Synergistic effects of tetramethylpyrazine and astragaloside IV on spinal cord injury via alteration of astrocyte A1/A2 polarization through the Sirt1-NF-κB pathway.

OBJECTIVE: Reactive astrocytes are hallmarks of traumatic spinal cord injury (T-SCI) and are associated with neuropathic pain (NP). Mediating the functional phenotype of reactive astrocytes helps neural repair and ameliorates NP in T-SCI. Here, we aimed to explore the role of tetramethylpyrazine (TMPZ) and astragaloside IV (AGS-IV) in astrocyte polarization and the underlying molecular mechanism in T-SCI. METHODS: Primary cultured astrocytes from mice were treated with LPS or conditioned medium from "M1" polarized microglia (M1-CM), followed by TMPZ and/or AGS-IV administration. The expression levels of "A1" astrocyte-specific markers (including C3, GBP2, Serping1, iNOS), "A2" astrocyte-specific markers (including S100a10 and PTX3), Sirt1 and NF-κB were detected via western blotting. TNF-α and IL-1ß levels were detected via ELISA. RT-PCR was used to evaluate OIP5-AS1 and miR-34a expression. si-OIP5-AS1 or the Sirt1 inhibitor EX-527 was administered to astrocytes. A spinal cord injury (SCI) model was constructed in Sprague-Dawley (SD) rats. Alterations in astrocytic "A1/A2" polarization in the spinal cord tissues were evaluated. RESULTS: LPS and M1-CM induced "A1" polarization of primary astrocytes. TMPZ and ASG IV could substantially reduce the expression of "A1"-related biomarkers but enhance "A2"-related biomarkers. OIP5-AS1 and Sirt1 levels were reduced in "A1"-polarized astrocytes, while miR-34a and p-NF-κB p65 were elevated. TMPZ and ASG IV enhanced OIP5-AS1 and Sirt1 levels and, in contrast, attenuated the changes in miR-34a and p-NF-κB p65 levels. Notably, the TMPZ and ASG IV combination had stronger effects on astrocyte polarization than the single treatment with TMPZ or ASG IV. OIP5-AS1 knockdown and Sirt1 inhibition both reversed the regulatory effects of TMPZ and ASG IV in astrocytic polarization. According to the in vivo experiments, the expression of "A1"-associated markers was enhanced in the spinal cords of SCI rats. The TMPZ and ASG IV combination reduced astrocytic "A1" polarization and enhanced astrocytic "A2" polarization. The expression of lncRNA OIP5-AS1 and Sirt1 was enhanced by TMPZ and ASG IV, while that of miR-34a and p-NF-κB p65 was inhibited. CONCLUSION: The combination of TMPZ and ASG IV can ameliorate dysregulated astrocytic polarization induced by spinal cord injury by affecting the lncRNA OIP5-AS1-Sirt1-NF-κB pathway.

Tetramethylpyrazine and Astragaloside IV have synergistic effects against spinal cord injury-induced neuropathic pain via the OIP5-AS1/miR-34a/Sirt1/NF-κB axis.

BACKGROUND: Both Tetramethylpyrazine (TMPZ) and Astragaloside IV (AGS-IV) can ameliorate neuronal apoptosis and neuroinflammation in CNS diseases. This study revolves around the underlying mechanism of TMPZ and AGS-IV in spinal cord injury (SCI)-associated neuropathic pain (NP). MATERIALS AND METHODS: An in-vivo NP model was constructed in Sprague-Dawley (SD) rats via SCI. qRT-PCR was employed to detect OIP5-AS1 and miR-34a. The paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) of the rats were evaluated. Neuronal apoptosis in the spinal cord of rats was examined by Nissl staining and TUNEL staining. The interactions between OIP5-AS1 and miR-34a as well as miR-34a and Sirt1 were investigated through dual luciferase assay and RIP assay. The protein expressions of Bad, Bax, Caspase-3, iNOS, COX2, NF-κB, and Sirt1 were examined by western blot. RESULTS: TMPZ and AGS-IV combination relieved behavioral symptoms of neuropathic pain in the SCI rat model, enhanced the levels of OIP5-AS1 and Sirt1, and lowered the profile of miR-34a. OIP5-AS1 downregulation weakened the neuroprotective function of TMPZ and AGS-IV in SCI rats and reversed their anti-inflammatory and anti-apoptotic effects on LPS-elicited primary spinal cord neurons. miR-34a was identified as a target of OIP5-AS1. Upregulated miR-34a partly abated the protective functions of TMPZ and AGS-IV in primary spinal cord neurons. Additionally, miR-34a targeted and repressed Sirt1, thus activating the NF-κB pathway and inflammatory reactions. Sirt1 inhibition reduced the protective effects mediated by OIP5-AS1. CONCLUSION: TMPZ and AGS-IV ameliorate SCI-elicited NP via the OIP5-AS1/miR-34a/Sirt1/NF-κB pathway.

[Treatment of locked lower cervical fracture and dislocation with anterior cervical fusion and internal fixation combined with the release of interlocking facet through the Luschka joint and anterior lamina space].

OBJECTIVE: To investigate the effectiveness of treatment of locked lower cervical fracture and dislocation with anterior cervical fusion and internal fixation combined with the release of the interlocking facet through the Luschka joint and anterior lamina space. METHODS: Twelve patients with lower cervical interlocking fracture and dislocation were analyzed retrospectively between January 2013 and June 2015. There were 7 males and 5 females, aged 25-59 years with an average age of 38.4 years. The disease duration was 9.6 hours to 100 days with an average of 7.3 days. There were 8 cases of unilateral locking and 4 cases of bilateral locking; 4 cases of old injury and 8 cases of fresh injury. The injured segments were 2 cases of C 3, 4, 5 cases of C 4, 5, 3 cases of C 5, 6, and 2 cases of C 6, 7. According to Meyerding classification, there were 9 cases of grade Ⅰ and 3 cases of grade Ⅱ. According to the functional classification of American Spinal Injury Association (ASIA), there were 2 cases of grade C, 6 cases of grade D, and 4 cases of grade E. The interlocking facet was released through the Luschka joint and anterior lamina space, and the anterior cervical fusion and internal fixation were used to treat the fracture and dislocation of the lower cervical spine. The recovery of spinal cord function was judged by the functional classification of ASIA; visual analogue scale (VAS) score, neck disability index (NDI) score, modified Japanese Orthopaedic Association (m-JOA) score were used to evaluate the clinical efficacy; the Cobb angle of fusion segment were observed by X-ray film. The intervertebral bone graft fusion was evaluated at 6 months after operation. RESULTS: The average operation time was 78.30 minutes, the average intraoperative blood loss was 167.30 mL, and the average postoperative drainage volume was 58.12 mL. No blood transfusion was given during or after operation. During the operation, there was no accidental injury of large blood vessels, esophagus, and trachea; no laryngo edema, dysphagia, hoarseness, and cerebrospinal fluid leakage occurred after operation; no spinal cord injury or nerve root injury aggravated; the incision healed by first intention, and no infection occurred. All 12 cases were followed up 15-20 months, with an average of 16.5 months. The symptoms and function of the nerve injury were significantly improved when compared with that before operation. Re-examination of the cervical spine X-ray film at 6 months after operation showed that the Cage or bone graft was not displaced or broken, the screw was not loosened or detached, and the intervertebral graft fusion rate was up to 100%. At last follow-up, the ASIA grade, Cobb angle of fusion segment, neck pain VAS score, m-JOA score, and NDI score were significantly improved when compared with preoperative one ( P<0.05). CONCLUSION: The effectiveness of treatment of locked lower cervical fracture and dislocation with anterior cervical fusion and internal fixation combined with the release of the interlocking facet through the Luschka joint and anterior lamina space is clear, which not only can make the injured segment get satisfactory reduction, immediate stability and reconstruction, and full decompression, but also can effectively prevent the secondary injury of spinal cord.

Effect of hypoxia-inducible factor-1/vascular endothelial growth factor signaling pathway on spinal cord injury in rats.

The aim of the present study was to evaluate the expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 (HIF-1), and to investigate the role of the HIF-1/VEGF signaling pathway following spinal cord injury (SCI). A total of 90 12-week-old Sprague Dawley rats were randomly divided into the following three groups: Sham group (operation without SCI); control group (SCI without ML228 treatment); and treatment group (SCI receiving ML228 treatment). ML228 was administered as it is an activator of HIF-1α. The control and treatment groups were subjected to spinal cord hemisection and motor activity was evaluated using the Basso, Beattie and Bresnahan (BBB) scoring system. Expression of HIF-1α and VEGF in each injured spinal cord section was assessed using immunohistochemistry. Prior to SCI, there were no significant differences in the BBB score among the three groups (P>0.05). However, one day after the operation, the BBB score of the sham group was significantly higher than that of the other two groups (P<0.05) and the BBB scores of the control and treatment groups did not differ significantly (P>0.05). BBB scores 3 and 7 days following surgery were significantly higher in the sham group than the other two groups (P<0.05) and the BBB scores of the treatment group were significantly higher than those of the control group (P<0.05). The expression of HIF-1α and VEGF proteins in all groups were measured 1, 3 and 7 days after the operation, and it was observed that their expression was higher in the treatment group than in the control group (P<0.05). Therefore, the results of the current study suggest that ML228 may effectively activate the HIF-1α/VEGF signaling pathway to promote the expression of HIF-1α and VEGF proteins within the injured segment of the spinal cord, which promotes neural functional recovery following SCI in rats. Therefore, treatment with ML228 may be developed as a novel therapeutic strategy to treat SCI.

Inhibition of telomerase activity by dominant-negative hTERT retards the growth of breast cancer cells.

BACKGROUND: Telomerase, a ribonucleoprotein enzyme mainly consisted of a catalytic protein subunit human telomerase reverse transcriptase (hTERT) and a human telomerase RNA component, is responsible for maintaining telomeres. Telomerase over-expression correlates significantly with tumors and is a prognostic marker. However, telomerase over-expression in breast cancers and the effect of telomerase inhibition as a candidate cancer therapy are unknown. METHODS: We used the dominant-negative mutant of hTERT (DN-hTERT) to inhibit telomerase activity on human breast adenocarcinoma cell line MCF-7 by transfection. Telomeric repeat amplification protocol assays and real-time quantitative RT-PCR were performed to investigate telomerase activity as well as expression of hTERT. Telomere length was measured by the flow-fluorescence in situ hybridization assay. Cell proliferation was assessed by the WST-8 assay, and apoptosis was evaluated by flow cytometry. The tumor formation ability of MCF-7 cells was investigated by transplanting cells subcutaneously into BALB/c nude mice. RESULTS: Ectopic expression of DN-hTERT caused dramatically inhibition of telomerase activity and reduction of telomere length. Telomerase inhibition induced growth arrest and apoptosis of MCF7 cells in vitro and loss of tumorigenic properties in vivo. CONCLUSION: This study shows that telomerase inhibition by DN-hTERT can effectively inhibit the cell viability and tumorigenicity of MCF7 cells and is an attractive approach for breast cancer therapy.

Long-term outcome of olfactory ensheathing cell transplantation in six patients with chronic complete spinal cord injury.

The aim of the study was to analyze the clinical efficacy and safety of olfactory ensheathing cell (OEC) transplantation for treating patients with chronic, complete spinal cord injury (SCI). Six patients with six chronic complete spinal cord injuries were recruited and treated with autologous OEC transplantation and followed for 24 months. The scores from before and after transplantation were analyzed. This was a self-control experiment. There was significant amelioration in the scores of the standard neurological classification of spinal cord injury made by the America Spinal Cord Injury Association (ASIA) and the International Association of Neurorestoratology-Spinal Cord Injury Functional Rating Scale (IANR-SCIFRS) following OEC transplantation with 24 months of follow-up. No clinical complications were observed. OEC transplantation would appear to be clinically safe and may promote the neurofunctional recovery of SCI based on data from six patients. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Clinical application of olfactory ensheathing cells in the treatment of spinal cord injury.

OBJECTIVES: To investigate the safety and therapeutic efficacy of autologous olfactory ensheathing cell (OEC) transplantation in cervical spinal cord injury (SCI). METHODS: Patients with cervical SCI of >6 months' duration were treated with autologous OECs, injected into the area surrounding the SCI under magnetic resonance imaging guidance, twice a week for 4 weeks. Patients were evaluated before treatment and at 3, 6, 12 and 24 months post-treatment, using the American Spinal Injury Association (ASIA) Impairment Scale, the ASIA sensory and motor score and the Functional Independence Measure (FIM) score. RESULTS: Eight patients were recruited to the study. Three months after treatment, ASIA and FIM scores had improved significantly compared with pretreatment, though by 1 year no further significant improvements in the ASIA score were seen. The return of substantial sensation and motor activity in various muscles below the injury level was observed in three patients during follow-up. In addition, bladder function was restored in two patients. There were no serious complications postoperatively or during the follow-up period. CONCLUSIONS: This study provides preliminary evidence of the safety and possible efficacy of autologous OEC transplantation.

Inhibition of cell growth and telomerase activity in osteosarcoma cells by DN-hTERT.

In order to study the effects of dominant negative human telomerase reverse transcriptase (DN-hTERT) on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 (DN) or IRES2-EGF (I, blank vector) with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells (MG63/DN). The telomerase activity was 2.45-0.11 in MG63/DN cells, while 3.40+/-0.12 in the cells transfected with blank vector (MG63/I), (P<0.05); DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I (P<0.01). It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.