BACKGROUND:Forskolin,a diterpenoid natural compound extracted from Coleus forskohlii,has a crucial regulatory role in skeletal muscle repair.However,the regulatory role of forskolin on myogenic differentiation of C2C12 skeletal muscle cells has not been fully explored.OBJECTIVE:To explore the effects of forskolin on the differentiation of C2C12 myoblast cell line and probe into the underlying molecular mechanisms.METHODS:C2C12 cells were treated with 0,0.1,0.25,0.5,1,5,10 and 20 μmol/L forskolin during growth,and cell proliferation was detected by cell counting kit-8 and qRT-PCR.C2C12 cells were treated with 0,0.25,0.5 and 1 μmol/L forskolin during the induction of myogenic differentiation.Immunofluorescence staining and qRT-PCR were used to detect C2C12 cells differentiation.Western blot was used to detect the expression level of myogenic differentiation-related signaling pathway proteins.RESULTS AND CONCLUSION:(1)The viability of C2C12 cells was decreased and cell proliferation was inhibited after treatment with high concentrations(＞1 μmol/L)of forskolin.(2)The qRT-PCR results showed that forskolin up-regulated the expression of Myh2,Myh4,Myomaker,but down-regulated the expression of Myh7 compared with the 0 μmol/L group,when C2C12 cells were differentiated for 4 days.Immunofluorescence staining results showed that the fusion index and myotube diameter of C2C12 cells were increased after forskolin treatment,and the number of myotubes was also increased.(3)Western blot results showed that the phosphorylated extracellular signal-regulated kinase 1/2 expression was inhibited;however,the phosphorylated protein kinase B was promoted after treatment with forskolin.The protein expression level of the myogenic differentiation transcription factor Myogenin was significantly up-regulated after treatment with forskolin.The above results demonstrate that forskolin may promote myogenic differentiation of C2C12 skeletal muscle cells through the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathway.

ObjectiveTo analyze the epidemiological characteristics of 8 major respiratory pathogens in influenza-like illness (ILI) cases with acute respiratory infections at fever clinics in Huangpu District, Shanghai from 2015 to 2024, and to provide a scientific basis for the prevention and treatment of respiratory diseases. MethodsA retrospective study was conducted in Huangpu District. Individuals meeting the case definition of ILI from 2015 to 2024 was registered. Their nasopharyngeal swabs were collected for pathogen detection. A total of 8 respiratory viruses were tested, including Influenza A virus (Flu A), Influenza B virus (Flu B), adenovirus (ADV), enterovirus/human rhinovirus (EV/HRV), human parainfluenza virus (HPIV), human coronavirus (HCoV), respiratory syncytial virus (RSV), and human metapneumovirus (HMPV). ResultsFrom 2015 to 2019, a total of 344 ILI cases were tested, of which 192 out of 344 cases (55.81%) were tested positive for single respiratory pathogen. From 2023 to 2024, 1 557 ILI cases were tested, with 572 out of 1 557 cases (36.74%) being positive for single pathogen. From 2023 to 2024, the positive rate of single pathogen in ILI cases was significantly lower than that in 2015‒2019 (χ²=42.66, P<0.001). Specifically, the positive rate of Flu A (χ²=74.43, P<0.001) decreased, while that of HPIV (χ²=8.66, P=0.003) increased, both with statistically significant differences. According to the seasonal pattern, the epidemic intensity of Flu A decreased in summer, while that of HPIV increased in summer and autumn. Demographic results showed statistically significant differences in the positive rates of EV/HRV between genders (χ²=22.38, P<0.001), with males exhibiting a higher positive rate than females. No statistically significant differences were identified in the positive rates of single pathogen among different age groups (χ²=4.42, P=0.110). Nevertheless, statistically significant differences were noted when comparing the positive rates of EV/HRV, Flu A, Flu B and HPIV across different age groups (P<0.05). EV/HRV was more commonly detected in the 15‒<25 age group (10.93%), while Flu A and HPIV had the highest positive rates in the ≥60 age group (21.24% and 4.77%). Flu B had the highest positive rate in the 25‒<60 age group (11.26%). 52.63% of cases with co-infections occurred during winter, with the primary pathogens involved being EV/HRV (9 cases) and HCoV (6 cases). The most prevalent combination of co-infection was Flu A with EV/HRV. ConclusionThe prevalence of respiratory pathogens among ILI cases from 2023 to 2024 exhibited notable fluctuations compared to that from 2015 to 2019. Therefore, influenza surveillance should be strengthened, and attention should also be paid to the prevalence of respiratory pathogens such as HPIV. These findings have profound implications for future research, surveillance, vaccine planning, and public health policy making.

BACKGROUND:Forskolin,a diterpenoid natural compound extracted from Coleus forskohlii,has a crucial regulatory role in skeletal muscle repair.However,the regulatory role of forskolin on myogenic differentiation of C2C12 skeletal muscle cells has not been fully explored.OBJECTIVE:To explore the effects of forskolin on the differentiation of C2C12 myoblast cell line and probe into the underlying molecular mechanisms.METHODS:C2C12 cells were treated with 0,0.1,0.25,0.5,1,5,10 and 20 μmol/L forskolin during growth,and cell proliferation was detected by cell counting kit-8 and qRT-PCR.C2C12 cells were treated with 0,0.25,0.5 and 1 μmol/L forskolin during the induction of myogenic differentiation.Immunofluorescence staining and qRT-PCR were used to detect C2C12 cells differentiation.Western blot was used to detect the expression level of myogenic differentiation-related signaling pathway proteins.RESULTS AND CONCLUSION:(1)The viability of C2C12 cells was decreased and cell proliferation was inhibited after treatment with high concentrations(＞1 μmol/L)of forskolin.(2)The qRT-PCR results showed that forskolin up-regulated the expression of Myh2,Myh4,Myomaker,but down-regulated the expression of Myh7 compared with the 0 μmol/L group,when C2C12 cells were differentiated for 4 days.Immunofluorescence staining results showed that the fusion index and myotube diameter of C2C12 cells were increased after forskolin treatment,and the number of myotubes was also increased.(3)Western blot results showed that the phosphorylated extracellular signal-regulated kinase 1/2 expression was inhibited;however,the phosphorylated protein kinase B was promoted after treatment with forskolin.The protein expression level of the myogenic differentiation transcription factor Myogenin was significantly up-regulated after treatment with forskolin.The above results demonstrate that forskolin may promote myogenic differentiation of C2C12 skeletal muscle cells through the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathway.

Objective: To explore the association between serum nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), NIMA-related kinase 7 (NEK7) and pulmonary fibrosis among patients with coal workers' pneumoconiosis, so as to provide a basis for the assessment of the degree of pulmonary fibrosis. Methods: Coal workers with pneumoconiosis hospitalized in the Second Affiliated Hospital of Xuzhou Medical University from July 2022 to July 2023 were selected by simple random sampling. Data such as age, stage of pneumoconiosis, and dust-exposure duration were collected through the hospital's electronic medical record management system. Venous blood was collected to detect the levels of serum NLRP3 and NEK7. High-resolution computed tomography (HRCT) image data of the chest were obtained through the hospital's imaging reporting system. The left and right lungs were divided into 6 pulmonary regions according to the upper, middle, and lower parts. The pulmonary fibrosis score was quantified according to the proportion of the pulmonary area occupied by HRCT manifestations of pulmonary fibrosis, including reticular shadows, pleural and interlobular septal thickening, traction bronchiectasis, and honeycombing. The association between the levels of serum NLRP3, NEK7, and pulmonary fibrosis was analyzed using a multiple linear regression model. Results: A total of 81 patients with coal workers' pneumoconiosis were included, all of whom were male, with a mean age of (71.46±11.69) years. There were 48, 28, and 5 cases in stage Ⅰ, stage Ⅱ, and stage Ⅲ of pneumoconiosis pathological staging, accounting for 59.26%, 34.57%, and 6.17%, respectively. There were 45 cases of tunneling and coal mining, accounting for 55.56%. There were 41 cases with dust exposure years of ≥30 years, accounting for 50.62%. The median serum NLRP3 and NEK7 in patients with coal workers' pneumoconiosis were 2.01 (interquartile range, 2.33) ng/mL and 0.98 (interquartile range, 0.83) ng/mL. The median score of pulmonary fibrosis was 5.00 (interquartile range, 5.50) points. After adjusting for age, stage of pneumoconiosis, type of work and dust-exposure duration, multiple linear regression analysis showed that serum NLRP3 (β'=0.649) and NEK7 (β'=0.346) were positively correlated with the pulmonary fibrosis score. Conclusion The increase in the levels of serum NLRP3 and NEK7 in patients with coal workers' pneumoconiosis is related to the increase in the degree of pulmonary fibrosis.

BACKGROUND: Salvianolate is a compound mainly composed of salvia magnesium acetate, which is extracted from the Chinese herb Salvia miltiorrhiza . In recent years, salvianolate injection has been widely used in the treatment of cardiovascular diseases, but the mechanism of how it can alleviate cardiotoxicity remains unclear. METHODS: The cardiac injury model was constructed by treatment with doxorubicin (Dox) or azithromycin (Azi) in zebrafish larvae. Heart phenotype, heart rate, and cardiomyocyte apoptosis were observed in the study. RNA-sequencing (RNA-seq) analysis was used to explore the underlying mechanism of salvianolate treatment. Moreover, cardiomyocyte autophagy was assessed by in situ imaging. In addition, the miR-30a/becn1 axis regulation by salvianolate was further investigated. RESULTS: Salvianolate treatment reduced the proportion of pericardial edema, recovered heart rate, and inhibited cardiomyocyte apoptosis in Dox/Azi-administered zebrafish larvae. Mechanistically, salvianolate regulated the lysosomal pathway and promoted autophagic flux in zebrafish cardiomyocytes. The expression level of becn1 was increased in Dox-induced myocardial tissue injury after salvianolate administration; overexpression of becn1 in cardiomyocytes alleviated the Dox/Azi-induced cardiac injury and promoted autophagic flux in cardiomyocytes, while becn1 knockdown blocked the effects of salvianolate. In addition, miR-30a, negatively regulated by salvianolate, partially inhibited the cardiac amelioration of salvianolate by targeting becn1  directly. CONCLUSION This study has proved that salvianolate reduces cardiomyopathy by regulating autophagic flux through the miR-30a/becn1 axis in zebrafish and is a potential drug for adjunctive Dox/Azi therapy.
Animals ; Zebrafish ; MicroRNAs/genetics* ; Autophagy/drug effects* ; Myocytes, Cardiac/metabolism* ; Cardiomyopathies/metabolism* ; Beclin-1/genetics* ; Apoptosis/drug effects* ; Plant Extracts/therapeutic use* ; Doxorubicin

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

The plant F-box protein more axillary growth 2 (MAX2) is a key factor in the signal transduction of strigolactones (SLs) and karrinkins (KARs). As the main component of the SKP1-CUL1-FBX (SCF) complex ubiquitin ligase E3, MAX2 is responsible for specifically recognizing the target proteins, suppressor of MAX2 1/SMAX1-like proteins (SMAX1/SMXLs), which would be degraded after ubiquitination. It can thereby regulate plant morphogenesis and stress responses. There exist homologous genes of MAX2 in the important grain and oil crop soybean (Glycine max). However, its role in plant defense responses has not been investigated yet. Here, GmMAX2b, a homologous gene of MAX2, was successfully cloned from stressed soybean. Bioinformatics analysis revealed that there were two MAX2 homologous genes, GmMAX2a and GmMAX2b, with a similarity of 96.2% in soybean. Their F-box regions were highly conserved. The sequence alignment and cluster analysis of plant MAX2 homologous proteins basically reflected the evolutionary relationship of plants and also suggested that soybean MAX2 might be a multifunctional protein. Expression analysis showed that plant pathogen infection and salicylic acid treatment induced the expression of GmMAX2b in soybean, which is consistent with that of MAX2 in Arabidopsis. Ectopic expression of GmMAX2b compensated for the susceptibility of Arabidopsis max2-2 mutant to pathogen, indicating that GmMAX2b positively regulated plant disease resistance. In addition, yeast two hybrid technology was used to explore the potential target proteins of GmMAX2b. The results showed that GmMAX2b interacted with SMXL6 and weakly interacted with SMXL2. In summary, GmMAX2b is a positive regulator in plant defense responses, and its expression is induced by pathogen infection and salicylic acid treatment. GmMAX2b might exert its effect through interaction with SMXL6 and SMXL2. This study expands the theoretical exploration of soybean disease resistant F-box and provides a scientific basis for future soybean disease resistant breeding.
Glycine max/metabolism* ; Disease Resistance/genetics* ; Plant Diseases/immunology* ; Plant Proteins/genetics* ; Cloning, Molecular ; Gene Expression Regulation, Plant ; F-Box Proteins/genetics* ; Arabidopsis/genetics* ; Phylogeny

The catalytic activity of 3-mercaptopyruvate (3MP) sulfurtransferase (MPST) converts 3MP to hydrogen sulfide (H₂S). However, the regulatory mechanisms governing MPST and its impact on the brain remain largely unexplored. Our study reveals the neuroprotective role of endothelial MPST-generated H₂S, regulated by protein phosphatase 2A (PP2A). Bioinformatics analysis and RNA sequencing demonstrated that endothelial PP2A is associated with neurodegenerative disease pathways. Cerebral ischemic mice exhibited significant inactivation of endothelial PP2A, evidenced by the reduction of PP2Acα in the brain endothelium. Mice with endothelium-specific null PP2A (PP2A^EC-cKO) exhibited neuronal loss, cognitive dysfunction, and long-term potentiation deficits. Postnatal inactivation of endothelial PP2A also contributes to cognitive dysfunction and neuronal loss. However, regaining endothelial PP2A activity by overexpressing Ppp2ca rescued neuronal dysfunction. Mechanistically, PP2A deficiency is intricately linked to the MPST-H₂S signaling pathway. A robust reduction in endothelial MPST-dependent H₂S production followed PP2A deficiency. Exogenous H₂S treatment and AAV-mediated overexpression of MPST in brain endothelial cells significantly mitigated neuronal dysfunction in PP2A^EC-cKO mice. Furthermore, PP2A deficiency promotes an increase in calcium influx and calpain2 phosphorylation, subsequently leading to MPST degradation. The PP2A activator (FTY720) and MPST activator (3MP sodium) both remarkably restored endothelial MPST-dependent H₂S production, subsequently rescuing ischemia-induced neurological deficits. In conclusion, our study demonstrates that endothelial PP2A deficiency leads to MPST degradation by activating calpain2, thus damaging neuronal function.

Acetaminophen (APAP) is the primary cause of drug-induced acute liver failure. Ovarian tumor deubiquitinase 6A (OTUD6A), a recently discovered deubiquitinase of the OTU family, has been primarily studied in tumor contexts. However, its role in APAP-induced liver injury (AILI) remains unclear. Therefore, this study aimed to investigate the involvement of OTUD6A in the pathogenesis of AILI. Our findings demonstrated a substantial upregulation of OTUD6A in both the liver tissue and isolated hepatocytes of mice following APAP stimulation. OTUD6A knockout exacerbated APAP-induced inflammation, hepatocyte necrosis, and liver injury, whereas OTUD6A overexpression alleviated these pathologies. Mechanistically, OTUD6A directly interacted with the enhancer of zeste homolog 2 (EZH2) and selectively removed K48-linked polyubiquitin chains from EZH2, enhancing its stability. This resulted in increased protein levels of EZH2 and H3K27me3, as well as reduced endoplasmic reticulum (ER) stress and cell death in hepatocytes. Collectively, our research uncovers a novel role for OTUD6A in mitigating APAP-induced liver injury by promoting EZH2 stabilization.

OBJECTIVES: To investigate the therapeutic mechanism of nodakenin for Crohn's disease (CD)-like colitis in mice. METHODS: Using a colonic organoid model with lipopolysaccharide (LPS)- and ATP-induced pyroptosis, we investigated the effects of nodakenin on pyroptosis, intestinal barrier function and inflammatory response by detecting key pyroptosis-regulating factors and assessing changes in permeability and pro-inflammatory factors. In a mouse model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis, the therapeutic effect of nodakenin was evaluated by measuring changes in body weight, DAI score, colonic histopathologies, inflammation score, intestinal barrier function and intestinal epithelial cell pyroptosis. The mechanism of nodakenin protection against pyroptosis of intestinal epithelial cells was explored using network pharmacology analysis and in vivo and in vitro experiments. RESULTS: In LPS- and ATP-induced colonic organoids, treatment with nodakenin significantly inhibited the expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11, improved intestinal FITC-dextran (FD4, 4000) permeability, and decreased the levels of IL-1β and IL-18. In the mouse model of TNBS-induced colitis, nodakenin treatment significantly alleviated weight loss, reduced DAI score, inflammatory cell infiltration and inflammation score, and decreased serum FD4 and I-FABP levels and bacteria translocation to the mesenteric lymph nodes, spleen and liver. The mice with nodakenin treatment had also lowered expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11 in the intestinal mucosa. Network pharmacology analysis suggested that the inhibitory effect of nodakenin on colitis was associated with the PI3K/Akt pathway. In both the colonic organoid model and mouse models of colitis, nodakenin effectively inhibited the activation of the PI3K/Akt pathway, and the application of IGF-1, a PI3K/Akt pathway activator, strongly attenuated the protective effect of nodakenin against intestinal epithelial cell pyroptosis and intestinal barrier dysfunction. CONCLUSIONS Nodakenin protects intestinal barrier function and alleviates CD-like colitis in mice at least partly by inhibiting PI3K/Akt signaling to reduce intestinal epithelial cell pyroptosis.
Animals ; Pyroptosis/drug effects* ; Mice ; Trinitrobenzenesulfonic Acid ; Colitis/drug therapy* ; Epithelial Cells/drug effects* ; Intestinal Mucosa/cytology* ; Disease Models, Animal ; Coumarins/pharmacology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Crohn Disease/drug therapy*