This study aims to comprehensively analyze the material basis of toad visceral oil(hereafter referred to as toad oil), and explore the pharmacological effect of toad oil on atopic dermatitis(AD). Ultra-high performance liquid chromatography-linear ion trap/orbitrap high-resolution mass spectrometry(UHPLC-LTQ-Orbitrap-MS) and gas chromatography-mass spectrometry(GC-MS) were employed to comprehensively identify the chemical components in toad oil. The animal model of AD was prepared by the hapten stimulation method. The modeled animals were respectively administrated with positive drug(0.1% hydrocortisone butyrate cream) and low-and high-doses(1%, 10%) of toad oil by gavage. The effect of toad oil on AD was evaluated with the AD score, ear swelling rate, spleen index, and pathological section results as indicators. A total of 99 components were identified by UHPLC-LTQ-Orbitrap-MS, including 14 bufadienolides, 7 fatty acids, 6 alkaloids, 10 ketones, 18 amides, and other compounds. After methylation of toad oil samples, a total of 20 compounds were identified by GC-MS. Compared with the model group, the low-and high-dose toad oil groups showed declined AD score, ear swelling rate, and spleen index, alleviated skin lesions, and reduced infiltrating mast cells. This study comprehensively analyzes the chemical composition and clarifies the material basis of toad oil. Meanwhile, this study proves that toad oil has a good therapeutic effect on AD and is a reserve resource of traditional Chinese medicine for external use in the treatment of AD.
Animals ; Dermatitis, Atopic/immunology* ; Disease Models, Animal ; Mice ; Male ; Gas Chromatography-Mass Spectrometry ; Humans ; Bufonidae ; Oils/administration & dosage* ; Chromatography, High Pressure Liquid ; Female ; Mice, Inbred BALB C

Following the release of the Technical Requirements on Quality Control and Standard Establishment of Traditional Chinese Medicine Formula Granules by the National Medical Products Administration in 2021, Chinese Pharmacopoeia Commission has promulgated 296 national drug standards so far, and most provinces have started the work of establishing provincial standards as supplements. The promulgation of standards fostered high-quality development of the industry. Since the implementation of national and provincial standards for more than three years, enterprises have gained deep understanding and hands-on experiences on the characteristics, technical requirements, production process, and quality control of traditional Chinese medicine(TCM) formula granules. Meanwhile, challenges have emerged restricting the high-quality development of this industry, including how to formulate quality control strategies for medicinal materials and decoction pieces, how to reduce manufacturing costs, and how to improve the pass rate and product stability under high standards. Based on the work experiences from standard management and process research, this article analyzed the distribution of sources, processing methods, dry extract rate ranges, process requirements for volatile oil-containing decoction pieces, control measures of safety indices, characteristics and trends of setting characteristic chromatograms or fingerprints, characteristics and trends of setting content ranges, and main differences between national standards and provincial standards. On the one hand, this article aims to present main characteristics for deeply understanding different indicators in standards and provide basic ideas for establishing quality and process control systems. On the other hand, from the perspective of industrial practice, suggestions are put forward on the important aspects that need to be focused on in the quality and process control of TCM formula granules.
Drugs, Chinese Herbal/chemistry* ; Quality Control ; Medicine, Chinese Traditional/standards* ; China ; Drug Industry/standards*

This study investigated the effects and underlying mechanisms of vanillic acid(VA) against cardiac fibrosis(CF) induced by isoproterenol(ISO) in mice. Male C57BL/6J mice were randomly divided into control group, VA group(100 mg·kg~(-1), ig), ISO group(10 mg·kg~(-1), sc), ISO + VA group(10 mg·kg~(-1), sc + 100 mg·kg~(-1), ig), ISO + dynamin-related protein 1(Drp1) inhibitor(Mdivi-1) group(10 mg·kg~(-1), sc + 50 mg·kg~(-1), ip), and ISO + VA + Mdivi-1 group(10 mg·kg~(-1), sc + 100 mg·kg~(-1), ig + 50 mg·kg~(-1), ip). The treatment groups received the corresponding medications once daily for 14 consecutive days. On the day after the last administration, cardiac functions were evaluated, and serum and cardiac tissue samples were collected. These samples were analyzed for serum aspartate aminotransferase(AST), lactate dehydrogenase(LDH), creatine kinase-MB(CK-MB), cardiac troponin I(cTnI), reactive oxygen species(ROS), interleukin(IL)-1β, IL-4, IL-6, IL-10, IL-18, and tumor necrosis factor-α(TNF-α) levels, as well as cardiac tissue catalase(CAT), glutathione(GSH), malondialdehyde(MDA), myeloperoxidase(MPO), superoxide dismutase(SOD), total antioxidant capacity(T-AOC) activities, and cytochrome C levels in mitochondria and cytoplasm. Hematoxylin-eosin, Masson, uranium acetate and lead citrate staining were used to observe morphological and mitochondrial ultrastructural changes in the cardiac tissues, and myocardial injury area and collagen volume fraction were calculated. Flow cytometry was applied to detect the relative content and M1/M2 polarization of cardiac macrophages. The mRNA expression levels of macrophage polarization markers [CD86, CD206, arginase 1(Arg-1), inducible nitric oxide synthase(iNOS)], CF markers [type Ⅰ collagen(Coll Ⅰ), Coll Ⅲ, α-smooth muscle actin(α-SMA)], and cytokines(IL-1β, IL-4, IL-6, IL-10, IL-18, TNF-α) in cardiac tissues were determined by quantitative real-time PCR. Western blot was used to detect the protein expression levels of Coll Ⅰ, Coll Ⅲ, α-SMA, Drp1, p-Drp1, voltage-dependent anion channel(VDAC), hexokinase 1(HK1), NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein(ASC), caspase-1, cleaved-caspase-1, gasdermin D(GSDMD), cleaved N-terminal gasdermin D(GSDMD-N), IL-1β, IL-18, B-cell lymphoma-2(Bcl-2), B-cell lymphoma-xl(Bcl-xl), Bcl-2-associated death promoter(Bad), Bcl-2-associated X protein(Bax), apoptotic protease activating factor-1(Apaf-1), pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved-caspase-9, poly(ADP-ribose) polymerase-1(PARP-1), and cleaved-PARP-1 in cardiac tissues. The results showed that VA significantly improved cardiac function in mice with CF, reduced myocardial injury area and cardiac index, and decreased serum levels of AST, CK-MB, cTnI, LDH, ROS, IL-1β, IL-6, IL-18, and TNF-α. VA also lowered MDA and MPO levels, mRNA expressions of IL-1β, IL-6, IL-18, and TNF-α, and mRNA and protein expressions of Coll Ⅰ, Coll Ⅲ, and α-SMA in cardiac tissues, and increased serum levels of IL-4 and IL-10, cardiac tissue levels of CAT, GSH, SOD, and T-AOC, and mRNA expressions of IL-4 and IL-10. Additionally, VA ameliorated cardiac pathological damage, inhibited myocardial cell apoptosis, inflammatory infiltration, and collagen fiber deposition, reduced collagen volume fraction, and alleviated mitochondrial damage. VA decreased the ratio of F4/80~+CD86~+ M1 cells and the mRNA expressions of CD86 and iNOS in cardiac tissue, and increased the ratio of F4/80~+CD206~+ M2 cells and the mRNA expressions of CD206 and Arg-1. VA also reduced protein expressions of p-Drp1, VDAC, NLRP3, ASC, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, IL-1β, IL-18, Bad, Bax, Apaf-1, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP-1, and cytoplasmic cytochrome C, and increased the expressions of HK1, Bcl-2, Bcl-xl, pro-caspase-3, pro-caspase-9 proteins, as well as the Bcl-2/Bax and Bcl-xl/Bad ratios and mitochondrial cytochrome C content. These results indicate that VA has a significant ameliorative effect on ISO-induced CF in mice, alleviates ISO-induced oxidative damage and inflammatory response, and its mechanism may be closely related to the inhibition of Drp1/HK1/NLRP3 and mitochondrial apoptosis signaling pathways, suppression of myocardial cell inflammatory infiltration and collagen fiber deposition, reduction of collagen volume fraction and CollⅠ, Coll Ⅲ, and α-SMA expressions, thus mitigating CF.
Animals ; Isoproterenol/adverse effects* ; Male ; Mice ; Signal Transduction/drug effects* ; Vanillic Acid/administration & dosage* ; Dynamins/genetics* ; Mice, Inbred C57BL ; Fibrosis/genetics* ; Apoptosis/drug effects* ; Mitochondria/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Myocardium/metabolism* ; Humans

OBJECTIVE: To analyze the MYH9 gene sequence of a patient with hereditary thrombocytopenia and diffuse large B-cell lymphoma and his family members, and to explore the relationship between MYH9 gene and tumors. METHODS: Peripheral blood samples were collected from the patients and their family members for complete blood count analysis. The platelet morphology was observed under microscope. The MYH9 gene sequence was analyzed by Whole Exon Sequencing and Sanger Sequencing. RESULTS: The mutation site c.279C>A:p.(Asn93Lys) in exon 2 of the MYH9 gene were found in patient and his family members, both presenting as thrombocytopenia. The platelet count was significantly increased after the administration of Avatrombopag. CONCLUSION A novel mutation of MYH9 was found in this study, and the case was sensitive to Avatrombopag, by exploring the relationship between the MYH9 gene and tumors, suggesting that the MYH9 gene may be associated with the development of diffuse large B-cell lymphoma.
Humans ; Myosin Heavy Chains/genetics* ; Thrombocytopenia/genetics* ; Mutation ; Male ; Lymphoma, Non-Hodgkin/genetics* ; Lymphoma, Large B-Cell, Diffuse/genetics* ; Molecular Motor Proteins/genetics* ; Pedigree

OBJECTIVE: To evaluate the efficacy and safety of Shexiang Tongxin Dropping Pill (STDP) in treating stable angina patients with phlegm-heat and blood-stasis syndrome by exercise duration and metabolic equivalents. METHODS: This multicenter, randomized, double-blind, placebo-controlled clinical trial enrolled stable angina patients with phlegm-heat and blood-stasis syndrome from 22 hospitals. They were randomized 1:1 to STDP (35 mg/pill, 6 pills per day) or placebo for 56 days. The primary outcome was the exercise duration and metabolic equivalents (METs) assessed by the standard Bruce exercise treadmill test after 56 days of treatment. The secondary outcomes included the total angina symptom score, Chinese medicine (CM) symptom scores, Seattle Angina Questionnaire (SAQ) scores, changes in ST-T on electrocardiogram and adverse events (AEs). RESULTS: This trial enrolled 309 patients, including 155 and 154 in the STDP and placebo groups, respectively. STDP significantly prolonged exercise duration with an increase of 51.0 s, compared to a decrease of 12.0 s with placebo (change rate: -11.1% vs. 3.2%, P<0.01). The increase in METs was significantly greater in the STDP group than in the placebo group (change: -0.4 vs. 0.0, change rate: -5.0% vs. 0.0%, P<0.01). The improvement of total angina symptom scores (25.0% vs. 0.0%), CM symptom scores (38.7% vs. 11.8%), reduction of nitroglycerin consumption (100.0% vs. 11.3%), and all domains of SAQ, were significantly greater with STDP than placebo (all P<0.01). The changes in Q-T intervals at 28 and 56 days from baseline were similar between the two groups (both P>0.05). Twenty-five participants (16.3%) with STDP and 16 (10.5%) with placebo experienced AEs (P=0.131), with no serious AEs observed. CONCLUSION STDP could improve exercise tolerance in patients with stable angina and phlegm-heat and blood stasis syndrome, with a favorable safety profile. (Registration No. ChiCTR-IPR-15006020).
Humans ; Double-Blind Method ; Drugs, Chinese Herbal/adverse effects* ; Male ; Female ; Middle Aged ; Angina, Stable/physiopathology* ; Aged ; Syndrome ; Treatment Outcome ; Placebos ; Tablets

OBJECTIVE: Humans are exposed to complex mixtures of environmental chemicals and other factors that can affect their health. Analysis of these mixture exposures presents several key challenges for environmental epidemiology and risk assessment, including high dimensionality, correlated exposure, and subtle individual effects. METHODS: We proposed a novel statistical approach, the generalized functional linear model (GFLM), to analyze the health effects of exposure mixtures. GFLM treats the effect of mixture exposures as a smooth function by reordering exposures based on specific mechanisms and capturing internal correlations to provide a meaningful estimation and interpretation. The robustness and efficiency was evaluated under various scenarios through extensive simulation studies. RESULTS: We applied the GFLM to two datasets from the National Health and Nutrition Examination Survey (NHANES). In the first application, we examined the effects of 37 nutrients on BMI (2011-2016 cycles). The GFLM identified a significant mixture effect, with fiber and fat emerging as the nutrients with the greatest negative and positive effects on BMI, respectively. For the second application, we investigated the association between four pre- and perfluoroalkyl substances (PFAS) and gout risk (2007-2018 cycles). Unlike traditional methods, the GFLM indicated no significant association, demonstrating its robustness to multicollinearity. CONCLUSION GFLM framework is a powerful tool for mixture exposure analysis, offering improved handling of correlated exposures and interpretable results. It demonstrates robust performance across various scenarios and real-world applications, advancing our understanding of complex environmental exposures and their health impacts on environmental epidemiology and toxicology.
Humans ; Environmental Exposure/analysis* ; Linear Models ; Nutrition Surveys ; Environmental Pollutants ; Body Mass Index

Drug impurity control is essential for ensuring pharmaceutical quality.In this study,an unknown impurity at relative retention time(RRT)of 0.45 in Torasemide injection was systematically investigated using high performance liquid chromatography-ion trap time-of-flight mass spectrometry(LC-IT-TOF-MS/MS),forced degradation studies,and nuclear magnetic resonance(NMR)spectroscopy,aiming to elucidate its chemical structure,propose a formation mechanism,and establish a control strategy.By analysis of the ultraviolet absorption spectrum and high-resolution MS1 and MS2 data of the impurity,its possible structure was postulated.Subsequently,the target impurity was prepared via forced degradation experiment,and its structure was confirmed by NMR spectroscopy.A detailed analysis of its formation mechanism revealed that Torasemide first hydrolyzed in solution to form Torasemide sulfonamide,and then underwent condensation,dehydration,and cyclization reactions with trace amounts of formaldehyde in the excipient polyethylene glycol(PEG)400 to generate the impurity.It was experimentally proven that the target impurity was related to the residual oxygen content in Torasemide injection.Ultimately,by optimizing the prescription process,the target impurity level was controlled below the individual impurity limit of the final product(≤0.3%).This study provided valuable insights for enhancing the quality control of Torasemide injection.

Ankylosing spondylitis (AS) combined with lower cervical fracture is often categorized into unstable fracture, with a high incidence of neurological injury and a high rate of disability and morbidity. As factors such as shoulder occlusion may affect the accuracy of X-ray imaging diagnosis, it is often easily misdiagnosed at the primary diagnosis. Non-operative treatment has complications such as bone nonunion and the possibility of secondary neurological damage, while the timing, access and choice of surgical treatment are still controversial. Currently, there are no clinical practice guidelines for the treatment of AS combined with lower cervical fracture with or without dislocation. To this end, the Spinal Trauma Group of Orthopedics Branch of Chinese Medical Doctor Association organized experts to formulate Clinical guidelines for the treatment of ankylosing spondylitis combined with lower cervical fracture in adults ( version 2024) in accordance with the principles of evidence-based medicine, scientificity and practicality, in which 11 recommendations were put forward in terms of the diagnosis, imaging evaluation, typing and treatment, etc, to provide guidance for the diagnosis and treatment of AS combined with lower cervical fracture.

Objective To evaluate the characteristics of different antigen-specific T cell immune responses to severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)after inoculation with 2 doses of SARS-CoV-2 inactivated vaccine for 12 months.Methods Fifteen healthy adults were enrolled in this study and blood samples collected at 12 months after receiving two doses of SARS-CoV-2 inactivated vaccine.The level and phenotypic characteristics of SARS-CoV-2 antigen-specific T lymphocytes were detected by activation-induced markers(AIM)based on polychromatic flow cytometry.Results After 12 months of inoculation with 2 doses of SARS-CoV-2 inactivated vaccine,more than 90%of adults had detectable Spike and Non-spike antigen-specific CD4+ T cells immune responses(Spike:14/15,P=0.0001;Non-spike:15/15,P<0.0001).80%of adults had detectable Spike and Non-spike antigen-specific CD8+ T cells immune responses(Spike:12/15,P=0.0463;Non-spike:12/15,P=0.0806).Antigen-specific CD4+ T cells induced by SARS-CoV-2 inactivated vaccination after 12 months were composed of predominantly central memory(CM)and effector memory 1(EM1)cells.On the other hand,in terms of helper subsets,antigen-specific CD4+ T cells mainly showed T helper 1/17(Th1/17)and T helper 2(Th2)phenotypes.Conclusions SARS-CoV-2 inactivated vaccination generates durable and extensive antigen-specific CD4+ T cell memory responses,which may be the key factor for the low proportion of severe coronavirus disease 2019(COVID-19)infection in China.

Aim To investigate the effect of total glu-cosides of paeony on inflammatory injury and TLR4/NF-κB/NLRP3 pathway in autoimmune thyroiditis(AIT)rats.Methods The experiment was divided into control group,model group,total glucosides of pae-ony(TGP),TLR4 inhibitor group and TGP+TLR4 ag-onist group,with 10 animals in each group.Except for the control group,the rats in other groups were subcu-taneously injected with thyroglobulin and Freund's ad-juvant to induce the AIT rat model.After six weeks of administration,thyroid histopathological changes were observed using hematoxylin-eosin(HE)staining;ser-um levels of TPOAb,TgAb,TSH,T3,T4,TNF-α,INF-γ,IL-1 β and IL-1 β were detected by enzyme-linked immunosorbent assay(ELISA);TLR4/NF-κB/NLRP3 pathway mRNAs and proteins expression in thyroid tis-sues were detected by RT-qPCR and Western blot.Re-sults Compared with the control group,the thyroid follicular epithelium of rats was significantly damaged,and the serum levels of TPOAb,TgAb,TSH,T3,T4,TNF-α,INF-γ,IL-1 β and IL-1 β increased(P＜0.01).The expression of TLR4/NF-κB/NLRP3 path-way mRNAs and proteins increased in the model group(P＜0.01).Compared with the model group,the damage of thyroid follicular epithelium was alleviated,and the serum levels of TPOAb,TgAb,TSH,T3,T4,TNF-α,INF-γ,IL-1 β and IL-1 β were reduced(P＜0.01),the expression of TLR4/NF-κB/NLRP3 path-way mRNAs and proteins were down-regulated in the TGP group and TLR4 inhibitor group(P＜0.01).Compared with TGP group,the damage of thyroid follic-ular epithelium was aggravated,and the levels of serum TPOAb,TgAb,TSH,T3,T4,TNF-α,INF-γ,IL-1 β and IL-1 β were elevated(P＜0.05 or P＜0.01),the pro-tein expressions of TLR4/NF-κB/NLRP3 pathway mR-NAs and proteins were up-regulated in TGP+TLR4 ag-onist group(P＜0.05 or P＜0.01).Conclusions TGP may play a protective role in thyroid by inhibiting the TLR4/NF-κB/NLRP3 pathway and improving the inflammatory injury of thyroid tissues.