Multi-omics reveals that alkaline mineral water improves the respiratory health and growth performance of transported calves.

BACKGROUND: Long-distance transportation, a frequent practice in the cattle industry, stresses calves and results in morbidity, mortality, and growth suppression, leading to welfare concerns and economic losses. Alkaline mineral water (AMW) is an electrolyte additive containing multiple mineral elements and shows stress-mitigating effects on humans and bovines. RESULTS: Here, we monitored the respiratory health status and growth performance of 60 Simmental calves subjected to 30 hours of road transportation using a clinical scoring system. Within the three days of commingling before the transportation and 30 days after the transportation, calves in the AMW group (n = 30) were supplied with AMW, while calves in the Control group (n = 29) were not. On three specific days, namely the day before transportation (day -3), the 30th day (day 30), and the 60th day (day 60) after transportation, sets of venous blood, serum, and nasopharyngeal swab samples were collected from 20 calves (10 from each group) for routine blood testing, whole blood transcriptomic sequencing, serology detection, serum untargeted metabolic sequencing, and 16S rRNA gene sequencing. The field data showed that calves in the AMW group displayed lower rectal temperatures (38.967 ℃ vs. 39.022 ℃; p = 0.004), respiratory scores (0.079 vs. 0.144; p < 0.001), appetite scores (0.024 vs. 0.055; p < 0.001), ocular and ear scores (0.185 vs. 0.338; p < 0.001), nasal discharge scores (0.143 vs. 0.241; p < 0.001), and higher body weight gains (30.870 kg vs. 7.552 kg; p < 0.001). The outcomes of laboratory and high throughput sequencing data revealed that the calves in the AMW group demonstrated higher cellular and humoral immunities, antioxidant capacities, lower inflammatory levels, and intestinal absorption and lipogenesis on days -3 and 60. The nasopharynx 16S rRNA gene microbiome analysis revealed the different composition and structure of the nasopharyngeal microflora in the two groups of calves on day 30. Joint analysis of multi-omics revealed that on days -3 and 30, bile secretion was a shared pathway enriched by differentially expressed genes and metabolites, and there were strong correlations between the differentially expressed metabolites and the main genera in the nasopharynx. CONCLUSIONS: These results suggest that AMW supplementation enhances peripheral immunity, nutrition absorption, and metabolic processes, subsequently affecting the nasopharyngeal microbiota and improving the respiratory health and growth performance of transported calves. This investigation provided a practical approach to mitigate transportation stress and explored its underlying mechanisms, which are beneficial for the development of the livestock industry. Video Abstract.

Molecular Regulatory Mechanism of Nano-Se Against Copper-Induced Spermatogenesis Disorder.

Selenium nanoparticle (Nano-Se) is a new type of selenium supplement, which can improve the deficiency of traditional selenium supplements and maintain its physiological activity. Due to industrial pollution and irrational use in agriculture, Cu overexposure often occurs in animals and humans. In this study, Nano-Se alleviated CuSO4-induced testicular Cu accumulation, serum testosterone level decrease, testicular structural damage, and decrease in sperm quality. Meanwhile, Nano-Se reduced the ROS content in mice testis and enhanced the activities of T-AOC, GSH, SOD, and CAT compared with CuSO4 group. Furthermore, Nano-Se alleviated CuSO4-induced apoptosis by increasing the protein expression of Cleaved-Caspase-3, Cleaved-Caspase-9, Cleaved-Caspase-12, and Bax/Bcl-2 compared with CuSO4 group. At the same time, Nano-Se reversed CuSO4-induced increase of γ-H2AX protein expression in mice testis. In conclusion, this study confirmed that Nano-Se could alleviate oxidative stress, apoptosis, and DNA damage in the testis of mice with Cu excess, thereby protecting the spermatogenesis disorder induced by Cu.

Effects of CuSO4 on hepatic mitochondrial function, biogenesis and dynamics in mice.

Copper is an essential trace element for animal. Excessive intake of copper will cause a large accumulation of copper in the body, especially in the liver, and induce hepatotoxicity, however, there are few studies on the effects of copper on hepatic mitochondrial biogenesis and mitochondrial dynamics. In this study, mice were treated with different doses of CuSO4 (0, 10, 20, and 40 mg/kg) for 21 and 42 days by gavage. The results verified that CuSO4 decreased the content of mitochondrial respiratory chain complexes I-IV in mouse liver. CuSO4 treatment resulted the decrease in the protein and mRNA expression levels of PGC-1α, TFAM, and NRF1, which were the mitochondrial biogenesis regulator proteins. Meanwhile, the proteins involved in mitochondrial fusion were reduced by CuSO4 , such as Mfn1 and Mfn2, however, mitochondrial fission proteins Drip1 and Fis1 were significantly increased. Abovementioned results show that CuSO4 could induce mitochondria damage in the liver of mice, and mitochondrial biogenesis and mitochondrial dynamics are involved in the molecular mechanism of CuSO4 -induced hepatotoxicity.

Apoptosis and DNA damage mediated by ROS involved in male reproductive toxicity in mice induced by Nickel.

Nickel (Ni) is the most important environmental pollution in the world. Ni has been confirmed to have multi-organ toxicology and carcinogenicity. Recently, Ni also can impair the male reproductive system, however, its precious mechanism still has not been clarified. The current work found that nickel chloride (NiCl2) induced histopathological lesions in testis. And, the Johnsen's score, seminiferous tubule diameter, and spermatogenic epithelium thickness were decreased in NiCl2-treated mice. The number of spermatogonium, primary spermatocyte, and round spermatid also were significantly reduced after Ni treatment. Next the potential molecular mechanism was measured. NiCl2 treatment elevated ROS production in the testis. Additionally, NiCl2 was found to induce apoptosis with features including up-regulation of Bax, cleaved-caspase-3, cleaved-caspase-8, caspase-9, and caspase-12, while down-regulation of Bcl-2 expression. In the meantime, the marker protein of DNA damage γ-H2AX was significantly increased in NiCl2-primed mice testis. To clarify effects of reactive oxygen species (ROS) in apoptosis and DNA damage induced by NiCl2, NiCl2 was used to co-treat antioxidant NAC (N-Acetyl-L-cysteine). NAC weakened ROS production induced by NiCl2, and played an inhibition role in apoptosis and DNA damage. Moreover, co-treatment using NiCl2 and NAC group also eliminated spermatogenesis disorders. In summary, research results reveal the relations of spermatogenesis disorder induced by NiCl2 with apoptosis and DNA damage mediated by ROS and apoptosis in the testis.

Induction of mitophagy via ROS-dependent pathway protects copper-induced hypothalamic nerve cell injury.

Copper (Cu) is one of the essential trace elements in the body, but excessive amounts of Cu harm multiple organs and tissues such as liver, kidneys, testis, ovaries, and brain. However, the mechanism of hypothalamic neurotoxicity induced by Cu is still unknown. This study examined the relationship between reactive oxygen species (ROS) and mitophagy in mouse hypothalamus treated with high Cu. The results demonstrated that high levels of copper sulfate (CuSO4) could cause histopathological and neuronal changes in the mouse hypothalamus, produce a large amount of ROS, induce mitophagy, and lead to an imbalance of mitochondrial fusion/fission. The main manifestations are an increase in the expression levels of LC3-II/LC3-I, p62, DRP1, and FIS1, and a decrease in the expression levels of MFN1 and MFN2. Cu can induce mitophagy also was confirmed by LC3 co-localization with TOMM20 (mitochondrial marker). Next, the effect of oxidative stress on CuSO4-induced mitophagy was demonstrated. The results showed that ROS inhibitor N-acetylcysteine (NAC) diminished CuSO4-induced mitophagy and reversed the disturbance of mitochondrial dynamics. Additionally, a study was carried out to evaluate the role of mitophagy in CuSO4-induced hypothalamic injury. The inhibition of mitophagy using mitophagy inhibitor (Mdivi-1) decreased cell viability and promoted CuSO4-inhibited mitochondrial fusion. The aforementioned results suggested that CuSO4 induced mitophagy via oxidative stress in N38 cells and mouse hypothalamus, and that the activation of mitophagy might generate protective mechanisms by alleviating Cu-induced mitochondrial dynamics disorder. This study provided a novel approach and theoretical basis for studying and preventing Cu neurotoxicity.

Nickel induces blood-testis barrier damage through ROS-mediated p38 MAPK pathways in mice.

Nickel (Ni) is an essential common environmental contaminant, it is hazardous to male reproduction, but the precise mechanisms are still unknown. Blood-testis barrier (BTB), an important testicular structure consisting of connections between sertoli cells, is the target of reproductive toxicity caused by many environmental toxins. In this study, ultrastructure observation and BTB integrity assay results indicated that NiCl2 induced BTB damage. Meanwhile, BTB-related proteins including the tight junction (TJ), adhesion junction (AJ) and the gap junction (GJ) protein expression in mouse testes as well as in sertoli cells (TM4) were significantly decreased after NiCl2 treatment. Next, the antioxidant N-acetylcysteine (NAC) was co-treated with NiCl2 to study the function of oxidative stress in NiCl2-mediated BTB deterioration. The results showed that NAC attenuated testicular histopathological damage, and the expression of BTB-related proteins were markedly reversed by NAC co-treatment in vitro and vivo. Otherwise, NiCl2 activated the p38 MAPK signaling pathway. And, NAC co-treatment could significantly inhibit p38 activation induced by NiCl2 in TM4 cells. Furthermore, in order to confirm the role of the p38 MAPK signaling pathway in NiCl2-induced BTB impairment, a p38 inhibitor (SB203580) was co-treated with NiCl2 in TM4 cells, and p38 MAPK signaling inhibition significantly restored BTB damage induced by NiCl2 in TM4 cells. These results suggest that NiCl2 treatment destroys the BTB, in which the oxidative stress-mediated p38 MAPK signaling pathway plays a vital role.

A Clinical Trial on the Welfare Effects of Administering Meloxicam to 10 to 21 Day Dairy Calves Following Caustic Paste Disbudding.

Caustic paste disbudding (CPD) is widely utilized for calves, which has been known to result in adverse effects on the calves and ethical concerns related to animal welfare, despite the use of local anesthetics. The administration of meloxicam has been demonstrated to provide benefits in alleviating pain and inflammation in juvenile calves under 9 d old and subjected to CPD. Nonetheless, there is a scarcity of literature documenting the beneficial impact of meloxicam in alleviating pain in calves aged over 9 d that have undergone CPD. Therefore, the objective of this clinical trial was to evaluate the efficacy of administering meloxicam and lidocaine for cornual nerve block together in mitigating the deleterious effects of CPD, as opposed to using lidocaine alone in calves older than 9 d. Thirty Holstein calves, aged between 10 and 21 d, were enrolled and randomly assigned to 1 of 2 treatments: lidocaine alone (Placebo), lidocaine and normal saline treatment before CPD, and lidocaine plus meloxicam, lidocaine and 0.5 mg/kg of meloxicam treatment prior to CPD. The researchers were blind to the treatment of calves to control the subjective error. The occurrences of actions associated with pain, which included head shaking, head rubbing, ear flicking, tail flicking, kicking, and head passing through the fence, were recorded. Physiological performance, including the respiration rate, heart rate, rectal temperature, mechanical nociceptive threshold (MNT), food intake, and daily activity level, was monitored. Hematological conditions were ascertained through the use of routine blood tests and enzyme-linked immunosorbent assay. The generalized linear mixed model was employed to analyze the data. The research findings revealed that applying the CPD procedure significantly elevated the frequencies of tail flicking, head shaking, and kicking, resulted in increases in respiratory rate, heart rate, daily active steps, and food intake and a decrease in MNT, and led to alterations in hematological markers, including platelet counts, mean platelet volume, prostaglandin E2, constitutive nitric oxide synthase, and hydroxyl radical. Considerable benefits, such as lower heart rates, higher food intake, and MNTs, as well as lower levels of white blood cell counts, lymphocyte counts, hemoglobin, mean platelet volume, prostaglandin E2, tumor necrosis factor-α, constitutive nitric oxide synthase, malondialdehyde, and hydroxyl radical, were observed in the calves that received meloxicam treatment in response to CPD. The findings of the study indicate that the co-administration of lidocaine and meloxicam provides obvious benefits in mitigating pain, inflammation, and oxidative stress in calves aged over 9 d and undergoing CPD. This endorses the use of meloxicam during the disbudding and dehorning procedures of calves.

Caustic paste disbudding (CPD) is a widely used practice in the cattle industry, yet there is a shortage of literature on the effects of meloxicam on calves aged 10 to 21 d who have undergone this procedure. In this clinical trial, we conducted a comparative analysis of the pain-related behavioral, physiological, and hematological performance of calves that were administered with either lidocaine plus normal saline (n = 15) or lidocaine plus meloxicam (n = 15) before undergoing disbudding operations. The findings demonstrated that the CPD operation had a significant impact on the pain-related behavior, physiological functions, and serum anti-inflammatory and antioxidative markers of the calves. On the other hand, the administration of meloxicam had notable advantages for the calves by enhancing the physiological and hematological parameters.

Low Glucose plus ß-Hydroxybutyrate Induces an Enhanced Inflammatory Response in Yak Alveolar Macrophages via Activating the GPR109A/NF-κB Signaling Pathway.

Yaks are often subject to long-term starvation and a high prevalence of respiratory diseases and mortality in the withered season, yet the mechanisms that cause this remain unclear. Research has demonstrated that ß-hydroxybutyrate (BHB) plays a significant role in regulating the immune system. Hence, we hypothesize that the low glucose and high BHB condition induced by severe starvation might have an effect on the pro-inflammatory response of the alveolar macrophages (AMs) in yaks. To validate our hypothesis, we isolated and identified primary AMs from freshly slaughtered yaks and cultured them in a medium with 5.5 mM of glucose or 2.8 mM of glucose plus 1-4 mM of BHB. Utilizing a real-time quantitative polymerase chain reaction (RT-qPCR), immunoblot assay, and enzyme-linked immunosorbent assay (ELISA), we evaluated the gene and protein expression levels of GPR109A (G-protein-coupled receptor 109A), NF-κB p65, p38, and PPARγ and the concentrations of pro-inflammatory cytokines interleukin (IL)-1ß and IL-6 and tumor necrosis factor (TNF)-α in the supernatant. The results demonstrated that AMs exposed to low glucose plus BHB had significantly higher levels of IL-1ß, IL-6, and TNF-α (p < 0.05) and higher activity of the GPR109A/NF-κB signaling pathway. A pretreatment of either pertussis toxin (PTX, inhibitor of GPR109A) or pyrrolidinedithiocarbamic (PDTC, inhibitor of NF-κB p65) was effective in preventing the elevated secretion of pro-inflammatory cytokines induced by low glucose plus BHB (p < 0.05). These results indicated that the low glucose plus BHB condition would induce an enhanced pro-inflammatory response through the activation of the GPR109A/NF-κB signaling pathway in primary yak AMs, which is probably the reason why yaks experience a higher rate of respiratory diseases and mortality. This study will offer new insight into the prevention and treatment of bovine respiratory diseases.

Autophagy-mediated ferroptosis involved in nickel-induced nephrotoxicity in the mice.

Nickel, as a widely polluted metal, has been shown nephrotoxicity. Ferroptosis is a new type of cell death driven by iron-dependent lipid peroxidation. Our study found that nickel chloride (NiCl2) induced ferroptosis in mouse kidney and TCMK-1 cells. The iron content was significantly increased in the kidney and TCMK-1 cells after NiCl2 treatment. Lipid peroxidation and MDA content were significantly increased, and GSH content and T-SOD activity were significantly decreased after exposure to NiCl2. Moreover, NiCl2 increased COX-2 protein levels, decreased SLC7A11 and GPX4 protein levels, and elevated Ptgs2 mRNA levels. Next, the mechanism of Ni-induced ferroptosis was investigated. The results showed that NiCl2 induced autophagy in TCMK-1 cells, which promoted ferroptosis induced by NiCl2. Furthermore, the data of autophagy activation or inhibition experiment showed that autophagy facilitated ferroptosis through the degradation of the iron regulation protein NCOA4 and FTH1. Otherwise, iron chelator DFOM treatment inhibited ferroptosis induced by NiCl2. Finally, ferroptosis inhibitor Fer-1 treatment significantly alleviated cytotoxicity induced by NiCl2. To sum up, our above results showed that ferroptosis is involved in NiCl2-induced nephrotoxicity, and NiCl2 induces autophagy-dependent ferritin degradation, releases iron ions, leads to iron overload, and induces ferroptosis. This study supplies a new theoretical foundation for the study of nickel and renal toxicity.

RAGE-TLR4 Crosstalk Is the Key Mechanism by Which High Glucose Enhances the Lipopolysaccharide-Induced Inflammatory Response in Primary Bovine Alveolar Macrophages.

The receptor of advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) are important receptors for inflammatory responses induced by high glucose (HG) and lipopolysaccharide (LPS) and show crosstalk phenomena in inflammatory responses. However, it is unknown whether RAGE and TLR4 can influence each other's expression through a crosstalk mechanism and whether the RAGE-TLR4 crosstalk related to the molecular mechanism of HG enhances the LPS-induced inflammatory response. In this study, the implications of LPS with multiple concentrations (0, 1, 5, and 10 µg/mL) at various treatment times (0, 3, 6, 12, and 24 h) in primary bovine alveolar macrophages (BAMs) were explored. The results showed that a 5 µg/mL LPS treatment at 12 h had the most significant increment on the pro-inflammatory cytokine interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor (TNF)-α levels in BAMs (p < 0.05) and that the levels of TLR4, RAGE, MyD88, and NF-κB p65 mRNA and protein expression were upregulated (p < 0.05). Then, the effect of LPS (5 µg/mL) and HG (25.5 mM) co-treatment in BAMs was explored. The results further showed that HG significantly enhanced the release of IL-1ß, IL-6, and TNF-α caused by LPS in the supernatant (p < 0.01) and significantly increased the levels of RAGE, TLR4, MyD88, and NF-κB p65 mRNA and protein expression (p < 0.01). Pretreatment with FPS-ZM1 and TAK-242, the inhibitors of RAGE and TLR4, significantly alleviated the HG + LPS-induced increment of RAGE, TLR4, MyD88, and NF-κB p65 mRNA and protein expression in the presence of HG and LPS (p < 0.01). This study showed that RAGE and TLR4 affect each other's expression through crosstalk during the combined usage of HG and LPS and synergistically activate the MyD88/NF-κB signaling pathway to promote the release of pro-inflammatory cytokines in BAMs.

Nickel induces hepatotoxicity by mitochondrial biogenesis, mitochondrial dynamics, and mitophagy dysfunction.

Nickel (Ni) is an important and widely hazardous chemical industrial waste. Excessive Ni exposure could cause multi-organs toxicity in human and animals. Liver is the major target organ of Ni accumulation and toxicity, however, the precise mechanism is still unclear. In this study, nickel chloride (NiCl2 )-treatment induced hepatic histopathological changes in the mice, and, transmission electron microscopy results showed mitochondrial swollen and deformed of hepatocyte. Next, the mitochondrial damages including mitochondrial biogenesis, mitochondrial dynamics, and mitophagy were measured after NiCl2 administration. The results showed that NiCl2 suppressed mitochondrial biogenesis by decreasing PGC-1α, TFAM, and NRF1 protein and mRNA expression levels. Meanwhile, the proteins involved in mitochondrial fusion were reduced by NiCl2 , such as Mfn1 and Mfn2, however, mitochondrial fission proteins Drip1 and Fis1 were significantly increased. The up-regulation of mitochondrial p62 and LC3II expression indicated that NiCl2 increased mitophagy in the liver. Moreover, the receptor-mediated mitophagy and ubiquitin (Ub)-dependent mitophagy were detected. NiCl2 promoted PINK1 accumulation and Parkin recruitment on mitochondria. And, the receptor proteins of mitophagy Bnip3 and FUNDC1 were increased in the NiCl2 -treated mice liver. Overall, these results show that NiCl2 could induce mitochondria damage in the liver of mice, and, dysfunction of mitochondrial biogenesis, mitochondrial dynamics and mitophagy involved in the molecular mechanism of NiCl2 -induced hepatotoxicity.

The Dysregulation of Inflammatory Pathways Triggered by Copper Exposure.

Copper (Cu) is an essential micronutrient for both human and animals. However, excessive intake of copper will cause damage to organs and cells. Inflammation is a biological response that can be induced by various factors such as pathogens, damaged cells, and toxic compounds. Dysregulation of inflammatory responses are closely related to many chronic diseases. Recently, Cu toxicological and inflammatory effects have been investigated in various animal models and cells. In this review, we summarized the known effect of Cu on inflammatory responses and sum up the molecular mechanism of Cu-regulated inflammation. Excessive Cu exposure can modulate a huge number of cytokines in both directions, increase and/or decrease through a variety of molecular and cellular signaling pathways including nuclear factor kappa-B (NF-κB) pathway, mitogen-activated protein kinase (MAPKs) pathway, JAK-STAT (Janus Kinase- signal transducer and activator of transcription) pathway, and NOD-like receptor protein 3 (NLRP3) inflammasome. Underlying the molecular mechanism of Cu-regulated inflammation could help further understanding copper toxicology and copper-associated diseases.

Mfn2 is responsible for inhibition of the RIG-I/IRF7 pathway and activation of NLRP3 inflammasome in Seneca Valley virus-infected PK-15 cells to promote viral replication.

Seneca Valley virus (SVV), a non-enveloped positive single-stranded virus can cause vesicular disease in swine. However, the mechanisms by which SVV activates an innate immune response remain unknown. Mitofusin-2 (MFN2), a mitochondria-shaping protein regulating mitochondrial fusion and fission, plays a crucial role in innate immune responses. But, the roles of Mfn2 in SVV infection have not been elucidated. Here, we show that SVV inhibited Mfn2 expression and NLRP3 inflammasome, activating RIG-I/IRF7 signaling pathway to increase IFN-λ3 expression. Overexpression of Mfn2 inhibited RIG-I/IRF7 signaling pathway, thus decreasing IFN-λ3 expression and promoting SVV replication. Interestingly, overexpression of Mfn2 also activated NLRP3 inflammasome but did not inhibit SVV proliferation. That may mean the RIG-I/IRF7 signaling pathway plays a more important role in SVV proliferation in PK-15 cells. This study could provide important insights into the modulation of host metabolism during SVV infection and provide a strong theoretical basis for a better understanding of the pathogenic mechanism and immune activation mechanism of SVV.

The combination of high glucose and LPS induces autophagy in bovine kidney epithelial cells via the Notch3/mTOR signaling pathway.

BACKGROUND: Aside respiratory diseases, beef cattle may also suffer from serious kidney diseases after transportation. Hyperglycemia and gram-negative bacterial infection may be the main reasons why bovine is prone to severe kidney disease during transportation stress, however, the precise mechanism is still unclear. The purpose of the current study is to explore whether the combined treatment of high glucose (HG) and lipopolysaccharide (LPS) could induce madin-darby bovine kidney (MDBK) cells injury and autophagy, as well as investigate the potential molecular mechanisms involved. RESULTS: As we discovered, the combined effect of HG and LPS decreased MDBK cells viability. And, HG and LPS combination also induced autophagy in MDBK cells, which was characterized by increasing the expression of LC3-II/I and Beclin1 and decreasing p62 expression. LC3 fluorescence signal formation was also significantly increased by HG and LPS combination treatment. Furthermore, we measured whether the mammalian target of rapamycin (mTOR) and the Notch3 signaling pathways were involved in HG and LPS-induced autophagy. The results showed that the combination of HG and LPS significantly increased the protein expression of Notch3 and decreased protein expression of p-mTOR, indicating that Notch3 and mTOR signaling pathways were activated. However, co-treatment with the Notch3 inhibitor (DAPT) could reverse the induction of autophagy, and increased the protein expression of p-mTOR. CONCLUSIONS: This study demonstrated that the combination effect of HG and LPS could induce autophagy in MDBK cells, and the Notch3/mTOR signaling pathway was involved in HG and LPS-induced autophagy.

Beta-Hydroxybutyrate: A Dual Function Molecular and Immunological Barrier Function Regulator.

Ketone bodies are crucial intermediate metabolites widely associated with treating metabolic diseases. Accumulating evidence suggests that ketone bodies may act as immunoregulators in humans and animals to attenuate pathological inflammation through multiple strategies. Although the clues are scattered and untrimmed, the elevation of these ketone bodies in the circulation system and tissues induced by ketogenic diets was reported to affect the immunological barriers, an important part of innate immunity. Therefore, beta-hydroxybutyrate, a key ketone body, might also play a vital role in regulating the barrier immune systems. In this review, we retrospected the endogenous ketogenesis in animals and the dual roles of ketone bodies as energy carriers and signal molecules focusing on beta-hydroxybutyrate. In addition, the research regarding the effects of beta-hydroxybutyrate on the function of the immunological barrier, mainly on the microbiota, chemical, and physical barriers of the mucosa, were outlined and discussed. As an inducible endogenous metabolic small molecule, beta-hydroxybutyrate deserves delicate investigations focusing on its immunometabolic efficacy. Comprehending the connection between ketone bodies and the barrier immunological function and its underlining mechanisms may help exploit individualised approaches to treat various mucosa or skin-related diseases.

Sodium fluoride suppresses spleen development through MAPK/ERK signaling pathway in mice.

Numerous studies have documented that excessive fluoride intake could cause pathological damage and functional disorder in organisms. Nevertheless, the systemic mechanism of fluorosis inhibiting the proliferation and development of splenic cell is still scarce. The preliminary studies have confirmed that high-dose NaF could inhibit splenic lymphocytes proliferation in vitro and cause toxic effects on spleen development in vivo. Here this study continued to explore the signaling pathway with the methods of quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB), revealing the mechanism of fluorosis in the growth system. Mice in 4 groups (control, 12 mg/kg, 24 mg/kg, 48 mg/kg) were gavage administrated with NaF solution continuously for 42 days. The results suggested that NaF more than 12 mg/kg slowed down the growth of mice, inhibited spleen growth and development, which was characterized by decreasing spleen volume, and inducing splenic cell apoptosis. For the Ras-Raf-MEK-ERK signaling pathway, the mRNA and protein expression levels of Ras were significantly elevated, and the phosphorylated protein expression levels of Raf (B-Raf, C-Raf) were increased. Meanwhile, mice mRNA expression levels were increased in a time and dose-dependent manner on the 21st and 42nd days of the experiment. Additionally, the mRNA and protein levels of MEK1/2 were increased on the 21st day of the experiment, while reduced on the 42nd day. The ERK1/2 levels were significantly decreased at both 21st and 42nd days of the experiment. This study showed that NaF activated Ras to induce downstream Raf-MEK-ERK cascade reaction, but failed to activate ERK eventually, the proliferation signal from the cell surface could not transmit to the nucleus, interfering with the regulation of cell proliferation, differentiation, meiosis, and suppressed spleen development ultimately.

Notch3-Mediated mTOR Signaling Pathway Is Involved in High Glucose-Induced Autophagy in Bovine Kidney Epithelial Cells.

It is reported that Notch3 and mTOR signaling pathways are involved in autophagy, and both can be activated by high glucose (HG). However, the relationship between Notch3 and mTOR and how Notch3 affects mTOR to regulate HG-induced autophagy in bovine kidney epithelial cells is still unclear. The purpose of this study is to explore how Notch3 affects mTOR to modulate HG-induced autophagy in bovine kidney cells. Our results showed that HG treatment significantly decreased the cell viability of MDBK cells in a dose-dependent manner. HG treatment significantly increased the expression of LC3-II/I ratio and Beclin1 protein and significantly decreased the expression of p62 protein. Consistently, LC3 fluorescence signal formation was detected by immunofluorescence in both dose and time-dependent manners. In addition, HG treatment significantly increased the expression of Notch3 protein and decreased the expression of the p-mTOR protein in both dose and time-dependent manners. Inhibition of Notch3 upregulated the expression of p-mTOR and p62 protein, and downregulated the expression of LC3-II/I ratio and Beclin1 protein. Besides, the function of Notch3 was investigated. In this study, inhibition of Notch3 activity significantly increased the viability of HG-stimulated MDBK cells. In summary, our results revealed that the Notch3-mediated mTOR signaling pathway was involved in HG-induced autophagy in MDBK cells.

Induction of autophagy via the ROS-dependent AMPK-mTOR pathway protects copper-induced spermatogenesis disorder.

Copper (Cu) is a necessary micronutrient at lower concentration, while excessive Cu exposure or Cu homeostasis disorders can lead to toxicity. The mechanism of male reproductive toxicity induced by Cu is still unknown. This study aims to investigate whether autophagy plays an important role in copper-induced spermatogenesis disorder in vivo and vitro. The present study showed that copper sulfate (CuSO4) might significantly promote autophagy level in the testis and mouse-derived spermatogonia cell line GC-1 spg cells. Concurrently, CuSO4 could induce autophagy via AMPK-mTOR pathway that downregulated p-mTOR/mTOR and subsequently upregulated p-AMPKα/AMPKα as well as p-ULK1/ULK1. In the meanwhile, CuSO4 treatment could also increase expression levels of the autophagy-related proteins. Then, the role of oxidative stress in CuSO4-induced autophagy was investigated. The findings demonstrated that oxidative stress inhibitor (NAC) attenuated CuSO4-induced autophagy in vivo and vitro, reversing the activation for AMPK-mTOR pathway. Additionally, the study also investigated how autophagy worked under the spermatogenesis disorder induced by CuSO4. Inhibition of autophagy could decrease cell viability, and enhance the ROS accumulation and apoptosis in the GC-1 cells, meanwhile, the spermatogenesis disorder, oxidative stress and histopathological changes were increased in the testis. Furthermore, co-treatment with the apoptosis inhibitor (Z-VAD-FMK) could decrease the spermatogenesis disorder but not influence autophagy. Besides, the crosslink between autophagy and ferroptosis were also measured, the data showed that inhibition of autophagy could suppress CuSO4-induced ferroptosis in in vivo and vitro. Altogether, abovementioned results indicated that CuSO4 induced autophagy via oxidative stress-dependent AMPK-mTOR pathway in the GC-1 cells and testis, and autophagy activation possibly led to the generation of protection mechanism through oxidative damage and apoptosis inhibition, however, autophagy also aggravate CuSO4 toxicology through promoting ferroptosis. Overall, autophagy plays a positive role for attenuating CuSO4-induced testicular damage and spermatogenesis disorder. Our study provides a possible targeted therapy for Cu overload-induced reproduction toxicology.

Activated Nrf-2 Pathway by Vitamin E to Attenuate Testicular Injuries of Rats with Sub-chronic Cadmium Exposure.

Cadmium (Cd), a heavy metal element, cumulates in the testis and can cause male reproductive toxicity. Although vitamin E (VE) as one of potential antioxidants protects the testis against toxicity of Cd, the underlying mechanism remained uncompleted clear. The aim of this study was to investigate whether the Nrf-2 pathway is involved with the protective effect of VE on testicular damages caused by sub-chronic Cd exposure. Thirty-two SD rats were divided into four groups and orally administrated with VE and/or Cd for 28 consecutive days: control group, VE group (100 mg VE/kg), Cd group (5 mg CdCl2/kg), and VE + Cd group (100 mg VE/kg + 5 mg CdCl2/kg). The results showed that 28-day exposure of Cd caused accumulation of Cd, histopathological lesions, and alternations of sperm parameters (elevated rate of abnormal sperm, decreased count of sperm, declined motility, and viability of sperm). Moreover, the rats exposed to Cd showed significant oxidative stress (increased contents of MDA and decreased levels or activities of T-AOC, GSH, CAT, SOD and GSH-Px) and inhibition of Nrf-2 signaling pathway (downregulation of Nrf-2, HO-1, NQO-1, GCLC, GCLM and GST) of the testes. In contrast, VE treatment significantly reduced the Cd accumulation, alleviated histopathological lesions and dysfunctions, activated Nrf-2 pathway, and attenuated the oxidative stress caused by Cd in the testes of rats. In conclusion, VE, through upregulating Nrf-2 pathway, could protect testis against oxidative damages induced by sub-chronic Cd exposure.