Osteoarthritis (OA) is a degenerative disease characterized by matrix degradation and cell death leading to a gradual loss of articular cartilage integrity. As a bacterial synthesis of quinine, pyrroloquinoline quinone (PQQ) is a strong redox cofactor with a variety of biological benefits, including antioxidant, anti-inflammation-induced mitochondrial metabolism regulation. This study was designed to investigate the effect of PQQ on TNF-α-induced mitochondrial damage in chondrocytes. Chondrocytes isolated from C57BL/6 mice were exposed to TNF-α 50 ng/mL, TNF-α 50 ng/mL + PQQ 10 µmol/L for 24 h. Then, morphological study, functional study and mechanism study were taken. The results revealed TNF-α-induced chondrocyte mitochondrion damage could be reduced by application of PQQ, evidenced by elevated number of mitochondria, well-kept mtDNA integrity, preserved ATP level, reestablished mitochondrial membrane potential, and prevented mitochondrial function. The present work strongly suggests that the mitochondrion is an important target for OA chondrocyte damage induced by TNF-α and the PQQ protection from this damage ameliorates mitochondrial dysfunction induced by TNF-α. PQQ might be a potential chemical for OA intervention.

Chondrocyte apoptosis activated by the mitochondrial dependent pathway serves a crucial role in cartilage degeneration of osteoarthritis (OA). In the present study, the protective effects of CMCS against sodium nitroprusside (SNP)-induced chondrocyte apoptosis were evaluated and the underlying molecular mechanisms were elucidated. Chondrocytes were isolated from articular cartilage of SD rats and identified by type II collagen immunohistochemistry. The chondrocytes stimulated with or without SNP to induce apoptosis, were treated by CMCS for various concentrations. The cell viability were determined by MTT and LDH assays. Cell apoptotic ratio was determined by Annexin V-FITC/PI staining. Mitochondrial membrane potential (ΔΨm) was detected by using Rhodamine123 (Rho123) staining. To understand the mechanism, the mRNA expression levels of Bcl-2, Bax, cytochrome c (Cyt c) and cleaved caspase-3 were detected by real-time PCR and western blot analysis, respectively. It was shown using the MTT and LDH assays that CMCS protected the viability of chondrocyte against SNP damage. Annexin V-FITC/PI and Rho123 staining showed that CMCS not only inhibited the cell apoptosis but also restored the reduction of the ΔΨm in chondrocytes. In SNP-induced chondrocytes, CMCS down-regulated the expression of Bax, Cyt c and cleaved caspase-3 but upregulated the expression of Bcl-2, as shown by real-time PCR and western blot. Taken together, these results indicated that CMCS has the protective effect on chondrocytes against SNP-induced apoptosis, at least partly, via inhibiting the mitochondrial dependent apoptotic pathway. Thus, CMCS may be potentially used as a biological agent for prevention and treatment of OA.

This present study was conducted to investigate the effects of Carboxymethylated chitosan (CMCS) on Schwann cells (SCs) proliferation, biosynthesis of neurotrophic factors and its possible involvements of protein kinases A (PKA) and protein kinase C (PKC) signaling pathways. SCs were treated with CMCS alone or in the presence of PKA inhibitor (H89) or PKC inhibitor (Staurosporine). Cell proliferation was assessed by MTT and immunocytochemistry of BrdU staining. The contents and activities of cAMP, PKA and PKC were analyzed by ELISA assay. Western blot analysis and immunofluorescence staining were used to detect the expression of CREB and p-CREB. Real-time PCR was used to determine the expression of NGF, CNTF, BDNF and GDNF mRNA. The results showed CMCS could promote SCs proliferation, promote PKA and PKC activation, increases cAMP and expression of NGF, CNTF, BDNF and GDNF mRNA. These stimulating effects were inhibited by H89 and Staurosporine. Taken together, Data from this present study indicated CMCS may stimulate proliferation and biosynthesis of cultured SCs by activation of PKA and PKC signaling pathways.

Carboxymethylated chitosan (CMCS) has many beneficial effects, including anti-oxidant and anti-apoptotic actions. However, the mechanisms by which CMCS protect against oxidative stress induced damage to Schwann cells (SCs) remains unclear. The present study aimed to investigate the mechanism by which CMCS protects SCs against hydrogen peroxide (H2O2) induced damage. H2O2 was used to establish a model of oxidative stress injury in SCs to mimic the development of nerve injury in vitro. Different concentrations (50, 100 and 200 µg/ml) of CMCS were added to test whether CMCS was capable of protecting SCs from H2O2 induced damage. MTT, LDH release and Annexin V/FITC assays were then performed. Levels of reactive oxygen species were detected using a reactive oxygen species assay kit, the mitochondrial membrane potential (ΔΨm) of SCs was analyzed by rhodamine123 fluorescence staining, the synthesis of Bcl-2, Bax, cytochrome c and caspase-3 were analyzed by real-time PCR and Western blot analysis. The results showed that CMCS protected SCs from apoptosis, decreased LDH release and enhanced cell viability, also decreased reactive oxygen species levels and increased ΔΨm. Additional experiments demonstrated that CMCS could decrease protein expression of Bax, cytochrome c and caspase-3, while promote Bcl-2 protein expression induced by H2O2. Taken together, the finding of this study indicated that CMCS prevented H2O2-induced damage to SCs through the mitochondrial dependent pathway.

Objective:To investigate the mechanisms of Pyrroloquinoline quinone (PQQ) against oxidative stress induced apoptosis in Schwann cells (SCs).Methods:SCs were cultured in vitro, identified by S-100 immunofluorence staining. SCs were divided into control group,H2O2 induced group,H2O2 + PQQ treated group.CCK-8 assay was used to detect cell proliferation. Apoptosis was detected by flow cytometry with Annecin V-FITC/PI staining, mitochondrial transmembrane potential was detected by flow cytometry with JC-1 labeled staining, cytochrome C (CytC), Bax and Caspase-9 protein levels was detected by Western blot analysis.Results:In this study, the S-100 positive cells were more than 95％,cell proliferation was decreased in H2O2 induced SCs,apoptotic rate was increased, mitochondrial transmnembrane potential was decreased,CytC, Bax and Caspase-9 protein levels were increased. After PQQ added, cell proliferation was increased, apoptotic rate decreased, mitochondrial transmembrane potential increased, CytC, Bax and Caspase-9 protein levels decreased.Conclusions:PQQ protects SCs from oxidative induced apoptosis by inhibiting mitochondrial signaling pathway.

Osteosarcoma, which is the most common type of primary bone tumor in adolescents, is characterized by complex genetic alterations and frequent resistance to conventional treatments. MicroRNAs (miRs) have emerged as fundamental regulators in gene expression through their ability to silence gene expression at post-transcriptional and translational levels. The present study investigated the role of miR‑410 in the progression of osteosarcoma. The results demonstrated that the expression of miR‑410 was markedly downregulated in human osteosarcoma tissues, and U2OS and MG‑63 osteosarcoma cell lines. Clinicopathological significance suggested that miR‑410 may be a potential biomarker for chemotherapy‑resistant osteosarcoma. Furthermore, overexpression of miR‑410 exhibited a limited effect on cell viability in U2OS and MG‑63 cells. Target prediction algorithms (TargetScan and miRanda) indicated that autophagy related 16‑like 1 (ATG16L1) was a potential target gene of miR‑410. A luciferase reporter assay demonstrated that miR‑410 directly decreased ATG16L1 expression by targeting its 3'‑untranslated region. In addition, the results revealed that miR‑410 was able to markedly inhibit autophagy. Accordingly, autophagy was activated as a protective mechanism when osteosarcoma cells were exposed to three common anticancer drugs, including rapamycin, doxorubicin and cisplatin. Furthermore, the autophagy inhibitor 3‑methyladenine and miR‑410 expression were able to improve the therapeutic response of the cells to chemotherapy drugs (rapamycin, doxorubicin and cisplatin), thus indicating that miR‑410 enhanced chemosensitivity through autophagy inhibition in osteosarcoma cells. In conclusion, studies regarding the function of miR‑410 on autophagy provided insight into the biological function of miR‑410 in osteosarcoma and may offer a promising approach for the treatment of osteosarcoma.

The protective and promotion effects of Carboxymethylated chitosan (CMCS) on peripheral nerve and cultured Schwann cells (SCs) have been demonstrated, but few studies discussed the protective roles of CMCS on SCs apoptosis. We explored the anti-apoptotic activities of CMCS in SCs to enhance cells survival in this present study. Rat SCs were isolated and cultured in vitro, hydrogen peroxide (H2O2) was used to establish the apoptosis models of SCs. Cells proliferative activity was assessed by CCK-8 assay. The apoptosis of SCs was detected by flow cytometry (FCM) analysis. Superoxide dismutase (SOD) and malondialdehyde (MDA) activities were detected by the corresponding assay kit. The nuclear appearance of apoptotic SCs was observed by nuclear staining with Hoechst 33342. The real-time PCR was performed to detect the levels of Bcl-2, Bax, Caspase-3 and -9 mRNA. Detection of caspase-3 and -9 was fulfilled by using Western blot analysis. FCM assay and Hoechst33342 staining results indicated that CMCS could protect SCs from apoptosis with dose and time-dependent manner. SOD and MDA analysis results indicated that CMCS could promote SOD activity and reduce the MDA levels in H2O2 induced SCs. The decreased caspase-3, -9 and Bax activities and increased Bcl-2 activity were observed in CMCS treated SCs. The present study indicates CMCS has the neuroprotective effect on peripheral nerves and inhibit SCs apoptosis.

The proliferation of Schwann cells around injured peripheral nerves supports the process of Wallerian degeneration and is critical for axonal regeneration. In this publication, carboxymethylated chitosan (CMCS) was studied to determine its capacity (i) to induce proliferation and secretion of nerve growth factor (NGF) and (ii) to activate Wingless-type(Wnt) protein/β-catenin signaling pathways in rat Schwann cells. CMCS was found to induce Schwann cell proliferation and NGF synthesis in Schwann cell in a dose and time dependent manner. CMCS was shown to activate factors in the Wnt/β-catenin signaling pathway, including Dvl-1, β-catenin, Tcf4, Lef1, C-myc, and Cyclin D1 which are active in the proliferation of Schwann cells and biosynthesis of NGF of Schwann cell. Overall, this study suggests that CMCS can promote the proliferation of cultured Schwann cells and synthesis of NGF by activating the Wnt/β-catenin signaling pathway.

The effects of tacrolimus postconditioning on protein-serine-threonine kinases (Akt) phosphorylation and apoptotic cell death in rats after spinal cord ischemia-reperfusion injury were investigated. Ninety male SD rats were randomly divided into sham operation group, ischemia-reperfusion group and tacrolimus postconditioning group. The model of spinal cord ischemia was established by means of catheterization through femoral artery and balloon dilatation. The spinal cord was reperfused 20 min after ischemia via removing saline out of balloon. The corresponding spinal cord segments were excised and determined for Akt activity in spinal cord tissue by using Western blotting at 5, 15, and 60 min after reperfusion respectively. Spinal cord tissue sections were stained immunohistochemically for detection of the phosphorylated Akt expression at 15 min after reperfusion. Flow cytometry was applied to assess apoptosis of neural cells, and dry-wet weights method was employed to measure water content in spinal cord tissue at 24 h after reperfusion. The results showed that the activities of Akt in tarcolimus postconditioning group were significantly higher than those in ischemia-reperfusion group at 5, 15, and 60 min after reperfusion (P<0.05, P<0.01). The Akt activities reached the peak at 15 min after reperfusion in ischemia-reperfusion group and tacrolimus postconditioning group. The percentage of apoptotic cells and water content in spinal cord tissue were significantly reduced (P<0.01) in tacrolimus postconditioning group as compared with those in ischemia-reperfusion group at 24 h after reperfusion. It is concluded that tacrolimus post-conditioning can increase Akt activity in spinal cord tissue of rats, inhibit apoptosis of neural cells as well as tissue edema, and thereby alleviate spinal cord ischemia-reperfusion injury.

The purpose was to study the effect of human cerebrospinal fluid (CSF) on differentiation of rat neural stem cells (NSCs), and thus explore the feasibility of transplanting stem cells via lumbar puncture clinically. Rat NSCs derived from fetal brain were divided into two groups, and cultured in DMEM/F12 supplemented with 10 % FBS and human CSF, respectively. Cellular growth was observed with an inverted microscope, and immunostaining was used to analyze differentiation of NSCs in both groups. Cells of fetal brain showed shapes of spindle or star with minor sprouts at fifth day post-culture, and stained with nestin. NSCs in the control group differentiated into neurons, with positive staining to NSE, when cultured further in DMEM/F12 supplemented with 10 % FBS. While NSCs in the experiment group, cultured in CSF, differentiated into astroglia on eighth day, with positive immunostaining to GFAP. The new neurons dissolved rapidly when they were cultured in CSF. Human CSF cannot promote NSCs to differentiation toward neuron, nor support newborn neurons survival. It seems an inappropriate approach to transplant stem cells through CSF.

Objective: To investigate the degeneration time of chondrocytes cultured in vitro derived from knee joint of rats in order to offer appropriate cells for the study on osteoarthritis.Methods: The chondrocytes derived from knee joint of rats were cultured and subcultured in vitro.Then the cultured cells were identified with toluidine blue staining and type Ⅱ collagen immunohistochemistry assay.The phenotype of chondrocytes were determined by assaying Type Ⅱ collagen with Western blot.Results: The synthesis of Type Ⅱ collagen as well as the cell proliferation rate descended notably in chondrocytes of genneration Ⅴ as compared with those of the first four generations(P0.05).Conclusion: The degeneration of chondrocytes cultured in vitro derived from knee joint of rats were occurred after generation Ⅳ.The phenotype of chondrocytes from generationⅠto Ⅳcould sustain,and these chondrocytes could be served as suitable target cells to study ostearthritis.

OBJECTIVE:To investigate the effects of pyrroloquinoline quinine (PQQ) on proliferation and expression of c-fos, c-jun, CREB and PCNA in cultured Schwann cells. METHODS:Schwann cells were cultured and purified in vitro. The purity of Schwann cells was identified by immunofluorescence of S-100. After synchronization of cell cycle by serum-free medium, different concentration of PQQ (0,1, 10, 100, 1,000, 10,000 nmol/L) were added into culture medium for 72 h. Flow cytometry was used to determine cell cycle. The content of c-fos, c-jun, and CREB mRNA were detected by RT-PCR, and the expression of PCNA protein was detected by Western blot. RESULTS:After PQQ treatment, the percentage of cells in G0/G1 phase decreased and the percentage of cells in S and G2/M phase increased. After treated by PQQ at concentration of 1-10,000nmol/L, content of c-fos,c-jun,CREB mRNA was increased by 0.33,0.42 and 0. 52 fold (P < 0. 05). However, at concentration of 1 000 nmol/L, there was no difference in mRNAs content when compare to control (P >0.05). And it showed a decline at concentration of 10,000 nmol/L (P < 0.05). PCNA protein expression was up-regulated at PQQ concentration of 1-100 nmol/L. At 100 nmol/L, the expression increased by 1.17 fold (P < 0.05); However, at 1,000 nmol/L, there was no difference in PCNA expression when compared to control. And 10,000 nmol/L of PQQ inhibited the expression of PCNA (P < 0.05). CONCLUSIONS:When treated with PQQ at concentration of 10-100 nmol/L, the proliferation of Schwann cells increased and the expression of c-fos,c-jun, CREB and PCNA was up-regulated.