Sam68 Promotes NF-κB Activation and Apoptosis Signaling in Articular Chondrocytes during Osteoarthritis.

OBJECTIVES: To investigate the expression of Sam68 in articular cartilage of knee osteoarthritis (OA) and the relationship between Sam68 and NF-κB activation and apoptosis signaling in OA articular chondrocytes. METHODS: Sam68 expression in normal and osteoarthritic cartilage was assessed by immunohistochemistry and RT-PCR on both meniscal/ligamentous injury (MLI)-induced OA rat model and the clinical human OA cartilage tissues. Sam68 expression in chondrocytes under tumor necrosis factor-alpha (TNF-α) stimuli was also assessed by immunoblot. Inhibiting Sam68 in chondrocytes under TNF-α stimuli was conducted using small interfering RNA (siRNA) and its influence on the expression of apoptotic marker and catabolic genes was examined by immunoblot. The mechanism of how Sam68 stimulates NF-κB activity was determined by co-immunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of TNF-α-treated chondrocytes for p65 and Sam68. RESULTS: Sam68 expression was increased in OA cartilage tissues and chondrocytes under TNF-α stimuli. Inhibition of Sam68 by siRNA significantly decreased the expression of apoptotic markers (cleaved caspase-3 and cleaved PARP) in chondrocytes following TNF-α-stimulation. Sam68 knockdown suppressed Iκ-B degradation and p65 nuclear transportation in TNF-α-treated chondrocytes, indicating a suppressed NF-κB activation. Upon TNF-α exposure, the nuclear transportation of Sam68 and its interaction with p65 was detected in chondrocytes. Furthermore, Sam68 knockdown also alleviated the TNF-α-induced catabolic marker (MMP13, ADAMTS5, iNOS and IL-6) expression. CONCLUSIONS: The highly expressed Sam68 promotes NF-κB signaling activation, catabolic gene expression and cellular apoptosis in TNF-α-treated chondrocytes, which may provide better insights into the pathophysiology of OA and a potential target for its treatment.

KPNA2 interacts with P65 to modulate catabolic events in osteoarthritis.

OBJECTIVE: Karyopherin alpha 2 (KPNA2) is a member of the importin α family, which acts as an adaptor to deliver P65 to the nucleus by recognizing the classic nuclear localization signal (NLS) of the cargo protein, and which has been reported as being involved in the pathogenesis of many diseases. This study was undertaken to determine the expression and possible functions of KPNA2 in osteoarthritis (OA). METHODS: KPNA2 expression in cartilage tissues of OA patients and normal controls was detected by RT-PCR and immunohistochemistry. SW1353 cells were stimulated with IL-1ß to establish the chondrocyte injury model in vitro. The expression of KPNA2 and catabolic genes in IL-1ß-treated SW1353 cells were determined by Western blot. The interaction between KPNA2 and P65 was analyzed by co-immunoprecipitation, the subcellular distribution and transportation of P65 were detected by the subcellular fractionation followed by immunoblot analysis and immunofluorescence. Furthermore, we used RNA interference to analyze the role of KPNA2 in IL-1ß-induced P65 nuclear importation and MMP13, ADAMTS-5 expression in SW1353 cells. RESULTS: Cartilage expression of KPNA2 was higher in patients with OA compared with normal controls and mainly locating in chondrocytes. In IL-1ß-treated SW1353 cells, up-regulation of KPNA2 was accompanied by the elevated expression of the catabolic marker protein levels, including MMP13 and ADAMTS-5, and increased NF-κB P65 nuclear importation. Knock-down of KPNA2 resulted in decreased catabolic marker protein levels in IL-1ß-treated SW1353 cells. KPNA2 interacted with p65, and loss of KPNA2 caused decreased nuclear translocation of the active p50/p65 NF-κB complex. CONCLUSIONS: These findings suggested that KPNA2 may promote NF-κB activation via facilitating P65 nuclear transportation, and thus subsequently accelerate the catabolic events of osteoarthritis.

Biomechanical analysis of a new cannulated screw for unstable femoral neck fractures.

Background: The treatment of unstable femoral neck fractures (FNFs) remains a challenge. In this study, a new cannulated screw for unstable FNFs was designed to provide a new approach for the clinical treatment of these injuries, and its biomechanical stability was analyzed using finite element analysis and mechanical tests. Methods: An unstable FNF model was established. An internal fixation model with parallel inverted triangular cannulated screws (CSs) and a configuration with two superior cannulated screws and one inferior new cannulated screw (NCS) were used. The biomechanical properties of the two fixation methods were compared and analyzed by using finite element analysis and mechanical tests. Results: The NCS model outperformed the CSs model in terms of strain and stress distribution in computer-simulated reconstruction of the inverted triangular cannulated screw fixation model for unstable FNFs. In the biomechanical test, the NCS group showed significantly smaller average femoral deformation (1.08 ± 0.15 mm vs. 1.50 ± 0.37 mm) and fracture line displacement (1.43 ± 0.30 mm vs. 2.01 ± 0.47 mm). In the NCS group, the mean stiffness was significantly higher than that in the CSs group (729.37 ± 82.20 N/mm vs. 544.83 ± 116.07 N/mm), and the mean compression distance was significantly lower than that in the CSs group (2.87 ± 0.30 mm vs. 4.04 ± 1.09 mm). Conclusion: The NCS combined with two ordinary cannulated screws in an inverted triangle structure to fix unstable FNFs can provide better biomechanical stability than CSs and exhibit a length- and angle-stable construct to prevent significant femoral neck shortening.

Meta-analysis of the efficacy of pancreatoduodenectomy with extended lymphadenectomy in the treatment of pancreatic cancer.

BACKGROUND: The purpose of this meta-analysis is to compare the efficacy of pancreatoduodenectomy (PD) with extended lymphadenectomy (PD/ELND) versus standard PD in the treatment of pancreatic cancer, with the hope of providing evidence for clinical practice. METHODS: The retrieval of relevant literature published before September 2012 was carried out on PubMed, Medline, Embase, and Cochrane Central Register of Controlled Trials (CENTRAL) by computer. Information was extracted according to Cochrane systematic review methods, and analyzed using software Stata 11.0. RESULTS: Five prospective randomized controlled trials (RCTs) were included in this meta-analysis of 555 cases (278 in the PD/ELND group and 277 in the standard PD group). The PD/ELND group showed a significantly lower 3-year survival rate (relative risk (RR) = 1.46, 95% confidence interval (CI) 1.03 to approximately 2.06, P = 0.034), prolonged operative time (weighted mean difference WMD = -1.03, 95% CI -1.96 to approximately -0.10, P = 0.029) and higher incidence of postoperative complications (RR = 0.56, 95% CI 0.42 to approximately 0.77, P = 0.000) by comparing with standard PD group. Besides, no significant difference was observed in the 1-year survival rate (RR = 0.87, 95% CI 0.60 to approximately 1.25, P = 0.69), 5-year survival rate (RR = 1.04, 95% CI 0.68 to approximately 1.58, P = 0.854), postoperative mortality (RR = 1.14, 95% CI 0.43 to approximately 3.00, P = 0.789), length of stay (WMD = -0.32, 95% CI -2.57 to approximately 1.94 , P = 0.784) and the amount of blood transfusions (WMD = -0.14, 95% CI -0.36 to approximately 0.08, P = 0.213). CONCLUSIONS: PD/ELND does not have an advantage over standard PD in the survival rate for patients with pancreatic cancer, but does increase operative time and incidences of postoperative complications.

[A comparison of efficacies between transjugular intrahepatic portosystemic shunt versus portoazygos devascularization in the treatment of portal hypertension with variceal bleeding].

OBJECTIVE: To compare the efficacies between transjugular intrahepatic portosystemic shunt (TIPS) and portoazygos devascularization (PAD) in the treatment of portal hypertension with variceal bleeding. METHODS: From December 1993 to December 2010, 309 patients with portal hypertension and variceal bleeding were admitted. According to their general conditions and Child-Pugh grades, they were assigned to undergo TIPS (group A, n = 235) or PAD (group B, n = 74). Before operation, compared with the PAD group, the TIPS group possessed worse liver functions, more severe ascites and a greater frequency of bleeding. After operation, the therapeutic efficacies and changes of portal hemodynamics, recurrent variceal bleeding, post-operative encephalopathy and long-term survival were evaluated between two groups. RESULTS: The postoperative portal pressure in the TIPS group ((42.6 ± 7.0) vs (26.3 ± 4.1) cm H2O) decreased much more than that in the PAD group ((38.7 ± 5.2) vs (33.5 ± 5.8) cm H2O, P < 0.01). The rebleeding rates during early postoperation were 0.85% (2/235) and 6.76% (5/74) in TIPS and PAD groups respectively, the occurring rates of hepatic encephalopathy 4.68% (11/235) and 4.05% (3/74) and the rates of operative mortality 1.70% (4/235) and 6.76% (5/74) respectively. Survival rates of 1, 3, 5 and 10 years were 98.30% (231/235) vs 92.24% (69/74), 92.41% (146/158) vs 88.06% (59/67), 80.77% (84/104) vs 79.25% (42/53), 51.43% (36/79) vs 51.85% (14/27) in TIPS and PAD groups respectively. CONCLUSIONS: As compared with PAD, TIPS offers the such advantages as less trauma, wider indication, faster hemostasis and satisfactory therapeutic efficacies. Especially for the emergency treatment of a patient with massive variceal bleeding and Child-Pugh C grade liver function, TIPS is a better option than PAD.

Leiomyosarcoma of the inferior vena cava: case report and treatment of recurrence with repeat surgery.

Leiomyosarcoma of the inferior vena cava (IVC) is an extremely rare malignancy with poor prognosis due to late diagnosis. Surgical resection currently remains the best treatment; however, recurrence frequently occurs and the 5-year survival rate is only 31%. The aim of this study is to report a case of IVC leiomyosarcoma and treatment of recurrence with repeat surgery. A 36-year-old woman with a high-grade leiomyosarcoma originating from the infrahepatic IVC underwent an en bloc excision of the tumor. Eleven months after the initial operation, two metastases to the omentum were observed. Since the patient showed no response to adjuvant chemotherapy (i.e., a combination of 5-fluorouracil and gemcitabine), repeat operations were used as the main treatment modality for recurrence. The median time to recurrence was 15 months (range 8-27). The middle and upper IVC segments were involved in the local recurrence, and metastatic lesions occurred in multiple sites including the stomach, omentum, mesentery, left liver, and pelvic cavity. Repeat operations to remove the recurrent and metastatic tumors led to a long-term (at least 7 years) survival, and the patient is still alive. Postoperative recoveries were uneventful. Neither complication related to the venous blood flow in the IVC nor renal impairment was noted. Our results suggest that in the setting of chemotherapy-refractory IVC leiomyosarcoma repeat surgery may be an alternative treatment for recurrence and improve survival time.

[Observation on experimental liver fibrosis and hepatic carcinogenesis of HBV gene knock-in transgenic mice induced by CCl4/ethanol].

OBJECTIVE: To study the effects of carbon tetrachloride (CCl4)/ethanol induction upon experimental liver fibrosis and hepatic carcinogenesis of HBV transgenic mice. METHODS: The wild-type mice, p21-HBx transgenic mice with integration of p21 locus by HBx gene and p21-HBsAg transgenic mice with integration of p21 locus by HBsAg gene were induced separately by CCl4/ethanol twice weekly for 20 weeks. The investigators observed the development of liver fibrosis and hepatic carcinogenesis in three groups and detected the gene expressions of HBx and HBsAg by RT-PCR. RESULTS: The expression of HBx or HBsAg mRNA existed in both control and induced transgenic mice, but in none of wild-type mice. Comparing with wild-type mice, p21 genes was not expressed in livers of transgenic mice. After induction by CCl4/ethanol, the fibrotic degrees of liver were not significantly different among wild-type mice, p21-HBx transgenic mice and p21-HBsAg transgenic mice, as well as between male and female mice. Reversely, the incidence rates of hepatic carcinogenesis of two HBV gene knock-in transgenic mouse lines (p21-HBx & p21-HBsAg) were higher than that of wild-type mice. And the incidence rate of hepatic carcinogenesis in males was also markedly higher than that in females. Induction by CCl4/ethanol markedly promoted and accelerated hepatic carcinogenesis in transgenic mice. CONCLUSIONS: Integration of HBsAg and HBx genes into the murine p21 locus can significantly promote the progression of hepatic carcinogenesis, but failed to promote the progression of liver fibrosis. The male mouse is more likely to develop experimental hepatocellular carcinoma than the female mouse. Experimental hepatocellular carcinoma induced by CCl4/ethanol in p21-HBx and p21-HBsAg transgenic mice is a feasible animal model.

[Effects of Smad4 on liver fibrosis and hepatocarcinogenesis in mice treated with CCl4/ethanol].

To study the effects of Smad4 on liver fibrosis and hepatocarcinogenesis in mice treated with CCl(4)/ethanol. The wild-type mice (Smad4 +/+) and the Smad4 knockout mice (Smad4 +/-) were injected subcutaneously with carbon tetrachloride(CCl(4))/ethanol twice a week for twenty weeks. The expression of Smad4, TGFbeta1, Smad2, Smad3, Smad6, TIMP1, MMP2 and MMP9 was detected by RT-PCR. In the cirrhotic liver, the expression of Smad4 mRNA was significantly higher than that in the normal liver. Comparing with wild-type mice (Smad4 +/+), the TGFbeta1-Smad4 signaling was markedly attenuated in the Smad4 knockout mice (Smad4 +/-). After induction by CCl(4)/ethanol, the hepatic fibrosis in the Smad4 knockout mice (Smad4 +/-) was obviously alleviated compared with the wild-type mice (Smad4 +/+), and the incidence rate of hepatocarcinogenesis of the former was also lower than that of the latter(32.0% vs 41.9%). These results indicate that knocking out Smad4 can delay the progression of liver fibrosis and liver cancer.

PDK1 promotes apoptosis of chondrocytes via modulating MAPK pathway in osteoarthritis.

3-Phosphoinositide dependent protein kinase-1 (PDK1), a serine threonine kinase, belongs to the AGC kinase family and is associated with apoptosis. The aim of this study was to investigate the expression of PDK1 (3-Phosphoinositide dependent protein kinase-1) in articular cartilage with osteoarthritis (OA) and to analyze the relationship between PDK1 and chondrocyte apoptosis. Immunohistochemistry and RT-PCR analysis showed that the expression of PDK1 in articular cartilage of OA patients and healthy controls. IL-1ß-stimulated SW1353 cells were used to imitate the OA-like chondrocyte injury in vitro, and IL-1ß-induced the expression of PDK1, apoptotic markers(PARP, caspase-3), and phosphorylated p38 were detected by Western blot. The co-localization of PDK1 and Cleaved-caspase3 was confirmed through immunofluorescence. Knocking down PDK1 expression through PDK1 siRNA. Western blot was performed to detect the knockdown efficiency of PDK1 and the impact of PDK1 knockout on IL-1ß-induced expression of apoptotic markers and phosphorylated p38 in SW1353 cells. Flow Cytometry-Based Annexin V/PI Staining was used to exam chondrocyte apoptosis. Our experimental results suggested that PDK1 may promote chondrocyte apoptosis in OA via p38 MAPK signaling pathway.

Upregulation of AEG-1 Involves in Schwann Cell Proliferation and Migration After Sciatic Nerve Crush.

Astrocyte-elevated gene-1 (AEG-1), also known as metadherin (MTDH) and lysine-rich CEACAM1 coisolated (LYRIC), has emerged as an important oncogene that regulates key cellular processes including apoptosis, migration, invasion, proliferation, and differentiation. It was reported that AEG-1 enhanced breast cancer cells migration in a NF-κB-dependent manner. Also, AEG-1 contributed cell proliferation through the PI3K-Akt/cyclin pathway. Besides, AEG-1 is implicated in diverse physiological and pathological diseases of the central nervous system (CNS), such as brain tumors, neuroblastomas, neurodegeneration, and neuronal development. However, the role of AEG-1 in the process of peripheral nervous regeneration after injury remains virtually unknown. In this study, we investigated the spatiotemporal expression of AEG-1 in a rat sciatic nerve crush model. At its peak expression, AEG-1 was expressed mainly in Schwann cells of the distal sciatic nerve segment from injury, but had few colocalizations in axons. Besides, the peak expression of AEG-1 was in parallel with proliferating cell nuclear antigen (PCNA). In vitro, we detected the increased expression of AEG-1 during the process of tumor necrosis factor α (TNF-α)-induced Schwann cell proliferation. Meanwhile, interference of AEG-1 inhibited both proliferation and migration of Schwann cells. In conclusion, we speculated that AEG-1 is involved in biochemical and physiological responses after sciatic nerve crush (SNC).

Pyrroloquinoline Quinone Decelerates Rheumatoid Arthritis Progression by Inhibiting Inflammatory Responses and Joint Destruction via Modulating NF-κB and MAPK Pathways.

Pyrroloquinoline quinone (PQQ) is a naturally occurring redox cofactor that acts as an essential nutrient and antioxidant and has been reported to exert potent immunosuppressive effects. However, the therapeutically potential of PQQ on rheumatoid arthritis (RA) has not been explored. In the present study, the anti-inflammatory effects of PQQ were investigated in interleukin (IL)-1ß-treated SW982 cells, a RA-like fibroblast-like synoviocytes (FLSs) injury model. Our observations showed that pretreatment with PQQ significantly inhibited the expression of matrix metalloproteinase (MMP)-1 and MMP-3 and suppressed the production of proinflammatory mediators such as TNF-α and IL-6 in IL-1ß-treated SW982 cells. The nuclear translocation of nuclear factor kappa B (NF-κB) and the phosphorylation level of p65, p38, and JNK MAP kinase pathways were also inhibited by PQQ in IL-1ß-stimulated SW982 cells. To further confirm the therapeutic effects of PQQ on RA in vivo, a collagen-induced arthritis (CIA) model was used. Mice treated with PQQ demonstrated marked attenuation of arthritic symptoms based on histopathology and clinical arthritis scores. These results collectively suggested that PQQ might be a promising therapeutic agent for alleviating the progress of RA.

SGTB Promotes the Caspase-Dependent Apoptosis in Chondrocytes of Osteoarthritis.

The purpose of this study is to investigate the expression of small glutamine-rich tetratricopeptide repeat (TPR)-containing ß (SGTB) in articular cartilage of osteoarthritis (OA) and analyze the relationship between SGTB and chondrocyte apoptosis. We established an OA rat model by the meniscal/ligamentous injury (MLI) modeling method and observed the expression of SGTB in articular cartilage by immunohistochemistry and RT-PCR. Human SW1353 chondrosarcoma cells were treated with interleukin-1ß (IL-1ß) to mimic the OA-like chondrocyte injury in vitro, and Western blot was employed to examine the IL-1ß-induced expression of SGTB and active caspase-3. The co-localization of SGTB and active caspase-3 was confirmed by immunofluorescence. We knocked down SGTB expression by RNA interference (RNAi) and overexpressed SGTB by plasmid transfection. Western blot was carried out to detect the knockdown/overexpressing efficiency of SGTB and evaluate its effects on IL-1ß-stimulated expression of active caspase-3 in SW1353 cells. Annexin V/propidium iodide staining was used to detect chondrocyte apoptosis. Then, Western blot was carried out to examine the IL-1ß-induced expression of Hsp70 and evaluate SGTB effects on IL-1ß-stimulated expression of Hsp70 in SW1353 cells. SGTB expression was significantly up-regulated in articular cartilage of OA rat model. IL-1ß stimulation increased the expression of SGTB and active caspase-3 in SW1353 cells. SGTB co-localized with active caspase-3 in IL-1ß-treated SW1353 cells. SGTB inhibition significantly reduced IL-1ß-stimulated expression of active caspase-3 in SW1353 cells. In line with this, overexpressing SGTB via Myc-SGTB transfection increased the active caspase-3 level in IL-1ß-stimulated SW1353 cells. Moreover, flow cytometry assay demonstrated that SGTB knockdown alleviated IL-1ß-induced apoptosis, but it was increased in SW1353 cells that overexpressed SGTB. Overexpressing SGTB via Myc-SGTB transfection decreased the Hsp70 level in IL-1ß-stimulated SW1353 cells. Our results suggested that SGTB positively regulate the activation of caspase-3 by negatively regulating the activity of Hsp70 and might promote chondrocyte apoptosis in OA. This study may provide a novel insight into the pathophysiology of OA and a potential therapeutic target for its treatment.

Clinical analysis of surgical treatment of portal hypertension.

AIM: To review the experience in surgery for 508 patients with portal hypertension and to explore the selection of reasonable operation under different conditions. METHODS: The data of 508 patients with portal hypertension treated surgically in 1991-2001 in our centers were analyzed. Of the 508 patients, 256 were treated with portaazygous devascularization (PAD), 167 with portasystemic shunt (PSS), 62 with selective shunt (SS), 11 with combined portasystemic shunt and portaazygous devascularization (PSS+PAD), 9 with liver transplantation (LT), 3 with union operation for hepatic carcinoma and portal hypertension (HCC+PH). RESULTS: In the 167 patients treated with PSS, free portal pressure (FPP) was significantly higher in the patients with a longer diameter of the anastomotic stoma than in those with a shorter diameter before the operation (P < 0.01). After the operation, FPP in the former patients markedly decreased compared to the latter ones (P < 0.01). The incidence rate of hemorrhage in patients treated with PAD, PSS, SS, PSS+PAD, and HCC+PH was 21.09% (54/256), 13.77 (23/167), 11.29 (7/62), 36.36% (4/11), and 100% (3/3), respectively. The incidence rate of hepatic encephalopathy was 3.91% (10/256), 9.58% (16/167), 4.84% (3/62), 9.09% (1/11), and 100% (3/3), respectively while the operative mortality was 5.49% (15/256), 4.22% (7/167), 4.84% (3/62), 9.09% (1/11), and 66.67% (2/3) respectively. The operative mortality of liver transplantation was 22.22% (2/9). CONCLUSION: Five kinds of operation in surgical treatment of portal hypertension have their advantages and disadvantages. Therefore, the selection of operation should be based on the actual needs of the patients.

[Effects of portaazygous disconnection, portocaval shunt and selective shunts on experimental rat liver cirrhosis].

OBJECTIVE: To evaluate the effects of portaazygous disconnection (PAD), portacaval shunt (PCS) and distal splenocaval shunt (DSCS) on the portosytemic shunting (PSS), hepatic function (HF), hepatic mitochondrial respiratory function (HMRF), oral glucose tolerance test (OGTT) and arterial ketone body ratio (KBR) in order to provide a sound basis for selecting suitable operations for patients. METHODS: Using a cirrhotic portal hypertensive model induced by CCl4/ethanol in Wistar rats, the PSS, HF, HMRF, OGTT and KBR were determined three weeks after PCS, DSCS and PAD. RESULTS: It was revealed that: (1) In the cirrhotic portal hypertension rats, the PSS increased significantly, HMRF and hepatic reserve function (HRF) decreased significantly when compared with the control rats. (2) At the time of first postoperative week, the mean blood glucose value in the 120-minute OGTT in each PAD, PCS and DSCS groups had significant differences compared with the cirrhotic control group. But during the second and third postoperative weeks, the mean blood glucose values in the 120-minute OGTT in both PAD and DSCS groups had no significant differences compared with the cirrhotic control group except for the PCS group. The values of KBR in the three operative groups decreased significantly compared with the cirrhotic control group during the two postoperative weeks. In the third postoperative week, only the values of KBR in the PCS group had a significant difference compared with the cirrhotic control group. (3) After PCS, the PSS was further increased; HF and HMRF were significantly decreased. Little improvement was found in the third postoperative week. (4) After DSCS and PAD, the above mentioned indices were less influenced, and they were restored more quickly than those in the PCS group. CONCLUSION: We found that PAD and DSCS are more desirable than PCS.

Nur77 Was Essential for Neurite Outgrowth and Involved in Schwann Cell Differentiation After Sciatic Nerve Injury.

Nur77, together with Nurr1 and NOR-1, constitutes the NR4A subgroup of orphan nuclear receptors and plays critical roles in cell proliferation, differentiation, migration, and apoptosis. Among them, Nur77 is universally well known to contribute to neurite outgrowth. However, information regarding its regulation and possible function in the peripheral nervous system is still limited. In this study, we performed a sciatic nerve injury model in adult rats and detected an increased expression of Nur77 in the sciatic nerve, which was similar to the expression of Oct-6. Immunofluorescence indicated that Nur77 was located in both axons and Schwann cells. In vitro, we observed enhanced expression of Nur77 during the process of both basic fibroblast growth factor (bFGF)-induced Schwann cells differentiation and nerve growth factor (NGF)-induced PC12 cell neurite outgrowth. In vitro and in vivo experiments indicated that inhibiting the function of Nur77 by specific short hairpin RNA could depress Schwann cells myelinization and axons regeneration. Collectively, all these results suggested that upregulation of Nur77 might be involved in Schwann cells differentiation and neurite elongation following sciatic nerve crush.

KPNA2 Contributes to the Inflammatory Processes in Synovial Tissue of Patients with Rheumatoid Arthritis and SW982 Cells.

Karyopherin-α2 (KPNA2) functions as an adaptor that transports several proteins to the nucleus. We investigated the function and possible mechanisms of KPNA2 involved in rheumatoid arthritis (RA). Western blotting and immunohistochemistry showed the protein expression of KPNA2 increased in synovial tissue of RA patients compared with the healthy controls. Double immunofluorescent staining indicated that KPNA2 co-localized with T cells, macrophage-like synoviocytes, fibroblast-like synoviocytes, and neutrophils in synovial tissue of RA patients. Moreover, the expression of KPNA2 in SW982 cells was increased in a time-dependent manner in response to TNFα stimulation. Both Western blotting and immunofluorescent staining assay revealed the co-localization of KPNA2 and P65 and their translocation from cytoplasma in TNFα-treated SW982 cells. Furthermore, knocking down the expression of KPNA2 by siRNA inhibited TNFα-induced expression of IL-6, MMP-1, and MMP-13 and, more importantly, decreased the P65 phosphorylation in SW982 cells. We therefore suggested that KPNA2 may play a key role in the inflammation process of RA via NF-κB P65 signal transduction pathway.

Pyrroloquinoline Quinone Slows Down the Progression of Osteoarthritis by Inhibiting Nitric Oxide Production and Metalloproteinase Synthesis.

Osteoarthritis (OA) is the most common arthritis and also one of the major causes of joint pain in elderly people. The aim of this study was to investigate the effects of pyrroloquinoline quinone (PQQ) on degenerated-related changes in osteoarthritis (OA). SW1353 cells were stimulated with IL-1ß to establish the chondrocyte injury model in vitro. PQQ was administrated into SW1353 cultures 1 h before IL-1ß treatment. Amounts of MMP-1, MMP-13, P65, IκBα, ERK, p-ERK, P38, and p-P38 were measured via western blot. The production of NO was determined by Griess reaction assay and reflected by the iNOS level. Meniscal-ligamentous injury (MLI) was performed on 8-week-old rats to establish the OA rat model. PQQ was injected intraperitoneally 3 days before MLI and consecutively until harvest, and the arthritis cartilage degeneration level was assessed. The expressions of MMP-1 and MMP-13 were significantly downregulated after PQQ treatment compared with that in IL-1ß alone group. NO production and iNOS expression were decreased by PQQ treatment compared with control group. Amounts of nucleus P65 were upregulated in SW1353 after stimulated with IL-1ß, while PQQ significantly inhibited the translocation. In rat OA model, treatment with PQQ markedly decelerated the degeneration of articular cartilage. These findings suggested that PQQ could inhibit OA-related catabolic proteins MMPs expression, NO production, and thus, slow down the articular cartilage degeneration and OA progression. Owing to its beneficial effects, PQQ is expected to be a novel pharmacological application in OA clinical prevention and treatment in the near future.

Inhibitory effect of retroviral vector containing anti-sense Smad4 gene on Ito cell line, LI90.

BACKGROUND: Transforming growth factor-beta1 (TGF-beta1) exerts strong fibrogenic potential in culture-activated HSCs. Smad4 is a key intracellular mediator for the transforming growth factor-beta (TGF-beta) superfamily of growth factors. The aim of this study was to assess the effects of the antisense Smad4 gene on Ito cell line, LI90. METHODS: The recombinant retroviral vector pLXSN-Smad4 was constructed by cloning the rat antisense Smad4 cDNA into the retroviral vector pLXSN. Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad4 and pLXSN vectors into PA317 cells. Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418. The expression of Smad4 was detected by Northern and Western blots. Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed. RESULTS: mRNA and protein expressions of Smad4 in LI90 cells transfected with retrovirus containing the antisense Smad4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells. Cells hypoexpressing the Smad4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline (P < 0.01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus. CONCLUSIONS: The antisense Smad4 gene can suppress the expression of the Smad4 gene, reduce endogenous production of Smad4 mRNA and protein, block TGF-beta1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM). Our results may provide a basis for the development of antifibrotic gene therapy.

[Obstruction of TGF-beta1 signal transduction can decrease the process of hepatocellular carcinoma in mice induced by CCl4/ethanol].

OBJECTIVE: To study obstruction of the TGF-beta(1) signal transduction by antisense RNA of Smad(4) and its effects on experimental hepatic carcinoma of mice. METHODS: We used the mouse model of primary hepatic carcinoma induced by CCl(4)/ethanol, and transferred antisense Smad(4)cDNA with retrovirus-mediated via portal vein infusion into liver. Southern Blot confirmed that the antisense Smad(4)cDNA had been integrated into the liver. The antisense Smad(4) gene could down-regulate the expression of Smad(4) in fibrotic liver observed by Northern and Western Blot. RESULTS: In the non-therapeutic cirrhotic liver, the expression of Smad(4) mRNA was significantly increased than normal liver. After antisense Smad(4) gene was transferred, the expression of Smad(4) mRNA in the therapeutic liver was significantly decreased compared with non-therapeutic cirrhotic liver. The fibrotic degree of therapeutic liver was alleviated compared with the non-therapeutic fibrotic liver. No significant difference was found between the rates of carcinogenesis of non-therapeutic cirrhotic liver and that of therapeutic cirrhotic liver. But the diameters and numbers of the liver cancers in the therapeutic cirrhotic group were less than that in the non-therapeutic cirrhotic group. CONCLUSION: These results indicate that the antisense Smad(4) gene not only can obstruct the progression of liver fibrosis, but also can inhibit the progression of liver cancer, by obstructing the signal transduction of TGF-beta1.

[Effects of anti-sense Smad4 gene on the biological characteristics of the fat-storing cell line CFSC].

OBJECTIVE: To investigate the effects of antisense Smad(4) on the biological characteristics of the fat-storing cell line CFSC. METHODS: Fat-storing cells of line CFSC from rat with liver fibrosis were cultured and transfected with 50 MOI of recombinant adenoviral vector carrying antisense Smad(4) (AdvATSmad(4)) or the control empty adenovirus (Adv0), both produced by 293 packaging cells, respectively. Two, four, and six days after the transfection the cultured cells were collected to undergo trypan blue staining and cell counting. The growth curves were drawn. The presence of antisense Smad(4) was detected by RT-PCR and Western blotting. (3)H-TdR was added into the culture media to be co-cultured for 6 hours. Then the cells were collected to examine the (3)H-TdR incorporation rate. RT-PCR and immunohistochemistry were used to examine the expression of COL1A1 and type I collagen, kinds of extracellular matrix (ECM). RESULTS: Compared with the control CFSC and the Adv0-transfected CFSC cells, the cell growth curve, (3)H-TdR incorporation rate, proline incorporation rate, expression of Smad(4), and expression of extracellular matrix were markedly decreased in the AdvATSmad(4)-transfected CFSCs. CONCLUSION: The antisense Smad(4) gene inhibits the expression of Smad(4) mRNA and protein, proline incorporation and cell growth, thus down-regulating the production of ECM. Antisense Smad(4) gene may be used as a choice of gene therapy for liver fibrosis.