Objective: To propose an improved method for constructing the internal quality control (IQC) framework for ELISA assays and validate its efficacy by statistically analyzing IQC data from nine blood center laboratories. Methods: 1) IQC data was collected from nine blood centers and analyzed using a domestic HBsAg ELISA detection kit as an example. 2) Differences between IQC values across batches within Blood Center 1 were assessed. 3) Statistical analyses were performed on batch usage, number of batches used, days of use, number of QC points, batch-specific means, and coefficients of variation (CV) across all nine centers. 4) Using the improved construction method for IQC framework, provisional and permanent frames were established for batches within Blood Center 1 and Blood Center 9, followed by outlier determination. Results: 1) Statistically significant differences were observed in IQC data between batches within Blood Center 1 (P<0.01). It is recommended that both the control material/reagents and the control chart framework be replaced simultaneously. 2) There were substantial differences among 9 blood centers regarding the control material/reagent lot numbers used, the number of QC runs per batch, and the QC values for identical lots. Therefore, individual laboratories should establish their own IQC chart frameworks. 3) The improved IQC framework construction method for ELISA assays is as follows: provisional frames are established via frame-shifting, using the pre-experimental mean and cumulative coefficient of variation (CV) from the preceding batch. For batches used >20 days with >20 QC points, permanent frames are constructed by aggregating in-control data accumulated over ≥20 days with ≥20 points to calculate cumulative mean and standard deviation. The provisional and permanent frames constructed by this method identified all 26 extreme outliers across Blood Centers 1 and 9 as out-of-control. Among the 218 general outliers, 10 were classified as normal by the provisional frames, while the remainder were designated as warnings or out-of-control. This method effectively monitors assay stability. Conclusion: Based on the statistical analysis of IQC practices across blood centers of varying scales, combined with the inherent characteristics of ELISA assays and the batch-to-batch instability of reagents/QC materials, it is recommended to reconstruct QC charts upon lot changes. The proposed method—utilizing frame-shifting for provisional frames and establishing permanent frames based on cumulative data—is applicable to blood center laboratories of differing sizes and effectively monitors the stability of the ELISA assay process.

ObjectiveTo investigate the mechanism of ferroptosis mediated by long-chain acyl-CoA synthetase 4 （ACSL4） signalling pathway in rats with coronary heart disease with blood stasis syndrome and the intervention effect of Xuefu Zhuyutang. MethodsSPF male SD rats were randomly divided into normal group， sham-operation group， model group， trimetazidine group （5.4 mg·kg^-1）， low-， medium-， and high-dose group （3.51， 7.02，14.04 g·kg^-1） of Xuefu Zhuyutang. The coronary artery left anterior descending ligation method was used to prepare a model of coronary heart disease with blood stasis syndrome， and continuous treatment for 7 d was conducted， while the sham-operation group was only threaded and not ligated. The general macroscopic symptoms of the rats were observed， and indicators such as electrocardiogram， echocardiography， and blood rheology were detected. The pathological morphology of myocardial tissue was observed by hematoxylin-eosin （HE） staining， and the changes in mitochondria in myocardial tissue were observed by transmission electron microscopy. The level of iron deposition in myocardial tissue was observed by Prussian blue staining. The levels of 12-hydroxyeicosatetraenoic acid （12-HETE） and 15-HETE were detected in serum by enzyme-linked immunosorbent assay. A biochemical colourimetric assay was used to detect the levels of Fe²⁺， lipid peroxidation （LPO）， glutathione （GSH）， and T-GSH/glutathione disulfide （GSSG） in myocardial tissue. DCFH-DA fluorescence quantitative assay was employed to detect the levels of reactive oxygen species （ROS）. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was adopted to detect the protein and mRNA expressions of glutathione peroxidase 4 （GPX4）， ferritin heavy chain 1 （FTH1）， ACSL4， and ly-sophosphatidylcholine acyltransferase3 （LPCAT3） in myocardial tissue. ResultsCompared with those in the normal group， the rats in the model group were poor in general macroscopic symptoms. The electrocardiogram showed widened QRS wave amplitude and increased voltage， bow-back elevation of the ST segments， elevated T waves， J-point elevation， and accelerated heart rate. Echocardiography showed a significant reduction in left ventricular ejection fraction （LVEF） and left ventricular fraction shortening （LVFS） （P<0.01）. Blood rheology showed that the viscosity of the whole blood （low， medium， and high rate of shear） was significantly increased （P<0.01）. HE staining showed an abnormal structure of myocardial tissue. There was a large area of myocardial necrosis and inflammatory cell infiltration and a large number of connective tissue between myocardial fibers. Transmission electron microscopy showed that the mitochondria were severely atrophy or swelling. The cristae were reduced or even broken， and the matrix was flocculent or even vacuolated. Prussian blue staining showed that there were a large number of iron-containing particles， and the iron deposition was obvious. The content of 12-HETE and 15-HETE in the serum was significantly increased （P<0.01）. The content of Fe²⁺， LPO， and ROS in myocardial tissue was significantly increased （P<0.01）. The content of GSH was significantly decreased （P<0.01）， and T-GSH/GSSG was decreased （P<0.01）. The protein and mRNA expressions of GPX4 and FTH1 in myocardial tissue were both significantly decreased （P<0.05， P<0.01）， while those of ACSL4 and LPCAT3 increased significantly （P<0.01）. Compared with the model group， the general macroscopic symptoms and electrocardiogram results of rats in low-， medium- and high-dose groups of Xuefu Zhuyutang were alleviated， and the differences in LVEF/LVFS ratios were all significantly increased （P<0.05， P<0.01）. The differences in whole-blood viscosity （low， medium， and high rate of shear） were all significantly decreased （P<0.01）. The results of HE staining and transmission electron microscopy showed that the morphology， structure， and mitochondria of cardiomyocytes were improved. The content of 12-HETE and 15-HETE in serum was reduced to different degrees in low-， medium-， and high-dose groups of Xuefu Zhuyutang （P<0.05， P<0.01）. The content of Fe²⁺， LPO， and ROS was significantly reduced in the medium- and high-dose groups of Xuefu Zhuyutang （P<0.05， P<0.01）， and the content of GSH and T-GSH/GSSG was significantly increased （P<0.05， P<0.01）. The protein and mRNA expressions of GPX4 and FTH1 were significantly increased to varying degrees in the medium- and high-dose groups of Xuefu Zhuyutang （P<0.05， P<0.01）， and ACSL4 and LPCAT3 were decreased to different degrees in the low-， medium-， and high-dose groups of Xuefu Zhuyutang （P<0.05， P<0.01）. ConclusionXuefu Zhuyutang can regulate iron metabolism and anti-lipid oxidation reaction to mediate ferroptosis through the ACSL4 signalling pathway， thus exerting a protective effect on rats with coronary heart disease with blood stasis syndrome.

The circadian rhythm regulates the 24-hour physiological and behavioral cycles through endogenous molecular clocks governed by core clock genes via the transcription-translation feedback loop (TTFL). In mammals, the suprachiasmatic nucleus (SCN) serves as the central pacemaker, coordinating the timing of physiological processes throughout the body by regulating clock genes such as CLOCK, BMAL1, PER, and CRY. The molecular clocks of peripheral tissues and cells are synchronized by the SCN through TTFLs to regulate metabolism, immunity, and energy homeostasis. Numerous studies indicate that circadian rhythm disruption is closely related to obesity, type 2 diabetes, metabolic syndrome and other diseases, and the mechanism involves the dysregulation of glucose and lipid metabolism, abnormal insulin signaling and low-grade inflammation. In recent years, small-molecule compounds targeting the core clock components such as CRY, REV-ERB, and ROR have been identified and shown potential to modulate metabolic diseases by stabilizing or inhibiting the activity of key clock proteins. This review summarizes the mechanisms and advances in these compounds, and explores the challenges and future directions for their clinical translation, providing insights for chronotherapy-based metabolic disease interventions.
Humans ; Metabolic Diseases/physiopathology* ; Animals ; Circadian Rhythm/physiology* ; Biological Clocks/drug effects* ; CLOCK Proteins/physiology* ; Circadian Clocks/physiology* ; Suprachiasmatic Nucleus/physiology*

This study aims to investigate the molecular mechanism by which naringin alleviates cerebral ischemia/reperfusion(CI/R) injury through DRP1/LRRK2/MCU signaling axis. A total of 60 SD rats were randomly divided into the sham group, the model group, the sodium Danshensu group, and low-, medium-, and high-dose(50, 100, and 200 mg·kg~(-1)) naringin groups, with 10 rats in each group. Except for the sham group, a transient middle cerebral artery occlusion/reperfusion(tMCAO/R) model was established in SD rats using the suture method. Longa 5-point scale was used to assess neurological deficits. 2,3,5-Triphenyl tetrazolium chloride(TTC) staining was used to detect the volume percentage of cerebral infarction in rats. Hematoxylin-eosin(HE) staining and Nissl staining were employed to assess neuronal structural alterations and the number of Nissl bodies in cortex, respectively. Western blot was used to determine the protein expression levels of B-cell lymphoma-2 gene(Bcl-2), Bcl-2-associated X protein(Bax), cleaved cysteine-aspartate protease-3(cleaved caspase-3), mitochondrial calcium uniporter(MCU), microtubule-associated protein 1 light chain 3(LC3), and P62. Mitochondrial structure and autophagy in cortical neurons were observed by transmission electron microscopy. Immunofluorescence assay was used to quantify the fluorescence intensities of MCU and mitochondrial calcium ion, as well as the co-localization of dynamin-related protein 1(DRP1) with leucine-rich repeat kinase 2(LRRK2) and translocase of outer mitochondrial membrane 20(TOMM20) with LC3 in cortical mitochondria. The results showed that compared with the model group, naringin significantly decreased the volume percentage of cerebral infarction and neurological deficit score in tMCAO/R rats, alleviated the structural damage and Nissl body loss of cortical neurons in tMCAO/R rats, inhibited autophagosomes in cortical neurons, and increased the average diameter of cortical mitochondria. The Western blot results showed that compared to the sham group, the model group exhibited increased levels of cleaved caspase-3, Bax, MCU, and the LC3Ⅱ/LC3Ⅰ ratio in the cortex and reduced protein levels of Bcl-2 and P62. However, naringin down-regulated the protein expression of cleaved caspase-3, Bax, MCU and the ratio of LC3Ⅱ/LC3Ⅰ ratio and up-regulated the expression of Bcl-2 and P62 proteins in cortical area. In addition, immunofluorescence analysis showed that compared with the model group, naringin and positive drug treatments significantly decreased the fluorescence intensities of MCU and mitochondrial calcium ion. Meanwhile, the co-localization of DRP1 with LRRK2 and TOMM20 with LC3 in cortical mitochondria was also decreased significantly after the intervention. These findings suggest that naringin can alleviate cortical neuronal damage in tMCAO/R rats by inhibiting DRP1/LRRK2/MCU-mediated mitochondrial fragmentation and the resultant excessive mitophagy.
Animals ; Rats, Sprague-Dawley ; Reperfusion Injury/genetics* ; Flavanones/administration & dosage* ; Rats ; Dynamins/genetics* ; Male ; Brain Ischemia/genetics* ; Protein Serine-Threonine Kinases/genetics* ; Signal Transduction/drug effects* ; Humans ; Drugs, Chinese Herbal/administration & dosage*

To explore the common irritant components in different base sources of Polygonati Rhizoma(PR). A rabbit eye irritation experiment was conducted to compare the irritant effects of raw products of Polygonatum kingianum, P. officinale, and P. multiflorum. The irritant effects of different solvent extraction parts and needle crystals of PR were compared, and the irritant components were screened. The morphology and structure of the purified needle crystal of PR were observed by microscope and scanning electron microscope and characterized by X-ray diffraction. Rabbit eye irritation and mouse abdominal inflammation model were used to evaluate rabbit eye irritation scores, inflammatory mediators, inflammatory factors levels in the peritoneal exudate of mice, with the peritoneal pathological section used as indicators. The inflammatory effect of needle crystals of PR was studied, and the content of calcium oxalate in three kinds of PR was determined by HPLC. The common protein in three kinds of PR was screened and compared by double enzymatic hydrolysis in solution combined with mass spectrometry. The results showed that three kinds of PR raw products had certain irritant effects on rabbit eyes, among which P. kingianum had the strongest irritant effect. There were no obvious irritant effects in the different solvent extraction parts of P. kingianum. Compared with the blank group, the needle crystal of PR had a significant irritant effect on rabbit eyes, and the inflammatory mediators and inflammatory factors in the peritoneal exudate were significantly increased(P<0.05) in a dose-dependent manner. Meanwhile, the peritoneal tissue of mice was damaged with significant inflammatory cell infiltration after intraperitoneal injection of needle crystal, indicating that needle crystal had an inflammatory effect. Microscope and scanning electron microscope observations showed that the needle crystals of PR were slender, with a length of about 100-200 μm and sharp ends. X-ray diffraction analysis showed that the needle crystals of PR were calcium oxalate monohydrate crystals. The results of HPLC showed that the content of calcium oxalate in P. kingianum was the highest among the three kinds of PR. It was speculated that the content of needle crystal in P. kingianum was higher than that in P. officinale and P. multiflorum, which was consistent with the results of the rabbit eye irritation experiment. The results of mass spectrometry showed that ribosome inactivating protein and mannose/sialic acid binding lectin were related to inflammation and cell metabolism in all three kinds of PR. There was no obvious irritant effect in different solvent extracts of PR. The calcium oxalate needle crystal contained was the main irritant component of PR, and three kinds of PR contained common ribosome inactivating protein and mannose/sialic acid binding lectin, which may be related to the inflammatory irritant effect of PR.
Animals ; Rabbits ; Mice ; Polygonatum/chemistry* ; Drugs, Chinese Herbal/toxicity* ; Rhizome/chemistry* ; Male ; Eye/drug effects* ; Female ; Humans

OBJECTIVE: To explore and verify the effect and potential mechanism of Brucea javanica Seed Oil Emulsion Injection (YDZI) and Shengmai Injection (SMI) on peripheral microcirculation dysfunction in treatment of gastric cancer (GC). METHODS: The potential mechanisms of YDZI and SMI were explored through network pharmacology and verified by cellular and clinical experiments. Human microvascular endothelial cells (HMECs) were cultured for quantitative real-time polymerase chain reaction, Western blot analysis, and human umbilical vein endothelial cells (HUVECs) were cultured for tube formation assay. Twenty healthy volunteers and 97 patients with GC were enrolled. Patients were divided into surgical resection, surgical resection with chemotherapy, and surgical resection with chemotherapy combining YDZI and SMI groups. Forearm skin blood perfusion was measured and recorded by laser speckle contrast imaging coupled with post-occlusive reactive hyperemia. Cutaneous vascular conductance and microvascular reactivity parameters were calculated and compared across the groups. RESULTS: After network pharmacology analysis, 4 ingredients, 82 active compounds, and 92 related genes in YDZI and SMI were screened out. β-Sitosterol, an active ingredient and intersection compound of YDZI and SMI, upregulated the expression of vascular endothelial growth factor A (VEGFA) and prostaglandin-endoperoxide synthase 2 (PTGS2, P<0.01), downregulated the expression of caspase 9 (CASP9) and estrogen receptor 1 (ESR1, P<0.01) in HMECs under oxaliplatin stimulation, and promoted tube formation through VEGFA. Chemotherapy significantly impaired the microvascular reactivity in GC patients, whereas YDZI and SMI ameliorated this injury (P<0.05 or P<0.01). CONCLUSIONS YDZI and SMI ameliorated peripheral microvascular reactivity in GC patients. β-Sitosterol may improve peripheral microcirculation by regulating VEGFA, PTGS2, ESR1, and CASP9.
Humans ; Microcirculation/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Stomach Neoplasms/physiopathology* ; Emulsions ; Male ; Plant Oils/administration & dosage* ; Brucea/chemistry* ; Middle Aged ; Female ; Drug Combinations ; Human Umbilical Vein Endothelial Cells/metabolism* ; Seeds/chemistry* ; Injections ; Vascular Endothelial Growth Factor A/metabolism* ; Aged ; Network Pharmacology

OBJECTIVE: To evaluate the dynamic changes of glucocorticoid (GC) dose and the feasibility of GC discontinuation in rheumatoid arthritis (RA) patients under the background of Chinese medicine (CM). METHODS: This multicenter retrospective cohort study included 1,196 RA patients enrolled in the China Rheumatoid Arthritis Registry of Patients with Chinese Medicine (CERTAIN) from September 1, 2019 to December 4, 2023, who initiated GC therapy. Participants were divided into the Western medicine (WM) and integrative medicine (IM, combination of CM and WM) groups based on medication regimen. Follow-up was performed at least every 3 months to assess dynamic changes in GC dose. Changes in GC dose were analyzed by generalized estimator equation, the probability of GC discontinuation was assessed using Kaplan-Meier curve, and predictors of GC discontinuation were analyzed by Cox regression. Patients with <12 months of follow-up were excluded for the sensitivity analysis. RESULTS: Among 1,196 patients (85.4% female; median age 56.4 years), 880 (73.6%) received IM. Over a median 12-month follow-up, 34.3% (410 cases) discontinued GC, with significantly higher rates in the IM group (40.8% vs. 16.1% in WM; P<0.05). GC dose declined progressively, with IM patients demonstrating faster reductions (median 3.75 mg vs. 5.00 mg in WM at 12 months; P<0.05). Multivariate Cox analysis identified age <60 years [P<0.001, hazard ratios (HR)=2.142, 95% confidence interval (CI): 1.523-3.012], IM therapy (P=0.001, HR=2.175, 95% CI: 1.369-3.456), baseline GC dose ⩽7.5 mg (P=0.003, HR=1.637, 95% CI: 1.177-2.275), and absence of non-steroidal anti-inflammatory drugs use (P=0.001, HR=2.546, 95% CI: 1.432-4.527) as significant predictors of GC discontinuation. Sensitivity analysis (545 cases) confirmed these findings. CONCLUSIONS RA patients receiving CM face difficulties in following guideline-recommended GC discontinuation protocols. IM can promote GC discontinuation and is a promising strategy to reduce GC dependency in RA management. (Trial registration: ClinicalTrials.gov, No. NCT05219214).
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Arthritis, Rheumatoid/drug therapy* ; Glucocorticoids/therapeutic use* ; Medicine, Chinese Traditional ; Retrospective Studies

Objective To investigate the relationship of aberrant SMAD family member 7(SMAD7)signaling pathway and ferroptosis in myelodysplastic syndromes(MDS)and evaluate the effect of asiaticoside(AC)-modulating SMAD7 up-regulation to suppress ferroptosis in MDS cell lines.Methods Publicly available MDS-related datasets from the Gene Expression Omnibus(GEO)database were analyzed to identify differentially expressed genes(DEGs)between MDS patients and healthy controls.These DEGs were cross-referenced with ferroptosis-associated genes from the Ferroptosis Database(FerrDb)to identify potential ferroptosis-related targets in MDS.Bone marrow mononuclear cells(BMMNCs)were isolated from 18 MDS patients freshly diagnosed in the First Medical Center of Chinese PLA General Hospitaland and from 16 healthy donors during October 2022 and November 2024.RT-qPCR was employed to detect the expression of SMAD7 and ferroptosis-related genes.Immunomagnetic bead sorting was applied to purify CD33+cells,and then qPCR and Western blotting were utilized to measure the expression of SMAD7 and ferroptosis-related biomarkers at mRNA and protein levels.Human normal bone marrow cells(HS-5)and MDS cell lines(MUTZ-1,SKM-1)were treated with gradient concentrations of AC(SMAD7 activator)and ferrostatin-1(Fer-1,ferroptosis inhibitor),and SMAD7 overexpression plasmids were transfected into MDS cells.qPCR and Western blotting were utilized to measure the expression of SMAD7 and ferroptosis-related biomarkers at mRNA and protein levels,and the contents of glutathione(GSH),malondialdehyde(MDA),superoxide dismutase(SOD),and reactive oxygen species(ROS)were detected.Flow cytometry of CD11b+was performed to measure cellular differentiation.Results ①Bioinformatics analysis revealed significant down-regulation of SMAD7 in MDS patients,correlating with ferroptosis activation.Compared to the healthy controls,MDS patients exhibited decreased SMAD7 expression(P<0.05),reduced levels of negative regulators of ferroptosis,glutathione peroxidase 4(GPX4)and ferritin heavy chain(FTH1)(P<0.05),and elevated expression of its positive regulator transferrin receptor protein 1(TFRC)(P<0.05).Consistent with this,when compared with the normal human bone marrow stromal cell line HS-5,the MDS cell lines MUTZ-1 and SKM-1 exhibited declined expression of SMAD7,GPX4,and FTH1,alongside elevated expression of TFRC(P<0.05).② Treatment with gradient concentrations of the ferroptosis inhibitors ferrostatin-1(Fer-1),the expression levels of GPX4 and FTH1 in MDS cell lines were significantly upregulated in a concentration-dependent manner(P<0.05),while TFRC was markedly downregulated(P<0.05).Additionally,GSH content and SOD activity were enhanced,whereas ROS levels and MDA content were significantly reduced(P<0.05).These results suggest that Fer-1 effectively suppresses ferroptosis in MDS cells.③SMAD7 overexpression led to up-regulation of GPX4 and FTH1 ih MDS cell lines,while downregulation of TFRC,improved anti-oxidative ability and reduced ferroptosis,with enhanced CD11b+expression and myeloid differentiation.④ Following AC treatment,the expression levels of GPX4 and FTH1 in MDS cell lines were significantly upregulated in a concentration-dependent manner(P<0.05),whereas the downregulation of TFRC did not reach statistical significance.Additionally,AC treatment effectively enhanced the antioxidant capacity of the cells,increased the proportion of CD11b+cells(P<0.05),and facilitated cellular differentiation.Conclusion AC activates SMAD7 in MDS cell lines,up-regulating GPX4 and FTH1 while suppressing TFRC expression.This mechanism alleviates oxidative damage and lipid peroxidation,thereby inhibiting ferroptosis in MDS cells.Concurrently,SMAD7 activation promotes cellular differentiation.

Objective To analyze the medication rules of Chinese herbal concentrated-granule for the treatment of hypertension nationwide using data mining and knowledge graph methods,thus to provide evidence for expanding its application in treating hypertension.Methods From January 2022 to March 2023,Chinese herbal concentrated-granule prescriptions for hypertension prescribed by traditional Chinese medicine experts nationwide were collected.Microsoft Excel was used to analyze the frequency,properties and flavors,meridian tropism,and efficacy categories of the medicinals.Hierarchical clustering was applied for cluster analysis,and the Neo4j graph database was utilized to construct an information knowledge graph illustrating the relationships between regions and medicinals.Results A total of 2 600 Chinese herbal concentrated-granule prescriptions were included,involving 370 medicinals.The top six frequently-used medicinals were Poria(Fuling),Glycyrrhizae Radix et Rhizoma(Gancao),Atractylodis Macrocephalae Rhizoma(Baizhu),Citri Reticulatae Pericarpium(Chenpi),Paeoniae Radix Alba(Baishao),and Angelicae Sinensis Radix(Danggui).The properties of the medicinals were mainly warm and mild,and their flavors were usually sweet,pungent,and bitter.The medicinals frequently have the meridian tropism of the spleen,lung,and liver meridians.Hierarchical clustering yielded seven clusters.The information knowledge graph of region-medicinals relationships revealed that medicinals used in five or more regions were Baishao,Baizhu,Chenpi,Pinelliae Rhizoma(Banxia),Fuling,Gastrodiae Rhizoma(Tianma),Bupleuri Radix(Chaihu),Chuanxiong Rhizoma(Chuanxiong),Salviae Miltiorrhizae Radix et Rhizoma(Danshen),Danggui,Gancao,and Astragali Radix(Huangqi).Conclusion Chinese herbal concentrated-granule for hypertension usually consist of tonifying medicinals,with sweet and warm properties,and having the meridian tropism of the spleen meridian.And the medicinals composed of the prescriptions often have the actions of calming the liver and suppressing yang,strengthening the spleen and removing dampness,and nourishing the liver and kidney.