Osteoprotegerin inhibit osteoclast differentiation and bone resorption by enhancing autophagy via AMPK/mTOR/p70S6K signaling pathway in vitro.

Osteoclasts are highly differentiated terminal cells formed by fusion of hematopoietic stem cells. Previously, osteoprotegerin (OPG) inhibit osteoclast differentiation and bone resorption by blocking receptor activator of nuclear factor-κB ligand (RANKL) binding to RANK indirect mechanism. Furthermore, autophagy plays an important role during osteoclast differentiation and function. However, whether autophagy is involved in OPG-inhibited osteoclast formation and bone resorption is not known. To elucidate the role of autophagy in OPG-inhibited osteoclast differentiation and bone resorption, we used primary osteoclast derived from mice bone marrow monocytes/macrophages (BMM) by induced M-CSF and RANKL. The results showed that autophagy-related proteins expression were upregulated; tartrate-resistant acid phosphatase-positive osteoclast number and bone resorption activity were decreased; LC3 puncta and autophagosomes number were increased and activated AMPK/mTOR/p70S6K signaling pathway. In addition, chloroquine (as the autophagy/lysosome inhibitor, CQ) or rapamycin (as the autophagy/lysosome inhibitor, Rap) attenuated osteoclast differentiation and bone resorption activity by OPG treatment via AMPK/mTOR/p70S6K signaling pathway. Our data demonstrated that autophagy plays a critical role in OPG inhibiting osteoclast differentiation and bone resorption via AMPK/mTOR/p70S6K signaling pathway in vitro.

Promotion of autophagy at the maturation step by IL-6 is associated with the sustained mitogen-activated protein kinase/extracellular signal-regulated kinase activity.

Increased autophagic vacuoles (AVs) occur in injured or degenerating neurons, under both developmental and pathological situations. Although an induced autophagy has been shown in inflammation response to cell factors, the underlying mechanism(s) remain(s) unknown. Here, we show that both cell factor IL-6 and environmental toxin MPP(+) promote the formation of vacuolation in SHSY5Y cells. By electron and immunofluorescent microscopy analyses, we showed that these structures are acid autolysosomes, containing cellular debris, and labeled by LC3 or LAMP1, markers of autophagosomes or lysosomes, respectively. Combining MPP(+) and IL-6 do not further increase vacuolation of SHSY5Y cells, and the vacuolation is less than that in the MPP(+)-treated group. MPP(+)-induced vacuolation results from significant increase in autophagy formation and delay in autophagy degradation, in relation to a decline of the lysosomal activity of arylsulfatase A. At molecular level, we show that this defect in autolysosomal maturation is independent of mammalian target of rapamycin and p38 inhibitions. Most importantly, we provide the first evidence that activation of ERK pathway is sufficient to commit cell to autophagic vacuolation. The sustained activation is required for MPP(+) to disrupt the autophagic pathway. IL-6 also induces a temporary and significant activation of ERK, but not sustained activation, and change sustained activation in MPP(+)-treated group into temporary activation. Taken together, these findings strongly support that IL-6 promotes the maturation of autophagosomes into functional autolysosomes by regulating ERK.

Aerobic Glycolysis Induced by mTOR/HIF-1α Promotes Early Brain Injury After Subarachnoid Hemorrhage via Activating M1 Microglia.

M1 microglial activation is crucial for the pathogenesis of early brain injury (EBI) following subarachnoid hemorrhage (SAH), and there is growing evidence that glucose metabolism is frequently involved in microglial activation. However, the molecular mechanism of glycolysis and its role in M1 microglial activation in the context of EBI are not yet fully understood. In this study, firstly, the relationship between aerobic glycolysis and M1 microglial activation as well as SAH-induced EBI was researched in vivo. Then, intervention on mammalian target of rapamycin (mTOR) was performed to investigate the effects on glycolysis-dependent M1 microglial activation and EBI and its relationship with hypoxia-inducible factor-1α (HIF-1α) in vivo. Next, Hif-1α was inhibited to analyze its role in aerobic glycolysis, M1 microglial activation, and EBI in vivo. Lastly, both in vivo and in vitro, mTOR inhibition and Hif-1α enhancement were administered simultaneously, and the combined effects were further confirmed again. The results showed that aerobic glycolysis and M1 microglial polarization were increased after SAH, and glycolytic inhibition could attenuate M1 microglial activation and EBI. Inhibition of mTOR reduced glycolysis-dependent M1 microglial polarization and EBI severity by down-regulating HIF-1α expression, while enhancement had the opposite effects. Blockading HIF-1α had the similar effects as suppressing mTOR, while HIF-1α agonist worked against mTOR antagonist when administered simultaneously. In conclusion, the present study showed new evidence that aerobic glycolysis induced by mTOR/HIF-1α might promote EBI after SAH by activating M1 microglia. This finding provided new insights for the treatment of EBI.

Selenoprotein P mRNA expression in human hepatic tissues.

AIM: To investigate the expression of selenoprotein P mRNA (SePmRNA) in tissues of normal liver, liver cirrhosis and hepatocellular carcinoma (HCC), and its relationship with HCC occurrence and development. METHODS: The expression of SePmRNA in tissues of normal liver, liver cirrhosis and HCC were detected by in situ hybridization using a cDNA probe. RESULTS: The enzyme digesting products of PBluescript-Human Selenoprotein P were evaluated by electrophoresis. The positive expression of SePmRNA was found in the tissues of normal liver, liver cirrhosis and HCC. The expression of SeP mRNA was found in hepatic interstitial substance, especially in endothelial cells and lymphocytes of vasculature. The positive rate of SePmRNA in normal liver tissue was 84.6% (11/13) and the positive signals appeared in the nucleus and cytoplasm, mostly in the nucleolus, and the staining granules were larger in the nucleolus and around the nucleus. The positive rate of SePmRNA in liver cirrhosis tissue was 45.0% (9/20) and the positive signals were mainly in the nucleolus and cytoplasm, being less around the nucleus and inner nucleus than that in normal liver tissue. The positive rate of SePmRNA in HCC tissue was 30.0% (9/30) and the positive signals were in the cytoplasm, but less in the nucleus. CONCLUSION: SePmRNA expression in the tissues of normal liver and HCC is significantly different (84.6% vs 30.0%, P=0.003), suggesting that SeP might play a role in the occurrence and development of HCC.

The effects and mechanism of estrogen on rats with Parkinson's disease in different age groups.

OBJECTIVE: In order to investigate the effect and mechanism of estrogen in rotenone-induced Parkinson's disease (PD) rats in different age groups. METHODS: we established rat models of PD by rotenone at different interventions. Then, behavioral tests, immunohistochemistry, western blot, high-performance liquid chromatography-electrochemical detector (HPLC-ECD) and electron microscopy were performed. RESULTS: Results revealed the following: (1) Rotenone significantly reduced rotarod latencies in senile rats, prolonged their climbing pole time, and decreased TH positive cells, DA and its metabolite, DOPAC. Estrogen ameliorated this effect, in which weaker effects were observed in younger rats compared with older rats. (2) Rotenone increased the expression of LC3-II in older rats, but estrogen and tamoxifen did not show the same effect. (3) Rotenone increased the number of autophagosomes, but estrogen increased the proportion of autolysosomes/autophagosomes in the rotenone-treated group. (4) U0126 could reduce the number of autophagosomes in the rotenone-treated group, but this did not change the proportion of autolysosome/autophagosome in combining rotenone with the estrogen group. Rapamycin did not increase the number of autophagosomes in the rotenone-treated group, but combining rapamycin with estrogen and rotenone was able to further increase the proportion of autolysome/autophagosomes. Therefore, we speculate that the senile rat model of PD was more reliable than that in young rats. CONCLUSIONS: In addition, estrogen could promote autophagy maturation through the ERK pathway, and had an obvious therapeutic effect on the rat model of PD.

Expression and clinical significance of TAp73alpha, p53, PCNA and apoptosis in hepatocellular carcinoma.

AIM: To study the prognostic role of TAp73alpha, p53, proliferating cell nuclear antigen (PCNA) and apoptosis in patients with hepatocellular carcinoma (HCC) after surgical tumor ablation. METHODS: Forty-seven human resected HCC tissues and 42 adjacent non-cancerous tissues were studied with 10 normal liver tissues as control group. TAp73alpha, p53, and PCNA were detected with Elivision immunohistochemistry. Terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick-end labeling (TUNEL) method was used to detect the apoptosis cells. All clinical and pathological materials were analyzed by SPSS10.0 statistical package. RESULTS: TAp73alpha overexpressed in HCC tissues (36.2%) when compared with adjacent non-cancerous tissues (2.38%, P<0.005) and normal liver tissues (0, P<0.01). Mutant type p53 (mt-p53) overexpressed in HCC tissues (38.3%) when contracted with adjacent non-cancerous tissues (16.7%, P<0.05) and normal liver tissues (0, P<0.01). Proliferation index (PI) level in HCC tissues was significantly higher than that in adjacent non-cancerous tissues (30.34%+/-4.46% vs 27.88%+/-5.89%, t, P = 0.028). Apoptosis index (AI) level in HCC tissues was higher than that in adjacent non-cancerous tissues (8.62%+/-2.28% vs 7.38%+/-2.61%, t, P = 0.019). Expression of TAp73alpha was associated with lymph node metastasis and mt-p53, with r = 0.407 and 0.265, respectively. Expression of mt-p53 was associated with Edmondson's stage and AFP, with r = 0.295 and -0.357, respectively. In Kaplan-Meier univariant analysis, TAp73alpha, AFP, TNM stage, portal vein invasion, liver membrane invasion and HBsAg correlated with prognosis (log rank, P = 0.039, 0.012, 0.002, 0.000, 0.014, 0.007, respectively). Multivariant Cox regression analysis showed that TAp73alpha, AFP, TNM stage, portal vein invasion, liver membrane invasion and age were independent factors of prognosis. CONCLUSION: These results suggest that TAp73alpha can be used as a prognostic indicator of patients with HCC undergoing surgical tumor ablation. AFP, TNM, portal vein invasion, liver membrane invasion and age also have a potency of predicting the prognosis of HCC.

Upregulation of cell surface estrogen receptor alpha is associated with the mitogen-activated protein kinase/extracellular signal-regulated kinase activity and promotes autophagy maturation.

Recently, accumulating evidence has implicated the dysregulation of autophagy as underlying the pathophysiology of several neurodegenerative diseases. The human neuronal cell line SH-SY5Y was exposed to 1-Methyl-4-phenylpyridinium (MPP(+)). The mechanism is that the sustained activation of the MAPK/ERK pathway by MPP(+) alters autophagy selectively at the maturation step, significant increasing in autophagy formation and delaying in autophagy degradation in SHSY5Y cells. In this study, we provided evidences that estrogen was capable of promoting SHSY5Y cells survival in MPP(+)-treated group. In particular, the up-regulation of mERα, but not mERß, was associated with a rapid and transient activation of ERK phosphorylation compatible with promoting autophagy maturation. The up-regulation of mERα changed the sustained activation of ERK phosphorylation in MPP(+)-treated group into a temporary activation. Taken together, these findings strongly support that the expression of mERα promotes the maturation of autophagosomes into functional autolysosomes by regulating ERK, determining SHSY5Y cells survival.

Effects of depression on parameters of cell-mediated immunity in patients with digestive tract cancers.

AIM: To evaluate the effects of depression on parameters of cell-mediated immunity in patients with cancers of the digestive tract. METHODS: One hundred and eight adult patients of both sexes with cancers of the digestive tract admitted between March 2001 and February 2002 in the Department of Medical Oncology, First Affiliated Hospital of Xi'an Jiaotong University were randomly enrolled in the study. The Zung self-rating depression scale (SDS), Zung self-rating anxiety scale (SAS), numeric rating scale (NRS) and social support rating scale (SSRS) were employed to evaluate the degree of depression and their contributing factors. In terms of their SDS index scores, the patients were categorized into depression group (SDS> or =50) and non-depression group (SDS<50). Immunological parameters such as T-lymphocyte subsets and natural killer (NK) cell activities in peripheral blood were determined and compared between the two groups of patients. RESULTS: The SDS index was from 33.8 to 66.2 in the 108 cases, 50% of these patients had a SDS index more than 50. Similarly, the SAS index of all the patients ranged from 35.0 to 62.0 and 46.3% of the cases had a SAS index above 50. Cubic curve estimation showed that the depression was positively correlated with anxiety and negatively with social support. Furthermore, the depression correlated with the tumor type, which manifested in a descending order as stomach, gallbladder, pancreas, intestine, esophagus, duodenum and rectum, according to their correlativity. Step-wise regression analysis suggested that hyposexuality, dispiritment, agitation, palpitation, low CD56 and anxiety were the significant factors contributing to depression. More severe anxiety (49.7 +/- 7.5 vs 45.3 +/- 6.9, P<0.05), pain (6.5 +/- 2.8 vs 4.6 +/- 3.2, P<0.05), poor social support (6.8 +/- 2.0 vs 7.6 +/- 2.1, P<0.05), as well as decline of lymphocyte count (0.33 +/- 0.09 vs 0.39 +/- 0.87, P<0.05) and CD56 (0.26 +/- 0.11 vs 0.29 +/- 0.11, P<0.05) were noted in the depression group compared with those of the non-depression patients. However, fewer obvious changes in CD4/CD8 ratio and other immunological parameters were found between the two groups. CONCLUSION: Depression occurs with a high incidence in patients with cancers of the digestive tract, which probably is not the sole factor leading to the impairment of immunological functions in these cases. However, comprehensive measures including psychological support should be taken in order to improve the immunological function, quality of life and clinical prognosis of these patients.

Cloning of human 15ku selenoprotein gene from H9 T cells.

AIM: To clone human 15ku selenoprotein gene. METHODS: H9 human T cells were cultured in RPMI1640 medium supplemented with 100 mL /L fetal calf serum. mRNA was isolated from the cells. cDNA library was constructed by RT-PCR. The human 15ku selenoprotein gene was obtained by PCR and cloned into T vector and sequenced. RESULTS: A unique cDNA fragment about 1 244 bp was obtained. Sequence analysis identified an open reading frame within the cDNA. The gene had an in-frame TGA, which encoded selenocysteine (Sec), and a 3'-UTR SECIS element, which was required for synthesis of selenoprotein. The predicted protein molecular mass was about 15ku (162 residues). The result was identical with human liver 15ku selenoprotein gene published in Genbank. CONCLUSION: Human 15ku selenoprotein gene can be successfully obtained from T cell line.