Fatty Liver Tissue Research Articles

Fluorescence lifetime imaging: A novel approach to simultaneous visualizing lipid droplets and endoplasmic reticulum polarity in live cells and fatty liver

Cellular polarity plays an indispensable role in both physiological functions and the progression of diseases, with organelles demonstrating specific polarities that are integral to their roles. The advent of a single fluorescent probe capable of concurrently and selectively visualizing two distinct organelles through distinct fluorescence emission spectra has emerged as a powerful approach for investigating their interactions within cellular mechanisms. However, the use of changes in dual fluorescence lifetimes to monitor polarity changes in two organelles has rarely been employed, despite its potential benefits for more accurate detection of subtle changes in organelle polarity. Herein, two polarity-sensitive probes (ER-LDs-1 and ER-LDs-2) were developed. ER-LDs-2 exhibited high sensitivity to polarity alterations in both lipid droplets (LDs) and endoplasmic reticulum (ER) under various physiological and pathological conditions, including ferroptosis and endoplasmic reticulum stress, as demonstrated by distinct changes in fluorescence lifetime. ER-LDs-2 enables the monitoring of polarity changes in mouse and human normal and fatty liver tissues by fluorescence lifetime imaging. Collectively, this study presents innovative tools that hold great potential for advancing our understanding of cellular organelle polarity dynamics and the progression of associated diseases.

Transcriptome responses to benzo[a]pyrene in liver slices of sub-arctic fish species

Due to the expanding oil-related activities, the arctic and sub-arctic marine environments are increasingly vulnerable to oil-related pollution such as accidental oil spills. These cold-water ecosystems harbor many fish species that are both ecologically and economically important such as the pelagic polar cod (Boreogadus saida), capelin (Mallotus villosus), and benthic long rough dab (Hippoglossoides platessoides). The latter two are much less studied and it is crucial to characterize their responses to oil-related contaminants and develop molecular biomarkers and genomic resources for future monitoring. In this study, liver slice preparation and culture methods were used to characterize the transcriptome responses (using RNA-seq) in capelin and long rough dab to exposures of the polycyclic aromatic hydrocarbon (PAH) compound benzo[a]pyrene (BaP). The liver slice culture and exposure experiments were performed onboard a research vessel in the Barents Sea. Strong up-regulation of genes involved in biotransformation, particularly the aryl hydrocarbon receptor signaling pathway was observed in both species. A comparison of the number of differentially expressed genes (DEGs) with previously published polar cod exposures indicates that the latter responded more strongly (higher number of genes), suggesting higher uptake and bioconcentration of BaP in the fatty liver tissue, although other factors such as differences in clearance rate could potentially affect the responses. This study provides new genomic resources and gene expression biomarkers in capelin and long rough dab, enhancing our understanding of their response mechanism to oil-related contaminants.

Highly sensitive near-infrared fluorescent probe for monitoring peroxynitrite in nonalcoholic fatty liver disease: Toward early diagnosis and therapeutic evaluation

Nonalcoholic fatty liver disease (NAFLD) poses a significant global health concern, necessitating precise diagnostic tools and effective treatment strategies. Peroxynitrite (ONOO−), a reactive oxygen species, plays a pivotal role in NAFLD pathogenesis, highlighting its potential as a biomarker for disease diagnosis and therapeutic evaluation. This study reports on the development of a near-infrared (NIR) fluorescent probe, designated DRP-O, for the selective detection of ONOO− with high sensitivity and photostability. DRP-O exhibits rapid response kinetics (within 2 min) and an impressive detection limit of 2.3 nM, enabling real-time monitoring of ONOO− dynamics in living cells. Notably, DRP-O demonstrates excellent photostability under continuous laser irradiation, ensuring reliable long-term monitoring in complex biological systems. We apply DRP-O to visualize endogenous ONOO− in living cells, demonstrating its potential for diagnosing and monitoring NAFLD-related oxidative stress. Furthermore, DRP-O effectively evaluates the efficacy of therapeutic drugs in NAFLD cell models, underscoring its potential utility in drug screening studies. Moreover, we confirm DRP-O to enable selective identification of fatty liver tissues in a mouse model of NAFLD, indicating its potential for the early diagnosis of NAFLD. Collectively, DRP-O represents a valuable tool for studying ONOO− dynamics, evaluating drug efficacy, and diagnosing NAFLD, offering insights into novel therapeutic strategies for this prevalent liver disorder.

Near-infrared polarity fluorescent probe for wash-free imaging lipid droplets and the application in the diagnosis of non-alcoholic fatty liver tissue

Lipid droplets (LDs), as primary cellular energy storage organelles, play essential roles not only in energy conservation but also in inflammatory responses, apoptosis, and the progression of diseases such as non-alcoholic fatty liver disease (NAFLD). Studies indicate that alterations in the polarity of LDs are closely linked to pathological factors like oxidative stress, ferroptosis, and lipophagy. Therefore, developing biological probes for precisely monitoring LDs polarity is crucial for understanding these biological phenomena and devising new therapeutic strategies. In this context, we synthesized four fluorescent probes (CNL1-CNL4) to precisely monitor the polarity of LDs using triphenylamine and carbazole derivatives as electron donors and isophorone and benzopyranonitrile units as electron acceptors, employing intramolecular charge transfer (ICT) mechanism. Our findings demonstrated that all four probes exhibited high sensitivity and specificity for polarity changes. Probes CNL2 and CNL4 were sensitive to polarity, and their maximum fluorescence intensity and maximum emission wavelength demonstrated a good linear relationship with polarity parameters within a certain range. Biological experiments demonstrated that both CNL2 and CNL4 possess excellent wash-free imaging capabilities and superior LDs targeting abilities but also enable dynamic monitoring of LDs and respond well to changes in LDs polarity under varying concentrations of oleic acid stimulation. Due to its longer emission wavelength (>650 nm), boasting excellent photostability, and featuring a larger Stokes shift (≈168 nm in acetonitrile), probe CNL4 was selected for further studies. Furthermore, probe CNL4 effectively monitors changes in LDs polarity under conditions of ferroptosis and oxidative stress. It has shown potential in the diagnosis of NAFLD tissue and during the process of lipophagy, highlighting its value in disease diagnostics and biological research. These findings not only deepen our understanding of the mechanisms underlying NAFLD progression but also provide new tools for diagnosing and treating related diseases.

Mitochondria-Targetable Near-Infrared Fluorescent Probe for Visualization of Hydrogen Peroxide in Lung Injury, Liver Injury, and Tumor Models.

Hydrogen peroxide (H2O2) overexpressed in mitochondria has been regarded as a key biomarker in the pathological processes of various diseases. However, there is currently a lack of suitable mitochondria-targetable near-infrared (NIR) probes for the visualization of H2O2 in multiple diseases, such as PM2.5 exposure-induced lung injury, hepatic ischemia-reperfusion injury (HIRI), nonalcoholic fatty liver (NAFL), hepatic fibrosis (HF), and malignant tumor tissues containing clinical cancer patient samples. Herein, we conceived a novel NIR fluorescent probe (HCy-H2O2) by introducing pentafluorobenzenesulfonyl as a H2O2 sensing unit into the NIR hemicyanine platform. HCy-H2O2 exhibits good sensitivity and selectivity toward H2O2, accompanied by a remarkable "turn-on" fluorescence signal at 720 nm. Meanwhile, HCy-H2O2 has stable mitochondria-targetable ability and permits monitoring of the up-generated H2O2 level during mitophagy. Furthermore, using HCy-H2O2, we have successfully observed an overproduced mitochondrial H2O2 in ambient PM2.5 exposure-induced lung injury, HIRI, NAFL, and HF models through NIR fluorescence imaging. Significantly, the visualization of H2O2 has been achieved in both tumor-bear mice as well as surgical specimens of cancer patients, making HCy-H2O2 a promising tool for cancer diagnosis and imaging-guided surgery.

Detraining after short-term exercise induces hyperphagia and obesity with fatty liver and brown adipose tissue whitening in young male OLETF rats.

This study examined the effects of exercise and detraining at a young age on fat accumulation in various organs. Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats were assigned to either the non-exercise sedentary (OLETF Sed) or exercise groups. The exercise group was subdivided into two groups: exercise between 4 and 12 weeks of age (OLETF Ex) and exercise between 4 and 6 weeks of age followed by non-exercise between 6 and 12 weeks of age (OLETF DT). Body weight was significantly lower in the OLETF Ex group than in the OLETF Sed group at 12 weeks of age. Fat accumulation in the epididymal white adipose tissue, liver, and brown adipose tissue was suppressed in the OLETF Ex group. During the exercise period, body weight and food intake in the OLETF DT group were significantly lower than those in the OLETF Sed group. However, food intake was significantly higher in the OLETF DT group than in the OLETF Sed group after exercise cessation, resulting in extreme obesity with fatty liver and brown adipose tissue whitening. Detraining after early-onset exercise promotes hyperphagia, causing extreme obesity. Overeating should be avoided during detraining periods in cases of exercise cessation at a young age.

Photoactivated theranostic nanomaterials based on aggregation‐induced emission luminogens for cancer photoimmunotherapy

AbstractIn this study, a series of high liposoluble near‐infrared emissive aggregation‐induced emission luminogens with triphenylamine derivatives as donor units and 1‐indanone as electron acceptor units are developed. Specifically, MTTOI has promising lipid droplets targeting ability and viscosity‐response characteristics, and it can monitor the viscosity fluctuations in living cells in real time. It is found that MTTOI is able to fulfill the activation of the apoptosis‐related signaling pathways under white light, and it can also activate the ferroptosis to synergize apoptosis resulting in tumor elimination. Interestingly, the occurrence of ferroptosis is positively related to the enhancement of immunogenic cell‐death effect, thus boosting the tumor infiltration of CD8+ T cells and reducing the proportion of regulatory T cells by cooperating with dendritic cells, which can not only carry out primary antitumor treatment, but also prevent tumor metastasis through strong immunological memory effect. Moreover, in vivo experimental results confirm that MTTOI successfully effectuates fatty liver tissue imaging. MTTOI fluorescence signals can be captured at the tumor site after 3 days of administration due to its high fat‐solubility and ease of fusion with tumors. This classic example could promote the further development of phototherapeutic agents in preclinical research and clinical applications.

Relationship between transmission/reception conditions of high-frequency plane wave compounding and evaluation accuracy of extended amplitude envelope statistics

The double–Nakagami (DN) model provides a method for analyzing the amplitude envelope statistics of quantitative ultrasound (QUS). In this study, the relationship between the sound field characteristics and the robustness of QUS evaluation was evaluated using five HF linear array probes and tissue-mimicking phantoms. Compound plane-wave imaging (CPWI) was used to acquire echo data. Five phantoms containing two types of scatterers were used to mimic fatty liver tissue. After clarifying the relationship between the sound field characteristics of the probes and QUS parameters, DN QUS parameters in 10 rat livers with different lipidification were evaluated using one HF linear array probe. For both phantom and in situ liver analyses, correlations between fat content and multiple QUS parameters were confirmed, suggesting that the combination of CPWI using a HF linear array probe with the DN model is a robust method for quantifying fatty liver and has potential clinical diagnostic applications.

Rational design of a red-emission fluorescent probe for imaging mitochondrial viscosity and fatty liver tissues

Abnormal mitochondrial viscosity is closely associated with various diseases. Thus, it is imperative to design and synthesized fluorescent probes for monitoring changes in mitochondrial viscosity. In this work, two series of fluorescence probes with triphenylamine unit and 2-hydroxyphenylbenzothiazole unit respectively were constructed. The impact of varying chain lengths on the photophysical properties of these probes was investigated. The results indicated that probe TPAC12 with dodecane group exhibited the largest fluorescence enhancement. The results of cell imaging experiments demonstrated that all these probes exhibited specific targeting towards mitochondria and were not influenced by changes in mitochondrial membrane potential. Additionally, probe TPAC12 facilitated the monitoring of viscosity alterations within mitochondria. Importantly, probe TPAC12 has been effectively utilized for the identification of both fatty liver and normal liver tissues. These results suggested that probe TPAC12 holds great potential as a valuable tool for detecting mitochondrial viscosity in biological systems.

Systematic analysis of the relationship between non-alcoholic fatty liver disease and tissue iron overload: promising areas for the use of polypeptide therapy

Iron overload in non-alcoholic fatty liver disease (NAFLD) is a fairly common phenomenon that receives very little attention in clinical practice. However, iron overload, leading to hemosiderosis (deposition of “indigestible” nanodispersed iron oxides in various tissues) significantly aggravates NAFLD, stimulating increased chronic inflammation, insulin resistance and hemosiderosis of other organs. As a result, ferroptosis of hepatocytes occurs (apoptosis caused by iron overload and hemosiderosis), which accelerates the transformation of non-alcoholic steatosis into non-alcoholic steatohepatitis (NASH) and, subsequently, into liver cirrhosis. Iron overload is aggravated by micronutrient deficiencies and pathogenic intestinal microbiota. The paper presents the results of a systematic analysis of this issue, describes the prospects for therapy using micronutrients and human placenta hydrolysates (HPP), which contribute not only to the regeneration of liver tissue, but also to the normalization of iron homeostasis.

Isophorone-based AIEgens fluorescent probe with red emission for targeting lipid droplets and identifying non-alcoholic fatty liver disease

Due to the disorder of lipid metabolism, the excessive accumulation of lipid droplets (LDs) in liver cells can result in the occurrence of non-alcoholic fatty liver disease (NAFLD). Therefore, it is great of significance to design and synthesized LDs-specific fluorescent probes for the early diagnosis of NAFLD. Herein, we developed a series of aggregation-induced emission (AIE) probes ISO-LD1, ISO-LD2 and ISO-LD3 based on isophorone group for LDs-specific imaging in living cells. The photophysical properties demonstrated that all the probes with red emission (λem > 600 nm) exhibited a strong fluorescence in high polarity solvents. In particular, probe ISO-LD3 has a highest fluorescence quantum yield (except for 1,4-dioxane) and a larger Stokes shift. Confocal laser scanning microscopy experiments indicated that probe ISO-LD3 could specifically stain LDs via a “washing-free” procedure within 10 s, and monitor the dynamic behaviors of LDs exhibiting a high signal/noise ratio. Importantly, given the satisfactory performance of probe ISO-LD3, it has been successfully used for the detection of the normal liver tissues and fatty liver tissues, respectively. This work illustrated that ISO-LD3 is a promising tool for the detection of LDs and LDs-related diseases.

Menin orchestrates hepatic glucose and fatty acid uptake via deploying the cellular translocation of SIRT1 and PPARγ

BackgroundMenin is a scaffold protein encoded by the Men1 gene, which interacts with various transcriptional proteins to activate or repress cellular processes and is a key mediator in multiple organs. Both liver-specific and hepatocyte-specific Menin deficiency promotes high-fat diet-induced liver steatosis in mice, as well as insulin resistance and type 2 diabetic phenotype. The potential link between Menin and hepatic metabolism homeostasis may provide new insights into the mechanism of fatty liver disease.ResultsDisturbance of hepatic Menin expression impacts metabolic pathways associated with non-alcoholic fatty liver disease (NAFLD), including the FoxO signaling pathway, which is similar to that observed in both oleic acid-induced fatty hepatocytes model and biopsied fatty liver tissues, but with elevated hepatic Menin expression and inhibited FABP1. Higher levels of Menin facilitate glucose uptake while restraining fatty acid uptake. Menin targets the expression of FABP3/4/5 and also CD36 or GK, PCK by binding to their promoter regions, while recruiting and deploying the cellular localization of PPARγ and SIRT1 in the nucleus and cytoplasm. Accordingly, Menin binds to PPARγ and/or FoxO1 in hepatocytes, and orchestrates hepatic glucose and fatty acid uptake by recruiting SIRT1.ConclusionMenin plays an orchestration role as a transcriptional activator and/or repressor to target downstream gene expression levels involved in hepatic energy uptake by interacting with the cellular energy sensor SIRT1, PPARγ, and/or FoxO1 and deploying their translocations between the cytoplasm and nucleus, thereby maintaining metabolic homeostasis. These findings provide more evidence suggesting Menin could be targeted for the treatment of hepatic steatosis, NAFLD or metabolic dysfunction-associated fatty liver disease (MAFLD), and even other hepatic diseases.Graphical

Dual-channel fluorescent probe for simultaneously detecting H2S and viscosity/polarity and its application in non-alcoholic fatty liver, tumor tissue, and food spoilage

Intracellular hydrogen sulfide (H2S) and micro-environments (e.g. viscosity and polarity) are tightly correlated with various physiological/pathological processes, making them potential biomarkers of many diseases. However, the simultaneously detecting H2S, viscosity, and polarity in inflammation, non-alcoholic fatty liver (NAFL) and tumor tissues containing clinical cancer patient samples has not been achieved yet due to the lack of effective tools. Herein, we presented a mitochondria-targeting near-infrared (NIR) fluorescent probe MQA-DNP) for simultaneously monitoring H2S, viscosity/polarity through dual channels. The probe could specifically recognize H2S via far-red emission (λem = 634 nm), and exhibited high sensitivity toward micro-environmental viscosity/polarity in the NIR channel (λem > 714 nm). Facilitated with the probe, we revealed for the first time that inflammation cells and NAFL tissues are accompanied by an up-regulation of H2S, as well as an increase in viscosity level (and/or decrease in polarity degree). Surprisingly, the simultaneous visualization of H2S, viscosity/polarity has also been achieved not only at the cancer cells and tumor models, but also in clinical cancer patients. Compared to detecting a single biomarker, the simultaneous tracking multi-markers through multi-channels seems to be more reliable for visual medical diagnosis of mitochondria-related diseases. Furthermore, inspired by the significant color changes and high sensitivity of MQA-DNP reaction with H2S, MQA-DNP has been successfully used to detect H2S released during food spoilage through test strips, and has great potential for application in complex environments systems.

Engineering of a NIR fluorescent probe for high-fidelity tracking of lipid droplets in living cells and nonalcoholic fatty liver tissues

LDs (Lipid droplets) are key organelles for lipid metabolism and storage, which are closely related to ferroptosis and fatty liver. Due to its small size and highly dynamic nature, developing high-fidelity fluorescent probes for imaging of LDs is crucial for observing the dynamic physiological processes of LDs and investigating LDs-associated diseases. Herein, we synthesized three dicyanoisophorone-based fluorescent probes (DCIMe, DCIJ, and DCIQ) with different electron-donating groups and studied their imaging performance for LDs. The results show that DCIQ is highly polarity sensitive and can perform high-fidelity imaging for LDs, with significantly better performance than DCIMe, DCIJ, and commercial LD probe BODIPY 493/503. Based on this, DCIQ was successfully applied to real-time observe the interplays between LDs and other organelles (mitochondria, lysosomes, and endoplasmic reticulum), and to image the dynamics of LDs with fast scanning mode (0.44 s/frame) and the generation of oleic acid-induced LDs with high-fidelity. Finally, DCIQ was used to study the changes of LDs in the ferroptosis process and nonalcoholic fatty liver disease tissues. Overall, this study provided a powerful tool for high-fidelity imaging of LDs in cells and tissues.

A lipid droplets-targetable fluorescent probe for polarity detection in cells of iron death, inflammation and fatty liver tissue

Abnormal accumulation and metabolism of lipid droplets can lead to a variety of diseases. Polarity, a key parameter of the microenvironment, is closely associated with many diseases and dysfunctions in the body. It is important to elucidate the relationship between the physiological activity of lipid droplets (LDs) and the polarity of the microenvironment. In this work, based on push-pull mechanism, a fluorescent probe (E)-3-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-1-(2-hydroxyphenyl)prop‑2-en-1-one (PPTH) with aggregation-induced emission (AIE) properties for the detection of polarity changes in cells was synthesized. PPTH not only visualize intracellular polarity fluctuation of iron death and inflammation but also distinguish between normal and fatty liver tissue.

Dielectric and Biological Characterization of Liver Tissue in a High-Fat Diet Mouse Model.

Hepatic steatosis may be caused by type 2 diabetes or obesity and is one of the origins of chronic liver disease. A non-invasive technique based on microwave propagation can be a good solution to monitor hepatic tissue pathologies. The present work is devoted to the dielectric permittivity measurements in healthy and fatty liver in the microwave range. A mouse model following normal and high sugar/glucose (HFS) diets was used. We demonstrated the change in the triglyceride and glucose concentration in the hepatic tissue of HFS diet mice. The difference in the dielectric permittivity of healthy and fatty liver was observed in the range from 100 MHz to 2 GHz. The dielectric permittivity was found to be 42 in the healthy tissue and 31 in the fatty liver tissue at 1 GHz. The obtained results demonstrate that dielectric permittivity can be a sensitive tool to distinguish between healthy and fatty hepatic tissue.

Raman imaging of rat nonalcoholic fatty liver tissues reveals distinct biomolecular states.

An essential challenge in diagnosing states of nonalcoholic fatty liver disease (NAFLD) is the early prediction of progression from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) before the disease progresses. Histological diagnoses of NAFLD rely on the appearance of anomalous tissue morphologies, and it is difficult to segment the biomolecular environment of the tissue through a conventional histopathological approach. Here, we show that hyperspectral Raman imaging provides diagnostic information on NAFLD in rats, as spectral changes among disease states can be detected before histological characteristics emerge. Our results demonstrate that Raman imaging of NAFLD can be a useful tool for histopathologists, offering biomolecular distinctions among tissue states that cannot be observed through standard histopathological means.

Red-emissive Dual-state Fluorogenic Probe for Wash-free Imaging of Lipid Droplets in Living Cells and Fatty Liver Tissues.

Lipid droplet (LD) dysfunction can result in various diseases, such as nonalcoholic fatty liver disease. Imaging agents built on dual-state emission (DSE) molecules that fluoresce in both dilute solutions and the aggregated state are receiving attention as this type of probe could provide bright fluorescence signals at variable concentrations, avoiding false signal readout caused by the concentration fluctuation in living systems. Herein, we identified a red emissive molecule featuring DSE, from three newly synthesized molecules, for specific detection of LDs in live cells. The bioimaging abilities have been well confirmed by optical spectroscopies, theoretical calculations, cell experiments, as well as animal studies. The DSE probe is effective for LD detection at concentrations ranging from 1 μM to 100 μM while retaining high brightness and signal fidelity. This study provides a knowledge base for the future design of DSE-active fluorescent probes for understanding LD-related diseases.

Solvatochromic Near-Infrared Aggregation-Induced Emission-Active Acrylonitriles by Acceptor Modulation for Low-Power Stimulated Emission Depletion Nanoscopy

Stimulated emission depletion (STED) nanoscopy has broadened our horizons to unravel mysterious functions of cellular structures on an unparalleled nanometer scale. Nevertheless, an intense depletion laser power is a general prerequisite for STED super-resolution imaging with a satisfactory resolution, inevitably leading to severe photobleaching of fluorophores, irreparable photodamage to biosamples, and impaired imaging quality. Herein, by modulating distinct acceptor units, a series of donor–acceptor–acceptor structured fluorescent acrylonitriles featured with aggregation-induced emission (AIE) for STED super-resolution imaging at a low depletion power were systematically developed. These AIE luminogens (AIEgens) exhibited tunable near-infrared emissions (650–733 nm) and high fluorescence quantum yields (QYs of up to 26.8%) in the solid state, significant Stokes shifts, and large two-photon absorption cross-sections. They also exhibited a typical solvatochromic effect and ultrahigh QYs of up to 98.4% in low-polarity solvents. Furthermore, these lipophilic solvatochromic acrylonitriles specifically lit up lipid droplets (LDs) with exceptionally high photostability in a wash-free manner. By taking TPA-BT-ANBI as an example, the STED super-resolution imaging of LDs with excellent resolution of 62 and 80 nm for cytosolic and nuclear LDs, respectively, at a low saturation depletion power of 0.83 MW/cm2 and extended time-lapse imaging of LD dynamics was achieved. Subsequent use of TPA-BT-ANBI for the optical discrimination of fatty liver tissues and two-photon deep-tissue imaging was also demonstrated. This study opens new avenues for versatile photostable materials at low depletion powers for target-specific STED super-resolution imaging.

Characteristics of the microstructure and the key components of white kidney bean sourdough bread induced by mixed-strain fermentation and its influence on gut microbiota.

In this study, the effect of mixed-strain fermentation using Kluyveromyces marxianus with either Lactobacillus plantarum or Pediococcus pentosaceus on the physiochemical and nutritional properties of white kidney bean flour sourdough was investigated. The results indicated that mixed-strain fermentation reduced the anti-nutritional factors produced from the white kidney bean flour, especially in the sourdough fermented by L. plantarum and K. marxianus (WKS-LK) compared to that by P. pentosaceus and K. marxianus (WKS-JK). Meanwhile, the content of lactic acid and acetic acid and the proportion of peptides with molecular weights ranging from <500 to 5000 Da were increased in the sourdoughs (WKS-LK > WKS-JK). Compared to the control (WK), microstructural characteristics of the dough seemed to be improved in WKS-LK followed by WKS-JK in terms of their corresponding gluten network consistency. Moreover, mixed fermentation led to a reduced starch digestibility accompanied by a higher content of resistant starch and slowly digestible starch. In contrast, protein digestibility was enhanced in WKS-LK and WKS-JK sourdough breads. More importantly, the changes in gut microbiota composition, short-chain fatty acid (SCFA) production, systemic inflammation, glucose tolerance and liver tissue histopathology following 21-day consumption of the sourdough bread were also evaluated via an animal model. The intake of sourdough breads reduced the abundance of the pathogenic microbiota Escherichia shigella. In contrast, the corresponding abundance of Rikenellaceae, Akkermansiaceae, Erysipelotrichaceae, Prevotellaceae and Eubacterium coprostanoligenes was increased, followed by enhanced SCFA generation, with the highest in WKS-LK and then WKS-JK. Meanwhile, a reduced level of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in the serum and improved glucose tolerance and liver tissue histopathology following the bread consumption were also achieved in the order of WKS-LK, then WKS-JK mice compared to WK.