摘要
目的:观察当归补血汤对高糖刺激小鼠永生化足细胞MPC5内质网应激通路蛋白激酶样内质网激酶(PERK)/活化转录因子4(ATF4)/CCAAT增强子结合蛋白同源蛋白(CHOP)及凋亡蛋白B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、切割型胱天蛋白酶-3(cleaved Caspase-3)的影响,通过体外研究阐明当归补血汤调控糖尿病肾病(DKD)足细胞凋亡的具体机制及靶点。方法:制备好含药血清、筛选好最适干预浓度后,将小鼠永生化足细胞MPC5分为5组:正常组(NG)、高糖组(HG)、空白血清组(KB)、当归补血汤含药血清组(DBT)、ERS抑制剂组(4-PBA)。NG组中足细胞用含5.5 mmol·L^(-1)葡萄糖的完全培养基培养;HG组中足细胞用含30 mmol·L^(-1)葡萄糖的完全培养基培养;KB组即HG组+10%空白血清;DBT组即HG组+10%当归补血汤含药血清;4-PBA组即HG组+4-PBA(2.5 mmol·L^(-1))。各组予对应药物进行干预刺激,作用48 h,然后将细胞收集。原位末端标记法(TUNEL)测细胞DNA损伤,免疫荧光检测葡萄糖调节蛋白78(GRP78)、磷酸化(p)-PERK、ATF4、CHOP、足细胞膜蛋白(Podocin)、突触足蛋白(Synaptopodin)表达,同时提取足细胞蛋白,蛋白免疫印迹法(Western blot)检测GRP78、PERK、p-PERK、真核翻译起始因子2α(eIF2α)、p-eIF2α、ATF4、CHOP、Bcl-2、Bax、cleaved Caspase-3蛋白表达。结果:与正常组比较,高糖组GRP78、p-PERK、ATF4和CHOP荧光强度显著增加,Podocin、Synaptopodin荧光强度减弱,足细胞TUNEL荧光阳性细胞核明显增多,GRP78、p-PERK/PERK、p-eIF2α/eIF2α、ATF4、CHOP、Bax、cleaved Caspase-3蛋白表达显著上升,Bcl-2表达显著下降(P<0.01)。与高糖组比较,当归补血汤含药血清组及ERS抑制剂组GRP78、p-PERK、ATF4和CHOP的荧光强度明显减弱,Podocin、Synaptopodin荧光表达增强,TUNEL阳性染色细胞核的数量明显减少,细胞核损伤减轻,GRP78、p-PERK/PERK、p-eIF2α/eIF2α、ATF4、CHOP、Bax、cleaved Caspase-3蛋白表达显著下调,Bcl-2蛋�
Objective:To observe the effect of Danggui Buxuetang on the endoplasmic reticulum stress(ERS)of high glucose-stimulated mouse podocyte cells MPC5 via the protein kinase R-like endoplasmic reticulum kinase(PERK)/activating transcript factor 4(ATF4)/CCAAT enhancer-binding protein homologous protein(CHOP)pathway and the expression B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),and cleaved Caspase-3 and to elucidate the specific mechanism and targets of Danggui Buxuetang in regulating podocyte apoptosis by cell experiments.Method:After the preparation of drug-containing serum and screening of the optimal intervention concentration,the mouse podocyte cells MPC5 were classified into five groups:normal control(NG),high glucose(HG),blank serum control(KB,HG+10% blank serum),Danggui Buxuetang-containing serum(DBT,HG+10% drug-containing serum),and ERS inhibitor(4-PBA,HG+2.5 mmol·L^(-1)4-PBA).The podocytes in the NG and HG groups were cultured with the complete media containing 5.5 mmol·L^(-1) and 30 mmol·L^(-1) glucose,respectively.Each group was treated with the corresponding drug for 48 h,and then the cells were collected.The expression of glucose-regulated protein 78(GRP78),p-PERK,ATF4,CHOP,podocin,and synaptopodin was detected by immunofluorescence.DNA damage was examined by the terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)assay.The proteins of podocytes were extracted,and the protein levels of GRP78,PERK,p-PERK,eukaryotic initiation factor 2α(eIF2α),p-eIF2α,ATF4,CHOP,Bcl-2,Bax,and cleaved Caspase-3 were determined by Western blot.Result:Compared with the NG group,the HG group showed increased fluorescence intensities of GRP78,p-PERK,ATF4,and CHOP,weakened fluorescence intensities of podocin and synaptopodin,increased TUNEL-positive nuclei of podocytes,up-regulated protein levels of GRP78,p-PERK/PERK,p-eIF2α/eIF2α,ATF4,CHOP,Bax,and cleaved Caspase-3,and down-regulated protein level of Bcl-2(P<0.01).Compared with the HG group,the DBT and 4-PBA groups presented weakened fluore
作者
顾悦
申宇航
丁鑫
王逸凡
张圆圆
郑琳琳
郭登洲
GU Yue;SHEN Yuhang;DING Xin;WANG Yifan;ZHANG Yuanyuan;ZHENG Linlin;GUO Dengzhou(Hebei University of Chinese Medicine,Shijiazhuang 050091,China;Lucheng Community Health Center of Tongzhou District,Beijing 101100,China;The First Affiliated Hospital of Hebei University of Chinese Medicine,Hebei Provincial Hospital of Traditional Chinese Medicine,Shijiazhuang 050011,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第16期10-18,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
河北省自然科学基金项目(H2022423367)
河北中医学院研究生创新能力培养项目(XCXZZBS2022018)。
