ObjectiveThrough multimodal research methods including medication rule mining， network pharmacology， molecular docking and dynamics simulation， and in vivo animal experiments， this study aims to speculate and verify the core composition （Ginseng Radix et Rhizoma Rubra-Salviae Miltiorrhizae Radix et Rhizoma-Notoginseng Radix et Rhizoma） and efficacy （Qi-invigorating and blood-activating） of the drug combination in Yitangkang Compound for improving diabetic cardiomyopathy （DCM）， investigate the interaction relationship and binding strength between core active ingredients of the drug combination and key signaling pathway targets， and further explore the mechanism by which the Qi-invigorating and blood-activating drug combination regulates the calcium signaling pathway to improve cardiac function in DCM rats. MethodsThe Ancient and Modern Medical Cases Cloud Platform was used to construct a DCM prescription database， and the "Analysis Method" module of the platform was applied to mine and summarize medication rules， thereby determining the core composition of the Qi-invigorating and blood-activating drug combination in Yitangkang. Drug-active ingredient-signaling pathway-core target-disease analysis and visualization were conducted by combining network pharmacology with the Traditional Chinese Medicine System Pharmacology Platform （TCMSP） database， SwissTargetPrediction platform， GeneCards database， MetaScape database， CytoScape software， etc. Then， molecular docking was performed via the CB-Dock2 platform， and molecular dynamics simulation of the high-binding-strength docking complexes was carried out by Gromacs software. Finally， in vivo animal experiments were carried out. Twenty-eight Sprague Dawley （SD） rats meeting the research criteria were divided into a normal group， a model group， a drug combination group （3.3 g·kg^-1）， and a Yitangkang group （20 g·kg^-1）. A type 2 diabetes mellitus （T2DM） rat model was established by high-fat diet feeding combined with intraperitoneal injection of streptozotocin （STZ）， followed by continuous feeding for eight weeks until the DCM model was successfully established. During this period， the traditional Chinese medicine （TCM） compound and drug combination were administered for prevention and treatment intervention. Meanwhile， changes in blood glucose， body weight， and heart index of each group were monitored. Cardiac function was assessed by echocardiography， and electrophysiological signals were detected by an electrocardiogram. The heart tissue was observed for pathological changes by hematoxylin-eosin （HE） and Masson staining， and the expression of L-type calcium channel （CACNA1C）， calmodulin （CALM1）， calcium/calmodulin-dependent protein kinase Ⅱδ （CAMK2D）， and neuronal nitric oxide synthase （NOS1） proteins in the calcium signaling pathway of myocardial tissue was detected by Western blot. ResultsIn 62 DCM prescriptions， Ginseng Radix et Rhizoma Rubra， Salviae Miltiorrhizae Radix et Rhizoma， and Notoginseng Radix et Rhizoma were used most frequently. Their meridian tropism mainly involved the spleen， heart， and lung， and their sweet and warm properties were prominent. The drugs for tonifying or blood-activating and stasis-resolving ranked top. In association rule analysis， （Ginseng Radix et Rhizoma Rubra， Salviae Miltiorrhizae Radix et Rhizoma）-Notoginseng Radix et Rhizoma had the highest lift. Network pharmacology obtained 75 active ingredients of the drug combination， 714 drug combination action targets， 2 702 disease targets， and 286 intersection targets. Protein-protein interaction （PPI） network predicted nine interaction component-targets （nine active ingredients and four calcium signaling pathway target genes）. Molecular docking showed the four complexes with the lowest binding energy were 2f3z-ginsenoside Re， 1cll-quercetin， 9blh-（6S）-6-（hydroxymethyl）-1，6-dimethyl-8，9-dihydro-7H-naphtho［8，7-g］benzofuran-10，11-dione， and 5vv0-miltionone Ⅱ. Dynamics simulation showed the CALM1-quercetin complex had the strongest binding affinity. The animal experiment results revealed that compared with the normal group， the model group showed significant changes in blood glucose， body weight， myocardial tissue morphology， heart index， cardiac function， electrophysiological indexes， and the expression levels of CACNA1C， CALM1， CAMK2D， and NOS1 proteins （P<0.05， P<0.01）. Compared with the model group， the Yitangkang group had a certain improvement effect on the above indexes （P<0.05， P<0.01）. Compared with the Yitangkang group， the drug combination group showed no significant difference in improving myocardial tissue morphology， heart index， cardiac function， electrophysiological indexes， and the expression of CACNA1C， CALM1， CAMK2D， and NOS1 proteins， except for blood glucose and body weight. ConclusionGinseng Radix et Rhizoma Rubra， Salviae Miltiorrhizae Radix et Rhizoma， and Notoginseng Radix et Rhizoma are the core Qi-invigorating and blood-activating drug combination in Yitangkang Compound. They have a good preventive and therapeutic effect on STZ-induced DCM in rats， and their mechanism of action may be related to the regulation of the calcium signaling pathway.

BACKGROUND: Doxorubicin hydrochloride (DOX) is extensively used in the treatment of various tumors. However, its clinical application is limited due to dose-dependent cardiotoxicity. Currently, few effective strategies exist to mitigate or eliminate DOX-induced cardiomyopathy (DIC). Although ferroptosis is implicated in DIC and its inhibition partially alleviates the condition, the direct targets of DOX in the progression of cardiotoxicity remain unclear. This study aimed to discover the direct targets of DOX in ferroptosis-mediated DIC. METHODS: A DOX pulldown assay was performed to identify proteins specifically binding to DOX in murine hearts, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify candidate proteins. A cardiac injury mouse model was established by DOX treatment. Based on this, multiple ferroptosis biomarkers were detected by flow cytometry, quantitative real-time polymerase chain reaction, western blotting, immunochemistry, etc. Besides, specific activator and inhibitor of signaling pathways were applied to illuminate molecular mechanisms. RESULTS: Glutathione S-transferase P1 (GSTP1) was identified as a DOX target. GSTP1 activity was inhibited in DOX-treated cardiomyocytes, while its overexpression significantly alleviated DIC. Moreover, GSTP1 overexpression inhibited acyl-CoA synthetase long-chain family member 4 (ACSL4)-dependent ferroptosis. Mechanistically, GSTP1 overexpression suppressed c-Jun N-terminal kinase (JNK) phosphorylation, thereby reducing reactive oxygen species (ROS) production and inhibiting ferroptosis in DIC. CONCLUSIONS This study identifies the DOX/GSTP1/JNK axis as a critical pathway mediating ACSL4-dependent ferroptosis in DIC. GSTP1 is highlighted as a potential key mediator of ferroptosis and a promising therapeutic target for DIC.

The incidence of cardiovascular diseases (CVD) together with their associated risk factors increasingly impact public health. According to the data provided in the 2024 Annual Report on Cardiovascular Health and Diseases in China, the crude incidence of cardiovascular and cerebrovascular diseases among Chinese residents aged ≥18 years, including acute myocardial infarction (AMI), angina pectoris treated by percutaneous transluminal coronary angioplasty/stent implantation and/or coronary artery bypass grafting, stroke, and sudden cardiac death was 620.33 per 100,000 population, with 87.6 for AMI. Stroke had an incidence of 491.0 per 100,000 population. The prevalence of adult coronary heart disease (CHD, aged ≥18 years) was 758 per 100,000 population. The CVD mortality in 2021 remained the highest, exceeding that of cancer and other causes. The crude mortality rate of CVD in 2021 was 364.16 per 100,000 population in rural areas and 305.39 per 100,000 population in urban areas. The crude mortality rates of cerebrovascular diseases and CHD among urban and rural residents in 2021 were 140.02 and 175.58, 135.08 and 148.19 per 100,000 population, respectively. In addition to interpreting the key findings of CVD incidence and mortality in China, we also update the data of associated risk factors, including tobacco use, physical activity, diet and nutrition, overweight and obesity, hypertension, dyslipidemia, diabetes, chronic kidney disease, sleep and psychological factors, environmental factors, and the diagnostic conditions of CVD diseases, aiming to provide a scientific foundation for advancing CVD prevention and control, and to inform relevant public health policy development.
Humans ; China/epidemiology* ; Cardiovascular Diseases/mortality* ; Risk Factors ; Adult ; Male ; Female ; Middle Aged ; Incidence ; Cerebrovascular Disorders/epidemiology* ; Aged

Objective To investigate the efficacy and safety of camrelizumab monoclonal antibody combined with molecular-targeted therapy in elderly patients with unresectable or advanced hepatocellular carcinoma(HCC).Methods A retrospective analysis was performed for the patients with unresectable/advanced HCC who attended six hospitals from January 1,2019 to March 31,2021,and all patients received camrelizumab monoclonal antibody treatment,among whom 84.8%also received targeted therapy.According to the age of the patients,they were divided into elderly group(≥65 years)and non-elderly group(＜65 years).The two groups were assessed in terms of overall survival(OS),progression-free survival(PFS),objective response rate(ORR),disease control rate(DCR),and immune-related adverse events(irAE).The chi-square test or the Fisher's exact test was used for comparison of categorical data between groups;the independent samples t-test was used for comparison of normally distributed continuous data,and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups.The Kaplan-Meier method was used for survival analysis,and the log-rank test was used for comparison of survival curves.Univariate and multivariate Cox proportional hazards regression analyses were used to determine the independent influencing factors for PFS and DCR at 6 months.Results A total of 99 HCC patients were enrolled,with 27 in the elderly group and 72 in the non-elderly group.The elderly group had an OS rate of 67.8%,an ORR of 44.4%,and a DCR of 74.1%at 12 months and a median PFS of 6.4(95%confidence interval[CI]:3.0-12.4)months,with no significant differences compared with the non-elderly group(all P＞0.05).The median OS was unavailable for the elderly group,while the non-elderly group had an OS of 18.9(95%CI:13.0-24.8)months;there was no significant difference between the two groups(P=0.485).The univariate and multivariate Cox regression analyses showed that major vascular invasion(MVI)was an independent risk factor for PFS(hazard ratio[HR]=2.603,95%CI:1.136-5.964,P=0.024)and DCR(HR=3.963,95%CI:1.671-9.397,P=0.002)at 6 months,while age,sex,etiology of HBV infection,presence of extrahepatic metastasis,Child-Pugh class B,and alpha-fetoprotein＞400 ng/mL were not associated with PFS or DCR at 6 months.For the elderly group,the incidence rates of any irAE and grade 3/4 irAE were 51.9%and 25.9%,respectively,with no significant differences compared with the non-elderly group(P＞0.05),and skin disease was the most common irAE in both groups(39.4%).Conclusion Camrelizumab monoclonal antibody combined with molecular-targeted therapy has similar efficacy and safety in patients with unresectable/advanced HCC aged≥65 years and those aged＜65 years.MVI is associated with suboptimal response to immunotherapy and poor prognosis.

BACKGROUND: Mutations in the structural domain of the epidermal growth factor receptor (EGFR) kinase represent a critical pathogenetic factor in non-small cell lung cancer (NSCLC). Small-molecule EGFR-tyrosine kinase inhibitors (TKIs) serve as first-line therapeutic agents for the treatment of EGFR-mutated NSCLC. But the resistance mutations of EGFR restrict the clinical application of EGFR-TKIs. In this study, we constructed a clinically relevant PC-9 EGFRD19/T790M/C797S cellular model featuring the mutation type within the EGFRD19/T790M/C797S. This model aims to investigate the inhibitory effects of small-molecule EGFR-TKIs and to provide a cellular platform for developing a new generation of innovative drugs that target resistance associated with EGFR mutations. METHODS: Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology was employed to knock in the EGFRT790M/C797S mutant fragment into NSCLC PC-9 cells, originally harboring the EGFRD19 mutation, to generate the PC-9 EGFRD19/T790M/C797S cell model. This model, with the EGFRD19/T790M/C797S mutant, was used to investigate the inhibitory effects of EGFR-TKIs on cell proliferation through MTS assay. Additionally, Western blot analysis was conducted to assess the regulation of EGFR protein expression and the phosphorylation levels of downstream signaling molecules, including protein kinase B (AKT) and mitogen-activated protein kinase (MAPK). RESULTS: PC-9 EGFRD19/T790M/C797S cells, with the EGFRD19/T790M/C797S mutation, were successfully generated using CRISPR/Cas9 technology. In terms of proliferation inhibition, the marketed first-, second-, and third-generation EGFR-TKIs that were ineffective against the EGFRD19/T790M/C797S mutation showed weak proliferation inhibitory activity against this cell line, and the proliferation inhibition (half maximal inhibitory concentration, IC50)>1000 nmol/L; in contrast, the fourth-generation EGFR-TKIs in development, which have better efficacy against the EGFRD19/T790M/C797S mutation, showed strong proliferation inhibition in this cell model. On mechanistic validation, the first-, second-, and third-generation EGFR-TKIs had weak inhibitory activity on the phosphorylation of EGFR and the downstream AKT/MAPK signaling pathway in this cell line, whereas the fourth generation of EGFR-TKIs under development significantly inhibited the phosphorylation of EGFR and the downstream AKT/MAPK signaling pathway in this cell line. CONCLUSIONS Using CRISPR/Cas9 technology, the EGFRT790M/C797S mutant fragment was successfully knocked into PC-9 cells to create cell lines harboring the EGFRD19/T790M/C797S mutation. The study demonstrated that the EGFR-TKIs showed different sensitivities to whether the EGFRD19/T790M/C797S mutation was effective or not and different inhibitory effects on the phosphorylation of EGFR and downstream pathways, which demonstrated that this cell line depended on the activation of the EGFRD19/T790M/C797S mutation and EGFR/AKT/MAPK signaling pathway for proliferation. This study provides a clinically relevant cellular evaluation and mechanism validation system for the development of a new generation of innovative drugs targeting EGFR mutation resistance.
ErbB Receptors/metabolism* ; Carcinoma, Non-Small-Cell Lung/metabolism* ; Humans ; Drug Resistance, Neoplasm/genetics* ; Lung Neoplasms/metabolism* ; Protein Kinase Inhibitors/pharmacology* ; Mutation ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Antineoplastic Agents/pharmacology*

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (M^pro) and papain like protease (PL^pro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851M^pro inhibitors and 205 PL^pro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both M^pro and PL^pro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of M^pro inhibitors and over 20% of PL^pro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 M^pro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.
Humans ; Antiviral Agents/chemistry* ; COVID-19 ; COVID-19 Drug Treatment ; High-Throughput Screening Assays ; Molecular Docking Simulation ; Protease Inhibitors/chemistry* ; SARS-CoV-2/enzymology* ; Viral Nonstructural Proteins

Objective:To study the intestinal flora specific differences with different lesional stages of metabolic (disorder) associated fatty liver disease (MAFLD), namely simple steatosis and steatohepatitis, so as to provide a new direction for MAFLD-related intestinal flora transplantation and targeted therapy.Methods:Mice were fed with normal diet, methionine-choline deficient diet (MCD) and a high-fat high-fructose diet (HFHF) for 12 weeks to construct simple steatosis and steatohepatitis models. HE and Sirius scarlet staining was performed to observe the liver pathological changes. The qPCR method was used to evaluate inflammation and liver fibrosis factors. A fully automatic biochemical analyzer was used to detect changes in liver transaminase and blood lipids. 16S rRNA sequencing method was used to observe the intestinal flora differences in the feces of each group of mice. The comparison of means between two groups was performed by t-test, and the comparison of means between multiple groups was performed by one-way analysis of variance. Kruskal-Wallis rank sum test was used for non-normally distributed data.Results:NAFLD scores were determined with pathological sections (HE and Sirius scarlet staining) of mice liver, which showed that the inflammation and liver fibrosis scores of the MCD and HFHF groups were 2.12 ± 0.18 and 1.06 ± 0.24, and 2.22 ± 0.16 and 0.46 ± 0.10, respectively. The degree of liver inflammation and fibrosis was significantly higher in the MCD than the HFHF group ( P < 0.001 and P < 0.01). Lipid deposition was higher in the HFHF than the MCD group ( P < 0.001), and the scores were 2.36 ± 0.17 and 1.60 ± 0.24 respectively. Simultaneously, the inflammatory [tumor necrosis factor-A (TNF-a), chemokine factor-2 (CXCL-2)] and hepatic fibrosis indicators [vascular smooth muscle actin alpha (a-SMA) and connective tissue growth factor (CTGF)] had confirmed the above-mentioned results at the transcription level. Moreover, the intestinal flora diversity was reduced ( P < 0.05) in the MCD group than the HFHF group, and the Simpson and Shannon index were 0.31 ± 0.10 and 0.42 ± 0.05, and 2.03 ± 0.33 and 1.70 ± 0.28, respectively, and the differences were significant between different intestinal flora groups. The levels of Desulfovibrio, Odoribacter, and Roseburia flora were significantly increased in the HFHF than the MCD group, and the levels of Faecalibaculum, Parasutterella, Alipis, Butyricimonas_virosa, Turicibacter_sp, and Romboutsia_ilealis were significantly increased in the MCD than the HFHF group, and the difference was statistically significant ( P < 0.05). Conclusion:There are significant differences in intestinal flora diversity between simple steatosis and steatohepatitis models. Therefore, clarifying the difference between the two may provide a new direction for the stage manner treatment of MAFLD.

Objective To explore the curative effect and influencing factors of percutaneous radiofrequency ablation(PRFA)for benign thyroid nodules in order to improve the effect of thyroid benign nodule ablation treatment.Methods The study included 482 benign thyroid nodules.The largest diameter of benign thyroid nodules ranges from 0.7 to 9.2 cm.Two hundred and fifty-eight nodules were solid nodules,224 nodules were cystic-solid nodules,and 96 thyroid nodules combined with the background of Hashimoto's thyroiditis.The patients of this study included 356 females and 126 males,the ages of the study population ranged from 14 to 82 years old.In order to observe the local pain,swelling,skin infection,hoarseness and other complications of the thyroid nodules with PRFA,we conducted conventional ultrasound and contrast-enhanced ultrasound immediately and followed up in 1,3,6,12,18 and 24 months after the treatment.Besides,the nodule volume reduction ratio was calculated,and the effects of gender,age,nodule size,cystic proportion,and the background of Hashimoto's thyroiditis on the treatment were analyzed.Results The volume of benign thyroid nodules after the PRFA treatment was significantly reduced after 1,3,6,12,18,24 months,and their nodule volume reduction ratio(VRR)was(51.2±5.7)%(1 months),(69.7±4.3)%(3 months),(84.6±3.7)%(6 months),(89.3±2.9)%(12 months),(93.7±1.6)%(18 months)and(94.9±1.4)%(24 months),respectively.The thyroid nodules were divided into 3 groups according to the cystic portion,significant differences were found in the volume reduction rate at 1,3,6,12,18 and 24 months after PRFA(F=66.858,69.101,19.410,49.559,146.653 and 309.950 respectively,all P＜0.001),the more cystic portion was,the faster the lesion shrinked.The nodules with the cystic portion ＜ 20%were grouped according to the maximum diameter(≤3 cm/＞3 cm),and the background of Hashimoto's disease; and significant differences were found in the volume reduction rate at 1,3,6,12,18 and 24 months after PRFA(the t values for different size groups were 9.710,8.925,9.899,12.734,17.226 and 42.580,respectively,the t values for whether there is a background of Hashimoto's disease were 66.858,69.101,19.410,49.559,146.653,309.950,respectively,all P ＜ 0.001).The VRR of nodules of which the maximum diameter ≤ 3 cm,or without Hashimoto's thyroiditis was larger after radiofrequency ablation.The nodules of which the maximum diameter≤3 cm and with the cystic portion ＜20%were grouped according to different genders(male or female)and different ages(≤40 or＞40 years old),and the mean VRR of the nodules between different gender and age groups are not significantly different(P＞0.05).Conclusions Ultrasound guided PRFA is a safe and effective minimally invasive therapy for benign thyroid nodules.

Objective To compare the ultrasound features of papillary thyroid carcinoma with or without cervical lymph node metastasis (LNM). Methods Patients suspected of thyroid cancer underwent the conventional ultrasound and contrast-enhanced ultrasound (CEUS) examinations. Patients' age,sex,and ultrasound characteristics of lesions were recorded. With the surgical pathology as the golden standard,the ultrasound features were compared between the cervical LNM group and non-LNM group. Results Of 144 patients,51 had cervical LNM and 93 did not. Patients' ages,sex and number of lesions had no significant difference between two groups (all P>0.05). Tumor with LNM had maximum size greater than 0.85 cm,ill-defined margin (P=0.000),irregular shape (P=0.007),internal heterogeneous echogenicity (P=0.007),microcalcification (P=0.020),internal heterogeneous low-enhancement (P=0.002),peripheral non-enhancement ring (P=0.030),and extracapsular extension (P=0.000). Conclusion Conventional ultrasound and CEUS are helpful for predicting the cervical LNM of PTC. CEUS can obtain more accurate diagnostic results for the extracapsular extension,which contributes to the prediction of cervical LNM.

Objective To investigate the differential diagnostic value for thyroid rich blood supply lesions by contrast-enhanced ultrasound (CEUS).Methods From February 2016 to January 2015,patients who suffered with thyroid nodules underwent conventional ultrasound and CEUS before biopsy.Sixty-two lesions with high-enhancement features were included in the final data.The enhancement patterns within and around lesions of CEUS were analyzed,and the high-enhancement ranges were compared with lesions sizes displayed on conventional ultrasound imaging.When pathology was regarded as the golden standard,39 nodules of 62 were benign,and 23 were malignant.The difference of CEUS characteristics between benign and malignant lesion was compared using x2 test.Results The homogeneity or not had significant difference between benign and malignant lesions with high-enhancement features (x2=9.78,P=0.002).The features of high-enhancement with cystic area and uneven distribution of high-enhancement area had significant difference between benign and malignant lesions (x2=10.77,P=0.001),and the former were frequently seen in benign lesions.Peripheral regular and irregular rings had significant difference between the benign and malignant lesions (x2=24.33,P ＜ 0.001).All lesions showed large high-enhancement area were malignant (x2=6.52,P=0.01).The diagnostic sensitivity,specificity and accuracy of CEUS were 95.83％,78.95％ and 85.48％ for thyroid rich blood supply lesions,which were better than those of conventional ultrasound (87.5％,68.42％,75.81％).Conclusions There are different features in CEUS between the benign and malignant thyroid lesions with rich blood supply.Heterogeneous high-enhancement,peripheral irregular rings and extensive high-enhancement area were malignant features.