Active and passive maternal smoking during pregnancy is associated with numerous adverse pregnancy outcomes, including premature birth, intrauterine growth restriction, and neonatal respiratory system underdevelopment. These outcomes often result in pulmonary dysplasia in offspring and other lifelong respiratory consequences (McEvoy and Spindel, 2017; Ding et al., 2022; Tarasi et al., 2022). Nicotine is the principal component responsible for damaging the developing lung (Kurihara et al., 2023). Despite the well-documented harmful effects of nicotine, the global prevalence of active smoking among pregnant and lactating women is estimated to range between 5 and 40% (McEvoy and Spindel, 2017), while rates of passive smoking (secondhand smoke exposure) reach up to 75% (Zhang et al., 2015; Reece et al., 2019). Perinatal nicotine exposure (PNE) disrupts pulmonary alveolar development (Blaskovic et al., 2020) and impairs pulmonary function (Thacher et al., 2018) in offspring, leading to pulmonary dysplasia, increased susceptibility to respiratory infections (Cheemarla et al., 2019), and asthma (Ding et al., 2022). Alarmingly, these adverse effects extend to subsequent generations (Liu et al., 2020), underscoring the urgent need to address this issue.

PNE affects lung development through multiple pathways, one of which may involve the maternal gut microbiome and short-chain fatty acids (SCFAs). Prenatal nicotine exposure has been shown to alter the abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria in maternal gut microbiota, and to reduce SCFA levels in the blood and amniotic fluid of dams (Zubcevic et al., 2022). Altered maternal gut microbiota can influence offspring lung development by regulating SCFA levels. For instance, depletion of the gut microbiome in pregnant rats using antibiotics led to decreased maternal SCFA levels, impaired placental development, and pulmonary hypoplasia in offspring (Parsons et al., 2022; Pronovost et al., 2023). Additionally, SCFAs can bind to G protein-coupled receptors (GPRs) in the lungs, thereby influencing lung development in offspring. Acetic acid supplementation was found to upregulate GPR43 gene expression in the lungs of mice with pulmonary dysplasia, reduce inflammatory factors, and mitigate lung damage (Zhang et al., 2020). Based on this evidence, we hypothesize that PNE-induced pulmonary dysplasia in offspring is at least partially due to alterations in maternal gut microbiota, SCFA levels, and GPRs expression in offspring lung.

Glucocorticoids are commonly used to prevent neonatal lung damage (Crowther et al., 2015); however, this approach has not been proven to be universally effective and may be associated with significant long-term neurodevelopmental and respiratory adverse consequences (Wang et al., 2022). Electroacupuncture (EA), a traditional Chinese medicine therapy, has been shown to regulate general well-being, immunity, and endocrine and metabolic functions, as well as to influence intestinal flora (Ye et al., 2024). Our previous studies found that EA at “Zusanli (ST36)” acupoints in PNE dams could protect offspring from pathological abnormalities in pulmonary function and lung morphology. Furthermore, we found that EA at ST36 mitigates PNE-induced pulmonary dysplasia in offspring by modulating the hypothalamic–pituitary–adrenal axis in both mother and offspring, reducing maternal glucocorticoid overexposure, and decreasing the expression of glucocorticoid receptors in the offspring’s lung (Ji et al., 2016; Liu et al., 2018; Dai et al., 2020; Lu et al., 2020). However, EA is known to improve diseases such as functional constipation (Xu H. et al., 2023; Xu M. M. et al., 2023), chronic colitis (Wang et al., 2020), myocardial ischemia reperfusion injury (Bai et al., 2021), and type 2 diabetes (An et al., 2022) by regulating the gut microbiota. Currently, it remains unclear whether EA at ST36 in perinatal nicotine-exposed dams protects offspring pulmonary dysplasia by modulating maternal gut microbiota and SCFA levels. This study aims to determine whether EA exerts its protective effect by regulating the gut microbiota of PNE dams and SCFA levels in both dams and their offspring. Additionally, we depleted the gut microbiota in PNE dams using antibiotics to observe whether the protective effect of EA on pulmonary function and lung morphology in offspring is diminished.

In addition to controlling nutrient supply for growth, a mother can influence the development of her offspring’s lungs through various mechanisms. These include regulation of the HPA axis (Liu et al., 2018), gut microbiota and its metabolites (Rodrigues et al., 2021), genetics (Shorey-Kendrick et al., 2017), and immune mechanisms (Zhao et al., 2019). Recent studies have shown that nicotine, the primary harmful component of tobacco smoke, can alter the maternal gut microbiota and the levels of SCFA in both mother and offspring. These changes may affect the developing lungs of the offspring (Zhang et al., 2020; Zubcevic et al., 2022). In our previous work, we demonstrated that applying conventional EA at ST36 acupoints in the PNE dam protects their offspring from PNE-induced pulmonary dysplasia (Liu et al., 2018; Lu et al., 2020). Notably, this protective effect was also observed when EA was applied at a shallow depth on the ST36 acupoints (unpublished data). The pulmonary function data from this study supports these findings. Consistent with recent research suggesting that EA’s therapeutic effects may be due to its modulation of gut microbiota and SCFA levels (Xu H. et al., 2023; Xu M. M. et al., 2023), our current work highlights the significant role of maternal gut microbiota and the SCFA levels in both mother and offspring in mediating EA’s protective effects against PNE-induced pulmonary dysplasia.

Considered as the largest “microbial organ” in the human body, the gut microbiota consists of beneficial bacteria, pathogenic bacteria, and conditional pathogenic bacteria. It plays a crucial role in maintaining host health and contributing to disease development (Hou et al., 2022). Numerous studies have revealed that maternal gut microbiota may be involved in placental morphogenesis, offspring growth and development, and have a significant impact on the long-term respiratory health of offspring (Lange et al., 2012; Thorburn et al., 2015; Sato et al., 2019; Lopez-Tello et al., 2022; Pronovost et al., 2023). In a study involving pregnant rats, nicotine exposure (6 mg/kg) resulted in notable changes in the relative abundance of microbial taxa in the cecum. Specifically, there was an increased relative abundance of Actinobacteria and Firmicutes, a downward trend in the relative abundance of Bacteroides and Proteobacteria, and a significant enrichment of the genus SMB53 within the Firmicutes species observed on day 19 of gestation (Zubcevic et al., 2022). These findings suggest that nicotine exposure during pregnancy leads to alterations in the maternal gut microbiota, which we hypothesize may contribute to pulmonary dysplasia in the offspring.

In this study, we employed a well-established PNE dam model to induce offspring pulmonary dysplasia. Maternal fecal microbiota was analyzed on PD21 to evaluate the impact of PNE on gut microbiota. Our findings revealed that PNE altered gut microbiota, as evidenced by a reduction in the community gut microbiota richness, reflected by the Sobs index. In addition, there was a downward trend in the relative abundance of Firmicutes and Actinobacteria, while the relative abundance of Bacteroidetes and Proteobacteria increased slightly, though not significantly. At the genus level, we observed a significant enrichment of Enterococcus in the family Enterococcaceae of Firmicutes, alongside a reduction in the abundance of Dorea, Ligilactobacillus, and Blautia compared to the CON group. Notably, Enterococcus is a conditionally pathogenic bacterium found in higher concentrations in the gut microbiota of smokers (Northrup et al., 2021). Its enrichment has been associated with various pathological conditions, including an increased predisposition to infections and wheezing (Lange et al., 2012; Fiore et al., 2019). Conversely, we observed a decrease in the abundance of Dorea, a genus linked to the inhibition of nicotine addiction and its reduced prevalence in children with allergic rhinitis (Chen et al., 2023; Chiu et al., 2019). Ligilactobacillus and Blautia are known to produce beneficial metabolites such as acetic acid and lactic acid, making them popular candidates for probiotics (Zheng et al., 2020; Liu et al., 2021). Overall, our data demonstrate that PNE alters the maternal gut microbiota, reducing its richness, and significantly enriching the conditionally pathogenic bacteria, while decreasing the abundance of beneficial bacterial genera. Notably, the changes we observed in the fecal microbiota of PNE dams differ somewhat from those reported in a previous study (Zubcevic et al., 2022). These differences may be attributable to differences in nicotine dosage (1 mg/kg vs. 6 mg/kg), delivery method (subcutaneous injection vs. osmotic minipump), exposure duration (E6 to PD21 vs. E6 to E21), and timing of microbiota determination (PD21 vs. E19). Additionally, there were differences in the testing methods employed, such as the use of different primer sets (338F-806R vs. 515F-806R) for sequence identification.

Shallow EA needling at ST36 in PNE dams partially restored gut microbiota structure in the dams. This restoration was evidenced by an increase in the Sobs index and a reversal in phylum-level variations induced by PNE, indicating a partial reduction of PNE-induced alterations in maternal gut microbiota. Moreover, the resultant phylum-level changes resulting from EA were similar to those in the gut microbiota of individuals who had quit smoking (Biedermann et al., 2014). Additionally, EA promoted the enrichment of o__RF39, Clostridia, and Oscillospirales within the Firmicutes phylum of the maternal gut microbiota affected by PNE. At the genus level, EA decreased the abundance of Enterococcus while increasing the abundance of Ligilactobacillus, Blautia, and Dorea. The order RF39, a recently defined order of Bacilli, has limited relevant reports available (Kim et al., 2022), but one study demonstrated a negative correlation of Bacilli abundance and the occurrence of asthma (Li et al., 2024). Clostridium, a member of the Clostridia class, and Oscillospira, belonging to the Oscillospirales family, are known to upregulate butyric acid levels and are considered potential candidates for probiotics (Guo et al., 2020; Yang et al., 2021). In a broiler lung injury model induced by PM2.5 exposure, a decrease in the abundance of Oscillospirales in the gut microbiota was observed, along with a reduction in SCFAs production, which may be associated with lung injury (Zhou et al., 2024). Based on these findings, we conclude that shallow EA needling at ST36 in PNE dams can exert a protective effect against offspring pulmonary dysplasia. This protective effect is likely mediated through the regulation of the maternal gut microbiota composition and structure, including an increase in community richness and abundance of beneficial bacteria, and reduced prevalence of opportunistic pathogenic bacteria.

SCFAs are among the most abundant microbial metabolites in the gut, consisting primarily of acetic, propionic, and butyric acids. Adverse changes in maternal serum SCFA levels may affect developing offspring and predispose them to respiratory diseases (Priyadarshini et al., 2014; Thorburn et al., 2015). Nicotine exposure in pregnant rats has previously been shown to decrease propionic acid levels in cecum and cause a downward trend in plasma acetic, propionic and butyric acids concentrations (Zubcevic et al., 2022). In our study, we also found decreased serum levels of acetic acid, butyric acid, and total SCFAs in PNE dams, with a downward trend in propionic, isobutyric and valeric acid levels. However, shallow EA needling at ST36 in PNE dams increased serum levels of acetic acid, isobutyric acid, butyric acid, and total SCFAs, while showing an upward trend in propionic and valeric acid concentrations. These results indicate that EA may protect offspring from PNE-induced pulmonary dysplasia by improving maternal SCFA levels.

Correlation network analysis between maternal gut microbiota and serum SCFAs revealed the following associations: Enterococcus was negatively correlated with propionic acid; g__unclassified_o__Oscillospirales was positively correlated with butyric acid; g__unclassified_o__RF39 was positively correlated with acetic, butyric, isobutyric and propionic acids; and Dorea was positively correlated with acetic, butyric, isobutyric, propionic and valeric acids. Since Enterococcus is not a major producer of SCFAs (Akhtar et al., 2022; Zhang et al., 2023) and the levels of acetic, butyric, and total SCFAs in the serum of PNE dams significantly altered without a statistically significant decrease in propionic acid levels, it suggests that PNE impairs offspring lung development likely by increasing the abundance of conditionally pathogenic bacteria and reducing the abundance of acetic or butyric acid producing bacteria. As a result, the protective effects of EA may be related to its ability to reverse these gut microbiota changes in PNE dams.

Moreover, changes in SCFA levels in early life of offspring may influence the development of later respiratory diseases. For example, a clinical trial found that infants with higher levels of butyric and propionic acid in their stool had a lower risk of future asthma (Roduit et al., 2019). SCFAs can bind to GPRs in the lung, influencing offspring lung development, with acetic acid and propionic acid preferentially activating GPR43, and propionic acid and butyric acid activating GPR41 (Mishra et al., 2020; Zhang et al., 2020). In the present study, we found that PNE led to decreased serum levels of acetic acid and total SCFAs, as well as a downward trend in the levels of propionic, isobutyric, butyric and valeric acids in the offspring of exposed dams. Correlation analysis indicated that this may be related to the reduced levels of SCFA in the serum of the dams. Additionally, PNE also downregulated GPR41 and GPR43 expression in offspring lungs. Therefore, reduced receptor binding of already diminished SCFAs provides an additional strong plausible contributor to PNE-induced offspring pulmonary dysplasia. Reversal of these effects by EA suggests that EA mediated amelioration of PNE-induced pulmonary dysplasia in offspring may involve modulation of serum SCFA levels in both the dams and offspring and the increased expression of SCFA receptors in offspring lung tissue.

Finally, since antibiotics can result in gut microbiome depletion, we used an antibiotic cocktail (Abx), consisting of ampicillin, neomycin, metronidazole and vancomycin, to further clarify the role of maternal gut microbiota in EA-mediated protection against PNE-induced offspring lung injury. Notably, a rodent study showed that, along with the reduction in gut microbiome abundance and diversity, Abx also reduced serum acetic, propionic and butyric acid levels (Ray et al., 2021). We observed that Abx administration, when combined with EA, blocked the beneficial effects of EA on pulmonary function and lung morphology in PNE offspring. These findings suggest that the protective effects of EA against PNE-induced pulmonary impairments in offspring may be related to maternal gut microbiota.

These data show that shallow EA needling at ST36 in PNE dams effectively improves pulmonary dysplasia in offspring rats. The protective mechanism of EA is likely associated with the regulation of changes in the gut microbiota of PNE dams. This mechanism primarily involves enhancing the composition of the maternal gut microbiota by increasing the community richness, boosting the abundance of beneficial bacteria, reducing the abundance of conditionally pathogenic bacteria, and upregulating the levels of acetic acid, butyric acid and total SCFAs in serum of dams and offspring. Additionally, EA treatment promotes the expression of GPR41 and GPR43 in offspring lung tissue. However, several limitations exist in our study. We failed to perform fecal transplantation to further elucidate the relationship between the therapeutic effect of EA and the gut microbiota and SCFAs. Moreover, we also did not use untargeted metabolomics or other methods to determine whether EA caused changes in other metabolites in the serum. Furthermore, the long-term effects of EA on the lung development of offspring were not investigated in our study, which is a long process that can be extended into until adolescence. For this reason, further studies are required needed to fully comprehend the specific mechanisms underlying the effects of EA treatment on gut microbiota and SCFAs, as well as the long-term efficacy of EA.