Timosaponin AIII mitigates zearalenone-induced kidney injury in weaned piglets in association with TLR/MAPK signaling suppression.

Abstract

Introduction: Zearalenone (ZEN) is a common Fusarium mycotoxin that frequently contaminates feed and can induce kidney dysfunction in pigs. Timosaponin AIII (TSA), a steroidal saponin isolated from Anemarrhena asphodeloides Bunge, possesses anti-inflammatory and antioxidant properties. This study evaluated whether TSA supplementation alleviates ZEN-induced kidney injury in weaned piglets and examined its association with TLR4/MAPK signaling.

Methods: Eighteen weaned female piglets were randomly assigned to three groups (n = 6 per group): Control (basal diet), ZEN (basal diet supplemented with 2 mg/kg ZEN), and ZEN + TSA (basal diet supplemented with 2 mg/kg ZEN and 20 mg/kg TSA). After 28 days, growth performance and kidney index were recorded. Serum CRE, BUN, and UA concentrations were measured. Kidney histopathology was assessed by H&E staining, and apoptosis was evaluated by TUNEL staining. Renal cytokines (IL-1β, IL-6, IL-10, and TNF-α) were quantified by ELISA. Expression of TLR/MAPK signaling-related genes and proteins was analyzed by qPCR and Western blot. Western blot densitometry used three biological replicates per group (n = 3), whereas ELISA, qPCR, serum biochemistry, and TUNEL quantification used all six animals per group (n = 6).

Results: ZEN exposure significantly reduced final body weight, ADFI, ADG, and kidney index, and increased F/G, serum CRE and BUN concentrations, and renal histopathological lesions (P < 0.05). ZEN also elevated renal IL-1β, IL-6, TNF-α, and IL-10 levels and increased TUNEL-positive apoptotic cells. TSA co-administration significantly ameliorated ZEN-induced growth impairment, kidney dysfunction, histopathological damage, cytokine elevation, and apoptosis (P < 0.05). At the molecular level, ZEN upregulated TLR4, NF-κB, IL-1β, and JNK expression, while TSA co-administration was associated with reduced IL-1β, JNK, and p-JNK/total JNK ratio. However, the difference in TLR4 protein abundance between the ZEN and ZEN + TSA groups was not statistically significant, and MAP3K7 remained elevated while IL-1R1 was not restored, indicating selective modulation of downstream MAPK effectors rather than uniform inhibition of the entire pathway.

Discussion: TSA supplementation is associated with the alleviation of ZEN-induced kidney injury in weaned piglets, concomitant with reduced inflammatory and apoptotic responses and selective modulation of TLR/MAPK downstream signaling, particularly JNK. However, due to the absence of a TSA-only control group, the lack of analytical verification of dietary ZEN and TSA concentrations, and the absence of direct pathway inhibitor, knockdown, or rescue experiments, these findings are correlative and cannot establish causality. Further studies incorporating TSA-only groups, verified feed concentrations, and pathway-specific interventions are needed to confirm the protective mechanism and safety of TSA.