BACKGROUND: Peste des petits ruminants (PPR) is a contagious and fatal disease of sheep and goats. PPR virus (PPRV) infection induces endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The activation of UPR signaling pathways and their impact on apoptosis and virus replication remains controversial.
OBJECTIVE: To investigate the role of PPRV-induced ER stress and the IRE1-XBP1 and IRE1-JNK pathways and their impact on apoptosis and virus replication.
METHODS: The cell viability and virus replication were assessed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, immunofluorescence assay, and Western blot. The expression of ER stress biomarker GRP78, IRE1, and its downstream molecules, PPRV-N protein, and apoptosis-related proteins was detected by Western blot and quantitative reverse transcription-polymerase chain reaction, respectively. 4-Phenylbutyric acid (4-PBA) and STF-083010 were respectively used to inhibit ER stress and IRE1 signaling pathway.
RESULTS: The expression of GRP78, IRE1α, p-IRE1α, XBP1s, JNK, p-JNK, caspase-3, caspase-9, Bax and PPRV-N were significantly up-regulated in PPRV-infected cells, the expression of Bcl-2 was significantly down-regulated. Due to 4-PBA treatment, the expression of GRP78, p-IRE1α, XBP1s, p-JNK, caspase-3, caspase-9, Bax, and PPRV-N were significantly down-regulated, the expression of Bcl-2 was significantly up-regulated. Moreover, in PPRV-infected cells, the expression of p-IRE1α, p-JNK, Bax, and PPRV-N was significantly decreased, and the expression of Bcl-2 was increased in the presence of STF-083010.
CONCLUSIONS: PPRV infection induces ER stress and IRE1 activation, resulting in apoptosis and enhancement of virus replication through IRE1-XBP1s and IRE1-JNK pathways.

Objective: To establish a method for the determination of n-butylamine in the air of the workplace by ion chromatography. Methods: In February 2022, on-site sampling was carried out using an atmospheric sampler. N-butylamine was adsorbed by a neutral silica gel tube and then performed for qualitative and quantitative determination by ion chromatography after ultrasonic desorption with 10 mmol/L sulfuric acid solution. Results: The linear range of the method was 0.0375-100.0 μg/ml, the linear equation of the standard curve was y=0.0713x-0.0327, the correlation coefficient was 0.9992. The detection limit of the method was 11.25 μg/L, and the lower limit of quantification was 37.50 μg/L, the lowest quantitative concentration was 0.025 mg/m(3) (in term of sampling 7.5 L). The average desorption efficiency of the method was 91.50%-95.38%, the precision was 1.10%-2.30%, the standard recovery was 83.83%-100.02%, sampling efficiency was 100.00%. Conclusion: This method is fast, sensitive and accurate, and can be used for the determination of n-butylamine in the air of workplace.
目的： 建立工作场所空气中正丁胺的离子色谱测定方法。 方法： 于2022年2月，利用大气采样器进行现场采样，正丁胺经中性硅胶管吸附，加10 mmol/L硫酸溶液超声解吸后由离子色谱法进行定性和定量测定。 结果： 该方法的线性范围为0.037 5~100.0 μg/ml，标准曲线线性方程为y=0.071 3x-0.032 7，相关系数为r=0.999 2；方法检出限为11.25 μg/L，定量下限为37.50 μg/L，最低定量浓度为0.025 mg/m(3)（以采样7.5 L计）；方法解吸效率为91.50%~95.38%，精密度为1.10%~2.30%，加标回收率为83.83%~100.02%，采样效率均为100.00%。 结论： 离子色谱法快速、灵敏、准确，可用于工作场所空气中正丁胺的测定。.

The antitumor effect of norcantharidin (NCTD) has been widely reported. However, whether NCTD can inhibit cervical cancer remains unknown. In the present study, it was shown that NCTD inhibited the viability of cervical cancer cells and caused cell cycle arrest in a concentration‑dependent manner. Further analysis revealed that the NCTD‑induced reduction in cell viability could be reversed by the inhibitor of apoptosis z‑VAD‑FMK and by the inhibitor of endoplasmic reticulum (ER) stress, 4‑phenylbutyric acid (4‑PBA). Additionally, NCTD led to the accumulation of reactive oxygen species as well as a decrease in the mitochondrial membrane potential in cervical cancer cells, whereas 4‑PBA pre‑treatment attenuated these alterations. In addition, NCTD increased the expression of the apoptosis‑related proteins Bip, activating transcription factor (ATF) 4 and C/EBP homologous protein in a concentration‑dependent manner. Moreover, NCTD significantly increased the expression of the ER stress‑related signaling molecules protein kinase R‑like ER kinase, inositol‑requiring enzyme 1 and ATF6, but 4‑PBA abolished these effects. In vivo experiments showed that NCTD significantly inhibited the growth of subcutaneous tumors in mice. Additionally, the expression of ER stress‑related molecules and apoptosis‑related proteins increased significantly after NCTD treatment. In conclusion, NCTD induces apoptosis by activating ER stress and ultimately curtails the progression of cervical cancer.

OBJECTIVE: To study the effects of different calcium ion concentrations on epithelial mesenchymal transformation (EMT) of human peritoneal mesothelial cell (HPMC) via endoplasmic reticulum stress (ERS).
METHODS: HPMC cell line HMrSV5 was cultured in vitro and treated in groups. The cells in the control group, high calcium group 1, and high calcium group 2 were treated with medium containing calcium ion concentrations of 1.25, 1.75, and 2.25 mmol/L, respectively. The solvent control group was treated with medium containing 1.25 mmol/L physiological calcium ion concentration and 0.1% dimethyl sulfoxide (DMSO), the high calcium+solvent group was treated with medium containing 2.25 mmol/L calcium ion concentration and 0.1% DMSO, the high calcium+4-phenylbutyric acid (4-PBA) group was treated with medium containing 2.25 mmol/L calcium ion concentration and 1 mmol/L ERS inhibitor 4-PBA, and each group was treated for 48 hours. Morphological changes of cells in each group were observed under light microscope. The expressions of epithelial cell phenotype marker zonula occluden-1 (ZO-1) and mesenchymal cell phenotype marker α-smooth muscle actin (α-SMA) in the cells were observed by immunofluorescence staining. The expressions of EMT marker genes E-cadherin, ZO-1, α-SMA and Vimentin were detected by fluorescence quantitative polymerase chain reaction (PCR). The expressions of ERS marker proteins phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), transcription activating factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by Western blotting.
RESULTS: Compared with the control group, the morphology of HMrSV5 cells became slender and fibrotic, the fluorescence intensity of ZO-1 increased, and the fluorescence intensity of α-SMA decreased in high calcium 1 and high calcium 2 groups, indicating that the cells transformed from epithelial cells to mesenchyme cells. The mRNA expressions of E-cadherin and ZO-1 were significantly decreased, while the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly increased, moreover, the expressions of the above marker genes or proteins in the high calcium 2 group was more obvious than those in the high calcium 1 group [E-cadherin mRNA (2-ΔΔCt): 0.53±0.05 vs. 0.75±0.09, ZO-1 mRNA (2-ΔΔCt): 0.42±0.06 vs. 0.69±0.06, α-SMA mRNA (2-ΔΔCt): 1.81±0.16 vs. 1.32±0.14, Vimentin mRNA (2-ΔΔCt): 2.05±0.22 vs. 1.48±0.16, p-PERK protein (p-PERK/β-actin): 0.81±0.09 vs. 0.59±0.06, p-eIF2α protein (p-eIF2α/β-actin): 0.87±0.10 vs. 0.50±0.06, ATF4 protein (ATF4/β-actin): 0.93±0.10 vs. 0.72±0.06, CHOP protein (CHOP/β-actin): 0.79±0.09 vs. 0.46±0.04, all P < 0.05]. Compared with the solvent control group, the morphological changes of cells, the expressions of EMT marker genes and ERS marker proteins after high calcium ion concentration of 2.25 mmol/L were consistent with those in the high calcium 2 group than control group. Compared with the high calcium+solvent group, the cell morphology recovered the characteristics of polygonal and pebble-like epithelial cells in the high calcium+4-PBA group, the fluorescence intensity of ZO-1 increased, the fluorescence intensity of α-SMA decreased, and the mRNA expressions of E-cadherin and ZO-1 in the cells were significantly increased [E-cadherin mRNA (2-ΔΔCt): 0.86±0.09 vs. 0.57±0.04, ZO-1 mRNA (2-ΔΔCt): 0.81±0.06 vs. 0.48±0.05, both P < 0.05], the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly decreased [α-SMA mRNA (2-ΔΔCt): 1.21±0.13 vs. 1.77±0.15, Vimentin mRNA (2-ΔΔCt): 1.30±0.14 vs. 1.94±0.20, p-PERK protein (p-PERK/β-actin): 0.38±0.04 vs. 0.92±0.11, p-eIF2α protein (p-eIF2α/β-actin): 0.34±0.05 vs. 1.05±0.13, ATF4 protein (ATF4/β-actin): 0.57±0.06 vs. 0.97±0.11, CHOP protein (CHOP/β-actin): 0.51±0.04 vs. 0.90±0.12, all P < 0.05].
CONCLUSIONS: High calcium ion concentrations of 1.75 mmol/L and 2.25 mmol/L promote EMT of HPMC via activating ERS.

BACKGROUND: Neuroinflammation assumes a pivotal role in both the etiological underpinnings and the dynamic progression of sepsis-associated encephalopathy (SAE). The occurrence of cognitive deficits with SAE is associated with neuroinflammation. 4-phenyl butyrate (4-PBA) may control inflammation by inhibiting endoplasmic reticulum stress (ERS). The primary objective of this investigation is to scrutinize the effectiveness of 4-PBA in mitigating neuroinflammation induced by lipopolysaccharides (LPS) and its consequent impact on cognitive function decline.
METHODS: LPS-injected mice with SAE and LPS-treated BV2 cell were established to serve as experimental paradigms, both contributing to the investigative framework of the study. Cognitive functions were assessed by behavioral tests. Hippocampal neuronal damage was assessed using Golgi staining and Nissl staining. Quantitative PCR assay and immunofluorescence were used to analyze neuroinflammation. Mitochondrial function was examined using transmission electron microscopy. Protein expression analysis was conducted through the application of western blotting methodology, serving as the investigative approach to elucidate molecular signatures in the experimental framework. Endoplasmic reticulum and mitochondrial calcium flow were detected using flow cytometry. To delve deeper into the mechanistic intricacies, the administration of 4μ8c was employed to selectively impede the IRE1α/Xbp1s pathway, constituting a strategic intervention aimed at elucidating underlying regulatory processes.
RESULTS: Expression levels of ERS-related proteins exhibited a significant upregulation in hippocampal tissues of LPS-treated mice when compared to wild-type (WT) counterparts. The administration of 4-PBA notably ameliorated memory deficits in LPS-treated mice. Furthermore, 4-PBA treatment was found to alleviate oxidative stress and neuroinflammation. Mechanistically, the IRE1α/Xbp1s-Ca2+ signaling pathway played a crucial role in mediating the beneficial effects of mitigating oxidative stress and maintaining mitochondrial calcium homeostasis, with inhibition of the IRE-related pathway displaying opposing effects.
CONCLUSIONS: Our results suggest that administration of 4-PBA treatment significantly attenuates ERS, alleviates cognitive decline, reduces inflammatory damage, and restores mitochondrial dynamics via the IRE1α/Xbp1s-Ca2+-associated pathway, which provides a new potential therapeutic approach to SAE.

Increasing evidence demonstrated that mitophagy and endoplasmic reticulum stress (ERS) was closely associated with memory decline in elderly type 2 diabetes mellitus (T2DM). Tea polyphenols (TP), an excellent natural antioxidant, has been reported to have neuroprotective properties in aging and diabetes, but the underlying mechanisms are still not fully understood. This study targets ERS-mitophagy in hippocampal neurons to investigate the improvement effect of memory in aged T2DM rats by TP. Rats were randomly divided into the control group, the aged group, the aged T2DM model group, the TP 75, 150, 300 mg/kg groups. TP 300 mg/kg ameliorated mitophagy by decreasing the levels of p-mTOR (S2448), P62 and HSP60 and increasing the levels of PINK1 and Parkin, the ratio of LC3Ⅱ/LC3Ⅰ, co-localization of LC3 and HSP60 and the number of autophagosomes and autolysosomes. TP 300 mg/kg attenuated ERS by downregulating the levels of p-PERK, p-eIF2α, ATF4, GRP78 and restoring the ER structure. To further verify epigallocatechin gallate (EGCG), which is the main active component of TP, enhanced mitophagy by inhibiting ERS, PC12 cells were pretreated with ERS activator tunicamycin (TM) or ERS inhibitor 4-phenylbutyric acid (4-PBA). The results showed that the improvement of mitophagy by EGCG was inhibited by TM and promoted by 4-PBA. Collectively, ERS-mitophagy in hippocampal neurons plays a key role in the improvement of memory by TP in aged T2DM rats. This study will provide a new perspective and strategy for the prevention of memory decline in elderly with T2DM.

This study analyzed the formation of goose fatty liver due to endoplasmic reticulum stress (ERS) caused by 3 types of sugar. Transcriptome analysis was performed for liver tissues from geese fed a traditional diet (maize flour), geese overfed with traditional diet, and geese overfed with diet supplemented with glucose, fructose, or sucrose. Correlation analysis of the liver tissue transcriptomes showed that differentially expressed genes (DEGs) involved in ERS were significantly negatively correlated with DEGs involved in inflammation response in the sucrose overfeeding group, and significantly positively correlated with the DEGs involved in lipid metabolism in fructose overfeeding group. Goose primary hepatocytes were isolated in vitro and then treated with glucose or fructose. Some were also treated with ERS inhibitor 4-phenylbutyric acid (4-PBA). In the hepatocytes, mRNA expression of X-Box Binding Protein 1 (XBP1), activating transcription factor 6 (AFT6) and glucose-regulated protein 78 (GRP78) genes increased in the two sugar groups (glucose and fructose), but were suppressed by adding 4-PBA. The mRNA expression data, protein kinase contents, and triglyceride (TG) and very low-density lipoprotein (VLDL) concentrations all suggest that ERS regulates lipid deposition induced by glucose and fructose via elevating lipid synthesis, inhibiting fatty acid oxidation, and decreasing lipid transportation. In conclusion, glucose, or fructose cause ERS and then ERS causes lipid deposition in goose primary hepatocytes. Three types of sugar cause lipid accumulation and then lipid accumulation prevents ERS during goose fatty liver formation, which suggests a potential mechanism protects goose livers from ERS. The different sugars may induce lipid deposition in different ways.

Colorectal cancer (CRC) is the main cause of cancer-associated death. Herein, we treated SW620 and HT-29 CRC cells with different curcumin concentrations, followed by treatment with the half maximal inhibitory concentration (IC50) curcumin/endoplasmic reticulum stress (ERS) inhibitor 4-phenyl butyric acid (4-PBA)/activating transcription factor 6 (ATF6) interference plasmid (si-ATF6). We detected cell proliferation/apoptosis, ATF6 cellular localization/nuclear translocation, ion concentration, ATF6 protein/apoptotic protein (Bax/Bcl-2/Cleaved Caspase-3) levels, and ERS-related proteins (glucose-regulated protein 78 [Grp78]/C/EBP homologous protein [CHOP]). We discovered inhibited cell proliferation/growth, enhanced cell apoptosis/(Bax/Bcl-2) ratio/Cleaved Caspase-3 levels/Ca2+ concentration in the cytoplasm/ERS-related protein (Grp78/CHOP) levels, and activated ERS following treatment with IC50 curcumin. 4-PBA partially reversed the inhibitory effect of curcumin on SW620 cells by restraining ERS. Curcumin stimulated ATF6 expression and its nuclear translocation to activate ERS. ATF6 silencing partly annulled the inhibitory effect of curcumin on SW620 cells. Our study explored the molecular mechanism of curcumin affecting CRC cell apoptosis through ATF6.

Endoplasmic reticulum (ER) stress refers to a condition where the normal functioning of the ER is disrupted due to a variety of cellular stress factors. As a result, there is an accumulation of unfolded and misfolded proteins within the ER. Numerous studies have shown that ER stress can exacerbate inflammatory reactions and contribute to the development of various inflammatory diseases. However, the role of ER stress in the stability of atherosclerotic plaques remains poorly understood. In this study, we aimed to explore the potential impact of a specific ER stress inhibitor known as 4-phenyl butyric acid (4-PBA) on atherosclerosis in mice. The mice were fed a high-fat diet, and treatment with 4-PBA significantly improved the stability of the atherosclerotic plaques. This was evidenced by a reduction in oxidative stress and an increase in circadian locomotor output cycles kaput (CLOCK) protein and mRNA expression within the plaques. Additionally, 4-PBA reduced the expression of ER stress-related proteins and decreased apoptosis in the atherosclerotic plaques. In vitro investigation, we observed the effect of 4-PBA on vascular smooth muscle cells (VSMCs) that were exposed to oxidized low-density lipoprotein (ox-LDL), a significant contributor to the development of atherosclerosis. 4-PBA reduced reactive oxygen species (ROS) production and attenuated apoptosis, GRP78 and CHOP protein expression in ox-LDL-Induced VSMCs via up-regulating CLOCK expression. However, when the short hairpin RNA against CLOCK (sh-CLOCK) was introduced to the VSMCs, the protective effect of 4-PBA was abolished. This suggests that the up-regulation of CLOCK expression is crucial for the beneficial effects of 4-PBA on atherosclerotic plaque stability. This finding suggests that targeting ER stress and modulating CLOCK protein levels might be a promising way to enhance the stability of atherosclerotic plaques.

Fluoride can be widely ingested from the environment, and its excessive intake could result in adverse effects. Dental fluorosis is an early sign of fluoride toxicity which can cause esthetic and functional problems. Though apoptosis in ameloblasts is one of the potential mechanisms, the specific signal cascade is in-conclusive. High-throughput sequencing and molecular biological techniques were used in this study to explore the underlying pathogenesis of dental fluorosis, for its prevention and treatment. A fluorosis cell model was established. Viability and apoptosis rate of mouse ameloblast-derived cell line (LS8 cells) was measured using cell counting kit-8 (CCK-8) assay and flow cytometry analysis. Cells were harvested with or without 2-mM sodium fluoride (NaF) stimulation for high-throughput sequencing. Based on the sequencing data, subcellular structures, endoplasmic reticulum stress (ERS), and apoptosis related biomarkers were verified using transmission electron microscopy, quantitative real-time polymerase chain reaction, and Western blotting techniques. Expression of ERS markers, apoptosis related proteins, and enamel formation enzymes were detected using Western blotting after addition of 4-phenylbutyrate (4-PBA). NaF-inhibited LS8 cells displayed time- and dose- dependent viability. Additionally, apoptosis and morphological changes were observed. RNA-sequencing data showed that protein processing in endoplasmic reticulum was obviously affected. ERS and apoptosis were induced by excessive NaF. Downregulation of kallikrein-related peptidase 4 (KLK4) was also observed. Inhibition of ERS by 4-PBA rescued the apoptotic and functional protein changes in cells. Excessive fluoride induces apoptosis by activating ERS, which is mediated by GRP-78/PERK/CHOP signaling. Key proteinase is present in maturation-stage enamel; KLK4 was also affected by fluoride, but rescued by 4-PBA. This study presents a possibility for therapeutic strategies for dental fluorosis, while further exploration is required.