[Objective] To investigate the effects of pyrroloquinoline quinine(PQQ)on proliferation and expression of NF-?B of Schwann cells.[Methods]Schwann cells were cultured from sciatic nerves of SD rats in vitro.The Schwann cells were identified and purified by immunofluorescence of S-100 and Ara-C.Experimental group with was 100 nmol/L of PQQ and control group were set up.The expression of NF-?B in Schwann cells was determined by RT-PCR and Western blotting.[Results]The expression of NF-?B were up-ragulated by PQQ in cultured Schwann cells(P

[Objective] To investigate the effects of pyrroloquinoline quinine (PQQ) on proliferation and expression of NF - kB of Schwann cells. [Methods] Schwann cells were cultured from sciatic nerves of SD rats in vitro. The Schwann cells were identified and purified by immunofluorescence of S-100 and Ara-C. Experimental group with was 100 nmol/L of PQQ and control group were set up. The expression of NF-kB in Schwann cells was determined by RT-PCR and Western blotting. [ Results] The expression of NF - kB were up - ragulated by PQQ in cultured Schwann cells (P<0.05).[Conclusion ] PQQ could promote the proliferation of Schwann cells and up-regulation of NF-rd3 play an important role in this process.

37 ℃, cell growth became poor, and clone formation decreased. At 39 ℃, cells could not adhere. Under incubator CO2 concentration between 3% and 8%, there was no significant difference in cell clone formation. CONCLUSION: The optimized culture condition of Schwann cells were 7.2 pH, 10% fetal bovine serum, 37 ℃ of temperature and 5% of CO2(v/v) of incubator.

ObjectiveTo investigate the effects of carboxymethyl-chitosan(CMCS) on chondrocyte apoptosis induced by sodium nitroprusside (SNP),and the effects of CD44 in the process.MethodsA small interference RNA (siRNA) targeting to CD44 mRNA (siRNA-1,siRNA-2,siRNA-3) was constructed.The siRNA was transfected into chondrocytes in vitro with LipofectamineTM 2000.The efficacy of transfection was detected by transfecting fluorescence siRNA into cells.The mRNA expression of CD44 in vitro was detected by RT-PCR.The protein level of CD44 was detected by Western blotting.The apoptosis rate of the transfected and non-transfected cells induced by SNP was detected by flow cytometry.Statistical analysis was conducted with one-way ANOVA and SNK-q test.ResultsThe efficacy of transfection was about 60％.As compared with the control group,the mRNA expression was specifically inhibited after transfecting CD44 siRNA-1 for 24,48 and 72 h(0.198±0.007 vs 0.429±0.053 at 24 h,0.211±0.016 vs 0.501±0.037 at 48 h,0.153±0.005 vs 0.341±0.009 at 72h,q=5.93,7.01,11.23,P＜0.01 ),and the protein level of cells was inhibited after transfecting CD44 siRNA-1 for 24 h compared with the control group (0.231±0.064 vs 0.675±0.113,q=13.09,P＜0.01 ).The FCM results showed that 3 mmol/L SNP could induce chondrocytes apoptosis(70±6)％,and 50,100,200 μg/ml C MCS could affect the inhibitory effect of SNP-induced apoptosis of chondrocyte [ (51 ±7)％,(30±4)％,(15±4)％,q=5.08,6.97,9.73,P＜0.01 ],but it had milder inhibitory effect on CD44-siRNA-1 transfected chondrocytes when compared with those of the non-transfected chondrocytes [ (34±6)％ vs(15±4)％,q=6.95,P＜0.01 ].ConclusionThe data of this study has suggest that siRNA-1 against CD44 gene can significantly inhibit the expression of CD44 in chondrocyte of rats in vitro after transfection.The CD44 may play an important role in chondrocyte apoptosis induced by SNP and protected by CMCS.

[Objective]To investigate the effects of Wnt/?-catenin signal pathway on Schwann cells proliferation promoted by pyrroloquinoline quinine (PQQ) and its molecular mechanisms.[Method]Schwann cells were cultured and purified in vitro. The purity was identified by S-100. PQQ of 10 nmol/L and 100 nmol/L were added into culture medium for 24 hours,respectively. Then the morphological changes promoted by PQQ were observed by inverted microscope. The expression of ?-catenin was detected by RT-PCR and Western blot in Schwann cells promoted by PQQ of different concentration for 72 hours.[Result]Morphological change was observed in Schwann cells treated by PQQ of 10 nmol/L and 100 nmol/L. The most obvious morphological changes took place in the Schwann cells treated by 100 nmol/L of PQQ,the RT-PCR and Western blot results showed that PQQ of 1-1000 nmol/L could up-regulate the expression of ?-catenin,especially when Schwann cells was treated by PQQ of 100 nmol/L(P

BACKGROUND:Carboxymethylated chitosan is shown to promote some kinds of cells proliferation, but its effects on proliferation of Schwann cells need further studies.
OBJECTIVE:To investigate the effects of carboxymethylated chitosan on proliferation of Schwann cells and expression of nuclear factor-κB in cultured Schwann cells.
METHODS:Schwann cells from Sprague-Dawley rats at logarithmic growth phase were seeded in 96-wel plates, and cultured respectively with PBS, 0, 10, 50, 100, 200, 500, 1 000 mg/L carboxymethyl chitosan for 24 hours. cellproliferation was detected using the cellcounting kit-8 assay. After trypsin digestion, Schwann cells from Sprague-Dawley rats at logarithmic growth phase were used to prepare cellsuspensions, which were seeded in 6-wel cellculture plates and cultured respectively with 50, 100 and 200 mg/L carboxymethyl chitosan and PBS for 24 hours. Then, 5-bromo-2-deoxyuridine, real-time PCR and western blot assay were performed.
RESULTS AND CONCLUSION:cellcounting kit-8 and 5-bromo-2-deoxyuridine detection results showed that carboxymethyl chitosan at 50-1000 mg/L, especial y at 200-500 mg/L, could promote Schwann cellproliferation. Real-time PCR and western blot results showed 50-200 mg/L carboxymethyl chitosan could promote nuclear factorκB mRNA and protein expression in Schwann cells in a dose-dependent manner, suggesting carboxymethyl chitosan can promote Schwann cellproliferation and expression of nuclear factor-κB in Schwann cells cultured in vitro.

OBJECTIVETo investigate the effects of Pyrroloquinoline quinine (PQQ) on hydrogen peroxide-induced apoptosis of Schwann cells (SCs) and its mechanism.

METHODSSCs were isolated and cultured in vitro, and identified by S-100 immunofluorescence staining. The cultured SCs were divided into control group, hydrogen peroxide-treated group, hydrogen peroxide and PQQ treated groups. The intracellular superoxide dismutase (SOD) and malondialdehyde (MDA) content was detected; the apoptotic rate of SCs induced by hydrogen peroxide was determined by flow cytometry assay. The Hoechst33342 staining was used to detect the nuclear fragmentation and apoptotic nuclear condensation of SCs; the Rhodamine123 staining was used to detect the changes of mitochondrial membrane potential in SCs, the Western blot analysis was used to detect the expression of Bcl-2 in hydrogen peroxide induced SCs.

RESULTSThe SOD activity was significantly decreased and MDA level was increased in H2O2 induced SCs (P < 0.05), after addition of PQQ, the SOD content increased and MDA content decreased (P < 0.05). Flow cytometry results showed that the early apoptotic rate was 58.8% in H2O2 induced SCs, which has significant difference compared with the control group (P < 0.05), after addition of 10, 50, 100 nmol/L PQQ, the apoptotic rates were reduced to 33.7%, 18.7%, 3.9% respectively, showing significantly different with injured group (P < 0.05). Hoechst 33342 staining showed that H2O2 induced SCs had typical morphological characteristics, such as uptake of nuclear chromatin, nuclear shrinkage, nuclear fragmentation phenomenon. The proportion of apoptotic cells after PQQ treatment reduced. Rhodamine staining results showed that the H2O2 induced mitochondrial membrane potential reduction in SCs, which was reversed by addition of PQQ. Western blot analysis showed that the expression of Bcl-2 was decreased in H2O2 induced SCs, while it increased significantly after addition of PQQ (P < 0.05).

CONCLUSIONPQQ has a protective effect on oxidative stress-induced apoptosis of SCs.

Apoptosis ; drug effects ; Benzimidazoles ; Cell Nucleus ; drug effects ; DNA Fragmentation ; Fluorescent Dyes ; Humans ; Hydrogen Peroxide ; pharmacology ; Malondialdehyde ; metabolism ; Oxidants ; pharmacology ; Oxidative Stress ; Pyrroles ; pharmacology ; Quinine ; pharmacology ; Quinolines ; pharmacology ; Schwann Cells ; cytology ; drug effects ; Superoxide Dismutase ; metabolism

BACKGROUND: Traumatic osteoarthritis (OA) resulted from the injury of joints and postoperation of joints is commonly observed. Intra-articular injection of sodium hyaluronate (Na-HA) has been considered as effective method for OA. OBJECTIVE: To observe the influence of intra-articular injection of NaHA on mRNA expression of inducible nitric oxide synthase (iNOS) in cartilage of traumatic OA induced by transection of anterior cruciate ligament. DESIGN: Randomized controlled animal experiment. SETTING: Department of Orthopaedics, Renmin Hospital, Wuhan Uni ersity. MATERIALS: The experiment was performed in Laboratory of Depart ment of Orthopaedics, Renmin Hospital, Wuhan University from April to December 2003, in which, 16 clean healthy flat-eared white rabbits, aged 5-6 months were employed. The rabbits were randomly divided into Na- HA injection group and saline control group with 8 rabbits in each group. Na-HA (2000, No 366095) was provided by Shanghai Jiahua Fine Biologi- cal Products Co. METHODS: ①OA model was established in rabbits of the two groups. Each rabbit was anesthetized intravenously with 1.0 mg/kg ketamine hy- drochloride and underwent unilateral anterior cruciate ligament transection. ②5 weeks after transection, Na-HA injection group rabbits received 0.3 mL of intra-articular 10 g/L Na-HA injection, once a week for 5 weeks. Ani mals in saline controlled group were treated with saline of the same vol ume. ③The rabbits were killed at week 10 after operation, general morphology and histopathological changes of articular cartilage degeneration of medial femoral condyle were evaluated (0 points as smooth articular surface with normal color and luster; 1 point as minimal fibrillation or a slight crevice and dark grey color of the surface; 2 points as erosion extending into superficial or middle layers of cartilage; 3 points as ulceration and erosion extending into the deep layers, and 4 points as denudation of cartilage, erosion extending to the sub-chondral bone). The mRNA expression of iNOS in cartilages was examined with reverse transcription-polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURES: ①Observation of articular cartilage degeneration of medial femoral condyle, ②observation of articular cartilage degeneration of medial femoral condyle at light microscopic level, ③expression of iNOS in cartilages of each group. RESULTS: A total of 16 clean healthy rabbits entered the result analysis. ① Result of articular cartilage degeneration of medial femoral condyle: Pathological change of articular surface of femoral condyle was observed under anatomic microscope. Cartilage degradation in experimental group was significantly less severe than that in saline control group. ②Histological changes of articular cartilage degeneration of medial femoral condyle at light microscopic level: The Na-HA injection group showed cartilage changes: Membrane of cartilage presented denaturalization and abscission. Chondrocytes of superficial zone presented denaturalization, necrosis, turbulence and erosion. Animals treated with saline showed denaturalization, necrosis and disorder of chondrocytes, ulceration penetrating into the middle or deep zone of the cartilage. New hyperplasia of capillary vessels and fibroblasts were more obvious. Proliferation of fibrous tissue appeared at the bottom of ulcer. ③Expression of iNOS in cartilages of two group:The gene expression of iNOS in cartilage of Na-HA injection group was(1.09±0.18) and the expression of saline control group was (1.26±0.23). Nosignificant difference of iNOS expression was found between the two groups(P ＞ 0.05).CONCLUSION: Intra-articular injection of Na-HA has protective and repairing effect on cartilage with early OA and can significantly reduce the severity of cartilage degradation during early stage of traumatic OA. Intra-articular injection of Na-HA does not down-regulated iNOS expression in cartilage.

Objective To observe the effect of intra-articular injection of CM-chitosan on nuclear factor κB (NF-κB) activation and nitric synthase expression in rat osteoarthritis cartilage,and to explore the mechanism of inhibition of joint destruction.Methods Thirty-six SD rats were randomly divided into three groups:A,B,C,12 in each group.Group A was the sham group,group B,C rats had the medial collateral ligaments cut off and part of medial meniscus were removed to establish osteoarthristis model.Group C rats were injected with 3％ carboxymethyl chitosan intra-articularly 0.15 mg/kg 5 weeks later,and then repeated injection every 1 week.Animals were sacrificed 11 weeks after surgery.The gross changes of cartilage and the expression of NF-κB (P65) were compared by immunohistochemistry,the protein expression of I-κB and P65 in nucleus were detected by Western-bloting.Inducible nitric oxide synthase (iNOS) mRNA and protein expres-sion were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and western blot.The general score of cartilage was analyzed by H test,the rest data was analyzed by one-way ANOVA.Results The cartilage degeneration scores pf group C (intra-articular injection of CM-chitosan group) were significantly less than those of group B.The protein expression of NF-κB of the articular cartilage in group C (106±7)was significantly lower than group B (147±8),the I-κB degradation was inhibited significantly in group C,and the expression of iNOS mRNA and iNOS protein were reduced in OA art~icular cartilage of arthritis rat chondrocytes,therefore,it had protective effect on articular cartilage.Conclusion CMC may inhibit NF-κB signaling pathway by inhibiting the degradation of I-κB in cartilage,which reduces iNOS mRNA and protein expression in rat osteoarthritis cartilage,thereby protects rat osteoarthritis cartilage cells.