Regulator of G protein signalling 18 promotes osteocyte proliferation by activating the extracellular signal­regulated kinase signalling pathway.

Osteocyte function is critical for metabolism, remodelling and regeneration of bone tissue. In the present study, the roles of regulator of G protein signalling 18 (RGS18) were assessed in the regulation of osteocyte proliferation and bone formation. Target genes and signalling pathways were screened using the Gene Expression Omnibus (GEO) database and Gene Set Enrichment Analysis (GSEA). The function of RGS18 and the associated mechanisms were analysed by Cell Counting Kit 8 assay, 5­ethynyl­2'­deoxyuridine assay, flow cytometry, reverse transcription­quantitative PCR, western blotting and immunostaining. Overlap analysis of acutely injured subjects (AIS) and healthy volunteers (HVs) from the GSE93138 and GSE93215 datasets of the GEO database identified four genes: KIAA0825, ANXA3, RGS18 and LIPN. Notably, RGS18 was more highly expressed in peripheral blood samples from AIS than in those from HVs. Furthermore, RGS18 overexpression promoted MLO­Y4 and MC3T3­E1 cell viability, proliferation and S­phase arrest, but inhibited apoptosis by suppressing caspase­3/9 cleavage. Silencing RGS18 exerted the opposite effects. GSEA of GSE93138 revealed that RGS18 has the ability to regulate MAPK signalling. Treatment with the MEK1/2 inhibitor PD98059 reversed the RGS18 overexpression­induced osteocyte proliferation, and treatment with the ERK1/2 activator 12­O­tetradecanoylphorbol­13­acetate reversed the effects of RGS18 silencing on osteocyte proliferation. In conclusion, RGS18 may contribute to osteocyte proliferation and bone fracture healing via activation of ERK signalling.

Anatomical Analysis of the Cervical Sympathetic Ganglion and Spinal Ganglion and its Physiological Significance in the Pathogenesis of Cervical Vertigo.

Objective: Investigating the anatomical connections between cervical sympathetic ganglia and spinal ganglia in rabbits and assessing the role of Neuropeptide Y in the pathogenesis of cervical vertigo. Method: Part 1: 32 adult healthy male New Zealand white rabbits (whose skin is very sensitive, so rabbits are generally used for stimulation experiments) were randomly divided into the upper cervical sympathetic ganglia (SCSG) stimulation group and the lower cervical sympathetic ganglia (ICSG) stimulation group, with 16 rabbits in each group. The two groups were divided into an experimental group and a control group, with 8 rabbits in each group. The cervical ganglia of each group of white rabbits were injected with 4% FluoroGold solution and observed under a section microscope. Part 2: Sixty New Zealand white rabbits were randomly divided into a blank control (n = 12), SCSG stimulation group (n = 12), SCSG sham surgery control (n = 12), ICSG stimulation group (n = 12), and ICSG sham surgery control group (n = 12). The SCSG group and ICSG group were subjected to electrical stimulation (i.e. 30.0Hz, 10.0V, 5-minute pulse width of 0.5 ms square wave pulse), and specimens were made. The expression of NPY was detected using immunohistochemical methods. Result: Neuropeptide Y was weakly expressed in all cervical ganglia (C1-C8). Compared with the sham surgery group, the superior cervical sympathetic ganglion stimulation group showed an increase in Neuropeptide Y positive cells in C2, C3, C4, and C5, with C2 and C3 showing the most significant increase. The number of C6, C7, and C8 Neuropeptide Y positive cells in the 3 C、3D and 4B, lower cervical sympathetic ganglion stimulated groups was higher than in the sham sham-operated group, and C6 and C7 significantly increased. Neuropeptide Y is like immunoreactive neurons in the cervical spinal ganglia, and the immunoreactive products are small brown particles distributed in the cytoplasm after electrical stimulation of the cervical sympathetic ganglia. The Neuropeptide Y content in the corresponding segment of the cervical spinal ganglia is significantly increased compared to the control group (P < .05). Conclusion: In New Zealand white rabbits, nerve fibers are interconnected between the cervical sympathetic ganglion and the cervical spinal ganglion, and this neural fiber connection has a certain segmental nature, providing experimental basis for the existence of the cervical spinal cord external nerve reflex arc and elucidating the pathogenesis of cervical vertigo in terms of neural anatomy. By using neuroelectrophysiological methods, it has been confirmed that electrical stimulation in the cervical spinal ganglia can reach the corresponding cervical sympathetic ganglia on the same side through a certain conduction pathway, providing experimental basis in neuroelectrophysiology for the existence of the cervical extraspinal nerve reflex arc and elucidating the pathogenesis of cervical vertigo. NPY may be involved in the pathogenesis of cervical vertigo, providing a theoretical basis for the clinical diagnosis of cervical vertigo.

Knockdown of exosome­mediated lnc­PVT1 alleviates lipopolysaccharide­induced osteoarthritis progression by mediating the HMGB1/TLR4/NF­κB pathway via miR­93­5p.

Osteoarthritis is a chronic degenerative joint disease. Long non­coding RNA plasmacytoma variant translocation 1 (PVT1) is involved in the progression of osteoarthritis and exosomes serve a central role in intercellular communication. However, whether PVT1 can be mediated by exosomes in osteoarthritis has not been reported. Whole blood was drawn from osteoarthritis patients and healthy volunteers. Lipopolysaccharide (LPS) was used to stimulate human normal chondrocytes (C28/I2) to construct a cell damage model in vitro. Protein levels were examined via western blot analysis. eThe expression of PVT1, microRNA (miR)­93­5p and high mobility groupprotein B1 (HMGB1) was evaluated through reverse transcription­quantitative PCR. Cell viability and apoptosis were determined through CCK­8 or flow cytometric assay. Inflammatory cytokines were measured via ELISA. The relationship between PVT1 or HMGB1 and miR­93­5p was confirmed by dual­luciferase reporter assay. PVT1, HMGB1 and exosomal PVT1 were upregulated while miR­93­5p was downregulated in osteoarthritis patient serum and LPS­induced C28/I2 cells. Exosomes from osteoarthritis patient serum and LPS­treated C28/I2 cells increased PVT1 expression in C28/I2 cells. PVT1 depletion reversed the decrease of viability and the increase of apoptosis, inflammation responses and collagen degradation of C28/I2 cells induced by LPS. PVT1 regulated HMGB1 expression via sponging miR­93­5p. miR­93­5p inhibition abolished PVT1 silencing­mediated viability, apoptosis, inflammation responses and collagen degradation of LPS­stimulated C28/I2 cells. HMGB1 increase overturned miR­93­5p upregulation­mediated viability, apoptosis, inflammation responses and collagen degradation of LPS­stimulated C28/I2 cells. Furthermore, PVT1 modulated the Toll­like receptor 4/NF­κB pathway through an miR­93­5p/HMGB1 axis. In summary, exosome­mediated PVT1 regulated LPS­induced osteoarthritis progression by modulating the HMGB1/TLR4/NF­κB pathway via miR­93­5p, providing a new route for possible osteoarthritis treatment.

Functional Pathway Between Cervical Spinal and Sympathetic Ganglia: A Neurochemical Foundation Between Neck Pain and Vertigo.

BACKGROUND: Cervical vertigo commonly concurs in patients with neck pain, but the concurrent mechanism of these 2 symptoms still remains unclear. We previously reported a bidirectional segmental nerve fiber connection between cervical spinal and sympathetic ganglia, which provided a hypothesis that this connection between the 2 ganglia may be the anatomic basis for the concurrence of neck pain and cervical vertigo. However, this concurrent mechanism needs biochemical and functional evidence. OBJECTIVES: This study aimed to investigate a possible noradrenergic pathway between cervical spinal and sympathetic ganglia. STUDY DESIGN: We performed both clinical and laboratory research. Clinical observation was a prospective case-control study. SETTING: Clinical study took place in our hospital; laboratory study was in an orthopedic laboratory. METHODS: Cervical lamina block therapy used in patients with cervical vertigo was clinically evaluated; norepinephrine (NE) expressions in cervical sympathetic ganglia were analyzed using immunohistochemical staining after electrical stimulation to the cervical spinal ganglia; the influence of phentolamine local injection to the vertebrobasilar artery flow was experimentally measured. RESULTS: Cervical lamina block therapy could significantly shorten the clinical hospital stays of patients with cervical vertigo (P = 0.000) and improve vertebral artery flow (P < 0.05). NE expressions in superior cervical sympathetic ganglia (SCG) or inferior cervical sympathetic ganglia (ICG) increased significantly when ipsilateral C2 to C3 or C6 to C8 spinal ganglia were electrically stimulated, respectively. Adrenergic receptor block with phentolamine significantly inhibited the decrease of basilar artery (BA) flow induced by electrical stimulation of the cervical spinal ganglia. The change range of BA flow caused by stimulations of C2 to C3 and C6 to C8 spinal ganglia was more than that of C4 and C5. LIMITATIONS: The inpatients observed in this clinical study might be influenced by some factors including emotion, diet, sleep, and others. The limitations of the laboratory study included animal species and small sample size. CONCLUSIONS: Adrenergic system could play a part in cervical spinal ganglia altering the vertebrobasilar artery system. It could provide a neurochemical foundation between neck pain and vertigo, and that segmental functional connections exist between cervical spinal and sympathetic ganglia. KEY WORDS: Cervical vertigo, neck pain, cervical sympathetic ganglia, cervical spinal ganglia, noradrenaline.

The correlation between SNPs within the gene of adrenergic receptor and neuropeptide Y and risk of cervical vertigo.

BACKGROUND: The current investigation was aimed to explore the potential associations of SNPs within ADRB2, ADRB1, NPY, and ADRA1A with risk and prognosis of cervical vertigo. METHODS: Altogether 216 patients with cervical vertigo and 204 healthy controls were gathered, and their DNAs were extracted utilizing the whole-blood DNA extraction kit. Besides, the PCR reactions were conducted using the TaqManR single nucleotide polymorphism (SNP) genotyping assays, and the SNPs were detected on the 7900HT real-time fluorogenic quantitative polymerase chain reaction (PCR) instrument. Finally, the severity of cervical vertigo was classified according to the JOA scoring, and the recovery rate (RR) of cervical vertigo was calculated in light of the formula as: [Formula: see text] RESULTS: The SNPs within ADRA1A [rs1048101 (T>C) and rs3802241 (C>T)], NPY [rs16476 (A>C), rs16148 (T>C), and rs5574 (C>T)], ADRB1 [rs28365031 (A>G)] and ADRB2 [rs2053044 (A>G)] were all significantly associated with regulated risk of cervical vertigo (all P < .05). Haplotypes of ADRA1A [CT and TC] and NPY [CCT and ATT] were also suggested as the susceptible factors of cervical vertigo in comparison with other haplotypes. Furthermore, the SNPs within ADRA1A [rs1048101 (T>C)], NPY [rs16476 (A>C), rs16148 (T>C)], as well as ADRB1 [rs28365031 (A>G)] all appeared to predict the prognosis of cervical vertigo in a relatively accurate way (all P < .05). Ultimately, the haplotypes of ADRA1A (CC) and NPY (CCT) tended to decrease the RR. CONCLUSIONS: The SNPs within ADRB2, ADRB1, NPY, and ADRA1A might act as the diagnostic biomarkers and treatment targets for cervical vertigo.

The impact of CYP2D6 polymorphisms on the pharmacokinetics of codeine and its metabolites in Mongolian Chinese subjects.

PURPOSE: Codeine is an analgesic drug acting on µ-opioid receptors predominantly via its metabolite morphine formed almost exclusively by CYP2D6. Genetic polymorphisms in CYP2D6 are associated with diminished pain relief and/or severe opioid side effects. In Chinese individuals, CYP2D6*10 is the most common allele with reduced enzyme activity. In this study, we investigated the effect of this allele on the pharmacokinetics of codeine and its metabolites. METHOD: A blood sample was collected from healthy Mongolian volunteers for CYP2D6 genotyping using a PCR-RFLP assay. A pharmacokinetic study was then carried out in three groups with CYP2D6*1/*1 (n=10), CYP2D6*1/*10 (n=10) and CYP2D6*10/*10 (n=9) genotypes by collecting serial blood samples for determination of plasma levels of codeine and its metabolites, morphine, morphine 3-glucuronide (M3G) and morphine 6-glucuronide (M6G) before and after a single 30-mg oral dose of codeine phosphate. Codeine and its metabolites were measured by LC-MS/MS. RESULTS: No significant differences were observed in the pharmacokinetic parameters of codeine in the three genotype groups. However, the C( max) and AUC(0-∞) of morphine, M3G and M6G were significantly different between the study groups (P<0.05). Compared with the *1/*1 group, the AUC(0-∞) for morphine in the *1/*10 and *10/*10 groups decreased by ratios (95 % CI) of 0.93 (0.26-1.59) and 0.494 (0.135-0.853) respectively. Corresponding ratios for M3G were 0.791 (0.294-1.288) and 0.615 (0.412-0.818) and for M6G were 0.643 (0.39-0.957) and 0.423 (0.267-0.579). CONCLUSION: This study demonstrates that the CYP2D6*10 allele plays an important role in the pharmacokinetics of the O-demethylated metabolites of codeine after oral administration.

Neural reflex pathway between cervical spinal and sympathetic ganglia in rabbits: implication for pathogenesis of cervical vertigo.

BACKGROUND CONTEXT: A functional association between cervix and vertigo has been observed in patients with cervical vertigo, implicating correlation between cervical spinal and sympathetic ganglia. However, it is unclear where there is an anatomic connection between those two groups of ganglia. PURPOSE: This study aimed to investigate the existence of the neural connections between cervical spinal and sympathetic ganglia. STUDY DESIGN/SETTING: FluoroGold staining patterns in cervical spinal and sympathetic ganglia were evaluated using FluoroGold retrograde tracing in New Zealand rabbits. METHODS: New Zealand rabbits were randomly divided into superior cervical spinal ganglion injection groups, inferior cervical spinal ganglion injection groups, superior cervical sympathetic ganglion injection group, and inferior cervical sympathetic ganglion injection group. Four percent FluoroGold solution was injected into these ganglia. Distribution of FluoroGold in cervical spinal and sympathetic ganglia was observed under a microscope. RESULTS: When FluoroGold solution was injected into C2 and C3 superior cervical spinal ganglia or C5-C6 inferior cervical spinal ganglia, fluorescence was only observed in the ipsilateral superior or inferior cervical sympathetic ganglia, respectively. When FluoroGold solution was injected into superior or inferior cervical sympathetic ganglia, fluorescence was found mainly in the ipsilateral C3-C4 superior or C5-C8 inferior spinal ganglia. No fluorescence was observed in contralateral ganglia of experimental animals and all ganglia of matched control animals injected with physiological saline. CONCLUSIONS: Bidirectional nerve fiber connections between cervical spinal and sympathetic ganglia were observed, and these connections are arranged in a segmental distribution. This observation may provide a possible neuroanatomic basis for the pathogenesis of cervical vertigo.

CYP2C8 polymorphism frequencies among Han, Uighur, Hui, and Mongolian Chinese populations.

PURPOSE: To determine the allele frequency of three codon-changing variants (CYP2C8*2, CYP2C8*3, and CYP2C8*4) in the Han, Uighur, Hui, and Mongolian Chinese populations, and compare genetic polymorphism differences between the Han and minority Chinese ethnicities. METHODS: Five hundred seventy four healthy unrelated volunteers from four major nationalities (Han: 136; Uighur: 153; Hui: 158; Mongolian: 127) in China were recruited. The study was approved by the local research ethics committee. DNA was extracted from peripheral leukocytes using a standard protocol. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to detect three alleles of CYP2C8. PCR results were confirmed by subsequent direct DNA sequencing. RESULTS: The three minorities showed the CYP2C8 polymorphism with an allele frequency as follows: the allele frequencies of CYP2C8*2, CYP2C8*3, and CYP2C8*4 were 0.33%, 2.94%, and 2.29% in Uighur; 0.39%, 1.57%, and 1.18% in Mongolian, and 0%, 1.58%, and 0.63% in Hui, respectively. For Han, CYP2C8*2, CYP2C8*3, and CYP2C8*4 were absent. CONCLUSION: To the best of our knowledge, the current study described polymorphisms of CYP2C8 in Chinese minorities for the first time. The results showed that there were significant ethnic differences in the distribution of CYP2C8 in the Han and three minorities, that is, the Uighur, Hui, and Mongolian Chinese populations.

Genetic polymorphisms of drug-metabolizing phase I enzymes CYP3A4, CYP2C9, CYP2C19 and CYP2D6 in Han, Uighur, Hui and Mongolian Chinese populations.

We randomly evaluated 672 unrelated, healthy Chinese volunteers (136 Han, 214 Uighur, 164 Hui and 158 Mongolian) to compare CYP3A4, CYP2C9, CYP2C19 and CYP2D6 allele frequencies. Genomic DNA was extracted from peripheral leukocytes and genotyped for CYP3A4*5, CYP3A4*18, CYP2C9*2, CYP2C9*13, CYP2C19*2, CYP2C19*3 and CYP2D6*10 by PCR-restriction fragment length polymorphism analysis (PCR-RFLP). Our results showed that there is no significant difference in the distribution of CYP2C19*3 and CYP3A4*18 genotypes in the Han, Uighur, Hui and Mongolian Chinese populations. The CYP2C9*13/*13 and CYP3A4*5 genotypes were not observed in any of the four Chinese populations. We found a higher incidence of the CYP2C9*2 allele in Uighur populations, compared to the Han, Hui and Mongolian populations. The incidence of the CYP2C19*2 allele in the Han population was not significantly different from that in the Uighur, Hui or Mongolian populations; however, the Uighur population showed significantly lower rates of this allele than the Hui and Mongolian populations, and the Mongolian population had a significantly lower incidence of this allele than the Hui population. There was no significant difference in the presence of the CYP2D6*10 allele in the Mongolian, Han or Hui populations. However, the Uighur population showed significantly lower rates of this allele than the other three populations. These findings provide basic genetic information for further pharmacogenomic investigations in the Chinese population.

[Bone graft repair of adolescence spondylolysis with lag screw and tension band fixation].

OBJECTIVE: To investigate direct bone graft repair with lag screw and tension band fixation technique and its value for the treatment of adolescence spondylolysis. METHODS: Lysis was prepared and the fibrous tissue within the gap was removed in 12 patients from 12 to 26 years (average 18.4). 1 - 2 mm extra bony element was resected on both sides with fresh interface with the bone graft harvested from the iliac crest. From the entry point 8 mm away from the midline at the inferior margin of the lamina, titanium lag screws of 35 - 45 mm long and 3.5 mm in diameter were placed at 300 upward and outward and tighted through the lysis, bone graft and the superior and lateral aspect of the pedicle. Extra match bone graft was placed around the surface of defect, an high intensity Nilon wire tension band between the cap of the screw and the basis of transverse process was constructed before wound closure and suction drainage. The patients were immobilized with plaster brace of Paris for 2 months. RESULTS: Average operation time was (10 +/- 55) minutes, and average blood loss was 170 ml. Follow-up ranged from 12 to 36 months (mean 17 months), and cases of 22 lysis healed within 3 months. CONCLUSIONS: Technique of direct bone graft repair with lag screw and tension band fixation for the treatment of adolescence spondylolysis is simple, safe and reliable. Combined biomechanical and biological processes, it is less invasive but advantageous in preserving the motion in the affected segment.