Calcific aortic valve disease (CAVD) is a prevalent cardiovascular disorder characterized by calcium deposition in the aortic valve, associated with high morbidity and mortality. Mechanical stress plays a key role in its pathogenesis, highlighting the importance of identifying mechanosensitive ion channel-related genes (MICRGs). In this study, transcriptome data from GSE83453 of patients with CAVD and healthy controls were analyzed to identify differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) and MICRGs were used to pinpoint key genes. The intersection of DEGs, WGCNA modules, and MICRGs identified CACNA1H as a candidate gene. External validation with GSE51472 and GSE55492 confirmed these findings. Both in vitro and in vivo studies confirmed that CACNA1H inhibition alleviates CAVD progression by repressing the osteogenic response. Mechanistically, CACNA1H inhibition attenuated phosphorylation of P65, a key regulator of the NF-κB pathway. These results suggest that CACNA1H may serve as a promising biomarker and therapeutic target for CAVD.

BACKGROUND:Pro-EGCG has shown therapeutic promise for endometriosis, yet its immunoregulatory mechanisms remain unclear. Myeloid-derived suppressor cells (MDSCs) are key drivers of disease progression. This study investigates whether Pro-EGCG alleviates endometriosis by modulating MDSC-mediated immune and stromal dysregulation. METHODS:The therapeutic effects of Pro-EGCG were evaluated in an experimental endometriosis mouse model by assessing lesion burden, histology, and MDSC dynamics. The functional necessity of monocytic MDSCs (M-MDSCs) was validated via adoptive transfer. Clinical relevance was assessed in peripheral blood and lesions from women with or without endometriosis via flow cytometry and multiplex immunofluorescence staining. Furthermore, the direct effects of Pro-EGCG on human PBMC-derived M-MDSCs were examined in vitro, including their immunosuppressive function and their ability to promote fibrosis in a co-culture system with human endometriotic stromal cells. RESULTS:In the mouse model, Pro-EGCG treatment significantly reduced lesion weight, volume, and fibrosis, accompanied by consistent M-MDSC reduction systemically and locally. Lesion-infiltrating M-MDSCs, rather than polymorphonuclear MDSCs (PMN-MDSCs), positively correlated with disease severity. Adoptive transfer of M-MDSCs reversed Pro-EGCG's therapeutic effects. In clinical samples, data confirmed a significant expansion of M-MDSCs in patients with endometriosis compared to controls. In vitro, Pro-EGCG compromised human M-MDSC survival and impaired their suppressive capacity by inhibiting ROS, Arg-1, and NO production. Furthermore, M-MDSC-induced stromal cell proliferation and fibrotic gene expression were abolished by Pro-EGCG preconditioning. CONCLUSION:Pro-EGCG hinders endometriosis progression by inhibiting M-MDSC accumulation, immunosuppressive functions, and pro-endometriotic activities. These findings position Pro-EGCG as a potential immunotherapy for endometriosis and other M-MDSC-driven inflammatory disorders.

Gestational diabetes mellitus (GDM), the most common pregnancy-related metabolic disorder, is characterized by exacerbated oxidative stress (OS). The inhibition of phosphoglycerate kinase 1 (PGK1), the first ATP-generating enzyme in the glycolytic pathway, activates Keap1-Nrf2 antioxidant pathways and reduces OS. However, the detailed roles of PGK1 in GDM remain unexplored. Disruption of pro-oxidant/antioxidant homeostasis was observed in the placentas of GDM patients. PGK1 was significantly upregulated in both human GDM placentas and streptozotocin (STZ)-induced model mice. Pharmacological inhibition of PGK1 in vivo ameliorated placental dysfunction, attenuated excessive ROS production, and improved pregnancy outcomes. Lentivirus-mediated PGK1 knockdown in HTR8/SVneo trophoblasts increased Nrf2-dependent antioxidant protein expression while reducing ROS generation. Mechanistically, PGK1 inhibition elevated estradiol levels, facilitating Keap1 dimerization, and this dimerization destabilized the Keap1-Nrf2 complex, enabling Nrf2 accumulation and antioxidant activation. Exogenous estradiol supplementation recapitulated the effect of inhibiting PGK1 by enhancing Keap1 dimer formation, effectively mitigating placental OS and adverse pregnancy phenotypes in GDM models. This study elucidates the critical role of PGK1 in restoring redox homeostasis through the estradiol-Keap1-Nrf2 axis in the pathogenesis of GDM. PGK1/estradiol crosstalk represents a druggable target, and pharmacological PGK1 inhibition has translational potential for mitigating oxidative stress-related pregnancy complications.

Background The long-term effects of physical activity (PA) variability and cumulative PA (CPA) from young adulthood on cardiac remodeling remain controversial, and it is unclear whether these effects resemble those observed in elite athletes.Methods A total of 1465 participants with recorded PA spanning a 30-year period were included in this study. PA levels were assessed at 9 follow-up visits using the CARDIA (Coronary Artery Risk Development in Young Adults) questionnaire. At year 30, left ventricular structure and function were evaluated using standardized echocardiography. CPA was calculated as the sum of average PA levels between consecutive visits multiplied by the corresponding intervisit intervals (in years). PA variability was quantified using average real variability (ARVPA). Additionally, participants were categorized into 4 groups: sustained low CPA and ARVPA group, low CPA and high ARVPA group, high CPA and low ARVPA group, and high CPA and high ARVPA group.Results This study included 849 women (57.1%), 883 White participants (60.3%), and had a mean age of 55.3 years. Participants in the sustained high CPA group exhibited lower incidence of hypertension and diabetes. Multiple linear regression analyses showed that, compared with the sustained-low-CPA group, the sustained-high-CPA group and high-CPA and high-ARVPA group exhibited increased left ventricular end-diastolic dimension index, and the high-CPA and high-ARVPA group revealed an increased left ventricular mass index and left atrial volume index.Conclusions Our study suggests that high CPA, even when accompanied with high variability in PA levels, is associated with alterations in left ventricular structure in midlife.

Previous studies have reported that low-intensity pulsed ultrasound (LIPUS) can alleviate cartilage degradation in osteoarthritis (OA). However, the functions and mechanisms of LIPUS in synovial fibrosis with OA require further study. To investigate the role of the PI3K/AKT signaling pathway in synovial fibrosis and in LIPUS treatment in synovial fibrosis, a TGF-β stimulated rat FLS cell model and a rat OA animal model based on anterior cruciate ligament transection (ACLT) and partial medial meniscectomy (MMx) were used. The results revealed that LIPUS delayed the progression of OA. Masson staining revealed that LIPUS reduced the collagen deposition of synovial tissue in OA rats. Correspondingly, immunofluorescence demonstrated that LIPUS significantly downregulated the expression of α-SMA, Col1a1 and Col3a1 in OA rats. Moreover, TGF-β stimulation upregulated fibrosis markers at the mRNA and protein levels in FLS, as well as increased phosphorylation-dependent activation of the PI3K/Akt pathway. 740Y-P was found to promote the fibrotic change of FLS induced by TGF-β, but LY294002 reduced its expression. However, LIPUS inhibits the fibrotic change and activation of the PI3K/Akt pathway in FLS under stimulation of TGF-β. In conclusion, LIPUS alleviates synovial fibrosis by blocking the PI3K/AKT pathway.

Aims The study aimed to evaluate longitudinal adiposity exposure, assessed by body roundness index (BRI), from young adulthood and its association with the risk of coronary artery calcification (CAC) incidence in mid-life. Methods and results We included 2102 participants from the CARDIA study with available BRI measurements at eight follow-up visits over 25 years. Cardiac computed tomography at the year 25 exam was used to assess the presence of CAC (CAC > 0). The cumulative BRI (cBRI) was calculated as the mean of the BRI between consecutive visits multiplied by the number of years. Logistic regression models and restricted cubic spline (RCS) analyses were used to assess the relationship between cBRI and the risk of CAC incidence. A total of 598 participants developed CAC over the 25-year follow-up period. Higher cBRI was associated with an increased risk of CAC incidence. Participants were divided into four quartiles based on cBRI. In model 3, the odds ratio (OR) for CAC was 2.52(95%CI, 1.82-3.49) in the top quartile of cBRI. RCS analyses suggested a linear relationship between cBRI and the risk of CAC incidence. Furthermore, multivariable Cox regression showed that individuals in the higher cBRI had a higher risk of all-cause mortality (1.009; 95% CI, 1.003-1.016). Conclusion Greater cBRI from early adulthood was associated with an elevated risk of CAC incidence in mid-life. These findings uncover potential evidence to support that BRI may be a useful marker for identifying individuals at high risk of CAC.

The neutrophil-percentage-to-albumin ratio (NPAR) has emerged as a concise and effective biomarker for assessing systemic inflammatory status, with established prognostic value for mortality risk across various disease populations. This study aimed to elucidate the association between NPAR and both all-cause and cardiovascular mortality in patients with diabetes and prediabetes. A cohort of 8560 patients with diabetes and prediabetes was recruited from the National Health and Nutrition Examination Survey (NHANES), with mortality outcomes tracked through the National Death Index up to December 31, 2019. A weighted Cox regression model was used to evaluate the association of NPAR with all-cause and cardiovascular mortality. The restricted cubic spline (RCS) curve was used to assess the nonlinear relationship between NPAR and mortality outcomes. Subgroup analysis was conducted to evaluate the differences in the predictive performance of various NPAR features across different subgroups. Cox regression analysis indicated that participants in the highest quartile of NPAR exhibited a substantially increased risk of all-cause mortality (hazard ratio [HR] 1.54; 95% confidence interval [CI] 1.28-1.87) and cardiovascular death (HR 2.04; 95% CI 1.47-2.84) compared to the reference group. The RCS curve further illustrated a significant nonlinear correlation between NPAR and outcomes in patients with diabetes and prediabetes. NPAR can serve as a robust nonlinear predictor for both all-cause and cardiovascular mortality in individuals diagnosed with diabetes or prediabetes.

Prevascularization is the key challenge for large-scale tissue engineering. Nevertheless, none of the engineered vasculature simultaneously recapitulates the multi-layered heterogeneous characterizations and functions yet. The recent studies reveal that matrix dynamics play an important role in vasculature morphogenesis. In this study, an extracellular matrix-mimicking bioink is developed by the interpenetrated dynamic-covalent crosslinking orthogonal design. The dynamic covalent crosslinking network of the bioink allows an adaptable microenvironment contributing to the functional compartmentalization of endothelial cells and smooth muscle cells toward histological vasculature configurations. Focal adhesion kinase pathway participates in the morphogenesis process by coupling the microscopically adaptable environment to the vasculature organization via upregulation of integrin-mediated adhesion and glycolysis. The engineered vasculature exhibits in vitro contraction in response to angiotensin II and significantly improves blood perfusion in the mouse hind limb ischemia model. In addition, the vascular network successfully prolongs the survival and function of surrounding human dermal fibroblasts postimplantation, which enhances the healing of large full-thickness wounds. Altogether, this work presents a one-step bioprinting strategy of prevascularization in predesigned architecture for vascular tissue engineering.

Advanced glycation end product-modified low-density lipoprotein (AGE-LDL) is related to inflammation and the development of atherosclerosis. Additionally, it has been demonstrated that receptor for advanced glycation end products (RAGE) has a role in the condition known as calcific aortic valve disease (CAVD). Here, we hypothesized that the AGE-LDL/RAGE axis could also be involved in the pathophysiological mechanism of CAVD. Human aortic valve interstitial cells (HAVICs) were stimulated with AGE-LDL following pre-treatment with or without interleukin 37 (IL-37). Low-density lipoprotein receptor deletion (Ldlr−/−) hamsters were randomly allocated to chow diet (CD) group and high carbohydrate and high fat diet (HCHFD) group. AGE-LDL levels were significantly elevated in patients with CAVD and in a hamster model of aortic valve calcification. Our in vitro data further demonstrated that AGE-LDL augmented the expression of intercellular cell adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6) and alkaline phosphatase (ALP) in a dose-dependent manner through NF-κB activation, which was attenuated by nuclear factor kappa-B (NF-κB) inhibitor Bay11-7082. The expression of RAGE was augmented in calcified aortic valves, and knockdown of RAGE in HAVICs attenuated the AGE-LDL-induced inflammatory and osteogenic responses as well as NF-κB activation. IL-37 suppressed inflammatory and osteogenic responses and NF-κB activation in HAVICs. The vivo experiment also demonstrate that supplementation with IL-37 inhibited valvular inflammatory response and thereby suppressed valvular osteogenic activities. AGE-LDL promoted inflammatory responses and osteogenic differentiation through RAGE/NF-κB pathway in vitro and aortic valve lesions in vivo. IL-37 suppressed the AGE-LDL-induced inflammatory and osteogenic responses in vitro and attenuated aortic valve lesions in a hamster model of CAVD.

Objective: Methylmalonic acid (MMA) buildup has recently been suggested to contribute to the onset of both age-related conditions and cardiovascular disorders. This research was aimed at examining the link between MMA and abdominal aortic calcification (AAC). Methods: Data from the 2013–2014 National Health and Nutrition Examination Survey (NHANES) were analyzed. Serum MMA levels were determined through LC-MS/MS, and MMA levels 250 nmol/L or above were considered high. Dual-energy X-ray absorptiometry was used to assess the presence of AAC. Logistic regression analysis was performed after propensity score matching (PSM) to study the relationship between MMA and AAC. Results: A total of 2483 participants were involved in this study. To eliminate large differences between the AAC and non-AAC groups, 1:1 PSM was performed. Logistic regression analysis indicated that participants with high MMA levels had a significantly greater likelihood of experiencing AAC than those with low MMA levels (OR: 1.38, 95% CI: 1.01–1.90, P = 0.046). No statistically interaction effects between AAC and BMI or the estimated glomerular filtration rate (eGFR) were observed in subgroup analyses. Conclusion: Our study indicated a significant association between high serum MMA levels and AAC incidence.

Abnormal subchondral bone remodeling plays a pivotal role in the progression of osteoarthritis (OA). Here, we analyzed subchondral bone samples from OA patients and observed a significant upregulation of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) specifically in subchondral bone osteoclasts. Notably, we found a strong correlation between UCHL1 expression and osteoclast activity in the subchondral bone during OA progression in both human and murine models. Conditional UCHL1 deletion in osteoclast precursors exacerbated OA progression, while its overexpression, mediated by adeno-associated virus 9, alleviated this process in male mice. Mechanistically, RANKL stimulates UCHL1 expression in osteoclast precursors, subsequently stabilizing CD13, augmenting soluble CD13 (sCD13) release, and triggering an autocrine inhibitory effect on the MAPK pathway, thereby suppressing osteoclast formation. These findings unveil a previously unidentified negative feedback loop, RANKL-UCHL1-sCD13, that modulates osteoclast formation and presents a potential therapeutic target for OA. Abnormal subchondral bone remodeling plays a pivotal role in the progression of osteoarthritis. Here, the authors reveal a negative feedback loop, RANKL-UCHL1-sCD13, which limits osteoclastogenesis in the subchondral bone to prevent osteoarthritis progression.

BACKGROUND:Painful diabetic neuropathy (PDN) is closely linked to inflammation, which has been demonstrated to be associated with pyroptosis. Emerging evidence has implicated TANK-binding kinase 1 (TBK1) in various inflammatory diseases. However, it remains unknown whether activated TBK1 causes hyperalgesia via pyroptosis. METHODS:PDN mice model of type 1 or type 2 diabetic was induced by C57BL/6J or BKS-DB mice with Lepr gene mutation. For type 2 diabetes PDN model, TBK1-siRNA, Caspase-1 inhibitor Ac-YVAD-cmk or TBK1 inhibitor amlexanox (AMX) were delivered by intrathecal injection or intragastric administration. The pain threshold and plantar skin blood perfusion were evaluated through animal experiments. The assessments of spinal cord, dorsal root ganglion, sciatic nerve, plantar skin and serum included western blotting, immunofluorescence, ELISA, and transmission electron microscopy. RESULTS:In the PDN mouse model, we found that TBK1 was significantly activated in the spinal dorsal horn (SDH) and mainly located in microglia, and intrathecal injection of chemically modified TBK1-siRNA could improve hyperalgesia. Herein, we described the mechanism that TBK1 could activate the noncanonical nuclear factor κB (NF-κB) pathway, mediate the activation of NLRP3 inflammasome, trigger microglia pyroptosis, and ultimately induce PDN, which could be reversed following TBK1-siRNA injection. We also found that systemic administration of AMX, a TBK1 inhibitor, could effectively improve peripheral nerve injury. These results revealed the key role of TBK1 in PDN and that TBK1 inhibitor AMX could be a potential strategy for treating PDN. CONCLUSIONS:Our findings revealed a novel causal role of TBK1 in pathogenesis of PDN, which raises the possibility of applying amlexanox to selectively target TBK1 as a potential therapeutic strategy for PDN.

AbstractPreeclampsia (PE) is considered as a disease of placental origin. However, the specific mechanism of placental abnormalities remains elusive. This study identified thrombospondin‐1 (THBS1) is downregulated in preeclamptic placentae and negatively correlated with blood pressure. Functional studies show that THBS1 knockdown inhibits proliferation, migration, and invasion and increases the cycle arrest and apoptosis rate of HTR8/SVneo cells. Importantly, THBS1 silencing induces necroptosis in HTR8/SVneo cells, accompanied by the release of damage‐associated molecular patterns (DAMPs). Necroptosis inhibitors necrostatin‐1 and GSK′872 restore the trophoblast survival while pan‐caspase inhibitor Z‐VAD‐FMK has no effect. Mechanistically, the results show that THBS1 interacts with transforming growth factor B‐activated kinase 1 (TAK1), which is a central modulator of necroptosis quiescence and affects its stability. Moreover, THBS1 silencing up‐regulates the expression of neuronal precursor cell‐expressed developmentally down‐regulated 4 (NEDD4), which acts as an E3 ligase of TAK1 and catalyzes K48‐linked ubiquitination of TAK1 in HTR8/SVneo cells. Besides, THBS1 attenuates PE phenotypes and improves the placental necroptosis in vivo. Taken together, the down‐regulation of THBS1 destabilizes TAK1 by activating NEDD4‐mediated, K48‐linked TAK1 ubiquitination and promotes necroptosis and DAMPs release in trophoblast cells, thus participating in the pathogenesis of PE.

Hemodynamic overload and dysregulation of cellular metabolism are involved in development of calcific aortic valve disease (CAVD). However, how mechanical stress relates to metabolic changes in CAVD remains unclear. Here, we show that Piezo1, a mechanosensitive ion channel, regulated glutaminase 1 (GLS1)-mediated gluta-minolysis to promote osteogenic differentiation of valve interstitial cells (VICs). In vivo, two models of aortic valve stenosis were constructed by ascending aortic constriction (AAC) and direct wire injury (DWI). Inhibition of Piezo1 and GLS1 in these models respectively mitigated aortic valve lesion. In vitro, Piezo1 activation induced by Yoda1 and oscillatory stress triggered osteogenic responses in VICs, which were prevented by Piezo1 inhi-bition or knockdown. Mechanistically, Piezo1 activation promoted calcium-dependent Yes-associated protein (YAP) activation. YAP modulated GLS1-mediated glutaminolysis, which enhanced osteogenic differentiation through histone acetylation of runt-related transcription factor 2 (RUNX2) promoters. Together, our work pro-vided a cross-talk between mechanotransduction and metabolism in the context of CAVD.

Objective To explore the association between dietary fiber and heart failure (HF). Methods Data were collected from the 2009–2018 National Health and Nutrition Examination Survey. Dietary fiber intake data were obtained from two 24-h dietary recall interviews. Logistic regression and restricted cubic spline models were used to explore the association of dietary intakes of total, cereal, fruit, and vegetable fiber with HF prevalence. Results A total of 21869 adults were included in this study. After adjusting for multiple confounding factors, the odds ratios (OR) and 95% confidence intervals (CI) for HF was 0.49 (0.28 to 0.87, P for trend = 0.016) for the highest tertile versus lowest tertile of total fiber intake. Similar results were observed for cereal but not fruit and vegetable fiber intake. Dose-response analysis indicated that dietary intake of total and cereal fiber were inversely associated with HF in a linear manner. Conclusion Intakes of total and cereal fiber were inversely associated with HF in adults.

Calcific aortic valve disease (CAVD) is a valvular disease frequently in the elderly individuals that can lead to the valve dysfunction. Osteoblastic differentiation of human aortic valve interstitial cells (HAVICs) induced by inflammation play a crucial role in CAVD pathophysiological processes. To date, no effective drugs for CAVD have been established, and new agents are urgently needed. Piericidin glycosides, obtained from a marine-derived Streptomyces strain, were revealed to have regulatory effects on mitochondria in previous studies. Here, we discovered that 13-hydroxypiericidin A 10-O-α-D-glucose (1→6)-β-D-glucoside (S18), a specific piericidin diglycoside, suppresses lipopolysaccharide- (LPS) induced inflammatory responses of HAVICs by alleviating mitochondrial stress in an interleukin (IL)-37-dependent manner. Knockdown of IL-37 by siRNA not only exaggerated LPS-induced HAVIC inflammation and mitochondrial stress but also abrogated the anti-inflammatory effect of S18 on HAVICs. Moreover, S18 alleviated aortic valve lesions in IL-37 transgenic mice of CAVD model. Microscale thermophoresis (MST) and docking analysis of five piericidin analogues suggested that diglycosides, but not monoglycosides, exert obvious IL-37-binding activity. These results indicate that S18 directly binds to IL-37 to alleviate inflammatory responses in HAVICs and aortic valve lesions in mice. Piericidin diglycoside S18 is a potential therapeutic agent to prevent the development of CAVD.

Upregulation of Brf1 (TFIIB-related factor 1) and Pol III gene (RNA polymerase III-dependent gene, such as tRNAs and 5S rRNA) activities is associated with cell transformation and tumor development. Alcohol intake causes liver injury, such as steatosis, inflammation, fibrosis, and cirrhosis, which enhances the risk of HCC development. However, the mechanism of alcohol-promoted HCC remains to be explored. We have designed the complementary research system, which is composed of cell lines, an animal model, human samples, and experiments in vivo and in vitro, to carry out this project by using molecular biological, biochemical, and cellular biological approaches. It is a unique system to explore the mechanism of alcohol-associated HCC. Our results indicate that alcohol upregulates Brf1 and Pol III gene (tRNAs and 5S rRNA) transcription in primary mouse hepatocytes, immortalized mouse hepatocyte-AML-12 cells, and engineered human HepG2-ADH cells. Alcohol activates MSK1 to upregulate expression of Brf1 and Pol III genes, while inhibiting MSK1 reduces transcription of Brf1 and Pol III genes in alcohol-treated cells. The inhibitor of MSK1, SB-747651A, decreases the rates of cell proliferation and colony formation. Alcohol feeding promotes liver tumor development of the mouse. These results, for the first time, show the identification of the alcohol-response promoter fragment of the Pol III gene key transcription factor, Brf1. Our studies demonstrate that Brf1 expression is elevated in HCC tumor tissues of mice and humans. Alcohol increases cellular levels of Brf1, resulting in enhancement of Pol III gene transcription in hepatocytes through MSK1. Our mechanism analysis has demonstrated that alcohol-caused high-response fragment of the Brf1 promoter is at p-382/+109bp. The MSK1 inhibitor SB-747651A is an effective reagent to repress alcohol-induced cell proliferation and colony formation, which is a potential pharmaceutical agent. Developing this inhibitor as a therapeutic approach will benefit alcohol-associated HCC patients.

Objective To examine the effects of p38 mitogen-activated protein kinase (MAPK) inhibitor on the behavioral response to zebrafish larvae after hypoxia/reoxygenation brain injury and to identify whether the protective effect is mediated by inhibiting apoptosis and protein,and mRNA related to apoptosis.Methods The 5-day post-fertilization zebrafish larvae were randomly assigned to 3 groups:control group,model group and intervention group.Fishes in the intervention group were separated into 3 subgroups according to p38 MAPK inhibitor concentration (5,10,20 μmol/L).The activity levels of the larvae were analyzed by using quantization mode of ZebraLab software,swimming distance and moving speed were recorded.Terminal transferase dUTP nick end labeling (TUNEL) assays of brain assays were performed.The protein levels of phosphorylation of p38 MAPK,apoptosis related proteins of B-cell lymphoma-2 (Bcl-2),Bcl-2 associated X protein(Bax) and Caspase-3 were determined by Western blot.The mRNA expressions of Bcl-2,Bax,and caspase-3 were also analyzed by reverse transcription-quantitative PCR (RT-qPCR).Results The activity movement analysis of the intervention group 2 and 3 demonstrated a significantly increase in the swimming distance compared with the model group(P ＜ 0.05).After irradiation under strong light,all groups showed dramatically increasing in the moving speed.After removal of strong light,a significant decrease in moving speed was found in the control group and intervention group 2 and intervention group 3.The TUNEL assay showed that apoptosis index decreased in the intervention group (21.7 ±2.0,12.8 ± 1.9,17.7 ±2.6) compared with model group (46.8 ±5.3) (all P ＜0.01).Western blot assays demonstrated a significant increase protein level of phosphorylation of p38 MAPK after hypoxia and reoxygenation,and the inhibitor reduced the p-p38 MAPK expression.Compared with the model group,p38 MAPK inhibitor increased the protein and mRNA expression level of Bcl-2,whereas reduced the Bax and caspase-3 expression in the brain.Conclusions Under the influences of p38 MAPK inhibitor,zebrafish larvae improved the behavioral changes after hypoxia-induced brain injury.The inhibitor (10 μmol/L) optimally reduces hypoxia-induced apoptosis in brain by up-regulating Bcl-2,down-regulating Bax/caspase-3 protein and their mRNA level.

Recent studies have shown that the phosphorylation and dephosphorylation of ULK1 and ATG13 are related to autophagy activity. Although ATG16L1 is absolutely required for autophagy induction by affecting the formation of autophagosomes, the post-translational modification of ATG16L1 remains elusive. Here, we explored the regulatory mechanism and role of ATG16L1 phosphorylation for autophagy induction in cardiomyocytes. We showed that ATG16L1 was a phosphoprotein, because phosphorylation of ATG16L1 was detected in rat cardiomyocytes during hypoxia/reoxygenation (H/R). We not only demonstrated that CSNK2 (casein kinase 2) phosphorylated ATG16L1, but also identified the highly conserved Ser139 as the critical phosphorylation residue for CSNK2. We further established that ATG16L1 associated with the ATG12-ATG5 complex in a Ser139 phosphorylation-dependent manner. In agreement with this finding, CSNK2 inhibitor disrupted the ATG12-ATG5-ATG16L1 complex. Importantly, phosphorylation of ATG16L1 on Ser139 was responsible for H/R-induced autophagy in cardiomyocytes, which protects cardiomyocytes from apoptosis. Conversely, we determined that wild-type PPP1 (protein phosphatase 1), but not the inactive mutant, associated with ATG16L1 and antagonized CSNK2-mediated phosphorylation of ATG16L1. Interestingly, one RVxF consensus site for PPP1 binding in the C-terminal tail of ATG16L1 was identified; mutation of this site disrupted its association with ATG16L1. Notably, CSNK2 also associated with PPP1, but ATG16L1 depletion impaired the interaction between CSNK2 and PPP1. Collectively, these data identify ATG16L1 as a bona fide physiological CSNK2 and PPP1 substrate, which reveals a novel molecular link from CSNK2 to activation of the autophagy-specific ATG12-ATG5-ATG16L1 complex and autophagy induction.