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

Objective To study the effects of carboxymethylated chitosan (CMCS) to nitric oxide (NO)-induced apoptosis on rat chondrocytes,and explore p38MAPK signal transduction pathway in the process and its mechanism.Methods The rat articular cartilage cells were cultured in vitro,collagen type-2 (collagen-2) immunohistochemical staining was used to identify the cartilage cells.The model of chondrocyte apoptosis was built by different concentrations of sodium nitroprusside (SNP) induction.The cells were divided into the control group,the SNP treated group SNP+CMCS treated group,and the SNP+p38 MAPK inhibitor SB203580 treated group.The apoptotic rate of chondrocytes was calculated by FCM,apoptotic nuclei was identified by Hoechst33342 stain,the mitochondrial membrane potential changes was detected by Rhodamine123 (Rho123) stain,the expression of p38 and p-p38 were detected by Western blotting analysis.Results 1-3 mmol/L SNP could induce chondrocyte apoptosis,the apoptotic rate was increased with the SNP increasing,the most obvious apoptosis was occurred in 3 mmol/L SNP treated chondrocytes,which was 69.8％ (P＜0.05).SNP could increase the nuclear fragmentation of chondrocytes,the cells with nuclear fragmentation was significantly higher than that in the control group.SNP could reduce mitochondrial membrane potential in chondrocytes,which decreased significantly compared with the control group.SNP could increase the p-p38 expression in chondrocytes,which was 4.3 times compared to the control group.CMCS of different concentrations could reduce the apoptotic rate of SNP-induced chondrocytes,which was 51.0％,29.9％ and 15.2％,which was decreased significantly (P＜0.05) when compared with 3 mmol/L SNP induced group,CMCS decreased the cells number of SNP-induced nuclear fragmentation.CMCS increased the mitochondrial membrane potential in SNP-induced chondrocytes.CMCS reduced the expression levels of p-p38 in SNP-induced chondrocytes.Conclusion CMCS has protective effect on SNP-induced apoptosis of chondrocytes.This process is completed by inhibiting the activity of p38 MAPK signal pathway.

Objective To discuss the diagnosis and treatment of iatrogenic urethral injuries and preventive measures.Methods Various clinical parameters in 26 cases of iatrogenic urethral injury between January 2006 to January 2014 were analyzed retro-spectively.Results Twenty-five cases were confirmed in the operation,1 case of confirmed 2 days postoperatively.All patients were follow-up visit for 3-24 months normal renal function,only 1 case of urethral stricture.Conclusion Through effective prevention, early diagnosis and treatment,close follow-up visit can minimize the patient′s occurrence of iatrogenic urethral injury.

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.

BACKGROUND:Recently, non-fusion technology representing as artificial cervical disc replacement continues to improve. On the basis of reconstruction of disc structure and function of involved segments, cervical spine structure of surgery area segment is significantly close to dynamic and static load stress distribution required by natural physiological systems. It effects are apparent in protecting intervertebral facet joints of degenerated segment and structure and function of the cervical spine of adjacent segments and in maintaining cervical dynamic stability, which presented obvious methodological strengths compared with segmental fusion technology.
OBJECTIVE:To evaluate the clinical outcomes of anterior cervical discectomy and fusion and Bryan artificial cervical disc replacement in the treatment of single-level cervical spondylotic myelopathy or radiculopathy.
METHODS:A total of 43 middle and old age patients with single-level cervical spondylotic myelopathy or radiculopathy, who were treated from March 2010 to March 2012, were enrol ed in this study. They were randomly assigned to anterior cervical discectomy and fusion group (fusion group) and Bryan artificial cervical disc replacement group. Range-of-motion of cervical overal and adjacent intervertebral area near the intervertebral space was observed with radiography. During fol ow-up, postoperative recovery of neurological function was evaluated using Japanese Orthopaedic Association scale, visual analog scale and neck disability index.
RESULTS AND CONCLUSION:None patients experienced complications of neurovascular injury during and after the surgery. Range-of-motion of postoperative overal cervical vertebra and adjacent joint was improved in the Bryan artificial cervical disc replacement group compared with the fusion group. Neurological function was apparently improved after surgery in each group. At 3 months after surgery, scores of Japanese Orthopaedic Association, visual analog scale and neck disability index were significantly improved in the Bryan artificial cervical disc replacement group compared with the fusion group (P<0.05). During final fol ow-up, there were significant differences in visual analog scale scores between the two groups. Japanese Orthopaedic Association scale score and neck disability index score were similar between the two groups. During fol ow-up, no prosthesis sinking, displacement or heterotopic ossification were detected. These data indicated that artificial cervical disc replacement could effectively keep the range of motion of cervical segments and protect disc degeneration of adjacent segment. Mid-term fol ow up obtained similar improvement of neurological function of fusion surgery. The moderate-term and short-term efficacies of non-fusion technology were better than fusion technology in the treatment of single-level cervical spondylopathy.

AIM:To construct eukaryotic expression vector of Sirt2 and detect its expression in HEK293 cells. METHODS: Total RNA was isolated from brain tissue of a-dult SD rat. A 1 130 bp fragment containing the coding region of Sirt2 was amplified by RT-PCR and the resulting PCR product was subcloned into PMD20-T vector and se-quenced. Coding region of Sirt2 was generated with PCR by using the PMD20-T-Sirt2 as template, the amplified PCR fragment was inserted into the EcoR I and Hind Ⅲ sites of the pcDNA3. 1myc-his(-)A expression vector, and the sequence was confirmed by DNA sequencing. The expression of new construct pcDNA3.1 myc-his(-) A-Sirt2 in HEK293 cells was detected by immunofluorescence. RESULTS: The full length coding region of Sirt2 was obtained and confirmed by sequencing, the expression of Sirt2 was detected successfully in HEK293 cells. CONCLUSION: The eukaryotic expression vector of Sirt2 has been successfully constructed, which will provide a useful tool for designing an in-depth investigation of the role of Sirt2.

Objective To observe the therapeutic effect of rhubarb combined with Probiotics for treatment of patients with severe acute pancreatitis (SAP) accompanied by acute lung injury (ALI). Methods A prospective randomized controlled trial was conducted. Sixty patients with SAP and ALI were randomly divided into a rhubarb group and a combination of rhubarb and Probiotics group (combined group), 30 cases in each group. Both groups were treated with conventional therapy. On that therapeutic base, the patients in rhubarb group were treated with rhubarb 3 g·kg-1·d-1 (Raw rhubarb in boiling water 200 mL was cooked for 30 minutes, then the decoction was divided into two parts to be infused through nasal jejunal tube, retaining for 1 hour);while in the combined group, beside the above rhubarb therapy, the patients were additionally given Probiotics [Clostridium butyric acid:Chang Lekang capsule, once 1 grain (0.42 g), twice a day] through nasal jejunal tube. The therapeutic course was 14 days in the two groups. The plasma endotoxin levels, oxygenation index, and acute physiology and chronic health evaluationⅡ(APACHEⅡ) score were observed before and after treatment for 7 days and 14 days, respectively. After treatment for 30 days, the time of intestine functional recovery and mechanical ventilation, number of damaged organs, number of infectious sites, number of pancreatic pseudocysts, length of intensive care unit (ICU) stay and 30-day fatality rate were observed in the two groups. Results With the prolongation of treatment, the plasma level of endotoxin and APACHEⅡscore in the two groups were gradually reduced, and the oxygenation index was gradually increased after treatment for 7 days and 14 days in the two groups compared with those before treatment, and there were statistically significant differences in above indexes on the 14th day after treatment between combined group and rhubarb group [endotoxin (μg/L): 19.16±1.90 vs. 21.20±1.05, oxygenation index (mmHg, 1 mmHg = 0.133 kPa): 369.50±5.45 vs. 341.50±7.36, APACHE Ⅱscore:11.84±0.60 vs. 13.00±0.86, P<0.05 or P<0.01]. The length of intestine functional recovery in the combined group was longer than that in the rhubarb group (days: 5.63±1.75 vs. 5.02±1.54), the number of damaged organs (2.08±0.74 vs. 2.70±1.11), the number of infectious sites (1.93±0.64 vs. 2.23±0.83), the number of pancreatic pseudocysts (1.22±0.34 vs. 1.63±0.11) and the mortality rate [10.0%(3/30) vs. 13.3%(4/30)] in the combined group were lower than those of the rhubarb group, but there were no statistically significant differences between two groups (all P > 0.05). The times of mechanical ventilation (days: 13.40±1.76 vs. 15.60±1.28) and the length of ICU stay (days:16.13±1.25 vs. 17.63±1.30) were significantly shorter in combined group than those in rhubarb group (both P<0.05). Conclusion Rhubarb combined with Probiotics for treatment of patients with SAP and ALI can promote their recovery of lung function and shorten their length of ICU stay.

OBJECTIVE:To systematically review the efficacy and safety of reinforcing kidney and invigorating spleen princi-ple in the treatment of primary osteoporosis (POP) in middle and elderly aged patients,and provide evidence-based reference for clinic. METHODS:Retrieved from VIP,Wanfang Database,CJFD,Medline,PubMed,Elsevier,Springer,Web of Science and Ovid,randomized control trials (RCT) about reinforcing kidney and invigorating spleen principle (test group) versus other meth-ods(no reinforcing kidney and invigorating spleen principle,control group)in the treatment of POP were collected. Meta-analysis was performed by using Rev Man 5.3 software provided by Cochrane after data extraction and quality evaluation modified by Jadad scale. RESULTS:Totally 28 RCTs were enrolled,involving 2849 patients. Results of Meta-analysis showed,the total effective rate [OR=3.80,95%CI(2.76,5.23),P<0.001],BGP level [MD=2.23,95%CI(1.80,2.66),P<0.001],BMD level of femoral neck [MD=0.04,95%CI(0.00,0.08),P=0.05] and BMD level of lumbar spine [MD=0.07,95%CI(0.03,0.11),P=0.001] in test group were significantly higher than control group,ALP level [MD=-5.59,95%CI(-10.51,-0.66),P=0.03] and VAS score [MD=-1.38,95%CI(-1.87,-0.89),P<0.001] were significantly lower than control group,with statistical significance. There were no significant differences in U-Ca/Cr [MD=-0.07,95%CI(-0.17,0.02),P=0.12] and serum Ca2+ level [MD=0.16,95%CI (-0.07,0.39),P=0.17] in 2 groups. CONCLUSIONS:Reinforcing kidney and invigorating spleen principle can improve patients' osteogenesis during bone metabolism,reduce the activity of the osteoclastic process and relief patients'pain in the treatment of POP.

BACKGROUND:It has been confirmed that carboxymethylated chitosan has an promoting effect on Schwann cel proliferation and secretion, but its impact on the cyclic adenosine monophosphate-mediated protein kinase A signaling pathway in schwann cel stil needs further study. OBJECTIVE:To investigate the effect of carboxymethylated chitosan on cyclic adenosine monophosphate/ protein kinase A signaling pathway in rat schwann cels. METHODS:The Schwann cels of the second generation neonatal rats were obtained and seeded in 6-wel plate at a concentration of 1×109/L. These Schwann cels were cultured and divided into four groups. The Schwann cels in the control group were cultured by adding PBS. The Schwann cels in the experimental groups were cultured by adding 50, 100 and 200 mg/L of carboxymethyl chitosan solution, respectively. After 24 hours, the concentration of cyclic adenosine monophosphate, protein kinase A activity and cyclic adenosine monophosphate response element binding protein mRNA expression were detected. RESULTS AND CONCLUSION:Compared with the control group, carboxymethyl chitosan increased cyclic adenosine monophosphate concentrations, the activity of protein kinase A and cyclic adenosine monophosphate response element binding protein mRNA expression within the Schwann cels in a dose-dependent manner (P < 0.05). These results demonstrate that carboxymethyl chitosan can increase the concentration of cyclic adenosine monophosphate within the Schwann cels and promote protein kinase A activity, thereby activating cyclic adenosine monophosphate/protein kinase A signaling pathway.

NSP4, as the diarrhea-related protein of rotavirus, is becoming an attractive candidate for vaccine development. To compare the immunogenicity of NSP4 from different genetic groups, we constructed eukaryotic expression plasmids comprising the NSP4 genes from four different genetic types using the pCI vector. The recombinant vectors were designated as pCI-97B6, pCI-97S36, pCI-97S34 and pCI-97SZ8, respectively. Following the conformation of the transient expression of the constructs in 293 cells, the plasmids were respectively subjected to the 5 round i. m. inoculation of BALB/c mice. The specific antibodies against NSP4 as well as the IgG1/IgG2a subclasses of immunoglobulin in mice sera were examined with indirect ELJSA after each immunization. The results showed that the immunization of plasmids expression NSP4s could elicit not only humoral but also cellular immunity, but the humoral immune response is dominant. There is a difference of immunogenecity among the NSP4 of different genetic type. Further studies were needed to focus on the relationship between the immunogenicity and protection effect.