摘要
目的探讨补肾活血方含药血清对过氧化氢诱导大鼠髓核细胞焦亡的作用。方法随机选取5只SD大鼠,灌胃给予3.597 g/kg补肾活血方,眼球取血,分离得含药血清;其余9只大鼠分离提取髓核细胞。用含0,5%,10%,20%,40%,80%,100%补肾活血方含药血清与髓核细胞孵育24 h,采用细胞计数试剂盒8(CCK-8)测定细胞活力。将髓核细胞随机分为空白对照组(A组)、模型组(B组)及含药血清低、中、高剂量组(C_(1)组、C_(2)组、C_(3)组)。A组加入无血清培养基;B组加入400μmol/L过氧化氢;C_(1)组、C_(2)组、C_(3)组分别预先用5%,10%,20%含药血清处理2 h,再用400μmol/L过氧化氢孵育4 h。采用乳酸脱氢酶(LDH)试剂盒测定其细胞活性,采用酶联免疫吸附法测定细胞上清液中白细胞介素1β(IL-1β)的表达水平,采用蛋白免疫印迹法测定细胞中p17、p20、半胱天冬蛋白酶1(caspase-1)、含NLR家族Pyrin域蛋白(NLRP3)、Gasdermin D(GSDMD)、Beclin 1、Rab5、Rab7、LC_(3)Ⅱ/Ⅰ蛋白的表达水平,采用丙二醛(MDA)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)试剂盒分别测量细胞中MDA和GSH的含量及SOD的活性。结果与补肾活血方含药血清的培养基比较,5%,10%,20%含药血清培养基中细胞活力差异不显著(P>0.05)。与B组比较,C_(1)组、C_(2)组、C_(3)组细胞上清液中IL-1β和GSDMD-NT,p17,p20,LC_(3)Ⅱ/Ⅰ蛋白的表达水平均显著减低,MDA含量显著减少(P<0.05);与B组比较,C_(2)组、C_(3)组细胞上清液中SOD活性和Rab5,Rab7蛋白的表达水平均显著升高,GSH含量显著增加(P<0.05)。结论补肾活血方抑制了过氧化氢诱导大鼠髓核细胞的焦亡、NLRP3炎性小体的活化及氧化应激,促进了细胞自噬。但其物质基础复杂,主要活性成分和作用机制仍不清楚,有待进一步深入研究。
Objective To investigate the effect of serum-containing Bushen Huoxue Recipe on the pyroptosis of rat nucleus pulposus cells induced by hydrogen peroxide.Methods Five SD rats were randomly selected and given 3.597 g/kg of Bushen Huoxue Recipe by gavage,and their blood was taken from eyeballs to obtain serum-containing drug.Nucleus pulposus cells were isolated from other rats.The nucleus pulposus cells were incubated with 0,5%,10%,20%,40%,80%and 100%of serum-containing Bushen Huoxue Recipe for 24 h,and the cell viability was measured by cell counting kit 8(CCK-8)method.The nucleus pulposus cells were randomly divided into the blank control group(group A),model group(group B),serum-containing drug low-,medium-and high-dose groups(groups C_(1),C_(2) and C_(3)).The cells in group A were added with serum-free medium,and those in group B were added with 400μmol/L of hydrogen peroxide(H_(2)O_(2)).The cells in groups C_(1),C_(2) and C_(3) were pretreated with 5%,10%,and 20%of serum-containing drug for 2 h,then incubated with 400μmol/L of H_(2)O_(2) for 4 h.The cell activity was measured by lactate dehydrogenase(LDH)kit,the expression level of interleukin-1β(IL-1β)in cell supernatant was measured by enzyme-linked immunosorbent assay(ELISA),and the levels of p17,p20,caspase-1,NL-RP3,Gasdermin D(GSDMD),Beclin 1,Rab5,Rab7 and LC3Ⅱ/Ⅰprotein were measured by Western blot.The contents of malondialdehyde(MDA),glutathione(GSH)and the activity of superoxide dismutase(SOD)were measured by the MDA,GSH and SOD kits.Results There was no significant difference in cell viability between 5%,10%and 20%medium with serum-containing Bushen Huoxue Recipe and medium without serum-containing Bushen Huoxue Recipe(P>0.05).Compared with those in group B,the expression levels of IL-1β,GSDMD-NT,p17,p20 and NLRP3 protein in the cell supernatant decreased significantly,while the content of MDA decreased significantly in groups C_(1),C_(2) and C_(3)(P<0.05).Compared with those in group B,the activity of SOD and the expression levels of Rab5,Ra
作者
张超
李兆勇
ZHANG Chao;LI Zhaoyong(The First Affiliated Hospital of Hunan University of Traditional Chinese Medicine,Changsha,Hunan,China 410007)
出处
《中国药业》
CAS
2022年第5期49-54,共6页
China Pharmaceuticals
基金
湖南省自然科学基金青年基金项目[2020JJ5443]。
关键词
补肾活血方
髓核细胞
焦亡
自噬
炎性小体
Bushen Huoxue Recipe
nucleus pulposus cells
pyroptosis
autophagy
inflammasome