CAJ | 학술논문

目的：从细胞水平探讨灯盏生脉胶囊（ DZSM ）含药血清对原代大鼠皮质神经元氧糖剥夺/复氧（ OGD/R ）损伤模型的保护作用及其可能机制。方法将出生24 h内SD大鼠的皮质神经元原代培养7 d后随机分为空白对照组、OGD模型组、正常血清对照组、低剂量DZSM组、高剂量DZSM组。空白对照组不进行预处理及造模，其他各组在OGD/R前分别采用0.9%氯化钠溶液、正常大鼠血清、低剂量DZSM含药血清、高剂量DZSM含药血清预处理，在OGD/R后24.0 h用光镜观察神经元形态学变化，采用XTT法测定细胞存活率、流式细胞术测定细胞凋亡率、Western blotting测定缝隙连接蛋白43（Cx43）表达量，并观察OGD/R后0.5、6.0、12.0、24.0 h Cx43表达变化。结果OGD/R后24.0 h，OGD模型组神经元呈现损伤特征，细胞存活率〔（54.6±6.4）%〕较空白对照组（100.0%）降低（P<0.05），细胞凋亡率升高〔（48.6±4.6）%与（8.8±1.0）%〕（P<0.05）；OGD/R模型组Cx43在OGD/R后0.5 h表达量升高，并持续整个观察过程（ P<0.05）。低、高剂量DZSM组光镜下可见神经元损伤，较OGD模型组减轻；低剂量DZSM细胞存活率为（69.6± 7.7）%，高剂量DZSM细胞存活率为（76.1±8.8）%，均较OGD模型组细胞存活率高（P<0.05）；低剂量DZSM组细胞凋亡率为（25.0 ± 5.9）%，高剂量DZSM组为（18.2±4.9）%，与OGD模型组〔（48.6± 9.2）%〕比较，差异有统计学意义（ P<0.05）。OGD模型组、正常血清对照组、低剂量DZSM组和高剂量DZSM组Cx43表达量分别为（72.8± 6.9）、（63.1±6.6）、（21.1±3.2）、（24.4 ± 3.8），低、高剂量DZSM组与OGD模型组相比，Cx43表达均下降（P<0.05）。结论 DZSM含药血清可抑制OGD/R模型神经元凋亡，提高细胞存活率，其机制可能与抑制Cx43的表达有关。
목적：종세포수평탐토등잔생맥효낭（ DZSM ）함약혈청대원대대서피질신경원양당박탈/복양（ OGD/R ）손상모형적보호작용급기가능궤제。방법장출생24 h내SD대서적피질신경원원대배양7 d후수궤분위공백대조조、OGD모형조、정상혈청대조조、저제량DZSM조、고제량DZSM조。공백대조조불진행예처리급조모，기타각조재OGD/R전분별채용0.9%록화납용액、정상대서혈청、저제량DZSM함약혈청、고제량DZSM함약혈청예처리，재OGD/R후24.0 h용광경관찰신경원형태학변화，채용XTT법측정세포존활솔、류식세포술측정세포조망솔、Western blotting측정봉극련접단백43（Cx43）표체량，병관찰OGD/R후0.5、6.0、12.0、24.0 h Cx43표체변화。결과OGD/R후24.0 h，OGD모형조신경원정현손상특정，세포존활솔〔（54.6±6.4）%〕교공백대조조（100.0%）강저（P<0.05），세포조망솔승고〔（48.6±4.6）%여（8.8±1.0）%〕（P<0.05）；OGD/R모형조Cx43재OGD/R후0.5 h표체량승고，병지속정개관찰과정（ P<0.05）。저、고제량DZSM조광경하가견신경원손상，교OGD모형조감경；저제량DZSM세포존활솔위（69.6± 7.7）%，고제량DZSM세포존활솔위（76.1±8.8）%，균교OGD모형조세포존활솔고（P<0.05）；저제량DZSM조세포조망솔위（25.0 ± 5.9）%，고제량DZSM조위（18.2±4.9）%，여OGD모형조〔（48.6± 9.2）%〕비교，차이유통계학의의（ P<0.05）。OGD모형조、정상혈청대조조、저제량DZSM조화고제량DZSM조Cx43표체량분별위（72.8± 6.9）、（63.1±6.6）、（21.1±3.2）、（24.4 ± 3.8），저、고제량DZSM조여OGD모형조상비，Cx43표체균하강（P<0.05）。결론 DZSM함약혈청가억제OGD/R모형신경원조망，제고세포존활솔，기궤제가능여억제Cx43적표체유관。
Objective To investigate the protective effect of drug serum contained in Dengzhan Shengmai( DZSM) capsule on the oxygen and glucose deprivation/reoxygenation injury model of cortical nerve cells of primary rats and its possible mechanism. Methods The primary culture of cortical nerve cells of newborn SD rats within 24 hours was conducted. On day 7 of the culture,the cells were divided into five groups:blank control group,OGD model group,normal serum control group, low-dose DZSM group and high-dose DZSM group. The blank control group was not administrated with any pretreatment and model building,the other four groups were respectively administrated with 0. 9% sodium chloride solution infection,the serum of normal rats,low -dose drug serum of DZSM and high -dose drug serum of DZSM before OGD/R. At 24h after OGD/R, morphological changes of the nerve cells were observed by light microscope,cell viability rates were determined by XTT methods, apoptosis rates were determined by flow cytometry,the expression levels of connexin 43(Cx43)were determined by Western blotting method,and the protein expression changes of Cx43 were observed at 0. 5h,6. 0h,12. 0h and 24. 0h after OGD/R. Results At 24. 0h after OGD/R,we noted injury characters in the nerve cells of the OGD model group,and the OGD group was lower in cell viability〔(54. 6 ± 6. 4)% vs. 100. 0%;P<0. 05〕and higher in apoptosis rate〔(48. 6 ± 4. 6)% vs. (8. 8 ± 1. 0)%;P<0. 05〕than the blank control group;the OGD model group had kept an increasing trend(P <0. 05)in Cx43 expression 0. 5h after OGD/R through the whole observation process. We observed nerve cell injury in low-dose DZSM group and high-dose DZSM group under light microscope,which was slighter than OGD model group;the cell viability rates of low-dose DZSM group and high-dose DZSM group were(69. 6 ±7. 7)% and(76. 1 ±8. 8)%,both higher(P<0. 05)than that of OGD model group;the apoptosis rates of low -dose DZSM group and high -dose DZSM group were( 25. 0 ± 5. 9 )% and (18. 2 ±4. 9)% respectively,which were significantly different(P <0. 05)from that of OGD group,which was(48. 6 ± 9. 2)%. The expression levels of Cx43 of OGD model group,normal serum control group,low-dose DZSM group and high-dose DZSM group were(72. 8 ± 6. 9),(63. 1 ± 6. 6),(21. 1 ± 3. 2)and(24. 4 ± 3. 8),with low-dose DZSM group and high-dose DZSM group lower(P <0. 05)than OGD group. Conclusion The drug serum contained in DZSM may inhibit the apoptosis of nerve cells after OGD/R,increase the viability rate of cells. The mechanism may be related with the inhibition of Cx43 expression.