[Target protein candidates of hypothalamus in aging rats with intervention by Qiongyugao].

To explore the associated proteins of the hypothalamus in aging rat models with intervention by Qiongyugao(QYG) based on iTRAQ technology, find out the target protein candidates and investigate the mechanism of delaying aging for Qiongyugao. The results showed that Qiongyugao increased GSH-Px activity in serum and SOD activity in liver; the total protein count identified by iTRAQ was 3 522, FDR<1%. There were 20 kinds of differential proteins between the blank group and model group; there were 295 kinds of differential proteins between model group and QYG group, and 40 kinds of them had a difference multiple ≥1.30 (the maximum value was 1.47). Compared with blank group, there were 14 kinds of proteins that were down-regulated in model group and up-regulated in QYG group. Combined with literature search and gene function search, 12 kinds of target protein candidates were screened out ： ST18, Ptprc, PSMB8, INPP4B, Shc3, Pik3r1, PIP5K1C, Nampt, Rasgrp2, Asah2, Pdpk1, and Map2k7. The expression of nuclear factor-kappa B (NF-κB) in the hypothalamic inflammatory pathway was detected by Western blot and the results showed that its expression level in model group(0.96) was higher than that in control group(0.85), while its expression level in QYG group(0.89) was lower than that in model group. Q-PCR results showed that the relative mRNA expression levels of PIP5K1C and Ptprc in model group were significantly lower than those in blank group(P<0.01); while compared with the model group, the mRNA expression levels of PIP5K1C and Ptprc in QYG group were significantly increased(P<0.01) . This result was consistent with proteomics data. QYG may delay aging by regulating hypothalamic inflammatory reaction.

A double-negative feedback loop between Wnt-ß-catenin signaling and HNF4α regulates epithelial-mesenchymal transition in hepatocellular carcinoma.

Wnt-ß-catenin signaling participates in the epithelial-mesenchymal transition (EMT) in a variety of cancers; however, its involvement in hepatocellular carcinoma (HCC) and downstream molecular events is largely undefined. HNF4α is the most prominent and specific factor maintaining the differentiation of hepatic lineage cells and a potential EMT regulator in HCC cells. However, the molecular mechanisms by which HNF4α maintains the differentiated liver epithelium and inhibits EMT have not been completely defined. In this study, we systematically explored the relationship between Wnt-ß-catenin signaling and HNF4α in the EMT process of HCC cells. Our results indicated that HNF4α expression was negatively regulated during Wnt-ß-catenin signaling-induced EMT through Snail and Slug in HCC cells. In contrast, HNF4α was found to directly associate with TCF4 to compete with ß-catenin but facilitate transcription co-repressor activities, thus inhibiting expression of EMT-related Wnt-ß-catenin targets. Moreover, HNF4α may control the switch between the transcriptional and adhesion functions of ß-catenin. Overexpression of HNF4α was found to completely compromise the Wnt-ß-catenin-signaling-induced EMT phenotype. Finally, we determined the regulation pattern between Wnt-ß-catenin signaling and HNF4α in rat tumor models. Our studies have identified a double-negative feedback mechanism controlling Wnt-ß-catenin signaling and HNF4α expression in vitro and in vivo, which sheds new light on the regulation of EMT in HCC. The modulation of these molecular processes may be a method of inhibiting HCC invasion by blocking Wnt-ß-catenin signaling or restoring HNF4α expression to prevent EMT.

Roles of autophagy in androgen-induced benign prostatic hyperplasia in castrated rats.

The present study investigated the role of androgen in the process of androgen-induced prostate hyperplasia in castrated rats and assessed the role of the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/Akt/mTOR) pathway in this process. Furthermore, the extent to which autophagy may affect the level of androgen-induced benign prostatic hyperplasia was also explored. A total of 40 Sprague Dawley rats were randomly divided into four groups: Testosterone group, rapamycin group, 3-methyladenine (3-MA) group, and control group. The extent of hyperplasia in prostate tissue the apoptosis and autophagy were assayed. The prostate wet weight, volume and index in the testosterone group were significantly higher compared with the control group (P<0.05) and these factors were significantly lower in the rapamycin group compared with the testosterone group (P<0.05). HE staining demonstrated that prostate hyperplasia was obvious in the testosterone group. Western blotting revealed that caspase-3 levels were higher in the 3-MA group compared with the control group and Bcl-2 was higher in the testosterone group compared with the control group (P<0.05). Furthermore, in the rapamycin group, Bcl-2 protein expression levels were significantly lower than those in the testosterone group (P<0.05). The prostate tissue was analyzed using electron microscopy and autophagy bodies were identified in the rapamycin group. In the process of androgen-induced prostatic hyperplasia in castrated rats, the role of androgen may be related to the PI3K/Akt/mTOR signaling pathway. Rapamycin was able to inhibit the effect of testosterone and promoted prostate tissue hyperplasia by inhibiting the PI3K/Akt pathway. In addition to inhibiting apoptosis in prostate cells, androgen was able to induce rat prostate hyperplasia and may also be related to the promotion of the proliferation of prostate cells.

Crosstalk between apoptosis and autophagy in prostate epithelial cells under androgen deprivation.

The present study investigated the molecular mechanism of apoptosis and autophagy in prostate epithelial cells under androgen deprivation (AD). BPH-1 cells were divided into four groups as follows: Control (Cont), AD, autophagy inhibition (AI) and AD + AI groups. Cells in the four groups were treated accordingly, and the level of apoptosis was subsequently measured via flow cytometry. The expression of the microtubule-associated proteins 1A/1B light chain 3 (LC3), caspase-3, poly (ADP-ribose) polymerase 1 (PARP-1) and Beclin-1 proteins of BPH-1 cells was detected at different time points following culture in androgen-deprived medium. Western blotting revealed that the basal levels of the LC3-II protein were detected at 0 h. At 4 h, LC3-II was significantly increased compared with 0 h (P<0.05). Beginning at 20 h, the expression level of the LC3-II protein decreased significantly (P<0.05). Western blotting revealed that beginning at 24 h, the expression level of the PARP-1 protein decreased significantly (P<0.001) and the cleavage fragments of the PARP-1 protein appeared. These results further imply that autophagy serves a cell protective function by mutual inhibition with apoptosis in BPH-1 cells in the removal of androgen conditions. Furthermore, the fragments of the cleaved Beclin-1 protein appeared as 35 and 37 kDa bands. Flow cytometry analysis demonstrated that the rate of cell apoptosis in the AD, AI and AD + AI groups was significantly increased compared with the Cont group (P<0.01). Compared with the AD or the AI groups individually, the rate of cell apoptosis in the AD + AI group was significantly increased (P<0.001). These findings suggest that in the early stage of AD, autophagy has a compensatory function in the cell, whereas in the whole process, autophagy and apoptosis share a mutual antagonism. The Beclin-1-C protein fragment contributed positive feedback to the process of apoptosis, which may be a potential mechanism of AD therapy. Therefore, AD and AI exhibit a synergistic effect to further improve the level of apoptosis.

Preparation of Rat Whole-kidney Acellular Matrix via Peristaltic Pump.

PURPOSE: To design a whole-kidney a cellular matrix scaffold using peristaltic pump perfusion and to ascertain the retention of extra cellular proteins by the scaffold. MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats weighing 200-250 g were used. Intravenous catheters were inserted into the renal artery followed by perfusion of decellularization solution using a peristaltic pump. After decellularization, the acellular matrix was observed under a microscope after hematoxylin and eosin (H&E) staining and a fluorescence microscope after 4',6-diamidino-2-phenylindole (DAPI) staining. Immunohistochemistry was used to identify the composition of kidney acellular matrix. RESULTS: The result of H&E and DAPI staining demonstrate the removal of cellular material in kidney a cellular matrix. Immunohistochemistry confirmed the conservation of the natural expression of extra cellular matrix proteins including collagen types I and IV, fibrin and laminin. CONCLUSION: Peristaltic pump perfusion enables successful preparation of renal a cellular matrix, to retainthe criticalproteins of natural extra cellular matrix. The resulting kidney a cellular matrix represents an ideal natural scaffold for renal tissue engineering.