ObjectiveTo screen suitable packaging and storage conditions for small packaged Alismatis Rhizoma decoction pieces， laying the foundation for developing standardized storage， maintenance techniques and determining shelf life. MethodsUsing the accelerated stability test method， the small packaged decoction pieces of Alismatis Rhizoma were placed in polyethylene plastic bags， aluminum foil polyethylene composite bags， and cowhide coated paper bags under temperature of （40±2） ℃ and relative humidity of （75±5）% conditions， the quality testing was conducted at the end of the 0^th， 1^st， 2^nd， 3^rd， and 6^th month， respectively. Using long-term stability test method， an orthogonal experiment was designed to investigate storage conditions， packaging materials， and packaging methods. At the end of the 0^th， 1^st， 3^rd， 6^th， 9^th， 12^th， 18^th， and 24^th month， the quality of small packaged Alismatis Rhizoma decoction pieces was tested under different packaging and storage conditions（including 2 packaging methods：vacuum packaging and sealed packaging， 3 storage conditions：room temperature， cool， and modified atmosphere， 3 packaging materials：cowhide coated paper bag， aluminum foil polyethylene composite bag， and polyethylene plastic bag）. Then， the G1-entropy weight method combined with orthogonal experiment was used to analyze the quality changes of the decoction pieces under different packaging and storage conditions to identify optimal packaging and storage conditions. The quality testing indicators for Alismatis Rhizoma decoction pieces were expanded beyond those specified in the 2020 edition of the Pharmacopoeia of the People's Republic of China. In addition to the existing indicators（characteristics， moisture content， extractives， and the total content of 23-acetyl alisol B and 23-acetyl alisol C）， new indicators including color value， water activity， total triterpenoid content， and alisol B content have been added. ResultsThe accelerated stability test results indicated that the quality of small packaged Alismatis Rhizoma decoction pieces was more stable when packaged in aluminum foil-polyethylene composite materials compared to cowhide-coated paper bags and polyethylene plastic bags. Analysis of the long-term stability test results using the G1-entropy weight method combined with orthogonal experiment revealed that storage conditions had the greatest impact on both raw and salt-processed products， followed by packaging materials， while the packaging method had the least influence. For both types of small packaged Alismatis Rhizoma decoction pieces， modified atmosphere storage demonstrated superior efficacy compared to cool storage or room temperature storage. Storage in aluminum foil-polyethylene composite bags was superior to polyethylene plastic bags or cowhide-coated paper bags. However， the stability of sealed raw products was better than vacuum-packed ones， whereas vacuum-packed salt-processed products exhibited greater stability than their sealed counterparts. ConclusionBased on the results of the quality changes of small packaged Alismatis Rhizoma decoction pieces under different storage conditions， it is recommended that the suitable storage packaging conditions for small packaged raw products are sealed packaging with aluminum foil polyethylene composite bags and controlled atmosphere storage， and the suitable storage and packaging conditions for small packaged salt-processed products are vacuum packaging with aluminum foil polyethylene composite bags and controlled atmosphere storage.

ObjectiveTo construct a large language model （LLM）-based intelligent pre-consultation system for traditional Chinese medicine （TCM） to improve efficacy of clinical practice. MethodsA TCM large language model was fine-tuned using DeepSpeed ZeRO-3 distributed training strategy based on YAYI 2-30B. A weighted undirected graph network was designed and an agent-based syndrome differentiation model was established based on relationship data extracted from TCM literature and clinical records. An agent collaboration framework was developed to integrate the TCM LLM with the syndrome differentiation model. Model performance was comprehensively evaluated by Loss function， BLEU-4， and ROUGE-L metrics， through which training convergence， text generation quality， and language understanding capability were assessed. Professional knowledge test sets were developed to evaluate system proficiency in TCM physician licensure content， TCM pharmacist licensure content， TCM symptom terminology recognition， and meridian identification. Clinical tests were conducted to compare the system with attending physicians in terms of diagnostic accuracy， consultation rounds， and consultation duration. ResultsAfter 100 000 iterations， the training loss value was gradually stabilized at about 0.7±0.08， indicating that the TCM-LLM has been trained and has good generalization ability. The TCM-LLM scored 0.38 in BLEU-4 and 0.62 in ROUGE-L， suggesting that its natural language processing ability meets the standard. We obtained 2715 symptom terms， 505 relationships between diseases and syndromes， 1011 relationships between diseases and main symptoms， and 1 303 600 relationships among different symptoms， and constructed the Agent of syndrome differentiation model. The accuracy rates in the simulated tests for TCM practitioners， licensed pharmacists of Chinese materia medica， recognition of TCM symptom terminology， and meridian recognition were 94.09%， 78.00%， 87.50%， and 68.80%， respectively. In clinical tests， the syndrome differentiation accuracy of the system reached 88.33%， with fewer consultation rounds and shorter consultation time compared to the attending physicians （P＜0.01）， suggesting that the system has a certain pre- consultation ability. ConclusionThe LLM-based intelligent TCM pre-diagnosis system could simulate diagnostic thinking of TCM physicians to a certain extent. After understanding the patients' natural language， it collects all the patient's symptom through guided questioning， thereby enhancing the diagnostic and treatment efficiency of physicians as well as the consultation experience of the patients.

This study aims to elucidate the role and mechanism of clematichinenoside AR(CAR) in protecting bone marrow mesenchymal stem cells(BMSCs) from hypoxia-induced apoptosis. BMSCs were isolated by the bone fragment method and identified by flow cytometry. Cells were cultured under normal conditions(37℃, 5% CO_2) and hypoxic conditions(37℃, 90% N_2, 5% CO_2) and treated with CAR. The BMSCs were classified into eight groups: control(normal conditions), CAR(normal conditions + CAR), hypoxia 24 h, hypoxia 24 h + CAR, hypoxia 48 h, hypoxia 48 h + CAR, hypoxia 72 h, and hypoxia 72 h + CAR. The cell counting kit-8(CCK-8) assay and terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) were employed to measure cell proliferation and apoptosis, respectively. The number of mitochondria and mitochondrial membrane potential were measured by MitoTracker®Red CM-H2XRo staining and JC-1 staining, respectively. The level of reactive oxygen species(ROS) was measured with the DCFH-DA fluorescence probe. The protein levels of B-cell lymphoma-2 associated X protein(BAX), caspase-3, and optic atrophy 1(OPA1) were determined by Western blot. The results demonstrated that CAR significantly increased cell proliferation. Compared with the control group, the hypoxia groups showed increased apoptosis rates, reduced mitochondria, elevated ROS levels, decreased mitochondrial membrane potential, upregulated expression of BAX and caspase-3, and downregulated expression of OPA1. In comparison to the corresponding hypoxia groups, CAR intervention significantly decreased the apoptosis rate, increased mitochondria, reduced ROS levels, elevated mitochondrial membrane potential, downregulated the expression of BAX and caspase-3, and upregulated the expression of OPA1. Therefore, it can be concluded that CAR may exert an anti-apoptotic effect on BMSCs under hypoxic conditions by regulating OPA1 to maintain mitochondrial homeostasis.
Mesenchymal Stem Cells/metabolism* ; Apoptosis/drug effects* ; Mitochondria/metabolism* ; Animals ; Rats ; Cell Hypoxia/drug effects* ; Homeostasis/drug effects* ; Reactive Oxygen Species/metabolism* ; Rats, Sprague-Dawley ; Membrane Potential, Mitochondrial/drug effects* ; Saponins/pharmacology* ; Caspase 3/genetics* ; Male ; bcl-2-Associated X Protein/genetics* ; Bone Marrow Cells/metabolism* ; Cell Proliferation/drug effects* ; Protective Agents/pharmacology* ; Cells, Cultured

Emodin is a hydroxyanthraquinone compound that is widely distributed and has multiple pharmacological activities, including anti-diarrheal, anti-inflammatory, and liver-protective effects. Research indicates that emodin may be one of the main components responsible for inducing hepatotoxicity. However, studies on the mechanisms of liver injury are relatively limited, particularly those related to bile acids(BAs) metabolism. This study aims to systematically investigate the effects of different dosages of emodin on BAs metabolism, providing a basis for the safe clinical use of traditional Chinese medicine(TCM)containing emodin. First, this study evaluated the safety of repeated administration of different dosages of emodin over a 5-week period, with a particular focus on its impact on the liver. Next, the composition and content of BAs in serum and liver were analyzed. Subsequently, qRT-PCR was used to detect the mRNA expression of nuclear receptors and transporters related to BAs metabolism. The results showed that 1 g·kg~(-1) emodin induced hepatic damage, with bile duct hyperplasia as the primary pathological manifestation. It significantly increased the levels of various BAs in the serum and primary BAs(including taurine-conjugated and free BAs) in the liver. Additionally, it downregulated the mRNA expression of farnesoid X receptor(FXR), retinoid X receptor(RXR), and sodium taurocholate cotransporting polypeptide(NTCP), and upregulated the mRNA expression of cholesterol 7α-hydroxylase(CYP7A1) in the liver. Although 0.01 g·kg~(-1) and 0.03 g·kg~(-1) emodin did not induce obvious liver injury, they significantly increased the level of taurine-conjugated BAs in the liver, suggesting a potential interference with BAs homeostasis. In conclusion, 1 g·kg~(-1) emodin may promote the production of primary BAs in the liver by affecting the FXR-RXR-CYP7A1 pathway, inhibit NTCP expression, and reduce BA reabsorption in the liver, resulting in BA accumulation in the peripheral blood. This disruption of BA homeostasis leads to liver injury. Even doses of emodin close to the clinical dose can also have a certain effect on the homeostasis of BAs. Therefore, when using traditional Chinese medicine or formulas containing emodin in clinical practice, it is necessary to regularly monitor liver function indicators and closely monitor the risk of drug-induced liver injury.
Emodin/administration & dosage* ; Bile Acids and Salts/metabolism* ; Animals ; Male ; Liver/injuries* ; Chemical and Drug Induced Liver Injury/genetics* ; Drugs, Chinese Herbal/adverse effects* ; Humans ; Rats, Sprague-Dawley ; Mice ; Rats

The chemical components of Carthami Flos were investigated by using macroporous resin, silica gel column chromatography, reversed-phase octadecylsilane(ODS) column chromatography, Sephadex LH-20, and semi-preparative high-performance liquid chromatography(HPLC). The planar structures of the compounds were established based on their physicochemical properties and ultraviolet-visible(UV-Vis), infrared(IR), high-resolution electrospray ionization mass spectrometry(HR-ESI-MS), and nuclear magnetic resonance(NMR) spectroscopic technology. The absolute configurations were determined by comparing the calculated and experimental electronic circular dichroism(ECD). Six flavonoid C-glycosides were isolated from the 30% ethanol elution fraction of macroporous resin obtained from the 95% ethanol extract of Carthami Flos, and identified as saffloquinoside F(1), 5-hydroxysaffloneoside(2), iso-5-hydroxysaffloneoside(3), isosafflomin C(4), safflomin C(5), and vicenin 2(6). Among these, the compounds 1 to 3 were new chalcone C-glycosides. The compounds 1, 2, 4, and 5 could significantly increase the viability of H9c2 cardiomyocytes damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) at a concentration of 50 μmol·L~(-1), showing their good cardioprotective activity.
Glycosides/pharmacology* ; Flowers/chemistry* ; Drugs, Chinese Herbal/pharmacology* ; Carthamus tinctorius/chemistry* ; Chalcones/pharmacology* ; Animals

Testicular descent occurs in two consecutive stages: the transabdominal stage and the inguinoscrotal stage. Androgens play a crucial role in the second stage by influencing the development of the gubernaculum, a structure that pulls the testis into the scrotum. However, the mechanisms of androgen actions underlying many of the processes associated with gubernaculum development have not been fully elucidated. To identify the androgen-regulated genes, we conducted large-scale gene expression analyses on the gubernaculum harvested from luteinizing hormone/choriogonadotropin receptor knockout ( Lhcgr KO) mice, an animal model of inguinoscrotal testis maldescent resulting from androgen deficiency. We found that the expression of secreted protein acidic and rich in cysteine (SPARC)-related modular calcium binding 1 ( Smoc1 ) was the most severely suppressed at both the transcript and protein levels, while its expression was the most dramatically induced by testosterone administration in the gubernacula of Lhcgr KO mice. The upregulation of Smoc1 expression by testosterone was curtailed by the addition of an androgen receptor antagonist, flutamide. In addition, in vitro studies demonstrated that SMOC1 modestly but significantly promoted the proliferation of gubernacular cells. In the cultures of myogenic differentiation medium, both testosterone and SMOC1 enhanced the expression of myogenic regulatory factors such as paired box 7 ( Pax7 ) and myogenic factor 5 ( Myf5 ). After short-interfering RNA-mediated knocking down of Smoc1 , the expression of Pax7 and Myf5 diminished, and testosterone alone did not recover, but additional SMOC1 did. These observations indicate that SMOC1 is pivotal in mediating androgen action to regulate gubernaculum development during inguinoscrotal testicular descent.
Animals ; Male ; Mice ; Testis/growth & development* ; Mice, Knockout ; Androgens/pharmacology* ; Testosterone/pharmacology* ; Receptors, LH/metabolism* ; Calcium-Binding Proteins/metabolism*

OBJECTIVE: To investigate the protective effects of stir-fried Semen Armeniacae Amarum (SAA) against aristolochic acid I (AAI)-induced nephrotoxicity and DNA adducts and elucidate the underlying mechanism involved for ensuring the safe use of Asari Radix et Rhizoma. METHODS: In vitro, HEK293T cells overexpressing Flag-tagged multidrug resistance-associated protein 3 (MRP3) were constructed by Lentiviral transduction, and inhibitory effect of top 10 common pairs of medicinal herbs with Asari Radix et Rhizoma in clinic on MRP3 activity was verified using a self-constructed fluorescence screening system. The mRNA, protein expressions, and enzyme activity levels of NAD(P)H quinone dehydrogenase 1 (NQO1) and cytochrome P450 1A2 (CYP1A2) were measured in differentiated HepaRG cells. Hepatocyte toxicity after inhibition of AAI metabolite transport was detected using cell counting kit-8 assay. In vivo, C57BL/6 mice were randomly divided into 5 groups according to a random number table, including: control (1% sodium bicarbonate), AAI (10 mg/kg), stir-fried SAA (1.75 g/kg) and AAI + stir-fried SAA (1.75 and 8.75 g/kg) groups, 6 mice in each group. After 7 days of continuous gavage administration, liver and kidney damages were assessed, and the protein expressions and enzyme activity of liver metabolic enzymes NQO1 and CYP1A2 were determined simultaneously. RESULTS: In vivo, combination of 1.75 g/kg SAA and 10 mg/kg AAI suppressed AAI-induced nephrotoxicity and reduced dA-ALI formation by 26.7%, and these detoxification effects in a dose-dependent manner (P<0.01). Mechanistically, SAA inhibited MRP3 transport in vitro, downregulated NQO1 expression in vivo, increased CYP1A2 expression and enzymatic activity in vitro and in vivo, respectively (P<0.05 or P<0.01). Notably, SAA also reduced AAI-induced hepatotoxicity throughout the detoxification process, as indicated by a 41.3% reduction in the number of liver adducts (P<0.01). CONCLUSIONS Stir-fried SAA is a novel drug candidate for the suppression of AAI-induced liver and kidney damages. The protective mechanism may be closely related to the regulation of transporters and metabolic enzymes.
Aristolochic Acids/toxicity* ; Animals ; Humans ; NAD(P)H Dehydrogenase (Quinone)/genetics* ; HEK293 Cells ; Kidney/pathology* ; Cytochrome P-450 CYP1A2/genetics* ; Mice, Inbred C57BL ; DNA Adducts/drug effects* ; Male ; Kidney Diseases/drug therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Mice ; Prunus armeniaca ; Plant Extracts

OBJECTIVES: To investigate the inhibitory effect of multimerin-2 (MMRN2) overexpression on growth and metastasis of cutaneous melanoma cells. METHODS: Clinical data of patients with cutaneous melanoma were obtained from the GEO database to compare MMRN2 expressions between normal and tumor tissues. A protein-protein interaction network was constructed using the STRING database, and the intersecting genes from GEPIA2.0 were subjected to GO and KEGG enrichment analysis. The prognostic relevance of MMRN2 expression level was assessed using Cox regression and "timeROC". The correlations of MMRN2 expression level with immune infiltration and angiogenesis-related genes were analyzed using GSCA database and the ssGSEA algorithm. Colony-forming assay, Transwell assay, and wound healing assay were used to examine the changes in proliferation and migration of cultured cutaneous melanoma cells following MMRN2 knockdown. In a mouse model bearing cutaneous melanoma xenograft, the effect of MMRN2 knockdown on vital organ pathologies, survival of the mice and GM-CSF, CXCL9, and TGF‑β1 protein expressions were analyzed. RESULTS: MMRN2 was significantly upregulated in metastatic cutaneous melanoma (P<0.001). Protein interaction network analysis identified 15 intersecting genes, which were enriched in endothelium development and cell-cell junctions. In patients with cutaneous melanoma, a high MMRN2 expression was correlated with a poor prognosis, an advanced T stage, a greater Breslow depth, and ulceration (P<0.05). MMRN2 expression level was strongly correlated with 24 immune cell types (P<0.001), fibroblasts, endothelial cells, and expressions of the pro-angiogenic genes (KCNJ8, SLCO2A1, NRP1, and COL3A1; P<0.001). In cultured B16F10 cells, MMRN2 knockdown significantly suppressed cell proliferation, migration and invasion and caused remo-deling of the immunosuppressive microenvironment. CONCLUSIONS MMRN2 overexpression drives progression of cutaneous melanoma by enhancing tumor metastasis, angiogenesis and immune evasion, highlighting its potential as a therapeutic target for melanomas.
Humans ; Melanoma/metabolism* ; Animals ; Cell Movement ; Prognosis ; Skin Neoplasms/metabolism* ; Mice ; Cell Proliferation ; Neoplasm Invasiveness ; Cell Line, Tumor ; Protein Interaction Maps

OBJECTIVE: Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection frequently develop central nervous system damage, yet the mechanisms driving this pathology remain unclear. This study investigated the primary pathways and key factors underlying brain tissue damage induced by the SARS-CoV-2 beta variant (lineage B.1.351). METHODS: K18-hACE2 and C57BL/6 mice were intranasally infected with the SARS-CoV-2 beta variant. Viral replication, pathological phenotypes, and brain transcriptomes were analyzed. Gene Ontology (GO) analysis was performed to identify altered pathways. Expression changes of host genes were verified using reverse transcription-quantitative polymerase chain reaction and Western blot. RESULTS: Pathological alterations were observed in the lungs of both mouse strains. However, only K18-hACE2 mice exhibited elevated viral RNA loads and infectious titers in the brain at 3 days post-infection, accompanied by neuropathological injury and weight loss. GO analysis of infected K18-hACE2 brain tissue revealed significant dysregulation of genes associated with innate immunity and antiviral defense responses, including type I interferons, pro-inflammatory cytokines, Toll-like receptor signaling components, and interferon-stimulated genes. Neuroinflammation was evident, alongside activation of apoptotic and pyroptotic pathways. Furthermore, altered neural cell marker expression suggested viral-induced neuroglial activation, resulting in caspase 4 and lipocalin 2 release and disruption of neuronal molecular networks. CONCLUSION These findings elucidate mechanisms of neuropathogenicity associated with the SARS-CoV-2 beta variant and highlight therapeutic targets to mitigate COVID-19-related neurological dysfunction.
Animals ; COVID-19/genetics* ; Mice ; Brain/metabolism* ; Apoptosis ; Mice, Inbred C57BL ; SARS-CoV-2/physiology* ; Pyroptosis ; Gene Expression Profiling ; Transcriptome ; Male ; Female

Objective To study the apoptosis and mechanism of lung epithelial cells induced by exposure to ozone oxidized black carbon(OBC)and explore the therapeutic effect of dexamethasone.Methods A549 cells were divided into seven groups:control group,dexamethasone group and 0,6.5,12.5,25.0,50.0 μg·mL-1OBC treatment groups.All cells were cultured for 24 h.Cell counting kit-8(CCK-8)was used to detect the survival rate of cells.Immunofluorescence was used to detect the activity of reactive oxygen species.Annexin V-FITC/PI two-color fluorescent dye Notation was used to detect apoptotic cells.Real-time quantitative polymerase chain reaction(PCR)was used to detect the expression level of P53,B-cell lymphoma-2(Bcl-2),cysteinyl aspartate specific proteinase-3(caspase-3)mRNA.Western blot was used to detect the expression level of P53,Bcl-2,caspase-3 protein.Results The cell survival rates of 50.0 μg·mL-1 OBC treatment group,control group and dexamethasone group were(12.64±2.45)％,(100.00±0.00)％and(42.39±2.50)％;the reactive oxygen species were(147.00±5.65),(42.82±2.50)and(94.40±4.30)MFI;the apoptosis rates of epithelial cells were(40.50±3.52)％,(5.02±1.15)％and(21.58±2.35)％;the expression of P53 mRNA were 3.81±0.22,1.04±0.02 and 2.03±0.08;the equivalent expression levels of P53 protein were 0.54±0.06,0.14±0.02 and 0.33±0.03.The above indicators in 50.0 μg·mL-1 OBC treatment group showed statistically significant differences compared with control group and dexamethasone group(all P＜0.05).Conclusion OBC exposure leads to oxidative damage of lung epithelial cells and activates their apoptosis related pathways,while dexamethasone has shown good therapeutic effects in vitro experiments.