American journal of physiology. Gastrointestinal and liver physiology最新文献

Primary cilia have been considered tumor-suppressing organelles in cholangiocarcinoma (CCA), though the mechanisms behind their protective role are not fully understood. This study investigates how the loss of primary cilia affects DNA damage response (DDR) and DNA repair processes. Human cholangiocyte cell lines were used to examine the colocalization of DNA repair proteins at the cilia and assess the impact of experimental deciliation on DNA repair pathways. Deciliation was induced using shRNA knockdown or CRISPR knockout of IFT20, IFT88, or KIF3A, followed by exposure to the genotoxic agents cisplatin, methyl methanesulfonate (MMS), or irradiation. Cell survival, cell cycle progression, and apoptosis rates were evaluated, and DNA damage was assessed using comet assays and phosphorylated H2AX (γH2AX) quantification. An in vivo liver-specific IFT88 knockout model, generated using Albumin-Cre/Lox recombination, was used to study the loss of primary cilia in the liver. Results showed that RAD51 localized predominantly at the base of the cilium, whereas Ataxia Telangiectasia and Rad3-related protein (ATR), PARP1, CHK1, and CHK2 were also detected within the ciliary shaft. Deciliated cells displayed dysregulation in critical DNA repair pathways. These cells also showed reduced survival and increased S-phase arrest after genotoxic challenges as compared with ciliated cells. Enhanced DNA damage was observed via increased γH2AX signals and comet assay results. An increase in γH2AX expression was also observed in our in vivo model, indicating elevated DNA damage. In addition, key DDR proteins such as Ataxia Telangiectasia Mutated protein (ATM), p53, and p21, were downregulated in deciliated cells after irradiation. This study underscores the crucial role of primary cilia in regulating DNA repair and suggests that targeting cilia-related mechanisms could present a novel therapeutic approach for CCA.NEW & NOTEWORTHY Our study reveals a novel link between primary cilia and DNA repair in cholangiocytes. We show that DNA damage response (DDR) and repair proteins localize to cilia, and deciliation impairs survival and induces S-phase arrest under genotoxic stress. Deciliated cells exhibit increased DNA damage after cisplatin, irradiation, or methyl methanesulfonate (MMS) challenge. Following irradiation, Ataxia Telangiectasia Mutated protein (ATM), p53, and p21 are downregulated in deciliated cells. Similarly, IFT88 knockout mice show heightened DNA damage, highlighting the role of primary cilia in genome stability.

The hypothesis of the development and origin of health and disease (DOHaD) highlights the relationship between exposure to harmful stimuli during pregnancy and the increased cardiometabolic risk in the offspring's adulthood. It is believed that the renin-angiotensin system (RAS) plays a central role in stress-induced hepatic programming. To determine the effects of prenatal stress, sex, and age on hepatic RAS of the offspring, pregnant Wistar rats were divided into control and stress groups. The unpredictable stress protocol was performed in the last week of pregnancy. The offspring were divided according to sex, age, and intervention. At 90 and 120 days, the offspring's blood and liver were collected to measure hepatic enzyme activity, isoprostane levels, and protein expression of the RAS and redox balance. At 90 days old, stress similarly reduced NOX4 in both sexes, whereas NOX2, NOX2/NOX4 ratio, and isoprostane were increased only in female offspring. Stress responses of NOX2 were still higher in females when compared with males. At 120 days, prenatal stress increased the activity of aspartate transaminase (AST) and alanine transaminase (ALT) in females, whereas it decreased AST in males. Furthermore, stress enhances the expression of angiotensin II type 1 receptor, ACE, and Mas receptor in males, whereas reducing NOX2/NOX4 ratio. Still, stress reduced ACE2 expression and continued to increase NOX2/NOX4 ratio in females at 120 days. Prenatal stress induces hepatic programming in offspring in a sex- and age-specific way, altering the RAS and NOX4 pathways at 120-day-old males while inducing early redox changes in females at 90 days and remaining at 120 days.NEW & NOTEWORTHY Prenatal stress may contribute to sex- and age-specific programming of the liver in adult offspring. Our results showed that stress during pregnancy alters the hepatic renin-angiotensin system only at 120-days old and specially in male offspring. Furthermore, the prenatal stress leads to redox imbalances in females at 90 days and remaining at 120 days old independently of the renin-angiotensin system.

Armillariella tabescens polysaccharides (ATPS) were investigated for their protective effects against 5-fluorouracil (5-FU)-induced intestinal mucositis in intestinal epithelial cell 6 (IEC-6) cells and a murine model, with a focus on the role of β-arrestin1 (ARRB1) in endoplasmic reticulum stress (ERS) suppression. The study evaluated cell viability, apoptosis, inflammatory cytokine secretion (interleukin-1β, interleukin-6, tumor necrosis factor-α), and lactate dehydrogenase (LDH) release in IEC-6 cells, whereas diarrhea severity, body weight loss, intestinal histopathology, and tight junction protein expression were assessed in C57BL/6 mice. Western blot, immunohistochemistry, and transmission electron microscopy were used to investigate the underlying mechanisms of ATPS-mediated ERS inhibition. ATPS significantly improved cell survival and proliferation, reduced inflammatory cytokines and apoptosis, alleviated diarrhea severity, mitigated weight loss, and preserved intestinal barrier integrity by upregulating tight junction protein. Mechanistically, ATPS suppressed ERS activation by reducing glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic translation initiation factor 2α subunit (p-eIF2α), and C/EBP homologous protein (CHOP) expression, effects that were significantly attenuated in ARRB1-knockdown IEC-6 cells and Arrb1^-/- mice, indicating that ARRB1 is essential for ATPS-mediated ERS suppression and intestinal protection. These findings suggest that ATPS protects against 5-FU-induced intestinal mucositis by modulating ARRB1 and inhibiting ERS, highlighting its potential as a novel therapeutic strategy for chemotherapy-induced intestinal injury.NEW & NOTEWORTHY This study provides new insights into the therapeutic potential of ATPS in alleviating 5-FU-induced intestinal mucositis by modulating ARRB1 and suppressing ERS. The protective effects of ATPS were validated in both IEC-6 cells and a murine model, demonstrating its ability to enhance intestinal barrier integrity, inhibit apoptosis, and reduce inflammation. These findings suggest that ARRB1-mediated ERS suppression is a critical mechanism underlying ATPS-induced intestinal protection, presenting a novel strategy for mitigating chemotherapy-induced gastrointestinal toxicity.

Gut motility is partly driven by underlying rhythmic electrical activity called slow waves. The origin and propagation of these electrical events are studied extensively in anesthetized animal models. However, the effects of anesthesia on slow waves remain unclear. This study examined how propofol and isoflurane influence spatiotemporal features of gastric slow waves, anterior-posterior coupling, and the efficacy of gastric pacing. Pigs were anesthetized with propofol (n = 7) and isoflurane (n = 8), and baseline electrical activity was measured using high-resolution surface-contact electrode arrays placed on the anterior and posterior gastric serosa. Following baseline recordings, pacing was applied to assess its effects. Slow wave propagation patterns were quantified, and the efficacy of spatial entrainment during pacing was compared under propofol and isoflurane. Under propofol, antegrade propagation was observed with 86% symmetry between anterior and posterior gastric surfaces, whereas isoflurane reduced symmetry to 25% (P = 0.0187) with propagation patterns frequently changing. Slow wave period (18.8 ± 5.1 vs. 28.1 ± 14.3 s, P = 0.016), amplitude (1.5 ± 0.7 vs. 0.7 ± 0.4 mV, P = 0.002), and speed (4.4 ± 1.1 vs. 3.5 ± 0.7 mm/s, P = 0.018) differed significantly between anesthetic groups at baseline, whereas only amplitude and speed differed during pacing. Spatial entrainment success was higher with propofol (83%) than with isoflurane (57%), but pacing effects remained localized to the paced surface without propagation across the greater curvature. Isoflurane induced more gastric dysrhythmias than propofol, making propofol preferable for studying normal activity and isoflurane preferred for investigating therapies. The uncoupling of anterior and posterior surfaces suggests a potential electrical barrier at the greater curvature, warranting further investigation.NEW & NOTEWORTHY The influence of propofol and isoflurane on the spatial propagation of gastric slow wave activity under baseline and pacing conditions was defined for the first time. Slow waves were significantly ordered and coupled across the anterior and posterior surfaces of the stomach under propofol compared with isoflurane. Slow waves entrained during pacing were confined to the surface where pacing was applied, suggesting an electrical barrier along the greater curvature of the stomach.

Ethanolamine phosphate phospholyase (ETNPPL) is an enzyme that irreversibly degrades phosphoethanolamine (p-ETN), an intermediate in the Kennedy pathway of phosphatidylethanolamine (PE) synthesis. Whole body knockout Etnppl mice were fed a high-fat diet (HFD) containing 45% kcal fat for 10 wk. Etnppl^-/- female mice were resistant to HFD-induced obesity and had decreased liver weight compared with Etnppl^+/+ mice. Furthermore, Etnppl^-/- female mice had improved glucose sensitivity and increased energy expenditure compared with Etnppl^+/+ mice. Plasma triglyceride (TG) levels were elevated in Etnppl^-/- female mice, although the rate of very low-density lipoprotein (VLDL) secretion was not increased. The hepatic expression of PCSK9 was elevated, indicating a possible decrease in VLDL uptake. Interestingly, both plasma and hepatic cholesterol levels were reduced in Etnppl^-/- relative to Etnppl^+/+ mice. No difference in hepatic phosphatidylcholine, PE, or TG was detected between groups. Histopathological examination of hepatic tissues revealed decreased lipid deposition in Etnppl^-/- mice that may be explained by the lower hepatic cholesterol level. Additionally, RNA sequencing analysis showed upregulation in genes related to cholesterol metabolism in Etnppl^-/- female mice. In male mice, a slight decrease in weight gain was observed in Etnppl^-/- mice compared with Etnppl^+/+ mice. No change in plasma and hepatic lipid levels was detected in Etnppl^-/- male mice. To conclude, ETNPPL impacts whole body energy expenditure, weight gain, cholesterol metabolism, and hepatic lipoprotein metabolism without altering hepatic phospholipid levels.NEW & NOTEWORTHY Etnppl^-/- female mice resisted diet-induced obesity with enhanced energy expenditure and less adipose tissue. In addition, Etnppl^-/- female mice fed an HFD showed decreased liver cholesterol deposition. RNA sequencing revealed changes in genes related to cholesterol and lipid metabolism in Etnppl^-/- female mice. Etnppl^-/- female mice fed an HFD supplemented with cholesterol had no difference in plasma and hepatic cholesterol levels compared with Etnppl^+/+ mice.

Epithelial cell death and compromised barrier function are key features of inflammatory bowel disease pathogenesis. Previous studies suggest that the nuclear bile acid receptor, farnesoid X receptor (FXR), promotes intestinal barrier function and protects against inflammation. Here, we investigated potential mechanisms involved. T₈₄ cell monolayers were treated with a combination of IFNγ and TNFα to model cytokine-induced barrier dysfunction in vitro. Apoptosis and necroptosis were assessed by measuring caspase 3/PARP cleavage and RIP3 phosphorylation, respectively. Epithelial permeability was determined by measuring 4-kDa fluorescein isothiocyanate-dextran (FD4) flux. Effects of FXR on barrier function in dextran sulfate sodium (DSS)-treated mice were assessed by measuring plasma levels of orally administered FD4. Treatment with IFNγ and TNFα enhanced FD4 flux and increased apoptosis in T₈₄ monolayers, as evidenced by increased cleaved PARP and caspase 3 levels. Pretreatment with the FXR agonist, GW4064, significantly inhibited cytokine-induced FD4 flux, but not apoptosis. Treatment with IFNγ and TNFα in the presence of the apoptosis inhibitor, Q-VD-OPh, induced necroptosis, as evidenced by increased RIP3 phosphorylation and enhanced FD4 flux, whereas a necroptosis inhibitor, necrostatin, inhibited these effects. GW4064 also inhibited cytokine-induced RIP3 phosphorylation and FD4 flux in the presence of Q-VD-OPh. In mice, treatment with the FXR agonist, obeticholic acid, attenuated DSS-induced disease activity and mucosal FD4 flux, but not levels of cleaved caspase 3 or phospho-RIP3. FXR activation inhibits cytokine-induced barrier dysfunction by inhibiting epithelial necroptosis rather than apoptosis in vitro. How such effects contribute to the protective actions of FXR in vivo requires further elucidation.NEW & NOTEWORTHY These studies demonstrate for the first time that FXR activation inhibits cytokine-induced necroptosis in vitro, an effect that may underlie protection against dysregulated barrier function in the setting of intestinal inflammation. These data support the potential for targeting FXR to promote epithelial barrier function in treatment of IBD.

Probiotics have been suggested to ameliorate intestinal epithelial homeostasis and barrier function. They also modulate several mediators and receptors of the expanded endocannabinoid system, or endocannabinoidome (eCBome), potentially explaining their beneficial effects on intestinal function. We aimed to study the effects of probiotic strains on gut barrier functions and the possible involvement of the eCBome in these effects. We cocultured three strains of Lactiplantibacillus plantarum with murine small intestine epithelial organoids and explored the involvement of eCBome signaling and inflammation in mediating the beneficial effects of the probiotics on the epithelial barrier function. All three L. plantarum strains reduced the transepithelial permeability of organoids and increased mRNA expression of several tight junction proteins (Clnd1, Clnd2, Ocln, Tjp1, and Cdh1) and intestinal barrier proteins (Muc2, Lyz1, Reg3a, and Defa20). Concomitantly, the three strains increased the expression of genes encoding eCBome receptors while decreasing the expression of two catabolic enzymes (Faah and Naaa), and increasing one anabolic enzyme (Daglb). Altogether, these changes led to an overall increase in levels of eCBome mediators, namely N-acyl-ethanolamines (NAEs) and, particularly, 2-monoacylglycerols (2-MAGs), as measured by LC-MS/MS. URB 597 and JZL 184, two selective inhibitors of NAE and 2-MAG catabolism, reduced the transepithelial permeability of organoids, as observed with L. plantarum strains. Interestingly, both inhibitors also reversed inflammation-induced transepithelial permeability in organoids. Elevated endogenous levels of NAEs or 2-MAGs promote improvement in small intestine transepithelial permeability, and L. plantarum strains may exploit this mechanism to exert this same beneficial effect.NEW & NOTEWORTHY Lactiplantibacillus plantarum strains improve transepithelial permeability and concomitantly increase the levels of eCBome mediators in murine small intestine epithelial organoids. Pharmacological elevation of NAE or 2-MAG levels enhances the expression of intestinal epithelial barrier genes and reduces the transepithelial permeability of murine small intestine epithelial organoids, suggesting that L. plantarum may exploit eCBome signaling to exert its beneficial effects.

Glia maturation factor-β (Gmfb), an actin filament debrancher, was initially identified in brain and recently linked to liver diseases. To investigate the role of hepatocyte Gmfb (hep-Gmfb) in liver reparative regeneration, hepatocyte-specific gmfb knockout (HepGKO) and overexpression (HepGOE) zebrafish strains were constructed. Both transgenic and wild-type (WT) zebrafish underwent partial hepatectomy (PHX) or were fed high-fat, high-cholesterol diets to model metabolism-associated steatotic liver disease (MASLD). Under physiological conditions, the HepGKO, HepGOE, and WT fish displayed similar survival, gross appearance, and liver histology. Following PHX, WT liver gmfb levels positively correlated with cell proliferation and proinflammatory cytokine levels. HepGOE showed enhanced regeneration and reduced liver steatosis compared with WT, whereas HepGKO exhibited opposite effects. In MASLD, WT liver gmfb increased with disease progression. HepGKO experienced worsening liver enlargement, steatosis, ballooning, inflammation, and endoplasmic reticulum stress, whereas HepGOE showed improvements. HepGOE liver had the highest cell proliferation, but all three groups showed similar levels of cell apoptosis. Moreover, elevated proinflammatory cytokines were observed across MASLD groups, being the highest in HepGKO and lowest in HepGOE. However, signal transducer and activator of transcription 3 (stat3) activation was the lowest in HepGKO and highest in HepGOE, whereas jnk and mapk/extracellularly regulated kinase (erk) activation was consistent across the MASLD groups. In il6-treated primary hepatocytes, gmfb abundance influenced stat3 activation, and hep-gmfb abundance significantly affected actin filaments distribution in hepatocytes both in vivo and vitro. Hep-Gmfb boosts regenerative processes by enhancing hepatocyte proliferation, alleviating fatty liver histological abnormalities, and modulating the Il6/Stat3 signaling, potentially through remodeling of actin-filament network within hepatocytes.NEW & NOTEWORTHY Glia maturation factor-β (Gmfb) has shown important implications in liver disease. Using transgenic zebrafish models, our research demonstrates that Gmfb in hepatocytes confers protective benefits for liver regeneration and repair. It promotes hepatocyte proliferation, alleviates steatosis and ballooning, and modulates Il6/Stat3 signaling in response to liver injuries, potentially through remodeling of actin-filament network. This submission represents the first in vivo observation of the phenotypic effects of Gmfb in hepatocytes during liver injury.