ABSTRACT
Objective The activation of P2X7 receptor in ventrolateral periaqueductal gray (vlPAG) is involved in the formation and maintenance of bone cancer pain (BCP). This study will establish a rat model of BCP and observe the effect of the activation of P2X7 receptor in vlPAG on D-serine level through brain microdialysis combined with ELISA. Methods Forty-two female SD rats were divided into four groups by random number table: normal control group (n=12), sham group (n=12), BCP group (n=12) and P2X7 receptor antagonist group (n= 6). The model of metastatic BCP in the tibias of the rats was established in the BCP group, and 20μL of RPMI-1640 medium cell suspension containing SHZ-88 breast cancer cells was injected (1×107 cancer cells/0.5 mL). The sham group was injected with treated cancer cells of the same volume (SHZ-88 breast cancer cells were kept in boiling water at 90 ℃ for 20 min), and the rest of the operation was the same as the BCP group. The normal control group received no treatment. The P2X7 receptor antagonist group was treated the same as the BCP group, except that the P2X7 receptor-specific antagonist A-438079 was added to the perfusion solution. The thermal pain threshold and mechanical pain threshold were detected at the same time in the normal control group, the sham group and the BCP group. The positive expression of P2X7 receptor in vlPAG of rats was detected by immunohistochemistry in each group in 21 days. The changes of D-serine in vlPAG dialysate were detected by ELISA in each group. Results The mechanical pain threshold and thermal pain threshold of the rats in BCP group on Day 5, 7, 10, 14, 18 and 21 were lower than those of the normal control group and sham group (P<0.01). The positive expression of P2X7 was scattered in vlPAG in normal control group and sham group. The number of P2X7 receptor positive cells in the BCP group was significantly higher than that in the control group and sham group (P<0.01). The content of D-serine in vlPAG of the rats in BCP group [(220.28±63.38)ng/mL] was significantly higher than that in the control group [(148.09±46.89)ng/mL] and the sham group [(147.32±51.44)ng/mL] (P<0.05). The content of D-serine in vlPAG [(134.20±41.77)ng/mL] in P2X7 receptor antagonist group was significantly lower than that in BCP group (P<0.05). Conclusion The activation of the P2X7 receptor in ventrolateral periaqueductal gray promotes D-serine release and participates in the mechanisms of BCP in rats .
ABSTRACT
Objective The main intracellular signal of P2X7 receptor activation is the increasing of Ca2+, which then presents the diversity of its physiological and pathological functions through multiple intracellular signal transduction. To observe the effect of activation of P2X7 receptor on intracellular calcium（Ca2+）level in lateral midbrain periaqueductal gray (lPAG) neurons of primary cultured rat. Methods The primary cultured lPAG neurons were randomly divided into 4 groups: control group(no drug added), only for control; BzATP group(100 μmol/L); A-740003+ BzATP group(incubate with 100 nmol/L A-740003 for 10 min, then add 10 μmol/L ofBzATP); BzATP control group(add in Ca2+-free solution for 20 min, then add BzATP). The incubation solution of control group, BzATP group and A-740003+ BzATP group are DMEM/F12 medium, and the BzATP control group is Ca2+-free. The laser scanning confocal microscopy (LSCM) was used to detect : the changes of cultured neuron Ca2+ levels by different concentrations of BzATP; the effects of A-740003 and Ca2+-free medium preincubation on BzATP-induced Ca2+ level alterations in cultured neurons. Results BzATP dose-dependently increased the Ca2+ levels in cultured lPAG neurons; A-740003 and Ca2+-free medium inhibited the BzATP-induced increasing of Ca2+ level in cultured lPAG neurons. LSCM showed: The intracellular calcium fluorescence insensity(2.48±1.05) in the BzATP group was significantly higher than that in the blank control group, BzATP control group and A-740003+ BzATP group[(1.12±0.03), (1.09±0.03), (1.14±0.08)](P<0.01). Conclusion The activation of P2X7 receptor can increase the level of lPAG neurons Ca2+ , and is associated with the extracellular Ca2+ influx.
ABSTRACT
Objective To establish a method for the determination of the content and dissolution of isophenylcyclopentyl?amine hydrochloride capsules. Methods The HPLC analysis was performed on a Diamonsil C18 column(150 mm×4.6 mm,5μm). A mixture of 0.02 mol/L KH2PO4 solution containing 0.1%triethylamine with pH adjusted to 3.0 by phosphoric acid-methanol-acetonitrile (30:35:35)was used as the mobile phase with the flow rate at 1.0 ml/min. The detection wavelength was 224 nm. Dissolution was de?termined by the basket method,using the 500 ml of 0.1 mol/L hydrochloric acid solution,pH 4.5 acetic acid buffer,water and pH 6.8 phosphoric acid buffer as dissolution media under the 50,75 and 100 r/min rotation speeds to select the dissolution condition. Re?sults This method had high specificity. The linear range for the quantitative determination was 20.74-155.58 μg/ml(r=1.0000), and the average recovery was 100.1%. The linear range for the determination of dissolution was 2.08-24.90μg/ml(r=0.9998),with the average recovery of 98.9%. The method of dissolution tests was established:0.1 mol/L hydrochloric acid solution was used as disso?lution medium and rotation speed was 50 r/min. The determined content and dissolution of three batches of capsules fulfilled the re?quirements. Conclusion The method is simple,accurate and reproducible for the determination of the content and dissolution of iso?phenylcyclopentylamine hydrochloride capsules.