Line gene pcr instrument

Total RNA was extracted from fresh-frozen neural tissue using an Ultrapure RNA Kit (CWbio Co. Ltd., China) and then reverse-transcribed with a HiFi-MMLV cDNA Kit (CWbio Co. Ltd., China). Real time PCR was performed on a Bioer line gene PCR instrument (BIOER, China) using Invitrogen primers. The PCR amplification procedure includes 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 59°C for 60 s. Data were normalized to Beta actin.

Three-month-old male Wistar rats with a body weight of 200–220 g were purchased from Shanghai Slaccas Experimental Animal Co., Ltd. (Shanghai, China; Certificate no. SCXK 2015-0012). Moxibustion was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All of the rats were provided free access to water and food and maintained in a 12 h:12 h light/dark cycle at 22 ± 2 °C and 65–69% relative humidity for 8 weeks. This study was approved by the ethics committee of Hubei University of Chinese Medicine (Wuhan, China). The animal research protocol was conducted in accordance with the European Community guidelines for the use of experimental animals. STZ was purchased from Hangzhou Baitong Biological Technology Co., Ltd. (Hangzhou, China). IL-1β, IL-6, and IL-8 ELISA commercially available kits (R&D Systems, Minneapolis, MN, USA) were used. Rabbit antibody against β-actin (ab189073), rabbit anti-NF-κB polyclonal antibody (ab7971), and rabbit anti-Nrf2 polyclonal antibody (ab31163) were purchased from Abcam (Cambridge, MA, USA). Total RNA was extracted from freshly frozen neural tissues by using an Ultrapure RNA kit (CWbio Co., Ltd., China) and then reverse-transcribed with a HiFi-MMLV cDNA kit (CWbio Co., Ltd., China). Real-time PCR was performed in a Bioer line gene PCR instrument (BIOER, China) by using Invitrogen primers.