ObjectiveA mouse model of vaccinia virus Tiantan strain (VTT)-induced encephalopathy was developed using AG6 mice. MethodsVTT was amplified by infecting Vero cells at a multiplicity of infection (MOI) of 0.01, followed by concentration and titration. After 72 h of incubation, virus-containing cells were collected and subjected to concentration. The concentrated viral suspension was serially diluted (10-fold dilutions) and added to 6-well plates containing confluent Vero cell monolayers for plaque assay. The number of plaques formed in each well was counted, and the virus titer was calculated based on the dilution factor. Fourteen 5-6-week-old AG6 mice (half male and half female, housed separately by sex) were randomly divided into a control group (n=3, PBS), a low-dose group (n=6, 1×10⁵ PFU), and a high-dose group (n=5, 5×10⁵ PFU). The mice were anesthetized by isoflurane inhalation and then infected via intranasal instillation. The mental state of the mice in each group was observed daily, and the body weight and mortality were recorded. On day 13 post-infection, 2% Evans Blue (4 mL/kg body weight) was administered via tail vein injection to assess blood-brain barrier (BBB) disruption. Subsequently, brain tissue samples were collected for immunofluorescence analysis to evaluate the activation of astrocytes and microglia. ResultsThe titer of purified VTT was 1×10⁷ PFU/mL. Compared with the control group, mice in the low-dose group showed no significant change in body weight, and no lethality was observed. In contrast, mice in the high-dose group exhibited significant weight loss starting on day 5 post-infection (P<0.05), accompanied by lethality. On day 13 post-infection, no Evans Blue extravasation was detected in the brain tissues of the low-dose group, while the olfactory bulb region of the high-dose group displayed distinct blue staining, indicating disruption of the BBB. Immunofluorescence analysis revealed no significant proliferation of astrocytes and microglia in the olfactory bulb region of the low-dose group on day 13 post-infection. In contrast, marked activation of glial cells was observable in the high-dose group. ConclusionAn animal model of VTT-induced encephalopathy in AG6 mice is successfully established, characterized by BBB disruption and reactive gliosis specifically localized to the olfactory bulb region, manifested as astrocytic and microglial proliferation.

ObjectiveThe widespread adoption of portable fundus cameras for primary care and community screening is hindered by limitations in current autofocus(AF) technologies. Image-based methods relying on sharpness evaluation require iterative searches, resulting in slow convergence, while projection-based techniques are susceptible to optical artifacts and calibration errors. To address these challenges, this study introduces a novel AF system based on direct wavefront sensing, designed to deliver simultaneous high speed, high precision, and operational robustness within the compact form factor essential for portable ophthalmic devices. MethodsOur approach fundamentally reimagines the AF process by directly measuring the ocular wavefront aberration. We developed a custom portable fundus camera integrating a miniaturized Shack-Hartmann wavefront sensor (SHWS) into the optical path. An 850 nm laser diode projects a point source onto the retina via oblique illumination to minimize corneal reflections. Light scattered from this spot carries the eye’s refractive error through the imaging optics and is directed to the SHWS, positioned at a plane optically conjugate to the primary color CMOS imaging sensor. A microlens array within the SHWS samples the incident wavefront, generating a pattern of focal spots on a CCD. Real-time centroid analysis of these spots provides a map of local wavefront slopes. These measurements are processed through a singular value decomposition (SVD) algorithm to fit a Zernike polynomial basis set, enabling real-time reconstruction of the wavefront phase. The defocus component (S) is extracted from the second-order Zernike coefficients, providing a direct, quantitative measure of the refractive error in diopters. This value serves as a precise error signal in a closed-loop control system, which commands a voice-coil actuated focusing lens to its null position in a single, deterministic step, eliminating the need for iterative search algorithms. ResultsComprehensive evaluation demonstrated the system’s high performance. Testing on a calibrated model eye (OEMI-7) established a highly linear relationship between the computed defocus S and the focusing lens position across a ±20 Diopter (D) compensation range, achievable within a 5 mm mechanical travel. The system achieved a focusing precision of 0.08 D, corresponding to an 18-fold improvement over a conventional projection spot-size method tested under identical conditions. The total focus acquisition time, encompassing wavefront measurement, computation, and lens actuation, averaged under 0.5 s. Clinical validation with 25 human volunteers (50 eyes, refractive range -15 D to +10 D) confirmed practical efficacy. The wavefront-sensing AF succeeded in 92% of attempts with a mean time of 0.5 s, substantially outperforming a projection-based benchmark which achieved only a 32% success rate with an average time of 4.25 s. The system provided instantaneous directional guidance and maintained stability during minor ocular movements. Objective assessment of image quality, via amplitude contrast of retinal vasculature, showed consistent and significant enhancement following AF correction across the entire tested diopter range. ConclusionThis work successfully implements and validates a direct wavefront-sensing autofocus paradigm for portable fundus cameras. By directly quantifying and compensating for the optical defocus aberration, this method bypasses the fundamental limitations of image-processing and projection-based techniques, enabling rapid, precise, and deterministic diopter compensation. The developed system delivers an exceptional combination of a wide operational range (±20 D), high accuracy (0.08 D), fast convergence (0.5 s), and a compact physical footprint. This technology provides a practical and high-performance focusing solution capable of enhancing the reliability, throughput, and diagnostic utility of portable retinal imaging in large-scale screening applications. Future efforts will be directed towards system cost optimization and performance adaptation for diverse ocular conditions.

ObjectiveMigraine is a leading neurological disorder and the fourth most common cause of years lived with disability worldwide, affecting nearly 116 million individuals. Although pharmacological treatments are available, their efficacy is often limited by side effects and variable response rates. Repetitive transcranial magnetic stimulation (rTMS) over the dorsolateral prefrontal cortex (DLPFC) offers a safe, non-invasive alternative for migraine management. However, the neurophysiological mechanisms, particularly how rTMS modulates local cortical excitability and distributed pain-related circuits, remain poorly understood. Elucidating these mechanisms is essential for optimizing treatment protocols and improving clinical outcomes. MethodsThis study employed concurrent transcranial magnetic stimulation and electroencephalography (TMS-EEG) to investigate neuroplastic and neurocircuitry mechanisms of DLPFC-rTMS in migraine. Study 1 compared 30 migraineurs and 28 healthy controls to identify abnormalities in TMS-evoked potentials (TEPs) and significant current density (SCD) within sensory-discriminative regions including the primary somatosensory cortex (S1) and posterior insula (pINS), cognitive-affective regions including the anterior insula (aINS) and midcingulate cortex (MCC), and a descending modulatory region, the periaqueductal gray (PAG). Study 2 used a single-blind, crossover, sham-controlled design in 34 healthy participants. Each participant received both active (10 Hz, 80% RMT, 1 500 pulses) and sham DLPFC-rTMS in counterbalanced order. TMS-EEG and cold pain tolerance were assessed before and after each session. ResultsIn Study 1, migraineurs showed a significantly less negative N120 amplitude compared to healthy controls (P=0.027, Cohen’s d=0.60), indicating local intracortical disinhibition. No group differences were observed for N40, P60, or P180 components. At the source level, migraineurs exhibited significantly higher SCD in the S1, pINS, aINS, and MCC (allQ<0.05), but not in the ventroposterior thalamus (vpTHAL), mediodorsal thalamus (mdTHAL), or PAG. In Study 2, active rTMS significantly reduced SCD from pre- to post-stimulation in the S1, aINS, and MCC (all Q<0.05). Sham stimulation also reduced SCD in the S1 (Q<0.05) but not in the aINS or MCC. Although no significant group-level analgesic effect was observed between active and sham conditions (P=0.107), correlation analyses revealed that greater SCD reductions in the S1 and MCC were significantly associated with higher post-rTMS pain tolerance (R=-0.487 and -0.495, both Q<0.01) and larger improvements in pain tolerance(R=-0.487 and -0.451, both Q<0.05). No such correlations were found following sham stimulation, suggesting that the behavioural relevance of neural changes is specific to active rTMS. ConclusionThis study provides novel evidence that migraineurs exhibit both local neuroplastic abnormalities (reduced N120 amplitude) and hyperactivity in key pain-processing regions (S1, pINS, aINS, MCC). A single session of DLPFC-rTMS reduced hyperactivity in the aINS, MCC, and S1. Notably, greater reductions in the S1 and MCC were associated with improved pain tolerance. These findings identify distinct cortical circuitries, particularly within the cognitive-affective pain network, that may serve as potential biomarkers for optimizing rTMS treatment in migraine and other chronic pain conditions. Future studies should validate these results in patient populations experiencing spontaneous migraine attacks and explore multi-session or accelerated rTMS protocols.

Objective To explore the application value of Laennec's membrane space in dealing with complex anatomical variations and dense adhesions during repeated liver transplantation (Re-LT). Methods The clinical data of 8 cases of Re-LT recipients treated in the Department of Hepatobiliary Surgery of the First Hospital Affiliated to Harbin Medical University were retrospectively analyzed. During the operation, based on the Laennec's membrane anatomical concept, the remaining Laennec space and the free technique within the hepatic portal plate were flexibly applied for dissection. Specifically, the first hepatic portal main ducts (biliary and portal veins) were dissected by lowering the hepatic portal plate inside the Laennec's membrane space, and the second hepatic portal was exposed to perform the modified piggyback liver transplantation to avoid the injury to the right adrenal gland and right kidney caused by the free inferior vena cava below the liver. Moreover, the dense adhesions between the liver and hollow organs were separated by a combination of blunt and sharp dissection within the subcapsular space of the liver capsule (Glisson capsule). Results All 8 cases of recipients successfully completed the surgery. Among them, 7 cases underwent end-to-end biliary anastomosis with a T-tube left in place, and 1 underwent choledochoenteric anastomosis, 2 cases underwent modified piggyback reconstruction, 2 cases used the subcapsular dissection technique to handle organ adhesions. One recipient died of generalized drug-resistant Klebsiella pneumoniae infection combined with septic shock after the operation, while the remaining 7 cases were discharged after the main complications (such as delayed graft function recovery, pulmonary infection, bile leakage, etc.) were cured or significantly improved. Two cases of recipients who used the subcapsular dissection technique effectively avoided the secondary injury of hollow organs, and no digestive tract leakage complications occurred after the operation. Conclusions By flexibly applying Laennec's membrane space and the intrahepatic portal plate free technique in Re-LT, it may effectively cope with the loss of anatomical layers and vascular dense adhesions, reducing the surgical difficulty and the risk of secondary injury, providing important technical support for improving the prognosis of recipients.

ObjectiveTo investigate the mechanism by which Xianglian Huazhuo prescription （XLHZ） ameliorates chronic atrophic gastritis （CAG） through regulating glycolysis via the microRNA-34b-5p （miR-34b-5p）/6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase 1 （PFKFB1） signaling pathway. MethodsBioinformatic screening was performed to identify the target genes of miR-34b-5p. In the animal experiments， a mouse model of CAG was established through a combination of N-methyl-N′-nitro-N-nitrosoguanidine （MNNG）， ranitidine， and irregular feeding. The mice were randomized into the following groups： normal， model， Morodan group （2 g·kg^-1）， and high-， medium-， and low-dose XLHZ groups（7.2，3.8，1.9 g·kg^-1）. In the cell experiments， GES-1 cells were treated with MNNG for the modeling of CAG and grouped as follows： GES-1， MNNG+GES-1， MNNG+GES-1+XLHZ（10%）， MNNG+GES-1+miR-34b-5p mimics， MNNG+GES-1+miR-34b-5p inhibitor， MNNG+GES-1+PFKFB1 inhibitor， and MNNG+GES-1+PFKFB1 mimics+XLHZ（10%）. The general conditions of mice were observed. Real-time PCR was adopted to measure the expression levels of miRNA-34b-5p and PFKFB1 mRNA in the gastric mucosa of mice in each group. Dual luciferase reporter gene was used to verify the targeting interaction between miR-34b-5p and PFKFB1 in GES-1 cells exposed to MNNG. The cell-counting kit-8 （CCK-8） was used to screen the drug-containing serum concentration. Real-time PCR was employed to verify the transfection efficiency. Fluorescent probes were used to detect the expression and nuclear translocation of hypoxia-inducible factor-1α （HIF-1α） in each group of cells. The protein levels of HIF-1α， hexokinase 2 （HK2）， PFKFB1， phosphofructokinase 1 （PFK1）， and lactate dehydrogenase （LDHA） in the gastric mucosa of mice were determined by Western blot. The levels of glucose consumption and lactate in the serum and cells were measured by biochemical methods. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the content of adenosine triphosphate （ATP） in the gastric mucosa tissue and cells. ResultsThe animal experiments showed that compared with the normal group， the model group exhibited symptoms including mental fatigue， lethargy， sparse and dull fur， a tendency to curl up， sluggish responses， reduced food and water intake， and slow body weight gain. In addition， the model group showed decreased expression of miRNA-34b-5p （P<0.01）， increased mRNA level of PFKFB1 （P<0.01）， upregulated protein levels of HIF-1α， HK2， PFKFB1， PFK1， and LDHA （P<0.01）， and increased glucose consumption， lactate content， and ATP content （P<0.01）. Compared with the model group， the XLHZ group and Morodan group showed improved mental state， gradually improved fur， increased activity， food and water intake， and body weight. HE showed that each treatment group exhibited well arranged and increased gastric mucosal glands， reduced inflammatory cells， and recovered gastric pits. Transmission electron microscopy showed that the size of mitochondria in each treatment group returned to normal， with no obvious irregular shape， complete and clear cristae， and uniform matrix. Furthermore， each treatment group showed increased expression of miRNA-34b-5p （P<0.05）， reduced mRNA level of PFKFB1 （P<0.05）， downregulated protein levels of HIF-1α， HK2， PFKFB1， PFK1， and LDHA （P<0.05）， and decreased glucose consumption， lactic acid， and ATP （P<0.05）. The cell experiments showed that the expression and nuclear translocation of HIF-1α were reduced in all groups after treatment （P<0.05）. The cell transfection efficiency was satisfactory （P<0.05）. miR-34b-5p exhibited an effect comparable to that of XLHZ， effectively inhibiting glycolysis， whereas PFKFB1 promoted glycolysis and reversed the protective effect of XLHZ in CAG mice （P<0.05）. ConclusionXLHZ could regulate the miR-34b-5p/PFKFB1 axis to inhibit glycolysis， thereby ameliorating MNNG-induced gastric mucosal atrophy.

ObjectiveTo construct a competing endogenous RNA （ceRNA） network involving long non-coding RNA NOTCH1 （lncRNA NOTCH1） and investigate whether Xianglian Huazhuo prescription （XLHZ） ameliorates gastric mucosal injury via regulating ferroptosis through the lncRNA NOTCH1/microRNA-138-5p （miR-138-5p）/silent information regulator 1 （SIRT1） axis. MethodsLncRNA sequencing and bioinformatics tools were used to construct the ceRNA network involving lncRNA NOTCH1. In the animal experiments， a chronic atrophic gastritis （CAG） model was induced by N-methyl-N'-nitro-N-nitrosoguanidine （MNNG） in mice. Mice were randomized into normal， model， Morodan （2.0 g·kg^-1）， and high-， medium-， and low-dose （7.6， 3.8， 1.9 g·kg^-1， respectively） XLHZ groups （n=10）. Transmission electron microscopy was adopted to assess gastric pathology. Real-time PCR was employed to measure the mRNA levels of lncRNA NOTCH1， miR-138-5p， SIRT1， nuclear factor erythroid 2-related factor 2 （Nrf2）， and glutathione peroxidase 4 （GPX4）. Western blot was adopted to quantify the protein levels of SIRT1， Nrf2， and GPX4 in the gastric tissue. In the cell experiment， dual-luciferase assay was employed to verify the targeting among lncRNA NOTCH1， miR-138-5p， and SIRT1. MNNG was used to treat GES-1 cells for disease modeling. The effects of NOTCH1 mimics on the expression of lncRNA NOTCH1， miR-138-5p， and SIRT1， as well as the effects of miR-138-5p inhibitor on the expression of miR-138-5p and SIRT1， were observed. The rescue experiment with XLHZ was designed with five groups： GES-1， GES-1+MNNG （Model）， Model+XLHZ， Model+NOTCH1 inhibitor+XLHZ， and Model+NOTCH1 inhibitor+miR-138-5p mimics+XLHZ. Real-time PCR and immunofluorescence assay were employed to measure the mRNA levels of lncRNA NOTCH1， miR-138-5p， SIRT1， Nrf2， and GPX4 and the Fe²⁺ content， respectively， in GES-1 cells. ResultsThe modeled mice showed mitochondrial damage with shrinkage and cristae loss， downregulated mRNA/protein levels of lncRNA NOTCH1， SIRT1， Nrf2， GPX4， and upregulated expression of miR-138-5p （P<0.01）. Compared with the model group， drug treatments reduced mitochondrial structural damage and the membrane density decreased， partially recovered the crista structure， downregulated the expression of miR-138-5p， and upregulated the mRNA and protein levels of lncRNA NOTCH1， SIRT1， Nrf2， and GPX4 （P<0.05， P<0.01）. In the cell experiments， the dual-luciferase assays confirmed lncRNA NOTCH1 bound to miR-138-5p and miR-138-5p bound to SIRT1， both suppressing luciferase activity （P<0.05， P<0.01）. Compared with the blank control group， the model group showed decreased expression of lncRNA NOTCH1， SIRT1， Nrf2， GPX4， increased expression of miR-138-5p， and enhanced Fe²⁺ fluorescence （P<0.01）. NOTCH1 mimics or miR-138-5p inhibitor reversed these changes （P<0.05， P<0.01）. Rescue experiments showed that NOTCH1 inhibitor reversed XLHZ-induced miR-138-5p decrease and SIRT1 increase （P<0.01）， while miR-138-5p overexpression suppressed SIRT1 expression （P<0.05， P<0.01）. The results confirmed that lncRNA NOTCH1 negatively regulated miR-138-5p， which in turn negatively regulated SIRT1. ConclusionXLHZ effectively alleviates gastric mucosal injury and curbs CAG progression by inhibiting ferroptosis through modulation of the lncRNA NOTCH1/miR-138-5p/SIRT1 axis.

ObjectiveTo explore the mechanism of Xianglian Huazhuo prescription （XLHZ） in ameliorating chronic atrophic gastritis （CAG） by regulating lysosomal iron metabolism. MethodsIn the animal experiments， a mouse model of CAG was established through a combined modeling approach， including free drinking of N-methyl-N′-nitro-N-nitrosoguanidine （MNNG， 100 mg·L^-1）， gavage of ranitidine （8 g·L^-1）， and irregular feeding. Mice were randomly assigned into the normal， model， Morodan （2.0 g·kg^-1）， and high-， medium-， and low-dose （7.2， 3.8， 1.9 g·kg^-1， respectively） XLHZ groups. In the cell experiments， GES-1 cells were treated with MNNG to construct a cell model of CAG， and cells were allocated into the GES-1， GES-1+MNNG （Model）， and Model+XLHZ groups. The general physical conditions of mice in each group were observed. Hematoxylin-eosin （HE） staining was used to detect the pathological morphology of the gastric mucosa. Transmission electron microscopy was employed to observe the ultrastructure of gastric mucosal cells. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the serum levels of interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and interleukin-10 （IL-10） in the serum. Western blot was used to determine the protein levels of lysosome-associated membrane protein 1 （LAMP1）， nuclear receptor coactivator 4 （NCOA4）， ferritin heavy chain 1 （FTH1）， six-transmembrane epithelial antigen of prostate 3 （Steap3）， and transient receptor potential mucolipin 1 （TRPML1） in the gastric tissue. Immunohistochemistry was employed to detect the positive expression of glutathione peroxidase 4 （GPX4） and 8-hydroxy-2′-deoxyguanosine （8-OHdG） in the gastric tissue. Fluorescent probe method was used to detect the levels of Fe²⁺ and reactive oxygen species （ROS）. Immunofluorescence assay was applied to observe the co-localization of FTH1 and LAMP1. ResultsThe animal experiments showed that compared with the normal group， the model group exhibited poor mental status and hair condition， accompanied by decreased locomotor activity， food and water intake， and even reduced body weight. HE staining results showed that the gastric mucosal epithelium of model mice presented poor continuity， significantly decreased mucosal thickness， irregular gastric fovea morphology with widened spacing， dilated glandular cavities， reduced and disarranged glands with obvious atrophy， and extensive inflammatory cell infiltration. Transmission electron microscopy revealed that the mitochondria were markedly shrunken with condensed and deeply stained matrix， drastically reduced and irregularly arranged cristae， and even vacuolization， and the outer mitochondrial membrane shrank and ruptured. In addition， the model group showed elevated serum levels of IL-6 and IL-1β （P<0.01）， declined serum level of IL-10 （P<0.01）， upregulated protein levels of LAMP1， NCOA4， Steap3， and TRPML1 （P<0.01）， downregulated protein level of FTH1 （P<0.01） in the gastric tissue， reduced positive expression of GPX4 （P<0.01）， and increased positive expression of 8-OHdG （P<0.01）. Compared with the model group， all the treatment groups showed improved mental state and hair appearance， increased locomotor activity， food and water intake， as well as gradual body weight gain in mice. HE staining demonstrated that the gastric mucosal epithelium of mice in each treatment group was continuous and intact， with normalized gastric fovea morphology， narrowed glandular cavities， increased and neatly arranged glands， and alleviated inflammatory cell infiltration. Transmission electron microscopy results showed that the mitochondria in mice from each treatment group possessed intact structures with recovered normal volume， increased and regularly arranged cristae， and homogeneous density of the mitochondrial membrane. The mice treated with high and medium doses of XLHZ exhibited declined serum levels of IL-6 and IL-1β （P<0.01） and elevated serum level of IL-10 （P<0.01）. Low-dose XLHZ reduced serum IL-1β level in mice （P<0.05）. The expression levels of LAMP1， NCOA4， Steap3 and TRPML1 protein in gastric tissue of mice in XLHZ high- and medium-dose groups were decreased （P<0.05， P<0.01）， and the expression level of FTH1 protein was increased （P<0.05， P<0.01）. The positive expression of GPX4 in the gastric tissue was increased in the high- and medium-dose XLHZ groups （P<0.05， P<0.01）， and that of 8-OHdG was decreased in all treatment groups （P<0.01）. The cell experiments showed that compared with the GES-1 group， the fluorescence intensities of Fe²⁺ and ROS， as well as the co-localization of FTH1 and LAMP1， were significantly increased in the GES-1 + MNNG group （P<0.01）. Compared with the GES-1 + MNNG group， the fluorescence intensities of Fe²⁺ and ROS were markedly decreased in the Model + XLHZ group （P<0.01）， while the co-localization of FTH1 and LAMP1 was reduced （P<0.05）. ConclusionXLHZ can effectively regulate lysosomal iron metabolism and ameliorate gastric mucosal and cellular injury induced by CAG.

ObjectiveTo investigate the mechanism by which Xianglian Huazhuo prescription （XLHZ） ameliorates chronic atrophic gastritis （CAG） through regulating glycolysis via the microRNA-34b-5p （miR-34b-5p）/6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase 1 （PFKFB1） signaling pathway. MethodsBioinformatic screening was performed to identify the target genes of miR-34b-5p. In the animal experiments， a mouse model of CAG was established through a combination of N-methyl-N′-nitro-N-nitrosoguanidine （MNNG）， ranitidine， and irregular feeding. The mice were randomized into the following groups： normal， model， Morodan group （2 g·kg^-1）， and high-， medium-， and low-dose XLHZ groups（7.2，3.8，1.9 g·kg^-1）. In the cell experiments， GES-1 cells were treated with MNNG for the modeling of CAG and grouped as follows： GES-1， MNNG+GES-1， MNNG+GES-1+XLHZ（10%）， MNNG+GES-1+miR-34b-5p mimics， MNNG+GES-1+miR-34b-5p inhibitor， MNNG+GES-1+PFKFB1 inhibitor， and MNNG+GES-1+PFKFB1 mimics+XLHZ（10%）. The general conditions of mice were observed. Real-time PCR was adopted to measure the expression levels of miRNA-34b-5p and PFKFB1 mRNA in the gastric mucosa of mice in each group. Dual luciferase reporter gene was used to verify the targeting interaction between miR-34b-5p and PFKFB1 in GES-1 cells exposed to MNNG. The cell-counting kit-8 （CCK-8） was used to screen the drug-containing serum concentration. Real-time PCR was employed to verify the transfection efficiency. Fluorescent probes were used to detect the expression and nuclear translocation of hypoxia-inducible factor-1α （HIF-1α） in each group of cells. The protein levels of HIF-1α， hexokinase 2 （HK2）， PFKFB1， phosphofructokinase 1 （PFK1）， and lactate dehydrogenase （LDHA） in the gastric mucosa of mice were determined by Western blot. The levels of glucose consumption and lactate in the serum and cells were measured by biochemical methods. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the content of adenosine triphosphate （ATP） in the gastric mucosa tissue and cells. ResultsThe animal experiments showed that compared with the normal group， the model group exhibited symptoms including mental fatigue， lethargy， sparse and dull fur， a tendency to curl up， sluggish responses， reduced food and water intake， and slow body weight gain. In addition， the model group showed decreased expression of miRNA-34b-5p （P<0.01）， increased mRNA level of PFKFB1 （P<0.01）， upregulated protein levels of HIF-1α， HK2， PFKFB1， PFK1， and LDHA （P<0.01）， and increased glucose consumption， lactate content， and ATP content （P<0.01）. Compared with the model group， the XLHZ group and Morodan group showed improved mental state， gradually improved fur， increased activity， food and water intake， and body weight. HE showed that each treatment group exhibited well arranged and increased gastric mucosal glands， reduced inflammatory cells， and recovered gastric pits. Transmission electron microscopy showed that the size of mitochondria in each treatment group returned to normal， with no obvious irregular shape， complete and clear cristae， and uniform matrix. Furthermore， each treatment group showed increased expression of miRNA-34b-5p （P<0.05）， reduced mRNA level of PFKFB1 （P<0.05）， downregulated protein levels of HIF-1α， HK2， PFKFB1， PFK1， and LDHA （P<0.05）， and decreased glucose consumption， lactic acid， and ATP （P<0.05）. The cell experiments showed that the expression and nuclear translocation of HIF-1α were reduced in all groups after treatment （P<0.05）. The cell transfection efficiency was satisfactory （P<0.05）. miR-34b-5p exhibited an effect comparable to that of XLHZ， effectively inhibiting glycolysis， whereas PFKFB1 promoted glycolysis and reversed the protective effect of XLHZ in CAG mice （P<0.05）. ConclusionXLHZ could regulate the miR-34b-5p/PFKFB1 axis to inhibit glycolysis， thereby ameliorating MNNG-induced gastric mucosal atrophy.

ObjectiveTo construct a competing endogenous RNA （ceRNA） network involving long non-coding RNA NOTCH1 （lncRNA NOTCH1） and investigate whether Xianglian Huazhuo prescription （XLHZ） ameliorates gastric mucosal injury via regulating ferroptosis through the lncRNA NOTCH1/microRNA-138-5p （miR-138-5p）/silent information regulator 1 （SIRT1） axis. MethodsLncRNA sequencing and bioinformatics tools were used to construct the ceRNA network involving lncRNA NOTCH1. In the animal experiments， a chronic atrophic gastritis （CAG） model was induced by N-methyl-N'-nitro-N-nitrosoguanidine （MNNG） in mice. Mice were randomized into normal， model， Morodan （2.0 g·kg^-1）， and high-， medium-， and low-dose （7.6， 3.8， 1.9 g·kg^-1， respectively） XLHZ groups （n=10）. Transmission electron microscopy was adopted to assess gastric pathology. Real-time PCR was employed to measure the mRNA levels of lncRNA NOTCH1， miR-138-5p， SIRT1， nuclear factor erythroid 2-related factor 2 （Nrf2）， and glutathione peroxidase 4 （GPX4）. Western blot was adopted to quantify the protein levels of SIRT1， Nrf2， and GPX4 in the gastric tissue. In the cell experiment， dual-luciferase assay was employed to verify the targeting among lncRNA NOTCH1， miR-138-5p， and SIRT1. MNNG was used to treat GES-1 cells for disease modeling. The effects of NOTCH1 mimics on the expression of lncRNA NOTCH1， miR-138-5p， and SIRT1， as well as the effects of miR-138-5p inhibitor on the expression of miR-138-5p and SIRT1， were observed. The rescue experiment with XLHZ was designed with five groups： GES-1， GES-1+MNNG （Model）， Model+XLHZ， Model+NOTCH1 inhibitor+XLHZ， and Model+NOTCH1 inhibitor+miR-138-5p mimics+XLHZ. Real-time PCR and immunofluorescence assay were employed to measure the mRNA levels of lncRNA NOTCH1， miR-138-5p， SIRT1， Nrf2， and GPX4 and the Fe²⁺ content， respectively， in GES-1 cells. ResultsThe modeled mice showed mitochondrial damage with shrinkage and cristae loss， downregulated mRNA/protein levels of lncRNA NOTCH1， SIRT1， Nrf2， GPX4， and upregulated expression of miR-138-5p （P<0.01）. Compared with the model group， drug treatments reduced mitochondrial structural damage and the membrane density decreased， partially recovered the crista structure， downregulated the expression of miR-138-5p， and upregulated the mRNA and protein levels of lncRNA NOTCH1， SIRT1， Nrf2， and GPX4 （P<0.05， P<0.01）. In the cell experiments， the dual-luciferase assays confirmed lncRNA NOTCH1 bound to miR-138-5p and miR-138-5p bound to SIRT1， both suppressing luciferase activity （P<0.05， P<0.01）. Compared with the blank control group， the model group showed decreased expression of lncRNA NOTCH1， SIRT1， Nrf2， GPX4， increased expression of miR-138-5p， and enhanced Fe²⁺ fluorescence （P<0.01）. NOTCH1 mimics or miR-138-5p inhibitor reversed these changes （P<0.05， P<0.01）. Rescue experiments showed that NOTCH1 inhibitor reversed XLHZ-induced miR-138-5p decrease and SIRT1 increase （P<0.01）， while miR-138-5p overexpression suppressed SIRT1 expression （P<0.05， P<0.01）. The results confirmed that lncRNA NOTCH1 negatively regulated miR-138-5p， which in turn negatively regulated SIRT1. ConclusionXLHZ effectively alleviates gastric mucosal injury and curbs CAG progression by inhibiting ferroptosis through modulation of the lncRNA NOTCH1/miR-138-5p/SIRT1 axis.

ObjectiveTo explore the mechanism of Xianglian Huazhuo prescription （XLHZ） in ameliorating chronic atrophic gastritis （CAG） by regulating lysosomal iron metabolism. MethodsIn the animal experiments， a mouse model of CAG was established through a combined modeling approach， including free drinking of N-methyl-N′-nitro-N-nitrosoguanidine （MNNG， 100 mg·L^-1）， gavage of ranitidine （8 g·L^-1）， and irregular feeding. Mice were randomly assigned into the normal， model， Morodan （2.0 g·kg^-1）， and high-， medium-， and low-dose （7.2， 3.8， 1.9 g·kg^-1， respectively） XLHZ groups. In the cell experiments， GES-1 cells were treated with MNNG to construct a cell model of CAG， and cells were allocated into the GES-1， GES-1+MNNG （Model）， and Model+XLHZ groups. The general physical conditions of mice in each group were observed. Hematoxylin-eosin （HE） staining was used to detect the pathological morphology of the gastric mucosa. Transmission electron microscopy was employed to observe the ultrastructure of gastric mucosal cells. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the serum levels of interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and interleukin-10 （IL-10） in the serum. Western blot was used to determine the protein levels of lysosome-associated membrane protein 1 （LAMP1）， nuclear receptor coactivator 4 （NCOA4）， ferritin heavy chain 1 （FTH1）， six-transmembrane epithelial antigen of prostate 3 （Steap3）， and transient receptor potential mucolipin 1 （TRPML1） in the gastric tissue. Immunohistochemistry was employed to detect the positive expression of glutathione peroxidase 4 （GPX4） and 8-hydroxy-2′-deoxyguanosine （8-OHdG） in the gastric tissue. Fluorescent probe method was used to detect the levels of Fe²⁺ and reactive oxygen species （ROS）. Immunofluorescence assay was applied to observe the co-localization of FTH1 and LAMP1. ResultsThe animal experiments showed that compared with the normal group， the model group exhibited poor mental status and hair condition， accompanied by decreased locomotor activity， food and water intake， and even reduced body weight. HE staining results showed that the gastric mucosal epithelium of model mice presented poor continuity， significantly decreased mucosal thickness， irregular gastric fovea morphology with widened spacing， dilated glandular cavities， reduced and disarranged glands with obvious atrophy， and extensive inflammatory cell infiltration. Transmission electron microscopy revealed that the mitochondria were markedly shrunken with condensed and deeply stained matrix， drastically reduced and irregularly arranged cristae， and even vacuolization， and the outer mitochondrial membrane shrank and ruptured. In addition， the model group showed elevated serum levels of IL-6 and IL-1β （P<0.01）， declined serum level of IL-10 （P<0.01）， upregulated protein levels of LAMP1， NCOA4， Steap3， and TRPML1 （P<0.01）， downregulated protein level of FTH1 （P<0.01） in the gastric tissue， reduced positive expression of GPX4 （P<0.01）， and increased positive expression of 8-OHdG （P<0.01）. Compared with the model group， all the treatment groups showed improved mental state and hair appearance， increased locomotor activity， food and water intake， as well as gradual body weight gain in mice. HE staining demonstrated that the gastric mucosal epithelium of mice in each treatment group was continuous and intact， with normalized gastric fovea morphology， narrowed glandular cavities， increased and neatly arranged glands， and alleviated inflammatory cell infiltration. Transmission electron microscopy results showed that the mitochondria in mice from each treatment group possessed intact structures with recovered normal volume， increased and regularly arranged cristae， and homogeneous density of the mitochondrial membrane. The mice treated with high and medium doses of XLHZ exhibited declined serum levels of IL-6 and IL-1β （P<0.01） and elevated serum level of IL-10 （P<0.01）. Low-dose XLHZ reduced serum IL-1β level in mice （P<0.05）. The expression levels of LAMP1， NCOA4， Steap3 and TRPML1 protein in gastric tissue of mice in XLHZ high- and medium-dose groups were decreased （P<0.05， P<0.01）， and the expression level of FTH1 protein was increased （P<0.05， P<0.01）. The positive expression of GPX4 in the gastric tissue was increased in the high- and medium-dose XLHZ groups （P<0.05， P<0.01）， and that of 8-OHdG was decreased in all treatment groups （P<0.01）. The cell experiments showed that compared with the GES-1 group， the fluorescence intensities of Fe²⁺ and ROS， as well as the co-localization of FTH1 and LAMP1， were significantly increased in the GES-1 + MNNG group （P<0.01）. Compared with the GES-1 + MNNG group， the fluorescence intensities of Fe²⁺ and ROS were markedly decreased in the Model + XLHZ group （P<0.01）， while the co-localization of FTH1 and LAMP1 was reduced （P<0.05）. ConclusionXLHZ can effectively regulate lysosomal iron metabolism and ameliorate gastric mucosal and cellular injury induced by CAG.