Effects of hypoxia on mRNA expression of housekeeping genes in rat brain tissue and primary cultured neural cells

Internal standards are critical for quantitative RNA analyses.Housekeeping genes are often used as internal standards with the assumption that their express-ion levels remain relatively constant in different experi-mental conditions.In this study,four commonly used housekeeping genes,Glyceraldehyde-3-phosphate dehy-drogenase(GAPDH),β-actin,28S rRNA and 18S rRNA were selected to test whether this assumption is tenable under hypoxic conditions.We tested the RNA expression level of these four genes in different hypoxic conditions.Rats subjected to acute hypoxia for 2 hours were used for tissue detection.Primary cultured neural stem cells from E13 fetal rats were treated with 3%O_(2) or 10%O_(2) for 24 hours for in vitro experiments.In both experiments,expression levels of 28S rRNA and 18S rRNA were constant,independent of hypoxia types.However,expression levels of GAPDH and b-actin were all changed in all kinds of hypoxic conditions.In particu-lar,the mRNA expression level of GAPDH was increased by 43.4%under 3%O_(2) hypoxic conditions.These results suggest that 18S rRNA and 28S rRNA are reliable internal controls for comparative analyses of transcrip-tion under hypoxia.GAPDH appears particularly unfa-vorable for this purpose in hypoxic conditions.

Effect of aqueous extract of Sanweitanxiang powder on calcium homeostasis protein expression in ischemic-reperfusion injury rat heart

OBJECTIVE: To investigate the underlying mechanism of reduced myocardial ischemia-reperfusion (I/R) injury in rats using the traditionalTibetan medicine Sanweitanxiang powder (SWTX). METHODS: Rats were randomly divided into six groups (n=10) as follows: (a) propranolol dinitrate control group, given propranolol dinitrate 0.02 g/kg for 10 days before I/R, (b) SWTX with a high dose group, given SWTX 1.5 g/kg for 10 days before I/R, (c) SWTX with a medium dose group, given SWTX 1.25 g/kg for 10 days before I/R, (d) sham group (Sham), in which the rat heart was exposed by pericardiotomy but without I/R, (e) SWTX with a low dose group, given SWTX 1.0 g/kg for 10 days before I/R, and (f) I/R injury group. Rats were intragastrically pretreated with propranolol dinitrate orSWTX. After that, the operation to cause ischemia and reperfusion was conducted.The histopathologic changes of rat hearts were observed by hematoxylin and eosin staining and transmission electron microscopy. Ca2+ homeostasis protein expression was determined by western blot. RESULTS: After SWTX pretreatment, the development of ultrastructural pathological changes from IR injury was attenuated. A decrease in the expression of B-cell lymphoma 2 associated X protein, and an increase in the expression of B-cell lymphoma 2 were observed. An increased activation of extracellular signal regulated kinases were found. Compared with the sham group, the expression of sarcoplasmic reticulum calcium-ATPase, phospholamban, and calsequestrin were all up-regulated after pretreatment with SWTX. CONCLUSION: The protective mechanism of SWTX pretreatment on myocardial I/R injury might be related to its effect on maintaining the balance of calcium homeostasis in rat heart.