title: Chuanxiongzine-astragaloside IV decreases IL-1β and Caspase-3 gene expressions in rat brain damaged by cerebral ischemia/reperfusion: a study of real-time quantitative PCR assay.
abstract: The purpose of this study was to establish an absolute quantitative method to detect IL-1β and Caspase-3 gene expressions in rat brain after cerebral ischemia-reperfusion (I/R) using real-time PCR. Rats were randomized into the following groups: sham operation group, model group (cerebral I/R group), astragaloside IV (AST IV) group, chuanxiongzine-AST IV group and nimodipine group (n = 10 in each group). The rats in all the groups except sham operation group were subjected to cerebral I/R treatment. Sham operation and model groups were treated by normal saline (5 mL/kg). AST IV, chuanxiongzine-AST IV and nimodipine groups received 20 mg/kg AST IV, 10 mg/kg chuanxiongzine plus 20 mg/kg AST IV, and 10 mg/kg nimodipine treatments, respectively. The administrations of drugs were performed with intraperitoneal injections at 0 and 12 h, 1 d, 2 d, 3 d, till to 7 d after I/R. A real-time quantitative PCR assay was developed for absolute quantification of the expressions of IL-1β and Caspase-3 genes. The absolute quantification approach relies on the construction of an accurate standard curve. Thus, two plasmids which contained rat IL-1β and Caspase-3 genes respectively were constructed. The cloned circular plasmids were then quantified using a spectrophotometer and used as standards. Standard curves were generated, and the copy numbers of IL-1β and Caspase-3 mRNA isolated from I/R-damaged brain tissue were also calculated by SYBR Green I dye method using specific primers. The results showed that melting curves exhibited sharp peaks, and PCR product also generated prominent band with expected size in agarose gel electrophoresis, which validated the optimization of the selected primer sets of IL-1β and Caspase-3 genes. The optimal annealing temperatures of IL-1β and Caspase-3 genes were 59 °C and 61.2 °C, respectively. Real-time PCR results showed that the expression of IL-1β and Caspase-3 genes in the model group was significantly elevated compared to that in the sham operation group. However, compared to those in the model group, IL-1β and Caspase-3 gene expressions were obviously decreased in AST IV, chuanxiongzine-AST IV and nimodipine groups. Especially in chuanxiongzine-AST IV group, those two genes showed the most significant expression down-regulation. These results suggest the absolute quantitative method established in the present study is capable of detecting the changes of IL-1β and Caspase-3 gene expressions in rat brain damaged by I/R.
authors.affiliation: Institute of Cardio-cerebrovascular Disease, Zhejiang Chinese Medical University, Hangzhou, China.
authors.forename: Zhen-Hong
authors.initials: ZH
authors.lastname: Zhu
authors.forename: Hai-Tong
authors.initials: HT
authors.lastname: Wan
authors.forename: Jin-Hui
authors.initials: JH
authors.lastname: Li
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002545:Brain Ischemia / Q000378:metabolism / Q000503:physiopathology; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism*; D008297:Male; D018696:Neuroprotective Agents / Q000494:pharmacology; D011719:Pyrazines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D015427:Reperfusion Injury / Q000378:metabolism / Q000517:prevention & control*; D012503:Saponins / Q000494:pharmacology*; D014315:Triterpenes / Q000494:pharmacology*
chemical_list: D053583:Interleukin-1beta; D018696:Neuroprotective Agents; D011719:Pyrazines; D012333:RNA, Messenger; D012503:Saponins; D014315:Triterpenes; C052064:astragaloside A; C505410:Casp3 protein, rat; D053148:Caspase 3; C017953:tetramethylpyrazine