Chronically socially isolated mice exhibit depressive-like behavior regulated by the gut microbiota.

Objectives: Chronic loneliness is a widespread issue, and the gut-brain axis is known to be crucial in facilitating communication between the gut and brain. However, the precise mechanism by which chronic loneliness affects the gut-brain axis remains uncertain. Methods: Fourteen 55-week-old Balb/c mice were used in the experiment, with seven mice being randomly assigned to the chronic social isolation (CSI) group. The CSI group mice underwent 12 weeks of isolation to simulate the psychiatric state of a population in prolonged social isolation. The mental state of the CSI mice was assessed through animal behavior analysis, while plasma cytokines were measured using ELISA. Additionally, the composition of the gut microbiota was analyzed using 16S rRNA sequencing, and the metabolite composition of the intestinal contents was examined using nontargeted metabolomics. The Student-T test was used to determine significant mean differences. Results: Mice that were exposed to the CSI exhibited increased immobility time lengths in forced swimming and hanging tail experiments, and decreased movement lengths and number of times traversing the intermediate region, compared to control mice. Additionally, CSI decreased the abundance of the probiotics Ruminococcaceae, Akkermansiaceae, and Christensenellaceae. Additionally, CSI reduced the production of the metabolites oleamide and tryptophan. Furthermore, IL-1ß, IL-4, and IL-6 were significantly increased, while TNF-α was significantly decreased. Conclusion: CSI induces a dysbiotic gut microbiota and the production of neurorelated metabolites, which in turn increase inflammatory responses and result in depressive behaviors in CSI mice. Therefore, these findings suggest that the gut microbiota may serve as a target for the treatment of long-term social isolation-induced mental disorders.

A SILAC-based accurate quantification of shrimp allergen tropomyosin in complex food matrices using UPLC-MS/MS.

The carryover of trace allergens in complex food matrices poses challenges for detection techniques. Here, we demonstrate an accurate UPLC-MS/MS quantification assay for the shrimp allergen tropomyosin with a full-length isotope-labelled recombinant tropomyosin (TM-I) internal standard in complex food matrices. The TM-I, expressed based on the SILAC technique, exhibited a high isotope labelling ratio (>99%), purity, and alignment with the natural sequence. This method determined the tropomyosin ranging from 0.2 to 100 ng/mL. Mean recoveries ranged from 89 to 116%, with intra- and inter-day RSDs below 12%, for three signature peptides across three types of commercially processed food matrices. The limits of quantitation were 1 µg/g in pop food and sauce, and 10 µg/g in surimi product, respectively. This study supports the use of recombinant full-length isotope-labelled proteins rather than stable-isotope labelling peptides as internal standards to achieve more accurate quantitation of food allergens as the digestion error is corrected.

[Determination of 80 pesticide residues in milk using NH_2-MWNTs by liquid chromatography-time of flight mass spectrometry].

OBJECTIVE: A method for the determination of 80 pesticide residues in milk by liquid chromatography-time-of-flight mass spectrometry(LC-QTof-MS) was developed. METHODS: The target compounds in milk were extracted with acetonitrile-methanol(9∶1, V/V) containing 1% acetic acid, and purified by aminated multi-walled carbon nanotubes(NH_2-MWNTs). The chromatographic column was Waters Acquity UPLC BEH C_(18 )(100 mm×2.1 mm, 1.7 µm). The 80 pesticides were detected by liquid chromatography-time-of-flight mass spectrometry and quantified using an external standard method by matrix matched calibration curve. RESULTS: The purification method showed a good linearity(r~2≥ 0.99) over the concentration range from 5 to 100 µg/L for the 80 pesticides in this study. The limits of detection(LODs) and quantification(LOQs) of the 80 pesticides in milk ranged from 0.01 to 0.50 µg/L and 0.03 to 1.50 µg/L, respectively. The mean recoveries of the three spiked levels ranged from 71.5% to 116.9% with the relative standard deviation ranging from 1.2% to 18.1%, indicating that the accuracy and precision of the method were good. Among the milk samples, no residues of the 80 pesticides in this study were found after screening. CONCLUSION: The method has good linearity, good sensitivity, accuracy and precision and is suitable for the simultaneous and rapid determination of 80 pesticide residues in milk.

Continuous exposure to bisphenol S increases the accumulation of endogenous metabolic toxicants by obstructing the glucuronic acid pathway.

Uridine diphosphate glucuronic acid (UDPGA) is an essential substrate in the glucuronidation of exogenous and endogenous lipophilic compounds via the liver glucuronic acid pathway, and its synthesis depends on glucose and energy in the body. Bisphenol S (BPS), as a lipophilic environmental pollutant, has been widely utilized in the manufacturing of daily necessities. The biological effect of BPS in interference with liver energy metabolism might affect UDPGA synthesis and the excretion of lipophilic compounds, but this was not clearly revealed. Here, female zebrafish that were exposed to BPS for 35 days exhibited a significant decrease in UDPGA in the liver with significant accumulation of exogenous BPS and endogenous bilirubin in the body. One vital reason may be that the exposure to BPS for 35 days promoted the lipid formation through PPARg signaling and reduced energy levels in the liver, resulting in the decreased raw materials for UDPGA production in glucuronic acid pathway. Meanwhile, transcriptome analysis showed that BPS inhibited the mRNA expression levels of genes related to the glucuronic acid pathway. The accumulation of endogenous and exogenous lipophilic compounds can trigger a variety of toxicological effect. Thus, weakened liver detoxification might be the primary cause of the toxicological effects of lipophilic pollutants.

High-throughput multitarget quantitative assay to profile the whole grain-specific phytochemicals alkylresorcinols, benzoxazinoids and avenanthramides in whole grain and grain-based foods.

Currently, there is a growing interest in using whole grain (WG)-specific phytochemicals to perform WG research, including research on dietary assessment, health mechanisms, and quality control. However, the current approaches used for WG-specific phytochemical analysis cannot simultaneously achieve coverage, specificity, and sensitivity. In the present study, a series of WG-specific phytochemicals (alkylresorcinols (ARs), benzoxazinoids (BXs) and avenanthramides (AVAs)) were identified, and their mass spectrometry (MS) fragmentation mechanism was studied by TOF MS. Based on diagnostic fragmentation ions and retention time prediction models, a LC-MS/MS method was developed. Through this method, 56 ARs, 13 BXs, and 19 AVAs in WGs and grain-based foods were quantified for the first time. This method was validated and yielded excellent specificity, high sensitivity and negligible matrix effects. Finally, we established WG-specific phytochemical fingerprints in a variety of WG and grain-based foods. This method can be used for WG quality control and WG precision nutrition research.

Multi-immunoaffinity column for the simultaneous analysis of bisphenol A and its analogues in Chinese foods by liquid chromatography tandem mass spectrometry.

Bisphenol A (BPA) and its four analogues have been receiving considerable attention owing to their potential endocrine disrupting effects. The European Food Safety Authority has proposed 0.04 ng/kg·body weight/day of thetemporary tolerable daily intake for BPA. Therefore, a more sensitive analytical method was urgently needed for the necessity of the risk reassessment of bisphenols (BPs). The matrix effect of Chinese foods is a challenge for the analysis of ultra-trace analytes due to the presence of various spices. A multi-immunoaffinity column (mIAC) was prepared for the purification of BPA, BPB, BPF, BPS, and BPAF in Chinese foods following ultra-high-performance liquid chromatography tandem mass spectrometry detection (UHLPC-MS/MS). The recoveries of each of BPs were ranged from 84.6% to 116.7%, and the intra-day precision and inter-day precision were ranged from 1.6% to 12.4%, and from 4.1% to 14.0%, respectively. This is the first report on the mIACs for simultaneous clean-up and analysis of BPs in complex Chinese foods.

Occurrence and migration of organophosphite and organophosphate esters into food simulants from single-use food packaging in China.

Organophosphite antioxidants (OPAs) and organophosphate esters (OPEs) are used as additives in food packaging. Because these chemicals have been found in various foods, they have caused increasing concern about potential health risks through food intake. Little information is available about the migration behaviors of OPAs and OPEs from single-use food packaging into food. In the present study, four OPAs and 23 OPEs were analyzed in paper and plastic single-use food packaging (n = 312), which are widely used for take-out food in China. The total concentrations of OPAs and OPEs in the packaging samples were 1966 and 189 ng/g, respectively. Tris (2,4-di-tert-butylphenyl) phosphite (AO168) was the dominant compound. OPAs and OPEs were present at higher concentrations in the plastic packaging than in the paper packaging. In a migration test, four OPAs and 15 OPEs were found in food simulants (4% acetic acid, 10% ethanol, and hexane). Higher levels of individual and total OPAs were found in hexane than the other food simulants, especially for AO168 migration from plastic packaging. The amounts of OPEs in the food simulants increased from the aqueous simulants (4% acetic acid and 10% ethanol) to the fatty food simulant (hexane). The migration efficiencies of the OPAs were higher than those of the OPEs. Preliminary calculations suggest that dietary exposure to OPAs and OPEs because of migration will be low for the population in China.

Occurrence and Estimated Daily Intake of Cortisone and Cortisol in Aquatic Food from China TDS.

Glucocorticoids (GCs) widely exist in animal food including aquatic food. This study aimed to survey the occurrences of cortisone and cortisol in aquatic food and the estimated daily intake (EDI) of cortisone and cortisol due to different habits of aquatic food consumption. The mean levels of cortisone and cortisol in freshwater fish purchased from market were 14.59 µg/kg and 69.15 µg/kg, respectively, which were markedly higher than the levels in marine fish. A test using Zebrafish was performed to compare the concentration of GCs by different killing methods. The results suggested that physically traumatic killing methods are one of the reasons why the levels of GCs in freshwater fish were higher than those in marine fish. The concentrations of cortisone and cortisol in composite aquatic food samples from 12 provincial districts of the fourth China Total Diet Study (TDS) were 0.72~15.75 µg/kg and 4.90~66.13 µg/kg, respectively, which were positively correlated with the distance from the coastline. Further, the correlation coefficient between the levels of cortisone and cortisol in aquatic food and the percentages of freshwater fish consumption were 0.758 (p < 0.01) and 0.908 (p < 0.01), respectively. There was a significant positive correlation between the levels of cortisone and cortisol in aquatic food in the fourth TDS and the percentages of freshwater fish consumption. The calculated average EDIs of cortisone and cortisol from aquatic food in the fourth TDS were 0.16 µg/d and 0.72 µg/d, respectively.

Combination of magnetic beads extraction and ultraperformance liquid chromatography tandem mass spectrometry detection for the clinical diagnosis of allergies.

The total amount of immunoglobulin E (IgE) in human serum is an important parameter in diagnosing allergies. To reduce the false diagnosis of allergies and better assist in therapy, clinical studies can be performed to obtain accurate and reliable measurements of IgE. A magnetic beads (MBs)-based ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for total IgE measurement and the diagnosis of food allergies in serum was developed in this study. First, IgE was extracted by MBs coupled with anti-IgE antibody from serum. The extracted IgE was quantified by a specific signal peptide after digestion. A spiked linear IgE concentration ranging from 400 to 5000 ng mL-1 was used for quantification. The limits of detection and quantification were 400 ng mL-1 and 800 ng mL-1, respectively, for the developed method. Additionally, the combined capacity of the extracted IgE with different allergic proteins was evaluated by a binding experiment in vitro. The combining capacity of IgE with different allergens was used to speculate the kind of allergens that induce allergies in patients. Overall, a new method was developed that could be used to quantify the amount of IgE and simultaneously diagnose which allergen causes an allergic reaction, and this method may provide a powerful new tool in the clinical detection of allergies. Moreover, the developed method was applied to analyze IgE in four samples of patient serum and four serum samples from healthy individuals.

Urinary analysis reveals high Alternaria mycotoxins exposure in the general population from Beijing, China.

Alternaria mycotoxins are of concern due to its adverse health effect, they affect various cereal crops and grain-based food along with modified forms that contribute to overall exposure. This study aimed to determine the frequency and level of exposure to Alternaria mycotoxins (tenuazonic acid, TeA; alternariol, AOH; alternariol monomethyl ether, AME; tentoxin, TEN; and altenuene, ALT) in human urine from Beijing adults. A total of 2212 urine samples were collected and analyzed for five mycotoxins using LC-ESI-MS/MS. More than 98% of the samples had at least one Alternaria mycotoxin detected. Among the mycotoxins, AME had the highest detection rate (96.0%), followed by TeA (70.5%). The calculated average daily intake values of AME (12.5 ng/kg b.w.) was 5 times the TTC value (2.5 ng/kg b.w.) set by the EFSA, indicating the potential health risks associated with mycotoxins. Immediate attention and subsequent actions should be taken to identify the sources of mycotoxins and the corresponding exposure pathways to humans in the investigated regions.

Characterization of the molecular properties and allergenicity (IgE-binding capacity) of ß-lactoglobulin by heat treatment using asymmetric-flow field-flow fractionation and ultra-performance liquid chromatography quadrupole time of flight mass chromatography.

In this study, the heat product (90 °C, 10 min) of ß-lactoglobulin (ß-LG) was analyzed by asymmetric-flow field-flow fractionation (AF4) to observe the effect of heat treatment. The changes in molar mass (M) and molar size induced by heat treatment were characterized by AF4, and changes in molar shape were observed by transmission electron microscopy (TEM). The results showed that ß-LG dissociated and aggregated into four fractions with different M values, sizes, and shapes after heat treatment. The vast aggregations with the highest allergenicity (IgE-binding capacity) might enhance the allergenicity of ß-LG. However, the number of characterized epitope peptides was decreased due to heat treatment. The above results provide some references for related studies of ß-LG and its allergenicity. Further separation and characterization of the high-allergenicity fractions and peptides will help to eliminate allergens in dairy products and reduce the occurrence of allergic reactions.

UPLC-MS/MS and Network Pharmacology-Based Analysis of Bioactive Anti-Depression Compounds in Betel Nut.

BACKGROUND: Betel nuts have long been used in traditional Chinese medicine. In our study, the bioactive components of betel nut were systematically investigated, and the main components and their target genes in the treatment of depression were predicted. METHODS: The metabolites of the kernels and peels were analyzed with a UPLC-MS/MS system. Mass spectrometry outcomes were annotated by MULTIAQUANT. "Compound-disease targets" were utilized to construct a pharmacology network. RESULTS: A total of 873 metabolites were identified, with a high abundance of flavonoids, alkaloids, and phenols. Moreover, the abundance of flavonoids, alkaloids, and phenols in the kernel was significantly higher than that in the peel. A high abundance of catechin, arginine, and phenylalanine was detected in the kernel, while a high abundance of arginine, arecoline, and aminobutyric acid was detected in the peel. Catechins and cyanoside were the most abundant flavonoids in the kernel and peel, respectively. Arecoline was the most abundant alkaloid. A total of 111 metabolites showed a significant difference between the kernels and peels. The relative abundance of 40 differential metabolites was higher than 100,000, including 14 primary metabolites, 12 flavonoids, 4 phenols, and 4 alkaloids. Among the 40 high abundance metabolites, 20 were higher in the kernel and 20 in the peel. In addition, the enrichment of metabolic pathways found that the kernel and peel of the fruit adopted different metabolic pathways for the synthesis of flavonoids and alkaloids. Network pharmacology prediction showed that 93 metabolites could target 141 depression-related genes. The main components of betel nut intervention in depression were predicted to include L-phenylalanine, protocatechuic acid, okanin, nicotinic acid, L-tyrosine, benzocaine, syringic acid, benzocaine, phloretic acid, cynaroside, and 3,4-dihydroxybenzaldehyde. CONCLUSION: Betel nuts are rich in natural metabolites, and some of these metabolites can participate in the intervention of depression. In addition, the metabolites showed distinct characteristics between the kernel and peel. Therefore, it is necessary to comprehensively and rationally use betel nuts.

Effect of chewing betel nut on the gut microbiota of Hainanese.

Betel nut chewing (BNC) is prevalent in South Asia and Southeast Asia. BNC can affect host health by modulating the gut microbiota. The aim of this study is to evaluate the effect of BNC on the gut microbiota of the host. Feces samples were obtained from 34 BNC individuals from Ledong and Lingshui, Hainan, China. The microbiota was analyzed by 16S rRNA gene sequencing. BNC decreased the microbial α-diversity. Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria were the predominant phyla, accounting for 99.35% of the BNC group. The Firmicutes-to-Bacteroidetes ratio was significantly increased in the BNC group compared to a control group. The abundances of the families Aerococcaceae, Neisseriaceae, Moraxellaceae, Porphyromonadaceae, and Planococcaceae were decreased in the BNC/BNC_Male/BNC_Female groups compared to the control group, whereas the abundances of Coriobacteriaceae, Streptococcaceae, Micrococcaceae, Xanthomonadaceae, Coxiellaceae, Nocardioidaceae, Rhodobacteraceae, and Succinivibrionaceae were increased. In general, the gut microbiome profiles suggest that BNC may have positive effects, such as an increase in the abundance of beneficial microbes and a reduction in the abundance of disease-related microbes. However, BNC may also produce an increase in the abundance of disease-related microbes. Therefore, extraction of prebiotic components could increase the beneficial value of betel nut.

Combination of polyvinylpolypyrrolidone extraction and standard addition strategy for the accurate determination of multiple allergen residues in red wine by UPLC-MS/MS.

During the winemaking process, fining materials derived from milk and egg products are traditionally used to remove undesirable substances to reduce bitterness and astringency. The possible residues of allergens in treated wine may pose a potential risk for allergy patients. In this study, we developed a method for the simultaneous quantification of eight allergens (αS1-casein, αS2-casein, ß-casein, κ-casein, ß-lactoglobulin, lysozyme, ovalbumin and ovotransferrin) in red wine by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). The sample was extracted with polyvinylpolypyrrolidone (PVPP) solution, following trypsin digestion and peptide-level purification by solid-phase extraction (SPE). A strategy based on standard addition was used for the accurate quantification of the target allergens in wine products. The limits of detection (LODs) were shown to be 0.003-0.015 µg/mL for milk allergens and 0.1 µg/mL for egg allergens. This economical and reliable method would be appropriate for routine analysis and further allergen label management for red wine.

Characterization and epitope prediction of phosphopyruvate hydratase from Penaeus monodon (black tiger shrimp).

Shellfish allergies constitute an important cause of food-induced anaphylactic reactions, which pose challenges to food safety and human health worldwide. In the present study, the specific IgE (sIgE) binding characteristics of different shrimp proteins of black tiger shrimp (Penaeus monodon) to the sera of eight shrimp-allergic patients from China were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and nanoliquid chromatography time-of-flight mass spectrometry. According to the PLGS scores (>2000) and the sequence coverage (>40%), eight proteins with sIgE binding activity were identified, including myosin heavy chain type 1 (K4Q4N8), hemocyanin (G1AP69 and Q95V28), phosphopyruvate hydratase (O96656), arginine kinase (C7E3T4), tropomyosin (A1KYZ2), sarcoplasmic calcium binding protein (H7CHW2) and glyceraldehyde-3-phosphate dehydrogenase (A0A097BQP2). Among these eight proteins, phosphopyruvate hydratase was a prevalent IgE-binding protein among these Chinese patients with binding observed in 100% of sera. Moreover, 13 peptides were predicted as epitopes of phosphopyruvate hydratase. These new details help us to understand the crustacean IgE-binding proteins especially Penaeus monodon IgE-binding proteins, that would cause allergic reaction to Chinese patients. And our findings may provide essential information to improve allergy prevention and clinical treatment to shrimp allergy in China. PRACTICAL APPLICATION: This research may have diagnostic and therapeutic value for shrimp allergies in China.

The amino - functionalized magnetic graphene oxide combined with graphite furnace atomic absorption spectrometry for determination of trace inorganic arsenic species in water samples.

A novel adsorbent of magnetic graphene oxide (GO) chemically modified by cysteamine hydrochloride (Fe3O4@SiO2/GO-NH2) through thiol-ene click chemistry reaction was synthesized. The prepared Fe3O4@SiO2/GO-NH2 exhibit selective adsorption to As(V) with high adsorption capacity (52.66 mg g-1). Taking Fe3O4@SiO2/GO-NH2 as the adsorbents, a new method of magnetic solid phase extraction (MSPE) combined with graphite furnace atomic absorption spectrometry (GFAAS) was developed in determining trace-level inorganic arsenic species (As(III) and As(V)) in environmental water and bottled water samples. Various experimental parameters affecting the MSPE have been optimized. Under the optimal experimental parameters, the limit of detection of the established method for As(V) was 1.02 ng L-1, the relative standard deviations were 7.9% (intra-day, c = 50 ng L-1, n = 5) and 4.6% (inter-day, c = 50 ng L-1, n = 7), respectively, and the enrichment factor of the method was 392. GBW08666 and GBW08667 (certified reference material) were analyzed to confirm the accuracy of the method, and the results were matched well with the certified values. The established MSPE-GFAAS method was successfully applied in analyzing trace/ultratrace As(III) and As(V) in real water samples.

Labeled Peptide-Free UHPLC-MS/MS Method Used for Simultaneous Determination of Shrimp and Soybean in Sauce Products.

Unintentional missing of shrimp and soybean allergen information on precautionary food allergen labeling often occurs in sauce products. To avoid food allergies, sensitive and time-saving analytical methods are urgently needed. However, the currently reported methods usually employed labeled peptides for isotope internal standard quantitation, and the matrix effect caused by protein extraction or digestion can not be corrected. In this study, a labeled peptide-free standard addition method was developed for simultaneous determination of shrimp and soybean in sauce products using ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Through the rational selection of stable peptides, satisfying mean recoveries and relative standard deviations of the chosen peptides are achieved. The limit of quantifications of each peptide ranged from 0.25 to 5 µg tropomyosin/g sauce and from 1 to 10 µg Gly m 6/g sauce, respectively. Using the labeled peptide-free UHPLC-MS/MS method, not only ideal experimental results were obtained surpassing those obtained with labeled peptides, but also the reagents were economized and shortening of the sample preparation time was achieved.

Determination of Sulfonamides in Milk by Cloud Point-Salting Out Extraction and Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry.

A method involving cloud point-salting out extraction (CPSOE) coupled with UHPLC-MS/MS was developed for the determination of eleven sulfonamides in milk. In this study, the type and concentration of the surfactant, de-emulsification condition, pH value, volume of n-butanol, equilibration temperature and time were optimized. For this developed method, the linear range of SAs was from 0.05 to 50 µg L-1, and the correlation coefficients were higher than 0.997. The average recoveries for SAs were from 61.32 to 91.67%, and the LOQs were less than 0.06 µg kg-1.

Development of a simple and rapid LC-MS/MS method for the simultaneous quantification of five Alternaria mycotoxins in human urine.

Alternaria mycotoxins, such as tenuazonic acid (TeA), altenuene (ALT), alternariol (AOH), tentoxin (TEN) and alternariol monomethyl ether (AME) are frequently found in foods and may pose a potential risk to human health. Human biomonitoring can help measure our exposure to these mycotoxins, and help us determine if the exposure is changing over time. In this study, a simple liquid-liquid extraction sample preparation procedure followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous analysis of five Alternaria mycotoxins in human urine. High recoveries (92.7-103.2%) were obtained for all the tested mycotoxins with relative standard deviations (RSDs, %) of less than 6.4%. The limits of quantification (LOQs) for the analytes in urine ranged from 0.001 to 0.05 ng/mL. The method was successfully applied to investigate the levels of five Alternaria mycotoxins from 135 volunteers. In all of the samples, at least one Alternaria mycotoxin was detected. TeA, AME and AOH were the predominant Alternaria mycotoxins, and the detection rates were 85.9%, 96.3% and 51.9%, respectively.

ROCK2 Confers Acquired Gemcitabine Resistance in Pancreatic Cancer Cells by Upregulating Transcription Factor ZEB1.

Resistance to chemotherapy is a major clinical challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC). Here, we provide evidence that Rho associated coiled-coil containing protein kinase 2 (ROCK2) maintains gemcitabine resistance in gemcitabine resistant pancreatic cancer cells (GR cells). Pharmacological inhibition or gene silencing of ROCK2 markedly sensitized GR cells to gemcitabine by suppressing the expression of zinc-finger-enhancer binding protein 1 (ZEB1). Mechanically, ROCK2-induced sp1 phosphorylation at Thr-453 enhanced the ability of sp1 binding to ZEB1 promoter regions in a p38-dependent manner. Moreover, transcriptional activation of ZEB1 facilitated GR cells to repair gemcitabine-mediated DNA damage via ATM/p-CHK1 signaling pathway. Our findings demonstrate the essential role of ROCK2 in EMT-induced gemcitabine resistance in pancreatic cancer cells and provide strong evidence for the clinical application of fasudil, a ROCK2 inhibitor, in gemcitabine-refractory PDAC.