Effects of sericin on oxidative stress and PI3K/AKT/mTOR signal pathway in cryopreserved mice ovarian tissue.

Ovarian tissue cryopreservation is an effective fertility protective strategy for preadolescent female cancer patients, whose tumor treatment cannot be delayed. In the present study, the effects of sericin, as an antioxidant, on mice ovarian tissue freezing and thawing were investigated. Mice ovarian tissues were cryopreserved and thawed in medium containing 0.5% or 1%sericin (w/v), and 0.1 mM melatonin. Then, the follicular morphology was observed. The levels of antioxidant enzymes were determined, including glutathione (GSH), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) and catalase (CAT). Moreover, the levels of nitric oxide (NO), malondialdehyde (MDA) and lactate dehydrogenase (LDH) were also tested. Besides, apoptosis-related proteins Bcl-2 and Bax were determined. Our results showed that 1% sericin maintained follicular morphology, inhibited apoptosis, decreased MDA and NO levels, and boosted endogenous antioxidant enzyme levels, while had no significant effect on LDH levels. Furthermore, these effects may be related with the activation of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of Rapamycin (mTOR) signaling pathway, as demonstrated by increased PI3K, p-AKT and mTOR levels. These findings demonstrate that 1% sericin may reduce oxidative stress and protect ovarian tissues during freezing and thawing via PI3K/AKT/mTOR signaling pathway.

Melatonin induction of HSP90 expression exerts cryoprotective effect on ovarian tissue.

Our previous study revealed that melatonin (MLT) protected the quality of cryopreserved ovarian tissues in mice. This work was carried out to examine the role of MLT in inducing HSP90 expression of ovarian tissue for achieving cryoprotection. Pieces of ovarian tissues were obtained from 50 female rats treated with MLT at 0, 0.001, 0.01, 0.1, and 1 mM, respectively. After cryopreservation-thawing, HSP90 mRNA and protein level were evaluated using qRT-PCR and western blot. The qRT-PCR results revealed that HSP90 mRNA expression was significantly (p < 0.01) upregulated in MLT-treated groups in comparison with the controls (0 mM). Western blot revealed higher HSP90 protein expression in MLT-treated groups compared to control group (0 mM), thus further confirming that MLT positively affected HSP90 expression. Moreover, 0.1 mM MLT had better effects than other concentrations of MLT. Conclusively, findings in the present work provide a feasible technology for improving cryopreserved ovarian tissue quality through the addition of MLT to elicit HSP90 expression.

Antioxidative effect of melatonin on cryopreserved ovarian tissue in mice.

Cryopreservation of ovarian tissues (OTs) has become the most effective way to preserve the fertility of female cancer patients. However, cryopreservation of OTs is still relatively at an experimental stage. The aim of study is to examine the effect of melatonin (MTL) on cryopreserved-thawed OTs. Fragments of OTs were cryopreserved in medium containing different concentrations (0 mM, 0.001 mM, 0.01 mM, 0.1 mM and 1 mM) of MLT. The endogenous enzymes (GSH-PX, GSH, SOD, CAT and T-AOC), MDA and ROS levels were all evaluated after cryopreservation. Our results showed that the 0.1 mM of MLT significantly improved the survival and diameter of follicles (P < 0.001). Meanwhile, the antioxidant enzymes activities (including GSH-PX, GSH, SOD, CAT and T-AOC) were enhanced and MDA content were significantly decreased in 0.1 mM of MLT group compared to other groups (P < 0.001). Additionally, compared to the control group, MTL of 0.1 mM resulted in a significantly lower ROS level. In conclusion, MLT protects the quality of cryopreserved OTs by decreasing oxidative stress level and the optimal concentration is 0.1 mM.

Melatonin Inhibits Oxidative Stress and Apoptosis in Cryopreserved Ovarian Tissues via Nrf2/HO-1 Signaling Pathway.

In the field of assisted reproductive technology, female fertility preservation, particularly ovarian tissue cryopreservation in adolescent cancer patients, has attracted much attention. Melatonin (MLT) is well known for its antioxidative and anti-apoptotic properties; however, whether it can ameliorate the cryoinjury and inhibit the generation of reactive oxygen species (ROS) in cryopreserved ovarian tissues (OTs) has not yet been reported. Here, we demonstrated that MLT could protect follicular integrity; prevent cell apoptosis; decrease ROS, malondialdehyde (MDA), and nitric oxide (NO) levels; and increase activities of glutathione peroxidases (GSH-Px), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) in cryopreserved OTs. Furthermore, these effects may be related with the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, as evidenced by increased mRNA levels of Nrf2 downstream genes, including heme oxygenase-1 (HO-1), glutathione S-transferase M1 (GSTM1), SOD, and CAT. In summary, MLT can not only directly scavenge ROS but also significantly induce the activation of antioxidative enzymes via the Nrf2 signaling pathway, which is a new mechanism underlying the protection effects of MLT on cryopreserved OTs.

[Mobilization and collection of autologous peripheral blood stem cells by CIE or IEV protocol in children with malignant solid tumors].

OBJECTIVE: Autologous peripheral blood stem cell transplantation (APBSCT) is an important method for treatment of malignant solid tumors in children. The mobilization and collection of blood stem cells is crucial for APBSCT. This study aimed to evaluate the clinical efficacy of mobilization and collection of blood stem cells by CIE or IEV chemotherapy protocol in APBSCT in children with neuroblastoma (NB) or rhabdomyosarcoma. METHODS: The protocols of CIE (cisplatin, etoposide) and IEV (vincristine, dosfamide, etoposide) were used as mobilization chemotherapy in 8 cases of NB with stage IV and 3 cases of rhabdomysacoma with stage III, respectively. The results of the mobilization of blood stem cells were observed. RESULTS: Of the 11 cases, mononuclear cells (MNC) and CD34+ cells were successfully collected and the volume of MNC and CD34 averaged (5.55 ± 1.43)× 10(8)/kg and (4.88 ± 2.48) × 10(6)/kg, respectively. No severe complications were observed during the mobilization and collection. A rapid hemopoietic reconstitution was observed in 10 children after APBSCT. One with NB out of the 10 children died of left heart failure 32 days after APBSCT. Others (9 cases) showed a nearly normal result of routine peripheral blood test 60 days after APBSCT. CONCLUSIONS: CIE or IEV protocol is effective and safe for the mobilization and collection of peripheral blood stem cells in children with NB or rhabdomysacoma.

[ATP-Sensitive potassium channel function during the progression of pressure overload heart failure in rats].

OBJECTIVE: To investigate K(ATP) channel function of cardiomyocytes isolated from the left ventricular wall of rats with or without abdominal aortic constriction at different time points under normal or simulated ischemic conditions. METHODS: Male Wistar rats were randomized into 4 groups (n = 10 - 13): 4-week sham-operated group (F4), 4-week aortic-banded group (T4), 12-week sham-operated group (F12), 12-week aortic-banded group (T12). Chronic pressure overload model was established by abdominal aortic constriction. Left ventricular myocytes were isolated by modified Langendorff perfusion method post in vivo hemodynamical measurements. The whole-cell patch-clamp technique was used to record transient outward current of K(ATP) channel on myocytes under normal and simulated ischemic perfusion conditions. The current densities of K(ATP) channel between F4 and T4 group, F12 and T12 group were compared under 0 mV of test potential. RESULTS: SBP, DBP and MBP were significantly increased in T4 group compared to F4 group, but were similar between T12 and F12 groups. LVEDP and +/- dp/dtmax were similar between T4 and F4 groups and LVEDP was significantly increased while +/- dp/dtmax significantly reduced in T12 group than that in F12 group. Whole-cell membrane current densities were similar between F4 and T4 group or F12 and T12 group under normoxic condition, the K(ATP) current densities increased dramatically in T12 group [(28.11 +/- 3.91) pA/pF vs (11.55 +/- 1.17) pA/pF, P < 0.01], but not in T4 group [(14.09 +/- 5.74) pA/pF vs (11.74 +/- 3.68) pA/pF, P > 0.05] in myocytes exposed to ischemic solution for 25 minutes. The total number of K(ATP) channel in ventricular myocytes was similar between F4 and T4 group or F12 and T12 group. CONCLUSIONS: The sarcolemmal K(ATP) channel was more sensitive to ischemia and the current magnitude was significantly increased at the stage of congestive heart failure. The functional change of K(ATP) channel occurred before the increase of total number of K(ATP) channel.

[Expression of odorant receptor genes on the olfactory epithelium following olfactory nerve disconnection].

OBJECTIVE: To constitute the animal model of unilateral olfactory nerve transection and observe the expression level and distribution of odorant receptors. METHODS: Thirty-two rats were divided into two groups: the olfactory nerve transection group (20) and the control group (12). The former group received the operation to transect the left olfactory nerve following the left olfactory bulb was exposed under microscope and the latter group did not give any disposal. At every stage of five days, two weeks, four weeks and six weeks after the operation, five rats from the nerve transection group and three from the control group were anaesthetized simultaneously, and olfactory epithelium were taken out after transcardial perfusion, then paraffin imbedding. Coronal sections were sliced for HE staining to observe the thickness changes of the olfactory epithelium, and for in situ hybridization (ISHs) to investigate the expression of olfactory receptor genes (Olr287, Olr226, Olr1493 and Olr1654) in the epithelium, also to evaluate the changes of the expression level and location of the selected receptors during the regeneration of olfactory epithelium. RESULTS: HE staining showed that 5 days after the operation cell quantity and thickness of the olfactory epithelium decreased obviously, which increased gradually 2 or 4 weeks after operation. After 6 weeks' recovery, the thickness of the epithelium could reach the control level. The pattern of cell staining by ISH showed a specific spatial distribution along the anteroposterior (AP) and dorsoventral (DV) axis. Evidence suggested that odorant receptors were distributed in continuous and multiple overlapping bands in the normal or nerve transected-recovered epithelium rather than in the conventionally accepted three or four zones. The data also demonstrated that the distribution of sensory neuron types, as identified and defined by odorant receptor expression, was restored to normal or nearly so by 6 weeks after operation. Likewise, the numbers of probe-labeled neurons in the nerve transected-recovered had an obvious decrease 5 days after olfactory nerve transection. Reactive cells (x(-) +/- s) of Olr1493 in the operated side was (53.9 +/- 19.9), compared with (419.0 +/- 21.2) in the unoperated side, there was statistic significance between them (t = 63.960, P < 0.01). Reactive cells increased gradually according to the regeneration of the epithelium, and were nearly equivalent to the normal side 6 weeks later without significant differentiation (t = 2.600, P > 0.05), according to the absolute positive cells in the operated and unoperated side of (417.8 +/- 32.4) and (445.3 +/- 10.0) respectively. CONCLUSION: The regeneration of the sensory neurons and receptors, both the number and the distribution, can recover to normal after olfactory nerve transection.

[Experimental study on effects of Heparin II domain of fibronectin on cultured human trabecular meshwork cells].

OBJECTIVE: To determine the effects of Heparin II (Hep II) domain on cultured human trabecular meshwork (HTM) cells. METHODS: HTM cells were cultured and treated with Hep II domain for 18 and 24 h. The morphological changes in HTM cells were assessed by light and electron microscopy. Changes in cell morphology and the organization of actin cytoskeleton, Vinculin, beta-Catenin were assessed by using immunofluorescence. RESULTS: Treatment of Hep II domains resulted in morphological changes from 10 to 24 h. In light microscopy, cells rounded up, retracted and detached from each other. In high performance liquid chromatography, Hep II domains-treated cells showed that actin fibre bundles were highly concentrated at the periphery of the cells with few actin filaments left in this area; decreased vinculin staining was observed toward the cell periphery; decreased beta-Catenin staining was also observed around the cell sub-membrane. Transmission electron microscopy showed expended intercellular space. After 24 h, changes of HTM cells were recovered. CONCLUSIONS: Hep II domains reversibly blocks actin cytoskeleton and cell-junction of HTM cells. Disorganization of actin cytoskeleton and cell-junction in trabecular meshwork through signal transduction may be a useful strategy for the decrease of intraocular pressure.

[Reversal effect of sodium selenite on multidrug resistance in K562/ADR cell line and its mechanisms].

This study was purposed to investigate the reversal effect of sodium selenite on multidrug resistance in adriamycin-resistant leukemic cell line K562/ADR and its mechanisms. The cytotoxicity and the reversal effect of sodium selenite on K562/ADR cells were assayed by MTT method; the apoptosis rate of K562 and K562/ADR cells were detected by flow cytometery, the mRNA expressions of mdr1 and bcl-2 were measured by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that 10 micromol/L sodium selenite significantly increased the cytotoxicity of adriamycin to K562/ADR cell and the reverse index (RI) was 2.31; the early apoptosis rate of K562 cells was elevated after treatment with 5 micromol/L Na(2)SeO(3) for 48 hours; and the medium-term and late apoptosis rate was elevated after treatment with both 5 and 10 micromol/L Na(2)SeO(3) for 48 and 72 hours. Both doses of 5 and 10 micromol/L Na(2)SeO(3) increased the early apoptosis rate of K562/ADR at 48 hours, and also increased the medium-term and late apoptosis rate after treating for 48 and 72 hours. The apoptosis rate was higher at dose of 10 micromol/L than that at 5 micromol/L, the apoptosis rate at 72 hours also was higher than that at 48 hours. The expressions of mdr1 mRNA and bcl-2 mRNA were decreased significantly by 10 micromol/L sodium selenite. It is concluded that sodium selenite can reverse the multidrug resistance in K562/ADR partially by down-regulating the expressions of mdr1 mRNA and bcl-2 mRNA, and increasing apoptosis rate of K562/ADR cells.

[Clinical features of primary ciliary dyskinesia].

OBJECTIVE: To investigate the clinical features, diagnosis and treatment of primary ciliary dyskinesia (PCD). METHODS: Three cases of PCD received endoscopic sinus surgery and were followed up for life quality and recovery. Among these 3 cases, two were twin brothers and the other girl was twin born with a healthy brother. The mucosa of inferior turbinate was extracted prior to the operation without narcotic and decongestant. The ultrastructure of mucosal cilia was detected with electron microscope. Nine exons of gene DNAH5 and chromosome in one case and her fraternal twin were evaluated. RESULTS: Nasal and sinus CT imaging of the 3 cases showed chronic pansinusitis (1 case accompanied with situs inversus according with the diagnosis of Kartagener syndrome). The nasal polyp was resected, and the sinuses were opened. The twin brothers received the adenoidectomy. All patients felt nasal ventilation improved while the surgical field still covered with thick discharges during follow-up for 2 - 4 years. Ciliary ultrastructures of the three cases showed lateral dynein absent, the sequence of 9 exons of DNAH5 and chromosome presented no change in the fraternal twins. CONCLUSIONS: Surgery could improve the symptoms of sinusitis in PCD. Change of ciliary ultrastructure was an important indication of its pathological changes and molecular biology evaluation needs further study.

[The effects of matrine on cell proliferation and telomerase activity in retinoblastoma cells in vitro].

OBJECTIVE: To evaluate the effects of matrine on cell proliferation and telomerase activity in retinoblastoma cells in vitro. METHODS: HXO-Rb44 cells were treated with 0.1 to 3.2 mmol/L matrine for 24 h, then cell viability was measured by MTT assay. Apoptosis rates were evaluated by flow cytometry and TUNEL assay after the cells were treated for various durations. The telomerase activity were measured by telomeric repeat amplification protocol. RESULTS: MA inhibited the growth of HXO-Rb44 cells. Ma induced the apoptosis and down-regulated the telomerase activity of HXO-Rb44 cells, which were time dependent. There was a positive correlation between the telomerase activity and the apoptosis (r = -0.961, P < 0.01). CONCLUSIONS: Ma can induce the apoptosis and down-regulate the telomerase activity of retinoblastoma cells, which may be one of important mechanisms to inhibit the cell proliferation. It suggests that Ma may be a potent drug for the treatment of retinoblastoma.

[Electrically stimulated olfactory evoked potential in olfactory-lesioned rabbit].

OBJECTIVE: To develop olfactory dysfunctioned animal model by injuring olfactory blub(s) and to investigate the characteristics of olfactory evoked potential. METHODS: The changes of OEP in rabbits were observed at 24 h, 48 h and 1 w after olfactory bulb(s) were electrolytically injured. The pathological and ultrastructural changes of olfactory mucosa and olfactory bulb were also observed. RESULTS: When unilateral olfactory bulb was damaged, N2 vanished with elongated latencies and lowered amplitudes of N1 and P1. When both olfactory bulbs were damaged, N1 and N2 were absent, only P1 could be recorded. The latency was extended obviously and the amplitude had a big change. Histopathology and ultrastructure showed that there were a lot of inflammatory cells and degenerated neurons scattered around OB 24 hours to 48 hours after injure. It was observed that the olfactory cilia had distraction perversion and colliquefaction changes. CONCLUSION: Different degree of olfactory bulb injury caused different effect on OEPs. These changes of potential were based on the histopathology and ultrastructure.