初始data.xlsx
PCR：Total RNA was extracted from rat brain tissue using Trizol reagent (Invitrogen, USA), while miRNA was isolated using a miRNA purification kit (CW0627S, CWBIO, China). RNA concentration and purity were measured using an NP80 UV spectrophotometer (NanoPhotometer, Germany), and samples with OD260/OD280 ratios between 1.8-2.0 were considered acceptable. miRNA was reverse transcribed into cDNA using miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (MR101-02, Vazyme, China). qPCR reactions were performed using miRNA Universal SYBR qPCR Master Mix (MQ101-02, Vazyme, China) on a CFX Connect™ fluorescence PCR system (Bio-Rad, USA). The reaction protocol was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 10 s; annealing at 58°C for 30 s; extension at 72°C for 30 s; for 40 cycles. Each sample was analyzed in triplicate using U6 as an internal reference. Relative gene expression was calculated using the 2^-ΔΔCT method. Primer sequences are listed in the Table1 below.
ELISA：ELISA was used to determine the levels of IL-1β and IL-18. Rat serum was collected and centrifuged at 1000g for 20 min. The assay was performed according to the manufacturer's instructions (ExCell Biology, Shanghai, China). The specific procedure was as follows The ELISA kit was equilibrated at room temperature. Standards were prepared according to the manufacturer's instructions. Blank, standard and sample wells were prepared. No samples or enzyme-labelled reagents were added to the blank wells. 50 μL of sample or standard was added to the remaining wells, followed by 100 μL of enzyme-labelled reagent. The plate was incubated for 1 hour at 37°C. After five washes, chromogenic substrate was added and incubated at 37°C for 15 minutes. The reaction was stopped by adding stop solution and the absorbance of each well was measured at a wavelength of 450 nm.
WB： 收集大鼠海马组织，并在含有蛋白酶和磷酸酶抑制剂的 RIPA 裂解缓冲液中匀浆，使用组织匀浆器提取总蛋白。将匀浆在 4°C 下以 12,000 rpm 离心 10 分钟，并收集上清液。使用 BCA 蛋白检测试剂盒进行蛋白定量。蛋白质变性后，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 （SDS-PAGE） 分离样品 1.5 h。将蛋白质以 300 mA 的恒定电流转移到 PVDF 膜 （Millipore） 上，持续不同的持续时间：NRG1 为 1 小时，ErbB4、TLR4 和 NLRP3 为 2 小时，ASC 为 0.5 小时。用脱脂牛奶封闭 PVDF 膜，然后在 4°C 下与一抗孵育过夜。 第二天，将膜与二抗在室温下孵育 2 小时。用化学发光试剂润湿膜，并使用高灵敏度化学发光成像系统进行可视化。使用 Image J 软件分析条带强度，β-肌动蛋白用作内部参考来计算靶蛋白的相对表达。
