Enhancement mechanism of glycation with l-arabinose and xylose on texture properties of silver carp mince gel.

BACKGROUND: Glycation is a green processing technology. Based on our previous studies, glycation with l-arabinose and xylose was beneficial to enhance the texture properties of silver carp mince (SCM) gels. However, the possible enhancement mechanism remained unclear. Therefore, in this study, SCM gels with different types of reducing sugar (glucose, l-arabinose, and xylose) were prepared based on our previous study. The possible mechanism of texture enhancement of SCM gels was analyzed by investigating the changes in water distribution, protein structures, and microstructure in the gel system. RESULTS: The glycation of l-arabinose and xylose enhanced the hardness, cohesiveness, chewiness, and resilience of SCM gels. Hardness increased from 1883.04 (control group) to 3624.54 (l-arabinose group) and 4348.18 (xylose group). Low-field nuclear magnetic resonance (LF-NMR) showed that glycation promoted the tight binding of immobilized water to proteins. Raman spectroscopic analysis showed that glycation increased the surface hydrophobicity and promoted the formation of disulfide bonds. Scanning electron microscopy (SEM) showed that glycation promoted the formation of uniform and dense three-dimensional network structure in SCM gels. CONCLUSION: In summary, glycation enhanced the binding ability of immobilized water to proteins, improved the surface hydrophobicity, promoted the formation of disulfide bonds, and led to a more uniform and dense gel network structure of proteins, thus enhancing the texture properties of SCM gels. This research provided a theoretical basis for a better understanding of the mechanism of the effect of glycation on the quality of gel products and also provided technical support for the application of l-arabinose and xylose in new functional gel foods. © 2024 Society of Chemical Industry.

ANXA1sp Protects against Sepsis-Induced Myocardial Injury by Inhibiting Ferroptosis-Induced Cardiomyocyte Death via SIRT3-Mediated p53 Deacetylation.

Sepsis-induced myocardial injury (SIMI), a common complication of sepsis, may cause significant mortality. Ferroptosis, a cell death associated with oxidative stress and inflammation, has been identified to be involved in SIMI. This study sought to investigate the role of ANXA1 small peptide (ANXA1sp) in SIMI pathogenesis. In this study, the mouse cardiomyocytes (H9C2 cells) were stimulated with lipopolysaccharide (LPS) to imitate SIMI in vitro. It was shown that ANXA1sp treatment substantially abated LPS-triggered H9C2 cell death and excessive secretion of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6). ANXA1sp pretreatment also reversed the increase of ROS and MDA generation as well as the decrease of SOD and GSH activity in H9C2 cells caused by LPS treatment. In addition, ANXA1sp considerably eliminated LPS-caused H9C2 cell ferroptosis, as revealed by the suppression of iron accumulation and the increase in GPX4 and FTH1 expression. Furthermore, the ameliorative effects of ANXA1sp on LPS-induced H9C2 cell damage could be partially abolished by erastin, a ferroptosis agonist. ANXA1sp enhanced SIRT3 expression in LPS-challenged H9C2 cells, thereby promoting p53 deacetylation. SIRT3 knockdown diminished ANXA1sp-mediated alleviation of cell death, inflammation, oxidative stress, and ferroptosis of LPS-treated H9C2 cells. Our study demonstrated that ANXA1sp is protected against LPS-induced cardiomyocyte damage by inhibiting ferroptosis-induced cell death via SIRT3-dependent p53 deacetylation, suggesting that ANXA1sp may be a potent therapeutic agent for SIMI.

Phloroglucinol inhibits oxytocin-induced contraction in rat gastric circular muscle and uterine smooth muscle.

Phloroglucinol is commonly used to alleviate dysmenorrhoea and stomach cramps. However, there is little evidence of phloroglucinol in the mechanism of primary dysmenorrhoea (PD) development. In this study, a PD rat model was established. The effects of phloroglucinol on the contraction of rat gastric circular muscle and uterine smooth muscle induced by oxytocin (OT) were investigated. The writhing response, and levels of oestradiol (E2), prostaglandin e2 (PGE2), and prostaglandin f2α (PGF2α) were determined. The protein and mRNA levels of OT receptor (OTR) were detected. OT showed a significant promoting effect on gastric circular muscle and uterine smooth muscle contraction. However, phloroglucinol strongly inhibited the contraction induced by 10-6 mol/L of OT. We also found that phloroglucinol reduced writhing response and attenuated uterine damage. Compared to the blank group, E2 and PGF2α were significantly increased, but PGE2 was significantly decreased in the PD model group. Phloroglucinol was found to reverse the changes of E2, PGF2α and PGE2. Moreover, phloroglucinol reduced the protein and mRNA levels of OTR. In conclusion, phloroglucinol could attenuate PD and inhibit the contraction of rat gastric circular muscle and uterine smooth muscle induced by OT. The mechanism might be related with the regulation of OTR expression.IMPACT STATEMENTWhat is already known on this subject? Phloroglucinol is commonly used to alleviate dysmenorrhoea and stomach cramps. However, there is little evidence of phloroglucinol in the mechanism of primary dysmenorrhoea (PD) development.What do the results of this study add? Phloroglucinol could attenuate PD and inhibit the contraction of rat gastric circular muscle and uterine smooth muscle induced by OT. The underlying mechanisms of phloroglucinol for PD treatment may be associated with OTR.What are the implications of these findings for clinical practice and/or further research? These findings provide novel ideas for the role of phloroglucinol in PD development.

NLRC5 protects neurons from oxygen-glucose deprivation-induced injury through activating the Nrf2/HO-1 pathway.

NLRC5 is a member of the Nod-like receptor (NLR) family that has been found to be associated with the hepatic ischemia/reperfusion (I/R) injury. However, the role of NLRC5 in cerebral I/R has not been fully understood. The aim of the current study was to evaluate the effects of NLRC5 on primary hippocampal neuronal cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R). Our results showed that the mRNA and protein levels of NLRC5 were significantly decreased in OGD/R-induced neurons. Overexpression of NLRC5 caused significant increase in cell viability, as well as decrease in ROS level. The bax expression was significantly decreased, while bcl-2 expression was increased in NLRC5-overexpressing neurons. Furthermore, increased nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression levels were observed in neurons transfected with pcDNA3.0-NLRC5. The mRNA levels of HO-1, NAD(P)H:quinone oxidoreductase 1 (NQO-1) and glutathione peroxidase 3 (GPx-3) were induced by NLRC5 overexpression in OGD/R-induced hippocampal neurons. Additionally, inhibition of Nrf2/HO-1 pathway abolished the protective effect of NLRC5 on cerebral I/R injury. In conclusion, these results indicated that NLRC5 protected hippocampal neurons from OGD/R-induced injury. The protective effects of NLRC5 were mediated by the Nrf2/HO-1 pathway. Thus, NLRC5 might serve as an effective target for the treatment of cerebral I/R injury.

Effect of bamboo shoot dietary fiber on gel properties, microstructure and water distribution of pork meat batters.

OBJECTIVE: To develop healthier comminuted meat products to meet consumer demand, the gel properties, rheological properties, microstructure and water distribution of pork meat batters formulated with various amounts of bamboo shoot dietary fiber (BSDF) were investigated. METHODS: Different levels of BSDF (0% to 4%) were added to pork batters, and the pH, color, water-holding capacity, texture and rheological properties of pork batters were determined. Then, pork batters were analyzed for their microstructure and water distribution using scanning electron microscopy (SEM) and low-field nuclear magnetic resonance (LF-NMR). RESULTS: Compared with the control, BSDF addition into meat batters showed a significant reduction in L*-value and a significant increase in b*-value (p<0.05). BSDF addition of up to 4% reduced the pH value of pork batters by approximately 0.15 units; however, the cooking loss and expressible water loss decreased significantly (p<0.05) with the increased addition of BSDF. The hardness and gel strength were noticeably enhanced (p<0.05) as the content of BSDF increased. The rheological results showed that BSDF added into pork batters produced higher storage modulus (G') and loss modulus (G″) values. The SEM images suggested that the addition of BSDF could promote pork batters to form a more uniform and compact microstructure. The proportion of immobilized water increased significantly (p<0.05), while the population of free water was decreased (p<0.05), indicating that BSDF improved the water-holding capability of pork batters by decreasing the fraction of free water. CONCLUSION: BSDF could improve the gel properties, rheological properties and water distribution of pork meat batters and decrease the proportion of free water, suggesting that BSDF has great potential as an effective binder in comminuted meat products.

Comparative study of thermal gelation properties and molecular forces of actomyosin extracted from normal and pale, soft and exudative-like chicken breast meat.

OBJECTIVE: The objectives of this study were to investigate the thermal gelation properties and molecular forces of actomyosin extracted from two classes of chicken breast meat qualities (normal and pale, soft and exudative [PSE]-like) during heating process to further improve the understanding of the variations of functional properties between normal and PSE-like chicken breast meat. METHODS: Actomyosin was extracted from normal and PSE-like chicken breast meat and the gel strength, water-holding capacity (WHC), protein loss, particle size and distribution, dynamic rheology and protein thermal stability were determined, then turbidity, active sulfhydryl group contents, hydrophobicity and molecular forces during thermal-induced gelling formation were comparatively studied. RESULTS: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that protein profiles of actomyosin extracted from normal and PSE-like meat were not significantly different (p>0.05). Compared with normal actomyosin, PSE-like actomyosin had lower gel strength, WHC, particle size, less protein content involved in thermal gelation forming (p<0.05), and reduced onset temperature (To), thermal transition temperature (Td), storage modulus (G') and loss modulus (G″). The turbidity, reactive sulfhydryl group of PSE-like actomyosin were higher when heated from 40°C to 60°C. Further heating to 80°C had lower transition from reactive sulfhydryl group into a disulfide bond and surface hydrophobicity. Molecular forces showed that hydrophobic interaction was the main force for heat-induced gel formation while both ionic and hydrogen bonds were different significantly between normal and PSE-like actomyosin (p<0.05). CONCLUSION: These changes in chemical groups and inter-molecular bonds affected protein-protein interaction and protein-water interaction and contributed to the inferior thermal gelation properties of PSE-like meat.

Immunological alterations in the endothelial barrier: a new predictive and therapeutic paradigm for sepsis.

INTRODUCTION: Despite the fact incidence and mortality vary widely among regions, sepsis remains a major cause of morbidity and cost worldwide. The importance of the endothelial barrier in sepsis and infectious diseases is increasingly recognized; however, the underlying pathophysiology of the endothelial barrier in sepsis remains poorly understood. AREAS COVERED: Here we review the advances in basic and clinical research for relevant papers in PubMed database. We attempt to provide an updated overview of immunological alterations in endothelial dysfunction, discussing the central role of endothelial barrier involved in sepsis to provide new predictive and therapeutic paradigm for sepsis. EXPERT OPINION: Given its physiological and immunological functions in infectious diseases, the endothelial barrier has been dramatically altered in sepsis, suggesting that endothelial dysfunction may play a critical role in the pathogenesis of sepsis. Although many reliable biomarkers have been investigated to monitor endothelial activation and injury in an attempt to find diagnostic and therapeutic tools, there are no specific therapies to treat sepsis due to its complex pathophysiology. Since sepsis is initiated by both hyperinflammation and immunoparalysis occurring simultaneously, a 'one-treatment-fits-all' strategy for sepsis-induced immune injury and immunoparalysis is bound to fail, and an individualized 'precision medicine' approach is required.

Characterization of aroma characteristics of silver carp mince glycated with different reducing sugars.

The purpose of this study was to investigate the volatile flavor changes in silver carp mince (SCM) gel glycated with different reducing sugars (glucose, L-arabinose, and xylose) based on E-nose, GC-IMS, and sensory evaluation. These results showed that glycation reduced the fishy smell of SCM gel and increased the meaty, toasty, and burnt smell. A total of 10 volatile compounds were considered as characteristic flavor compounds and potential markers. Among them, the main contributors of fishy included hexanal, heptanal, n-nonanal, octanal, etc. Toasty and burnt were mainly related to the production of 3-methylbutanal and furfurol. These results heralded that glycation could be used to improve the volatile flavor of SCM. This research provided a theoretical basis and technical support for glycation in aquatic food flavor quality control, aquatic pre-made food development, and aquatic leisure food processing.

A linear energy transfer distributions computation method for inhomogeneous medium by using the water equivalent ratio approximation.

Dose-averaged linear energy transfer (LET), LETd is widely used in proton therapy. Compared with analytical models, Monte Carlo (MC) simulations are more accurate in obtaining LETd distributions, but they are time-consuming. This study used the 3D LETd distributions of proton beam spots in water by MC simulations as a benchmark data set. Subsequently, by combining the water equivalent ratio of various human tissues, the 3D LETd distributions of clinical cases could be quickly obtained. Our method was applied to a single spot of 160 MeV proton beam in a water-bone phantom and a pelvic case. We also computed the 3D LETd distributions for multiple proton beam spots in the pelvic case and a lung case. The results of our method were compared with the results of MC simulations, demonstrating that our method can rapidly provide 3D LETd distributions of clinical cases with acceptable differences from MC simulations.

ANXA1sp attenuates sepsis-induced myocardial injury by promoting mitochondrial biosynthesis and inhibiting oxidative stress and autophagy via SIRT3 upregulation.

Sepsis-induced myocardial injury is one of the most difficult complications of sepsis in intensive care units. Annexin A1 (ANXA1) short peptide (ANXA1sp) protects organs during the perioperative period. However, the protective effect of ANXA1sp against sepsis-induced myocardial injury remains unclear. We aimed to explore the protective effects and mechanisms of ANXA1sp against sepsis-induced myocardial injury both in vitro and in vivo. Cellular and animal models of myocardial injury in sepsis were established with lipopolysaccharide. The cardiac function of mice was assessed by high-frequency echocardiography. Elisa assay detected changes in inflammatory mediators and markers of myocardial injury. Western blotting detected autophagy and mitochondrial biosynthesis-related proteins. Autophagic flux changes were observed by confocal microscopy, and autophagosomes were evaluated by TEM. ATP, SOD, ROS, and MDA levels were also detected.ANXA1sp pretreatment enhanced the 7-day survival rate, improved cardiac function, and reduced TNF-α, IL-6, IL-1ß, CK-MB, cTnI, and LDH levels. ANXA1sp significantly increased the expression of sirtuin-3 (SIRT3), mitochondrial biosynthesis-related proteins peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM). ANXA1sp increased mitochondrial membrane potential (△Ψm), ATP, and SOD, and decreased ROS, autophagy flux, the production of autophagosomes per unit area, and MDA levels. The protective effect of ANXA1sp decreased significantly after SIRT3 silencing in vitro and in vivo, indicating that the key factor in ANXA1sp's protective role is the upregulation of SIRT3. In summary, ANXA1sp attenuated sepsis-induced myocardial injury by upregulating SIRT3 to promote mitochondrial biosynthesis and inhibit oxidative stress and autophagy.

Annexin-A1 short peptide alleviates septic myocardial injury by upregulating SIRT3 and inhibiting myocardial cell apoptosis.

Septic myocardial injury is a common complication of severe sepsis, which occurs in about 50% of cases. Patients with this disease may experience varying degrees of myocardial damage. Annexin-A1 short peptide (ANXA1sp), with a molecular structure of Ac-Gln-Ala-Tyr, has been reported to exert an organ protective effect in the perioperative period by modulating sirtuin-3 (SIRT3). Whether it possesses protective activity against sepsis-induced cardiomyopathy is worthy of study. This study aimed to investigate whether ANXA1sp exerts its anti-apoptotic effect in septic myocardial injury in vitro and in vivo via regulating SIRT3. In this study, we established in vivo and in vivo models of septic myocardial injury based on C57BL/6 mice and primary cardiomyocytes by lipopolysaccharide (LPS) induction. Results showed that ANXA1sp pretreatment enhanced the seven-day survival rate, improved left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), and cardiac output (CO), and reduced the levels of creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and lactate dehydrogenase (LDH). Western blotting results revealed that ANXA1sp significantly increased the expression of SIRT3, Bcl-2, and downregulated Bax expression. TUNEL staining and flow cytometry results showed that ANXA1sp could attenuate the apoptosis rate of cardiomyocytes, whereas this anti-apoptotic effect was significantly attenuated after SIRT3 knockout. To sum up, ANXA1sp can alleviate LPS-induced myocardial injury by reducing myocardial apoptosis via SIRT3 upregulation.

[Investigation on the signaling pathways in the mechanism of hyperoxia-induced acute lung injury based on transcriptomics sequencing].

OBJECTIVE: To observe and verify the changes of transcriptome in hyperoxia-induced acute lung injury (HALI), and to further clarify the changes of pathways in HALI. METHODS: Twelve healthy male C57BL/6J mice were randomly divided into normoxia group and HALI group according to the random number table, with 6 mice in each group. The mice in the normoxia group were fed normally in the room, and the mice in the HALI group was exposed to 95% oxygen to reproduce the HALI animal model. After 72 hours of hyperoxia exposure, the lung tissues were taken for transcriptome sequencing, and then Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathway enrichment analysis was performed. The pathological changes of lung tissue were observed under light microscope after hematoxylin-eosin (HE) staining. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to verify the key molecules in the signal pathways closely related to HALI identified by transcriptomics analysis. RESULTS: Transcriptomic analysis showed that hyperoxia induced 537 differentially expressed genes in lung tissue of mice as compared with the normoxia group including 239 up-regulated genes and 298 down-regulated genes. Further KEGG pathway enrichment analysis identified 20 most significantly enriched pathway entries, and the top three pathways were ferroptosis signaling pathway, p53 signaling pathway and glutathione (GSH) metabolism signaling pathway. The related genes in the ferroptosis signaling pathway included the up-regulated gene heme oxygenase-1 (HO-1) and the down-regulated gene solute carrier family 7 member 11 (SLC7A11). The related genes in the p53 signaling pathway included the up-regulated gene tumor suppressor gene p53 and the down-regulated gene murine double minute 2 (MDM2). The related gene in the GSH metabolic signaling pathway was up-regulated gene glutaredoxin 1 (Grx1). The light microscope showed that the pulmonary alveolar structure of the normoxia group was normal. In the HALI group, the pulmonary alveolar septum widened and thickened, and the alveolar cavity shrank or disappeared. RT-RCR and Western blotting confirmed that compared with the normoxia group, the mRNA and protein expressions of HO-1 and p53 in lung tissue of the HALI group were significantly increased [HO-1 mRNA (2-ΔΔCt): 2.16±0.17 vs. 1.00±0.00, HO-1 protein (HO-1/ß-actin): 1.05±0.01 vs. 0.79±0.01, p53 mRNA (2-ΔΔCt): 2.52±0.13 vs. 1.00±0.00, p53 protein (p53/ß-actin): 1.12±0.02 vs. 0.58±0.03, all P < 0.05], and the mRNA and protein expressions of Grx1, MDM2, SLC7A11 were significantly decreased [Grx1 mRNA (2-ΔΔCt): 0.53±0.05 vs. 1.00±0.00, Grx1 protein (Grx1/ß-actin): 0.54±0.03 vs. 0.93±0.01, MDM2 mRNA (2-ΔΔCt): 0.48±0.03 vs. 1.00±0.00, MDM2 protein (MDM2/ß-actin): 0.57±0.02 vs. 1.05±0.01, SLC7A11 mRNA (2-ΔΔCt): 0.50±0.06 vs. 1.00±0.00, SLC7A11 protein (SLC7A11/ß-actin): 0.72±0.03 vs. 0.98±0.01, all P < 0.05]. CONCLUSIONS: HALI is closely related to ferroptosis, p53 and GSH metabolism signaling pathways. Targeting the key targets in ferroptosis, p53 and GSH metabolism signaling pathways may be an important strategy for the prevention and treatment of HALI.

Gpnmb silencing protects against hyperoxia-induced acute lung injury by inhibition of mitochondrial-mediated apoptosis.

Background: Hyperoxia-induced acute lung injury (HALI) is a complication to ventilation in patients with respiratory failure, which can lead to acute inflammatory lung injury and chronic lung disease. The aim of this study was to integrate bioinformatics analysis to identify key genes associated with HALI and validate their role in H2O2-induced cell injury model.Methods: Integrated bioinformatics analysis was performed to screen vital genes involved in hyperoxia-induced lung injury (HLI). CCK-8 and flow cytometry assays were performed to assess cell viability and apoptosis. Western blotting was performed to assess protein expression.Results: In this study, glycoprotein non-metastatic melanoma protein B (Gpnmb) was identified as a key gene in HLI by integrated bioinformatics analysis of 4 Gene Expression Omnibus (GEO) datasets (GSE97804, GSE51039, GSE76301 and GSE87350). Knockdown of Gpnmb increased cell viability and decreased apoptosis in H2O2-treated MLE-12 cells, suggesting that Gpnmb was a proapoptotic gene during HALI. Western blotting results showed that knockdown of Gpnmb reduced the expression of Bcl-2 associated X (BAX) and cleaved-caspase 3, and increased the expression of Bcl-2 in H2O2 treated MLE-12 cells. Furthermore, Gpnmb knockdown could significantly reduce reactive oxygen species (ROS) generation and improve the mitochondrial membrane potential.Conclusion: The present study showed that knockdown of Gpnmb may protect against HLI by repressing mitochondrial-mediated apoptosis.

Wedelolactone inhibits ferroptosis and alleviates hyperoxia-induced acute lung injury via the Nrf2/HO-1 signaling pathway.

Hyperoxia-induced acute lung injury (HALI) is a complication of oxygen therapy. Ferroptosis is a vital factor in HALI. This paper was anticipated to investigate the underlying mechanism of wedelolactone (WED) on ferroptosis in HALI. The current study used hyperoxia to injure two models, one HALI mouse model and one MLE-12 cell injury model. We found that WED treatment attenuated HALI by decreasing the lung injury score and lung wet/dry (W/D) weight ratio and alleviating pathomorphological changes. Then, the inflammatory reaction and apoptosis in HALI mice and hyperoxia-mediated MLE-12 cells were inhibited by WED treatment. Moreover, WED alleviated ferroptosis with less iron accumulation and reversed expression alterations of ferroptosis markers, including MDA, GSH, GPX4, SLC7A11, FTH1, and TFR1 in hyperoxia-induced MLE-12 cells in vitro and in vivo. Nrf2-KO mice and Nrf2 inhibitor (ML385) decreased WED's ability to protect against apoptosis, inflammatory response, and ferroptosis in hyperoxia-induced MLE-12 cells. Collectively, our data highlighted the alleviatory role of WED in HALI by activating the Nrf2/HO-1 pathway.

Comparative study of different dosages of grain-sized moxibustion on uterine artery blood flow in patients with cold and dampness primary dysmenorrhea. / ä¸åŒå‰‚é‡éº¦ç²’ç¸å¯¹å¯’æ¹¿å‡æ»žåž‹åŽŸå‘æ€§ç—›ç»æ‚£è€ å­å®«åŠ¨è„‰è¡€æµå½±å“çš„æ¯”è¾ƒç ”ç©¶.

OBJECTIVES: To observe the differences in the effects of different dosages of grain-sized moxibustion on uterine artery blood flow in patients with cold and dampness primary dysmenorrhea (PD). METHODS: A total of 60 patients with PD were randomly divided into 3 groups with 20 cases in each group. Acupoints Sanyinjiao (SP6), Diji (SP8) and Xuehai (SP10) were selected in all the 3 groups, and different dosages of grain-sized moxibustion were used (3 moxa cones, 6 moxa cones, 9 moxa cones) respectively. Treatment started 7 days before menstruation for 3 times, lasting for a total of 3 menstrual cycles. The values of uterine artery blood flow parameters including pulsatility index (PI), resistance index (RI), and systolic/diastolic ratio (S/D) were recorded before and after treatment. The visual analog scale (VAS) score and cox menstrual symptom scale (CMSS) score (including severity ［CMSS-S］ and time of duration ［CMSS-T］) were evaluated before treatment, at the end of each menstrual cycle, and one menstrual cycle after treatment. RESULTS: The values of uterine artery blood flow parameters (PI, RI, S/D) after treatment in the 9 moxa cones group were lower than those before treatment, as well as lower than those in the 3 and 6 moxa cones groups after treatment (P<0.05). The VAS scores of the 3 moxa cones group were lower than those before treatment in the first and second cycle (P<0.05). The VAS scores of the 6 and 9 moxa cones groups were lower than those before treatment at each observation point (P<0.05), and were lower than those of the 3 moxa cones group in the third cycle of treatment and follow-up period (P<0.05). And the VAS score of the 9 moxa cones group was lower than that of the 6 moxa cones group during the follow-up period (P<0.05). Compared with the scores before treatment, the CMSS-T scores at each observation point after treatment were lower in the 9 moxa cones group (P<0.05)；the CMSS-T scores in the second and third cycle after treatment, and follow-up period were lower in the 6 moxa cones group (P<0.05), with the CMSS-S scores in the second and third cycle after treatment, and follow-up period lower in the 6 and 9 moxa cones groups (P<0.05). The CMSS-T and CMSS-S scores of the 6 and 9 moxa cones groups were lower than those of the 3 moxa cones group in the third cycle and follow-up period (P<0.05). The CMSS-T and CMSS-S scores of the 9 moxa cones group were lower than those of the 6 moxa cones group during the follow-up period (P<0.05). CONCLUSIONS: Grain-Sized moxibustion has dose-effect relationship in the treatment of PD. Compared with 3 and 6 moxa cones groups, 9 moxa cones group has advantages in improving uterine artery blood flow parameters and alleviating dysmenorrhea symptoms in PD patients.

[Wedelolactone alleviates hyperoxia-induced acute lung injury by regulating ferroptosis].

OBJECTIVE: To study whether wedelolactone can reduce hyperoxia-induced acute lung injury (HALI) by regulating ferroptosis, and provide a basic theoretical basis for the drug treatment of HALI. METHODS: A total of 24 C57BL/6J mice were randomly divided into normal oxygen control group, HALI model group and wedelolactone pretreatment group, with 8 mice in each group. Mice in wedelolactone pretreatment group were treated with wedelolactone 50 mg/kg intraperitoneally for 6 hours, while the other two groups were not given with wedelolactone. After that, the HALI model was established by maintaining the content of carbon dioxide < 0.5% and oxygen > 90% in the molding chamber for 48 hours, and the normal oxygen control group was placed in indoor air. After modeling, the mice were sacrificed and lung tissues were collected. The lung histopathological changes were observed under light microscope and pathological scores were performed to calculate the ratio of lung wet/dry mass (W/D). The levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1ß), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) in lung tissues of mice in each group were determined. The protein expression of glutathione peroxidase 4 (GPX4) in lung tissue was detected by Western blotting. RESULTS: Under light microscope, the alveolar structure of HALI model group was destroyed, and a large number of neutrophils infiltrated the alveolar and interstitial lung, and the interstitial lung was thickened. The pathological score of lung injury (score: 0.75±0.02 vs. 0.11±0.01) and the ratio of lung W/D (6.23±0.34 vs. 3.68±0.23) were significantly higher than those in the normal oxygen control group (both P < 0.05). Wedelolactone pretreated mice had clear alveolar cavity and lower neutrophil infiltration and interstitial thickness than HALI group. Pathological scores (score: 0.43±0.02 vs. 0.75±0.02) and W/D ratio (4.56±0.12 vs. 6.23±0.34) were significantly lower than HALI group (both P < 0.05). Compared with the normal oxygen control group, the levels of SOD (kU/g: 26.41±4.25 vs. 78.64±3.95) and GSH (mol/g: 4.51±0.33 vs. 12.53±1.25) in HALI group were significantly decreased, while the levels of MDA (mmol/g: 54.23±4.58 vs. 9.65±1.96), TNF-α (µg/L: 96.32±3.67 vs. 11.65±2.03), IL-6 (ng/L: 163.35±5.89 vs. 20.56±3.63) and IL-1ß (µg/L: 72.34±4.64 vs. 15.64±2.47) were significantly increased, and the protein expression of GPX4 (GPX4/ß-actin: 0.44±0.02 vs. 1.00±0.09) was significantly decreased (all P < 0.05). Compared with the HALI group, the levels of SOD (kU/g: 53.28±3.69 vs. 26.41±4.25) and GSH (mol/g: 6.73±0.97 vs. 12.53±1.25) were significantly higher in the wedelolactone pretreatment group, and the levels of MDA (mmol/g: 25.36±1.98 vs. 54.23±4.58), TNF-α (µg/L: 40.25±4.13 vs. 96.32±3.67), IL-6 (ng/L: 78.32±4.65 vs. 163.35±5.89), and IL-1ß (µg/L: 30.65±3.65 vs. 72.34±4.64) were significantly lower (all P < 0.05), and protein expression of GPX4 was significantly higher (GPX4/ß-actin: 0.68±0.04 vs. 0.44±0.02, P < 0.05). CONCLUSIONS: Wedelolactone attenuates HALI injury by regulating ferroptosis.

[MicroRNA21-5p alleviates hyperoxia-induced acute lung injury in rats through activating phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway by regulating type II alveolar epithelial cell apoptosis].

OBJECTIVE: To investigate whether microRNA-21-5p (miR-21-5p) alleviates hyperoxia-induced acute lung injury (HALI) through activating the phosphatidylinositol 3 kinase/serine-threonine protein kinase (PI3K/Akt) signaling pathway by regulating apoptosis of type II alveolar epithelial cell (AEC II). METHODS: Seventy-two male Sprague-Dawley (SD) rats were divided into normozone-controlled group, HALI group, PI3K/Akt signaling pathway inhibitor LY294002+HALI group (LY+HALI group), miR-21-5p overexpression+LY294002+HALI group (miR-21-5p+LY+HALI group), miR-21-5p overexpression+HALI group (miR-21-5p+HALI group), and dimethyl sulfoxide (DMSO)+HALI group by random number table method with 12 rats in each group. Animal models of HALI were prepared using 95% concentrations of oxygen. The animals in the normozone-controlled group were fed normally under normoxia. Transfection of lung tissue by miR-21-5p adeno-associated viral vector AAV6-miR-21-5p was performed by instillation of 200 µL titer (1×1012 TU/mL) through a tracheal catheter 3 weeks prior to modeling. DMSO and LY294002 were administered via the tail vein at 0.3 mg/kg 1 hour before modeling. After 48 hours of modeling, carotid artery blood was collected to detect oxygenation index (OI) and respiratory index (RI), and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect miR-21-5p expression. Lung tissue was collected, and the levels of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1ß) were measured by enzyme-linked immunosorbent assay (ELISA), and the ratio of pulmonary wet/dry weight (W/D) was determined, and the pathological changes of lung histopathology were observed under the light microscopy after hematoxylin-eosin (HE) staining. Each group was purified AEC II cells from 6 rats, the apoptosis rate was detected by flow cytometry, and the expression levels of phosphatase and tensin homologous gene (PTEN), and proteins from the PI3K/Akt signaling pathway were detected by Western blotting. RESULTS: Compared with the normozone-controlled group, alveolar septal thickening and massive inflammatory cell infiltration were found after hyperoxia exposure, RI, inflammatory factors, lung W/D ratio, pathological score, AEC II cells early apoptosis rate, PTEN protein expression and phosphorylation level of Akt were increased, while OI and miR-21-5p expression were decreased, indicating the successful preparation of the model. After pretreatment, LY294002 could aggravate the pathological injury of lung tissue in HALI rats, RI, inflammatory factors and lung W/D ratio were further increased, and OI was further reduced compared with HALI group. At the same time, it could promote the AEC II cell apoptosis, further up-regulate the expression of PTEN, and reduce the phosphorylation of Akt. However, miR-21-5p pretreatment could negatively regulate PTEN, activate PI3K/Akt signal pathway, inhibit AEC II cell apoptosis, and reduce HALI, which was shown by the decreased level of inflammatory factors in miR-21-5p+LY+HALI group compared with LY+HALI group [TNF-α (µg/L): 100.33±3.48 vs. 116.55±2.53, IL-6 (ng/L): 141.06±3.70 vs. 161.31±3.59, IL-1ß (µg/L): 90.82±3.69 vs. 112.23±2.87, all P < 0.05], RI, lung injury pathology score, lung W/D ratio, and AEC II cell early apoptosis rate were significantly decreased [RI: 0.81±0.02 vs. 1.05±0.07, pathology score: 0.304±0.008 vs. 0.359±0.007, lung W/D ratio: 5.29±0.03 vs. 5.52±0.08, apoptosis rate: (27.20±2.34)% vs. (34.17±1.49)%, all P < 0.05], OI and expressions of miR-21-5p were significantly increased [OI (mmHg, 1 mmHg ≈ 0.133 kPa): 266.71±2.75 vs. 230.12±4.04, miR-21-5p (2-ΔΔCt): 2.21±0.13 vs. 0.33±0.03, both P < 0.05], and PTEN protein expression in AEC II cell was significantly reduced (PTEN/GAPDH: 0.50±0.06 vs. 0.93±0.06, P < 0.05), and phosphorylation level of Akt was significantly increased [phosphorylated Akt (p-Akt) protein (p-Akt/GAPDH): 0.86±0.05 vs. 0.56±0.06, P < 0.05]. CONCLUSIONS: miR-21-5p attenuates HALI by inhibiting AEC II cell apoptosis, possibly through negative regulation of PTEN to activate PI3K/Akt signaling pathway.

Hyperoxia can Induce Lung Injury by Upregulating AECII Autophagy and Apoptosis Via the mTOR Pathway.

Oxygen therapy is a crucial medical intervention, but it is undeniable that it can lead to lung damage. The mTOR pathway plays a pivotal role in governing cell survival, including autophagy and apoptosis, two phenomena deeply entwined with the evolution of diseases. However, it is unclarified whether the mTOR pathway is involved in hyperoxic acute lung injury (HALI). The current study aims to clarify the molecular mechanism underlying the pathogenesis of HALI by constructing in vitro and in vivo models using H2O2 and hyperoxia exposure, respectively. To investigate the role of mTOR, the experiment was divided into five groups, including normal group, injury group, mTOR inhibitor group, mTOR activator group, and DMSO control group. Western blotting, Autophagy double labeling, TUNEL staining, and HE staining were applied to evaluate protein expression, autophagy activity, cell apoptosis, and pathological changes in lung tissues. Our data revealed that hyperoxia can induce autophagy and apoptosis in Type II alveolar epithelial cell (AECII) isolated from the treated rats, as well as injuries in the rat lung tissues; also, H2O2 stimulation increased autophagy and apoptosis in MLE-12 cells. Noticeably, the experiments performed in both in vitro and in vivo models proved that the mTOR inhibitor Rapamycin (Rapa) functioned synergistically with hyperoxia or H2O2 to promote AECII autophagy, which led to increased apoptosis and exacerbated lung injury. On the contrary, activation of mTOR with MHY1485 suppressed autophagy activity, consequently resulting in reduced apoptosis and lung injury in H2O2-challenged MLE-12 cells and hyperoxia-exposed rats. In conclusion, hyperoxia caused lung injury via mTOR-mediated AECII autophagy.

ROS-mediated MAPK activation aggravates hyperoxia-induced acute lung injury by promoting apoptosis of type II alveolar epithelial cells via the STAT3/miR-21-5p axis.

Inhibition of type II alveolar epithelial (AE-II) cell apoptosis is a critical way to cure hyperoxia-induced acute lung injury (HALI). It has been reported that miR-21-5p could reduce H2O2-induced apoptosis in AE-II cells. However, the upstream molecular mechanism remains unclear. Herein, we established a cellular model of HALI by exposing AE-II cells to H2O2 treatment. It was shown that miR-21-5p alleviated H2O2-induced apoptosis in AE-II cells. ROS inhibition decreased apoptosis of H2O2-evoked AE-II cells via increasing miR-21-5p expression. In addition, ROS induced MAPK and STAT3 phosphorylation in H2O2-treated AE-II cells. MAPK inactivation reduces H2O2-triggered AE-II cell apoptosis. MAPK activation inhibits miR-21-5p expression by promoting STAT3 phosphorylation in H2O2-challenged AE-II cells. Furthermore, STAT3 activation eliminated MAPK deactivation-mediated inhibition on the apoptosis of AE-II cells under H2O2 condition. In conclusion, ROS-mediated MAPK activation promoted H2O2-triggered AE-II cell apoptosis by inhibiting miR-21-5p expression via STAT3 phosphorylation, providing novel targets for HALI treatment.

[Role and mechanism of signal transducer and activator of transcription protein STAT1/3/5 in hyperoxia-induced acute lung injury].

OBJECTIVE: To investigate whether signal transducer and activator of transcription (STAT1/3/5) have a protective effect on hyperoxia-induced acute lung injury (HALI) and its mechanism. METHODS: Seventy C57BL/6J mice were randomly divided into five groups: normoxia control group, HALI group, and STAT1/3/5 inhibitor groups, with 14 mice in each group. The HALI model was established by exposure to more than 90% hyperoxia for 48 hours; three STAT inhibitor groups were pretreated by intraperitoneal injection of STAT1 inhibitor 40 mg/kg and STAT3 inhibitor 5 mg/kg, and STAT5 inhibitor 10 mg/kg for 1 week. Six blood samples were randomly collected from each group, and microRNA-21 (miR-21) expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Lung tissue of the sacrificed mice was obtained, and enzyme linked immunosorbent assay (ELISA) was used to detect the contents of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1ß), superoxide dismutase (SOD), malonic dialdehyde (MDA), and matrix metalloproteinase 9 (MMP9). The water content of lung tissue was calculated. The pathological changes in lung tissue were observed under the light microscope, and the pathological score of lung injury was performed. Western blotting was used to detect the expression of phosphorylated STAT (p-STAT1, p-STAT3, p-STAT5) in lung tissue. The 7-day cumulative survival rates of the remaining 8 mice in each group were analyzed using Kaplan-Meier survival curves. RESULTS: Under the light microscope, the alveolar structures in the HALI group and the STAT1 inhibitor group were destroyed, a large number of neutrophils (NEU) infiltrated in the alveoli and lung interstitium, which were thickened. The pathological score of lung injury and the water content of the lung tissue was significantly increased. In STAT3 inhibitor and STAT5 inhibitor groups, the alveolar cavity was clear, the degree of NEU infiltration and the thickness of lung interstitium were lower than those in HALI group, the pathological score of lung injury and the water content of lung tissue were significantly decreased, especially in STAT3 inhibitor group. Compared with the normoxia control group, the contents of TNF-α, IL-6, IL-1ß, MDA, and MMP9, and the expression levels of p-STAT3 and p-STAT5 in the HALI group were significantly increased. In contrast, the content of SOD and the expression of miR-21 were significantly decreased. Compared with the HALI group, the contents of TNF-α, IL-6, IL-1ß, MDA, and MMP9 in the STAT3 inhibitor group and STAT5 inhibitor group were significantly decreased. At the same time, the content of SOD and the expression of miR-21 were significantly increased, especially in STAT3 inhibitor group [TNF-α (µg/L): 42.53±3.25 vs. 86.36±5.48, IL-6 (ng/L): 68.46±4.28 vs. 145.00±6.89, IL-1ß (µg/L): 28.74±3.53 vs. 68.00±5.64, MDA (µmol/g): 20.33±2.74 vs. 42.58±3.45, and MMP9 (ng/L): 128.55±6.35 vs. 325.13±6.65, SOD (kU/g): 50.53±4.19 vs. 22.53±3.27, miR-21 (2-ΔΔCt): 0.550±0.018 vs. 0.316±0.037, all P < 0.05]. Kaplan-Meier survival curve analysis showed that the 7-day cumulative survival rates of the STAT3 inhibitor group and STAT5 inhibitor group were significantly higher than those of the HALI group [62.5% (5/8), 37.5% (3/8) vs. 12.5% (1/8), both P < 0.05]. CONCLUSIONS: Inhibition of STAT3 hyperactivation may suppress the inflammatory response, regulate oxidative stress, improve lung permeability through regulating the expression of miR-21, which exert lung protection in HALI.