Sinomenine attenuates chronic inflammatory pain in mice.

Sinomenine, an alkaloid originally isolated from the roots of Sinomeniumacutum, is used as a traditional Chinese medicine for rheumatic arthritis. However, little is known about the neuronal mechanisms underlying the analgesic effects of sinomenine in animals with chronic inflammatory pain. In this study, we investigated the persistent inflammatory pain induced by hind paw injection of complete Freund's adjuvant (CFA) in mice, which was reversed by sinomenine administration. In the anterior cingulate cortex (ACC), a region highly associated with chronic pain processing, the upregulation of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors and Ca2+/calmodulin-dependent protein kinase II, total levels of GluA1, and phosphorylation of GluA1 at Ser831 (p-GluA1-Ser831) were reversed by systemically administrating sinomenine. Furthermore, sinomenine treatment downregulated the mammalian target of rapamycin (mTOR) pathway. Increases in p-mTOR, p-p70S6k, p-S6, and p-4EBP, which were induced by chronic inflammation, were all changed. However, sinomenine did not affect the levels of GluN2A-containing NMDA receptors and p-GluA1-Ser845, as well as the total levels of mTOR, p70S6k, S6, and 4EBP. In conclusion, results indicated that sinomenine reduced the chronic inflammatory pain induced by CFA, at least partially by regulating the GluN2B receptors and mTOR signals in the ACC.

Analgesic effects of adenylyl cyclase inhibitor NB001 on bone cancer pain in a mouse model.

BACKGROUND: Cancer pain, especially the one caused by metastasis in bones, is a severe type of pain. Pain becomes chronic unless its causes and consequences are resolved. With improvements in cancer detection and survival among patients, pain has been considered as a great challenge because traditional therapies are partially effective in terms of providing relief. Cancer pain mechanisms are more poorly understood than neuropathic and inflammatory pain states. Chronic inflammatory pain and neuropathic pain are influenced by NB001, an adenylyl cyclase 1 (AC1)-specific inhibitor with analgesic effects. In this study, the analgesic effects of NB001 on cancer pain were evaluated. RESULTS: Pain was induced by injecting osteolytic murine sarcoma cell NCTC 2472 into the intramedullary cavity of the femur of mice. The mice injected with sarcoma cells for four weeks exhibited significant spontaneous pain behavior and mechanical allodynia. The continuous systemic application of NB001 (30 mg/kg, intraperitoneally, twice daily for three days) markedly decreased the number of spontaneous lifting but increased the mechanical paw withdrawal threshold. NB001 decreased the concentrations of cAMP and the levels of GluN2A, GluN2B, p-GluA1 (831), and p-GluA1 (845) in the anterior cingulate cortex, and inhibited the frequency of presynaptic neurotransmitter release in the anterior cingulate cortex of the mouse models. CONCLUSIONS: NB001 may serve as a novel analgesic to treat bone cancer pain. Its analgesic effect is at least partially due to the inhibition of AC1 in anterior cingulate cortex.

Anxiolytic-like effects of translocator protein (TSPO) ligand ZBD-2 in an animal model of chronic pain.

The activation of Translocator protein (18 kDa) (TSPO) has been demonstrated to mediate rapid anxiolytic efficacy in stress response and stress-related disorders. This protein is involved in the synthesis of endogenous neurosteroids that promote γ-aminobutyric acid (GABA)-mediated neurotransmission in the central neural system. However, little is known about the functions and the underlying mechanisms of TSPO in chronic pain-induced anxiety-like behaviors. The novel TSPO ligand N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2) was used in the present study. We found that ZBD-2 (0.15 or 1.5 mg/kg) significantly attenuated anxiety-like behaviors in mice with chronic inflammatory pain induced by hindpaw injection of complete Freund's adjuvant (CFA). However, the treatment did not alter the nociceptive threshold or inflammation in the hindpaw. Hindpaw injection of CFA induced the upregulation of TSPO, GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and NR2B-containing N-methyl-D-aspartate (NMDA) receptors in the basolateral amygdala (BLA). ZBD-2 administration reversed the alterations of the abovementioned proteins in the BLA of the CFA-injected mice. Electrophysiological recording revealed that ZBD-2 could prevent an imbalance between excitatory and inhibitory transmissions in the BLA synapses of CFA-injected mice. Therefore, as the novel ligand of TSPO, ZBD-2 induced anxiolytic effects, but did not affect the nociceptive threshold of mice under chronic pain. The anxiolytic effects of ZBD-2 were related to the regulation of the balance between excitatory and inhibitory transmissions in the BLA.

Antidepressant-Like and Anxiolytic-Like Effects of ZBD-2, a Novel Ligand for the Translocator Protein (18 kDa).

Activation of translocator protein (18 kDa) (TSPO) plays an important role to mediate rapid anxiolytic efficacy in stress response and stress-related disorders by the production of neurosteroids. However, little is known about the ligand of TSPO on the anxiety-like and depressive behaviors and the underlying mechanisms in chronic unpredictable mild stress (UCMS) mice. In the present study, a novel ligand of TSPO, ZBD-2 [N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide] synthesized by our laboratory, was used to evaluate the anxiolytic and antidepressant efficacy and to elucidate the underlying mechanisms. ZBD-2 (3 mg/kg) significantly attenuated anxiety-like and depressive behaviors in the UCMS mice, which was blocked by TSPO antagonist PK11195 (3 mg/kg). Treatment of ZBD-2 reversed the decrease in biogenic amines (norepinephrine, dopamine, and serotonin) in the brain region of hippocampus in the UCMS mice. The decreases in TSPO, GluN2B-containing N-methyl-D-aspartate (NMDA) receptors, GluA1, p-GluA1-Ser831, p-GluA1-Ser845, PSD-95, and GABAA-a2 were integrated with the increases of CaMKII and iNOS levels in the hippocampus of the UCMS mice. ZBD-2 significantly reversed the changes of above proteins. However, ZBD-2 or PK11195 treatment did not affect the levels of GluN2A-containing NMDA receptors and the total levels of GAD67. Our study provides strong evidences that ZBD-2 has a therapeutic effect on chronic stress-related disorders such as depression and anxiety through regulating the biogenic amine levels and the synaptic proteins in the hippocampus.

G-1 exerts neuroprotective effects through G protein-coupled estrogen receptor 1 following spinal cord injury in mice.

Spinal cord injury (SCI) always occurs accidently and leads to motor dysfunction because of biochemical and pathological events. Estrogen has been shown to be neuroprotective against SCI through estrogen receptors (ERs), but the underlying mechanisms have not been fully elucidated. In the present study, we investigated the role of a newly found membrane ER, G protein-coupled estrogen receptor 1 (GPR30 or GPER1), and discussed the feasibility of a GPR30 agonist as an estrogen replacement. Forty adult female C57BL/6J mice (10-12 weeks old) were divided randomly into vehicle, G-1, E2, G-1 + G-15 and E2 + G-15 groups. All mice were subjected to SCI using a crushing injury approach. The specific GPR30 agonist, G-1, mimicked the effects of E2 treatment by preventing SCI-induced apoptotic cell death and enhancing motor functional recovery after injury. GPR30 activation regulated phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK/extracellular signal-regulated kinase (ERK) signalling pathways, increased GPR30 and anti-apoptosis proteins Bcl-2 and brain derived neurotrophic factor (BDNF), but decreased the pro-apoptosis factor Bax and cleaved caspase-3. However, the neuroprotective effects of G-1 and E2 were blocked by the specific GPR30 antagonist, G-15. Thus, GPR30 rather than classic ERs is required to induce estrogenic neuroprotective effects. Given that estrogen replacement therapy may cause unexpected side effects, especially on the reproductive system, GPR30 agonists may represent a potential therapeutic approach for treating SCI.

[The effect of inhibition of hyperplasia on spinal cord reactive astrocytes by mild hypothermia].

OBJECTIVE: To observe changes in the morphology and activity of astrocytes in spinal cord at different temperatures after scratches treatment, and to investigate the effect of mild hypothermia on hyperplasia of reactive astrocytes. METHODS: Spinal cord astrocytes were cultured from the neonatal SD rat, and reactive astrocytes were prepared by scratches treatment. Mild hypothermia choose 33℃, cell cul-ture 48 h. Cells were divided into control group、scratches group、mild hypothermia group and scratch+mild hypothermia group. Cell mor-phology of each group were observed in 0 h、6 h、12 h、24 h、48 h、3 d、5 d、7 d. Nestin positive rate were detected by using immunofluorescence staining method. Cell activity were observed by methyl thiazolyl tetrazolium salt (MTT) assay. The degree of apoptosis was observed by using PI staining method. RESULTS: Compared with control group and mild hypothermia group, cell body in both scratches group and scratch+mild hypothermia group was hypertrophy, the protrusion around was increased and extend, the cytoplasm was enrich, and the growth rate was signif-icantly increased. Compared with scratches group, scratch+mild hypothermia group cells grew slowly, the protrusion around was decreased, and cells of the scratched area ingrowth significantly slowly. Nestin and PI positive rate of cells were also significantly lower. All the results had statistically significant differences (P<0.01). CONCLUSIONS: After scratch injury, astrocytes were activated to be reactive astrocytes and hyper-plasia. Mild hypothermia significantly inhibited spinal cord reactive astrocytes overgrowth and restrain astrocytes apoptosis.

Osteoprotective Effect of Echinocystic Acid, a Triterpone Component from Eclipta prostrata, in Ovariectomy-Induced Osteoporotic Rats.

Echinocystic acid (EA) is a natural triterpone enriched in various herbs and has been used for medicinal purposes in China. In the present study, we systematically examined the effects of EA on ovariectomy-induced osteoporosis in rats for the first time. Three-month-old female ovariectomy (OVX) Sprague-Dawley rats were used to evaluate the osteoprotective effect of EA. Results showed that administration of EA (5 or 15 mg/kg/day) for 12 weeks prevented lower levels of maximum stress and Young's modulus of femur induced by OVX. EA also recovered bone metabolic biomarkers levels in OVX rats, including osteocalcin, alkaline phosphatese, deoxypyridinoline, and urinary calcium and phosphorus. EA (5 and 15 mg/kg/day) could prevent the alteration of total bone mineral density in the femur caused by OVX. However, only high dose (15 mg/kg/day) of EA significantly improved trabecular architecture, as evidenced by higher levels of bone volume/tissue volume, trabecula number, and trabecula thickness, and lower levels of trabecula separation and structure model index compared with OVX rats. In addition, EA treatment decresed the serum levels of IL-1ß and TNF-α in OVX rats. In conclusion, EA could prevent reduction of bone mass and strength and improve the cancellous bone structure and biochemical properties in OVX rats. Hence, EA may serve as a new candidate or a leading compound for anti-osteoporosis.

Polymorphisms in TP53 are associated with risk and survival of osteosarcoma in a Chinese population.

Osteosarcoma (OS) is the most frequent histological form of primary bone cancer in adolescence. TP53 is a tumor suppressor gene which is essential for regulating cell division and preventing tumor formation. The purpose of this study is to examine whether genetic mutations in the TP53 gene are associated with OS risk and survival in a Chinese population. Five polymorphisms in the TP53 gene were selected in a case-control study, including 210 OS patients and 420 cancer-free controls. We found that subjects carrying rs12951053 CC genotype and rs1042522 GG genotype were significantly associated with risk of OS [odds ratio (OR)=1.68, 95% confidence intervals (CI): 1.05-2.68; OR=1.89, 95% CI: 1.16-3.07] compared with subjects carrying the common genotypes. Results of haplotype analysis also showed that A-G-G-A-C haplotype (rs12951053, rs1042522, rs8064946, rs9895829 and rs12602273) conferred significant decreased risk of OS (OR=0.37, 95% CI: 0.19-0.72) compared with A-C-G-A-C haplotype. Besides, rs1042522 was an independent prognostic factor for OS with hazard radio (HR)=1.94 (95% CI: 1.03-3.65) in GG genotype than in CC genotype. Our data suggest that genetic mutations in the TP53 gene are associated with risk and survival of OS in Chinese population.

Exchanging reamed nailing versus augmentative compression plating with autogenous bone grafting for aseptic femoral shaft nonunion: a retrospective cohort study.

OBJECTIVE: The purpose of this study was to compare the outcomes of exchanging reamed nailing (ERN) and augmentative compression plating (ACP) with autogenous bone grafting (BG) for the treatment of aseptic femoral shaft nonunion secondary to the treatment of intramedullary nailing (IMN). METHODS: A multicenter retrospective study was performed for 178 patients (180 cases) of aseptic femoral shaft nonunion secondary to first treatment of IMN. All cases were fixed with either ERN (n=87) or ACP (n=93). In the ERN group, 42 cases (48.3%) were nonisthmal nonunions and 45 (51.7%) were isthmal nonunions. In the ACP group, 46 cases (49.5%) were nonisthmal nonunions, and 47 (50.5%) were isthmal nonunions. Operation time, blood loss, time to union, union rate, volume of drainage, time to renonunion, and complication rate were compared between the 2 groups. RESULTS: All patients were followed up, with a mean period of 4.1 years (range: 1-7.1 years). Bone union occurred in 93/93 cases (100%) in the ACP group versus 75/87 cases (86.2%) in the ERN group (odds ratio [OR]=3.28, 95% confidence interval [CI] 0.8-14). Of the 12 cases involved with renonunion in the ERN group, 10 were nonisthmal nonunions, and 2 were isthmal nonunions with cortical bone defect >3 cm. The union time, blood loss, and complication rate of the ERN group were significantly higher than those of the ACP group (p=0.028, p=0.035, and p=0.021, respectively). No significant difference was found in the average operation time of the 2 groups (p=0.151). However, for the nonisthmal nonunions, a significant difference was found between the ERN and ACP groups (p=0.018). CONCLUSION: ACP with autogenous BG can obtain a higher bone union rate and shorter time to union than ERN in the treatment of aseptic femoral shaft nonunion after failed IMN. Especially for nonisthmal femoral shaft nonunions or isthmal nonunions with larger bone defects, ACP with autogenous BG can be more advantageous than ERN for patients. A future prospective observational study should be conducted.

Osteoprotective effect of combination therapy of low-dose oestradiol with G15, a specific antagonist of GPR30/GPER in ovariectomy-induced osteoporotic rats.

Identified and cloned in 1996 for the first time, G protein-coupled oestrogen receptor (ER) 30 (GPR30/GPER) has been a hot spot in the field of sex hormone research till now. In the present study, we examined the effects of low-dose oestradiol (E2) combined with G15, a specific antagonist of GPR30 on ovariectomy (OVX)-induced osteoporosis in rats. Female Sprague-Dawley (SD) rats undergoing OVX were used to evaluate the osteoprotective effect of the drugs. Administration of E2 [35 µg/kg, intraperitoneally (ip), three times/week) combining G15 (160 µg/kg, ip, three times/week) for 6 weeks was found to have prevented OVX-induced effects, including increase in bone turnover rate, decrease in bone mineral content (BMC) and bone mineral density (BMD), damage of bone structure and the aggravation in biomechanical properties of bone. The therapeutic effect of these two drugs in combination was better than that of E2 alone. Meanwhile, the administration of G15 prevented body weight increase or endometrium proliferation in the rats. In conclusion, administration of low-dose E2 combining G15 had a satisfactory bone protective effect for OVX rats, without significant influence on body weight or the uterus. This combination therapy may be an effective supplement of drugs in prevention and treatment for postmenopausal osteoporosis.

Osteoprotective effects of estrogen membrane receptor GPR30 in ovariectomized rats.

G protein-coupled estrogen receptor 30 (GPR30) is expressed in bone tissue. However, little is known regarding the function of GPR30 in postmenopausal osteoporosis. In this study, we examined the effects of GPR30 on ovariectomy (OVX)-induced osteoporosis in rats, including the effects on proliferation, differentiation, and expression of proteins in osteoblasts. Administration of G1 (35 µg/kg, ip, 3 times/week for 6 weeks), a specific agonist of GPR30, prevented OVX-induced increase in bone turnover rate, decrease in bone mineral content and bone mineral density, damage to bone structure, and aggravation of bone biomechanical properties. In addition, G1 did not affect uterine weight in the OVX rats. Osteoblasts isolated from calvarias from newborn rats were used to explore the underlying mechanisms. G1 (150 pM) promoted proliferation and differentiation of osteoblasts through a positive feedback of GPR30, which then activated the PI3K-Akt, ERK, and CREB pathways. G15 (750 pM), a specific antagonist of GPR30, reversed the above effects initiated by G1 treatment. In conclusion, activation of GPR30 protected bones against osteoporosis in OVX rats and exerted no untoward effect on the uterus. We suggest that GPR30 can be used as an effective therapeutic target for the prevention and treatment of postmenopausal osteoporosis.