OBJECTIVE: To observe the distribution characteristics of sensitization areas on the body surface in the rat models with functional constipation and diarrhea, explore the regulatory patterns of electroacupuncture (EA) of different intensities at "Tianshu" (ST25) on distal colon motility, and clarify the roles of the neurons of different subtypes in the enteric nervous system (ENS) displayed in the regulatory effect. METHODS: Of 90 SD male rats of SPF grade, 15 rats were randomized into a normal group, a constipation group and a diarrhea group, 5 rats in each one. The stool form and fecal water content, as well as the distribution of the Evans blue (EB) extravasation on the body surface after the intravenous injection with EB on the tails were observed. Eighteen rats were randomized into a normal +2 mA group, a normal +4 mA group and a normal + 6 mA group, 6 rats in each one. Using physiological signal acquisition system, the area under the curve and the average amplitude of colon peristalsis were recorded and analyzed, and the immediate effect on distal colon peristalsis observed after EA with different intensities at "Tianshu" (ST25). Thirty rats were randomized into a normal group, a constipation group, a diarrhea group, a constipation +2 mA group, and a diarrhea +6 mA group, 6 rats in each one, so as to observe the cumulative effect on colon motility disorder in the rat models of constipation and diarrhea after EA at "Tianshu" (ST25). Twelve rats were randomized into a constipation +2 mA group and a diarrhea +6 mA group, 6 rats in each one, to observe the immediate effect on colon motility disorder in the rat models of constipation and diarrhea after EA at "Tianshu" (ST25). Fifteen rats were randomly divided into a normal group, a constipation group, a diarrhea group, a constipation +2 mA group, and a diarrhea + 6 mA group, 3 rats in each one. Using the whole-mount staining technique, the expression of vesicular acetylcholine transporter (VAChT)-positive neurons and nitric oxide synthase (nNOS)-positive neurons in ENS was detected. According to the group divisions, the functional constipation models were established by intragastric administration of loperamide hydrochloride (10 mg/kg, once daily, for consecutive 7 days), and the functional diarrhea models were prepared by intragastric administration of folium sennae decoction (10 mL/kg, once daily, for consecutive 2 days). The interventions were delivered with EA of different intensities (the electric current of 2, 4 or 6 mA) at bilateral "Tianshu" (ST25), separately, with the continuous wave and the frequency of 10 Hz used. RESULTS: Compared with the normal group, the fecal amount was decreased, and the fecal water content was reduced in the rats of the constipation group (P<0.001); and loose stool was presented and the fecal water content increased in rats of the diarrhea group (P<0.001). EB extravasation on the body surface happened in the region from T₆ to S₂ of the rats in the constipation and diarrhea groups, and it was more concentrated in the lower abdominal and the lower back regions from T₁₀ to L₃. Compared with the indexes before EA, in the normal +2 mA group and the normal +4 mA group, the areas under the curve and the average amplitude of the distal colon peristalsis were higher during EA delivery (P<0.01, P<0.05), showing a stimulatory immediate effect; and the post-effect was obtained after EA at 2 mA. Whereas, these two indexes were declined during EA in the rats of the normal +6 mA group (P<0.001), showing an inhibitory immediate effect. After many interventions with EA, when compared with those before EA, the above two indexes rose in the constipation +2 mA group (P<0.05, P<0.01), and they were dropped in the diarrhea +6 mA group (P<0.01, P<0.05). The area under the curve of the colon peristalsis in the constipation +2 mA group was higher than that of the constipation group (P<0.001), and that in the diarrhea +6 mA group was lower compared with that in the diarrhea group (P<0.001). The stimulatory effect of EA on colon motility in the constipation +2 mA group was stronger than that of the normal + 2 mA group (P<0.05), and its inhibitory effect was not different statistically in comparison between the normal +6 mA group and the diarrhea +6 mA group (P>0.05). In ENS of the distal colon, after EA at 2 mA, the proportion of VAChT-positive neurons was higher than that of the activated nNOS-positive neurons (P<0.001); and after EA at 6 mA, the activated nNOS-positive neurons were dominant (P<0.001). CONCLUSION In the functional constipation and diarrhea rat models, the sensitization areas on the body surface are centralized in the lower abdominal and the lower back regions of T₁₀ to L₃. Electroacupuncture at "Tianshu" (ST25) has a bidirectional regulatory effect on distal colon motility, and this effect is coordinated with the intensity of electroacupuncture, and may be mediated by ENS neurons of different subtypes.
Animals ; Electroacupuncture ; Male ; Rats ; Colon/innervation* ; Acupuncture Points ; Rats, Sprague-Dawley ; Constipation/physiopathology* ; Gastrointestinal Motility ; Humans ; Diarrhea/physiopathology*

Objective To investigate the impact of Qizhi Jiangtang Capsule (QZJT) on renal damage in diabetic nephropathy (DN) mice via NOD like receptors family pyrin domain containing 3/caspase-1/ Gasdermin D (NLRP3/caspase-1/GSDMD) signaling pathway. Methods Mice were randomly allocated into six experimental groups: a normal control group (NC), a diabetic nephropathy model group (DN), a low-dose QZJT treatment group (L-QZJT), a high-dose QZJT treatment group (H-QZJT), a positive control group administered Shenqi Jiangtang Granules (SQJT), and an ML385 group (treated with an inhibitor of nuclear factor erythroid 2-related factor 2, Nrf2). Upon successful model induction, therapeutic interventions were commenced. Renal function impairment in the mice was evaluated through quantification of fasting blood glucose (FBG), 24-hour urinary albumin (UAlb), serum creatinine (SCr), blood urea nitrogen (BUN), and the kidney-to-body mass ratio (K/B). Renal tissue pathology was evaluated using HE and PAS staining. Serum levels of inflammatory cytokines IL-1β and IL-18 were quantified by ELISA. Levels of podocyte markers and proteins involved in relevant pathways were assessed using Western blot analysis. Results Compared with the NC group, FBG, 24 h UAlb, SCr, and BUN were increased in the DN group, and the K/B mass ratio was also increased. In contrast, compared with the DN group, FBG, 24 h UAlb, SCr, and BUN in both the low-dose (L-QZJT) and high-dose Quanzhou Jintang (H-QZJT) groups were decreased, and the K/B mass ratio was decreased as well. The therapeutic efficacy of H-QZJT was comparable to that of Shenqi Jiangtang Granules. QZJT ameliorated renal histopathological injury in DN mouse, increased the protein levels of Nephrin (a podocyte marker), and decreased the protein levels of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), pro-caspase-1, and GSDMD-N. After ML385 treatment, renal cells exhibited swelling and morphological changes, the inflammatory infiltrate area was enlarged, the protein levels of NLRP3, ASC, pro-caspase-1, and GSDMD-N were up-regulated, and the levels of IL-1β and IL-18 were increased. Conclusion QZJT may inhibit podocyte pyroptosis by acting on the Nrf2 to regulate the NLRP3/caspase-1/GSDMD pathway, thus improving renal damage in DN mouse.
Animals ; Diabetic Nephropathies/pathology* ; Podocytes/pathology* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Pyroptosis/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Caspase 1/genetics* ; Signal Transduction/drug effects* ; Mice ; Phosphate-Binding Proteins/genetics* ; Male ; Intracellular Signaling Peptides and Proteins/metabolism* ; Mice, Inbred C57BL ; Kidney/pathology* ; Gasdermins

Drug resistance is one of the key factors affecting the effectiveness of cancer treatment methods, including chemotherapy, radiotherapy, and immunotherapy. Its occurrence is related to factors such as mRNA expression and methylation within cancer cells. If drug resistance in patients can be accurately identified early, doctors can devise more effective treatment plans, which is of great significance for improving patients' survival rates and quality of life. Cancer drug resistance prediction based on artificial intelligence (AI) technology has emerged as a current research hotspot, demonstrating promising application prospects in guiding clinical individualized and precise medication for cancer patients. This review aims to comprehensively summarize the research progress in utilizing AI algorithms to analyze multi-omics data including genomics, transcriptomics, epigenomics, proteomics, metabolomics, radiomics, and histopathology, for predicting cancer drug resistance. It provides a detailed exposition of the processes involved in data processing and model construction, examines the current challenges faced in this field and future development directions, with the aim of better advancing the progress of precision medicine.

Drug administration via hollow microneedles (HMN) have the advantages of painlessness, avoidance of first-pass effect, capability of sustained infusion, and no need for professional personnel operation. In addition, HMN can also be applied in the fields of body fluid extraction and biosensors, showing broad application prospects. However, traditional manufacturing technologies cannot meet the demand for low-cost mass production of HMN, limiting its widespread application. This paper reviews the main manufacturing technologies used for HMN in recent years, which include photolithography and etching, laser etching, sputtering and electroplating, micro-molding, three-dimensional (3D) printing and drawing lithography. It further analyzes the characteristics and limitations of existing manufacturing technologies and points out that the combination of various manufacturing technologies can improve production efficiency to a certain extent. In addition, this paper looks forward to the future trends of HMN manufacturing technology and proposes possible directions for its development. In conclusion, it is expected that this review can provide new ideas and references for follow-up research.
Printing, Three-Dimensional ; Needles ; Humans ; Drug Delivery Systems/methods* ; Equipment Design ; Microinjections/methods*

Objective : To identify autophagy-related genes involved in pulmonary infection caused by the highly drug-resistant and virulent methicillin-resistant Staphylococcus aureus strain USA300 ( USA300) ，and to explore the underlying molecular mechanisms ， thereby providing potential targets for immunotherapy． Methods: The GSE220943 dataset of a USA300-induced pulmonary infection mouse model was obtained from the GEO database． Differentially expressed genes ( DEGs ) were identified using the DESeq2 package． Autophagy-related genes ( ARGs) were retrieved from the MSigDB and Autophagy databases．Weighted gene co-expression network analysis ( WGCNA) was performed to construct gene co-expression modules．Genes overlapping among DEGs，ARGs，and WGCNA modules were identified as autophagy-related DEGs．Gene Ontology ( GO) enrichment analysis was con- ducted using the clusterProfiler R package，while Kyoto Encyclopedia of Genes and Genomes ( KEGG) pathway en- richment analysis was performed via the Metascape platform．Immune cell infiltration was analyzed using the Immu- CellAI-mouse website．A protein - protein interaction ( PPI) network was constructed using the STRING database， and hub genes were identified through topological analysis in Cytoscape． Receiver operating characteristic curve ( ROC) curves were plotted via the website https: / /www．bioinformatics．com．cn． Finally，key gene expression was validated in mouse lung tissues by real-time quantitative reverse transcription PCR ( RT-qPCR) ． Results: A total of 6 135，4 075，3 680，and 2 342 differentially expressed genes ( DEGs) were identified at 12，24，48，and 96 hours post-infection，respectively．By integrating DEGs，autophagy-related genes ( ARGs) ，and WGCNA mod- ules，19 autophagy-related DEGs were identified． GO and KEGG enrichment analyses indicated that these genes were mainly involved in CD4 + T cell activation and regulation，innate immune responses，and autophagosome mem- brane formation．Immune infiltration analysis revealed that innate immune cells such as neutrophils and dendritic cells predominated during the early phase of infection，while γδ T cells and M2 macrophages became more promi- nent in the later stages．PPI network analysis identified 12 hub autophagy-related genes，among which three upreg- ulated key genes ( Eif2ak2，Ikbke，and Nfkbiz) were further confirmed．The area under the ROC curve for all three genes was 1. 000．RT-qPCR validation demonstrated significantly elevated expression of these three genes in lung tissues at 24 hours post-infection ( all P＜0. 05) ． Conclusion Eif2ak2，Ikbke，and Nfkbiz may be involved in the pulmonary infection caused by USA300 by promoting autophagy and hold promise as potential targets for immuno- therapy．

The objective of this study was to investigate whether Stenotrophomonas maltophilia(S.maltophilia)induces ferroptosis,a form of iron-dependent cell death,leading to an inflamma-tory response in RAW 264.7 macrophages by elevating oxidative stress levels.RAW 264.7 cells were stimulated with varying concentrations of S.maltophilia.The concentrations of TNF-αand IL-1β were quantified using ELISA kits to assess the impact of S.maltophilia on the inflammatory response in RAW 264.7 cells.The activities of glutathione(GSH)and malondialdehyde(MDA)levels were measured using GSH and MDA assay kits to evaluate changes in oxidative stress.West-ern blot analysis was employed to detect the expression levels of COX-2,xCT,GPX4,and other proteins involved in ferroptosis signaling pathways,thereby investigating the effect of S.malto-philia on ferroptosis in RAW 264.7 cells.The results demonstrated that S.maltophilia induced concentration-dependent increases in inflammation and oxidative stress in RAW 264.7 cells,up-regulated the expression of COX-2 protein and down-regulated the expression of xCT and GPX4.Pretreatment with the ROS inhibitor N-acetylcysteine(NAC)significantly mitigated the S.malto-philia-induced oxidative stress and ferroptosis signaling activation,thereby alleviating the inflam-matory response.Furthermore,treatment with the ferroptosis inhibitor Fer-1 directly suppressed the activation of the ferroptosis signaling pathway and reversed the inflammation induced by S.maltophilia.These findings suggest that S.maltophilia triggers inflammation in RAW 264.7 cells by activating the ferroptosis signaling pathway via an increase in oxidative stress levels.

OBJECTIVE: To investigate the role and mechanism of the cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of interferon gene (cGAS/STING) pathway in oleic acid-induced acute lung injury (ALI) in mice. METHODS: Male wild-type C57BL/6J mice were randomly divided into five groups (each n = 10): normal control group, ALI model group, and 5, 50, 500 μg/kg inhibitor pretreatment groups. The ALI model was established by tail vein injection of oleic acid (7 mL/kg), while the normal control group received no intervention. The inhibitor pretreatment groups were intraperitoneally injected with the corresponding doses of cGAS inhibitor RU.521 respectively 1 hour before modeling. At 24 hours post-modeling, blood was collected, and mice were sacrificed. Lung tissue pathological changes were observed under light microscopy after hematoxylin-eosin (HE) staining, and pathological scores were assessed. Western blotting was used to detect the protein expressions of cGAS, STING, phosphorylated TANK-binding kinase 1 (p-TBK1), phosphorylated interferon regulatory factor 3 (p-IRF3), and phosphorylated nuclear factor-κB p65 (p-NF-κB p65) in lung tissue. Immunohistochemistry was performed to observe STING and p-NF-κB positive expressions in lung tissue. Serum interferon-β (IFN-β) levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with the normal control group, the ALI model group exhibited significant focal alveolar thickening, intra-alveolar hemorrhage, pulmonary capillary congestion, and neutrophil infiltration in the pulmonary interstitium and alveoli, along with markedly increased pathological scores (10.33±0.58 vs. 1.33±0.58, P < 0.05). Protein expressions of cGAS, STING, p-TBK1, p-IRF3, and p-NF-κB p65 in lung tissue significantly increased [cGAS protein (cGAS/β-actin): 1.24±0.02 vs. 0.56±0.02, STING protein (STING/β-actin): 1.27±0.01 vs. 0.55±0.01, p-TBK1 protin (p-TBK1/β-actin): 1.34±0.03 vs. 0.22±0.01, p-IRF3 protein (p-IRF3/β-actin): 1.23±0.02 vs. 0.36±0.01, p-NF-κB p65 protein (p-NF-κB p65/β-actin): 1.30±0.02 vs. 0.53±0.02, all P < 0.05], positive expressions of STING and p-NF-κB in lung tissue were significantly elevated [STING (A value): 0.51±0.03 vs. 0.30±0.07, p-NF-κB (A value): 0.57±0.05 vs. 0.31±0.03, both P < 0.05], and serum IFN-β levels were also significantly higher (ng/L: 256.02±3.84 vs. 64.15±1.17, P < 0.05). The cGAS inhibitor pretreatment groups showed restored alveolar structural integrity, reduced inflammatory cell infiltration, and decreased hemorrhage area, along with dose-dependent lower pathological scores as well as the protein expressions of cGAS, STING, p-TBK1, p-IRF3 and p-NF-κB p65 in lung tissue, with significant differences between the 500 μg/kg inhibitor group and ALI model group [pathological score: 2.67±0.58 vs. 10.33±0.58, cGAS protein (cGAS/β-actin): 0.56±0.03 vs. 1.24±0.02, STING protein (STING/β-actin): 0.67±0.03 vs. 1.27±0.01, p-TBK1 protein (p-TBK1/β-actin): 0.28±0.01 vs. 1.34±0.03, p-IRF3 protein (p-IRF3/β-actin): 0.32±0.01 vs. 1.23±0.02, p-NF-κB p65 protein (p-NF-κB p65/β-actin): 0.63±0.01 vs. 1.30±0.02, all P < 0.05]. Compared with the ALI model group, positive expressions of STING and p-NF-κB in lung tissue were significantly reduced in the 500 μg/kg inhibitor group [STING (A value): 0.40±0.01 vs. 0.51±0.03, p-NF-κB (A value): 0.43±0.02 vs. 0.57±0.05, both P < 0.05], and serum IFN-β levels were also markedly reduced (ng/L: 150.03±6.19 vs. 256.02±3.84, P < 0.05). CONCLUSIONS The cGAS/STING pathway is activated in oleic acid-induced ALI, leading to exacerbated inflammatory responses and increased lung damage. RU.521 can inhibit cGAS, thereby down-regulating the expression of pathway proteins and cytokines, and providing protection to lung tissue.
Animals ; Acute Lung Injury/chemically induced* ; Male ; Nucleotidyltransferases/metabolism* ; Mice ; Signal Transduction ; Mice, Inbred C57BL ; Membrane Proteins/metabolism* ; Oleic Acid/adverse effects* ; Transcription Factor RelA/metabolism* ; Lung/pathology* ; Interferon Regulatory Factor-3/metabolism* ; Disease Models, Animal

Triple-negative breast cancer (TNBC) represents an aggressive breast cancer subtype with poor prognosis and limited targeted treatment options. This investigation examined the anti-cancer potential of Caerulomycin A (Cae A), a natural compound derived from marine actinomycetes, against TNBC. Cae A demonstrated selective inhibition of viability and proliferation in TNBC cell lines, including 4T1, MDA-MB-231, and MDA-MB-468, through apoptosis induction. Mechanistic analyses revealed that the compound induced sustained endoplasmic reticulum (ER) stress and subsequent upregulation of C/EBP homologous protein (CHOP) expression, resulting in mitochondrial damage-mediated apoptosis. Inhibition of ER stress or CHOP expression knockdown reversed mitochondrial damage and apoptosis, highlighting the essential role of ER stress and CHOP in Cae A's anti-tumor mechanism. Both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) decreased in TNBC cells following Cae A treatment, indicating reduced mitochondrial respiratory and glycolytic capacities. This diminished energy metabolism potentially triggers ER stress and subsequent apoptosis. Furthermore, Cae A exhibited significant anti-tumor effects in the 4T1 tumor model in vivo without apparent toxicity. The compound also effectively inhibited human TNBC organoid growth. These results indicate that Cae A may serve as a potential therapeutic agent for TNBC, with its efficacy likely mediated through the disruption of glucose metabolism and the induction of ER stress-associated apoptosis.
Humans ; Endoplasmic Reticulum Stress/drug effects* ; Triple Negative Breast Neoplasms/genetics* ; Apoptosis/drug effects* ; Cell Line, Tumor ; Female ; Animals ; Glucose/metabolism* ; Mice ; Cell Proliferation/drug effects* ; Transcription Factor CHOP/genetics* ; Antineoplastic Agents/pharmacology* ; Mitochondria/metabolism* ; Mice, Inbred BALB C

BACKGROUND:In most species,a bone defect that is longer than 1.5 or 2 times its diameter can be considered a critical bone defect,and when the bone defect volume reaches the critical value,it cannot heal on its own.Currently,there is no uniform standard for the size of critical-sized defects in the osteoporotic femoral condyle of rabbits.OBJECTIVE:To establish a rabbit model with different sizes of bone defects in the osteoporotic femoral condyle and to determine the critical-sized defects of osteoporotic femoral condyle in rabbits.METHODS:Thirty-six 3-month-old female New Zealand white rabbits were randomly divided into ovariectomy group(n=30)and sham operation group(n=6).Rabbits in the ovariectomy group underwent bilateral ovariectomy to establish an osteoporosis model,and then femoral condyle bone defect models of different diameters(diameters were 4,5,6,and 7 mm,and depths were 8 mm)were further established;rabbits in the sham operation group did not undergo ovariectomy.At 8 and 12 weeks after modeling,3 rats were randomly selected from each group for CT scanning and three-dimensional reconstruction to evaluate the healing of bone defects.Afterwards,samples were taken for gross observation and hematoxylin-eosin staining to observe the growth of new bone in the femoral condyle bone defect area.RESULTS AND CONCLUSION:(1)All rabbits survived and moved well after modeling of osteoporosis and femoral condyle bone defect.(2)At 12 weeks after osteoporosis modeling,dual-energy X-ray absorptiometry results showed that the bone mineral density of lumbar vertebrae in ovariectomy group was significantly lower than that in sham operation group(P＜0.05).Hematoxylin-eosin staining showed that the bone trabeculae in the ovariectomy group became thinner and sparse.The proportion of bone tissue area in the ovariectomy group was significantly lower than that in the sham operation group(P=0.00).Micro-CT results showed that the bone tissue parameters of the femoral condyle in the ovariectomy group were significantly different from those in the sham operation group(P＜0.05),and the ovariectomy group showed obvious characteristics of osteoporosis.(3)CT showed that the bone defect in the 4 mm and 5 mm diameter groups was basically completely repaired at 12 weeks after surgery.There was more new bone tissue in the 6 mm diameter group,but the central part of the bone defect was not completely repaired.A small amount of new bone tissue grew in the 7 mm diameter group,and the bone defect was obvious.(4)Gross observation at 12 weeks after surgery showed that the femoral condyle bone defect in the 4 mm and 5 mm diameter groups was completely repaired.Obvious depression was seen in the bone defect area of the 6 mm and 7 mm diameter groups,and the bone defect was not completely repaired.(5)Histological observation at 12 weeks after surgery showed that the bone defect area of the 4 mm and 5 mm diameter groups was completely filled with new bone,and the trabecular structure was irregular;while there were new trabeculae in the periphery of the 6 mm and 7 mm diameter groups,and the bone defect in the central area was still obvious.(6)The results showed that during the 12-week experimental observation period of osteoporotic femoral condyle defects in rabbits,under the condition of the same defect depth of 8 mm,femoral condyle defects with a diameter ≥ 6 mm could not heal on their own,while femoral condyle defects with a diameter＜6 mm were completely repaired.A diameter of 6 mm and a depth of 8 mm can be used as the critical bone defect value of osteoporotic femoral condyle in rabbits.

Abstract Cachexia is one of the serious complications in patients with end-stage cancer.Progressive depletion of skeletal muscle is an important feature of cachexia.Previous studies have found that excessive activation of autophagy accelerates skeletal muscle wasting in cachexia,and glutamine released from excessive catabolism of muscle tissue can trigger the autophagy.Mammalian target of rapamycin complex 1(mTORC1) and AMP-activated protein kinase(AMPK) signaling regulate autophagy genesis;moreover,glutamine regulates the mTORC1 and AMPK signaling pathways.Therefore,it is deduced that higher level of glutamine may result in abnormal autophagy by regulating mTORC1 and AMPK in cancer cachexia,which contributes to the development of skeletal muscle wasting.Here,this review discusses the following three perspectives:firstly,autophagy hyperactivation is involved in skeletal muscle wasting in cancer cachexia.Secondly,how mTORC1 and AMPK signaling pathways regulate autophagy.Finally,glutamine is involved in skeletal muscle wasting in cancer cachexia by induced autophagy hyperactivation via regulating mTORC1/AMPK signaling.Our study will provide a scientific basis for the development of potential therapeutic strategies.