脊髓内源性硫化氢在大鼠糖尿病神经痛维持中的作用

Role of endogenous hydrogen sulfide of the spinal cord in the maintenance of diabetic neuropathicpain in rats

目的观察脊髓内源性硫化氢（H2S）在大鼠糖尿病神经痛维持中的作用。方法雄性SD大鼠（体质量150-170g）48只，其中24只大鼠腹腔注射链脲霉素（60mg/kg）制备成糖尿病神经痛（DNP）模型，其余24只大鼠腹腔注射等体积生理盐水作为对照。48只大鼠均于腹腔注射2周后行鞘内置管，然后将DNP模型大鼠随机分为两组（n=12）：D组和DM组，将正常对照大鼠分成两组（n=12）：c组和cM组；腹腔注射后3周，c组、D两组鞘内给予生理盐水10μl；CM、DM组鞘内给予10μmoL/L高铁血红蛋白10μl，每日1次，连续4d，分别于鞘内注射第l天（T1）、第2天（他）、第3天（T3）、第4天（T4）4个时间点，在给药前后30min测定各组大鼠50％机械缩足阈值（PwT），并于．r4测量结束后处死大鼠，取腰骶膨大处脊髓，测定脊髓背角N-甲基-D．天冬氨酸受体2B亚基（NR2B）mRNA和蛋白表达。结果与c组和cM组比较，D组和DM组大鼠PwT降低，NR2BmRNA和蛋f1表达上调（尸〈0．05）；与D组PwT比较，DM组大鼠在T2～T4时间点的给药前、T1～T4时间点的给约后PwT升高；与D组mRNA（O．89±O．09）和蛋白表达（1．28±O．17）比较，DM组大鼠脊髓背角NR2BmRNA（0．69±0．11）和蛋白表达（0．98±0．14）下调（P〈0．05）；与给药前比较（3．8±0．9、6．1±1．4），DM组大鼠在T1～T2时间点给药后PWT（7．5±1．8、8．7±1．6）升高（P〈0．05）。结论脊髓内源性H2S参与了大鼠糖尿病神经痛的维持，其机制与上调脊髓背角NR2B表达有关。

Objective To evaluate the role of endogenous hydrogen sulfide in the maintenance of diabetic neuropathic pain （DNP） in rats. Methods The DNP models were established by intraperitoneal injection of stretopzin （STZ） in 24 male SD rats aged 4 weeks （ 150-170 g）, and other 24 rats were intrap eritoneally injected with the same volume of normal saline as normal group. All the rats were implanted in trathecal catheter. The rats with DNP were randomly divided into 2 subgroups （n = 12 each） ： saline group （ group D） and methemoglobin group （ group DM）. The rats in normal group were randomly divided into 2 subgroups （n = 12 each）： saline group （group C） and methemoglobin group （group CM）. Three weeks after intraperitoneal injection, the rats were separately intratheeally administered methemoglobin 10 μmol/L （ 10 μl） （group DM）, saline 10 μl （group D）, methemoglobin 10 μmol/L （10 μl） （group CM） and sa line 10μl （group C）, respectively, once a day for 4 days. The paw withdrawal mechanical threshold （DWT） of the rats were measured before and after injection at 1 （ T1 ）, 2 （ T2 ）, 3 （ T3 ）, 4 （ T4 ） days, then the rats were sacrificed at T4 point after the measurement and the lumbar segments of the spinal cord were removed for determination of N-methyl-D-aspartate receptor subunit 2B （NR2B） protein expression （by Western blotting） and NR2B mRNA expression [ by reverse transcriptase-polymerase chain reaction （RT-PCR） Results As compared with group C and group CM, the PWT in group D and group DM was significantly reduced （ P 〈 0. 05 ） , while the expression of NR2B protein and mRNA was significantly in creased （P 〈0. 05）. As Compared with group D [ NR.2B mRNA （0. 89 ±0. 09）, NR2B protein （ 1.28 ± 0. 17） ] , the PWT in group DM was significantly increased at T2-T4 point before the injection and T1-T4 point ＇after the injection, while the expression of NR2B mRNA （0. 69 ±0. 11 ） and NR2B protein （0. 98 ± 0. 14） in group DM was significantly decreased （P 〈0. 05）. As compared with the point before the injection （ 3.8 ± 0. 9 and 6. 1 ± 1.4 ） , the PWT in group DM were significantly increased at T1 -T2 point after the injeetion （7.5 ± 1.8 and 8.7 ±1.6） （ P 〈 0. 05 ）. Conclusion Endogenous hydrogen sulfide in the spinal eord is involved in the maintenanee of DNP in rats and the possible meehanisms were related with up-regulation of NR2B in the spinal dorsal horn.