Andrographolide Suppresses Influenza A Virus-induced Pyroptosis via PI3K/AKT-mediated Caspase-3/GSDME Inactivation

The experimental timeline proceeded as follows: mice were euthanized on the fourth day following infection, and their lung tissues were immediately collected. Following this, total RNA was extracted from the homogenized lung samples utilizing TRIzol reagent (Invitrogen, 15596018CN). Subsequently, RNA-seq libraries were prepared specifically from high-integrity RNA that passed quality assessments, which is a critical step for reliable transcriptomic profiling (RNA Integrity Number > 7.0) by Annoroad Gene Technology Co., Ltd. Briefly, after second-strand cDNA synthesis, libraries were constructed using a heat-labile uracil-DNA glycosylase enzyme protocol (New England Biolabs) and amplified by PCR; fragments of 300 ± 50 bp were selected. Sequencing reads were aligned to the Mus musculus reference genome, and gene expression levels were quantified as fragments per kilobase of transcript per million mapped reads. Differential expression analysis was performed using the edgeR package, with thresholds set at |fold change| > 2 and a p value < 0.05.