ObjectiveTo explore the effect of serum containing Zhenwutang on myocardial mast cell apoptosis induced by angiotensin Ⅱ （AngⅡ） and the mechanism of the correlation between apoptosis and mitochondrial autophagy. MethodsIn this experiment， AngⅡ and serum containing Zhenwutang with different concentrations were used to interfere with H9C2 cardiomyocytes for 24 h， and the survival rate of H9C2 cardiomyocytes was detected by cell counting kit-8 （CCK-8） to screen the optimal concentration for the experiment. Enzyme-linked immunosorbent assay （ELISA） was used to detect the content of B-type natriuretic peptide （BNP） in cell culture supernatant， and immunofluorescence was used to detect the cell surface area to verify the construction of the myocardial mast cell model. Subsequently， the experiment was divided into a blank group （20% blank serum）， a model group （20% blank serum + 5×10^-5 mol·L^-1 AngⅡ）， low-， medium-， and high-dose （5%， 10% and 20%） serum containing Zhenwutang groups， an autophagy inhibitor group （1×10^-4 mol·L^-1 3-MA）， and autophagy inducer group （1×10^-7 mol·L^-1 rapamycin）. The apoptosis level of H9C2 cells and the changes of mitochondrial membrane potential were detected by flow cytometry. The lysosomal probe （Lyso Tracker） and mitochondrial probe （Mito Tracker） co-localization was employed to detect autophagy. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect Caspase-3， Caspase-9， B-cell lymphoma 2 （Bcl-2）， Bcl-2-related X protein （Bax）， and cytochrome C （Cyt C） in apoptosis-related pathways and the relative mRNA expression of ubiquitin ligase （Parkin）， phosphatase and tensin homolog （PTEN）-induced kinase 1 （PINK1）， and p62 protein in mitochondrial autophagy-related pathways. Western blot was used to detect cleaved Caspase-3， cleaved Caspase-9， Bax， Bcl-2， and Cyt C in apoptosis-related pathways， phosphorylated ubiquitin ligase （p-Parkin）， phosphorylated PTEN-induced kinase 1 （p-PINK1）， p62， and Bcl-2 homology domain protein Beclin1 in mitochondrial autophagy-related pathways， and the change of microtubule-associated protein 1 light chain 3 （LC3） Ⅱ/Ⅰ ratio. ResultsCCK-8 showed that when the concentration of AngⅡ was 5×10^-5 mol·L^-1， the cell activity was the lowest， and there was no cytotoxicity. At this concentration， the surface area of cardiomyocytes was significantly increased （P<0.01）， and the content of BNP in the supernatant of culture medium was significantly increased （P<0.05）. Therefore， AngⅡ with a concentration of 5×10^-5 mol·L^-1 was selected for the subsequent modeling of myocardial mast cells. Compared with the blank group， the model group and the autophagy inhibitor 3-MA group had a significantly increased apoptosis rate （P<0.01） and significantly decreased mitochondrial membrane potential （P<0.01）. The results of immunofluorescence co-localization showed that compared with the blank group， the model group had a significantly decreased number of red and green fluorescence spots. The results of Real-time PCR showed that compared with that in the blank group， the relative mRNA expression of Bax， Caspase-3， Caspase-9， Cyt C， and p62 in the model group was significantly up-regulated （P<0.01）， while the relative mRNA expression of Bcl-2， Parkin， and PINK1 was significantly down-regulated （P<0.01）. In addition， the relative protein expression of Bax， cleaved Caspase-3， cleaved Caspase-9， Cyt C， and p62 was significantly up-regulated （P<0.01）. The LC3Ⅱ/Ⅰ was significantly decreased， and the relative protein expression of Bcl-2， p-Parkin， p-PINK1， and Beclin1 was significantly down-regulated （P<0.01）. Compared with the model group， the serum containing Zhenwutang groups and the autophagy inducer group had significantly decreased apoptosis rate （P<0.01）， and the decrease ratio of mitochondrial membrane potential is significantly lowered （P<0.01） in a dose-dependent manner. Additionally， both red and green fluorescence spots became more in these groups. In the 3-MA group， the number of red and green fluorescence spots decreased significantly. The relative mRNA expression of Bax， Caspase-3， Caspase-9， Cyt C， and p62 was significantly down-regulated （P<0.05， P<0.01）， while that of Bcl-2， Parkin， and PINK1 was significantly up-regulated （P<0.01）. In the serum containing Zhenwutang groups， the relative protein expression levels of Bax， cleaved Caspase-3， cleaved Caspase-9， Cyt C， and p62 were significantly down-regulated （P<0.05，P<0.01）. The LC3Ⅱ/Ⅰ was significantly increased， and the relative protein expression levels of Bcl-2， p-Parkin， p-PINK1， and Beclin1 were significantly up-regulated （P<0.01）. ConclusionThe serum containing Zhenwutang can reduce the apoptosis of myocardial mast cells and increase mitochondrial autophagy. This is related to the inhibition of intracellular Bax/Bcl-2/Caspase-3 apoptosis pathway and regulation of Parkin/PINK1 mitochondrial autophagy pathway.

Objective:The phenotype and genotype of a pedigree with Glanzmann thrombasthenia caused by compound heterozygous mutation in the ITGA2B gene and its molecular pathogenesis were explored.Methods:The platelet aggregation rate of the proband and his family was detected by using a platelet aggregation test with adenosine diphosphate, collagen, epinephrine, arachidonic acid, and ristocetin. The expression levels of CD41 (αⅡb), CD61 (β3), and CD42b (GPⅠb) on the platelet surface was detected by flow cytometry. Gene sequencing technology was used for the genetic identification of the family. RT-PCR was used in the detection of mRNA splicing, and qRT-PCR was used in detecting the relative mRNA level of the ITGA2B gene. Bioinformatics analysis was used to evaluate the pathogenicity of mutation sites and their effects on protein structure and function. The expressions of total αⅡb and β3 in platelets were analyzed by Western blot.Results:Except ristocetin, the other four inducers could not induce platelet aggregation in the proband. Flow cytometry showed that the expression levels of αⅡb and β3 were only 0.25% and 9.76%, respectively, on the platelet surface of the proband, whereas GPⅠb expression was relatively normal. The expression levels of glycoproteins in the other family members were almost normal. c.480C>G and c.2929C>T mutations were detected in the proband through gene sequencing. The c.480C>G mutation was inherited from his mother, and the c.2929C>T mutation was inherited from his father. The RT-PCR and sequencing results showed that the c.480C>G mutation caused mRNA splicing in the proband and his mother, resulting in the deletion of 99 bases in c.476G-574A (p.S160-S192). qRT-PCR showed that the c.2929C>T variant reduced the mRNA level of the ITGA2B gene in the proband and his father. Bioinformatics analysis suggested that the c.480C>G mutation might form a binding sequence with hnRNP A1 protein and generate the 5′SS splice site. The three-dimensional structural model of the αⅡb subunit showed that the β-propeller domain of the p.S160-S192 deletion lost two β-strands and one α-helix in blade 2. The c.2929C>T nonsense mutation caused premature translation termination and produced a truncated protein with the deletion of p.R977-E1039, including the cytoplasmic domain, transmembrane domain, and a β chain of the extracellular Calf-2 domain. The total αⅡb expression of the proband was absent, and the relative expression of β3 was 11.36% of the normal level.Conclusion:The compound heterozygous mutation c.480C>G in exon 4 and c.2929C>T in exon 28 of the ITGA2B gene probably underlies Glanzmann thrombasthenia in this pedigree.

Objective:To analyze the F10 gene mutations in a Chinese pedigree affected with the deficiency of the hereditary coagulation factor X (FX), resulting from a new deletion mutation, and to study the associated molecular pathogenesis.Methods:Next generation sequencing (NGS) was performed to screen the genetic mutations in the proband which were then verified by Sanger sequencing. The FX activity (FX∶C) of probands and their family members was detected using the blood clotting method, and the mutation sites of the family members were analyzed using Sanger sequencing. The pathogenicity of the mutation site was predicted by using the online bioinformatics software, Mutation Taster. The SWISS-MODEL software was used for stimulating the three-dimensional models of the wild-type and mutant proteins for analyzing the influence of the mutation site on the structure and function of the proteins, and for analyzing the difference between the catalytic residues of the wild-type and the mutant proteins. The level of the F10 gene mRNA was quantitatively analyzed by qRT-PCR (quantitative reverse transcription polymerase chain reaction) method by constructing plasmids, transfecting human embryonic kidney 293T cells (HEK 293T), and analyzing the splicing of the mutated site by RT-PCR method. The levels of FⅩ∶Ag in cell lysates and cell culture media (both inside and outside the cells) were detected by the ELISA (enzyme linked immunosorbent assay) method.Results:A medium-grade factor X deficiency with a 36.42% FⅩ∶C ratio was detected in the proband by the coagulation method. NGS analysis demonstrated a heterozygous deletion mutation in exon 8:c.902_919del (p.Ala301_Glu306del) in the proband. Sanger sequencing analysis indicated that some members of the family (mother and grandfather) were also carriers of the corresponding deletion mutation. Online bioinformatics software predicted the pathogenic nature of the c.902_919del mutation, with a pathogenic score of 0.999. The 3D protein structure model analysis indicated that the c.902_919del mutation resulted in the disappearance of a segment of β-fold in the protein structure, thereby shortening the preceding segment of the β-fold and a subsequent loss of hydrogen bonds between adjacent amino acids with no significant difference in the side chain conformation of the key catalytic residues compared to the wild-type. mRNA splicing analysis indicated the absence of alternative splicing changes in the mutation, and qRT-PCR results indicated the absence of a statistically significant difference between the mRNA levels of F10 gene and wild-type mRNA in cells expressing c.902_919del mutant. The ELISA results indicated that there was no statistically significant difference in the FX∶Ag levels of the mutant cell culture medium and the lysate.Conclusions:In this pedigree, the heterozygous mutation in exon 8 of F10 gene (c.902_919del, p.Ala301_Glu306del) caused the hereditary factor Ⅹ deficiency.

Objective RNA sequencing(RNA-seq)and bioinformatic analysis were combined and used to explore the anti-inflammatory effects and mechanisms of naringenin(Nar)in lipopolysaccharide(LPS)-stimulated human peri-odontal ligament stem cells(hPDLSCs).Methods Cell counting kit-8,quantitative real-time reverse transcription poly-merase chain reaction(qRT-PCR),and enzyme-linked immunosorbent assay(ELISA)were adopted to detect the effects of Nar on the proliferation and expression of inflammatory factors in LPS-stimulated hPDLSCs,screening for the opti-mal anti-inflammatory concentration of Nar.Differentially expressed genes(DEGs)were screened using|log2FC|≥1 and P≤0.05 as criteria.Volcano plot analysis,Kyoto En-cyclopedia of Genes and Genomes(KEGG)pathway en-richment analysis,the String database,and the MCODE module of Cytoscape were utilized to select core genes and enriched pathways.The effects on the nuclear factor κB(NF-κB)signaling pathway were verified using ELISA,qRT-PCR,and Western blot.Results Appropriate concentrations of Nar could alleviate the expression of inflammatory fac-tors and promote the proliferation of hPDLSCs stimulated by LPS.The best anti-inflammatory effect was achieved with 20 μmol/L Nar.RNA-seq showed significant enrichment of inflammation-related signaling pathways.The anti-inflamma-tory effect of Nar was mediated by inhibiting the NF-κB signaling pathway,similar to the effect of the NF-κB inhibitor BAY 11-7802.Conclusion Nar could exert its anti-inflammatory effects by inhibiting the NF-κB signaling pathway,making it a potential therapeutic option for the adjuvant treatment of periodontitis.

Objective:To analyze the factors affecting delayed chemotherapy in children with Burkitt lymphoma (BL) and their influence on prognosis.Methods:Retrospective cohort study. Clinical data of 591 children aged ≤18 years with BL from May 2017 to December 2022 in China Net Childhood Lymphoma (CNCL) was collected. The patients were treated according to the protocol CNCL-BL-2017. According to the clinical characteristics, therapeutic regimen was divided into group A, group B and group C .Based on whether the total chemotherapy time was delayed, patients were divided into two groups: the delayed chemotherapy group and the non-delayed chemotherapy group. Based on the total delayed time of chemotherapy, patients in group C were divided into non-delayed chemotherapy group, 1-7 days delayed group and more than 7 days delayed group. Relationships between delayed chemotherapy and gender, age, tumor lysis syndrome before chemotherapy, bone marrow involvement, disease group (B/C group), serum lactate dehydrogenase (LDH) > 4 times than normal, grade Ⅲ-Ⅳ myelosuppression after chemotherapy, minimal residual disease in the interim assessment, and severe infection (including severe pneumonia, sepsis, meningitis, chickenpox, etc.) were analyzed. Logistic analysis was used to identify the relevant factors. Kaplan-Meier method was used to analyze the patients' survival information. Log-Rank was used for comparison between groups.Results:Among 591 patients, 504 were males and 87 were females, the follow-up time was 34.8 (18.6,50.1) months. The 3-year overall survival (OS) rate was (92.5±1.1)%,and the 3-year event-free survival (EFS) rate was (90.5±1.2)%. Seventy-three (12.4%) patients were in delayed chemotherapy group and 518 (87.6%) patients were in non-delayed chemotherapy group. The reasons for chemotherapy delay included 72 cases (98.6%) of severe infection, 65 cases (89.0%) of bone marrow suppression, 35 cases (47.9%) of organ dysfunction, 22 cases (30.1%) of tumor lysis syndrome,etc. There were 7 cases of chemotherapy delay in group B, which were seen in COPADM (vincristine+cyclophosphamide+prednisone+daunorubicin+methotrexate+intrathecal injection,4 cases) and CYM (methotrexate+cytarabine+intrathecal injection,3 cases) stages. There were 66 cases of chemotherapy delay in group C, which were common in COPADM (28 cases) and CYVE 1 (low dose cytarabine+high dose cytarabine+etoposide+methotrexate, 12 cases) stages. Multinomial Logistic regression analysis showed that the age over 10 years old ( OR=0.54,95% CI 0.30-0.93), tumor lysis syndrome before chemotherapy ( OR=0.48,95% CI 0.27-0.84) and grade Ⅲ-Ⅳ myelosuppression after chemotherapy ( OR=0.55,95% CI 0.33-0.91)were independent risk factors for chemotherapy delay.The 3-year OS rate and the 3-year EFS rate of children with Burkitt lymphoma in the delayed chemotherapy group were lower than those in the non-delayed chemotherapy group ((79.4±4.9)% vs. (94.2±1.1)%, (80.2±4.8)% vs. (92.0±1.2)%,both P<0.05). The 3-year OS rate of the group C with chemotherapy delay >7 days (42 cases) was lower than that of the group with chemotherapy delay of 1-7 days (22 cases) and the non-delay group (399 cases) ((76.7±6.9)% vs. (81.8±8.2)% vs. (92.7±1.3)%, P=0.002).The 3-year OS rate of the chemotherapy delay group (9 cases) in the COP (vincristine+cyclophosphamide+prednisone) phase was lower than that of the non-chemotherapy delay group (454 cases) ((66.7±15.7)% vs. (91.3±1.4)%, P=0.005). Similarly, the 3-year OS rate of the chemotherapy delay group (11 cases) in the COPADM1 phase was lower than that of the non-chemotherapy delay group (452 cases) ((63.6±14.5)% vs. (91.5±1.3)%, P=0.001). Conclusions:The delayed chemotherapy was related to the age over 10 years old, tumor lysis syndrome before chemotherapy and grade Ⅲ-Ⅳ myelosuppression after chemotherapy in pediatric BL. There is a significant relationship between delayed chemotherapy and prognosis of BL in children.

Objective:To investigate grey matter volume changes and their correlation with the severity of anxiety in adolescents with major depressive disorder (MDD) accompanied by anxious distress specifier (ADS).Methods:From June 2022 to June 2023, totally 71 inpatients with MDD in the child and adolescent psychiatry department of Anhui Mental Health Center were included. According to the definition of ADS in the DSM-5, MDD adolescents were divided into the group with anxious distress (MDD/ADS+ group, n=44) and the group without anxious distress (MDD/ADS- group, n=27). Healthy adolescents matched in terms of gender, age, education level were recruited as the control group (HC group, n=19). Voxel-based morphometry (VBM) was used to compare changes in grey matter volume among the three groups.Grey matter volume values of brain regions with significant differences between the MDD/ADS+ group and MDD/ADS- group were collected, and their correlation with Hamilton anxiety rating scale (HAMA) score were analyzed by Pearson correlation analysis using SPSS 26.0 software. Results:Compared to the MDD/ADS- group, the MDD/ADS+ group showed a significant decrease in grey matter volume in the right dorsolateral prefrontal cortex (MNI: x=16.5, y=34.5, z=52.5, t=4.48, P<0.05) and the right cerebellum (MNI: x=49.5, y=-69.0, z=-24.0, t=5.18, P<0.05). In MDD adolescents, the grey matter volumes of the right dorsolateral prefrontal cortex and the right cerebellum were negatively correlated with HAMA score ( r=-0.249, -0.491, both P<0.05). Conclusion:In adolescents with MDD accompanied by ADS, a decrease in gray matter volume is observed in the right dorsolateral prefrontal cortex and the right superior cerebellum. These brain regions may serve as potential biological markers for the severity of anxiety in adolescents with MDD.

Objective To analyze the trends of disease burden of cervical cancer,uterine cancer,and ovarian cancer among Chinese women from 1990 to 2019,and to provide a basis for formulating precise preven-tion and control measures in China.Methods The global disease burden data in 2019 were used to describe the changes in indicators such as incidence,mortality,years of life lost due to premature mortality(YLL),years lived with disability(YLD),and disability-adjusted life year(DALY)of cervical,uterine,and ovarian canc-ers in China from 1990 to 2019.Furthermore,the Bayesian age-period-cohort model was adopted to predict the incidence and mortality of the cancers from 2020 to 2030.Results From 1990 to 2019,the incidence rates and mortality of cervical,uterine,and ovarian cancers in Chinese women showed an upward trend,and the age-standardized incidence rate of ovarian cancer increased the most(0.78% ).In 2019,the incidence of cervical cancer and uterine cancer concentrated in the women of 55-59 years old,and ovarian cancer mainly occurred in the women of 70-74 years old.The DALY,YLL,and YLD of cervical,uterine,and ovarian cancers all pres-ented varying degrees of growth at all ages.The Bayesian age-period-cohort model predicted that from 2020 to 2030,the incidence and mortality of cervical cancer in China showed a decreasing trend,while those of uterine cancer and ovarian cancer showed an increasing trend.There was no significant change in the age with high inci-dence of the three cancers.Conclusions From 1990 to 2019,the overall disease burden of cervical,uterine,and ovarian cancers in China increased,while the disease burden of cervical cancer decreased after 2020.It is recom-mended that the efforts should be doubled for the prevention and control of cervical,uterine,and ovarian cancers.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons in the brain and spinal cord. One important aspect of ALS pathogenesis is superoxide dismutase 1 (SOD1) mutant-mediated mitochondrial toxicity, leading to apoptosis in neurons. This study aimed to evaluate the neural protective synergistic effects of ginsenosides Rg1 (G-Rg1) and conditioned medium (CM) on a mutational SOD1 cell model, and to explore the underlying mechanisms. We found that the contents of nerve growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor significantly increased in CM after human umbilical cord mesenchymal stem cells (hUCMSCs) were exposed to neuron differentiation reagents for seven days. CM or G-Rg1 decreased the apoptotic rate of SOD1^G93A-NSC34 cells to a certain extent, but their combination brought about the least apoptosis, compared with CM or G-Rg1 alone. Further research showed that the anti-apoptotic protein Bcl-2 was upregulated in all the treatment groups. Proteins associated with mitochondrial apoptotic pathways, such as Bax, caspase 9 (Cas-9), and cytochrome c (Cyt c), were downregulated. Furthermore, CM or G-Rg1 also inhibited the activation of the nuclear factor-kappa B (NF-κB) signaling pathway by reducing the phosphorylation of p65 and IκBα. CM/G-Rg1 or their combination also reduced the apoptotic rate induced by betulinic acid (BetA), an agonist of the NF-κB signaling pathway. In summary, the combination of CM and G-Rg1 effectively reduced the apoptosis of SOD1^G93A-NSC34 cells through suppressing the NF-κB/Bcl-2 signaling pathway (Fig. 1 is a graphical representation of the abstract).
Humans ; NF-kappa B/metabolism* ; Ginsenosides/pharmacology* ; Amyotrophic Lateral Sclerosis/genetics* ; Culture Media, Conditioned/pharmacology* ; Superoxide Dismutase-1 ; Neurodegenerative Diseases ; Neurons/metabolism* ; Apoptosis

Various c-mesenchymal-to-epithelial transition (c-MET) inhibitors are effective in the treatment of non-small cell lung cancer; however, the inevitable drug resistance remains a challenge, limiting their clinical efficacy. Therefore, novel strategies targeting c-MET are urgently required. Herein, through rational structure optimization, we obtained novel exceptionally potent and orally active c-MET proteolysis targeting chimeras (PROTACs) namely D10 and D15 based on thalidomide and tepotinib. D10 and D15 inhibited cell growth with low nanomolar IC₅₀ values and achieved picomolar DC₅₀ values and >99% of maximum degradation (D_max) in EBC-1 and Hs746T cells. Mechanistically, D10 and D15 dramatically induced cell apoptosis, G1 cell cycle arrest and inhibited cell migration and invasion. Notably, intraperitoneal administration of D10 and D15 significantly inhibited tumor growth in the EBC-1 xenograft model and oral administration of D15 induced approximately complete tumor suppression in the Hs746T xenograft model with well-tolerated dose-schedules. Furthermore, D10 and D15 exerted significant anti-tumor effect in cells with c-MET^Y1230H and c-MET^D1228N mutations, which are resistant to tepotinib in clinic. These findings demonstrated that D10 and D15 could serve as candidates for the treatment of tumors with MET alterations.