Caffeine intake during pregnancy is common, while its effect on gut microbiota composition of offspring and the relationship with susceptibility to adult diseases remains unclear. This study aimed to confirm the effects of prenatal caffeine exposure (PCE) on the gut microbiota composition and its metabolites in female offspring rats, and to further elucidate its underlying mechanism and intervention targets in adult non-alcoholic fatty disease (NAFLD). The results showed that the gut microbiota of PCE female offspring at multiple time points from infancy to adolescence were significantly changed with depletion of butyric acid-producing bacteria, leading to a decrease in butyric acid in adulthood. It was also found that PCE female offspring rats were sensitive to NAFLD induced by a postnatal high-fat diet (HFD), which is mainly related to the enhancement of hepatic triglyceride synthesis function. Through mechanism exploration, we found that HFD further reduced the fecal and serum butyric acid levels in the PCE female offspring, which was significantly negatively correlated with hepatic SREBP-1c/FASN mRNA expression and triglyceride level. In vivo and in vitro experiments confirmed that sodium butyrate (NaB) supplementation could reduce hepatic lipid accumulation through MCT1/GPR109A-AMPK, thereby effectively decreasing the susceptibility to NAFLD in the PCE female offspring rats.

Nicotine, ethanol, and caffeine are the most common exogenous substances in the men’s living environment, but their effects on the cartilage quality in the father and offspring have not been reported. According to the average daily intake of adult men, we constructed a male rat model of paternal mixed exposure (PME) to low-dose nicotine (0.1 mg/kg.d), ethanol (0.5 g/kg.d), and caffeine (7.5 mg/kg.d) for 8 weeks. Then, the male rats mated with normal female rats to obtain offspring. The results showed that PME reduced the cartilage quality of paternal and offspring rats. Among them, the paternal cartilage was damaged by enhancing matrix degradation, while the offspring cartilage was damaged by reducing matrix synthesis. The cartilage damage in male offspring rats was more evident than in female offspring. It was further confirmed that differential GC regulation mechanisms were the main reasons for the intergenerational differential damage of paternal/offspring cartilage quality caused by PME. In addition, the sex hormone receptor AR and ERβ mediated the sex difference of PME-induced fetal cartilage dysplasia by affecting the binding degree of GR/P300. This study provided a theoretical and experimental basis for guiding male healthy lifestyle and exploring early prevention and treatment strategies for paternal diseases.