Mechanistic Basis of Zhigancao Decoction Synergy with PD-1 Blockade in NSCLC

1. After the tumor-bearing mice were successfully established, the weight of the mice was recorded once every two days, and the volume of the tumor was measured and calculated. Finally, the weight of the tumor was measured.2. Transcriptome sequencing resultsUsing the RNA extraction kit from Sangon Biotech (Shanghai, China), total RNA was extracted from lung tumor tissues according to the manufacturer's instructions, and RNase-free DNase I was used to treat it to eliminate genomic DNA contamination. Qualified RNA samples were prepared using the VAHTS™ mRNA seq V2 library preparation kit (Vazyme, Nanjing, China) according to the manufacturer's protocol. Sequencing was performed on the Illumina HiSeq 3000 platform at Sangon Biotech (Shanghai, China). The raw sequencing data were quality-controlled using FastQC (version 0.11.2) and filtered using Trimmomatic (version 0.36) to remove low-quality reads and adapters.3. Gut microbiota sequencing resultsThe total community genomic DNA of bacteria 16S rRNA genes was extracted using the E.Z.N.A™ MagBind soil DNA kit (Omiga Biotechnology Company, Norcross, USA), following the manufacturer's instructions. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using specific primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′-GACTACHVGGGTATCTAATCC-3′). The amplification products were purified, mixed at equal molar concentrations, and used for library construction at the end of the library, using the Illumina library preparation kit (Illumina Inc., San Diego, California, USA). Sequencing was performed on the Illumina MiSeq (2×300bp) platform at Shanghai Sang Gen Biotechnology Company (Shanghai, China). The raw sequencing reads were processed for deduplication, quality filtering, and trimming (using Cutadapt software, version 2.7) to remove adapter sequences and low-quality bases.4. Network pharmacology and molecular dockingThe main chemical components of Zhikai Dihuang Pills with oral bioavailability ≥ 30% and drug-likeness ≥ 0.18 were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Then, the chemical structures of licorice, ginseng, rehmannia, palmetto, and peony were submitted to the PharmMapper database to predict potential molecular targets based on the standard of standardized fitting score > 0.5. The retrieved targets were converted to gene symbols using the UniProt database. Non-small cell lung cancer (NSCLC) related genes were obtained from the GeneCards database. The targets of Zhikai Dihuang Pills were overlapped with NSCLC genes using the bioinformatics and evolutionary genomics network tools to determine the key targets. These targets were then imported into the STRING 11.5 database (https://string-db.org) to construct a protein-protein interaction (PPI) network with a medium confidence level > 0.400. The core RNA sequencing differentially expressed genes (DEGs) and key targets' PPI network were visualized using Cytoscape (v3.8.2). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the R package clusterProfiler. 5. Use the commercial ELISA kit from Maixin (Jiangsu) to determine serum tumor markers (including carcinoembryonic antigen (CEA) and cytokeratin fragment 21-1 (CY211)), thyroid function markers (including thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4)), inflammatory cytokines (including tumor necrosis factor alpha (TNFα), interleukin 8 (IL8), and interleukin 6 (IL6)), and immune function markers (including CD3, CD4, and CD8).Use the commercial biochemical test kit from ABclonal (Wuhan) to determine serum biochemical parameters, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (γ-GT), creatine kinase (CK), creatine kinase MB (CK-MB), and lactate dehydrogenase (LDH).6. Mass spectrometry analysisWeigh 0.0261 grams of ZGCT powder sample, add 1 milliliter of 70% methanol (Merck, Germany) solution, and then add 100 micrograms per milliliter of internal standard solution (2-chlorobenzylalanine; Shanghai Yuanye Biotechnology Co., Ltd.). Use the Thermo Vanquish UHPLC coupled with the Q-Exactive HF LC system (Thermo Fisher Scientific, USA). The sample is separated on a Zorbax Eclipse C18 (1.8 micrometers × 2.1 millimeters × 100 millimeters) chromatographic column. The mobile phase consists of 0.1% formic acid (A phase) and 100% (volume ratio) acetonitrile (B phase). The gradient elution program is as follows: 0 - 2 minutes, 95% A phase; 2 - 7 minutes, 70% A phase; 7 - 14 minutes, 22% A phase; 14 - 20 minutes, 5% A phase. The flow rate is 0.3 milliliters per minute, and the column temperature is maintained at 30°C. The full scan range of MS1 is (m/z 100 - 1500), and data-dependent MS2 scanning (dd-MS2, TopN = 5) is adopted. The resolution of MS1 is set to 120,000, and the resolution of MS2 is set to 60,000. The collision mode is high-energy collision dissociation (HCD). The spray voltage is 3.5 kilovolts (positive ion mode) or 3.5 kilovolts (negative ion mode). The injection volume is 2 microliters.