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Background: There is an urgent need for new treatments to solve hair loss problem. As mesenchymal stem cells were proved to have effects on promoting tissue repair and regeneration, in which the exosome plays a vital role, we aim to investigate the influence of umbilical cord mesenchymal stem cells exosome (UCMSC-Exos) on hair growth and its mechanism., Methods: The hUCMSC-Exos were extracted by ultracentrifugation. Primary fibroblasts were cultured with or without hUCMSC-Exos and cell proliferation was evaluated by CCK-8 assay. C57BL/6 mice model of depilation-induced hair regrowth was treated with either hUCMSC-Exos (200 μg/mL) or PBS on one side of the dorsal back. Real time quantitative PCR, flow cytometry analysis, immunohistochemistry and Immunofluorescent staining were used to analyze the regulative effect of hUCMSC-Exos on hair follicle stem/progenitor cells and Wnt/β-catenin pathway., Results: The proliferation of fibroblasts incubated with hUCMSC-Exos at the concentration of 200 μg/mL was greater than other groups. Treatment with hUCMSC-Exos resulted in rapid reentry into anagen. Hair follicle stem/progenitor cell markers (K15, Lgr5, Lgr6, CD34 and Lrig1) and Wnt/β-catenin pathway related factors (Wnt5, Lef1, Lrp5 and β-catenin) were increased in hUCMSC-Exos-injected region., Conclusion: hUCMSC-Exos promote fibroblasts proliferation and accelerate mouse hair regrowth by upregulating hair follicle stem/progenitor cell and Wnt/β-catenin pathway, which suggests potential therapeutic approaches for hair loss disorders., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The in vivo functions of SerpinB2 in tumor cells and tumor-associated macrophages (TAMs) during breast cancer development and metastasis remain elusive. SerpinB2-deficient MMTV-PyMT mice (PyMT SB2-/- ) were previously produced to explore the biological roles of SerpinB2 in breast cancer. Compared with MMTV-PyMT wild-type (PyMT WT ) mice, PyMT SB2-/- mice showed delayed tumor progression and reduced CK8 + tumor cell dissemination to lymph nodes. RNA-Seq data revealed significantly enriched genes associated with inflammatory responses, especially upregulated M1 and downregulated M2 macrophage marker genes in PyMT SB2-/- tumors. Decreased CD206 + M2 and increased NOS2 + M1 markers were detected in the primary tumors and metastatic lymph nodes of PyMT SB2-/- mice. In an in vitro study, SerpinB2 knockdown decreased the sphere formation and migration of MDA-MB-231 cells and suppressed protumorigenic M2 polarization of RAW264.7 cells. The combination of low SerpinB2, high NOS2, and low CD206 expression was favorable for survival in patients with breast cancer, as assessed in the BreastMark dataset. Our study demonstrates that SerpinB2 deficiency delays mammary tumor development and metastasis in PyMT WT mice, along with reduced sphere formation and migration abilities of tumor cells and decreased macrophage protumorigenic polarization., (© 2024. The Author(s).)

Background: SerpinB2 is highly expressed in immune and tumor cells and is involved in multiple biological functions, including cell survival and remodeling for disease progression. This study prepared SerpinB2-deficient mice and analyzed the differentially expressed genes (DEGs) to determine if loss of this protein delays mammary tumor progression., Results: A total of 305 DEGs (75 upregulated and 230 downregulated; > 1.5-fold difference, P < 0.05) were identified in SB2-/-;PyMT tumors compared with PyMT tumors. The DEGs were mainly involved in immune and inflammatory responses related to T cell differentiation, IFN-γ production, and lymphocyte chemotaxis based on 61 enriched GO terms, hierarchical clustering, KEGG pathways, and a functionally grouped annotation network. The significantly changed DEGs (Anxa3, Ccl17, Cxcl13, Cxcr3, IFN-γ, Nr4a1, and Sema3a) annotated with at least two GO categories in SB2-/-;PyMT tumors was validated by qRT-PCR., Conclusions: SerpinB2 deficiency alters the expression of multiple genes in mammary tumors, which might cause a delay in PyMT-induced mammary tumor progression., (© 2022. The Author(s).)

We here evaluated the therapeutic effect of tumor cell-derived exosomes (TEXs)-stimulated dendritic cells (DCs) in a syngeneic orthotopic breast tumor model. The DC line DC2.4 and breast cancer cell line E0771 originally isolated from C57BL/6 mice were used. E0771 cells stably expressing the exosomal CD63-RFP or luciferase (Luc) and DC2.4 cells stably expressing GFP were produced using lentivirus. TEXs were purified from conditioned medium of E0771/CD63-RFP cells. Breast tumor model was established by injecting E0771/Luc cells into mammary gland fat pad of mice. TEXs contained immune modulatory molecules such as HSP70, HSP90, MHC I, MHC II, TGF-β, and PD-L1. TEXs were easily taken by DC2.4 cells, resulting in a significant increase in the in vitro proliferation and migration abilities of DC2.4 cells, accompanied by the upregulation of CD40. TEX-DC-treated group exhibited a decreased tumor growth compared with control group. CD8 + cells were more abundant in the tumors and lymph nodes of TEX-DC-treated group than in those of control group, whereas many CD4 + or FOXP3+ cells were localized in those of control group. Our results suggest a potential application of TEX-DC-based cancer immunotherapy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Purpose: The tools to trigger dendritic cell (DC) activation and to verify DC migration in vivo are important for directing DC immunotherapy toward successful treatment. We evaluated whether tumor cell-derived exosome (TEX)-stimulated DC migration into lymph node (LN) in mouse could be tracked using gold nanoparticle (GN)-labeling and ultrasound (US)-guided photoacoustic imaging (PAI)., Procedures: GFP-transduced DC2.4 cells were used. RFP-tagged TEXs were purified from a stable 4T1 cell line overexpressing the exosomal CD63-RFP fusion protein. The TEX uptake by DCs was visualized using confocal laser scanning microscopy. GNs with surface plasmon resonance at 808 nm were used for DC-labeling. DCs that migrated into axillary LN were longitudinally monitored by US-guided PAI and analyzed by silver staining and immunohistochemistry., Results: TEXs were easily internalized in DCs, increased proliferation and migration capacities, and upregulated co-stimulatory molecules, CCR7 and TNF-α without cytotoxicity. The GN-labeling exerted no adverse effects on the biological functions of DCs. US-guided PAI and DC-labeling allowed for sensitive and longitudinal monitoring of TEX-stimulated DC migration toaxillary LN., Conclusions: TEXs efficiently activated DCs and GN-labeled DC migration into LN was successfully monitored using US-guided PAI, suggesting that TEXs are a good source for DC activation and US-guided PAI is a cost-effective and easy-to-use imaging modality for noninvasive tracking of DCs.

Increased cancer risk and immune disorders linked with exposure to environmental endocrine disruptors like bisphenol A (BPA) have been steadily reported. Nevertheless, the impacts of BPA on the breast ductal carcinoma in situ (DCIS) progression and macrophage polarization remain to be elucidated. Here, we analyzed the differentially expressed genes in BPA-exposed DCIS cells and explored BPA effects on DCIS progression and macrophage polarization in vitro and in vivo. Two hundred and ninety-one genes were differentially expressed in 10-8 M BPA-exposed DCIS cells, in which the gene ontology terms of biological processes associated with negative regulation of cell death, cell adhesion, and immune response was enriched. 10-8 M BPA promoted the proliferation and migration of DCIS cells and the migration of macrophages, and upregulated the expression of M1 (NOS2) or M2 markers (Arg-1 and CD206) in macrophages. In coculture system, the migratory capacity of both cells and the expression levels of NOS2, Arg-1, and CD206 in macrophages were significantly enhanced upon 10-8 M BPA. In a DCIS xenograft model, oral exposure to an environmentally human-relevant low dose of 2.5 µg/l BPA for 70 days via drinking water led to an approximately 2-fold promotion in the primary tumor growth rate and a significant enhancement of lymph node metastasis along with increased protumorigenic CD206+ M2 polarization of macrophages. These results demonstrate that BPA acts as an accelerator to promote DCIS progression to invasive breast cancer by affecting DCIS cell proliferation and migration as well macrophage polarization toward a protumorigenic phenotype., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The feasibility of detecting breast cancer stem-like cells (BCSCs) with magnetic resonance imaging using extradomain-B of fibronectin (EDB-FN)-specific peptide (APT EDB )-conjugated thermally cross-linked superparamagnetic iron oxide nanoparticles (APT EDB -TCL-SPIONs) is previously demonstrated. Here, doxorubicin (Dox)-loaded APT EDB -TCL-SPIONs (Dox@APT EDB -TCL-SPIONs) are generated and their theranostic ability in a BCSC xenograft mouse model is assessed. The Dox@APT EDB -TCL-SPIONs enable more efficient delivery of Dox to tumors than nontargeted Dox@TCL-SPIONs. Much greater inhibition of BCSC tumor growth is observed after treatment with the Dox@APT EDB -TCL-SPIONs than with either Dox@TCL-SPIONs or free Dox. Hypointense signals are observed in the majority of the mice in postcontrast but not precontrast T2*-weighted MR images of tumors 7 days after treatment with Dox@APT EDB -TCL-SPIONs. An inverse correlation is observed between signal intensity and both EDB-FN expression and response to chemotherapy. The data indicate Dox@APT EDB -TCL-SPIONs can detect BCSCs within tumors by targeting EDB-FN-expressing cells. These nanoparticles thus have theranostic potential in breast cancer., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The function of preadipocytes in the progression of early stage breast cancer has not been fully elucidated at the molecular level. To delineate the role of preadipocytes in breast cancer progression, we investigated the cross-talk between human breast ductal carcinoma in situ (DCIS) cells and preadipocytes with both an in vitro culture and xenograft tumor model., Methods: GFP or RFP was transduced into human DCIS cell line MCF10DCIS.com cells or preadipocytes using lentivirus. Cell sorter was used to separate pure, viable populations of GFP- or RFP-transduced cells. Cell viability and proliferation was assessed by crystal violet assays and cell migration and invasion capability was assayed by the transwell strategy. Gene and protein levels were measured by western blot, RT-PCR and immunostaining. Adipokines and cytokines were quantified using ELISA. Human tumor xenografts in a nude mice model were used. Ultrasound imaging of tumors was performed to evaluate the therapeutic potential of a IL-6 neutralizing antibody., Results: In the co-culture system with the MCF10DCIS.com and preadipocytes, MCF10DCIS.com proliferation, migration and invasion were enhanced by preadipocytes. Preadipocytes exhibited in an increased IL-6 secretion and cancer-associated fibroblast markers expression, FSP1 and α-SMC in co-culture with MCF10DCIS.com or in MCF10DCIS.com conditioned media, whereas the adipocyte differentiation capacity was suppressed by co-culture with MCF10DCIS.com. A neutralizing antibody of IL-6 or IL-6R suppressed the promotion of MCF10DCIS.com proliferation and migration by co-culture with preadipocytes. In the xenograft tumor model, the tumor growth of MCF10DCIS.com was enhanced by the co-injection of preadipocytes, and the administration of IL-6 neutralizing antibodies resulted in potent effects on tumor inhibition., Conclusions: Our findings suggest that IL-6-mediated cross-talk between preadipocytes and breast DCIS cells can promote the progression of early stage breast cancer. Therefore, blocking IL-6 signaling might be a potential therapeutic strategy for breast DCIS characterized by pathological IL-6 overproduction.

Crosstalk between breast cancer and macrophages has potential implications for tumor metastasis. This study investigates macrophage polarization induced by triple-negative breast cancer (TNBC) cell-derived exosomes that promote lymph node (LN) metastasis in orthotopic TNBC models. The MDA-MB-231 cancer cell line expressing the exosomal CD63-red fluorescence (RFP) fusion protein was generated to noninvasively visualize exosome transfer into cancer cells and macrophages. Administration of RFP-tagged exosomes enhanced migration of macrophages and induced macrophage polarization in vitro . In orthotopic TNBC models, noninvasive bioluminescent imaging, ultrasound-guided photoacoustic imaging, and histological analysis revealed that intravenous injection of RFP-tagged exosomes promoted primary tumor growth and axillary LN metastasis in which expression of CD206, a marker or alternatively activated type 2 (M2) macrophages, was significantly higher than expression of NOS2, a marker of classically activated type 1 (M1) macrophages. These results suggest breast cancer cell-derived exosomes stimulate macrophage polarization that creates favorable conditions for LN metastatic processes in TNBC., Competing Interests: CONFLICTS OF INTEREST The authors declare no competing interests.

Current EGFR-targeted therapy for triple negative breast cancer (TNBC) has produced disappointing results. A rational therapeutic strategy to improve EGFR-targeted treatment for TNBC is therefore needed. In this study we evaluated the feasibility of treating TNBC using photoacoustic imaging (PAI)-guided near-infrared photothermal therapy (NIR-PTT) with anti-EGFR-conjugated gold nanorods (anti-EGFR-GN). NIR-PTT combined with anti-EGFR-GN exerted synergistic anti-proliferative and apoptotic actions through upregulation of HSP70 and cleaved caspase-3, downregulation of Ki-67 and EGFR, and inhibition of several intracellular signaling molecules (mTOR, AKT, ERK1/2 and FAK). These combined effects give this approach significant efficacy. Our findings suggest PAI-guided NIR-PTT using anti-EGFR-GN represent a novel and effective strategy for EGFR-targeted therapy in TNBC., Competing Interests: CONFLICTS OF INTEREST There are no conflicts of interest to disclose.

Squamous cell carcinoma (SCC) is a common skin cancer, of which the incidence is relatively high, ranking second among the non‑melanoma skin cancers. It is known that numerous intracellular signal regulators are involved in the pathogenesis of SCC. The Wnt/β-catenin signaling pathway serves an important role in cancer development. However, the downstream effectors of β‑catenin remain to be clearly elucidated yet. The present study investigated the functional importance of Wnt/β‑catenin signaling in cutaneous SCC. β‑catenin expression was reduced using recombinant adenovirus expressing specific microRNA (miR). Knockdown of β‑catenin resulted in a marked reduction of the colony-forming activity of the SCC cells, SCC12. In an attempt to identify the β‑catenin downstream genes, it was found that Sox9 was regulated by β‑catenin in SCC12 cells. Overexpression of a constitutively active form of β‑catenin led to the induction of Sox9, while knockdown of β‑catenin resulted in downregulation of Sox9. When the expression of Sox9 was reduced using specific miR, colony-forming activity of the SCC12 cells was significantly reduced. When Sox9 was overexpressed in cells where β‑catenin was knocked down, it partially restored the colony‑forming potential. Taken together, the present results suggested that Sox9 is a β-catenin downstream transcription factor and is positively involved in SCC development.

Genkwadaphnin is a daphnane diterpene ester molecule isolated from the flower buds of Daphne genkwa. In the present study, we investigated the apoptosis-inducing effect of genkwadaphnin in squamous cell carcinoma (SCC) cells. Apoptosis was triggered in SCC12 cells following genkwadaphnin treatment in a time- and concentration-dependent manner. Genkwadaphnin treatment increased phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Knockdown of JNK and p38 MAPK by recombinant adenovirus expressing microRNA (miR) resulted in significant inhibition of genkwadaphnin-induced apoptosis in SCC12 cells. Finally, pretreatment with the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) markedly reduced SCC12 cell apoptosis, concomitant with significant inhibition of MAPK activation. These results indicate that genkwadaphnin has the potential to induce apoptosis in SCC cells, providing information on which to base further research with the aim of developing a cure for SCC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A new training system that can adjust training intensity and indicate the center pressure of a subject was proposed by applying controlled electric current to the Magneto-Rheological damper. The experimental studying on the muscular activities were performed in lower extremities during maintaining and moving exercises, which were processed on an unstable platform with Magneto rheological dampers and recorded in a monitor. The electromyography (EMG) signals of the eight muscles in lower extremities were recorded and analyzed in certain time and frequency domain. Muscles researched in this paper were rectus femoris (RF), biceps femoris (BF), tensor fasciae latae (TFL), vastuslateralis (VL), vastusmedialis (VM), gastrocnemius (Ga), tibialis anterior (TA), and soleus (So). Differences of muscular activities during four moving exercises were studied in our experimental results. The rate of the increment of the muscular activities was affected by the condition of the unstable platform with MR dampers, which suggested the difference of moving exercises could selectively train each muscle with varying intensities. Furthermore, these findings also proposed that this training system can improve the ability of postural balance.

Quercetin, a flavonoid, is an inhibitor of P-glycoprotein-mediated efflux transport, and its oxidative metabolism is catalyzed by CYP enzymes. Thus, it is expected that the pharmacokinetics of both intravenous and oral doxorubicin can be changed by quercetin. The purpose of this study was to investigate the effect of oral quercetin on the bioavailability and pharmacokinetics of orally and intravenously administered doxorubicin in rats. The effects of quercetin on the P-glycoprotein (P-gp) and CYP3A4 activities were also evaluated. Quercetin inhibited CYP3A4 enzyme activity in a concentration-dependent manner with a 50% inhibition concentration (IC(50)) of 1.97 μM. In addition, quercetin significantly enhanced the intracellular accumulation of rhodamine-123 in MCF-7/ADR cells overexpressing P-gp. The pharmacokinetic parameters of doxorubicin were determined in rats after oral (50 mg/kg) or intravenous (10 mg/kg) administration of doxorubicin to rats in the presence and absence of quercetin (0.6, 3 or 15 mg/kg). Compared to control, quercetin significantly (p < 0.05 for 0.6 mg/kg, p < 0.01 for 3 and 15 mg/kg) increased the area under the plasma concentration-time curve (AUC(0-∞), 31.2-136.0% greater) of oral doxorubicin. Quercetin also significantly increased the peak plasma concentration (C(max)) of doxorubicin, while there was no significant change in T(max) and T(1/2) of doxorubicin. Consequently, the absolute bioavailability of doxorubicin was increased by quercetin compared to control, and the relative bioavailability of oral doxorubicin was increased by 1.32 to 2.36 fold. In contrast, the pharmacokinetics of intravenous doxorubicin were not affected by quercetin. These results suggest that the quercetin-induced increase in bioavailability of oral doxorubicin can be attributed to enhanced doxorubicin absorption in the gastrointestinal tract via quercetin-induced inhibition of P-gp and reduced first-pass metabolism of doxorubicin due to quercetin-induced inhibition of CYP3A in the small intestine and/or in the liver rather than reduced renal and/or hepatic elimination of doxorubicin. Therefore, it appears that the development of oral doxorubicin preparations is possible, which will be more convenient than the intravenous dosage forms. Therefore, concurrent use of quercetin provides a therapeutic benefit - it increases the bioavailability of doxorubicin administered orally.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. In this study, we investigated the role of the POU domain-containing transcription factor Brn2 in keratinocyte differentiation. Immunohistochemical analysis showed that Brn2 is expressed primarily in the upper granular layer. Consistent with its epidermal localization, Brn2 expression was highly induced at 14 days after calcium treatment of cultured normal human epidermal keratinocytes. When Brn2 was overexpressed by adenoviral transduction, Brn2 led to increased expression of the differentiation-related genes involucrin, filaggrin, and loricrin in addition to inhibition of their proliferation. Chromatin immunoprecipitation demonstrated that Brn2 bound to the promoter regions of these differentiation-related genes. We injected the purified Brn2 adenovirus into rat skin, which led to a thickened epidermis with increased amounts of differentiation related markers. The histopathologic features of adenovirus-Brn2 injected skin tissues looked similar to the features of lichen planus, a human skin disease showing chronic inflammation and well-differentiated epidermal changes. Moreover, Brn2 is shown to be expressed in almost all cell nuclei of the thickened epidermis of lichen planus, and Brn2 also attracts T lymphocytes. Our results demonstrate that Brn2 is probably a transcriptional factor playing an important role in keratinocyte differentiation and probably also in the pathogenesis of lichen planus lesions.

The purpose of this study is to develop an abnormal gait detection algorithm and a vibratory stimulation system on a lower limb to improve gait stability and prevent falls. The system consists of a gait measurement module, an abnormal gait detection module, and a vibratory stimulation module. The gait measurement module measures the vertical acceleration of the ankle during walking using an accelerometer. The measured acceleration values are sent to a portable microcontroller, which controls vibratory stimulations to the ankles based on an algorithm that detects the peak acceleration values. If the acceleration peaks are found to occur irregularly, the abnormal gait detection algorithm activates the vibratory stimulation module. To determine the effect of vibratory stimulations under dynamic condition, this study investigated the contribution of ankle muscle proprioception on the control of dynamic stability and lower limb kinematics while walking using vibratory stimulation to alter the muscle spindle output of individuals' left lower limb. Vibrators were attached to the left ankle joint (tibialis anterior, triceps surae). Participants were required to walk along a travel path and step over an obstacle placed in their way. There were four task conditions; an obstacle (10%, 20%, and 30% of the participants' height) was positioned at the midpoint of the walkway, or the participants' walking path remained clear. For each obstacle condition, participants experienced either no vibration, or vibration of the tibialis anterior muscle and the triceps surae muscle of the left lower limb. Vibration began upon detection of an abnormal gait and continued for one second. Vibrating the ankle muscles of the left lower limb while stepping over an obstacle resulted in significant changes in COM behavior on both the anterior/posterior (A/P) and medial/lateral (M/L) planes. The results provide strong evidence that the primary endings of the ankle muscle spindles play a significant role in the control of posture and balance during the swing phase of locomotion by providing information on the movement of the body's COM with respect to the support foot.

Objective: To report the morphometric characteristics of ultrastructure inside A549 cells., Methods: A549 cells were processed for inverted microscopy and transmission electron microscopy (TEM). Cell images were obtained randomly using inverted microscopy and TEM. The morphometric parameters of ultrastructure were tested using precise morphometric techniques by Image-Pro Plus analysis software., Results: (1) The diameter of A549 cells from inverted microscopy and TEM images was 14.93 μm and 10.59 μm. (2) By defining cell as reference space the volume densities (VV) of nucleus and cytoplasm were about 0.28 and 0.72; the surface densities (SV) of nucleus were 0.19 μm-1. By defining cell nucleus as reference space the VV of nucleoli, euchromatin and heterochromatin were 0.076, 0.72 and 0.20 respectively; the SV of nucleoli was 0.15 μm-1. By defining cytoplasm as reference space the VV of mitochondria, lamellar bodies and lysosomes were 0.046, 0.025 and 0.014; the SV of mitochondria, lamellar bodies and lysosomes were 0.60 μm-1, 0.36 μm-1, and 0.18 μm-1. (3) In individual A549 cell total volume and surface of mitochondria were 61.91 μm³ and 1001.67 μm²; Total volume and surface area of lamellar bodies were 76.82 μm³ and 428.68 μm²; Total volume and surface area of lysosomes were 21.69 μm³ and 212.04 μm²., Conclusions: The morphometric parameters of some ultrastructures within A549 cells were established using precise morphometric techniques by Image-Pro Plus analysis software.

The objective of this study was to determine the effects of chondroitin sulfate C (CS-C) on sperm capacitation and fertilization parameters in vitro in pigs. Frozen-thawed ejaculated pig sperm (semen S-484) were incubated with fertilization medium containing CS-C (0-2mg/ml) for 1h and the capacitation rate with chlorotetracycline (CTC) assay was examined, which showed that CS-C increased the rate of incapacitation F pattern spermatozoa converted to capacitation B pattern sperm cell in concentration-dependent manner and mostly increased capacitation B pattern sperm cell and decreased acrosome reaction AR pattern sperm cell in 1mg/ml concentration. When sperm was incubated for 1, 2 and 4h in fertilization medium containing 1mg/ml CS-C, it showed that the capacitated B pattern sperm cell was significantly (p<0.01) increased and the AR pattern sperm cell was significantly decreased at each time point in the presence than in the absence of CS-C. For identifying the validity of CS-C in sperm capacitation, sperm-oocyte was inseminated in fertilization medium containing CS-C (0-2mg/ml) and the rate of fertilized oocytes was examined, which showed that the penetration rates significantly (p<0.05) increased from 0.5 to 1.0mg/ml concentrations (87.4-96.3%) compared with control (74.9%). For identifying the universality of CS-C in sperm capacitation, four different semens (boar S-484, S-454, D-815 and D-748) were incubated in fertilization medium containing CS-C (1mg/ml) for 2h, respectively, which showed that CS-C increased the rate of capacitation B pattern sperm cell and decreased acrosome reaction AR pattern sperm cell in each semen. And it showed that CS-C yielded a higher promote effect (93.9%, 83.9%, 60.7% and 44.9%, respectively) on sperm penetration compared to unaddition control (63.4%, 22.0%, 3.3% and 3.3%, respectively). Sperm-oocyte binding analysis showed that CS-C increased the number of sperm bound to oocyte compared unaddition control in each semen. These results suggested that CS-C is the efficient factor on sperm capacitation in pigs, CS-C may promote sperm from the incapacitated to capacitated state and sequentially prevent sperm from spontaneous acrosome reaction, and thus facilitate the sperm-zona binding and sperm penetration to oocyte.

Etoposide is mainly metabolized by cytochrome P450 (CYP) 3A and is a substrate for P-glycoprotein (P-gp). This study examined the effects of verapamil, a CYP3A and P-gp inhibitor, on the pharmacokinetics of etoposide in rats. A single dose of etoposide was administered via oral (p.o.; 10 mg/kg) or intravenous (i.v.; 3.3 mg/kg) routes to rats alone (control animals) or together in combination with verapamil (2 or 6 mg/kg; experimental animals). The presence of verapamil significantly increased the area under the plasma concentration-time curve (AUC); (P < 0.05; 39.2-47.6%) and significantly reduced (P < 0.01; 27.8-31.2%) the total body clearance (CLt) of p.o. administered etoposide. The absolute bioavailability (F) of etoposide increased by 1.38- to 1.47-fold. The presence of verapamil significantly increased (P < 0.01; 38.3-38.9%) the AUC and significantly reduced (P < 0.01; approximately 27%) the total body clearance (CLt) of i.v. administered etoposide. This increased bioavailability suggests that verapamil inhibits metabolic activity and elimination etoposide. The increased bioavailability of etoposide in the presence of verapamil should be taken into consideration for dosage regimens due to a potential drug interaction (DI).

Objective: To evaluate the effect of human hair keratin (HHK) in peripheral nerve repair and explore the mechanism of sciatic nerve regeneration., Methods: Rat models of sciatic nerve damage was established by creating a 10-mm gap in the sciatic nerve, which was bridged with a HHK implant. Histological examinations of the nerve tissues were performed at different time points after the surgery., Results: During the period from 2 days to 2 weeks following HHK implantation, Schwann cells were found to undergo dedifferentiation and proliferate along the HHK implant. Three weeks after HHK implantation, numerous macrophages and megakaryocytes occurred around the HHK, and a large quantity of regenerated Schwann cells aligned in orderly fashion was seen between the fine filaments of partially degraded HHK, where axons and capillaries were also observed. Six weeks later, massive nerve fibers and capillaries developed around the HHK, and at 9 weeks, the HHK implant was substantially degraded and numerous regenerated nerve fibers occurred characterized by obvious epineurium and perineurium. Till 12 weeks after HHK implantation, HHK was almost completely degraded and replaced by the newly regenerated nerve fibers that had grown across the nerve defect., Conclusions: HHK is an ideal material for nerve injury repair. Apocytosis plays a key role in the differentiation process of highly differentiated Schwann cells into immature Schwann cells following nerve injury. As a protective mechanism, the axons undergo enclosure and dissociation following injuries, and the intact axons give rise to growth cones that extend fibers of growing buds to competitively bind the one or more Schwann cells, but only one such but finally develops into a complete axon. The nerve fiber barrier membrane is derived from the capillary menchymal stem cells and the outmost vascular barrier membrane. The regeneration of the Schwann cells, axons and the nerve membrane is the result of self-organization through a well synchronized and coordinated mechanism.

This study investigated the effects of orally administered morin, an inhibitor of cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp), on the pharmacokinetics of orally and intravenously administered nicardipine in rats. Nicardipine is reportedly a substrate for CYP3A4 and P-gp. Nicardipine was administered orally (12 mgkg(-1)) with or without orally administered morin (1.5, 7.5 and 15 mgkg(-1)), and intravenously (4 mgkg(-1)) with or without orally administered morin (7.5 and 15 mgkg(-1)). In the presence of morin, the pharmacokinetic parameters of nicardipine were significantly altered in the oral group but not in the intravenous group, suggesting that CYP3A-mediated metabolism of nicardipine in the liver is not significantly inhibited by morin. The presence of 7.5 and 15 mgkg(-1) of morin significantly increased (P < 0.01, 67.8-112%) the area under the plasma concentration-time curve and the peak plasma concentration (P < 0.01, 53.5-93.1%) of orally administered nicardipine. The presence of 7.5 and 15 mgkg(-1) of morin significantly decreased (P < 0.01, 40.4-52.8%) the total body clearance of orally administered nicardipine compared with the control group. The enhanced oral bioavailability of nicardipine suggests that intestinal-mediated CYP3A4 metabolism and P-gp-mediated efflux of nicardipine are inhibited by morin. Based on these results, concomitant use of morin or morin-containing dietary supplements with nicardipine may require close monitoring for potential drug interactions.

This study examined the effect of morin on the bioavailability and pharmacokinetics of tamoxifen and its metabolite, 4-hydroxytamoxifen, in rats. A single dose of tamoxifen was administered to rats intravenously (2 mg/kg) or orally (10 mg/kg), with or without morin (3 or 10 mg/kg). The presence of morin significantly altered the pharmacokinetics of the orally administered tamoxifen. Compared with the oral control group (given tamoxifen alone), the total body clearance (CL/F) of tamoxifen in the presence of morin was significantly reduced (by 35.9-40.8%, p<0.01). The area under the plasma concentration-time curve (AUC(0-infinity)) and the peak plasma concentration (Cmax) of tamoxifen significantly (p<0.05 for 3 mg/kg of morin, p<0.01 for 10 mg/kg of morin) increased by 50.6-68.9% and 65.1-80.9%, respectively. Consequently, the absolute bioavailability (AB) of tamoxifen in the presence of morin was 37.4-40.5%, which was enhanced significantly (p<0.05) compared with the oral control group (23.9%). The relative bioavailability (RB) of tamoxifen was 1.56 to 1.68 times higher than the control group. The increased bioavailability of tamoxifen is likely to be due to the decrease in the first-pass metabilism by the intestines and liver. Morin at a dose of 10 mg/kg significant increased the AUC(0-infinity), of 4-hydroxytamoxifen (by 50.9%, p<0.05) but the metabolite:parent ratio (MR) of 4-hydroxytamoxifen was not altered significantly, suggesting that the formation of 4-hydroxytamoxifen is not affected considerably by morin. The increased bioavailability of tamoxifen in the presence of morin should be taken into consideration for dosage regimens due to potential drug interaction.

The aim of this study was to investigate the effects of hesperidin on the pharmacokinetics of verapamil and its major metabolite, norverapamil, in rats. The pharmacokinetic parameters of verapamil and norverapamil in rats were measured after the oral administration of verapamil (9 mg/kg) in the presence or absence of hesperidin (3 or 10 mg/kg). Compared to the control group, the presence of hesperidin significantly (p<0.01) increased the area under the plasma concentration-time curve (AUC) of verapamil by 71.1-96.8% and the peak concentration (C(max)) of verapamil by 98.3-105.2%. Hesperidin significantly (p<0.01) decreased the total plasma clearance (CL/F) of verapamil by 41.6-49.2% in rats. However there was no significant change in the time to reach the peak plasma concentration (T(max)), the elimination rate constant (K(el)) and the terminal half-life (T(1/2)) of verapamil in the presence of hesperidin. The AUC and C(max) of norverapamil were significantly (p<0.05) higher in rats co-administrated with hesperidin than those of the control. Consequently hesperidin significantly enhanced bioavailability of verapamil in rats. These results might be due to the decreased efflux and metabolism of verapamil in the intestine. Drug interactions should be concerned in the clinical setting when verapamil is used concomitantly with hesperidin or hesperidin-containing dietary.

Background: Sphingosylphosphorylcholine (SPC) has been reported as a novel lipid mediator that exerts various actions on wound healing process., Objective: The aim of this study is to evaluate the involvement of interleukin-6 (IL-6) in SPC-induced wound healing acceleration., Methods: We performed immunohistochemical analysis to demonstrate the IL-6 induction by SPC. To analyze the signaling events, skin fibroblasts were treated with SPC, and then RT-PCR, ELISA and Western blot analyses were carried out., Results: SPC markedly induced interleukin-6 (IL-6) expression in rabbit ear wound. SPC also induced IL-6 expression at both the mRNA and protein levels in human dermal fibroblasts cultured in vitro. SPC rapidly phosphorylated p42/44 extracellular signal-regulated kinase (ERK). Pretreatment with PD 98059, a specific MAPK kinase 1/2 inhibitor, markedly suppressed SPC-induced IL-6 expression in a dose-dependent manner. Protein kinase C (PKC) activation by phorbol myristate acetate (PMA) potentiated IL-6 mRNA expression, whereas PKC inhibition by bisindolylmaleimide blocked SPC-induced p42/44 ERK phosphorylation and IL-6 expression. Over-expression of PKCalpha markedly induced the IL-6 expression and p42/44 ERK activation., Conclusion: These results suggest that SPC-induced IL-6 production is mediated by PKC-dependent p42/44 ERK activation in human dermal fibroblasts cultured in vitro.

Cyclophilin A (CypA) has been reported to be overexpressed in cancer cells, especially in solid tumors. To determine the role of CypA in tumorigenesis, we investigated the induction of CypA as well as the role it plays in cancer cells. Here, we have shown that induction of CypA is associated with hypoxia in a variety of cells, including DU145 human prostate cancer cell line. Our analysis of the CypA promoter clearly showed that CypA up-regulation is mediated by hypoxia-inducible factor-1alpha transcription factor. Interestingly, overexpression of CypA prevented hypoxia- and cisplatin-induced apoptosis, and this was associated with the suppression of reactive oxygen species generation and depolarization of mitochondrial membrane potential, whereas small interfering RNA-based CypA knockdown aggravated these factors. These results suggest that CypA is important in tumorigenesis, especially in tumor apoptosis.

The present study aimed to investigate the effect of fluvastatin on the pharmacokinetics of diltiazem in rats. Pharmacokinetic parameters of diltiazem were determined in rats following an oral administration of diltiazem (15 mg/kg) in the presence and absence of fluvastatin (0.6 and 2.0 mg/kg). Compared with the control given diltiazem alone, the C(max) and AUC of diltiazem increased by 30-70% in rats with the concurrent use of fluvastatin, while there was no significant change in T(max) and the plasma half-life (T(1/2)) of diltiazem. Consequently, absolute and relative bioavailability values of diltiazem in the presence of fluvastatin were significantly higher (p<0.05) than those from the control group, implying that fluvastatin could reduce the presystemic extraction of diltiazem. In conclusion, the concurrent use of fluvastatin significantly enhanced the oral exposure of diltiazem in rats.

Objective: To culture interleukin-1beta (IL-1beta)-activated Schwann cells (SCs) with human hair keratins (HHKs) for artificial nerve bridge construction., Methods: SCs purified by primary culture with or without IL-1beta activation were cultured with HHKs decorated by extracellular matrix (ECM), and the artificial nerve bridge was implanted into the defect of rat sciatic nerve. The morphology of the SCs cultured with HHKs was monitored by inverted microscope, scanning electron microscope and evaluated by immunocytochemical staining, and the expression of nerve growth factor (NGF) in the sciatic nerve was observed by in situ hybridization., Results: Activated SCs showed better ability to adhere to the HHKs and grew well. The HHKs component in the artificial nerve bridge underwent degradation in the sciatic nerve defect after 3 to 4 weeks, and IL-1beta activation resulted in enhanced NGF expression in the SCs., Conclusion: The constructed artificial nerve bridge by three-dimensional culture of IL-1beta-activiated SCs with HHKs decorated by ECM promotes the repair of sciatic nerve defects and accelerates sciatic nerve regeneration.

Rhesus bone marrow mesenchymal stem cells (MSCs) were transfected with the BMP12 gene by electroporation, and the phenotype of the transfected cells was identified by morphological observation and molecular biological assay. After transfection, cells became slender, and their processes became thinner and were interwoven into a network. There were more organelles in the transfected cells than in the parental MSCs. The transfected cells exhibited mRNA expressions of BMP12, collagen type I and scleraxis, but not collagen type III mRNA expression. Immunocytochemical analysis also showed the presence of collagen type I, but not collagen type III in the transfected cells. The transfected cells were positive for CD44 and negative for HLA-DR. Therefore, MSCs can be introduced to differentiate into tenocytes by BMP12 gene transfection, and bone marrow MSCs can serve as an alternative seed cell for application in tendon tissue engineering.

Objective: To culture Schwann cells (SCs) and human hair keratins (HHKs) for artificial nerve bridge construction., Methods: SCs were purified by primary culture and labeled with BrdU, which were then cultured with HHKs decorated by ECM. The artificial nerve bridge was implanted into the defect of sciatic nerve, beneath the skin, and in the skeletal muscles of SD rat, respectively. The morphology of the SCs cultured with HHKs was monitored by inverted microscope and evaluated by immunocytochemical staining. Growth of BrdU-labeled SCs in vivo was observed by immunocytochemical staining on paraffin sections., Results: In vitro cultured SCs were capable of adhering to HHKs and grew well four weeks after implantation. The HHK component in the artificial nerve bridge underwent degradation in the defect of the sciatic nerve, beneath the skin, and in the skeletal muscles of SD rat, and SC survival and proliferation were verified., Conclusion: SCs can survive in three-dimensional culture with HHKs for construction of artificial nerve bridge to repair nerve defects.

Sphingosylphosphorylcholine (SPC) has been shown to accelerate wound healing. As angiogenesis is fundamental to proper wound healing, we examined the effect of SPC on angiogenesis using a well-established rat aortic ring assay. SPC significantly stimulated the sprouting of endothelial cells from rat aortic ring. Recognizing its potential effect on angiogenesis, we further investigated the action of SPC using human umbilical vein endothelial cells (HUVECs) cultured in vitro. SPC significantly accelerated the closure of in vitro wound. In addition, SPC markedly enhanced the chemotactic migration and capillary-like tube formation. Subsequently, we examined whether SPC affected the production of urokinase-type plasminogen activator (uPA), an important regulator of angiogenesis, and found that SPC stimulated the expression of uPA at both the transcriptional and translational levels. Consistent with these results, SPC increased the activity of cell-surface-associated plasminogen activator. Pretreatment with antiuPA antibody significantly diminished both the chemotactic migration and capillary-like tube formation, indicating the potential importance of uPA in SPC-induced angiogenesis. Together, these results suggest that SPC may affect angiogenesis in the wound-healing process via regulation of uPA production.

The NF-kappaB/iNOS pathway stimulates muscle differentiation downstream of the PI 3-kinase/p38 MAPK pathway and diverse antioxidants block muscle differentiation. Therefore, we here investigated whether Nox 2 links those two myogenic pathways in H9c2 and C2C12 myoblasts. Compared with the proliferation stage, ROS generation was enhanced from the early stage of differentiation and gradually increased as differentiation progressed. Antioxidants suppressed the activated NF-kappaB/iNOS pathway during muscle differentiation. Nox 2 activity was also increased during muscle differentiation. Treatment with DPI and apocynin, two inhibitors of NADPH oxidase, and suppression of Nox 2 expression using siRNA, but not Nox 1, inhibited NADPH oxidase activity, muscle differentiation, and the NF-kappaB/iNOS pathway. Inhibition of PI 3-kinase and p38 MAPK suppressed the Nox 2/NF-kappaB/iNOS pathway. Nitric oxide restored muscle differentiation blocked by treatment with antioxidants or suppression of the Nox 2/NF-kappaB/iNOS pathway. In conclusion, Nox 2 stimulates muscle differentiation downstream of the PI 3-kinase/p38 MAPK pathway by activating the NF-kappaB/iNOS pathway via ROS generation.

Cyclosporin A (CsA) is a potent immunosuppressive agent, and can cause severe adverse effects including nephrotoxicity partly due to generation of reactive oxygen species (ROS). Glucocorticoids, which are widely used in combination with CsA, have been shown to reduce oxidative injuries in various cells, but its mechanism is not understood well. To investigate the effects of prednisolone (Pd) on CsA-induced cellular damage and ROS generation in Madin-Darby canine kidney (MDCK) tubular epithelial cells, cells were treated with CsA, CsA plus Pd, or CsA plus vitamin E. Pretreatment with Pd protected cells from CsA-induced apoptosis but not from G(0)/G(1) cell cycle arrest even at its maximal protective concentration (30 microM), whereas vitamin E almost completely inhibited both CsA-induced apoptosis and cell cycle arrest at 1 microM concentration. In addition, Pd reduced the amount of CsA-induced ROS and showed partly restored catalase which was down-regulated by 10 microM CsA at both the mRNA and protein levels. Vitamin E completely abolished CsA-induced ROS generation and catalase attenuation at 10 microM concentration. Finally, the effects of 1 microM vitamin E on CsA-induced ROS and apoptosis as well as cell cycle arrest were similar to those of 30 microM Pd. We conclude that, in MDCK cells, Pd protects against CsA-induced cytotoxicity by suppressing ROS generation, although its protective effect is weaker than that of vitamin E.

We performed experimental studies on the training of postural control using a training system which consists of an unstable platform, a computer, a computer interface, a monitoring device, and training programs. Using this system with the training programs that we have developed, we performed a variety of experiments of training the abilities of postural control of subjects. To evaluate the effects of the training, the parameters on how long a subject can maintain a focus on a target, the mean absolute deviation of the trace, and the fatigue of the muscles in lower limbs were measured. The experimental results showed that the training system can improve the ability of postural control of the subject. Therefore, the training system could be applied to clinical rehabilitation training for posture control as a new balance training system.

Objective: To study the effect of FK506 in promoting apoptosis of peripheral blood-derived macrophages activated by homogenate of allogenic nerve tissues., Methods: Homogenate of the allogenic nerve tissues was prepared using the sciatic nerve and injected in one-month-old SD rats, from which the macrophages activated by the homogenate were collected from the abdominal cavity and cultured in vitro. The cells were divided into 4 groups according to different concentrations of FK506 for treatment, namely 0 (group A, control group), 0.25 ng/ml (group B), 0.5 ng/ml (group C), and 1.0 ng/ml (group D). The cells of the 4 groups were inoculated into 96-well plate respectively for detecting the viability of the macrophages by MTT assay and for morphological evaluation of the cell apoptosis by transmission electron microscopy and fluorescence microscopy., Results: The cells in groups B and C exhibited reduced viability and signs of apoptosis, and necrosis was observed in group D. Transmission electron microscopy and fluorescence microscopy identified early apoptotic changes and the presence of apoptotic body in the macrophages. The apoptotic rates of groups B and C were much higher than that in group A found by flow cytometry., Conclusion: FK506 can promote the apoptosis of macrophage activated by allogenic nerve homogenate and reduce macrophage-mediated immunological rejection of peripheral nerve allograft.

Objective: To establish a fast and simple method for screening mutant yeast strains., Materials and Methods: Homologous recombination technique was used to detect mutant yeast strains in yeast genomic library, with the green fluorescent protein gene as the reporter gene in the transposon., Results: The strains that emitted green fluorescence were isolated, indicating that the gfp gene was inserted into the yeast genome by homologous recombination., Conclusion: This study established a useful method for functional genome study by homologous recombination technique, and provide an alternative for gene therapeutic drug development.

From our over a decade-long experience in experiments and clinical applications of human hair keratin (HHK) artificial tendon, a conclusion was drawn that HHK artificial tendon components and their degradation products could stimulate the proliferation and differentiation of tenoblasts in the neighboring tissues into tenocytes. With the regulation of this process by certain cytokines secreted by other cells, autotendons can be finally formed. We also found that after grafting for the tissue defects such as in the bone, nerve and muscle, "in situ construction" of the tissue/organ substitutes occurred, which inspired us to propose a wholly new theoretical system--in vivo tissue engineering, defined as in vivo reconstruction of the defected tissues or organs. The grafted absorbable scaffold biomaterial itself and its degradation products can activate the mitosis, proliferation, and differentiation of adult stem cells in the surrounding tissues, which organically interact with the material to form an organic complex under in vivo physiological conditions. Finally the matrix material is completely replaced by the complex, an almost identical structure with the normal tissue in terms of anatomy and histology. One of the advantages of in vivo tissue engineering is "in vivo construction" of the tissue/organ substitutes in anatomy, histology and function way of "in vivo cultivation" of the seed cells under in vivo microenvironment and in vivo precise regulation mechanism. It solves such problems as immune rejection against seed cells, variation and functional deterioration of the seed cells, complicated preservation procedure, transportation and high cost. The most prominent advantage of this technique is that it best meets the clinical need, not only in the sense of its powerful clinical potential, but also in its significant theoretical value.

Dedifferentiation is an important event in developmental biology and morphology but frequently neglected by the biologists. In the past, attention was given predominantly to its relationship with tumorigenesis. In our series of researches, we found important and inherent relations between dedifferentiation and regeneration of damaged cells and tissues in the emergence of large numbers of Schwann cells that digested their own myelin sheath components and senescent organelles by autophagic mechanism during regeneration of the peripheral nerves following injuries, when some apocytosomes (apo+cyto+sis), by means of apocytosis (apo+cyto+sis), budded from the cells containing such primitive organelles as free ribosomes and polyribosomes and retaining a very small quantity of active mitochondria and Golgi apparatus, all characterizing an immature state of the cells after dedifferentiation. Many autophagic bodies were found enwrapped by membranes L02 cells (a liver cell line) after vincristine-induced damage, with numerous apocytosomes dissociated following budding around the cells. The cells survived the treatment presented immature appearance, a phenomenon we termed as dedifferentiation, after which the metabolic burden of the cells was relieved, the senescent and damaged organelles were cleared, and the cells resumed their viability and proliferated vigorously to repair and reconstruct the damaged tissues. As most researchers have currently give their full attention to the important role of stem cells in the regeneration of damaged cells and tissues, we, after long-term observations and experiments, propose that mature cells also take part in the regeneration and reconstruction process after dedifferentiation.

Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid metabolite that can enhance wound healing. In a search for effectors downstream of SPC in the wound-healing process, we found that the expression of the gene for plasminogen activator inhibitor-1 (PAI-1) was significantly affected. ELISA and western blot analyses showed that SPC markedly induced PAI-1 production in human dermal fibroblasts cultured in vitro. Inhibition by pre-treatment with pertussis toxin (PTx), but not by tyrphostin A47 (a receptor tyrosine kinase inhibitor), indicated that PTx-sensitive G proteins were involved in SPC-induced PAI-1 expression. SPC elicited a rapid and transient increase in intracellular calcium levels ([Ca2+]i), measured using laser scanning confocal microscopy, which was partly mediated through PTx-sensitive G proteins. Pre-treatment with thapsigargin, but not with EGTA, abolished SPC-induced PAI-1 expression, indicating the importance of Ca2+ release from internal stores. Phorbol-12-myristate-13-acetate (PMA) induced the expression of PAI-1, and pre-treatment with Ro 31-8220 (a PKC inhibitor) markedly suppressed SPC-induced PAI-1 expression. SPC-induced PAI-1 expression was also significantly suppressed by PD98059 (a specific MAPK kinase 1/2 inhibitor). Consistent with this result, SPC stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase (ERK). Together, these results suggest that SPC induces PAI-1 production through a G protein-coupled calcium increase and downstream kinase signaling events in cultured human dermal fibroblasts.

Cyclosporin A (CsA) suppresses immune reaction by inhibiting calcineurin activity after forming complex with cyclophilins and is currently widely used as an immunosuppressive drug. Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed family member of cyclophilins. We previously showed that CsA toxicity is mediated by ROS generation as well as by inhibition of peptidyl-prolyl cis-trans isomerase (PPIase) activity of CypA in CsA-treated myoblasts [FASEB J. 16 (2002) 1633]. Since CsA-induced nephrotoxicity is the most significant adverse effect in its clinical utilization, we here investigated the role of CsA inhibition of CypA PPIase activity in its nephrotoxicity using transgenic mouse models. Transgenic mice of either wild type (CypA/wt) or R55A PPIase mutant type (CypA/R55A), a dominant negative mutant of CypA PPIase activity, showed normal growth without any apparent abnormalities. However, CsA-induced nephrotoxicity was virtually suppressed in CypA/wt mice, but exacerbated in CypA/R55A mice, compared to that of littermates. Also, life expectancy was extended in CypA/wt mice and shortened in CypA/R55A mice during CsA administration. Besides, CsA-induced nephrotoxicity was inversely related to the levels of catalase expression and activity. In conclusion, our data provide in vivo evidence that supplement of CypA PPIase activity allows animal's resistance toward CsA-induced nephrotoxicity.

Objective: To investigate the autophagic clearance of degenerated neuron axon during regeneration of rat sciatic nerves., Methods: Wallerian degeneration model was established in rats by sciatic nerve transection. Samples from the distal stump were collected at different time points after the transaction and ultrathin sections prepared for electron microscopic examination and acid phosphatase (AcPase) activity detection., Results: Neuron axon degeneration occurred after transection of rat sciatic nerve, presenting predominantly swelling of the axoplasm and separation of the axon from the myelin sheath seen 5 h to 2 d after the transection. On day 4, axoplasm condensation took place and the axons were completely separated from the myelin sheath to form dissociative axon body. Vacuoles of various sizes were identified in the axon in the early stage after operation and later when the axons were completely dissociated from the nerve sheath, larger dissociative axon bodies occurred. The axolemma surrounding the axon body was derived from the neuronal cytomembrane, and the condensed axoplasm contained numerous autophagic vacuoles at all levels along with large number of neurofilaments, microtubules and microfilaments arranged in a crisscross pattern. The autophagic vacuoles exhibited acid phosphatase (AcPase) activities. Since the day 7, the axon bodies were absorbed after degradation and macrophages could be spotted occasionally., Conclusion: The degenerated axons were cleared mainly through autophagy during regeneration of rat sciatic nerve and macrophages only assist in this process.

Objective: To clone the cDNA of the preprotein cDNA of human bone morphogenetic protein 12 (hBMP12)., Methods: Two primers were designed according to hBMP12 sequence reported in GenBank. The hBMP12 preprotein cDNA was obtained by reverse transcriptional (RT)-PCR from the mRNA extracted from human placenta, followed by cloning into pTARGETTM plasmid and sequence analysis of the plasmid pT(ARGE)T(TM)-BMP12., Results: DNA agarose gel electrophoresis showed that the product of RT-PCR was about 920 bp, as was consistent with the result of PCR detection of the recombinant plasmid. The result of sequence analysis was in agreement with the reported hBMP12 sequence in GenBank., Conclusion: The preprotein of hBMP12 cDNA has been successfully cloned with correct sequence.

Terminal differentiation in epidermal keratinocytes involves major biochemical changes including the expression of many new differentiation-specific genes. To further understand this process, we performed suppression-subtractive hybridization of keratinocytes cultured under high-calcium condition, known to induce differentiation in vitro. We randomly isolated 300 clones representing 90 different genes. By reverse Northern blot analyses, 20 different genes were found to be overexpressed, of which 13 were confirmed as differentially expressed genes during keratinocyte differentiation by Northern blot analysis. Of those, five genes, transglutaminase 1, keratin 6, interleukin-1 receptor antagonist, kallikrein 7, and heat shock protein 27, are known to be up-regulated during epidermal differentiation. Six genes, ferritin-L chain, ribosomal protein S6, tumor-associated calcium signal transducer 2, neuroendocrine secretory protein 55, phosphoserine aminotransferase, and neutrophil gelatinase-associated lipocalin, heretofore were not known to be up-regulated during keratinocyte differentiation. We also identified two novel genes. One of these maps to chromosome 1q21 of the epidermal differentiation complex, and its expression level was strongly increased in differentiating keratinocytes. These differentially expressed genes may provide significant opportunities for further understanding of the epidermal keratinocyte differentiation.

Objective: To investigate the autophagic effect of Schwann cells in the process of rat sciatic nerve regeneration., Methods: Wallerian degeneration model was established by transecting the rat sciatic nerve. Samples from the distal stump were obtained 0, 0.5, 1, 1.5, 2, 3, 4, 5, 7, 10, and 15 days respectively after the transaction, and ultrathin sections were prepared for examination with electron microscope., Results: Axons started to separate from the myelin sheath at day 0.5 after the transection, followed by rapid vacuolar degeneration. Since day 2, the myelin sheath folded and broke into fragments, and in the Schwann cells large cell membrane-bound myelin debris and many scattered small fragments could be seen to fuse with the lysosomes to form ACPase reaction-positive autophagic vacuoles. Immature cells were occasionally seen in the endoneural space, appearing in large amount a week later. After day 7, the number of autophagic vacuoles began to diminish. In the entire course of the observation, macrophages containing autophagic vacuoles, were spotted only occasionally., Conclusion: The degenerated myelin debris are cleared mainly through the mechanism of autophagy by the Schwann cells during regeneration of rat sciatic nerve. Schwann cells dedifferentiate into Schwann cell precursors, which then proliferate and differentiate to take part in the regeneration of the nerves.

Objective: To evaluate the relationship between receptor-mediated endocytosis, autophagosome formation and apoptosis by observing the morphological changes of mouse peritoneal macrophages in the course of receptor-mediated endocytotic activities, autophagy and apoptosis., Methods: Mouse peritoneal macrophages were incubated with horseradish peroxidase-labeled concanavalin A (ConA-HRP), and morphological examinations were performed at different time points after the incubation., Results: After the incubation of the macrophages with ConA-HRP, ConA-HRP was observed to enter the vesicles by way of receptor-mediated endocytosis. Three kinds of endosomes were observed, namely vesicles, tubes and double-membrane sheets. The double-membrane sheets enveloped a portion of the cytoplasm and organelles, thus giving rise to vesicles, or the autophagosomes, which later fused with lysosome, followed by the apoptosis of the macrophages., Conclusion: Receptor-mediated endocytosis of ConA-HRP is associated with autophagy and apoptosis.

Objective: To study the expression and distribution of nerve growth factor (NGF) and low-affinity neurophin receptor p75 during human hair keratin conduit-induced repair of rabbit tibial nerves., Methods: Rabbit tibial nerves were transected and connected by either routine suture or by conduits made of human hair keratin (HHK), and after different time periods, paraffin-embedded sections of the nerve tissue at the damaged sites and the adjacent tissues, with normal rabbit tibial nerve sections as control, were prepared for immunohistochemistry., Results: No positive NGF staining was observed in normal tibial nerve tissues, but 76 days after the surgery, strong NGF positivity was detected in the newly generated nerve tissue around the HHK implants until 100 days after the surgery, which was absent in the tissues around the suture. As for p75, there was no positive staining observed in normal tibial nerve tissue. Light positive p75 staining was found in the mature nerve tissues around the HHK implants, where the newly generated tissues were strongly p75-positive during the period between 76-and loo-days after surgery., Conclusions: HHK and its degenerative product, but not routine suture, can induce the production of NGF and p75 to create a favorable micro-environment for nerve regeneration. More NGF and p75 are produced in newly generated neurons than in mature ones.

Aim: To study the effects of resveratrol (Res) on secondary spinal cord edema, the activity of lactate dehydrogenase (LDH), Na+, K+-ATPase, and malondialdehyde (MDA) content in experimental spinal cord injured (SCI) rats., Methods: The weight-dropping method was used to produce the experimental SCI in adult rats. Res ( 50, 100 mg/kg) and methylprednisolone (MPSS) 100 mg/kg were injected ip immediately after the induction of SCI. The effects of Res on edema, LDH, Na+, K+-ATPase, and MDA were determined at 1 h, 24 h, and 48 h after SCI compared with MPSS. The electron microscope was employed to investigate the ultrastructural effects of Res on axons, neurons, and subcellular organelles after SCI., Results: Res obviously inhibited the secondary spinal cord edema with the most remarkable suppressing rate by 11.5 % at 48 h. Res significantly suppressed the activities of the lactate dehydrogenase with the highest suppressing rate > 40 % at 24 h. Res markedly improved the Na+, K+-ATPase activities that were promoted to the biggest extent of 60 % at 48 h. At the same time, Res (50 and 100 mg/kg) obviously reduced MDA production in the injured spinal cord tissue in comparison with the SCI model, the most remarkable effect of Res was detected at 48 h with the inhibitory rate >40 %. The ultrastructural findings suggested that SCI caused profound spinal cord damage, which could be protected or improved by injection of Res and MPSS., Conclusion: Both Res and MPSS effectively protected the spinal cord from secondary spinal cord injures. But the effects of Res 50 and 100 mg/kg were stronger in improving the energy metabolism system and inhibiting the lipid peroxidation in the local injured spinal cord after SCI than MPSS at the dose of 100 mg/kg. Res might have greatly potent therapeutic effects on SCI.

Objective: To investigate the changes of oligodendrocyte proliferation and differentiation in response to implantation of degradable human hair keratin (HHK) into the spinal cord with acute injury in adult rats., Method: Rat models of acute spinal cord injury were established by direct impact of the exposed spinal cord with a weight-dropping device, 12 of which received immediate HHK implant into the injured spinal cord. The rats of control injury group (n=12) were subjected to the injury but did not receive subsequent implant, and those in sham operation group (n=12) only had the spinal cord exposure without injury. Another 8 rats were used as the normal control group. Samples of the spinal cord were obtained 1, 4, 12, and 26 weeks respectively after the operations and serial sections were prepared for examination with light and electron microscope., Results: One week after the injury, few oligodendrocytes were observed among the large number of the infiltrating leukocytes in the injured spinal cord with HHK implants. Staining with hematoxylin eosin and Mallory's phosphotungstic acid-hematoxylin at the fourth week revealed oligodendrocyte proliferation around the HHK implant. The period from the 12th to 26th week was characterized by nerve regeneration and myelin sheath reconstruction, in the course of which empty cavity occurred within the sheath of the oligodendrocytes, and lamellar separation of the myelin sheath were observed. Phagocytized myelin sheath and axone fragment were detected in oligodendrocytes, with the newly generated oligodendrocytes scattering abound the rebuilt myelin sheath., Conclusion: HHK can be beneficial for the proliferation and differentiation of oligodendrocytes and myelin sheath rebuilding and repair in the course of neuronal regeneration after acute spinal cord injury.

Objective: To observe the morphological changes of collagen fibrils during the formation of autogenous tendon induced by human hair keratin (HHK) artificial tendon., Methods: Rabbit models of injured tendon were established in which implantation of HHK artificial tendon was performed to observe the formation of autogenous tendon under light microscope and electron microscope at 3, 6, 9, 12 and 16 weeks after HHK implantation., Results and Conclusion: During autogenous tendon formation induced by HHK artificial tendon, the tendon cells of the impaired end of the tendon and beneath the tendon membrane dedifferentiate and are capable of collagen secretion, followed by the formation of typesI,II and III collagen fibrils, and eventually, the majority of the tendon cells disappear with the collagenization of the tendon.

Objective: To investigate the ultrastructure of rat thymus tissues with rat coronavirus (RCV) infection for clarifying the mechanism responsible for the morphological changes of the cells infected by RCV., Methods: Routine electron microscopy was performed for observing RCV-infected rat thymus tissues., Results: Following RCV infection, endoplasmic reticulum (ER) pools of different dimensions were observed in the cytoplasm of the thymic epithelial reticular cells, merging subsequently with each other into larger ER lakes filled with particles of mature RCV, or viral inclusion bodies. After germination on the ER membrane, the viruses entered the matrix of the ER lake to mature and were eventually excreted to the extracellular space. The RCV particles were spherical in shape with a diameter of 100-130 nm and two distinct membranes, the outer one being the envelope and the inner one the nuclear capsid to enclose the viroplasm. Between the envelop and nuclear capsid was a electron-lucent middle layer comprising one to two thin membranous structures. Large quantity of short spike-like projections starting from the nucleus capsid penetrated the middle layer and the envelop to reach the glycoprotein coat and formed a corona-like structure. Mature RCV particles were distributed around the ER pools, cytoplasm, and intercellular space, and the RCVs in the endosome/lysosome were devoid of the envelop and nuclear capsid., Conclusion: The ER lakes are involved in the maturation of the viruses, and the envelop and nuclear capsid of the virus entering the cells from extracellular space are removed and degraded in the endosome/lysosome. Replications of virus occurs in plasma of the thymic epithelial reticular cells, and no RCV can be detected in the thymocytes.

Objective: To study the mechanism of direct lung injury by seawater and explore its possible management., Methods: To exclude the interference of hypoxia and acidosis during the study of seawater-induced direct lung injury, 18 normal hybrid dogs were randomly assigned into group A (with all lung lobes perfused with seawater), group R (with the right lung lobe perfused with seawater) and group D (with the diaphragmatic lobe of lung perfused with seawater), with 6 dogs in each group. The changes in blood gas dynamics, blood gas acid-base status and electrolytes, along with the histological changes in the lung tissues were comparatively analyzed between the 3 groups. Bronchial microscope was employed to observe the continuous changes in the bronchioles before and after seawater perfusion in group D, and the concentration of the bronchoalveolar fluid and blood LDH-L and ALP levels were tested., Results: The values of PaO(2), PaCO(2), pH, actual bicarbonate (AB), base excess (BE), tidal volume, and respiration rate in groups A and R were significantly different from those in group D (P < 0.01), and in groups A and R, the above measurements at every stage after seawater perfusion were significantly different from those before perfusion (P < 0.01). In group D, however, blood gas dynamics, blood gas acid-base status and electrolytes changed little after seawater perfusion (P > 0.05). In all the groups, obvious lung tissue injuries were observed under optical microscope after seawater perfusion. Observation with electron microscope revealed injuries to type II alveolar epithelial cells, broadened respiratory mucosa, and platelet adherence. Bronchial microscope in group D presented the bronchus filled with bronchoalveolar fluid, and blood LDH-L and ALP levels kept rising significantly (P < 0.01). Within 4 h after seawater perfusion, no pathological changes were seen in the lung tissues without direct contact with seawater., Conclusions: Seawater inspiration and retention in the lungs may lead to severe direct lung injury, and is the primary factor responsible for acute lung injury after drowning in the sea.