Dioscin improves fatty liver hemorrhagic syndrome by promoting ERα-AMPK mediated mitophagy in laying hens.

BACKGROUND: Mitochondria play a crucial role in upholding metabolic homeostasis. Mitochondrial damage closely associated with the pathogenesis of fatty liver hemorrhagic syndrome (FLHS), while mitophagy being among the most effective methods for eliminating the damaged mitochondria. Dioscin, a natural extract, can activate autophagy; however, its effects on FLHS regarding mitophagy regulation remain unelucidated. PURPOSE: We explored the impact of dioscin on FLHS induced by a high-energy and low-protein (HELP) diet in laying hens, mainly focused the protective effects of dioscin on mitochondrial injury. METHOD: To investigate the impact of dioscin on fatty liver syndrome in laying hens, we first induced the condition by feeding them a high-energy and low-protein diet. Then, we assessed lipid metabolism-related markers using oil red staining and a commercial detection kit. In addition, the role of dioscin on fatty liver syndrome in laying hens was confirmed by assessing the activation of hepatocyte fat deposition and hepatocyte apoptosis; and the mechanism of dioscin in FLHS was investigated through LMH cell experiment in vitro. Furthermore, CETSA and molecular docking were conducted for additional confirmation. RESULT: The results showed that dioscin alleviated mitochondrial damage, relieved the excessive deposition of hepatic lipid droplets and oxidative stress induced by HELP diet in laying hens. Furthermore, dioscin regulated the mitophagy by activating the estrogen receptor α (ERα)/adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway, thus mitigating mitochondria injury and apoptosis in hepatocytes. In addition, we found that dioscin promoted the translocation of nuclear transcription factor into nucleus by activating ERα-AMPK signaling, facilitating autophagic flux in the liver of laying hens and LMH cells. Furthermore, cells pretreated with the lysosomal acidification inhibitor bafilomycin A1 blocked the inhibitory effect of dioscin on the apoptosis induced by palmitic acid (PA)-stimulation in LMH cells, suggesting that dioscin reduces PA-induced apoptosis by activating mitophagy. Moreover, dioscin-induced lysosomal acidification and mitochondrial biogenesis were reversed in PA-induced LMH cells pretreated with ERα-specific inhibitor methylpiperidino pyrazole. CONCLUSION: This study firstly demonstrated that dioscin alleviates fatty liver syndrome induced by HELP diet in laying hens. The findings from this study illustrated that dioscin plays a significant role in reducing mitochondrial damage and apoptosis, and these beneficial effects mainly achieve through promotion of ERα-AMPK signaling, which mediates autophagy within the liver of laying hens fed a HELP-diets. These findings provide a theoretical basis for considering dioscin as a possible treatment option for mitigating FLHS in egg-laying hens.

Polygenic prediction of human longevity on the supposition of pervasive pleiotropy.

The highly polygenic nature of human longevity renders pleiotropy an indispensable feature of its genetic architecture. Leveraging the genetic correlation between aging-related traits (ARTs), we aimed to model the additive variance in lifespan as a function of the cumulative liability from pleiotropic segregating variants. We tracked allele frequency changes as a function of viability across different age bins and prioritized 34 variants with an immediate implication on lipid metabolism, body mass index (BMI), and cognitive performance, among other traits, revealed by PheWAS analysis in the UK Biobank. Given the highly complex and non-linear interactions between the genetic determinants of longevity, we reasoned that a composite polygenic score would approximate a substantial portion of the variance in lifespan and developed the integrated longevity genetic scores (iLGSs) for distinguishing exceptional survival. We showed that coefficients derived from our ensemble model could potentially reveal an interesting pattern of genomic pleiotropy specific to lifespan. We assessed the predictive performance of our model for distinguishing the enrichment of exceptional longevity among long-lived individuals in two replication cohorts (the Scripps Wellderly cohort and the Medical Genome Reference Bank (MRGB)) and showed that the median lifespan in the highest decile of our composite prognostic index is up to 4.8 years longer. Finally, using the proteomic correlates of iLGS, we identified protein markers associated with exceptional longevity irrespective of chronological age and prioritized drugs with repurposing potentials for gerotherapeutics. Together, our approach demonstrates a promising framework for polygenic modeling of additive liability conferred by ARTs in defining exceptional longevity and assisting the identification of individuals at a higher risk of mortality for targeted lifestyle modifications earlier in life. Furthermore, the proteomic signature associated with iLGS highlights the functional pathway upstream of the PI3K-Akt that can be effectively targeted to slow down aging and extend lifespan.

Activation of ARP2/3 and HSP70 Expression by Lipoteichoic Acid: Potential Bidirectional Regulation of Apoptosis in a Mastitis Inflammation Model.

Mastitis typically arises from bacterial invasion, where host cell apoptosis significantly contributes to the inflammatory response. Gram-positive bacteria predominantly utilize the virulence factor lipoteichoic acid (LTA), which frequently leads to chronic breast infections, thereby impacting dairy production and animal husbandry adversely. This study employed LTA to develop models of mastitis in cow mammary gland cells and mice. Transcriptomic analysis identified 120 mRNAs associated with endocytosis and apoptosis pathways that were enriched in the LTA-induced inflammation of the Mammary Alveolar Cells-large T antigen (MAC-T), with numerous differential proteins also concentrated in the endocytosis pathway. Notably, actin-related protein 2/3 complex subunit 3 (ARPC3), actin-related protein 2/3 complex subunit 4 (ARPC4), and the heat shock protein 70 (HSP70) are closely related. STRING analysis revealed interactions among ARPC3, ARPC4, and HSP70 with components of the apoptosis pathway. Histological and molecular biological assessments confirmed that ARPC3, ARPC4, and HSP70 were mainly localized to the cell membrane of mammary epithelial cells. ARPC3 and ARPC4 are implicated in the mechanisms of bacterial invasion and the initiation of inflammation. Compared to the control group, the expression levels of these proteins were markedly increased, alongside the significant upregulation of apoptosis-related factors. While HSP70 appears to inhibit apoptosis and alleviate inflammation, its upregulation presents novel research opportunities. In conclusion, we deduced the development mechanism of ARPC3, ARPC4, and HSP70 in breast inflammation, laying the foundation for further exploring the interaction mechanism between the actin-related protein 2/3 (ARP2/3) complex and HSP70.

Exploring the Mechanism of H2S Synthesis in Male Bactrian Camel Poll Glands Based on Data Independent Acquisition Proteomics and Non-Targeted Metabolomics.

During estrus, the poll glands of male Bactrian Camels (Camelus Bactrianus) become slightly raised, exuding a large amount of pale yellow watery secretion with a characteristic odor that may contain hydrogen sulfide (H2S). However, whether H2S can be synthesized in the poll glands of male Bactrian Camels and its role in inducing camel estrus remains unclear. This study aimed to identify differentially expressed proteins (DEPs) and signaling pathways in the poll gland tissues of male Bactrian Camels using data independent acquisition (DIA) proteomics. Additionally, gas chromatography-mass spectrometry (GC-MS) was performed to identify differentially expressed metabolites (DEMs) in the neck hair containing secretions during estrus in male Bactrian Camels, to explore the specific expression patterns and mechanisms in the poll glands of camels during estrus. The results showed that cystathionine-γ-lyase (CTH) and cystathionine-ß-synthase (CBS), which are closely related to H2S synthesis in camel poll glands during estrus, were mainly enriched in glycine, serine, and threonine metabolism, amino acid biosynthesis, and metabolic pathways. In addition, both enzymes were widely distributed and highly expressed in the acinar cells of poll gland tissues in camels during estrus. Meanwhile, the neck hair secretion contains high levels of amino acids, especially glycine, serine, threonine, and cystathionine, which are precursors for H2S biosynthesis. These results demonstrate that the poll glands of male Bactrian Camels can synthesize and secrete H2S during estrus. This study provides a basis for exploring the function and mechanism of H2S in the estrus of Bactrian Camels.

SLC34A2 Targets in Calcium/Phosphorus Homeostasis of Mammary Gland and Involvement in Development of Clinical Mastitis in Dairy Cows.

The type II Na/Pi co-transporter (NaPi2b), encoded by the solute carrier (SLC) transporter 34A2 (SLC34A2), is responsible for calcium (Ca) and phosphorus (P) homeostasis. Unbalanced Ca/P metabolism induces mastitis in dairy cows. However, the specific role of SLC34A2 in regulating this imbalance in Holstein cows with clinical mastitis (CM) remains unclear. The aim of this study was to investigate the role of SLC34A2 and identify differentially expressed proteins (DEPs) that interact with SLC34A2 and are associated with Ca/P metabolism in dairy cows with CM. Immunohistochemical and immunofluorescence staining results showed that SLC34A2 was located primarily in the mammary epithelial cells of the mammary alveoli in both the control (healthy cows, Con/C) and CM groups. Compared to the Con/C group, the relative expression of the SLC34A2 gene and protein were significantly downregulated in the CM group. We identified 12 important DEPs included in 11 GO terms and two pathways interacting with SLC34A2 using data-independent acquisition proteomics. The PPI (protein-and-protein interaction) network results suggested that these DEPs were associated with ion metabolism and homeostasis, especially SLC34A2. These results demonstrate that SLC34A2 downregulation is negatively correlated with the occurrence and development of CM in Holstein cows, providing a basis for exploring the function and regulatory mechanism of SLC34A2 in Ca/P metabolism and homeostasis in Holstein cows with CM.

Verification of Key Target Molecules for Intramuscular Fat Deposition and Screening of SNP Sites in Sheep from Small-Tail Han Sheep Breed and Its Cross with Suffolk.

Intramuscular fat (IMF) is vital for meat tenderness and juiciness. This study aims to explore the IMF deposition mechanism and the related molecular markers in sheep. Two populations, Small-tail Han Sheep (STH) and STH × Suffolk (SFK) F1 (SFK × STH), were used as the research object. Histological staining techniques compared the differences in the longissimus dorsi muscle among populations. A combination of transcriptome sequencing and biological information analysis screened and identified IMF-related target genes. Further, sequencing technology was employed to detect SNP loci of target genes to evaluate their potential as genetic markers. Histological staining revealed that the muscle fiber gap in the SFK × STH F1 was larger and the IMF content was higher. Transcriptome analysis revealed that PIK3R1 and PPARA were candidate genes. Histological experiments revealed that the expressions of PIK3R1 mRNA and PPARA mRNA were lower in SFK × STH F1 compared with the STH. Meanwhile, PIK3R1 and PPARA proteins were located in intramuscular adipocytes and co-located with the lipid metabolism marker molecule (FASN). SNP locus analysis revealed a mutation site in exon 7 of the PIK3R1 gene, which served as a potential genetic marker for IMF deposition. This study's findings will provide a new direction for meat quality breeding in sheep.

Dimethyl fumarate restores Ca2+ dyshomeostasis through activation of the SIRT1 signal to treat nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is characterized by an excessive lipid accumulation in the liver, with a global prevalence of approximately 25 %. While early-stage steatosis is reversible and can be intervened upon, it has the potential to progress to some serious complications, including cirrhosis and even liver cancer. Dimethyl fumarate (DMF), a derivative of fumaric acid shows promise in intervening in certain diseases. However, the precise effect and underlying mechanism of DMF on hepatic steatosis remain unclear. In this study, we demonstrated that DMF mitigates hepatic steatosis in mice subjected to high-fat/high-cholesterol (HFHC) diets. Meanwhile, our in vivo and in vitro results showed that DMF relieves lipid accumulation, oxidative stress, and endoplasmic reticulum (ER) stress. Mechanically, our findings revealed that the effect of DMF on reducing lipid accumulation is linked to the restoration of Ca2+ homeostasis. Furthermore, we found that activation of the SIRT1 signal by DMF plays an important role in correcting the mishandling of the Ca2+ signal, and knockdown of SIRT1 expression reverses the beneficial role of DMF PA-incubated AML12 cells. In conclusion, our results suggested DMF's amelioration of hepatic steatosis is related to the activation of SIRT1-mediated Ca2+ signaling.

Alleviative effect of melatonin against the nephrotoxicity induced by cadmium exposure through regulating renal oxidative stress, inflammatory reaction, and fibrosis in a mouse model.

Chronic cadmium (Cd) exposure causes severe adverse health effects on the human body, especially the kidney tissue. Studies have demonstrated oxidative stress to be involved in renal pathological variations after exposure to Cd, but few effective treatments are available for the disease yet. Therefore, the present study was carried out to investigate the potential therapeutic intervention and its underlying molecular mechanisms of melatonin (MT), a natural antioxidant with multiple biological activities, against renal injury caused by Cd exposure in mice. C57BL/6 male mice (eight-week-old) were intragastrically administered with CdCl2, MT, or both for 30 days. Biochemical analysis showed that MT intervention significantly improved the SOD, GSH, and CAT activities while markedly decreasing the kidney MDA content of the mice exposed to Cd. Histological examination indicated that Cd exposure resulted in the atrophy of the renal glomerular, the degeneration and dilation of tubules, and the accumulation of fibrocytes. By contrast, MT administration effectively ameliorated the histological outcome of the injured kidney tissue. Moreover, administrating MT significantly inhibited proinflammatory cytokines TNF-α and iNOS expression in Cd-treated mice. Further, MT treatment markedly suppressed the expressions of renal fibrosis-related factors TGF-ß1, α-SMA, and collagen Ⅰ in the injured renal tissue and the accumulation and development of renal fibrosis. In addition, the administration of MT significantly reduced the expression of caspase-3 and cell apoptotic death in the kidney tissue of Cd-exposed mice. In all, the data showed that MT has a compelling therapeutic potential in alleviating the pathological variations of renal injury caused by Cd exposure.

A High-Precision Quartz Resonant Ultra-High Pressure Sensor with Integrated Pressure Conversion Structure.

A quartz resonant pressure sensor is proposed for high-precision measurement of ultra-high pressure. The resonant unit realizes a push-pull differential layout, which restrains the common-mode interference factor, and the resonator is only subject to axial force. The pressure conversion unit is made in an integrated manner, avoiding output drift problems caused by residual stress and small gaps during assembly, welding, and other processes in sensor preparation. Theoretical and simulation analysis was conducted on the overall design scheme of the sensor in this paper, verifying the feasibility. Sensor prototypes were created and performance experiments were conducted. The experimental results show that the sensitivity of the ultra-high pressure sensor is 46.32 Hz/MPa at room temperature within the pressure range of 120 MPa, and the comprehensive accuracy is 0.0266%. The comprehensive accuracy of the sensor is better than 0.0288% FS in the full temperature range environment. This proves that the sensor scheme is suitable for high-precision and high-stability detection of ultra-high pressure, providing new solutions in special pressure measurement fields such as deep-sea and oil exploration.

Integrated multi-omics reveals the beneficial role of chlorogenic acid in improving the growth performance and immune function of immunologically stressed broilers.

Intensive production can cause immunological stress in commercial broilers. Chlorogenic acid (CGA) regulates the intestinal microbiota, barrier function, and immune function in chickens. As complex interrelations regulate the dynamic interplay between gut microbiota, the host, and diverse health outcomes, the aim of this study was to elucidate the immunoregulatory mechanisms of CGA using multi-omics approaches. A total of 240 one-day-old male broilers were assigned to a 2 × 2 factorial design with 2 CGA levels (0 or 500 mg/kg) either with or without dexamethasone (DEX) injection for a 21-day experimental period. Therefore, there were 4 dietary treatments: control, DEX, CGA, and DEX + CGA, with 6 replicates per treatment. CGA supplementation improved (P < 0.05) growth performance, jejunal morphology, jejunal barrier function, and immune function in DEX-treated broilers. Moreover, in DEX + CGA-treated broilers, the increase in gut microbiome diversity (P < 0.05) was consistent with a change in taxonomic composition, especially in the Clostridiales vadin BB60_group. Additionally, the levels of short-chain fatty acids increased remarkably (P < 0.01) after CGA supplementation. This was consistent with the Kyoto Encyclopedia of Genes and Genomes analysis results that the "pyruvate fermentation to butanoate" pathway was more enriched (P < 0.01) in the DEX + CGA group than in the DEX group. Proteomics revealed that CGA treatment increased the expression of several health-promoting proteins, thymosin beta (TMSB4X) and legumain (LGMN), which were verified by multiple reaction monitoring. Metabolomics revealed that CGA treatment increased the expression of health-promoting metabolites (2,4-dihydroxy benzoic acid and homogentisic acid). Proteomic and metabolic analyses showed that CGA treatment regulated the peroxisome proliferator-activated receptor (PPAR) and mitogen-activated protein kinase (MAPK) pathways. Western blotting results support these findings. Pearson's correlation analyses showed correlations (P < 0.01) between altered immune function, jejunal barrier function, different microbiota, proteins, and metabolites parameters. Overall, our data indicate that CGA treatment increased growth performance and improved the immunological functions of DEX-treated broilers by regulating gut microbiota and the PPAR and MAPK pathways. The results offer novel insights into a CGA-mediated improvement in immune function and intestinal health.

Gas/Liquid Chromatography-Mass Spectrometry Analysis of Key Functional Substances Regulating Poll Gland Secretion in Male Camels during Seasonal Estrus.

Increased poll gland secretion is a major characteristic and indicator of estrus in male Bactrian camels; however, research on these poll glands and their secretion is extremely rare. In this study, we determine the chemical composition of poll gland secretions and identify the key functional substances that regulate seasonal estrus in male camels. A GC/LC-MS dual platform was used to analyze ventral hair (control) and neck mane samples containing poll gland secretions from male Bactrian camels during estrus. Multidimensional and single-dimensional analyses were used to screen differentially expressed metabolites (DEMs) between groups. Functional prediction of enriched metabolites was performed using a Human Metabolome Database comparison and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, which were then compared with a behavioral analysis of male Bactrian camels in estrus. A total of 1172 DEMs and 34 differential metabolic pathways were identified. One metabolite group was found to relate to steroid synthesis and metabolism, and another metabolite group was associated with neural metabolism. Therefore, we speculate that steroids and neurochemicals jointly regulate estrous behavior in male Bactrian camels, thus providing theoretical insights into the development and function of poll glands in Bactrian camels.

Genome-wide single-nucleotide polymorphism data and mitochondrial hypervariable region 1 nucleotide sequence reveal the origin of the Akhal-Teke horse.

OBJECTIVE: The study investigated the origin of the Akhal-Teke horse using genome-wide single-nucleotide polymorphism (SNP) data and mitochondrial hypervariable region 1 (HVR-1) nucleotide sequences. METHODS: Genome-wide SNP data from 22 breeds (481 horses) and mitochondrial HVR-1 sequences from 24 breeds (544 sequences) worldwide to examine the origin of the Akhal- Teke horse. The data were analyzed using principal component analysis, linkage disequilibrium analysis, neighbor-joining dendrograms, and ancestry inference to determine the population relationships, ancestral source, genetic structure, and relationships with other varieties. RESULTS: A close genetic relationship between the Akhal-Teke horse and horses from the Middle East was found. Analysis of mitochondrial HVR-1 sequences showed that there were no shared haplotypes between the Akhal-Teke and Tarpan horses, and the mitochondrial data indicated that the Akhal-Teke horse has not historically expanded its group. Ancestral inference suggested that Arabian and Caspian horses were the likely ancestors of the Akhal- Teke horse. CONCLUSION: The Akhal-Teke horse originated in the Middle East.

Localization of FGF21 Protein and Lipid Metabolism-Related Genes in Camels.

With the ability to survive under drought and chronic hunger, camels display a unique regulation characteristic of lipid metabolism. Fibroblast growth factor (FGF) 21 is a peptide hormone that regulates metabolic pathways, especially lipid metabolism, which was considered as a promising therapeutic target for metabolic diseases. To understand the FGF21 expression pattern and its potential relationship with lipid metabolism in camels, this study investigated the distribution and expression of FGF21, receptor FGFR1, and two lipid metabolism markers, leptin and hormone-sensitive lipase (HSL), using an immunohistochemistry (IHC) assay. The results showed that FGF21 was widely expressed in camel central nerve tissue and peripheral organs but absent in lung and gametogenic tissue, including the testis, epididymis, and ovary. In striated muscle, FGF21 is only present at the fiber junction. FGFR1 is expressed in almost all tissues and cells, indicating that all tissues are responsive to FGF21 and other FGF-mediated signals. Leptin and HSL are mainly located in metabolic and energy-consuming organs. In the CNS, leptin and HSL showed a similar expression pattern with FGFR1. In addition, leptin expression is extremely high in the bronchial epithelium, which may be due to its role in the immune responses of respiratory mucosa, in addition to fat stores and energy balance. This study found that FGF21 showed active expression in the nervous system of camels, which may be related to the adaptability of camels to arid environments and the specific regulation of lipid metabolism. This study showed a special FGF21-mediated fat conversion pattern in camels and provides a reference for developing a potential therapeutic method for fat metabolism disease.

Highly efficient neutralizer-free l-malic acid production using engineered Saccharomyces cerevisiae.

In industrial bioproduction of organic acids, numerous neutralizers are required which substantially increases production costs and burdens the environment. To address this challenge, a Saccharomyces cerevisiae mutant (named TAMC) with a low pH tolerance (pH 2.3) was isolated by adaptive laboratory evolution. Taking the synthesis of l-malic acid as an example, the malate dehydrogenase 3 without signal peptide (MDHΔSKL) and pyruvate carboxylase 2 (PYC2) were overexpressed in cytoplasmic synthesis pathway, and the l-malic acid titer increased 5.6-fold. Subsequently, the malic acid transporter SpMae1 was designed, and the extracellular l-malic acid titer was increased from 7.3 to 73.6 g/L. Furthermore, by optimizing the synthesis of the precursor pyruvate, the titer reached 81.8 g/L. Finally, without any neutralizer, the titer in the 3-L bioreactor reached 232.9 g/L, the highest l-malic acid titer reported to date. Herein, the engineered l-malic acid overproducer paves the way for the large-scale green production of l-malic acid.

An oligosaccharide marker for rapid authentication of edible bird's nest.

Edible bird's nest (EBN) is a popular and expensive food material. The limited supply and great demand result in the use of adulterants. The authenticity concern is raised due to the lack of appropriate quality markers. Herein, this study aims to provide a specific oligosaccharide marker for rapid EBN authentication. Comparing the benzocaine (ABEE)-labeled saccharide profiles of multiple batches of EBN and adulterants indicates seven unique EBN oligosaccharides. The most abundant one, named BNM001, was selected as a marker and characterized to be Neu5Ac (2-3) Gal by MS and NMR spectra. This new oligosaccharide marker enables a rapid authentication of EBN within 10 min. ABEE labelling of this marker further upgraded the accuracy and sensitivity of the LC-qTOF-MS quantitative analysis. The relative marker content was associated with the quality of EBN products. These results suggest a specific and efficient quality marker for rapid authentication of EBN and related products.

Polygenic prediction of human longevity on the supposition of pervasive pleiotropy.

The highly polygenic nature of human longevity renders cross-trait pleiotropy an indispensable feature of its genetic architecture. Leveraging the genetic correlation between the aging-related traits (ARTs), we sought to model the additive variance in lifespan as a function of cumulative liability from pleiotropic segregating variants. We tracked allele frequency changes as a function of viability across different age bins and prioritized 34 variants with an immediate implication on lipid metabolism, body mass index (BMI), and cognitive performance, among other traits, revealed by PheWAS analysis in the UK Biobank. Given the highly complex and non-linear interactions between the genetic determinants of longevity, we reasoned that a composite polygenic score would approximate a substantial portion of the variance in lifespan and developed the integrated longevity genetic scores (iLGSs) for distinguishing exceptional survival. We showed that coefficients derived from our ensemble model could potentially reveal an interesting pattern of genomic pleiotropy specific to lifespan. We assessed the predictive performance of our model for distinguishing the enrichment of exceptional longevity among long-lived individuals in two replication cohorts and showed that the median lifespan in the highest decile of our composite prognostic index is up to 4.8 years longer. Finally, using the proteomic correlates of iLGS, we identified protein markers associated with exceptional longevity irrespective of chronological age and prioritized drugs with repurposing potentials for gerotherapeutics. Together, our approach demonstrates a promising framework for polygenic modeling of additive liability conferred by ARTs in defining exceptional longevity and assisting the identification of individuals at higher risk of mortality for targeted lifestyle modifications earlier in life. Furthermore, the proteomic signature associated with iLGS highlights the functional pathway upstream of the PI3K-Akt that can be effectively targeted to slow down aging and extend lifespan.

HMOX1 Promotes Ferroptosis in Mammary Epithelial Cells via FTH1 and Is Involved in the Development of Clinical Mastitis in Dairy Cows.

Ferroptosis is associated with inflammatory diseases as a lethal iron-dependent lipid peroxidation; its role in the development of clinical mastitis (CM) in dairy cows is not well understood. The aim of this study was to identify differentially expressed proteins (DEPs) associated with iron homeostasis and apoptosis, and to investigate further their roles in dairy cows with CM. The results suggested that ferroptosis occurs in the mammary glands of Holstein cows with CM. Using data-independent acquisition proteomics, 302 DEPs included in 11 GO terms related to iron homeostasis and apoptosis were identified. In particular, heme oxygenase-1 (HMOX1) was identified and involved in nine pathways. In addition, ferritin heavy chain 1 (FTH1) was identified and involved in the ferroptosis pathway. HMOX1 and FTH1 were located primarily in mammary epithelial cells (MECs), and displayed significantly up-regulated expression patterns compared to the control group (healthy cows). The expression levels of HMOX1 and FTH1 were up-regulated in a dose-dependent manner in LPS induced MAC-T cells with increased iron accumulation. The expression levels of HMOX1 and FTH1 and iron accumulation levels in the MAC-T cells were significantly up-regulated by using LPS, but were lower than the levels seen with Erastin (ERA). Finally, we deduced the mechanism of ferroptosis in the MECs of Holstein cows with CM. These results provide new insights for the prevention and treatment of ferroptosis-mediated clinical mastitis in dairy animals.

CTH/H2S Regulates LPS-Induced Inflammation through IL-8 Signaling in MAC-T Cells.

Hydrogen sulfide (H2S), as an endogenous gaseous signaling molecule, plays an important role in the inflammatory process. Our previous study found that Cystathionine-γ-lyase (CTH) and H2S are correlated with the occurrence and development of Clinical Mastitis (CM) in Holstein cows. However, the functions and regulatory mechanisms of CTH/H2S are still unknown. In this study, the inflammatory mammary cell model based on the MAC-T cell line was established by Lipopolysaccharide (LPS)-induced manner to further explore the function and regulatory mechanism of CTH/H2S in cows with CM. In the inflammatory MAC-T cell, the CTH expression and H2S production were both repressed in an LPS-dose dependent manner, which demonstrated that CTH/H2S is related to the progression of inflammation. The inhibition of CTH/H2S using a selective CTH inhibitor, ß-cyano-l-Alanine (BCA), promoted LPS-induced inflammation response and the expression of inflammatory cytokines. However, this was reversed by the H2S donor NaHS, demonstrating that H2S can protect cells from inflammatory damage. Intriguingly, interleukin-8 (IL-8) showed an inverse expression pattern correlated with the H2S-mediated cell protection effect during the inflammation process, and the inhibition test using a selective IL-8 receptor antagonist, SB225002, showed that IL-8 signaling plays a critical role in mediating endogenous H2S synthesis, and CTH/H2S exerts its anti-inflammation via IL-8-mediated signaling. This study provided support for the prevention and treatment of CM and the development of a novel anti-inflammatory strategy.

M cells of mouse and human Peyer's patches mediate the lymphatic absorption of an Astragalus hyperbranched heteroglycan.

The gut cell wall is considered an impenetrable barrier to orally administrated polysaccharides. We recently reported a selective lymphatic route for Radix Astragali polysaccharide RAP to enter Peyer's patches (PPs) to trigger immune responses. However, how RAP enters PPs is unclear. Herein, we screened the intestinal epithelial cells of mice and found that the follicle-associated epithelium cells were specifically bound with FITC-RAP. Further studies in vitro and in vivo revealed that RAP was efficiently transported by microfold (M) cells. We also confirmed that M cell-transported RAP directly contacted dendritic cells. More importantly, for the first time, we verified this interesting M cell-mediated transcytosis of RAP in the human distal ileum. Mechanistically, we identified M cells to be the transporter cells that independently deliver RAP into the lymphatic system to trigger immune responses. This interesting transcytosis mechanism might apply to many other immunomodulatory polysaccharides orally dosed to human body.