摘要
目的:探讨补肾启智方通过调控微小RNA-335(microRNA-335,miR-335)靶向Rho家族相关蛋白激酶2(Rho associated protein kinase 2,ROCK2)蛋白,对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤HT22细胞焦亡与应激颗粒的影响。方法:取对数生长期的HT22细胞,随机分为空白组、模型组、补肾启智方组、miR-335抑制组、空病毒对照组、补肾启智方+miR-335抑制组。除空白组外,其余各组细胞均建立OGD/R损伤模型。采用CCK-8法检测细胞存活率,qRT-PCR法检测细胞miR-335 mRNA表达水平,Western blot检测细胞ROCK2、T细胞胞内抗原1(T cell intracellular antigen 1,TIA1)、核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing CARD,ASC)、活化半胱氨酸天冬氨酸蛋白酶-1(c-Caspase-1)、消皮素D-N(gasdermin D-N,GSDMD-N)蛋白表达水平,ELISA法检测细胞上清液白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-18(interleukin-18,IL-18)水平。结果:与空白组比较,模型组细胞存活率、miR-335 mRNA水平降低(P<0.05)。与模型组比较,补肾启智方组细胞存活率、miR-335 mRNA水平升高,miR-335抑制组细胞存活率、miR-335 mRNA水平降低(P<0.01)。与空病毒对照组比较,miR-335抑制组细胞存活率、miR-335 mRNA水平降低(P<0.01)。与miR-335抑制组比较,补肾启智方+miR-335抑制组细胞存活率、miR-335 mRNA水平升高(P<0.01)。与空白组比较,模型组细胞ROCK2、NLRP3、ASC、c-Caspase-1、GSDMD-N蛋白表达水平升高,TIA1蛋白表达水平降低(P<0.01)。与模型组比较,补肾启智方组细胞ROCK2、NLRP3、ASC、c-Caspase-1、GSDMD-N蛋白表达水平降低,TIA1蛋白表达水平升高,而miR-335抑制组细胞上述蛋白的表达趋势与之相反(P<0.05)。与空病毒对照组比较,miR-335抑制组细胞ROCK2、NLRP3、ASC、c-Caspase-1、GSDMD-N蛋白表达水平�
Objective:To investigate the effect of Bushen Qizhi Formula(BSQZ)on oxygen-glucose deprivation/reoxygenation(OGD/R)injury induced HT22 cell pyroptosis and stress granules(SGs)by regulating microRNA-335(miR-335)targeting Rho-associated protein kinase 2(ROCK2).Methods:HT22 cells in logarithmic growth phase were randomly divided into a blank group,a model group,a BSQZ group,a miR-335 inhibition group,an empty virus control group,and BSQZ+miR-335 inhibition group.Except for the blank group,OGD/R injury models were established in the cells of the other groups.The cell survival rate was detected using CCK-8 method,the expression level of miR-335 mRNA was detected with qRT-PCR,and the expressions of ROCK2,T cell intracellular antigen 1(TIA1),nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein containing CARD(ASC),c-Caspase-1 and gasdermin D-N(GSDMD-N)protein were detected using Western blot.ELISA was used to detect interleukin-1β(IL-1β)and interleukin-18(IL-18)levels in cell supernatant.Results:Compared with that of the blank group,the cell survival rate and miR-335 mRNA level of the model group were decreased significantly(P<0.05).Compared with that of the model group,the cell survival rate and miR-335 mRNA level of the BSQZ group were significantly increased,while that of the miR-335 inhibition group were decreased significantly(P<0.01).Compared with that of the empty virus control group,the cell survival rate and miR-335 mRNA level of the miR-335 inhibition group were decreased significantly(P<0.01).Compared with that of the miR-335 inhibition group,the cell survival rate and miR-335 mRNA level of the BSQZ+miR-335 inhibition group were significantly increased(P<0.01).Compared with that of the blank group,the protein expression levels of ROCK2,NLRP3,ASC,c-Caspase-1 and GSDMD-N were significantly increased,while the TIA1 protein expression level of the model group was significantly decreased(P<0.01).Compared with that of the model group,the protein expressi
作者
傅晨
巩俐
王翎沣
马承平
刘雪梅
FU Chen;GONG Li;WANG Lingfeng;MA Chengping;LIU Xuemei(Dongfang Hospital Affiliated to Beijing University of Chinese Medicine,Beijing China 100078)
出处
《中医学报》
CAS
2024年第8期1583-1590,共8页
Acta Chinese Medicine
基金
国家自然科学基金青年科学基金项目(82104808)。
