OBJECTIVES: To explore the mechanism of cinnamic acid (CA) for improving doxorubicin-induced myocardial injury (DIC) in mice. METHODS: Network pharmacology analysis was used to obtain the key targets of CA and DIC. Male C57BL/6J mice were randomized into Sham, DOX, CA (25, 50 and 100 mg/kg)+DOX, and CA+Ferrostatin-1+DOX groups, and their myocardial function and pathology were examined by echocardiography and HE staining. Serum levels of CK-MB, LDH, MDA, IL-6, TNF‑α and myocardial ROS level were detected, and the expression levels of TLR4 and ferroptosis pathway proteins in myocardial tissue were detected by Western blotting. Cultured murine cardiomyocytes (HL-1 cells) with or without transfection with a small interfering RNA targeting TLR4 (si-TLR4) were treated with DOX or Erastin, and the cellular ROS content was measured by DCFH-DA staining; the expression level of GPX4 was detected using immunofluorescence staining. RESULTS: Network pharmacology analysis suggested that CA may improve DIC through TLR4 signaling. DOX treatment caused obvious myocardial injury in mice, which showed significantly increased serum levels of CK-MB, LDH, MDA, IL-6, TNF-α and myocardial ROS level with decreased myocardial levels of SLC7A11 and GPX4 proteins and increased levels of TLR4 and PTGS2 proteins. All these changes in the mouse models were significantly alleviated by treatment with CA, and the mice receiving CA or ferrostatin-1 treatment exhibited increased myocardial expressions of SLC7A11 and GPX4 proteins and lowered expressions of TLR4 and PTGS2 proteins. In cultured HL-1 cells, treatment with DOX and Erastin both obviously increased intracellular ROS level and decreased cellular GPX4 expression level, and these changes were strongly attenuated by TLR4 interference. CONCLUSIONS CA, as a potent herbal monomer, can effectively alleviate DIC in mice by inhibiting TLR4-mediated ferroptosis.
Animals ; Ferroptosis/drug effects* ; Toll-Like Receptor 4/metabolism* ; Myocytes, Cardiac/metabolism* ; Mice, Inbred C57BL ; Mice ; Male ; Doxorubicin/adverse effects* ; Cinnamates/pharmacology* ; Signal Transduction ; Reactive Oxygen Species/metabolism*

Objective To investigate the effects and mechanisms of high-fat diet(HFD)-induced obesity on male ze-brafish reproductive function.Methods Adult male zebrafish were divided into normal diet(ND)group and HFD group.Growth and metabolic conditions were evaluated by measuring body weight,body length,BMI,organ index,and glucose/lipid lev-els.Reproductive capacity was assessed via sperm concentration,motility,fertilization rate,and testosterone levels.Testicular tissues from both of groups were subjected to transeriptomic sequencing(RNA-seq).And qRT-PCR was used to validate the expression of genes.Results Compared to male zebrafish in ND group,the ones in HFD group exhibited hepatic steatosis and glucose/lipid meta-bolic disorders(P＜0.05).Testicular structural disorganization,along with reduced testosterone levels,decreased gonadosomatic in-dex,and impaired sperm concentration and motility occurred in HFD group(P＜0.05).GO analysis revealed that differentially ex-pressed genes were enriched in spermatogenesis and ciliary system,while KEGG analysis highlighted metabolic related pathways(pu-rine metabolism,thyroid hormone synthesis,mTOR signaling)and cell adhesion molecules.Twenty key differentially expressed genes were validated by qRT-PCR,which confirmed the reliability of RNA-Seq results.Conclusion Impairment of reproductive function induced by HFD in zebrafish may be associated with three regulatory mechanisms including ciliary system,metabolic dysregulation,and aberrant cell adhesion molecule signaling.This study provides mechanistic insights and identifies potential therapeutic targets for clinical management of diet-associated infertility.

Objective To investigate the effects and mechanisms of high-fat diet(HFD)-induced obesity on male ze-brafish reproductive function.Methods Adult male zebrafish were divided into normal diet(ND)group and HFD group.Growth and metabolic conditions were evaluated by measuring body weight,body length,BMI,organ index,and glucose/lipid lev-els.Reproductive capacity was assessed via sperm concentration,motility,fertilization rate,and testosterone levels.Testicular tissues from both of groups were subjected to transeriptomic sequencing(RNA-seq).And qRT-PCR was used to validate the expression of genes.Results Compared to male zebrafish in ND group,the ones in HFD group exhibited hepatic steatosis and glucose/lipid meta-bolic disorders(P＜0.05).Testicular structural disorganization,along with reduced testosterone levels,decreased gonadosomatic in-dex,and impaired sperm concentration and motility occurred in HFD group(P＜0.05).GO analysis revealed that differentially ex-pressed genes were enriched in spermatogenesis and ciliary system,while KEGG analysis highlighted metabolic related pathways(pu-rine metabolism,thyroid hormone synthesis,mTOR signaling)and cell adhesion molecules.Twenty key differentially expressed genes were validated by qRT-PCR,which confirmed the reliability of RNA-Seq results.Conclusion Impairment of reproductive function induced by HFD in zebrafish may be associated with three regulatory mechanisms including ciliary system,metabolic dysregulation,and aberrant cell adhesion molecule signaling.This study provides mechanistic insights and identifies potential therapeutic targets for clinical management of diet-associated infertility.

Objective To construct c-Met CAR-T cells secreting PD-1 antibodies to reduce immune inhibitory effect of tumor cells and enhance the efficacy of CAR-T cell therapy against pancreatic cancer.Methods Kaplan-Meier Plotter,GEPIA,and Timer 2.0 bioinformatics databases were used to analyze c-Met expression in pancreatic cancer and its correlation with survival and immune infiltration status.In clinical samples of pancreatic cancer and pancreatic cancer Aspc-1 cells,c-Met and PD-L1 expressions were detected using immunohistochemistry or flow cytometry.Using gene editing technology,PD-1 secretory antibodies and HIS tags were linked to second-generation c-Met CAR molecules to construct PD-1/c-Met CAR plasmids,which were then packaged into lentiviruses for infection of activated T cells.The positive rate and cell subset distribution of CAR-T cells were analyzed with flow cytometry,and secretory PD-1 antibodies in cell supernatants were detected using Western blotting.The target cell killing efficiency and proliferative activity of the modified CAR-T cells were evaluated after activation,and cytokine secretion was analyzed using ELISA.Results The expression of c-Met was significantly higher in pancreatic cancer than in normal tissues,and its expression level was negatively correlated with the patients'survival and positively correlated with immune cell infiltration.The clinical samples of pancreatic cancer tissues expressed significantly higher levels of c-Met and PD-L1 than the adjacent tissues,and 90.7%and 57.7%of Aspc-1 cells were positive for c-Met and PD-L1,respectively.The constructed PD-1/c-Met CAR-T cells were capable of secreting PD-1 antibodies and showed a significantly higher killing efficiency against tumor cells than c-Met CAR-T cells at an effector-to-target ratio of 20:1,with also a higher proliferative activity after target cell stimulation and higher levels of IL-2 and TNF-α secretin.Conclusion PD-1/c-Met CAR-T cells have higher killing efficiency against pancreatic cancer cells with also higher proliferative activity than c-Met CAR-T cells.

Objective To construct c-Met CAR-T cells secreting PD-1 antibodies to reduce immune inhibitory effect of tumor cells and enhance the efficacy of CAR-T cell therapy against pancreatic cancer.Methods Kaplan-Meier Plotter,GEPIA,and Timer 2.0 bioinformatics databases were used to analyze c-Met expression in pancreatic cancer and its correlation with survival and immune infiltration status.In clinical samples of pancreatic cancer and pancreatic cancer Aspc-1 cells,c-Met and PD-L1 expressions were detected using immunohistochemistry or flow cytometry.Using gene editing technology,PD-1 secretory antibodies and HIS tags were linked to second-generation c-Met CAR molecules to construct PD-1/c-Met CAR plasmids,which were then packaged into lentiviruses for infection of activated T cells.The positive rate and cell subset distribution of CAR-T cells were analyzed with flow cytometry,and secretory PD-1 antibodies in cell supernatants were detected using Western blotting.The target cell killing efficiency and proliferative activity of the modified CAR-T cells were evaluated after activation,and cytokine secretion was analyzed using ELISA.Results The expression of c-Met was significantly higher in pancreatic cancer than in normal tissues,and its expression level was negatively correlated with the patients'survival and positively correlated with immune cell infiltration.The clinical samples of pancreatic cancer tissues expressed significantly higher levels of c-Met and PD-L1 than the adjacent tissues,and 90.7%and 57.7%of Aspc-1 cells were positive for c-Met and PD-L1,respectively.The constructed PD-1/c-Met CAR-T cells were capable of secreting PD-1 antibodies and showed a significantly higher killing efficiency against tumor cells than c-Met CAR-T cells at an effector-to-target ratio of 20:1,with also a higher proliferative activity after target cell stimulation and higher levels of IL-2 and TNF-α secretin.Conclusion PD-1/c-Met CAR-T cells have higher killing efficiency against pancreatic cancer cells with also higher proliferative activity than c-Met CAR-T cells.

Objective To investigate the role of TLR4/NF-κB signal pathway in pathogenesis of brain inju-ry during deep hypothermia circulatory arrest(DHCA). Methods BV2 microglia cells were subjected to oxygen-glucose deprivation/reoxygenation(OGD/R),in vitro model for DHCA. The BV2 were randomly divided into the control group(C group)and the experimental group(O group). BV2 viability was determined by CCK-8 assay. TLR4 and its downstream signaling molecules ,MyD88 and phosphorylated NF-κB (p-p65) expressions were detected by Western blotting. TLR4 mRNA expression in BV2 microglial cells were determined by RT-PCR. Level of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in culture medium was detected by ELASA. Results Compared with the group C,BV2 microglia cell viability in experiment group was obviously weaker(P<0.05). Expressions of TLR4,MyD88 and phosphorylated NF-κB(p-p65)from the experiment group increased remarkedly than those from the group C (P < 0.05). TLR4 mRNA level was higher significantly in the group O than in the group C (P < 0.01). Production of IL-6 and TNF-α in the group O were up-regulated apparently compared to the group C(P<0.01). Conclusion TLR4/NF-κB signaling pathway contributed to activation of BV2 microglia cells treated by OGD/Reoxygenation ,which was probably the exactly way that involved in pathogenesis of brain injury during deep hypothermia circulatory arrest.