Ketosis correlates with increased mastitis risk, but the underlying mechanism remains unclear. Nuclear factor erythroid 2-related factor 1 (NFE2L1) regulates inflammatory responses, and hyperoside (Hyp) exerts anti-inflammatory/antioxidant effects. However, whether NFE2L1 participates in the Hyp-mediated protection of mammary epithelial cells remains unclear. We investigated Hyp's mechanism against high-concentration free fatty acid (FFA)-induced damage via NFE2L1. Clinical ketotic cows showed reduced mammary NFE2L1, zona occludens 1 (ZO-1), occludin, claudin 1, and elevated levels of interleukin (IL)-1B and IL-6. FFAs downregulated NFE2L1, ZO-1, occludin, and activated inflammatory signaling, which Hyp (20 μM) reversed. NFE2L1 silencing abolished Hyp's protective effects. Overall, these data suggested that Hyp may serve as a promising therapeutic agent to counteract inflammation and maintain barrier integrity in the mammary tissue of clinical ketotic cows, with its efficacy dependent on NFE2L1.

Endometritis, a major inflammatory cause of infertility, is driven by unresolved immune dysregulation in which macrophage polarization is critical. Yet, how inflammatory signaling is spatially propagated within the endometrial microenvironment remains unclear. Here, we report that exosomes—natural nanoscale extracellular vesicles—released from lipopolysaccharide (LPS)-injured endometrial epithelial cells (EECs) act as pathogenic nanocarriers that fuel macrophage-dependent inflammation. We demonstrate that LPS enhances exosome biogenesis and secretion via the AKT/ATG16L1 pathway. These exosomes efficiently deliver their molecular cargo to macrophages, triggering NF-κB activation and polarizing them toward a pro-inflammatory M1 phenotype. RNA sequencing identified lncRNA OTUD6B-AS1 as a highly enriched cargo in exosomes from inflamed EECs. Functional studies established that exosome-mediated transfer of lncRNA OTUD6B-AS1 is both necessary and sufficient to drive M1 polarization. Mechanistically, lncRNA OTUD6B-AS1 functions as a competing endogenous RNA (ceRNA), sequestering miR-128 to relieve its repression on Notch2, thereby amplifying NF-κB signaling. This axis was validated in clinical endometritis tissues, which exhibited elevated lncRNA OTUD6B-AS1 and Notch2 alongside reduced miR-128. Importantly, targeting this pathway—through genetic knockdown of lncRNA OTUD6B-AS1 or pharmacological inhibition of the miR-128/Notch2 node—abolished the pro-inflammatory effects. Our work not only delineates a new exosome-coordinated signaling circuit in endometritis but also highlights exosomes as druggable natural nanoparticles. These findings position exosome-based engineering—such as cargo modulation or designed vesicle delivery—as a promising nanomedicine strategy to intercept pathological cell-cell communication and treat inflammatory diseases.

Elevated circulating nonesterified fatty acids (NEFA) represent a key pathological feature in dairy cows with fatty liver. Palmitic acid (PA), a major component of NEFA, can be enzymatically attached to proteins via a reversible post-translational modification known as palmitoylation, which potently modulates protein activity and function. Studies have revealed that aberrant hepatic palmitoylation is a crucial mechanism promoting lipid accumulation in non-ruminants. Nevertheless, the extent and pathological relevance of hepatic protein palmitoylation in dairy cows with fatty liver have largely remained unexplored. Therefore, this study was conducted to determine the status of hepatic protein palmitoylation in dairy cows with fatty liver and to elucidate its functional role in the development of hepatic steatosis. Blood and liver samples were collected from 10 dairy cows with fatty liver (hepatic triglyceride [TG] content >5%) and 10 control cows (hepatic TG content <1%) that had a similar number of lactations (median: 3, range: 2 to 4) and days in milk (median: 9 d, range: 5 to 14 d). To determine the effects of NEFA on palmitoylation, hepatocytes isolated from calves were treated with 1.2 mM NEFA for 12 h. To investigate the effects of palmitoylation on lipid accumulation in bovine hepatocytes, the cells were treated with 1.2 mM NEFA for 12 h in the presence or absence of a palmitoylation inhibitor (2-bromohexadecanoic acid). The results revealed that dairy cows with fatty liver exhibited liver injury and elevated hepatic palmitoyl-CoA content. Moreover, fatty liver dairy cows showed higher hepatic mRNA abundance of ZDHHC4/5/14/20 and lower mRNA abundance of ZDHHC3/19/21/23/24. In contrast, the mRNA abundance of depalmitoylase-related genes, including lysophospholipase 1 (LYPLA1 and LYPLA2), palmitoyl-protein thioesterase 1 (PPT1 and PPT2) and abhydrolase domain containing 17 (ABHD17A, ABHD17B and ABHD17C), was lower in the liver of cows with fatty liver than in control cows. Consistently, a greater abundance of palmitoylated proteins was observed in the liver of dairy cows with fatty liver. In vitro, NEFA treatment induced lipid accumulation, cell injury, and aberrant protein palmitoylation in bovine hepatocytes. Additionally, the upregulation of palmitoyltransferases and downregulation of depalmitoylases observed in cows with fatty liver were recapitulated in NEFA-treated bovine hepatocytes. Importantly, pharmacological inhibition of palmitoylation significantly alleviated NEFA-induced lipid accumulation and cell damage in bovine hepatocytes. Overall, these findings establish protein palmitoylation as both a critical pathological mechanism and a promising therapeutic target for fatty liver in dairy cows.

Ketosis is a common metabolic disorder in dairy cows, typically occurring during early postpartum negative energy balance and characterized by hyperketonemia, hepatic lipid accumulation, and oxidative stress. Although acetoacetyl-CoA synthetase (AACS) plays a key role in ketone body metabolism, its association with acetoacetate (AcAc) utilization during ketosis remains unclear. This study aimed to assess the effects of AACS on fatty acid and cholesterol metabolic pathways in neonatal bovine hepatocytes exposed to high concentrations of non-esterified fatty acids (NEFA). Liver tissues were collected from healthy (n = 6; BHBA < 1.0mM) and ketotic (n = 6; BHBA > 3.0mM) dairy cows. Additionally, hepatocytes isolated from neonatal calves were treated with 1.2mM NEFA to establish an in vitro ketotic model. To evaluate the role of AACS, two separate experimental approaches were employed using NEFA-challenged hepatocytes: siRNA-mediated AACS knockdown and exogenous AcAc supplementation. Results demonstrated that liver tissues from ketotic cows and NEFA-treated cells exhibited upregulation of AACS and HMGCS2, along with key lipogenic proteins (SREBF1, ACACA, and FASN). In contrast, the expression of cholesterol synthesis and efflux factors (SREBF2, HMGCR, ABCA1, ABCG5, and ACAT2) was downregulated. NEFA challenge also reduced BDH1 and CPT1A levels, decreased intracellular total cholesterol, increased TAG accumulation, and induced oxidative stress and mitochondrial dysfunction. Silencing AACS partially attenuated NEFA-induced lipid accumulation but further suppressed cholesterol synthesis and efflux-related gene expression. Conversely, AcAc supplementation upregulated the expression of cholesterol synthesis and efflux-related genes but exacerbated lipid deposition, oxidative stress, and mitochondrial dysfunction. Overall, these findings indicate that NEFA-induced upregulation of AACS abundance in hepatocytes may modulate the partitioning of acetoacetate toward de novo lipogenesis and alter cholesterol synthesis and efflux-related gene expression.

Dietary hyperoside enriches gut Lactobacillus , boosting the metabolite HPLA. Circulating HPLA targets uterine TLR4, suppressing TLR4/NF-κB signaling and alleviating endometrial inflammation and damage.

Objective:This study aimed to investigate whether miR-27b-3p regulates oxidative stress and inflammatory responses through the Nrf2/HO-1 signaling pathway during the progression of fatty liver. Methods:Liver tissues from dairy cows with fatty liver and high-fat diet (HFD)-induced fatty liver mice were collected to evaluate lipid accumulation, oxidative stress, inflammation, and the expression of miR-27b-3p and Nrf2/HO-1 pathway components. An in vitro steatosis model was established in AML-12 hepatocytes using palmitic acid (PA). Oxidative stress markers (ROS, SOD, and MDA), inflammatory cytokines (IL-1β, IL-6, and TNF-α), and apoptosis-related proteins were assessed. Bioinformatic prediction and dual-luciferase reporter assays were used to determine the targeting relationship between miR-27b-3p and Nrf2. Gain- and loss-of-function experiments were performed using miR-27b-3p mimics and inhibitors to evaluate its regulatory effects on the Nrf2/HO-1 pathway and downstream cellular responses. Results:Fatty liver tissues from dairy cows and HFD-fed mice exhibited significant lipid deposition, enhanced oxidative stress, and elevated inflammatory responses, accompanied by increased miR-27b-3p expression and reduced Nrf2/HO-1 signaling activity. Similar alterations were observed in PA-treated AML-12 cells. Bioinformatic analysis and dual-luciferase assays confirmed that miR-27b-3p directly binds to the 3' untranslated region of Nrf2. Inhibition of miR-27b-3p restored Nrf2 and HO-1 expression, reduced ROS and MDA levels, increased SOD activity, suppressed NF-κB activation, decreased pro-inflammatory cytokine production, and alleviated apoptosis. Conversely, overexpression of miR-27b-3p further inhibited Nrf2/HO-1 signaling and exacerbated oxidative stress, inflammation, and cell injury. Conclusion:miR-27b-3p promotes the progression of fatty liver toward steatohepatitis by directly targeting Nrf2 and suppressing the Nrf2/HO-1 antioxidant pathway, thereby enhancing oxidative stress, inflammatory responses, and hepatocyte apoptosis. These findings identify miR-27b-3p as a key upstream regulator of redox imbalance in fatty liver and suggest that targeting miR-27b-3p may represent a promising strategy for the prevention and treatment of bovine fatty liver disease.

The supraphysiological levels of free fatty acids (FFA) during early lactation led to an inflammatory response in the mammary gland of ketotic cows, which may be attributed to the untimely clearance of apoptotic cells (AC). Given the engulfment role of mammary epithelial cells for AC and the regulation of TGF-β3 on phagocytosis, we speculated that TGF-β3-mediated phagocytic function is closely related to inflammatory response in the mammary gland of ketotic cows. The objective of this study was to elucidate (1) the inflammatory state and phagocytic function of mammary epithelial cells and activity of the TGF-β3 pathway in mammary gland of ketotic cows, and (2) the role of TGF-β3-mediated phagocytosis on the inflammatory response of bovine mammary epithelial cells in response to exogenous FFA. In this study, excessive proliferation of AC, NF-κB/MAPK-mediated inflammatory response, TLR4-mediated local natural immune dysfunction, phagocytic dysfunction of mammary epithelial cells, and activation of the TGF-β3 pathway were detected in the mammary gland of ketotic cows or in immortalized bovine mammary epithelial (MAC-T) cells in response to exogenous FFA. Knockdown of TLR4 attenuated the FFA-induced inflammatory response in MAC-T cells. Overexpression of TGF-β3 exacerbated FFA-induced TLR4-mediated local immune dysfunction and inflammatory response by aggravating phagocytic responses. Knockdown of TGF-β3 attenuated the overactivation of inflammation resulting from FFA challenge through inhibition of the TLR4 pathway, which improved phagocytic ability in MAC-T cells. Taken together, TGF-β3-mediated phagocytosis may be a promising therapeutic target for reducing the negative effect of FFA-induced inflammation in mammary gland of dairy cows with ketosis.

Pathogenic Escherichia coli (E. coli) is a widely distributed pathogen that can cause varying degrees of zoonotic diseases, and infected animals often experience intestinal inflammation accompanied by diarrhea and dysbiosis. Previously, for the first time, we isolated Escherichia coli primarily of type B2 from a large-scale dairy farm in Yunnan, China. The 16s rRNA sequencing showed significant differences in the gut microbiota of calves infected with B2 E. coli, with higher abundance of harmful bacteria and lower abundance of beneficial bacteria compared with healthy calves. The metabolomics indicated that the concentrations of oxoadipic acid, 16-oxopalmitate, oerillyl alcohol, palmitoleic acid, and 4-phenylbutyrate (4-PBA) were significantly higher in the healthy group than in the infected group. The mouse model was established to assess the regulatory effect of 4-PBA on E. coli-induced colitis. Both oral administration of 4-PBA and fecal microbiota transplantation (FMT) had strong resistance to E. coli infection, improved survival rate and body weight, reduced intestinal tissue damage, decreased the levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), and restrained TLR4/MyD88/NF-κB pathway. Our study demonstrated that 4-PBA could relieve E. coli-induced colitis by improving gut microbiota structure and inhibiting the expression of pro-inflammatory cytokines through the TLR4/MyD88/NF-κB pathway. The present finding reveals the therapeutic potential of the gut-microbiota-derived metabolite 4-PBA for the treatment of colitis caused by E. coli.

Endometritis in dairy cows cause severe economic losses in the dairy farming industry. Although bta-miR-200b expression is decreased in cows with endometritis, role of bta-miR-200b in moderation of endometritis in dairy cows is unknown. In the present study, bovine endometrial epithelial (BEND) cells stimulated with lipopolysaccharide (LPS, 0-100 μg/mL) for 24 h significantly decreased cell viability and the expression of bta-miR-200b consistent with the expression pattern of bta-miR-200b in endometritis. Subsequently, western blotting revealed that overexpression of bta-miR-200b significantly suppressed the LPS-induced increase in phosphorylated p65 protein levels in BEND cells, thereby decreasing the release of proinflammatory factors and inhibiting apoptosis. As predicted by KEGG analysis, LPS induced overexpression of bta-miR-200b significantly increased the protein abundance of phosphorylated AKT and the Bcl-2/Bax ratio. These findings suggested that the inhibition of inflammatory injury by bta-miR-200b is mediated by the PI3K/AKT pathway. The results of western blotting further revealed that the overexpression of bta-miR-200b decreased the protein abundance of PTEN, indicating that PTEN is a target of bta-miR-200b. Knocking down both bta-miR-200b and PTEN in BEND cells resulted in decreasing level of phosphorylated AKT and the Bcl-2/Bax ratio while promoting p65 nuclear transcription. Furthermore, immunofluorescence staining and flow cytometry verified that PTEN knockdown inhibited the effect of bta-miR-200b inhibition on LPS-induced apoptosis. Collectively, these findings suggest that bta-miR-200b attenuates LPS-induced inflammatory injury by targeting the PTEN/AKT/NF-κB axis. These findings provide a theoretical basis for further studies on the in vivo application of miRNA-based therapy to mitigate endometritis.

Bovine viral diarrhea virus (BVDV) is classified into cytopathic (CP) and noncytopathic (NCP) types. Previous studies confirmed that NCP BVDV infection is the main cause of persistent infection and immune suppression in cattle, and its molecular mechanism of using host biological processes to evade immunity remains unclear. We initially examined the replication of the AV303 strain (NCP BVDV) in MDBK cells at different time points. Proteomic analysis at the peak replication time point revealed that the TLR signaling pathway and the adaptor protein Myeloid differentiation factor 88 (MyD88) were upregulated. Subsequently, we found that AV303 infection initiated autophagy but had degradation barriers, meanwhile the proliferation rate of MDBK cells increased. After knocking down MyD88, the cell proliferation rate was restored, and autophagy flow was activated utterly. Mechanistically, AV303 regulated cell proliferation by promoting ERK1/2 and Akt/mTOR, and both activities were inhibited after MyD88 is knocked down. The activity of MyD88 affected the degradation stage of autophagic flux. Treatment enhance its replication. This study demonstrates that the activity of MyD88 mediated by the AV303 strain can regulate host cell autophagy and proliferation, creating advantageous conditions for its replication. This study identifies a novel mechanism in host-NCP BVDV interaction and highlights the potential of MyD88 as a target for anti-NCP BVDV drug development.

The heavy metal cadmium (Cd) affects the global livestock production economy mainly through the contamination of feed raw materials and secondary contamination in feed processing, and it also poses a serious threat to food safety and human health. The nucleotide-binding oligomerization domain-like pyrin-domain-containing protein 3 (NLRP3) inflammasome is a key regulatory element of pyroptosis, which is engaged in kidney injury. Meanwhile, autophagy is also involved in renal inflammation. Mammalian target of rapamycin (mTOR) plays an important role in pyroptosis and autophagy, but its function in Cd-induced kidney injury remains unclear. In this study, we explored the role of mTOR-mediated autophagy and pyroptosis in kidney injury caused by Cd exposure and elucidated its underlying mechanism. Our data showed that Cd exposure reduced the integrity of kidney cell membranes, increased the expression of pyroptosis-associated proteins, and promoted the release of inflammatory cytokines. Subsequently, a notable attenuation in Cd-induced pyroptosis was observed following the administration of CY-09, an NLRP3 inhibitor. In addition, Cd exposure promoted autophagy in kidney cells. Importantly, in both in vivo and in vitro experiments, rapamycin, an mTOR inhibitor, downregulated the expression of pyroptosis-related proteins, thereby significantly improving Cd-induced kidney injury. In summary, our results indicate that mTOR-mediated autophagy has a significant protective effect on NLRP3 inflammasome-dependent kidney injury induced by Cd exposure, thus providing new insights into the prevention and treatment of Cd poisoning.

Toxoplasma gondii (T. gondii) is a worldwide zoonotic parasite that can infect almost warm-blood animals, including humans, which seriously affect the health of host. Cats are known to be the only definitive host of T. gondii and continuously excrete highly infectious oocysts. This parasite carried by the companion animals leads to a great public health risk. However, there is little information on epidemiology of T. gondii in urban cats in Kunming, Southwest China. In the present study, a total of 231 serum and fecal samples were collected in Kunming aera, and then seroprevalence of T. gondii IgG antibodies in serum and molecular investigation in feces were analyzed to elucidate T. gondii infection in urban cats. The results revealed that 168 of 231 cats (72.7%) were positive for T. gondii antibodies, and 1 of 74 cat feces (1.4%) also showed a positive PCR for T. gondii DNA. The positive fecal sample was sequenced and then phylogenetically analyzed, and the isolate of T. gondii in the present study was closely related to T. gondii strain CN. In addition, the food, water and age of cats were identified as the risk factor for seropositivity. Overall, our findings indicate the widespread occurrence of T. gondii infection in urban cats in Kunming, Southwest China and identify food, water and age are the risk factors associated with T. gondii infection, which can provide effective information for developing strategies to prevent and control this zoonosis.

Endometritis is one of the most common causes of infertility in dairy cows, and is histopathologically characterized by inflammation and damage of endometrial epithelium. Interferon-tau (IFN-τ) is a novel type I interferon secreted by ruminant trophoblast cells with low cytotoxicity even at high doses. Previous studies suggested that IFN-τ plays an important role in inflammation. However, the mechanisms whereby IFN-τ may modulate the inflammatory responses in the bovine endometrium are unknown. In the present study, primary bovine endometrial epithelial cells (BEEC) isolated from fresh and healthy uterine horns were used for in vitro studies. The integrity of BEEC was assessed by immunofluorescence staining for cytokeratin 18 (CK-18, a known epithelial marker). For the experiments, BEEC were stimulated with different concentrations of lipopolysaccharide (LPS; 0–20 µg/mL) for different times (0–24 h). Cell viability and apoptosis were assessed via CCK-8 and flow cytometry. In a preliminary study, we observed that compared with the control group without LPS, 10 µg/mL of LPS stimulation for 24 h induced apoptosis. In a subsequent study, 20 or 40 ng/mL of IFN-τ alleviated LPS-induced apoptosis. Relative to the LPS group, western blotting further revealed that IFN-τ inhibited the protein abundance of TLR4 and phosphorylated (p-) p65 (p-p65) and Bax/Bcl-2 ratio, suggesting that IFN-τ can protect BEEC against inflammatory injury. Furthermore, the protein abundance of p-phosphoinositide 3-kinase (p-PI3K), p-protein kinase B (p-AKT), p-glycogen synthase kinase-3β (p-GSK3β), β-catenin, and p-forkhead box O1 (p-FoxO1) was lower in the LPS group, whereas IFN-τ upregulated their abundance. The use of LY294002, a specific inhibitor of PI3K/AKT, attenuated the upregulation of p-PI3K, p-AKT p-GSK3β, β-catenin, and p-FoxO1 induced by IFN-τ, and also blocked the downregulation of TLR4, p-p65, and Bax/Bcl-2 ratio. This suggested that the inhibition of TLR4 signaling by IFN-τ was mediated by the PI3K/AKT pathway. Furthermore, compared with the LPS group, the β-catenin agonist SB216763 led to greater p-FoxO1 and lower p-p65 and cell apoptosis. In contrast, knockdown of β-catenin using small interfering RNA had the opposite effects. To explore the role of FoxO1 on the inhibition of TLR4 by IFN-τ, we employed LY294002 to inhibit the PI3K/AKT while FoxO1 was knocked down. Results revealed that the knockdown of FoxO1 blocked the upregulation of TLR4 and p-p65 induced by LY294002, and enhanced the inhibition of IFN-τ on TLR4, p-p65, and cell apoptosis. Overall, these findings confirmed that IFN-τ can protect endometrial epithelial cells against inflammatory injury via suppressing TLR4 activation through the regulation of the PI3K/AKT/β-catenin/FoxO1 axis. These represent new insights into the molecular mechanisms underlying the anti-inflammatory function of IFN-τ in BEEC, and also provide a theoretical basis for further studies on the in vivo application of IFN-τ to help prevent negative effects of endometritis.

Introduction: High concentrations of nonesterified fatty acids (NEFA) is the key of characteristic of fatty liver in dairy cows. Therefore, the aim of this study was to investigate the effect of high concentration of NEFA on lipid metabolism in hepatocytes through the lipidomic approach and molecular biology techniques.Methods: Stimulate AML-12 cells with different concentrations of NEFA, observe the cellular lipid accumulation, and select 0.6 mM NEFA stimulation concentration for subsequent experiments. Collect cells for lipidomics analysis.Results: High concentration of NEFA (0.6–2.4 mM) significantly reduced the cell viability in a concentration-dependent manner, indicating that high concentrations of NEFA have lipotoxicity on hepatocytes. In addition, NEFA promoted triglycerides (TAG) accumulation, increased the mRNA expression of the lipogenic molecules SREBP1c and FASN, and decreased the mRNA expression of lipolytic molecules CPT1A and HSL in hepatocytes. Mechanistically, high concentration of NEFA induced lipid metabolism disorders in hepatocytes by regulating metabolic pathways such as glycerol phospholipid metabolism, glycosyl phosphatidylinositol anchored biosynthesis, triglyceride metabolism, sphingolipid metabolism, and inositol phosphate metabolism.Discussion: High concentration of NEFA is lipotoxic to cells, promoting lipid accumulation. LPE (18:2), LPE (18:3), LPE (18:1) via glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, glycerolipid metabolism, sphingolipid metabolism, and inositol phosphate metabolism, indicating their potential regulation role in the pathogenesis of fatty liver.

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In non-ruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, the objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. Four experiments were performed as follows: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated with 100 ng/mL lipopolysaccharide (LPS) and 100 ng/mL interferon-γ (IFN-γ) or 10 ng/mL interleukin-4 (IL4) and 10 ng/mL interleukin-10 (IL10) for 24 h; (2) Immortalized bovine macrophages were treated with 0, 0.3, 0.6, 1.2 mM FFA and LPS and IFN-γ or IL4 and IL10 for 24 h; (3) Macrophages were pretreated with 2 μM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86+, but lower MFI of CD206+ and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and mean fluorescence intensity of CD86, whereas it downregulated the protein abundance of arginase 1 (ARG1) and mean fluorescence intensity of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α (TNFA), interleukin-1B (IL1B), and interleukin-6 (IL6) mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate (LC3-II) protein abundance and autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows via impairing mTOR-mediated autophagy.

Exosomes are natural carriers of biological macromolecules that are involved in the pathogenesis of a wide variety of inflammatory diseases. The purpose of this study was to investigate the role of exosomes derived from injured endometrial epithelial cells (EECs) in the development of endometritis. We isolated exosomes derived from LPS-injured EECs and identified these exosomes as proinflammatory mediators that can be internalized by macrophages and thus induce proinflammatory macrophage activation. We further found that miR-331 expression was sharply downregulated in exosomes derived from LPS-injured EECs and that macrophages treated with these exosomes also presented a lower level of miR-331. Importantly, the pathogenic role of exosomal miR-331 in promoting endometrial inflammation was revealed by the ability of adoptively transferred EECs-derived exosomes to cause macrophage activation, and this was reversed by miR-331 overexpression. Mechanistically, overexpression of miR-331 in macrophages mitigated NF-κB p65 phosphorylation by inhibiting the Notch1/IKKα pathway, which in turn curbed macrophage activation. In vivo assays further unveiled that miR-331 expression is negatively correlated with proinflammatory macrophage activation and that miR-331 upregulation markedly slowed disease progression in mice with endometritis. The exosome/miR-331/Notch1 axis plays a critical pathological role in endometrial inflammation, representing a new therapeutic target for endometritis.

Oral infection with cysts is the main transmission route of Toxoplasma gondii (T. gondii), which leads to lethal intestinal inflammation. It has been widely recognized that T. gondii infection alters the composition and metabolism of the gut microbiota, thereby affecting the progression of toxoplasmosis. However, the potential mechanisms remain unclear. In our previous study, there was a decrease in the severity of toxoplasmosis after T. gondii α-amylase (α-AMY) was knocked out. Here, we established mouse models of ME49 and Δα-amy cyst infection and then took advantage of 16S rRNA gene sequencing and metabolomics analysis to identify specific gut microbiota-related metabolites that mitigate T. gondii-induced intestinal inflammation and analyzed the underlying mechanism. There were significant differences in the intestinal inflammation between ME49 cyst- and Δα-amy cyst-infected mice, and transferring feces from mice infected with Δα-amy cysts into antibiotic-treated mice mitigated colitis caused by T. gondii infection. 16S rRNA gene sequencing showed that the relative abundances of gut bacteria, such as Lactobacillus and Bacteroides, Bifidobacterium, [Prevotella], Paraprevotella and Macellibacteroides, were enriched in mice challenged with Δα-amy cysts. Spearman correlation analysis between gut microbiota and metabolites indicated that some fatty acids, including azelaic acid, suberic acid, alpha-linolenic acid (ALA), and citramalic acid, were highly positively correlated with the identified bacterial genera. Both oral administration of ALA and fecal microbiota transplantation (FMT) decreased the expression of pro-inflammatory cytokines and restrained the MyD88/NF-κB pathway, which mitigated colitis and ultimately improved host survival. Furthermore, transferring feces from mice treated with ALA reshaped the colonization of beneficial bacteria, such as Enterobacteriaceae, Proteobacteria, Shigella, Lactobacillus, and Enterococcus. The present findings demonstrate that the host gut microbiota is closely associated with the severity of T. gondii infection. We provide the first evidence that ALA can alleviate T. gondii-induced colitis by improving the dysregulation of the host gut microbiota and suppressing the production of pro-inflammatory cytokines via the MyD88/NF-κB pathway. Our study provides new insight into the medical application of ALA for the treatment of lethal intestinal inflammation caused by Toxoplasma infection.

Activated autophagy-lysosomal pathway (ALP) can degrade virtually all kinds of cellular components, including intracellular lipid droplets, especially during catabolic conditions. Sustained lipolysis and increased plasma fatty acids concentrations are characteristic of dairy cows with hyperketonemia. However, the status of ALP in adipose tissue during this physiological condition is not well known. The present study aimed to ascertain whether lipolysis is associated with activation of ALP in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes. In vivo, blood and subcutaneous adipose tissue (SAT) biopsies were collected from nonhyperketonemic (nonHYK) cows [blood β-hydroxybutyrate (BHB) concentration <1.2 mM, n = 10] and hyperketonemic (HYK) cows (blood BHB concentration 1.2-3.0 mM, n = 10) with similar days in milk (range: 3-9) and parity (range: 2-4). In vitro, calf adipocytes isolated from 5 healthy Holstein calves (1 d old, female, 30-40 kg) were differentiated and used for (1) treatment with lipolysis inducer isoproterenol (ISO, 10 µM, 3 h) or mammalian target of rapamycin inhibitor Torin1 (250 nM, 3 h), and (2) pretreatment with or without the ALP inhibitor leupeptin (10 μg/mL, 4 h) followed by ISO (10 µM, 3 h) treatment. Compared with nonHYK cows, serum concentration of free fatty acids was greater and serum glucose concentration, DMI, and milk yield were lower in HYK cows. In SAT of HYK cows, ratio of phosphorylated hormone-sensitive lipase to hormone-sensitive lipase, and protein abundance of adipose triacylglycerol lipase were greater, but protein abundance of perilipin 1 (PLIN1) and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC) was lower. In addition, mRNA abundance of autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), protein abundance of lysosome-associated membrane protein 1, and cathepsin D, and activity of β-N-acetylglucosaminidase were greater, whereas protein abundance of sequestosome-1 (p62) was lower in SAT of HYK cows. In calf adipocytes, treatment with ISO or Torin1 decreased protein abundance of PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes, but increased glycerol content in the supernatant of calf adipocytes. Moreover, the mRNA abundance of ATG5, ATG7, and MAP1LC3B was upregulated, the protein abundance of lysosome-associated membrane protein 1, cathepsin D, and activity of β-N-acetylglucosaminidase were increased, whereas the protein abundance of p62 was decreased in calf adipocytes treated with ISO or Torin1 compared with control group. Compared with treatment with ISO alone, the protein abundance of p62, PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes were higher, but the glycerol content in the supernatant of calf adipocytes was lower in ISO and leupeptin co-treated group. Overall, these data indicated that activated ALP is associated with increased lipolysis in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes.