Targeted Chemical Monitoring and Integrated Preclinical Evaluation of Zishui Qinggan Yin in 4-NQO-Induced Esophageal Precancer: Histopathological and PI3K/AKT/mTOR-Associated Changes

Esophageal squamous cell carcinoma frequently develops through a prolonged precancerous phase, creating an opportunity for intervention. Zishui Qinggan Yin (ZSQGY) is a multi-component botanical preparation, but its effects in esophageal precancer and its formulation-level chemical profile remain incompletely defined. We monitored a predefined 40-analyte panel by UPLC-Q Exactive/MS in three independently extracted aliquots from the same formulation batch administered to mice. A 4-nitroquinoline-1-oxide model was then evaluated by consensus histopathology, mouse-grouped whole-slide digital pathology, network-based target prioritization, molecular docking, exploratory drug affinity responsive target stability (DARTS), Western blotting, qPCR, and serial-section immunohistochemistry. Signals corresponding to 38 predefined analytes were reported. ZSQGY treatment was associated with a lower-grade lesion pattern in the evaluated mouse-level specimens. Exploratory DARTS was analyzed in two independent n = 6 tissue-background datasets. In the original 4-NQO model-background cohort, ex vivo ZSQGY (100 μg/mL) increased the AKT1-immunoreactive relative-protection signal in five of six lysates (mean 2.04 ± 1.15). In an additional cohort from mice that had received ZSQGY in vivo, ex vivo ZSQGY produced mean relative-protection values of 1.21, 1.75, and 1.92 at 50, 100, and 200 μg/mL, respectively. The nominal exact p value for the 100 μg/mL versus vehicle contrast in the additional exploratory dataset was 0.0313; This exploratory result should be interpreted cautiously. Neither dataset establishes direct binding or in vivo target occupancy. PI3K p110α DARTS was technically inconclusive. Endpoint tissues showed lower AKT, mTOR, S6K1, and 4E-BP1 phosphorylation, together with PTEN-associated changes, reduced Ki-67, increased cleaved caspase-3 staining, and selected downstream transcript changes, including a non-significant downward trend in PARP1. These findings support a preclinical association between ZSQGY treatment, attenuation of esophageal precancer progression, and AKT-centered PI3K/AKT/mTOR pathway modulation. They do not establish direct binding, in vivo target occupancy, pathway necessity, active constituents, or causal mediation.