title: [Effect of Tongxinluo on Apoptosis of Rat Cardiac Microvascular Endothelial Cells].
abstract: OBJECTIVE
  To observe the protective effects of Tongxinluo (TXL) on apoptosis of rat cardiac microvascular endothelial cells (RCMECs) resulting from homocysteine (Hcy) induced endoplasmic reticulum stress (ERS), and to determine the signaling pathway behind its protection.
  
  
  METHODS
  Primary cultured RCMECs were isolated from neonatal rats using tissue explant method. The morphology of RCMECs was observed using inverted microscope, identified and differentiated by CD31 immunofluorescence method. Selected were well growing 2nd-4th generations of RCMECs. The optimal action time was determined by detecting the expression of glucose regulated protein 78 (GRP78) using immunofluorescence method. In the next experiment RCMECs were divided into 5 groups, i.e., the blank control group, the Hcy induced group (Hcy 10 mmol/L, 10 h), the Hcy + TXL group (Hcy 10 mmol/L + TXL 400 µg/mL), the Hcy +LY294002 group (Hcy 10 mmol/L + LY294002 5 µmol/L, LY294002 as the inhibitor of PI3K), the Hcy + LY294002 + TXL group (Hcy 10 mmol/L + LY294002 5 µmol/L + TXL 400 µg/mL). The apoptosis rate of RCMECs was detected by flow cytometry. mRNA and protein expressions of GRP78, C/ EBP homologous protein (CHOP), and cysteinyl aspartate specific proteinase-12 (caspase12) were detected by real-time reverse transcription PCR (RT-PCR) and Western blot respectively. Expression levels of phosphorylation of phosphatidylinositol 3-kinase (P-PI3K), total phosphatidylinositol 3-kinase (T- P13K) , phosphorylation of kinase B (P-Akt) , and total kinase B (T-Akt) were detected by Western blot.
  
  
  RESULTS
  Ten hours Hcy action time was determined. Compared with the blank control group, the apoptosis rate was increased (22.77%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, protein expressions of P-PI3K and P-Akt,ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy induced group (P < 0.05, P < 0.01). Compared with the Hcy induced group, the apoptosis rate was decreased (10.17%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were decreased, and expression levels of P-PI3K, P-Akt, P-PI3K/T-PI3K, and P-Akt/T-Akt were increased in the Hcy + TXL group (P < 0.05, P < 0.01). Compared with the Hcy + TXL group, the apoptosis rate was increased (17.9%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, expression levels of P-PI3K and P-Akt, ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy + TXL + LY294002 group (P < 0.05, P < 0.01).
  
  
  CONCLUSIONS
  TXL could inhibit the apoptosis of RCMECs resulting from Hcy-induced ERS and its mechanism might be associated with activating PI3K/Akt signaling pathway.
authors.forename: Geng
authors.initials: G
authors.lastname: Wei
authors.forename: Hong-rong
authors.initials: HR
authors.lastname: Li
authors.forename: Hong-li
authors.initials: HL
authors.lastname: Liu
authors.forename: Bing
authors.initials: B
authors.lastname: Yao
authors.forename: Meng
authors.initials: M
authors.lastname: Chen
authors.forename: Jun-qing
authors.initials: JQ
authors.lastname: Liang
authors.forename: Zhen-hua
authors.initials: ZH
authors.lastname: Jia
authors.forename: Yi-ling
authors.initials: YL
authors.lastname: Wu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053454:Caspase 12 / Q000378:metabolism; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D059865:Endoplasmic Reticulum Stress; D042783:Endothelial Cells / Q000187:drug effects*; D009025:Morpholines / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D051742:Transcription Factor CHOP / Q000378:metabolism
chemical_list: D002867:Chromones; D004365:Drugs, Chinese Herbal; D009025:Morpholines; C513941:tongxinluo; D051742:Transcription Factor CHOP; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D053454:Caspase 12