| Objective: To evaluate the effects of DOR-GLT-1/GS-NMDA receptor pathway on remifentanil–induced hyperalgesia and explore the protective effects of hydrogen. Contents: To prove DOR-GLT-1/GS-NMDA receptor pathway play an important role in remifentanil-induced hyperalgesia, two modelswere made, one is the incisionremifentanil induced hyperalgesia rat models(in vivo study) and the other one is the spinal cord slices incubation mode. This study also explored the protective effects of hydrogenthrough repairing GLT-1, GS and NMDA receptor function. This finding provides new ideas for clinical prevention and treatment of hyperalgesia.Methods: The study was mainly divided into 3 parts: Part I: 40 SD rats(male) were divided into 5 groups randomly,and every group contains 8 rats: control group(C): Normal saline(NS) was infused; incision pain group(I): Made a incision with NS infusion; glycine group(G): glycine was infused at 15μg·kg-1·min-1 with incision; remifentanil group(R): remifentanil was infused; remifentanil+incision pain group(RI): remifentanil was infused with incision; NS or remifentanil infusion dose was 0.1ml·kg-1·min-1 or 1.0μg·kg-1·min-1, infusion time is 60 min. The PWT and PWL were detected at 1 day before and 2h, 6h, 24 h, 48 h after infusion. The L4-6 spinal cords were removed after the last behavioral test for measuring DOR and NMDA receptor membrane and total protein, Mn SOD, GLT-1, GS and NR1, NR2 A, NR2 B subunit protein nitration. Part II: In vivo experiments: 32 male SD rats were randomly divided into 4 groups(n=8): DOR agonist deltorphin II or inhibitor naltrindole+RI group(D+RI or N+RI group): 4n M deltorphin II or 30 n M naltrindole was injected intrathecally 10 minutes before remifentanil infusion, and then underwent incision; The expression of Mn SOD, GLT-1, GS protein nitration and NMDA receptor NR1, NR2 B protein nitration, membrane and total proteins were measured. In vitro experiments: 40 male SD rats(14-18 postnatal days) were randomly divided into 5 groups(n = 8): Group C: L4-6 slices were only incubated in ACSF(artificial cerebrospinal fluid) without drugs; Group G or group R: spinal cord slices were incubated in ACSF containing 0.24 μmol/L glycine or 4nmol/L remifentanil; RD or RN group: spinal cord slices were incubated in ACSF containing 4nmol/L remifentanil and 10nmol/L DeltorphinⅡ or 1nmol/L naltrindole. After 60 min incubation, NMDA receptor mediated m EPSC was measured. Part III: 48 male SD rats were randomly divided into 6 groups(n = 8): hydrogen enriched saline(HS group): 10ml/kg hydrogen enriched saline(HS) was administrated 10 minutes before 0.1 ml·kg-1·min-1 NS infusion. HS+RI group: 1ml/kg, 3ml/kg or 10ml/kg HS was infused intraperitoneally 10 minutes before remifentanil;Others are the same as above. The Mn SOD, GLT-1, GS and NR1, NR2 B subunit protein nitration were assessed. Results The PWT and PWL were markedly decreased in I, R and RI group comparing with group C; meanwhile, DOR and NR1, NR2 B membrane and total protein expression were increased, Mn SOD, GLT-1, GS and NR1, NR2 B subunit protein nitration. DOR inhibitor significantly inhibited hyperalgesia, decreased Mn SOD, GLT-1, GS and NR1, NR2 B subunit protein nitration, inhibited NR1 and NR2 B membrane trafficking. remifentanil induced NMDAR-mediated m EPSCs amplitude enhancement and increased frequency. HS attenuated remifentanil induced hyperalgesia in a dose-dependent manner, decreased Mn SOD, GLT-1, GS and NR1, NR2 B subunit protein nitration levels. Conclusion remifentanil may increase DOR membrane expression, causing GLT-1 and GS disfunction, and lead to changes in NMDA receptor expression and function, Eventually result in hyperalgesia. HS attenuates remifentanil induced hyperalgesia probably through repairing GLT-1, GS and NMDA receptor function alleviate hyperalgesia. |
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