Resistant Starch Supplementation Modulates Gut Microbiota, Short-Chain Fatty Acids, and Markers of Gut Barrier Integrity in Estrogen-Deficient Mice

Background Menopause-associated estrogen decline promotes gut dysbiosis and impaired gut health, but the effects of resistant starch (RS) under estrogen-deficient conditions remain unclear. Objectives Herein, we examine the effects of retrograded (RS3) and chemically modified (RS4) resistant starch on the gut microbiota and gut barrier integrity in estrogen-deficient mice. Methods 3-month-old female C57BL/6 mice received the ovotoxicant vinyl cyclohexene diepoxide (VCD, 160 mg/kg) or sesame oil (vehicle) for 30 days, followed by 16 weeks of dietary intervention with control (AIN-93M), 5% RS3, or 5% RS4 (n=15/group). Estrogen deficiency was confirmed by reduced uterine weight and increased serum FSH. Gut microbiota composition was assessed via 16S rRNA sequencing; jejunal and cecal gene expressions of inflammatory markers ( Tnf, Il22 ), tight junction proteins ( Tjp1 , Cldn1 ), and antimicrobial peptides ( Reg3b , Reg3g ) were evaluated by qPCR; and fecal short-chain fatty acids (SCFAs) were quantified by GC-FID. Jejunal and colon sections were examined by histological staining. Data were analyzed using a 2-way ANOVA with estrogen status and diet as factors, followed by post hoc tests when p<0.05. Results RS supplementation induced distinct clustering in bacterial β-diversity, enriched beneficial genera including Bifidobacterium , Dubosiella , Bacteroides , and Akkermansia , and reduced pro-inflammatory Clostridium sensu_stricto_1 in estrogen-deficient mice. Jejunal Tnf expression was elevated by VCD in the control diet but suppressed by RS4, with no significant change in RS3. Both RS3 and RS4 enhanced Tjp1 and Cldn1 expression and elevated fecal SCFAs, while RS3 additionally increased Reg3g expression. Histological analysis showed no significant effects of VCD and diet on most of the parameters, except for modest effect on gut inflammation with VCD which was improved by RS supplementation. Conclusions These findings suggest that RS supplementation modulates gut microbiota composition, SCFA production, and inflammatory markers, with RS4 showing stronger anti-inflammatory effects and RS3 preferentially enhancing antimicrobial peptide expression, in estrogen-deficient mice.