Subacute ruminal acidosis induces pyroptosis via the mitophagy-mediated NLRP3 inflammasome activation in the livers of dairy cows fed a high-grain diet.

High-grain (HG) feeding can trigger subacute ruminal acidosis (SARA) and subsequent liver tissue injury. This study investigated pyroptosis and NLRP3 inflammasome activation in SARA-induced liver injury, and the role of mitophagy during this process. Twelve mid-lactating Holstein cows equipped with rumen fistulas were randomly divided into 2 groups: a low-grain (LG) diet group (grain:forage = 4:6) and a HG diet group (grain:forage = 6:4). Each group had 6 cows. The experiment lasted for 3 wk. The ruminal fluid was collected through the rumen fistula on experimental d 20 and 21, and the pH immediately measured. At the end of the experiment, all animals were slaughtered, and peripheral blood and liver tissue were collected. The ruminal pH was lower in the HG group than that in the LG group at all time points. In addition, the ruminal pH in the HG group was lower than 5.6 at 3 consecutive time points after feeding (4, 6, and 8 h on d 20; 2, 4, and 6 h on d 21), indicating that HG feeding induced SARA. The content of lipopolysaccharide, IL-1ß, and apoptosis-related cysteine protease 1 (caspase-1) and the activity of alanine aminotransferase and aspartate aminotransferase in the blood plasma of the HG group were all significantly increased. Hepatic caspase-1 activity was increased in the livers of the HG group. The increased expression levels of pyroptosis- and NLRP3 inflammasome-related genes IL1B, IL18, gasdermin D (GSDMD), apoptosis-associated speck-like protein containing a card (ASC), NLR family pyrin domain-containing 3 (NLRP3), and caspase-1 (CASP1) in liver tissue of the HG group were detected. Furthermore, western blot analysis showed that HG feeding led to increased expression of pyroptosis- and NLRP3 inflammasome-related proteins GSDMD N-terminal (GSDMD-NT), IL-1ß, IL-18, cleaved-caspase-1, ASC, NLRP3, and cleaved-caspase-11 and upregulated expression of mitophagy-related proteins microtubule-associated protein 1 light chain 3 II (MAP1LC3-II), beclin 1 (BECN1), Parkin, and PTEN-induced kinase 1 (PINK1) in liver tissue. Collectively, our results revealed that SARA caused increased mitophagy and activated the NLRP3 inflammasome, causing pyroptosis and subsequent liver injury in dairy cows fed a HG diet.

Forkhead box protein A2 alleviates toll-like receptor 4-mediated inflammation, endoplasmic reticulum stress, autophagy, and apoptosis induced by lipopolysaccharide in bovine hepatocytes.

Lipopolysaccharide (LPS) is an important stimulus of inflammation via binding to toll-like receptor 4 (TLR4), but the role of TLR4 in LPS-induced cellular homeostasis disruption indicated by the increased level of endoplasmic reticulum (ER) stress, autophagy, and apoptosis is unknown in the liver of dairy cows. Previous studies show that forkhead box protein A2 (FOXA2) is an important transcriptional factor to maintain cellular metabolic homeostasis, but the mechanisms by which FOXA2 mediates cellular homeostasis disruption in response to LPS remains unclear. To achieve the aims, hepatocytes separated from dairy cows at ∼160 d in milk were pretreated with a specific TLR4 inhibitor TAK-242 for 12 h, followed by LPS treatment for another 12 h to investigate the role of TLR4 in LPS-induced disruption of cellular homeostasis. The results indicated that LPS-induced nuclear factor-κB (NF-κB)-mediated inflammatory cascades, ER stress, autophagy, and apoptosis via activating TLR4 and downregulating FOXA2 expression in bovine hepatocytes. The application of TLR4 inhibitor alleviated LPS-induced inflammation through inactivating NF-κB proinflammatory pathway, restored cell homeostasis by decreasing the level of ER stress, autophagy, and apoptosis, and upregulated FOXA2 expression. Furthermore, we also elevated FOXA2 expression with an overexpression plasmid to clarify its molecular role in response to LPS challenge. FOXA2 overexpression reduced LPS-caused inflammation by inhibiting NF-κB signaling pathway. Also, FOXA2 could alleviate ER stress to block unfolded protein response and suppress autophagic flux. In addition, FOXA2 enhanced mitochondrial membrane potential via reducing pro-apoptotic protein BAX, CASPASE3, and Cleaved CASPASE3 expression and elevating anti-apoptotic protein BCL-2 expression to mitigate LPS-induced apoptosis. Taken together, these findings suggested that FOXA2 is a mediator to alleviate TLR4-controlled inflammation, ER stress, autophagy, and apoptosis in LPS-treated bovine hepatocytes, it could serve as a potential target to intervene cell homeostasis disruption caused by LPS in the liver of dairy cows.

Subacute ruminal acidosis downregulates FOXA2, changes oxidative status, and induces autophagy in the livers of dairy cows fed a high-concentrate diet.

The purpose of this experiment was to investigate high-concentrate feeding-induced changed status of oxidative and autophagy in the livers of dairy cows. Hepatocyte nuclear factor 3ß (FOXA2) was reported in cases of liver fibrosis, glucolipid metabolism, and hepatocyte differentiation, but not in cases liver damage in cows fed a high-concentrate diet. Therefore, we also aimed to explore the potential role of FOXA2 in SARA-induced liver damage. We divided 12 mid-lactating Holstein cows into 2 groups and fed them a high-concentrate (HC group, forage:concentrate = 4:6) and a low-concentrate (forage:concentrate = 6:4) diet. After a 2-wk adaptation period and a 3-wk experimental period, peripheral blood was collected for determination of antioxidant enzyme activity, and liver tissue was collected to examine genes and proteins. On d 20 and 21 of the experiment, rumen fluid was collected, and the pH was measured. A significant difference in rumen fluid pH was found between the 2 groups (low-concentrate: 6.10 ± 0.07 vs. HC: 5.59 ± 0.09). The rumen fluid pH in the HC group was less than 5.6 at 2 time points, indicating that SARA was successfully induced. Lipopolysaccharide (0.24 ± 0.10 vs. 0.42 ± 0.12) and malondialdehyde (1.46 ± 0.25 vs. 2.94 ± 0.65) increased, whereas superoxide dismutase (14.06 ± 0.63 vs. 11.71 ± 0.64), reduced glutathione (14.48 ± 2.25 vs. 6.82 ± 0.67), and the total antioxidant capacity (0.43 ± 0.03 vs. 0.30 ± 0.03) decreased in the peripheral blood of the HC group. Moreover, in liver tissue from the HC group, catalase (0.71 ± 0.03 vs. 0.49 ± 0.03) and superoxide dismutase (27.46 ± 1.90 vs. 20.32 ± 1.54) were decreased, whereas malondialdehyde (0.21 ± 0.03 vs. 0.28 ± 0.03) was elevated. Meanwhile, we observed lower gene expression of CAT (1.00 ± 0.15 vs. 0.64 ± 0.17), NAD(P)H quinone dehydrogenase 1 (NQO1; 1.00 ± 0.09 vs. 0.47 ± 0.14), glutathione peroxidase 1 (GPX1; 1.03 ± 0.27 vs. 0.55 ± 0.09), SOD1 (1.01 ± 0.17 vs. 0.76 ± 0.17), and SOD3 (1.02 ± 0.21 vs. 0.55 ± 0.16) in the liver tissue of the HC group. Furthermore, western blot analysis showed that high-concentrate feeding led to decreased sirtuin-1 (SIRT1) (1.00 ± 0.10 vs. 0.62 ± 0.15) and FOXA2 (1.02 ± 0.19 vs. 0.68 ± 0.18), elevated autophagy-related protein microtubule associated protein 1 light chain 3 II (MAP1LC3-II; 1.00 ± 0.32 vs. 1.98 ± 0.83) and autophagy related 5 (ATG5; 1.00 ± 0.30 vs. 1.80 ± 0.27), and suppressed antioxidant signaling pathway-related protein nuclear factor erythroid 2-like 2 (NFE2L2; 1.00 ± 0.18 vs. 0.61 ± 0.30) and heme oxygenase 1 (HMOX1; 1.00 ± 0.48 vs. 0.38 ± 0.25) in liver tissue. Overall, these data indicated that SARA elevated systematic oxidative status and enhanced autophagy in the liver, and suppressed SIRT1 and FOXA2 may mediate enhanced oxidative damage and autophagy in the livers of dairy cows fed a high-concentrate diet.

AMPK-endoplasmic reticulum stress axis contributes to lipopolysaccharide-caused mitochondrial dysfunction by regulating mitochondria-associated membrane function in bovine hepatocytes.

Mitochondrial homeostasis is closely associated with cellular homeostasis process, whereas mitochondrial dysfunction contributes to apoptosis and mitophagy. Hence, analyzing the mechanism of lipopolysaccharide (LPS)-caused mitochondrial damage is necessary to understand how cellular homeostasis is maintained in bovine hepatocytes. Mitochondria-associated membranes (MAM), a connection between endoplasmic reticulum (ER) and mitochondria, is important to control mitochondrial function. To investigate the underlying mechanisms of the LPS-caused mitochondrial dysfunction, hepatocytes isolated from dairy cows at ∼160 d in milk (DIM) were pretreated with the specific inhibitors of adenosine 5'-monophosphate-activated protein kinase (AMPK), ER stress, RNA-activated protein kinase-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), c-Jun N-terminal kinase, and autophagy followed by a 12 I1/4g/mL LPS treatment. The results showed that inhibiting ER stress with 4-phenylbutyric acid decreased the levels of autophagy and mitochondrial damage with AMPK inactivation in LPS-treated hepatocytes. The AMPK inhibitor compound C pretreatment alleviated LPS-induced ER stress, autophagy and mitochondrial dysfunction by regulating the expression of MAM-related genes, such as mitofusin 2 (MFN2), PERK, and IRE1α. Moreover, inhibiting PERK and IRE1α mitigated autophagy and mitochondrial dynamic disruption by regulating the MAM function. Additionally, blocking c-Jun N-terminal kinase, the downstream sensor of IRE1α, could reduce the levels of autophagy and apoptosis and restore the balance of mitochondrial fusion and fission by modulating the B cell leukemia 2 (BCL-2)/BCL-2 interacting protein 1 (BECLIN1) complex in the LPS-treated bovine hepatocytes. Furthermore, autophagy blockage with chloroquine could intervene in LPS-caused apoptosis to restore mitochondrial function. Collectively, these findings suggest that the AMPK-ER stress axis is involved in the LPS-caused mitochondrial dysfunction by mediating the MAM activity in bovine hepatocytes.

[HUVECs extraction and UHPLC LTQ Orbitrap analysis for screening vascular endothelium protective components in Kudiezi injection].

An effective method has been employed as a tool for screening active components in Kudiezi injection by using cell chromatography and sensitive UHPLC-HR-MSn method. The potential bioactive components in Kudiezi injection could be selectively bound to the HUVECs target cells first. After cell target desensitization and inactivation, the chemical constituents with cell target affinity were identified by LC-MS, so as to screen the possible active components in Kudiezi injection. Based on the accurate mass measurements and the retention time, in total, 9 compounds were tentatively identified and characterized, including 4 sesquiterpene lactones, 3 phenolic acids and 2 flavonoids. HUVECs biospecific extraction coupled with UHPLC-LTQ-Orbitrap analysis could provide a rapid and efficient method for the identification of potential bioactive components in Kudiezi injection, and provide the reference for further research on its effective materials basis.

Conjugated Linoleic Acid Ameliorates Hydrogen Peroxide-Induced Mitophagy and Inflammation via the DRP1-mtDNA-STING Pathway in Bovine Hepatocytes.

Oxidative stress is tightly associated with liver dysfunction and injury in dairy cows. Previous studies have shown that cis-9, trans-11 conjugated linoleic acid (CLA) possesses anti-inflammatory and antioxidative abilities. In this study, the bovine hepatocytes were pretreated with CLA for 6 h, followed by treatment with hydrogen peroxide (H2O2) for another 6 h to investigate the antioxidative effect of CLA and uncover the underlying mechanisms. The results demonstrated that H2O2 treatment elevated the level of mitophagy, promoted mitochondrial DNA (mtDNA) leakage into the cytosol, and activated the stimulator of interferon genes (STING)/nuclear factor kappa B (NF-κB) signaling pathway to trigger an inflammatory response in bovine hepatocytes. In addition, the dynamin-related protein 1(DRP1)-mtDNA-STING-NF-κB axis contributed to the H2O2-induced oxidative injury of bovine hepatocytes. CLA could reduce mitophagy and the inflammatory response to attenuate oxidative damage via the DRP1/mtDNA/STING pathway in bovine hepatocytes. These findings offer a theoretical foundation for the hepatoprotective effect of CLA against oxidative injury in dairy cows.

Dietary disodium fumarate supplementation alleviates subacute ruminal acidosis (SARA)-induced liver damage by inhibiting pyroptosis via mitophagy-NLRP3 inflammasome pathway in lactating Hu sheep.

Liver damage is common in ruminants with subacute ruminal acidosis (SARA). Disodium fumarate (DF) could regulate rumen microbial community and neutralize ruminal organic acids. This study aimed to evaluate the effect of dietary DF supplementation on SARA-induced liver damage and investigate the underlying mechanism. The results showed that feeding a high-concentrate diet induced decreased rumen fluid pH and increased ruminal LPS. The rumen fluid pH in the HC group was less than 5.6 at 4 time points, indicating that SARA was successfully induced. The histopathological analysis showed that in the HC group, hemorrhage and inflammatory cell infiltration were observed in liver tissue. Using ELISA kits and biochemical analyzer, we identified that the contents of interleukin 1beta (IL-1ß), interleukin 18 (IL-18), caspase-1, and the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in hepatic vein were elevated in the HC group. However, DF supplementation increased rumen fluid pH value, decreased ruminal LPS, attenuated hemorrhage and inflammatory cell infiltration in the liver tissue, and decreased contents of IL-1ß, IL-18, caspase-1, AST, and ALT in the hepatic vein. Real-time PCR and western blot analysis displayed that SARA-induced increased expression of pyroptosis-related proteins (GSDMD-NT) was attenuated in the HCDF group. Meanwhile, SARA induced increased expression of mitophagy and inflammasome-related proteins (MAP1LC3-II, PINK1, Parkin, cleaved-caspase-11, cleaved-caspase-1, NLRP3, and ASC) and elevated expression of inflammasome-related genes (NLRP3, CASP1, and ASC), which was reversed by DF supplementation. Moreover, SARA activated toll-like receptor 4 (TLR4)-nuclear factor kappa B (NF-κB) signaling pathway and inhibited the entry of forkhead box A2 (FOXA2) into the nucleus, which was reversed by DF supplementation. Collectively, our data suggest that dietary DF supplementation inhibited hepatocyte pyroptosis by regulating the mitophagy-NLRP3 inflammasome pathway and the NF-κB signaling pathway, thus alleviating SARA-induced liver damage in Hu sheep.

A high-concentrate diet provokes inflammatory responses by downregulating Forkhead box protein A2 (FOXA2) through epigenetic modifications in the liver of dairy cows.

A high-concentrate diet has been reported to promote an inflammatory response in dairy cows. The purpose of this study was to clarify the effect of the high-concentrate (HC) diet on hepatic Forkhead box protein A2 (FOXA2) expression and uncover the molecular mechanisms in inflammatory responses in the liver. The results showed that the HC diet reduced the ruminal fluid pH and elevated the secretion of SAA3, IL-1α, and IL-8 and reduced that of IL-10 in peripheral blood plasma. Compared with the low-concentrate (LC) group, the concentration of myeloperoxidase (MPO) was higher in the liver of dairy cows in the HC group. In addition, the relative mRNA expression of acute phase proteins (HP, SAA3, and LBP), proinflammatory cytokines (TNFα, IL-1α, IL-1ß, IL-8), TLR4, MyD88, TRAF6, TRIF, IκBα, p65, p38 and JNK1 was upregulated and that of IL-10 was downregulated in the liver of the HC group. Consistently, the protein abundance of TLR4, TNFα and phosphorylation of proteins involved in NF-κB (IκBα and p65) and MAPK (p38 and JNK) pathways were significantly increased in the HC group compared with the LC group. And both the mRNA and protein abundance of FOXA2 were downregulated in the HC group. Further epigenetic analysis results demonstrated that chromatin compaction and DNA hypermethylation contributed to inhibiting FOXA2 expression, in which the demethylase ten-eleven translocation 1 (TET1) and histone deacetylase 3 (HDAC3) might participate. Overall, these findings demonstrated that the high-concentrate diet triggered inflammatory cascades and downregulated FOXA2 by epigenetic modifications in the liver of dairy cows.

Protective Effects of N-Acetylcysteine on Lipopolysaccharide-Induced Respiratory Inflammation and Oxidative Stress.

As the leading cause of bovine respiratory disease (BRD), bacterial pneumonia can result in tremendous losses in the herd farming industry worldwide. N-acetylcysteine (NAC), an acetylated precursor of the amino acid L-cysteine, has been reported to have anti-inflammatory and antioxidant properties. To explore the protective effect and underlying mechanisms of NAC in ALI, we investigated its role in lipopolysaccharide (LPS)-induced bovine embryo tracheal cells (EBTr) and mouse lung injury models. We found that NAC pretreatment attenuated LPS-induced inflammation in EBTr and mouse models. Moreover, LPS suppressed the expression of oxidative-related factors in EBTr and promoted gene expression and the secretion of inflammatory cytokines. Conversely, the pretreatment of NAC alleviated the secretion of inflammatory cytokines and decreased their mRNA levels, maintaining stable levels of antioxidative gene expression. In vivo, NAC helped LPS-induced inflammatory responses and lung injury in ALI mice. The relative protein concentration, total cells, and percentage of neutrophils in BALF; the level of secretion of IL-6, IL-8, TNF-α, and IL-1ß; MPO activity; lung injury score; and the expression level of inflammatory-related genes were decreased significantly in the NAC group compared with the LPS group. NAC also ameliorated LPS-induced mRNA level changes in antioxidative genes. In conclusion, our findings suggest that NAC affects the inflammatory and oxidative response, alleviating LPS-induced EBTr inflammation and mouse lung injury, which offers a natural therapeutic strategy for BRD.

STIM1-Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry.

(1) Background: The basic mechanism of store-operated Ca2+ entry (SOCE) in bovine hepatocytes (BHEC) is related to the activation of STIM1 and Orai1. The effect of STIM1- and Orai1-dependent calcium ion signaling on the NF-κB signaling pathway is unclear. (2) Methods: In this study, the expression of STIM1 and Orai1 in BHEC was regulated. RT-qPCR, Western blotting, and an immunofluorescence antibody (IFA) assay were performed to elucidate the effect of inflammation and endoplasmic reticulum stress (ERS) in BHEC. (3) Results: First of all, in this study, RT-PCR and Western blotting were used to detect the levels of IκB, NF-κB, and inflammatory factors (IL-6, IL-8, and TNF-α) and the expression of genes and proteins related to ERS (PERK, IRE1, ATF6, GRP78, and CHOP), which reached peak levels simultaneously when BHEC were treated with 16 µg/mL LPS for 1 h. For STIM1, we overexpressed STIM1 in BHEC by using plasmid transfection technology. The results showed that after overexpression of STIM1, the gene and protein expression of STIM1 levels were significantly upregulated, and the expression of Orai1 on the cell membrane was also upregulated, which directly activated the SOCE channel and induced inflammation and ERS in BHEC. The overexpression group was then treated with LPS, and it was found that the overexpression of STIM1 could enhance LPS-induced BHEC inflammation and ERS in BHEC. For Orai1, BHEC were pretreated with 8 µg/mL of the specific inhibitor BTP2 for 6 h. It was found that BTP2 could inhibit the expression of mRNA in Orai1, significantly reduce the gene expression of STIM1, inhibit the activation of the NF-κB signaling pathway, and alleviate inflammation and ERS in BHEC under LPS stimulation. (4) Conclusions: In conclusion, STIM1/Orai1 can intervene and exacerbate LPS-induced inflammation and ERS in bovine hepatocytes through SOCE.

MicroRNA-1249 Targets G Protein Subunit Alpha 11 and Facilitates Gastric Cancer Cell Proliferation, Motility and Represses Cell Apoptosis.

AIM: The purpose of our study was to investigate the effects of miR-1249 in gastric cancer. METHODS: By analyzing the data obtained from TCGA database, the expression and prognosis of miR-1249 in gastric cancer patients were analyzed. Then, CCK8, colony forming and transwell assays were used to test cell proliferation and motility. The cell apoptosis was detected by flow cytometry. The Pearson correlation coefficient analyzed was applied to analyze the correlation between GNA11 and miR-1249. qRT-PCR and Western blotting assays were employed to detect the mRNA and protein levels. RESULTS: We discovered that miR-1249 was highly expressed and was associated with a worse prognosis in gastric cancer patients. Besides, miR-1249 was up-regulated in gastric cancer cell lines (AGS, MKN45 and SNU1). More interestingly, miR-1249 exerted facilitating impacts on gastric cancer cell proliferation and motility, whereas miR-1249 acted as a suppressing effect on gastric cancer apoptosis. G protein subunit alpha 11 (GNA11) was a target gene of miR-1249 and was negatively correlated with miR-1249. Furthermore, GNA11 was negatively regulated by miR-1249. Additionally, GNA11 was lowly expressed in gastric cancer tissues and cell lines, as well as low GNA11 expression, was related to poor overall survival results in gastric cancer patients. The promoting influences of miR-1249 over-expression on AGS cell proliferation and motility was rescued by GNA11 over-expression, which might be achieved by regulating PI3K/AKT/mTOR signalling pathway. CONCLUSION: Above all, we concluded that miR-1249 was concerned with the progression of gastric cancer through regulating GNA11, suggesting that miR-1249 and GNA11 might serve as predictive biomarkers for gastric cancer therapy.

Cis-9, Trans-11 CLA Alleviates Lipopolysaccharide-Induced Depression of Fatty Acid Synthesis by Inhibiting Oxidative Stress and Autophagy in Bovine Mammary Epithelial Cells.

Lipopolysaccharide (LPS) is the dominating endotoxin of Gram-negative bacteria, which can cause mastitis. Bovine mammary epithelial cells (BMECs), as major components of the mammary gland, usually suffer LPS challenge. Cis-9, trans-11 conjugated linoleic acid (CLA) has been reported to have anti-inflammatory characteristics, while its anti-oxidative ability to maintain cellular homeostasis in BMECs under LPS challenge is limited. Therefore, we studied whether cis-9, trans-11 CLA can restore the disturbance of cellular homeostasis indicated by the redox status and autophagy level caused by LPS and have an effect on cellular function- milk fat metabolism. For oxidative stress, LPS challenge promoted the formation of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) and decreased the concentration of glutathione. Anti-oxidative signaling regulated by transcription factor nuclear factor, erythroid 2 like 2 (Nrf2) was also depressed by LPS at the mRNA and protein level. However, cis-9, trans-11 CLA pretreatment downregulated the formation of ROS and TBARS and upregulated the expression of antioxidative enzymes. As a part of innate immunity, autophagy was also motivated by LPS challenge, while CLA decreased the autophagy level. LPS and H2O2 inhibited milk fat synthesis-related transcription factor sterol regulatory element binding protein (SREBP1), peroxisome proliferator activated receptor gamma (PPARG) and their downstream enzymes. Furthermore, 50 uM cis-9, trans-11 CLA promoted the mRNA and protein abundance of milk fat synthesis-related genes and lipid droplet formation in BMECs. In conclusion, LPS challenge disturbed the cellular homeostasis and depressed milk fat synthesis in BMECs; while cis-9, trans-11 CLA alleviated oxidative stress and decreased autophagy level, thus promoting milk fat synthesis, which offers a natural therapeutic strategy for mastitis.