SCFAs对奶牛瘤胃上皮细胞Ca2+信号通路相关基因表达的影响

为探究短链脂肪酸(SCFAs)对奶牛瘤胃上皮细胞(BRECs)Ca~(2+)信号通路相关基因表达的影响,试验分为3个处理组,分别为野生型BRECs组,含20mmol/L SCFAs BRECs组,含20mmol/L SCFAs且通过CRISPR/Cas 9系统敲除GPR41基因的BRECs组,每个处理组3个重复,每组细胞均培养24h后,收集细胞提取总RNA,通过qRT-PCR对Ca~(2+)信号通路相关基因的mRNA表达量和细胞内Ca~(2+)浓度进行测定。结果表明,与野生型BRECs相比,添加20mmol/L SCFAs可极显著增加PLCB2的mRNA表达量(P0.01),显著增加IP3R1的mRNA表达量(P0.05),对PLCE1、PLCL1、PKCB和PKCG的mRNA表达量无显著差异(P0.05),可增加细胞内Ca~(2+)浓度但无显著差异(P0.05);敲除SCFAs的受体GPR41后,添加20 mmol/L SCFAs可显著降低IP3R1的mRNA表达量(P0.05),极显著上调PLCE1和PLCB2的mRNA表达量(P0.01),但对PLCL1、PKCB和PKCG的mRNA表达量无显著影响(P0.05),细胞内Ca~(2+)浓度有降低趋势但无显著差异(P0.05)。综上,SCFAs可以通过激活其受体GPR41来调控BRECs内Ca~(2+)信号通路中相关基因的表达和细胞内Ca~(2+)的释放。     

蜂胶对细菌脂多糖刺激下奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接蛋白的影响

本试验旨在研究中国蜂胶乙醇提取物（ethanol extract of Chinese propolis，EECP）对细菌脂多糖（lipopolysaccharide，LPS）刺激下体外培养奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接渗透性的影响。EECP中总酚酸和总黄酮含量测定采用福林酚法和硝酸铝法，并建立LPS诱导奶牛乳腺上皮细胞（bovine mammary epithelial cells，MAC-T）炎症模型，采用CCK-8法测定EECP对MAC-T相对增殖率的影响，利用实时荧光定量PCR（RT-qPCR）评估EECP对LPS诱导的MAC-T细胞炎症相关因子（IL-6、IL-8、TNF-α和IL-1β）相对mRNA转录水平；以及对紧密连接蛋白（occludin、ZO-1）相对mRNA转录水平进行检测，并进一步利用免疫荧光技术对紧密连接膜蛋白进行定位，确定EECP对LPS诱导MAC-T细胞炎症紧密连接渗透性的影响。结果显示：EECP中总酚酸含量为106.35 mg没食子酸当量（GAE）·g^-1、总黄酮含量为320.85 mg芦丁当量（RE）·g^-1；CCK-8结果显示EECP的安全浓度为0~15 μg·mL^-1，并可有效提高LPS刺激下MAC-T的活力；LPS刺激显著增加了细胞炎症相关因子IL-6、IL-8、TNF-α和IL-1β mRNA的转录量（P<0.001）；但2.5~15.0 μg·mL^-1 EECP预处理显著降低了IL-6、IL-8、TNF-α和IL-1β mRNA的转录量；与此类似，LPS刺激显著抑制了紧密连接蛋白基因（occludin、ZO-1）mRNA的转录量（P<0.01），而EECP预处理后紧密连接蛋白基因（occludin和ZO-1）mRNA的转录量显著增加（P<0.05）；免疫荧光染色试验也证实EECP能通过上调紧密连接蛋白（occludin、ZO-1）的表达，缓解LPS诱导的乳腺上皮细胞屏障功能紊乱。该结果证实，EECP对细菌脂多糖诱导奶牛乳腺上皮细胞炎症具有良好的保护作用，这为利用中国蜂胶预防奶牛乳腺炎提供了试验基础。     

Outer membrane protein A of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in RAW264.7 cells

AIM:To analyze the effects of outer membrane protein A (OmpA) from Acinetobacter baumannii ATCC 19606 on the autophagy of RAW264.7 cells. METHODS:The RAW264.7 cell model stimulated by OmpA was established. The effects of OmpA on the autophagy of RAW264.7 cells were detected by immunofluorescence, Western blot and transmission electron microscopy. RESULTS:The OmpA increased the expression of LC3B-Ⅱ and reduced the phosphorylation levels of Akt, mTOR and p70S6K. Rapamycin further reduced the phosphorylation levels of mTOR and p-70S6K, and increased the expression of LC3B-Ⅱ induced by OmpA. CONCLUSION:The OmpA of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in the RAW264.7 cells. This work provides a basis for further research on the molecular mechanism of autophagy induced by Acinetobacter baumannii to find a new method against the infection of Acinetobacter baumannii.     

Surplus spermine does not protect salmon cells against lipopolysaccharide (LPS)‐induced stress,but excess spermine is eliminated through spermidine/spermine N1‐acetyltransferase (SSAT)

The present trials tested the efficiency of surplus spermine to reduce inflammation and oxidative stress following LPS‐induced stress using an in vitro model of head kidney and liver cells isolated from Atlantic salmon. Spermine did not protect cells from LPS‐induced inflammatory response at either 0.3, 0.6 or 0.9 mM. However, as the gene expression of spermidine/spermine N1‐acetyltransferase (SSAT) increased with increasing spermine concentration, we addressed possible oxidative effects of the increased SSAT using its activator DENSPM or inhibitor of polyamine oxidation of the acetylated polyamines using MDL72527 at a spermine concentration of 0.6 mM. There was no significant effect of DENSPM, but MDL72527 decreased gene expression of GPX‐3 (p = .04), while gene expression of catalase and MnSOD was unaffected by treatment (p = .30 and p = .48, respectively). In conclusion, spermine did not protect cells from LPS‐provoked inflammation. The higher the spermine concentration, the more SSAT producing acetylated spermine occurred. Inhibiting the acetylated polyamine oxidases by MDL72527 improved oxidation status as expected due to a lower endogenous production of H₂O₂ by polyamine and acetylated polyamine oxidases. Probably care should be taken using polyamines or arginine as functional ingredients to avoid any increased oxidation within cells.     

Characterization and expression of SLO1 potassium channels during spermatogenesis in Eriocheir sinensis

SLO1 potassium channels are pivotal to many aspects of spermatogenic cell. Experiments were conducted to assess physiology and function of SLO1 potassium channels in different developmental stages of spermatogenic cell in Eriocheir sinensis. First, the expression of SLO1 protein was examined via Western blot, RT‐PCR, immunohistochemistry and immunofluorescence assays. The results showed that the expression of the SLO1 protein was not uniform in the spermatogenic cells of E. sinensis. Second, whole‐cell patch clamp technique was used to record the potassium current of spermatogenic cells and to analyse the electrophysiological characteristics of the potassium channels with the aid of inhibitors. It is proved that the potassium current in E. sinensis germ cells is associated with intracellular Ca²⁺, and the calcium‐activated potassium channel mediated by SLO1 protein is mainly large‐conductance Ca²⁺‐activated K⁺ channels (BK_Ca). Based on these researches, the cDNA of SLO1 from testis was cloned and sequenced. The SLO1 protein contained domains bound to calcium ions, and the spatial structure formed by its tetramers constituted potassium channels. Phylogenetic analysis revealed that SLO1 was much closer to Scylla paramamosain than other examined species. Finally, iberiotoxin (IbTX) and CdCl₂ were used to inhibit the acrosome reaction (AR) induced by A23187 and to explore the role of SLO1 potassium channels in the AR of E. sinensis. The experimental results showed that SLO1 potassium channels were expressed in E. sinensis spermatogenic cells and played an important role in the AR of crab sperm (SP).     

New angiotensin-converting enzyme inhibitory peptide from Coix prolamin and its influence on the gene expression of renin-angiotensin system in vein endothelial cells

Coix seed, which is a traditional Chinese medicine, has been used to treat hypertension for thousands of years. It has been shown that Coix prolamin peptides display high levels of angiotensin I converting enzyme (ACE) inhibitory activity. Hence, we purified the ACE inhibitory peptides from Coix prolamin hydrolysates and evaluated the influence of the most potent peptide on the renin-angiotensin system (RAS) genes expression in human umbilical vein endothelial cells (HUVECs). In this study, Coix prolamin peptides were sequentially separated by ultrafiltration, ion exchange chromatography, gel filtration chromatography and RP-HPLC, while the peptide structure was analyzed by mass spectrometry. Next, in silico proteolysis, pharmacophore and molecular docking were further applied to screen and optimize the structure of peptides. Finally, a novel ACE inhibitory peptide VDMF was obtained, in which its influence on the gene expression of RAS signaling pathway in AngⅡ-injury HUVECs was evaluated by quantitative real-time PCR. VDMF significantly down-regulated ACE, AngII type 1 receptor (AT1R) and ACE2 mRNA expression in comparation with model group, while up-regulating Mas gene expression. Hence, we obtained a novel antihypertensive candidate that was derived from the Coix peptides, which could involve a multi-modulation mechanism that regulates blood pressure.     

Seaweed polysaccharide mitigates intestinal barrier dysfunction induced by enterotoxigenic Escherichia coli through NF-κB pathway suppression in porcine intestinal epithelial cells

This study aimed to investigate the protective effects and underlying mechanism of seaweed polysaccharide (SWP) on intestinal epithelial barrier dysfunction induced by E. coli in an IPEC-J2 model. A preliminary study was done to screen optimum SWP concentrations by cell viability, cytotoxicity, apoptosis and proliferation evaluation. The regular study was conducted to evaluate the protective effects of SWP against E. coli challenge via the analysis of transepithelial electrical resistance (TEER), tight junction proteins, NF-κB signalling pathway, proinflammatory cytokines and the E. coli adhesion and invasion. Our results show that 4 h E. coli challenge down-regulated tight junction proteins expression, decreased TEER, activated NF-κB signalling pathway and increased proinflammatory response, which indicates that the E. coli infection model was well-established. Pre-treatment with 240 μg/ml SWP for 24 h alleviated the 4 h E. coli -induced intestinal epithelial barrier dysfunction, as evidenced by the up-regulated expression of Occludin, Claudin-1 and ZO-1 at both mRNA and protein level and the increased TEER of IPEC-J2 cells. Pre-incubation with 240 μg/ml SWP for 24 h inhibited the activation of the NF-κB signalling pathway by 4 h E. coli challenge, including the decreased mRNA expression of TLR-4, MyD88, IκBα, p-65, as well as the reduced ratio of protein expression of p-p65/p65. Also, pre-treatment with 240 μg/ml SWP for 24 h decreased proinflammatory response (IL-6 and TNF-α) induced by 4 h E. coli challenge and decreased the E. coli adhesion and invasion. In conclusion, SWP mitigated intestinal barrier dysfunction caused by E. coli through NF-κB pathway in IPEC-J2 cells and 240 μg/ml SWP exhibited better effect. Our results also provide a fundamental basis for SWP in reducing post-weaning diarrhoea of weaned piglets, especially under E. coli -infected or in-feed antibiotic-free conditions.     

Soluble Klotho protein suppresses THP-1-derived foam cell formation

AIMTo investigate the role of soluble Klotho protein in THP-1-derived foam cell formation. METHODSTHP-1 monocytes were induced into macrophages by treatment with 160 nmol/L phorbol myristate acetate for 48 h, and then were divided into 6 groups: negative control group (THP-1-derived macrophages), positive control group [THP-1-derived foam cells induced by oxidized low-density lipoprotein (ox-LDL) for 48 h], and 25, 50, 100 and 200 μg/L soluble Klotho protein groups (THP-1-derived macrophages pretreated with soluble Klotho protein at the indicat?ed concentraions for 2 h and then induced by ox-LDL for 48 h). Lipid droplets in cytoplasm were observed by oil red O staining. The cholesterol outflow rate was detected by scintillation counting technique. The content of intracellular total cholesterol, free cholesterol and cholesterol ester was detected by enzyme fluorescence analysis. The expression of acyl-coenzyme A:cholesterol acyltransferase 1 （ACAT1） and ATP-binding cassette transport?er A1 (ABCA1) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTSOil red O staining and lipid mass quantification showed that THP-1-derived foam cell formation was dose-dependently suppressed by soluble Klotho protein. The cholesterol efflux rate of THP-1-derived foam cells was increased by soluble Klotho protein in a dose-dependent manner (P<0.05). In addition, soluble Klotho protein decreased the expression of ACAT1 and increased the expression of ABCA1 in a dose-dependent manner (P<0.05). CONCLUSION The soluble Klotho protein inhibits THP-1-derived foam cell formation in a dose-dependent manner by down-regulating the expression of ACAT1 and up-regulating the expression of ABCA1.     

Replication of neurovirulent equine herpesvirus type 1 (EHV-1) in CD172a+ monocytic cells

Equine herpesvirus type 1 (EHV-1) is responsible for respiratory disorders, abortion and myeloencephalopathy (EHM) in horses. Two pathotypes of EHV-1 strains are circulating in the field: neurovirulent (N) and non-neurovirulent (NN). For both strains, CD172a⁺ monocytic cells are one of the main carrier cells of EHV-1 during primary infection, allowing the virus to invade the horse’s body. Recently, we showed that EHV-1 NN strains showed a restricted and delayed replication in CD172a⁺ cells. Here we characterize the in vitro replication kinetics of two EHV-1 N strains in CD172a⁺ cells and investigate if the replication of these strains is similarly silenced as shown for EHV-1 NN strains. We found that EHV-1 N replication was restricted to 7–8% in CD172a⁺ cells compared to 100% in control RK-13 cells. EHV-1 N replication was not delayed in CD172a⁺ cells but virus production was significant lower (10^3.0 TCID₅₀/10⁵ inoculated cells) than in RK-13 cells (10^8.5 TCID₅₀/10⁵ inoculated cells). Approximately 0.04% of CD172a⁺ cells produced and transmitted infectious EHV-1 to neighbour cells compared to 65% of RK-13 cells. Unlike what we observed for the NN strain, pretreatment of CD172a⁺ cells with histone deacetylases inhibitors (HDACi) did not influence the replication of EHV-1 N strains in these cells. Overall, these results show that the EHV-1 replication of N strains in CD172a⁺ cells differs from that observed for NN strains, which may contribute to their different pathogeneses in vivo.