitro. The proliferation and differentiation of HSC to CFU-E were interfered with 6 x 10(-8) mol/L of ATRA. The expression levels of genes hoxb2 and hoxb4 in blank control and ATRA groups were detected by FQ-RT-PCR on day 3, 7 and 10 of culture. The results showed that the expressions of genes Hoxb2 and hoxb4 were a little on day 3, obviously increased on day 7 and reached highest level on day 10 in 2 groups. The expression level of hoxb4 on day 3, 7 and 10 in blank control group was obviously higher than expression level of hoxb2. As compared with blank control group, the expressions of genes hoxb2 and hoxb4 in the ATRA group were significantly up-regulated. It is concluded that the genes hoxb2 and hoxb4 all expressed in process of proliferation and differentiation to erythroid progenitors, which suggests that hoxb2 and hoxb4 relate to erythroid hematopoiesis, and the hoxb4 has more great relevance to erythroid hematopoiesis as compared with hoxb2. The ATRA (6 x 10(-8) mol/L) can up-regulate the expression of hoxb2 and hoxb4 significantly.

Objective: To analyze the clinical manifestations and the methods and effects of surgical management on Duane's Retraction Syndrome (DRS)., Methods: 67 cases with DRS were summarized retrospectively. The data were recorded and analysed, including sex, age, type, clinical features, surgical methods and the results., Results: 63 cases had only monocular involvement. The number of Type I DRS was 47 cases (35 cases with esotropia, 12 cases with exotropia). 8 cases were Type II DRS with exotropia and 12 cases were Type III DRS (11 of them with exotropia). In 37 cases with upshoot and/or downshoot phenomenon, 31 of them were associated with exotropia. Ipsilateral medial rectus or lateral rectus recession, or bilateral medial rectus recession, improved the head compensation position, narrow palpebral fissure and globe retraction, with improvement or elimination of the deviation. After the operation, 60 cases (90%) showed horizontal tropia less than 10 prism diopters. Upshoot and/or downshoot were improved in all of the 37 cases after recessing medial or/and lateral recti, or simultaneously weakening of the inferior oblique muscle., Conclusion: Weakening medial and/or lateral recti can improve the clinical manifestations of DRS. Upshoot and downshoot are more common in eyes with exotropia. Lateral rectus recession is the main treatment method for relieving upshoot and downshoot in DRS with exotropia. We infer that the upshoot and downshoot phenomenon mainly related to the "bridle" effect. Recession of both horizontal recti can improve the serious clinical manifestations.

Objective: To investigate the stereoacuity before and after superior oblique sagittal transposition in patients with acquired excyclotorsion., Methods: 14 cases of acquired excyclotorsion who had complaints of forward and downward torsional diplopia were treated with superior oblique sagittal transposition. All patients were examined with Titmus test prior and after surgery., Results: Surgical treatment greatly improved torsional diplopia. The stereoacuity improved gradually from 800 to 60 seconds of arc, peripheral to macular stereoacuity, and even foveal stereoacuity., Conclusion: The superior oblique sagittal transposition is an effective method for primary and down gaze acquired excyclotorsion. The operation not only alleviated diplopia in the practical fixation field, but also improved stereoacuity.

Aim: To investigate the effect of tricyclodecane-9-yl-xanthogenate (D609) on cell differentiation in vascular endothelial cells (VECs) and marrow stromal cells (MSCs)., Methods: Morphological changes were observed under phase contrast microscope. Electron microscope and immunostaining were used for VECs identification. The expressions of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry., Results: After 6 h of induction with D609, some VECs showed morphological changes characteristic of neurones. 9 h later, more VECs became neuron-like cells. About 30.8% of VECs displayed positive NSE (P<0.01), while the expression of GFAP was negative. When MSCs were exposed to D609, the cells displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive networks at 3 h. 6 h later, almost all of the cells exhibited a typical neuronal appearance, and 85.6% of MSCs displayed intensive positive NSE, but GFAP did not express., Conclusion: D609 induces VECs and MSCs differentiation into neuron-like cells.

Two safrole oxide derivatives, 1-methoxy-3-(3,4-methylenedioxyphenyl)-2-propanol (MOD) and 1-ethoxy-3-(3,4-methylenedioxyphenyl)-2-propanol (EOD), were newly synthesized as promoters of apoptosis in vascular endothelial cells (VECs). The purpose of this study was to investigate the effects of these two safrole oxide derivatives on cell growth and apoptosis induced by deprivation of survival factors (serum and fibroblast growth factors, aFGF and bFGF) in VECs. Morphological changes were observed with light microscopy. Cell growth was determined by using MTT (3-[4, 5-dimethyl thiazol-2-yl]-2, 5-diphenytetrazolium) method. DNA fragmentation was analyzed by agarose gel electrophoresis and fluorescence microscopy. Apoptosis rate and cell cycle distribution were analyzed by flow cytometry (FCM). The cells deprived of FGF and serum were exposed to MOD 10-40 mg l(-1) for 24 h. Cell growth was suppressed (P<.01), while detachment and DNA fragmentation of these cells were promoted (P<.01). When the cells were treated with MOD30 mg l(-1) for 24 h, apoptosis rate was 21.43% (P<.01). The fact that 66.50% of the cells were trapped in S phase of cell cycle indicated that the cell cycle was blocked at S phase. Treated with EOD 10-40 mg l(-1) for 24 h, the cells were observed; the results showed that VEC growth was inhibited and the apoptosis was triggered (P<.01). At 30 mg l(-1) concentration, EOD blocked 55.22% of the cells at S phase. The data suggested that MOD and EOD might promote apoptosis of VEC by blocking the cell cycle at S phase.

Aim: To investigate effect of safrole oxide on cell growth and apoptosis induced by deprivation of survival factors (fibroblast growth factors, aFGF and bFGF) in vascular endothelial cells (VEC)., Methods: Morphological changes were observed by light microscopy. Cell growth was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium) method. DNA fragmentation was analyzed by agarose gel electrophoresis and fluorescence microscopy. Cell cycle distribution was analyzed by flow cytometry (FCM)., Results: The cells deprived of FGF were exposed to safrole oxide 5-25 mg/L for 24 h. Cells spreading and growth were promoted (P<0.01), detachment and DNA fragmentation of these cells were suppressed (P<0.01), safrole oxide 10 mg/L had no obvious effect on cell cycle distribution (P>0.05). When the cells were treated with safrole oxide 50-100 mg/L, detachment and DNA fragmentation of VEC were promoted (P<0.01). The cell cycle was blocked at G2-M phase by safrole oxide 100 mg/L., Conclusion: Safrole oxide 10 mg/L inhibited, but 100 mg/L promoted apoptosis of VEC. Safrole oxide might be an important compound that affects VEC growth and apoptosis.

23 patients with fresh or old rupture of the inferior rectus muscle were treated and the causes of injury, modalities of treatment and clinical results analyzed. It is emphasized that in cases of ocular trauma, especially those with laceration of the eyelid or bulbar conjunctiva, the position and motility of the eyeball should be examined so that rupture of the inferior rectus would not be neglected and reduction of the injured muscle be accomplished.