The aim of this study was to evaluate the effect of Fructus Ligustri Lucidi (FLL) supplementation on energy metabolism, immunity, and the rumen microbiome in peripartum cows. Twenty healthy multiparous Holstein dairy cows were enrolled in the trial based on expected calving date, parity, and previous lactation milk yield. They were randomly assigned to either a control diet (Con; n = 10) or the control diet supplemented with 150 g/d per cow of FLL (FLL; n = 10). The trial spanned 6 wk, from 3 wk prepartum to 3 wk postpartum. Cows were milked thrice daily at 0500, 1100, and 1700 h, with milk yield and SCC recorded. Blood samples were collected from each cow at -3, -2, -1, 0, 1, 2, and 3 weeks relative to calving to evaluate the metabolic, inflammatory, and oxidative profiles. Peripheral blood neutrophils were isolated to measure reactive oxygen species (ROS), phagocytic activity, and neutrophil extracellular traps (NET). Rumen liquid was obtained via esophageal tubing at -3, 0, and 3 wk to investigate microbial biodiversity. Results indicated that FLL supplementation did not significantly affect the milk yield or SCC within 3 wk postpartum. Notably, FLL ameliorated negative energy balance from 1 wk to 3 wk, mitigated inflammation from calving day to 3 wk, and reduced oxidative stress at both -1 and 1 wk. The FLL treatment enhanced neutrophil phagocytosis from calving day to 1 wk, increased NET release at -1 wk, and reduced neutrophil ROS levels on calving day. Moreover, FLL increased rumen microbial α-diversity on calving day. At the phylum level, Firmicutes abundance was higher in the Con group on calving day compared with 3 wk pre- and postpartum, whereas Bacteroidota exhibited the opposite trend; FLL maintained stable Firmicutes and Bacteroidota abundances throughout the peripartum period. At the genus level, Prevotella abundance decreased in both groups on calving day but rebounded by 3 wk, with significantly higher Prevotella levels in the FLL group on calving day. The level 2 Kyoto Encyclopedia of Genes and Genomes pathway prediction further indicated divergent metabolic profiles: lipid metabolism pathways were less active in the FLL group than in the Con group on calving day and at 3 wk, whereas immune-related pathways were enriched in the FLL group. Energy metabolism pathway activity was higher in the FLL group on calving day but lower by 3 wk compared with the Con group. Correlation analysis indicated that rumen microbiome shifts were associated with energy metabolism, immunity, and oxidative stress. In conclusion, this study elucidates the dynamic changes in rumen microbiome during the peripartum period and highlights the beneficial effects of FLL supplementation on energy metabolism, immune function, and rumen microbial homeostasis in peripartum dairy cows.

Subclinical ketosis (SCK), a common metabolic disorder in dairy cows during the peripartum period, is accompanied by systemic inflammation and elevated circulating BHB. β-Hydroxybutyrate contributes to the development of systemic inflammation by inhibiting the spontaneous apoptosis of neutrophils in SCK cows. However, the underlying molecular mechanism is still unclear. This study investigated the role of the degranulation-macrophage-1 antigen (Mac-1) axis in this process. The results demonstrated that BHB promoted the degranulation process and activated membrane Mac-1 (CD11b/CD18) on neutrophils both ex vivo and in vitro. Neutrophils were isolated from healthy cows and treated with 2 mM BHB, a specific degranulation inhibitor, or a Mac-1 blocking antibody in vitro. We found that the released granule contents during degranulation activated the intracellular Mac-1 signaling pathway, which was involved in BHB-mediated inhibition of bovine neutrophil apoptosis. Overall, our findings reveal that the degranulation-Mac-1 axis plays a critical role in regulating BHB-induced inhibition of spontaneous neutrophil apoptosis in dairy cows with SCK.

Serpin family H member 1 (SERPINH1) is responsible for encoding the protein known as heat shock protein 47, which functions as a molecular chaperone specific to collagen (COL). This protein has been identified as a potential therapeutic target for COL‐related disorders. In this study, we aimed to investigate the role of SERPINH1 in the tumorigenicity of gliomas. To achieve this, we utilized various bioinformatics tools to analyze gene expression, overall survival, protein–protein interactions, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and Gene Set Enrichment Analysis (GSEA). Based on The Cancer Genome Atlas database revealed that SERPINH1 and four COL family members (COL1A1, COL3A1, COL4A1, and COL4A2) expression are significantly upregulated in glioma tissues compared with normal nontumor tissues. GO, KEGG, and GSEA analyses exhibited that SERPINH1 is implicated in the establishment and degradation of COL‐containing extracellular matrix (ECM), focal adhesion, and ECM–receptor interaction in glioma. SERPINH1 is an independent prognostic factor, exhibiting a positive association with the augmentation of neutrophils and macrophages, as well as the manifestation of immune checkpoint molecules within glioma. Experimental assessments conducted both in vitro and in vivo demonstrated that the suppression of SERPINH1 impeded the migratory, invasive, and proliferative capacities of glioma cells, while concurrently fostering cellular apoptosis. Consequently, SERPINH1 emerges as an oncogenic gene and an independent prognostic marker for glioma, potentially facilitating the advancement of immunotherapeutic interventions for the treatment of glioma.

Osteoarthritis (OA) is a chronic degenerative joint disease, characterized by cartilage injury. Milk fat globule-epidermal growth factor 8 (MFG-E8) exhibited anti-inflammatory effects, with undefined mechanism in OA. Eighteen C57BL/6 J mice were randomized into Sham and destabilization of medial meniscus (DMM) groups, with DMM surgery for OA model establishment. Subsequently, DMM mice received rmMFG-E8 (50 ng/g) as rmMFG-E8 group (n = 6). HE staining and Safranin O/Fast green staining for cartilage tissue pathological damage, TUNEL staining for apoptosis, ELISA for pro-inflammatory factors, and immunohistochemistry were performed. Following extraction of primary mouse chondrocytes, cells were randomized into Control, OA (10 ng/mL IL-1β), OA + rmMFG-E8 (500 ng/mL), and OA + rmMFG-E8+3-MA (autophagy inhibitor, 5 mM) groups. Cell viability by CCK8, pro-inflammatory factors by qRT-PCR, and apoptosis by flow cytometry were detected. In vivo and in vitro, transmission electron microscopy for autophagy and Western blot for autophagy- and apoptosis-related expression were conducted. In vivo, DMM group showed severe cartilage tissue damage, higher matrix metalloproteinase 13 (MMP13), Cleaved caspase-3, Cleaved PARP, Bax, TNF-α, IL-1β, IL-6 levels, and lower Bcl-2, MFG-E8, Collagen II, LC3II/LC3I, Beclin1, and ATG5 expression. Further rmMFG-E8 intervention improved mobility and pathological damage in DMM mice, with lower MMP13 expression. In vitro, rmMFG-E8 in OA group reduced TNF-α, IL-1β, IL-6, Cleaved caspase-3, Cleaved PARP, Bax, and P62 levels, and enhanced cell viability, Bcl-2, LC3II/LC3I, Beclin1, and ATG5 expression. Further 3-MA treatment up-regulated apoptosis and decreased cell viability and autophagy. Therefore, MFG-E8 exerts anti-OA effects by inhibiting apoptosis and inducing autophagy, offering a new potential target for OA treatment.

Ketosis is often accompanied by a reduction in milk production in dairy cows, but the molecular mechanism has not been fully elucidated. Ketotic cows possess systemic oxidative stress (OS), which may implicate apoptosis in mammary glands. Sirtuin 3 (SIRT3) is a vital regulator of cellular redox homeostasis and is under the control of AMP-activated protein kinase (AMPK) signaling in nonruminants. Thus, we aimed to investigate (1) the AMPK-SIRT3 and apoptosis status of mammary glands from ketotic cows, (2) the effect of SIRT3 on OS-induced apoptosis in bovine mammary epithelial cells (BMEC), and (3) the role of AMPK signaling on SIRT3-mediated effects on apoptosis. Mammary gland samples were reused from a previous study, which contained healthy and ketotic cows (both n = 15). BMEC were incubated with 0, 0.3, 0.6, or 0.9 mM H2O2 for 6 h with/without a 30 min incubation of an antioxidant MitoQ (1 μM). Then BMEC were incubated with SIRT3 overexpression adenovirus (Ad-SIRT3) for 6 h followed by a 6 h incubation with 0.6 mM H2O2. Finally, BMEC were treated with the AMPK inhibitor Compound C (Cd C,10 μM) for 30 min before the H2O2 challenge, or cells were initially treated with the AMPK agonist MK8722 (10 μM) for 30 min followed by a 30-h culture with/without si-SIRT3 and eventually the H2O2 exposure. Ketotic cows displayed higher levels of Bax, Caspase-3 and Bax/Bcl-2 but lower levels of Bcl-2 in mammary glands. H2O2 incubation displayed similar results, exhibiting a dose-dependent manner between the H2O2 concentration and the apoptosis degree. Mito Q pretreatment reduced cellular reactive oxygen species and rescued cells from apoptosis. Ketotic cows had a lower mammary protein abundance of SIRT3. Similarly, H2O2 incubation downregulated both mRNA and protein levels of SIRT3 in a dose- and time-dependent manner. Ad-SIRT3 infection lowered levels of cellular reactive oxygen species, Bax, Caspase-3 and Bax/Bcl-2 but increased levels of Bcl-2. TUNEL assays confirmed that Ad-SIRT3 infection mitigated H2O2-induced apoptosis. Both ketotic cows and H2O2-induced BMEC had lower levels of p-AMPK and p-AMPK/AMPK. Additionally, Cd C pretreatment decreased SIRT3 and Bcl-2 expression but increased levels of Bax and Caspase-3. Contrary to the inhibitor, MK8722 had opposite effects and reduced the percentage of apoptotic cells. However, these effects of MK8722 were reversed upon SIRT3 silencing. In conclusion, in vivo data confirmed that ketosis is associated with greater apoptosis and restricted AMPK-SIRT3 signaling in mammary glands; in vitro data indicated that SIRT3 mitigates OS-induced apoptosis via AMPK signaling. As such, there may be potential benefits for targeting the AMPK-SIRT3 axis to help counteract the negative effects of mammary glands during ketosis.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is suggested as an alternative terminology to the previously termed non-alcoholic fatty liver disease. Despite its introduction, the relationships between MAFLD, non-MAFLD, and the albumin-bilirubin (ALBI) grade, an indicator of liver dysfunction, are yet to be thoroughly understood. This research delves into these relationships and strives to pinpoint factors that might contribute to a favorable prognosis. Utilizing a cross-sectional design, this investigation examined data extracted from the National Health and Nutrition Examination Survey, focusing on American adults aged 20 and above. The findings revealed a notably elevated incidence of liver dysfunction in individuals diagnosed with MAFLD. This elevated risk was particularly pronounced in females under the age of 65. Delving deeper with a multivariate logistic regression approach, it was discerned that high levels of physical activity inversely correlated with hepatic dysfunction, especially in MAFLD patients without cirrhosis manifestation. The conclusions drawn from this research underscore that MAFLD-diagnosed patients face a more daunting hepatic outlook compared to their non-MAFLD counterparts and healthy controls. Moreover, the therapeutic potential of regular physical activity in forestalling hepatic dysfunction in non-cirrhotic MAFLD patients stands affirmed.

Dairy cows have high incidence of ketosis during perinatal. According to our previous studies, elevated ketone bodies (mainly β-hydroxybutyrate, BHB) in the peripheral blood are believed to contribute to the impairment of neutrophils mobility and directionality thereby contributing to the immunosuppression and further infectious disease secondary to ketosis. However, the specific effect of BHB on the directionality of bovine neutrophils needs further study and the underlying molecular mechanisms are still unclear. According to the concentration of serum BHB, 40 multiparous cows (within 3 wk postpartum) were selected and divided into the control (n = 20, BHB <0.6 mM) or clinical ketosis (n = 20, BHB >3.0 mM) group. Blood samples were collected for baseline serum characteristics analysis and neutrophil mobility and directionality detection. Platelet activation factor was used as a chemoattractant in cell migration experiments. Our ex-vivo data showed ketotic cows, compared with control cows, were in a negative energy balance state, and their neutrophils had shorter migration distance, lower migration speed, and impaired migration directionality. Neutrophils from control cows were incubated with 3.0 mM BHB for 6 h in vitro. Similarly, BHB stimulation resulted in impaired mobility and directionality of bovine neutrophils. We further specifically studied the underlying molecular mechanism of BHB regulating neutrophil migration directionality in the present study. Cell division control protein 42 homolog (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1), 2 key markers in the regulation of migration directionality, were found increased after BHB treatment in their total and activated protein levels while decreasing in their transcription level, suggesting that an imbalance of the protein degradation system may be involved. Interestingly, transmission electron microscopy data revealed a decrease in autophagosome number in neutrophils from ketotic cows. Western blotting data showed the accumulation of sequestosome-1 (p62) protein and a decrease in microtubule-associated protein 1 light chain 3-II (LC3-II) protein abundance after BHB treatment, further confirming that the autophagy flux was inhibited in neutrophils from ketotic cows. Additionally, rapamycin (RAPA), a specific autophagy activator, was used with or without BHB treatment in vitro. Accordingly, the BHB-induced impairment of migration directionality but not mobility was relieved by RAPA. Furthermore, as verified by in vivo experiments, compared with the control cows, the protein abundance of total and activated Cdc42 and Rac1 increased and their mRNA abundance decreased in neutrophils from ketotic cows. Overall, the present study revealed that pathological concentration of BHB impairs neutrophil migration directionality through inhibiting the autophagy-mediated degradation of Cdc42 and Rac1. These findings help explain the immunosuppression caused by ketosis.

奶牛亚临床酮病(subclinical ketosis,SCK)是一种围产期高产奶牛高发的代谢紊乱性疾病,伴有高酮血症和系统性炎性反应.中性粒细胞(polymorphonuclear granulocyte,PMN)在调控系统性炎症反应中起重要作用.β-羟基丁酸(β-hydroxybutyric acid,BHBA)可能通过增强PMN黏附提高其促炎能力,进而导致系统性炎性反应的发生,然而其调控机制尚不清楚.本研究根据血清BHBA水平筛选出对照组(CON,n=15,BHBA＜0.6 mmol/L,Glu＞3.0 mmol/L)和 SCK 组(n=15,1.2 mmol/L＜BHB＜3.0 mmol/L,Glu＜2.8 mmol/L).奶牛血液免疫生化指标结果显示:SCK奶牛血清白介素 1β(interleukin-1,IL-1β)、白介素6(interleukin-6,IL-6)、肿瘤坏死因子(tumor necrosis factor,TNF-a)、触珠蛋白(haptoglobin,HP)和血清淀粉样蛋白(serum amyloid A,SAA)的浓度均显著增加.体外使用BHBA刺激健康奶牛分离的PMN显著增强了其黏附能力,显著增加了黏附相关分子淋巴细胞功能相关抗原-1(lymphocyte function association antigen-1,CD11a)和巨噬细胞功能相关抗原-1(macrophage-1,CD11b)的蛋白丰度;显著增加了炎性因子TNF-a、IL-1β和IL-6的mRNA丰度.以上数据表明,BHBA能够增加奶牛PMN黏附相关分子的表达,进而增强其黏附能力和促炎功能,为SCK奶牛的系统性炎性反应的发生和发展机制提供了新的理论解释.

酮病是围产期奶牛常见的代谢紊乱性疾病,其伴有系统性炎性反应.中性粒细胞嗜天青颗粒(azurophil gran-ules,AGs)含有大量促炎介质,其过度释放可能与系统性炎性反应有关.根据血清中β-羟基丁酸的水平,将30头多产奶牛(产后 3 周内)分为对照组(Control,n=15,BHB＜1.2 mmol/L)和酮病组(Ketosis,n=15,BHB＞3 mmol/L).检测血清炎性介质发现,与对照组奶牛相比,酮病奶牛血清中结合珠蛋白(HP)、血清淀粉样蛋白A(SAA)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)和肿瘤坏死因子-α(TNF-α)水平均显著升高;酮病奶牛血清AGs标记蛋白髓过氧化物酶(MPO)水平显著升高,且MPO与上述促炎介质水平呈强正相关性;从酮病奶牛分离的中性粒细胞(PMN)中MPO的蛋白含量显著降低,而血液中CH138A+/CD63high(脱颗粒的PMN)细胞比例升高、胞膜CD63的平均荧光强度增强,进一步证实酮病奶牛AGs的脱颗粒水平升高.总之,本研究发现酮病奶牛AGs脱颗粒水平升高与系统性炎性反应的发生相关,为酮病奶牛系统性炎性反应的防治提供了理论基础.

Ketosis occurs most frequently in the peripartal period and is associated with liver injury and steatosis. Lysosomes serve as the terminal degradative station and contribute to liver homeostasis through their role in the digestion of dysfunctional organelles and lipid droplets. Transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function. Thus, the objective of the present study was to investigate the status of lysosomal function and TFEB transcriptional activity and potential changes in abundance of upstream effectors of TFEB identified in nonruminants, including mechanistic target of rapamycin kinase complex 1 (mTORC1), protein kinase B (Akt), glycogen synthase kinase β (GSK3β), and extracellular signal-regulated kinase1/2 (ERK1/2), and to explore which factor induces the above changes. Liver and blood samples were collected from healthy cows (n = 10) and ketotic cows (n = 10) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9 d). Calf hepatocytes were isolated from Holstein calves and treated with 10 ng/mL growth hormone (GH), 3.0 mM β-hydroxybutyrate (BHB), 1.5 ng/mL interleukin-18 (IL-18), 0.15 ng/mL tumor necrosis factor-α (TNF-α), or 1.2 mM free fatty acid (FFA) for 12 h. Serum levels of FFA and activities of alanine aminotransferase and aspartate aminotransferase were greater in ketotic cows, whereas glucose was lower. Additionally, ketotic dairy cows exhibited higher serum concentrations of GH, IL-18, and TNF-α, and lower serum concentration of insulin. The lower protein abundance of lysosome-associated membrane protein 1 (LAMP1) and mRNA abundance of LAMP1 indicated that hepatic lysosomal mass was lower in ketotic cows. Furthermore, lower protein abundance of cathepsin D (CTSD) and mRNA abundance of CTSD and V0 domain of the vacuolar ATPase along with lower activity of β-N-acetylglucosaminidase indicated impairment in hepatic lysosomal function due to ketosis. The lower nuclear abundance, total protein, and mRNA abundance of TFEB and peroxisome proliferator-activated receptor γ coactivator 1 α along with greater phosphorylated (p)-TFEB in the liver of ketotic cows indicated an impairment of hepatic TFEB transcriptional activity. The protein abundances of phosphorylated mTOR (p-mTOR) and its downstream effectors ribosomal protein S6 kinase B (RPS6KB) and eukaryotic factor 4E-binding protein 1 (EIF4EBP1) were greater, whereas p-Akt, p-GSK3β, and p-ERK1/2 were lower in the liver of ketotic cows. Importantly, elevated phosphorylation of mTOR, RPS6KB, and EIF4EBP1 was observed in calf hepatocytes treated with GH, BHB, IL-18, TNF-α, and FFA. Moreover, BHB, TNF-α, and FFA, not GH and IL-18, reduced TFEB transcriptional activity and impaired lysosomal function in calf hepatocytes. Taken together, these data suggest that BHB, TNF-α, and FFA overactivate the hepatic mTORC1 signaling pathway during ketosis and further impaired TFEB transcriptional activity and lysosomal function, which may contribute to liver injury and steatosis.

酮病是高产奶牛常发的一种以高非酯化脂肪酸(non-esterified fatty acids,NEFA)为病理学特征的能量代谢障碍疾病.酮病奶牛脂解产生的大量NEFA被乳腺上皮细胞(bovine mammary epithelial cell,BMEC)所吸收,而高浓度的NEFA具有显著的细胞毒性,这可能与乳腺的非感染性炎性水平显著升高有关,但内在机制尚未明确.本研究利用BMEC进行体外试验,探讨高浓度NEFA对BMEC炎性通路的影响和作用机制.体外培养BMEC,添加不同浓度的NEFA和shIRE1α,通过免疫荧光法检测细胞角蛋白-18(CK-18)鉴定分离的BMEC,采用Western blot和qRT-PCR检测内质网应激关键分子和NF-κB炎性信号通路关键分子的基因转录水平和蛋白表达水平.结果显示,高NEFA可以显著增加IRE1α的磷酸化水平和葡萄糖调节蛋白(glucose regulated protein 78,GRP78)的表达,增加p65的磷酸化水平和下游炎性因子肿瘤坏死因子α(TNF-α)、白介素-6(IL-6)和白介素 1β(1L-1β)的mRNA表达水平.shIRE1α转染BMEC后,显著降低IRE1α的磷酸化水平,且沉默IRE1α能显著逆转高NEFA对BMEC的NF-κB信号通路的活化,使p65的磷酸化水平显著降低,且显著降低其下游炎性因子TNF-α、IL-6和IL-1β的mRNA表达水平.结果表明,NEFA能够诱导BMEC的内质网应激,并通过IRE通路激活NF-κB信号通路促进BMEC炎性因子的表达,增加乳腺非感染性炎性水平,本研究为进一步探究酮病奶牛乳腺损伤的分子机制奠定了基础.

通过比较工、Ⅱ型酮病奶牛血液中炎性因子和肝损伤等相关指标的差异,为奶牛不同类型酮病的诊断和防治提供数据支持.本研究选取Ⅰ、Ⅱ型酮病奶牛和健康奶牛各20头,采血后分离血浆或血清,分别检测炎性以及肝损伤相关指标.结果 显示,Ⅰ、Ⅱ型酮病奶牛血液非酯化脂肪酸(NEFA)含量,促炎因子如白介素-1(IL-1)、白介素-6(IL-6)以及肿瘤坏死因子α(TNF-α)的含量均显著高于健康奶牛,且Ⅱ型酮病奶牛血液促炎因子含量高于工型酮病奶牛.同时,工、Ⅱ型酮病奶牛中肝损伤标志物丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)的活性也显著高于健康奶牛,且Ⅱ型酮病奶牛ALT和AST的酶活显著高于Ⅰ型酮病奶牛.结果 表明,Ⅰ、Ⅱ型酮病奶牛均存在系统性炎症和肝损伤,且Ⅱ型酮病奶牛更加严重,本试验为奶牛酮病的综合防治奠定理论基础.

目的 研究微小RNA-124(microRNA-124,miR-124)对癫痫神经元Apelin(APLN)表达的调控作用及其对神经元损伤的影响.方法 采用低镁细胞外液培养建立癫痫神经元模型.双荧光素酶报告基因系统验证miR-124与APLN的靶标关系,实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测miR-124与APLN mRNA的表达,western blotting检测APLN、Bax、Bcl-2与caspase 3蛋白的表达,流式细胞仪检测神经元凋亡情况.结果 miR-124 mimics+APLN基因野生型组荧光素酶活性明显低于对照组,但尚未达到统计学差异.qRT-PCR与western blotting检测均提示上调miR-124表达可促进APLN表达,下调miR-124可抑制APLN mRNA表达.流式细胞仪检测发现,上调miR-124表达可促进癫痫神经元凋亡,其机制为上调促凋亡蛋白Bax、caspase 3表达,下调抗凋亡蛋白Bcl-2表达.结论 本研究结果提示miR-124与APLN基因可能存在靶标关系,可能正调控APLN表达;除APLN基因外,miR-124可能通过调控其他基因表达,最终表现为促进癫痫海马神经元凋亡.

亚急性瘤胃酸中毒(subacute rumen acidosis,SARA)奶牛存在系统性炎性反应,其分子机制尚不清楚.本研究通过检测健康奶牛(n=15)和SARA奶牛(n=15)外周血免疫生化指标、外周血中性粒细胞(PMN)黏附能力和自噬流的变化,以及体外病理浓度组胺(7 μmol/L)对健康奶牛分离培养的PMN黏附和自噬流的影响,探明SARA奶牛高组胺血症对PMN黏附的影响及其分子机制.结果 显示,SARA奶牛与健康奶牛相比平均日产奶量下降14.3％;SARA奶牛血清中脂多糖(LPS)的含量从不可检测升高到0.22 EU/mL左右,而组胺浓度从平均1.12 μmol/L增加到6.32 μmol/L;血清急性期反应蛋白内毒素结合蛋白(LBP)、触珠蛋白(Hp)和血清淀粉样蛋白(SAA)的浓度均有显著性增加,促炎因子肿瘤坏死因子(TNF-α)、白介素6(IL-6)和白介素1β(IL-1β)的平均浓度也有明显升高;免疫印迹结果显示,自噬标记分子微管相关蛋白1轻链3-β(LC3-Ⅱ)和自噬降解底物P62蛋白水平增多,表明自噬流受阻;黏附试验显示SARA奶牛PMN黏附能力增强,且SARA奶牛PMN表面主要黏附分子白细胞功能相关抗原-1(CD11a)表达增加了约0.5倍,而巨噬细胞功能相关抗原-1(CD11b)表达增加了约3倍,表明SARA奶牛PMN自噬流抑制会导致黏附增强;体外结果显示,组胺可以导致自噬流阻断以及PMN黏附能力的增强,说明SARA奶牛PMN黏附增强主要由高组胺血症引起.结果 表明,SARA奶牛高组胺血症可以通过抑制自噬流引起中性粒细胞黏附增强,进而导致系统性炎性反应.

Susceptibility to mastitis is highest during the peripartal (transition) period and is often concomitant with other comorbidities such as ketosis. Although infection with pathogenic microorganisms and immune-dysfunction around calving clearly play key roles in mastitis development, other metabolic factors also contribute. Sirtuin 3 (SIRT3), a mitochondrial deacetylase regulating energy and redox homeostasis, antagonizes the lipotoxic effects of nonesterified fatty acids (NEFA). Thus, we hypothesized that increases in circulating NEFA concentrations, as observed in the transition period, provokes inflammatory responses that can be reversed via activation of SIRT3. Here we aimed to study (1) proinflammatory NF-κB signaling and SIRT3 abundance in mammary tissue of ketotic cows and healthy controls, and (2) the effect of SIRT3 on NF-κB activation in bovine mammary epithelial cells (BMEC) treated with high levels of NEFA. The mammary gland biopsy samples were from a previous study, which included 15 healthy cows and 15 ketotic cows. Primary BMEC were isolated from 3 healthy Holstein cows with collagenase III digestion. Purified BMEC were incubated with or without SIRT3 overexpression adenovirus for 48 h, then treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 24 h. Mammary tissue of ketotic cows was associated with lower protein abundance of SIRT3 along with greater NF-κB P65 phosphorylation levels (p-NF-κB P65), p-NF-κB P65:NF-κB P65 ratio, and mRNA abundance of IL1B and IL6. In BMEC, exogenous NEFA dose-dependently reduced protein abundance of SIRT3, but increased p-NF-κB P65, p-NF-κB P65:NF-κB P65 ratio, and mRNA abundance of IL1B and IL6. Compared with green fluorescent protein adenovirus vector + NEFA, overexpression of SIRT3 in NEFA-treated BMEC downregulated p-NF-κB P65 and mRNA abundance of IL1B and IL6. Immunofluorescence indicated that overexpression of SIRT3 inhibited nuclear translocation of NF-κB P65. Overall, our data demonstrated that ketosis is associated with a reduction in SIRT3 abundance and activation of NF-κB signaling in the mammary gland. In vitro data provided evidence that high NEFA concentrations inhibit SIRT3, which contributes to enhanced NF-κB signaling including nuclear translocation and a pro-inflammatory response. The data suggest a promising role of SIRT3 as a target for helping alleviate localized inflammation of the mammary gland resulting from exposure to high concentrations of NEFA.

选择15头健康奶牛与15头酮病奶牛,采集血液与脂肪组织.检测两组奶牛血清中非酯化脂肪酸(nonestester-ified fatty acid,NEFA)、β-羟基丁酸(BHBA)、葡萄糖(glucose)含量.利用Western blot检测脂肪组织中脂解酶HSL的磷酸化水平以及ATGL蛋白表达,胰岛素信号通路关键分子IR和Akt的磷酸化水平;利用qRT-PCR检测脂肪组织中TNF-α和IL-1β的mRNA表达水平.随后从1日龄健康荷斯坦奶牛腹膜网膜和肠系膜脂肪组织分离培养原代脂肪细胞,在分化成熟的脂肪细胞中添加0、0.1、1.0、10.0 μg/L的肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)处理3 h,检测IR和Akt的磷酸化水平以及培养基上清中甘油(GC)含量.结果显示,与健康奶牛相比,酮病奶牛脂肪组织HSL磷酸化水平增加、ATGL蛋白表达量增加、IR和Akt的磷酸化水平降低,炎性因子TNF-α和IL-1β mRNA表达水平明显升高.此外,体外添加TNF-α显著增加HSL的磷酸化水平、ATGL的蛋白表达和培养基上清中GC含量,显著抑制IR和Akt的磷酸化水平.结果表明,TNF-α导致酮病奶牛脂肪组织脂解加剧并降低胰岛素敏感性.

Innate immune suppression and high blood fatty acid levels are the pathological basis of multiple metabolic diseases. Neutrophil vacuolation is an indicator of the immune status of patients, which is associated with autophagy-dependent granule degradation. Vacuolated neutrophils are observed in ethanol toxicity and septicemia patients due to the changes in their blood constituents, but how about the neutrophils in nonalcoholic fatty liver disease (NAFLD) patient is unknown. Here, we confirmed that an adhesion deficiency and an increased autophagy level existed in NAFLD neutrophils, and the three neutrophil granule subunits, namely, the azurophil granules, specific granules and gelatinase granules, could be engulfed by autophagosomes for degradation, and these autophagy-triggered granule degradation events were associated with vacuolation in palmitic acid (PA)-treated and NAFLD neutrophils. Concordantly, the adhesion-associated molecules CD11a, CD11b, CD18 and Rap1 on the three granule subunits were degraded during PA induced autophagy. Moreover, the cytosolic CD11a, CD11b, CD18 and Rap1 were targeted by Hsc70 and then delivered to lysosomal-like granules for degradation. Notably, in vitro and ex vivo , PA induced autophagy by inhibiting the p-PKCα/PKD2 pathway. Overall, we showed that high blood PA level inhibited the p-PKCα/PKD2 pathway to induce NAFLD neutrophil autophagy, which promoted the degradation of CD11a, CD11b, CD18 and Rap1 and further decreased the adhesion of neutrophils, thereby impairing the neutrophil function of NAFLD patients. This theory provides a new therapeutic strategy to improve the immune deficiency in NAFLD patients. Visual Abstract Key Points Vacuolation and adhesion deficiency of NAFLD neutrophils are associated with autophagy-dependent granule degradation PA inhibits p-PKCα/PKD2 to induce autophagy, which induces the degradation of CD11a, CD11b, CD18 and Rap1 and decreases neutrophil adhesion

为研究线粒体融合蛋白2(mitofusin 2,MFN2)对高β-羟丁酸(β-hydroxybutyrate,BHBA)活化NF-κB炎性通路的影响,体外培养奶牛肝细胞,添加不同浓度(0.0,1.2,2.4,4.8 mmol/L)的BHBA,并转染过表达MFN2的腺病毒,运用Western blot和qRT-PCR技术检测NF-κB炎性通路关键分子的基因和蛋白表达.结果显示,随着BHBA浓度的增加,IKBα和NF-κBp65的磷酸化水平以及IL-1β、IL-6和TNFα的mRNA表达均显著升高,MFN2的基因和蛋白表达水平则显著降低;过表达MFN2后,显著抑制了高BHBA诱导的IKBα和NF-κB p65磷酸化水平及IL-lβ、IL-6、TNFα mRNA表达水平的升高.结果表明,在奶牛肝细胞中过表达MFN2可以显著抑制高BHBA活化的NF-κB炎性通路.

[This corrects the article DOI: 10.3389/fcell.2020.00245.].

Background and Purpose Identifying safe and effective compounds that target to mitophagy to eliminate impaired mitochondria may be an attractive therapeutic strategy for non-alcoholic fatty liver disease. Here, we investigated the effects of cyanidin-3-O-glucoside (C3G) on non-alcoholic fatty liver disease (NAFLD) and the underlying mechanism. Experimental Approach Non-alcoholic fatty liver disease was induced by a high-fat diet for 16 weeks. C3G was administered during the last 4 weeks.In vivo, recombinant adenoviruses and AAV8 were used for overexpression and knockdown of PTEN-induced kinase 1 (PINK1), respectively. AML-12 and HepG2 cells were used for the mechanism study. Key Results C3G administration suppressed hepatic oxidative stress, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and steatosis and improved systemic glucose metabolism in mice with NAFLD. These effects of C3G were also observed in palmitic acid-treated AML-12 cells and hepatocytes from NAFLD patients. Mechanistic investigations revealed that C3G increased PINK1/Parkin expression and mitochondrial localization and promoted PINK1-mediated mitophagy to clear damaged mitochondria. Knockdown of hepatic PINK1 abolished the mitophagy-inducing effect of C3G, which blunted the beneficial effects of C3G on oxidative stress, NLRP3 inflammasome activation, hepatic steatosis and glucose metabolism. Conclusion and Implications These results demonstrate that PINK1-mediated mitophagy plays an essential role in the ability of C3G to alleviate NAFLD and suggest that C3G may be a potential drug candidate for NAFLD treatment.