Cystatin C alleviates H2O2-induced H9c2 cell injury.

OBJECTIVE: At present, the incidence of acute myocardial infarction is increasing year by year, and it has become one of the diseases with the highest mortality rate in humans. Myocardial ischemia-reperfusion injury (MIRI) is a major problem in the treatment of myocardial infarction, but clinically there is no effective way to treat MIRI. This study used Cystatin C (Cys C) to treat cardiomyocytes and rats to investigate the effect of Cys C on MIRI. MATERIALS AND METHODS: We used H2O2 to induce rat cardiomyocytes (H9c2 cells) injury and stimulated the cells with Cys C. Cell counting kit 8 (CCK8) assay was used to determine the optimal concentration of H2O2 and Cys C to stimulate H9c2 cells. We determined the effects of Cys C on oxidative stress and apoptosis levels in H9c2 cells by measuring the activity of dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA), and the expression of apoptosis-related molecules (caspase3/8/9, Bax and Bcl-2). Changes in the activity of the NF-κB signaling pathway in H9c2 cells were also detected. In addition, we made rat MIRI models by ligating the coronary arteries and used Cys C to treat rats to verify the effect of Cys C on MIRI. RESULTS: According to the results of the CCK8 assay, 1000 µM of H2O2 and 15 µM of Cys C were used to stimulate H9c2 cells. Cys C alleviated H2O2-induced H9c2 cell injury, manifested as a decrease in LDH and MDA activity and an increase in SOD activity. Cys C also reduced the apoptosis level in H9c2 cells. The activity of NF-κB signaling pathway in injured H9c2 cells was increased, and stimulation of Cys C could inhibit the NF-κB signaling pathway in H9c2 cells. The application of Cys C in MIRI rats also verified its therapeutic effect on MIRI. CONCLUSIONS: Cys C reduced the oxidative stress and apoptosis levels of cardiomyocytes by inhibiting the activity of NF-κB signaling pathway in cardiomyocytes, thereby reducing cardiomyocyte injury and treating MIRI.

[Two families of X-linked lymphoproliferative disease type 1 characterized by agammaglobulinemia].

Objective: To investigate the clinical and immunological laboratory features, mutations in SH2D1A gene and SAP protein expression in four children of two families with X-linked lymphoproliferative disease type 1(XLP-1). Method: Four patients (Family A including Patient 1 and Patient 2, Family B including Patient 3 and Patient 4) and their maternal relatives were enrolled in this study. The clinical manifestation, EBV infection status and chest CT scan were analyzed. The absolute and relative numbers of lymphocyte subsets, T lymphocyte proliferative response, SAP protein expression were assessed by flow cytometry. Quantification of signal joint TCR rearrangementexcision circle (sjTRECs), CDR3 spectratyping of TCRvß and gene mutation of SH2D1A were detected by PCR based on genomic DNA or cDNA. Result: Four male patients from two families were diagnosed with XLP-1. The ages of disease onset were more than 1 year, more than 1 year, more than 1 month and 6 months. The ages at diagnosis were nine years and ten months, sixteen years and eight months, fourteen years and ten months, four years and nine months. All patients had recurrent infections and EBV infection. Patients 1, 2, and 3 had agammaglobulinemia and Patient 4 had hypogammaglobulinemia. Chest CT scan showed all patients had atelectasis and pneumonia, and Patient 3 had bronchiectasis. Patient 3 was diagnosised as Burkitt lymphoma. For immunological function, all patients exhibited reduced CD4/CD8 ratios, increased numbers of exhausted T lymphocyte, decreased number of NK cell. The numbers of total B lymphocyte and naïve B lymphocyte were normal, but the number of memory B lymphocyte declined in all cases. Four patients' copy numbers of sjTRECs were low and CDR3 spectratypings of TCRvß showed mildly skewed. But their T lymphocyte proliferative response was normal. SAP protein expression in four cases were measured by flow cytometry. Two patients from Family A were absent and two patients from Family B showed decreased values. SH2D1A gene sequence analysis showed that the patients of Family A harbored a nonsense mutation (c.163 C>T; p.R55X) in exon 2. Their mother and two sisters were carriers. A missense mutation of SH2D1A gene (c.278 G>A; p.G93D) in exon 3 was found in the patients of Family B. The mother was carrier. Four patients remain survived, Patient 3 gave up treatment, other three patients received IVIG therapy. Conclusion: Four patients with XLP-1 from two families characterized by agammaglobulinemia have an extreme vulnerability to Epstein-Barr virus (EBV) infection. The functions of T cell, B cell and NK cell are impaired at different stages. The detection of SAP protein and SH2D1A gene are the key methods for diagnosis of XLP-1.

[Effects of extracellular regulated protein kinases protein and impairment of blood testis barriar stucturein of mice with exposure to decabromodiphenyl ether].

Objective: To study changes in expression of claudin-11 and proteins related to mitogen-activated protein kinase (MAPK) signaling pathways, as well as the ultrastructure of the blood testis barrier (BTB), in male ICR mice exposed to decabromodiphenyl ether (BDE-209). Methods: Fifty-two mice, 4 weeks of age, weighing 15-21 g, were provided with adaptive feeding for 1 week. Mice were randomly divided into 4 groups, named control, low-dose, medium-dose and high-dose groups. The treated groups received BDE-209, by intragastric gavage, at doses, respectively, of 100, 300 and 500 mg/kg. Mice were sacrificed after 6 weeks and organs harvested on ice, weighed and stored at -80 °C. The ultrastructure of testicular tissues was examined by electron microscopy. Western blotting was used to detect proteins related to the MAPK pathway, including p38 mitogen activated protein kinase (p38), phosphorylated p38 (p-p38), extracellular regulated protein kinase 1/2 (ERK1/2) , phosphorylated ERK1/2 (p-ERK1/2) , c-jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK) and the BTB tight junction protein claudin-11. Analyze the difference between each groups. Results: At sacrifice, the body weights in each treated group were compared with those in the control group weighing (41.14 ± 0.60) g. Compared with controls, body weights were significantly different (P<0.05) in the middle dose, at (39.97 ± 0.66) g and high dose, at (39.98± 0.55) g in control group. The coefficients of the testis were significantly lower (P<0.05) in each treated group than in controls, with values of (0.37±0.0)%, (0.31±0.05)% and (0.31±0.04)% for low-dose, medium-dose and high-dose groups, respectively. The epidymus coefficient values were also significantly lower than controls (P<0.05), with values of (0.16±0.06)%, (0.11±0.05)% and (0.07±0.03)%, respectively in the same three dose groups. Electron microscopy ultrastructure showed that, compared with the control group, the testes in the middle and high dose groups had closely connected fractures, cell edema and more vacuoles. Compared with in the control group, levels of p-p38 and p-JNK in testicular tissue were significantly increased (P<0.05). In the control group and in low-, medium- and high-dose groups, the p-p38/p38 ratios were 1.35±0.13, 3.46±0.10, 5.71±0.26 and 4.79±0.21, respectively. The corresponding p-JNK/JNK ratios were 2.07±0.0, 4.77±0.18, 3.63±0.06 and 4.85±0.15. Claudin-11 levels were significantly lower (P<0.05) than control values in each dosed group. The corresponding values in control, low-dose, medium-dose and high-dose groups were 8.33±0.36, 2.06±0.27, 3.37±0.27 and 1.55±0.19, respectively. Conclusion: BDE-209 increased expression of proteins in the MAPK pathway and decreased expression of the BTB tight junction protein claudin-11 in testicular tissue. It also caused ultrastructural damage to the Sertoli cell BTB tight junctions. This suggested that BDE-209 might damage Sertoli cells BTB through effects on the MAPK pathway.

Plasma thrombospondin-1 and clinical outcomes in traumatic brain injury.

OBJECTIVE: Thrombospondin-1 (TSP-1) acts as an anti-angiogenic factor, and its expression in rat brain is upregulated after intracerebral hemorrhage. The current study was designed to investigate the change of plasma TSP-1 levels and assess the prognostic predictive effect of plasma TSP-1 level and it is associated with head trauma severity in the patients with severe traumatic brain injury (STBI). MATERIALS AND METHODS: The plasma TSP-1 levels of 134 patients and 134 healthy controls were measured using enzyme-linked immunosorbent assay. The relationships between plasma TSP-1 levels and trauma severity reflected by Glasgow Coma Scale (GCS) scores as well as between plasma TSP-1 levels and short-term and long-term clinical outcomes were analyzed using multivariate analysis. RESULTS: Plasma TSP-1 levels were statistically significantly higher in patients than in healthy controls. The multivariate analysis demonstrated close association of TSP-1 with GCS scores and also identified TSP-1 as an independent predictor for 1-week mortality, 6-month mortality, and 6-month unfavorable outcome. Plasma TSP-1 levels had high prognostic predictive value based on receiver operating characteristic curve. The difference between its prognostic predictive value and GCS scores was not statistically significant. CONCLUSIONS: Plasma TSP-1 levels are elevated and are highly associated with head trauma severity and short-term and long-term outcomes of STBI. TSP-1 may be a good prognostic biomarker of STBI.

[Studies on injury-mechanism of trichosanthin on trophoblast cells and choriocarcinoma cells in culture].

For analysing the injurious mechanism of Trichosanthin (TCS) on trophoblast cells, cytotrophoblast cells were separated from placental villi in human early pregnancy and cultured on millipore filters coated with collagen in dual-environmental culture chambers. After 7-10 days culture the cells grew into confluent monolayer. A lot of multinucleated giant cells (synthetial like) could be found under light microscopy. Cytotrophoblast cells and the giant one were characterized as epithelial type by indirect immuno-fluorescent staining with anti-keratin. Because the procedure of separation of trophoblast cells is laborious and the choriocarcinoma cells (JAR) were as sensitive as trophoblast cells to trichosanthin, so the choriocarcinoma cells were used instead of the trophoblast cells in the later experiments. The internalization and distribution of TCS conjugated to 15 nm (in diameter) gold particles were examined. Electron microscopy showed that the TCS-gold particles were bound to the cell membrane and entered via coated pit and then internalized into coated vesicles (endosomes) within 30-60 minutes after treatment. Nevertheless, a number of free TCS-gold particles entered into the cell membrane nonspecifically. In the series of 60-120 minutes treatment, the TCS-gold particles presented in the multivesicular bodies. It is worthy to emphasize that the TCS-gold particles entered the cytosol from the endosomes and distributed nearby the rough endoplasmic reticulum (RER) and ribosomes within 120-180 minutes. In the meanwhile, the cells showed pathological changes markedly. With the same method, human liver carcinoma cells (BE 7402), kidney cells of African green monkey (CV-1) and rat embryonic liver epithelial cells (LW 13) were treated by TCS-gold particles as control. However, no particles had been found on the cell surface or in the cytosol within 60-120 minutes (see Table 1). In addition, trophoblast cells were treated by BSA-gold particles and transferrin-gold particles separately there still no particles could be found. (See Table 2). It is more interesting that TCS-Hepama-1-gold particles internalized into the choriocarcinoma cells with the same manner as TCS-gold entered, and it could not be affected by pretreatment with Hepama-1 an hour. But, TCS-Hepama-1-gold particles bound to the microvilli of human liver carcinoma cells, and it can be inhibited competitively by pretreatment with Hepama-1 for an hour (see Table 3). These results indicated consistently that trichosanthin possesses a high affinity to the membrane of trophoblast cells and choriocarcinoma cells, and the internalization of this plant toxic protein into its target cells characterized by receptor-mediated endocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)

[Appearance and distribution of retinoic acid in the limb regeneration of Cynops orientalis].

Urodeles are the unique vertebrate which can regenerate their limbs in larvae and adult. The pattern formation of blastema cells in the limb regeneration is a central problem. Many evidence have been established to support the hypothesis that RA may be the morphogen in limb development. The limb development and regeneration are so similar morphogenetic processes that we suspect RA may also play an important role in the latter process. In order to give an answer to this question, we tested the limbs in different stages of regeneration with the specific antibody prepared against RA. The results indicated that RA appeared on day six and arrived its peak on day eight after amputation. During this time, the dedifferentiation of the stump is proceeding to the strongest degree and the distribution of RA showed a posterior-anterior gradient. If the stump is treated with RA for 24 hours at this stage, proximal distal duplication of the regenerate can be induced. These results and the fact that the close connection between apical epidermal cap and mesenchymal cells and abundant RA outside the epidermal cells suggest that RA may play an important role in the establishment of positional information of the blastema.

Endocytic activity of Sertoli cells grown in bicameral culture chambers.

Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding 125I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous potentials of embryonic epithelium in urodeles (Cynops orientalis).

The spontaneous potentials (SPs) of nerveless embryonic epithelium of Cynops orientalis were observed by conventional intracellular micro-electrode technique. The amplitude and frequency of SPs of epithelial cells and the course of initiation and decline were recorded. The SPs can occur repeatedly at the stages when the embryonic epithelium is able to conduct excitation. The SP is very similar to the evoked propagatable potential, but has shorter duration. The amplitudes and frequencies of SPs differ in the different embryos and in different epithelial cells in the same embryo. The excitable epithelial cells of the embryo may lose their conductivity when the SPs come to a stable phase both in amplitude and frequency. And the conductivity can recover again after the subsiding of SPs. The SPs can be eliminated by tetrodotoxin (TTX), but they are not affected by the treatment of cobalt chloride.