Adenosine A2A Receptor Mediates Inhibition of Synovitis and Osteoclastogenesis after Electroacupuncture in Rats with Collagen-Induced Arthritis.

BACKGROUND: This study was to investigate the role of adenosine A2A receptors (A2AR) in inhibiting the effect of electroacupuncture (EA) on osteoclastogenesis in collagen-induced arthritis (CIA). METHODS: Wistar rats were divided into four groups: sham-control group, CIA-control group, CIA-EA group, and CIA-EA-SCH58261 (A2AR antagonist) group. We detected tumor necrosis factor-α (TNF-α), nuclear transcription factor-κB (NF-κB), receptor activator of NF-κB ligand (RANKL), protein kinase A (PKA), and extracellular regulatory protein kinase 1/2 (ERK1/2) in peripheral blood by ELISA. PKA, ERK1/2, and NF-κB in ankle joints were determined by western blotting. We evaluated the arthritis damage by histological examination and determined the number of osteoclasts by tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: EA treatment downregulated the expression of TNF-α, RANKL, PKA, ERK1/2, and NF-κB in peripheral blood but increased the levels of PKA and ERK1/2 in ankle joints. Importantly, EA treatment reduced bone erosion as evidenced by the histological findings and inhibited osteoclastogenesis as revealed by TRAP staining. All these effects of the EA treatment were reversed by combining EA treatment with the A2AR antagonist SCH58261. CONCLUSION: Our data suggest that EA treatment activated A2AR. The effects of the A2AR antagonist SCH58261 suggest that the inhibition of osteoclast formation, the inhibition of TNF-α, RANKL, and NF-κB expression, and the increase of ERK1/2 are all dependent on this EA-induced A2AR activation. It is therefore likely that these pathways with clearly defined roles in inflammation and bone erosion are at least partially involved in the mediation of the inhibition of synovitis and osteoclast formation induced by EA.

AMP-activated protein kinase-dependent induction of autophagy by erythropoietin protects against spinal cord injury in rats.

AIMS: Autophagy has been regarded as a promising therapeutic target for spinal cord injury (SCI). Erythropoietin (EPO) has been demonstrated to exhibit neuroprotective effects in the central nervous system (CNS); however, the molecular mechanisms of its protection against SCI remain unknown. This study aims to investigate whether the neuroprotective effects of EPO on SCI are mediated by autophagy via AMP-activated protein kinase (AMPK) signaling pathways. METHODS: Functional assessment and Nissl staining were used to investigate the effects of EPO on SCI. Expressions of proteins were detected by Western blot and immunohistochemistry. RESULTS: Treatment with EPO significantly reduced the loss of motor neurons and improved the functional recovery following SCI. Erythropoietin significantly enhanced the SCI-induced autophagy through activating AMPK and inactivating mTOR signaling. The inhibitor of AMPK, compound C, could block the EPO-induced autophagy and beneficial action on SCI, whereas the activator of AMPK, metformin, could mimic the effects of EPO. In the in vitro studies, EPO enhanced the hypoxia-induced autophagy in an AMPK-dependent manner. CONCLUSIONS: The AMPK-dependent induction of autophagy contributes to the neuroprotection of EPO on SCI.

Association between ankylosing spondylitis and the miR-146a and miR-499 polymorphisms.

miRNAs are small, non-coding RNAs that regulate the expression of multiple target genes at the post-transcriptional level. Single-nucleotide polymorphisms (SNPs) in miRNA sequences may alter miRNA expression and have been implicated in the pathogenesis of multiple forms of arthritis, including rheumatoid arthritis (RA) and osteoarthritis. The present study explored the association between ankylosing spondylitis (AS) and two single nucleotide polymorphisms (SNPs), miR-146a rs2910164G>C and miR-499 rs3746444T>C, in a Han Chinese population. A case-control study consisting of 102 subjects with AS and 105 healthy controls was designed. The two miRNA SNPs were identified by direct sequencing. Subsequently, their gene and genotype frequencies were compared with healthy controls. A significant difference was observed in the miR-146a rs2910164G>C SNP. The frequency of the G allele was markedly higher in the AS patients than in the healthy controls (P = 0.005, Pc = 0.01, OR = 1.787), and the frequency of the GG genotype was higher in AS patients than in controls (P = 0.014, Pc = 0.042, OR = 2.516). However, no significant association was found between the miR-499 rs3746444T>C variant and susceptibility to AS. This is the first study to address the association between the miR-146a rs2910164G>C and miR-499 rs3746444T>C polymorphisms and AS, and it suggests a potential pathogenic factor for AS. Further studies are needed to validate our findings in a larger series, as well as in other ethnic backgrounds.

[Establishment of a real-time PCR assay for simultaneously detecting human BKV and CMV DNA and its application in renal transplantation recipients].

To establish a fluorescent quantitative PCR method (FQ-PCR) with TaqMan probe for simultaneous detection of polyomavirus (BKV) and cytomegalovirus (CMV) and to evaluate its clinical application in the renal transplantation recipients. The conservative sequences of BKV and CMV were targeted and amplified by nested PCR technique. The PCR products were cloned into the plasmids pcDNA3. 1(+). The recombinant plasmid containing target sequences of BKV and CMV were constructed as external standards. The TaqMan-based assay was optimized. For evaluating the assay, the sensitivity was determinated by diluted standard (5 X 103-10icopies/mL), and the specificity was verified by negative control and positive control, and the precision was assessed by intra-assay coefficient of variation (ICV) through detecting standard repeatedly (20 times). A total of 480 blood samples of renal transplantation recipients were used to detect BKV and CMV DNA simultaneously with FQ-PCR, and the concentrations of FK506 were measured by ELISA. The association of DNA copy and concentrations of FK506 was analyzed. The cloned target BKV and CMV DNA was confirmed by sequencing and analysis. The sensitivity of the FQ-PCR assay reached 5 X 103 copies/ml in detecting BKV or CMV DNA. Control DNA verified the assay specifically detecting target DNA. The precision of the assay to quantif target DNA copies was acceptable (Intra-assay CV was 3.44% for BKV and 2.23% for CMV; Inter-assay CV was 4. 98% for BKV and 3.76% for CMV;). Of 480 samples, 130 samples (27. 08%) were CMV DNA positive, significantly higher than the BKV DNA positive (13.33%, 64/480, P<0.05). The positive BKV or CMV DNA was found to be associated with high concentrations of FK506 (P<0. 05). In conclusion, the developed real-time PCR assay for detecting both CMV and BKV DNA simultaneously was s high sensitive, precise and time-effectiveand could be applied in the monitoring of the CMV and BKV infection in the renal transplantation recipients.

[The application of genotyping in the complicated ABO blood group type].

OBJECTIVE: To investigate the application of ABO blood group genotyping in complicated ABO blood group type. METHODS: Ten specimens of complicated ABO blood group were genotyped by sequence specific primer PCR (PCR -SSP), and confirmed by DNA sequencing and alignment. Six hundred and ten blood samples typed by ABO immunoassay were as control of genotyping. RESULTS: Ten cases of complicated blood type were identified by high resolution PCR- SSP as rare ABO blood groups: cis-AB01 (3 cases), B(A)04 (2 cases), cisAB02, B(A)02, Bel03, Bw12 and Ael05, confirmed by DNA sequencing. Genotyping and serotype detected 610 cases ABO blood group were coincident, and the frequency of A, B, AB and O were as 28.69%, 27.54%, 8.2% and 35.57% respectively. According to the genotypes, the highest frequency subgroup was O1 (32.87%), the lowest was A2 (0.66%). CONCLUSION: PCR -SSP could type the ABO blood group accurately, but also the sub-group of blood type. However, special designed high resolution PCR -SSP or DNA sequencing is needed to identify the complicated blood groups.

[Experimental study of eliminat dampness resolv phlegm method in treating cervical spondylopathy of the vertebral artery type].

OBJECTIVE: To detect the velocity and viscosity of blood flow of the vertebral arteries, the apoptotic cell and apoptotic related protein in the brain in order to offer theoretical foundation for the treatment of cervical spondylopathy of the vertebral artery type with the eliminat dampness resolv phlegm method. METHODS: Sixty male Japanese big ear rabbits were divided randomly into normal sodium group (A), Flunarizine group (B), low dosage Wendantang group (C), large dosage Wendantang group (D), Flunarizine group combined with large dosage Wendantang group (E), normal group (F). Each group had 10 rabbits. Xiaozhiling injection was injected around the vertebral arteries of rabbits in group A, B C, D, E to make the model of the cervical spondylopathy of the vertebral artery type. Normal sodium (20 ml x kg(-1)d(-1)) was apply through intragastric administration in group A, F; Flunarizine (0.8 mg x kg(-1)d(-1)), low dosage Wendantang (1 g x ml(-1)d(-1)), large dosage Wendantang (2 g x ml(-1)d(-1)), Flunarizine combined with large dosage Wendantang were respectively apply through intragastric administration in group B, C, D, E. The velocity and viscosity of blood flow of the vertebral arteries, the apoptotic cell and apoptotic protein in the brain were detected before and after the treatment. RESULTS: Satisfactory animal model were obtained in group A, B, C, D, E. The rabbits of group E had the most improvement of the velocity and viscosity of blood flow of the vertebral arteries while at meantime, which had the lowest apoptotic index and apoptotic related protein expression in the brain. CONCLUSION: The routine treatment for the cervical spondylopathy of the vertebral artery combined with eliminat dampness resolv phlegm method could improve velocity and viscosity of blood flow of the vertebral arteries, which maybe relate with reduction of apoptosis in the brain.