Deregulation of endothelial nitric oxide synthase (eNOS) plays an important role in the development of multiple cardiovascular diseases. Our recent study demonstrated that genistein supplementation attenuates pulmonary arterial hypertension in broilers by restoration of endothelial function. In this study, we investigated the molecular mechanism by using broiler pulmonary arterial endothelial cells (PAECs). Our results showed that genistein stimulated a rapid phosphorylation of eNOS at Ser(1179) which was associated with activation of eNOS/NO axis. Further study indicated that the activation of eNOS was not mediated through estrogen receptors or tyrosine kinase inhibition, but via a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent signaling pathway, as the eNOS activity and related NO release were largely abolished by pharmacological inhibitors of PI3K or Akt. Thus, our findings revealed a critical function of Akt in mediating genistein-stimulated eNOS activity in PAECs, partially accounting for the beneficial effects of genistein on the development of cardiovascular diseases observed in animal models.
Animals ; Cell Line ; Chickens ; Disease Models, Animal ; Endothelium, Vascular/drug effects/*metabolism/pathology ; Enzyme Activation/drug effects ; Female ; Genistein/*pharmacology ; Humans ; Hypertension, Pulmonary/drug therapy/*metabolism/pathology ; Nitric Oxide Synthase Type III/genetics/*metabolism ; Oncogene Protein v-akt/*metabolism ; Phosphorylation ; Signal Transduction/drug effects

ObjectiveTo explore the mechanism of Cangxi Tongbi capsules （CXTB） in regulating the p38 mitogen-activated protein kinase （p38 MAPK）/NOD-like receptor protein 3 （NLRP3）/cysteine protease-1 （Caspase-1） signaling pathway to inhibit pyroptosis of cartilage cells in knee osteoarthritis （KOA）. MethodSixty male SD rats were randomly divided into a sham operation group， a model group， low， medium， and high dose CXTB groups， and a positive control group， with 10 rats per group. The modified Hulth method was employed to establish a rat model of KOA. According to their respective assignments， rats were administered CXTB （0.25， 0.5， 1.0 g·kg^-1） and Celecoxib （24 mg·kg^-1） by gavage. The sham operation and model groups were given an equivalent volume of physiological saline. Treatment was performed once daily for 28 days. Micro-computed tomography （Micro-CT） was used to assess bone volume/total volume （BV/TV） and trabecular separation （Tb.Sp）. Joint degeneration was evaluated through hematoxylin-eosin （HE） staining， safranin-fast green （SO） staining， and Osteoarthritis Research Society International （OARSI） scoring. Western blot analysis was conducted to measure the expression levels of p38 MAPK， phosphorylated p38 MAPK （p-p38 MAPK）， NLRP3， Caspase-1， and gasdermin D （GSDMD） proteins. Real-time PCR was used to assess mRNA expression levels of p38 MAPK， NLRP3， Caspase-1， and GSDMD genes. Enzyme-linked immunosorbent assay （ELISA） was used to measure serum concentrations of inflammatory cytokines including tumor necrosis factor-alpha （TNF-α）， interleukin-1 beta （IL-1β）， and interleukin-18 （IL-18）. After knee replacement surgery， cartilage tissue was analyzed using Western blot to assess the protein expression levels of p38 MAPK， p-p38 MAPK， NLRP3， Caspase-1， and GSDMD， and Real-time PCR was used to evaluate gene expression levels of p38 MAPK， NLRP3， Caspase-1， and GSDMD. ResultMicro-CT analysis revealed significant narrowing of the joint space and increased bone spur formation in KOA rats compared with the sham operation group， with a decrease in BV/TV ratio and an increase in Tb.Sp value （P<0.01）. Serum levels of TNF-α， IL-1β， and IL-18 were elevated （P<0.01）. The protein expression levels of p-p38 MAPK， NLRP3， Caspase-1， and GSDMD in cartilage were significantly increased （P<0.01）， and the mRNA expression levels of p38 MAPK， NLRP3， Caspase-1， and GSDMD were also enhanced （P<0.01）. Significant differences in protein expression of p-p38 MAPK， NLRP3， Caspase-1， and GSDMD were observed between normal and diseased cartilage tissues after knee replacement surgery （P<0.05）， and the gene expression of p38 MAPK， NLRP3， Caspase-1， and GSDMD were also significantly different （P<0.01）. HE and SO staining showed roughened joint surfaces， reduced cartilage thickness， and disordered cellular arrangement in KOA rats. OARSI scores were significantly higher （P<0.01）. Compared with the model group， treatment with low， medium， and high concentrations of CXTB resulted in increased BV/TV ratios and decreased Tb.Sp values in the knee joints of rats （P<0.01）. HE and SO staining indicated a trend towards smoother joint surfaces and reduced OARSI scores （P<0.01）. The protein expression levels of p-p38 MAPK， NLRP3， Caspase-1， and GSDMD were notably decreased （P<0.05）， as were the mRNA expression levels of p38 MAPK， NLRP3， Caspase-1， and GSDMD （P<0.01）. Additionally， serum concentrations of TNF-α， IL-1β， and IL-18 were significantly reduced （P<0.01）. ConclusionCXTB intervention may alleviate knee joint degeneration in KOA rats and inhibit the expression of inflammatory factors and pyroptosis of cartilage cells， thereby protecting cartilage. The underlying mechanism may involve modulation of the p38 MAPK/NLRP3/Caspase-1 signaling pathway.

Objective To explore the role of MAPK4 in cervical squamous cell carcinoma(CSCC)for the identification of candidate prognostic prediction biomarkers and molecular therapeutic targets.Methods The TCGA cohort was subjected to Kaplan-Meier survival analysis.Immunohistochemistry experiments were conducted on clinical samples to explore the correlation between MAPK4 and patient prognosis.A nomogram was constructed based on MAPK4 mRNA levels.Western blot,CCK-8,colony formation,and Transwell cell function experiments were performed to clarify the abnormal expression and role of MAPK4 in CSCC.DIA proteome sequencing was used to identify effector molecules regulated by MAPK4.Combined with the above cell function experiments,the knockdown of MAPK4 and the overexpression of effector molecules revealed that MAPK4 regulated effector molecules to mediate tumor progression.Results CSCC patients with elevated MAPK4 mRNA levels and high protein expression have a worse prognosis.The constructed nomogram based on MAPK4 can accurately predict the 1-,3-,and 5-year survival rates of patients.Compared with that in normal cervical tissues,MAPK4 protein expre-ssion was up-regulated in tumors.MAP-K4 knockdown substantially inhibited the proliferation,colony formation,mig-ration,and invasion of CSCC ME180 and SiHa cells.SLC3A2 is a downs-tream effector molecule of MAPK4.Overexpression SLC3A2 can weaken the inhibitory effect of MAPK4 knockdown on cell proliferation,colony formation,migration,and invasion.Conclusion MAPK4 is a candidate prognostic biomarker for patients with CSCC.MAPK4 positively regulates SLC3A2 protein expression and accelerates tumor progression,making it a potential molecular therapeutic target for patients with CSCC.