Atherosclerosis, a chronic inflammatory disease of the arterial wall, is a leading cause of cardiovascular diseases worldwide. The complex pathogenesis of atherosclerosis involves genetic predisposition, environmental factors, and immune responses. N-Methyl-d-aspartate receptors (NMDARs), a subclass of glutamate receptors, are critical for synaptic plasticity, learning, and memory in the central nervous system (CNS). Non-neuronal NMDARs are poorly understood compared to neuronal receptors, but there is a developing consensus that they have distinct structural and functional properties when activated by glutamate and NMDARs co-agonists. Emerging evidence indicates that non-neuronal NMDARs may participate in an array of physiological and pathophysiological processes, including but not limited to driving macrophage polarization, lipid dysregulation in macrophages, inflammation response, vascular smooth muscle cells phenotype switching and endothelial dysfunction, thereby fueling atherogenesis. This review discusses the association between NMDARs genes and atherosclerosis risk, molecular mechanisms underlying NMDARs-mediated regulation of atherosclerosis-related cells, and potential therapeutic implications. Besides, we introduce some pharmacological tools that can be used for studying NMDARs outside the CNS, which reflect modern subunit-selective agents to provide more precise insight into NMDARs mediate the various effects. Overall, the study of NMDARs may provide insights into the pathogenesis of atherosclerosis and lead to the development of more effective therapeutic strategies.

Pulmonary fibrosis (PF) is a progressive, chronic lung disease, which is accompanied by epithelial cell damage, fibroblast activation, and macrophage polarization; the fibrotic niche is primarily composed of these three cell types. The local microenvironment, composed of a fibrotic niche, participates in disease progression by altering epigenetic modifications. Lactylation is a novel post-translational modification involving the writing, removal, and recognition of lactyl groups. Studies have demonstrated that lactylation promotes profibrotic cellular phenotypes by altering the epigenetics. At the microenvironmental level, lactylation affects PF progression by influencing lung epithelial cells, fibroblasts, and macrophages in the fibrotic niche. Lactylation may promote the development of PF by affecting epithelial cell autophagy, cellular senescence, expression of proliferation or repair genes, apoptosis, ferroptosis, endoplasmic reticulum stress, and epithelial-mesenchymal transition and may influence the activation of fibroblasts and secretion of pro-fibrotic factors by macrophages. By focusing on the key cell types in the fibrotic microenvironment, in this review, we elaborate on the roles of lactic acid and lactylation in PF and the potential future research directions of lactylation in the field of PF. This review summarizes the potential pathways through which lactylation promotes PF, based on the current sites and roles of lactylation in epithelial cells, fibroblasts, and macrophages. In addition, by summarizing the current research methods of lactylation and their limitations, our review paves the way for future studies on lactylation and PF.

Idiopathic pulmonary fibrosis is a chronic, progressive disease in older adults with unclear pathogenesis and a lack of effective drugs. Columbianadin, a natural coumarin analog isolated from Angelicae pubescentis Radix, has a wide range of pharmacological effects; however, its effects on pulmonary fibrosis are unknown. This study investigates the anti-pulmonary fibrosis effects of columbianadin and their underlying mechanisms of action. An in vivo model of mouse lung fibrosis was established, and mice were randomly assigned to different doses of columbianadin. The effects of 5'-adenosine monophosphate-activated protein kinase (AMPK) on the anti-pulmonary fibrosis and anti-cellular senescence effects of columbianadin was observed by combining AMPK inhibitor and columbianadin. Cellular senescence was induced in vitro by hydrogen peroxide and treated with different concentrations of columbianadin, and we observed the effect of AMPK on the anti-cellular senescence effect of columbianadin by specifically silencing the AMPK gene. Columbianadin reduced the expression levels of collagen type I alpha 1 (col1-a1), alpha-smooth muscle actin (a-SMA), p21, and p16 in lung tissues of mice with pulmonary fibrosis, and these effects were inhibited by AMPK inhibitors. Similarly, Columbianadin reduced the expression levels of p21 and p16 in senescent cells. In addition, we found that columbianadin promoted Sirt1 and Sirt3 expression as well as AMPK phosphorylation, whereas the anti-cellular senescence effect of columbianadin and the effect of promoting the expression of Sirt1 and Sirt3 were suppressed by specific silencing of the AMPK gene. Columbianadin exerts its anti-pulmonary fibrosis effect by inhibiting cellular senescence via the AMPK-Sirt1/3 pathway. The present study provided new insight into a novel treatment of pulmonary fibrosis.

Renal fibrosis is a common pathological process associated with chronic kidney disease (CKD) progression. Intelectin-1, a newly identified adipokine, has been demonstrated to protect renal function in mice with type 2 diabetic nephropathy. However, the role of intelectin-1 in renal fibrosis and the underlying mechanisms remain unclear. This study aimed to: (1) investigate the effects of intelectin-1 on renal fibrosis in mice, and (2) explore the potential involvement of intelectin-1 in regulating renal tubular epithelial cells (TECs) senescence and mitochondrial dysfunction. To our knowledge, these findings represent the first demonstration that intelectin-1 treatment significantly attenuates renal fibrosis in unilateral ureteral obstruction (UUO) in mice by effectively inhibiting TECs senescence. Furthermore, intelectin-1 treatment alleviated mitochondrial dysfunction in TECs, as evidenced by improved mitochondrial membrane potential and decreased mitochondrial reactive oxygen species (mtROS) production. Mechanistically, intelectin-1 treatment activated AMPK signaling that subsequently inhibited the mTOR and p38 pathways. In conclusion, our findings suggest that intelectin-1 attenuates renal fibrosis in mice by inhibiting TECs senescence and alleviating mitochondrial dysfunction via AMPK/mTOR/p38MAPK signaling. These results provide a potential therapeutic target for the treatment of renal fibrosis in CKD. Further studies are warranted to explore the clinical relevance and translational potential of adipokines, including intelectin-1, in human renal fibrosis.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) can result from various factors, including sepsis, one of the high-risk causes of ALI/ARDS. Recent research emphasizes the role of Glutamate metabolism in ALI/ARDS. Our study found a strong correlation between the difference in serological Glutamate levels of arterial vs venous blood and the progression of lung injury. High arterial - venous (A-V) Glutamate discrepancies were significantly associated with severity in ALI/ARDS patients. Additionally, the subunit of Glutamate transporter system XC- was notably elevated in mouse lungs affected by sepsis and LPS-induced macrophages. Pharmacological inhibition of system XC- or knocking down xCT worsened sepsis-related lung injury in mice. We also showed that xCT in macrophages is essential for activating system XC- for Glutamate transport, offering protection against sepsis-related ALI. Our findings highlight the therapeutic potential of Glutamate transport in mitigating lung injury and provide a promising approach for predicting ALI/ARDS prognosis.

Rationale: Pulmonary fibrosis is a chronic progressive lung disease with limited therapeutic options. We previously revealed that there is iron deposition in alveolar epithelial type II cell (AECII) in pulmonary fibrosis, which can be prevented by the iron chelator deferoxamine. However, iron in the cytoplasm and the mitochondria has two relatively independent roles and regulatory systems. In this study, we aimed to investigate the role of mitochondrial iron deposition in AECII injury and pulmonary fibrosis, and to find potential therapeutic strategies. Methods: BLM-treated mice, MLE-12 cells, and primary AECII were employed to establish the mouse pulmonary fibrosis model and epithelial cells injury model, respectively. Mitochondrial transplantation, siRNA and plasmid transfection, western blotting (WB), quantitative real-time polymerase chain reaction (RT-qPCR), polymerase chain reaction (PCR), immunofluorescence, immunoprecipitation (IP), MitoSOX staining, JC-1 staining, oxygen consumption rate (OCR) measurement, and Cell Counting Kit -8 (CCK8) assay were utilized to elucidate the role of mitochondrial iron deposition in cell and lung fibrosis and determine its mechanism. Results: This study showed that prominent mitochondrial iron deposition occurs within AECII in bleomycin (BLM)-induced pulmonary fibrosis mouse model and in BLM-treated MLE-12 epithelial cells. Further, the study revealed that healthy mitochondria rescue BLM-damaged AECII mitochondrial iron deposition and cell damage loss. Mitoferrin-2 (MFRN2) is the main transporter that regulates mitochondrial iron metabolism by transferring cytosolic iron into mitochondria, which is upregulated in BLM-treated MLE-12 epithelial cells. Direct overexpression of MFRN2 causes mitochondrial iron deposition and cell damage. In this study, decreased ubiquitination of the ubiquitin ligase F-box/LRR-repeat protein 5 (FBXL5) degraded iron -reactive element -binding protein 2 (IREB2) and promoted MFRN2 expression as well as mitochondrial iron deposition in damaged AECII. Activation of the prostaglandin E2 receptor EP4 subtype (EP4) receptor signaling pathway counteracted mitochondrial iron deposition by downregulating IREB2-MFRN2 signaling through upregulation of FBXL5. This intervention not only reduced mitochondrial iron content but also preserved mitochondrial function and protected against AECII damage after BLM treatment. Conclusion: Our findings highlight the unexplored roles, mechanisms, and regulatory approaches of abnormal mitochondrial iron metabolism of AECII in pulmonary fibrosis. Therefore, this study deepens the understanding of the mechanisms underlying pulmonary fibrosis and offers a promising strategy for developing effective therapeutic interventions using the EP4 receptor activator.

This study aimed to compare the impact of the partially flipped physiology classroom (PFC) and the traditional lecture-based classroom (TLC) on students' learning approaches. The study was conducted over 5 mo at Xiangya School of Medicine from February to July 2022 and comprised 71 students majoring in clinical medicine. The experimental group (n = 32) received PFC teaching, whereas the control group (n = 39) received TLC. The Revised Two-Factor Study Process Questionnaire (R-SPQ-2F) was used to assess the impact of different teaching methods on students' learning approaches. After the PFC, students got significantly higher scores on deep learning approach (Z = -3.133, P < 0.05). Conversely, after the TLC students showed significantly higher scores on surface learning approach (Z = -2.259, P < 0.05). After the course, students in the PFC group scored significantly higher in deep learning strategy than those in the TLC group (Z = -2.196, P < 0.05). The PFC model had a positive impact on deep learning motive and strategy, leading to an improvement in the deep approach, which is beneficial for the long-term development of students. In contrast, the TLC model only improved the surface learning approach. The study implies that educators should consider implementing PFC to enhance students' learning approaches. NEW & NOTEWORTHY In this article, we compare the impact of the partially flipped classroom (PFC) and the traditional lecture classroom (TLC) in a physiology course on medical students' learning approaches. We found that the PFC benefited students by significantly enhancing their deep learning motive, strategy, and approach, which was good for them. However, the TLC model only improved the surface learning motive and approach.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disease for which there is a lack of effective pharmacological treatments. Hirudin, a natural peptide extracted from leeches, has been used for broad pharmacological purposes. In this study, we investigated the therapeutic effects of hirudin on IPF and its related mechanism of action. By constructing a mouse model of pulmonary fibrosis and treating it with hirudin in vivo, we found that hirudin exerted anti-fibrotic, anti-oxidative, and anti-fibroblast senescence effects. Moreover, using an in vitro model of stress-induced premature senescence in primary mouse lung fibroblasts and treating with hirudin, we observed inhibition of fibroblast senescence and upregulation of PGC1-alpha and Sirt3 expression. However, specific silencing of PGC1-alpha or Sirt3 suppressed the anti-fibroblast senescence effect of hirudin. Thus, the PGC1-alpha/Sirt3 pathway mediates the anti-fibroblast senescence effect of hirudin, potentially serving as a molecular mechanism underlying its anti-fibrosis and anti-oxidative stress effects exerted on the lungs.

Background Pulmonary fibrosis is a chronic progressive disease with complex pathogenesis, short median survival time, and high mortality. There are few effective drugs approved for pulmonary fibrosis treatment. This study aimed to evaluate the effect of praziquantel (PZQ) on bleomycin (BLM)-induced pulmonary fibrosis. Methods In this study, we investigated the role and mechanisms of PZQ in pulmonary fibrosis in a murine model induced by BLM. Parameters investigated included survival rate, lung histopathology, pulmonary collagen deposition, mRNA expression of key genes involved in pulmonary fibrosis pathogenesis, the activity of fibroblast, and M2/M1 macrophage ratio. Results We found that PZQ improved the survival rate of mice and reduced the body weight loss induced by BLM. Histological examination showed that PZQ significantly inhibited the infiltration of inflammatory cells, collagen deposition, and hydroxyproline content in BLM-induced mice. Besides, PZQ reduced the expression of TGF-β and MMP-12 in vivo and inhibited the proliferation of fibroblast induced by TGF-β in vitro. Furthermore, PZQ affected the balance of M2/M1 macrophages. Conclusions Our study demonstrated that PZQ could ameliorate BLM-induced pulmonary fibrosis in mice by affecting the balance of M2/M1 macrophages and suppressing the expression of TGF-β and MMP-12. These findings suggest that PZQ may act as an effective anti-fibrotic agent for preventing the progression of pulmonary fibrosis.

N-methyl-d-aspartate (NMDA) receptor (NMDAR) activation mediates glutamate (Glu) toxicity and involves bleomycin (BLM)-induced acute lung injury (ALI). We have reported that bone marrow-derived mesenchymal stem cells (BM-MSCs) are NMDAR-regulated target cells, and NMDAR activation inhibits the protective effect of BM-MSCs on BLM-induced pulmonary fibrosis, but its effect on ALI remains unknown. Here, we found that Glu release was significantly elevated in plasma of mice at d 7 after intratracheally injected with BLM. BM-MSCs were pretreated with NMDA (the selective agonist of NMDAR) and transplanted into the recipient mice after the BLM challenge. BM-MSCs administration significantly alleviated the pathological changes, inflammatory response, myeloperoxidase activity, and malondialdehyde content in the damaged lungs, but NMDA-pretreated BM-MSCs did not ameliorate BLM-induced lung injury in vivo. Moreover, NMDA down-regulated prostaglandin E2 (PGE2) secretion and cyclooxygenase (COX)-2 expression instead of COX-1 expression in BM-MSCs in vitro. We also found that NMDAR1 expression was increased and COX-2 expression was decreased, but COX-1 expression was not changed in primary BM-MSCs of BLM-induced ALI mice. Further, the cultured supernatants of lipopolysaccharide (LPS)-pretreated RAW264.7 macrophages were collected to detect inflammatory factors after co-culture with NMDA-pretreated BM-MSCs. The co-culture experiments showed that NMDA precondition inhibited the anti-inflammatory effect of BM-MSCs on LPS-induced macrophage inflammation, and PGE2 could partially alleviate this inhibition. Our findings suggest that NMDAR activation attenuated the protective effect of BM-MSCs on BLM-induced ALI in vivo. NMDAR activation inhibited COX-2 expression and PGE2 secretion in BM-MSCs and weakened the anti-inflammatory effect of BM-MSCs on LPS-induced macrophage inflammation in vitro. In conclusion, NMDAR activation attenuates the protective effect of BM-MSCs on BLM-induced ALI via the COX-2/PGE2 pathway. Keywords: Acute Lung Injury, BM-MSCs, NMDA receptor, COX-1/2, PGE2.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) has a high mortality rate and incidence of complications. The pathophysiology of ALI/ARDS is still not fully understood. The lipopolysaccharide (LPS)-induced mouse model of ALI has been widely used to study human ALI/ARDS. Sulfasalazine (SASP) has antibacterial and anti-inflammatory effects and is used for treating inflammatory bowel and rheumatic diseases. However, the effect of SASP on LPS-induced ALI in mice has not yet been reported. Therefore, we aimed to investigate the effect of SASP on LPS-induced ALI in mice. Mice were intraperitoneally injected with SASP 2h before or 4h after LPS modeling. Pulmonary pathological damage was measured based on inflammatory factor expression (malondialdehyde and superoxide dismutase levels) in the lung tissue homogenate and alveolar lavage fluid. The production of inflammatory cytokines and occurrence of oxidative stress in the lungs induced by LPS were significantly mitigated after the prophylactic and long-term therapeutic administration of SASP, which ameliorated ALI caused by LPS. SASP reduced both the production of inflammatory cytokines and occurrence of oxidative stress in RAW264.7 cells, which respond to LPS. Moreover, its mechanism contributed to the suppression of NF-κB and nuclear translocation. In summary, SASP treatment ameliorates LPS-induced ALI by mediating anti-inflammatory and antioxidant effects, which may be attributed to the inhibition of NF-κB activation and promotion of antioxidant defenses. Thus, SASP may be a promising pharmacologic agent for ALI therapy.

Idiopathic pulmonary fibrosis is a fatal interstitial lung disease for which effective drug therapies are lacking. Senegenin, an effective active compound from the traditional Chinese herb Polygala tenuifolia Willd, has been shown to have a wide range of pharmacological effects. In this study, we investigated the therapeutic effects of senegenin on pulmonary fibrosis and their associated mechanisms of action. We found that senegenin inhibited the senescence of epithelial cells and thus exerted anti-pulmonary-fibrosis effects by inhibiting oxidative stress. In addition, we found that senegenin promoted the expression of Sirt1 and Pgc-1α and that the antioxidative and antisenescent effects of senegenin were suppressed by specific silencing of the Sirt1 and Pgc–1α genes, respectively. Moreover, the senegenin-induced effects of antioxidation, antisenescence of epithelial cells, and antifibrosis were inhibited by treatment with Sirt1 inhibitors in vivo. Thus, the Sirt1/Pgc-1α pathway exerts its antifibrotic effect on lung fibrosis by mediating the antioxidative and antisenescent effects of senegenin.

Pulmonary fibrosis is a formidable challenge in chronic and age-related lung diseases. Myofibroblasts secrete large amounts of extracellular matrix and induce pro-repair responses during normal wound healing. Successful tissue repair results in termination of myofibroblast activity via apoptosis; however, some myofibroblasts exhibit a senescent phenotype and escape apoptosis, causing over-repair that is characterized by pathological fibrotic scarring. Therefore, the removal of senescent myofibroblasts using senolytics is an important method for the treatment of pulmonary fibrosis. Procyanidin C1 (PCC1) has recently been discovered as a senolytic compound with very low toxicity and few side effects. This study aimed to determine whether PCC1 could improve lung fibrosis by promoting apoptosis in senescent myofibroblasts and to investigate the mechanisms involved. The results showed that PCC1 attenuates bleomycin (BLM)-induced pulmonary fibrosis in mice. In addition, we found that PCC1 inhibited extracellular matrix deposition and promoted the apoptosis of senescent myofibroblasts by increasing PUMA expression and activating the BAX signaling pathway. Our findings represent a new method of pulmonary fibrosis management and emphasize the potential of PCC1 as a senotherapeutic agent for the treatment of pulmonary fibrosis, providing hope for patients with pulmonary fibrosis worldwide. Our results advance our understanding of age-related diseases and highlight the importance of addressing cellular senescence in treatment.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease of unknown origin and the most common interstitial lung disease. However, therapeutic options for IPF are limited, and novel therapies are urgently needed. Histone deacetylases (HDACs) are enzymes that participate in balancing histone acetylation activity for chromatin remodeling and gene transcription regulation. Increasing evidence suggests that the HDAC family is linked to the development and progression of chronic fibrotic diseases, including IPF. This review aims to summarize available information on HDACs and related inhibitors and their potential applications in treating IPF. In the future, HDACs may serve as novel targets, which can aid in understanding the etiology of PF, and selective inhibition of single HDACs or disruption of HDAC genes may serve as a strategy for treating PF.

Idiopathic pulmonary fibrosis is a progressive and age-related disease that results from impaired lung repair following injury. Targeting senescent myofibroblasts with senolytic drugs attenuates pulmonary fibrosis, revealing a detrimental role of these cells in pulmonary fibrosis. The mechanisms underlying the occurrence and persistence of senescent myofibroblasts in fibrotic lung tissue require further clarification. In this study, we demonstrated that senescent myofibroblasts are resistant to apoptosis by upregulating the proapoptotic protein BAX and antiapoptotic protein BCL-2 and BCL-XL, leading to BAX inactivation. We further showed that high levels of inactive BAX-mediated minority mitochondrial outer membrane permeabilization (minority MOMP) promoted DNA damage and myofibroblasts senescence after insult by a sublethal stimulus. Intervention of minority MOMP via the inhibition of caspase activity by quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (QVD-OPH) or BAX knockdown significantly reduced DNA damage and ultimately delayed the progression of senescence. Moreover, the BAX activator BTSA1 selectively promoted the apoptosis of senescent myofibroblasts, as BTSA1-activated BAX converted minority MOMP to complete MOMP while not injuring other cells with low levels of BAX. Furthermore, therapeutic activation of BAX with BTSA1 effectively reduced the number of senescent myofibroblasts in the lung tissue and alleviated both reversible and irreversible pulmonary fibrosis. These findings advance the understanding of apoptosis resistance and cellular senescence mechanisms in senescent myofibroblasts in pulmonary fibrosis and demonstrate a novel senolytic drug for pulmonary fibrosis treatment.

To the Editor: Type 2 diabetes (T2D) is an endocrine system disease, characterized by persistent hyperglycemia, and is one of the most common and fastest growing diseases in the world.[1] Although significant progresses have been made in understanding the pathology of T2D with abnormal insulin-secretion profiles, the molecular and cellular mechanisms of β cells remain largely uncertain. Moreover, the liver-expressed antimicrobial peptide 2 (LEAP2) may antagonize the inhibition effect of ghrelin on insulin secretion by inhibiting the ghrelin receptor (growth hormone secretagogue receptor, GHSR). The regulation of glucose homeostasis by the ghrelin–LEAP2–GHSR axis remains largely unknown. It is hypothesized that the ghrelin–LEAP2–GHSR axis may regulate the insulin–glucose homeostasis in diabetic condition. In this study, a high-fat diet (HFD) combined with multiple injections of low-dose streptozotocin (STZ) was used to construct a mouse model of T2D. The effects of exogenous LEAP2 in T2D and normal mice were tested. Eight-week-old C57BL/6J mice were provided by Department of Laboratory Animal, Central South University (Hunan, China). All animal experiments were approved by the Ethics Committee for Animal Experiments of Central South University. The in vitro effects of LEAP2 were studied in cultured MIN6 cells (passage from 10 to 20, kindly provided by Prof. Jingjing Zhang, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China), and the potential molecular mechanisms involved in altered insulin secretion were investigated. Firstly, mice were intraperitoneally injected with LEAP2 (30 μg/kg daily) for 14 days after establishment of the T2D mouse model [Figure 1A]. LEAP2 treatment had no significant effects on body weight and food intake in the control and T2D mice [Figure 1B and Supplementary Figure 1A, https://links.lww.com/CM9/C78]. The fasting blood glucose and basal insulin levels in T2D mice showed typical changes in overt diabetes with an increase in fasting blood glucose and a decrease in insulin levels. There were no significant changes in the fasting blood glucose and insulin levels between the T2D + LEAP2 mice and the T2D mice, suggesting that the treatment adopted in this experiment (30 μg/kg, treatment for 14 days) had no significant effects on the fasting blood glucose and the fasting insulin secretion from the pancreatic islets in such T2D mice [Supplementary Figure 1B,C, https://links.lww.com/CM9/C78]. LEAP2 treatment, although did not change fasting glucose and insulin levels, significantly improved glucose tolerance, without affecting the insulin sensitivity or final insulin levels in T2D mice [Figure 1C, D and Supplementary Figure 1D, E, https://links.lww.com/CM9/C78]. After HFD and STZ treatment, the pancreatic islets of T2D mice are damaged and shrinked compared to control mice [Figure 1E]. An histological examination of the pancreas revealed that the LEAP2 treatment partially reversed the loss of islet mass and structural damage in T2D mice, no significant changes in pancreatic islet structure were observed in LEAP2 treatment alone, indicating that a 2-week LEAP2 treatment improved the morphology of pancreatic islets in T2D mice [Figure 1E]. The LEAP2 treatment increased the mass of the islets relative to the total pancreas in T2D + LEAP2 mice [Supplementary Figure 1F,G, https://links.lww.com/CM9/C78]. These results demonstrate that LEAP2 improved glucose tolerance with probably better glucose-stimulated insulin secretion and reversed the remaining islets damaged by HFD and repeated STZ.Figure 1: LEAP2 improved the glucose tolerance and pancreatic islet morphology in vivo and promoted GSIS in vitro. (A) Experimental protocol for constructing the mouse T2D model and LEAP2 treatment. (B) Determination of rate of body weight change after LEAP2 treatment in T2D and control mice (n = 7). (C,D) Measurement of blood glucose during IGTT after LEAP2 treatment in T2D and control mice (n = 5–7). (E) Representative hematoxylin and eosin staining images of pancreases after LEAP2 treatment in T2D and control mice (n = 5). From left to right: Control group, standard chow diet mice were intraperitoneally injected with saline; LEAP2 group, standard chow diet mice were intraperitoneally injected with LEAP2 (30 μg/kg); T2D group, T2D mice were intraperitoneally injected with saline; T2D+LEAP2 group, T2D mice were intraperitoneally injection with LEAP2 (30 μg/kg). ​All treatments were carried out at the same time in the experiment, after 14 days of continuous treatment with LEAP2 or saline. Scale bars = 50 μm. (F) LEAP2 significantly promoted MIN6 cells insulin secretion in the conditions of 3 mmol/L (Control and LEAP2 group) or 18 mmol/L (HG and HG + LEAP2 group) glucose (n = 3). (G) Measurement of intracellular Gck levels treated by LEAP2 in MIN6 cells (n = 6). (H) Measurement of intracellular ATP level treated by LEAP2 in MIN6 cells (n = 6). (I) Measurement of intracellular Ca2+ level treated by LEAP2 in MIN6 cells (n = 6). (J,K) PPARγ level in MIN6 cells treated with LEAP2 by Western blotting (n = 3). (L) Measurement of insulin content in MIN6 cells treated with LEAP2 or GW9662 (n = 3). (M) Measurement of Gck content in MIN6 cells treated with LEAP2 or GW9662 (n = 3). (N) GHSR siRNA significantly downregulated GHSR expression in mRNA levels (n = 3). (O) Measurement of insulin content in MIN6 cells treated with LEAP2 after knockdown of GHSR (n = 3). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. All data are presented as the mean ± SD. AUC: Area under the curve; HFD: High-fat diet; HG: High glucose; GHSR: Growth hormone secretagogue receptor; IGTT: Intraperitoneal glucose tolerance test; LEAP2: Liver-expressed antimicrobial peptide 2; ns: Not significant; prot: Protein; PPAR: Poly (ADP-ribose) polymerase; siRNA: small interfering RNA; SD: Standard deviation; STZ: Streptozotocin; T2D: Type 2 diabetes.The above-mentioned observations were performed in vivo, and the direct effects of LEAP2 on the pancreatic β cells were then investigated in vitro. Using the MIN6 cell line (derived from tumors arising in transgenic mice expressing the SV40 T antigen under control of the insulin promoter) as an in vitro experimental model without ghrelin, the cytotoxic effects of LEAP2 were not observed at a concentration of 1 μmol/L for 2 h using the Cell Counting Kit-8 (CCK-8) [Supplementary Figure 2A, https://links.lww.com/CM9/C78]. To investigate the effects of LEAP2 on GSIS in the MIN6 cells, glucose concentrations of 3 mmol/L and 18 mmol/L were selected in this experiment. The results showed that insulin secretion in the high glucose (HG, 18 mmol/L) group increased significantly compared with that in the control (low glucose, 3 mmol/L) group. Compared with the HG group, the insulin secretion of the HG + LEAP2 group was significantly increased [Figure 1F]. Furthermore, we investigated the molecular mechanisms of LEAP2 in promoting the GSIS; The quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to measure the expression of insulin synthesis-related genes (Ins2, Pdx-1, Mafa) and insulin secretion-related genes (Glut2, Gck, Ucp2, Kir6.2, Sur1) in MIN6 cells. Compared with the control group, no significant differences were found on the mRNA expression of insulin synthesis-related genes by LEAP2 treatment [Supplementary Figure 2B–D, https://links.lww.com/CM9/C78]. However, LEAP2 significantly increased the mRNA expression of Sur1 and Gck, but not Glut2, Kir6.2, and Ucp2 in MIN6 cells [Supplementary Figure 2E–I, https://links.lww.com/CM9/C78]. Gck is the first rate-limiting enzyme in the process of glucose metabolism. Its hydrolysis rate directly affects the subsequent oxidative decomposition of glucose and the insulin secretion.[2,3] LEAP2 significantly increased the protein level of Gck in MIN6 cells [Figure 1G]. The functional analysis showed that 1 μmol/L of LEAP2 treatment led to a significant increase in adenosine 5′-triphosphate (ATP) content in MIN6 cells [Figure 1H]. The level of [Ca2+]i was detected by the Fluo-3-pentaacetoxymethyl ester (Fluo-3 AM) probe (catalog No. S1056, Beyotime, Shanghai, China) in MIN6 cells and was significantly increased by LEAP2 treatment [Figure 1I]. It was reported that activation of poly (ADP-ribose) polymerase (PPAR)γ increased the level of Gck in hepatocytes and islet β-cells.[4,5] Similarly, the LEAP2 (1 μmol/L) treatment of cells for 2 h increased the mRNA and protein levels of PPARγ in MIN6 cells [Figure 1J, K and Supplementary Figure 2J, https://links.lww.com/CM9/C78]. However, LEAP2 treatment had no effects on the protein expression of Akt and phosphorylated Akt in MIN6 cells [Supplementary Figure 2K,L, https://links.lww.com/CM9/C78] To investigate the activation of PPARγ in the secretory effects of LEAP2 on insulin secretion, MIN6 cells were incubated with the PPARγ-specific antagonist, GW9662 (catalog No. HY-16578, MedChemExpress, Shanghai, China), for 12 hours in the presence and absence of LEAP2. Compared with the control group, GW9662 significantly reduced the insulin and Gck content in MIN6 cells, and suppressed the increase in insulin secretion from MIN6 cells by LEAP2, and the increase in the protein level of Gck by LEAP2 [Figure 1L, M]. LEAP2 is a new endogenous antagonist of ghrelin receptor (GHSR) and the second ligand of GHSR.[6] In order to further confirm that the effect of LEAP2 on insulin secretion from the MIN6 cells was mediated by GHSR without ghrelin, small interfering RNA (siRNA) was used to suppress GHSR expression [Figure 1N and Supplementary Figure 3A,B, https://links.lww.com/CM9/C78]. After knockdown of GHSR in the MIN6 cells, the effect of LEAP2 on insulin secretion was significantly blocked [Figure 1O] with no significant differences in expression of the insulin-synthesis genes and insulin-secretion-related genes [Supplementary Figure 3C–I, https://links.lww.com/CM9/C78]. Interestingly, the basal insulin secretion of the MIN6 cells was also reduced by the knockdown of GHSR [Figure 1O]. In addition, the basal levels of PPARγ mRNA and protein expression without LEAP2 or ghrelin were also reduced by GHSR knockdown [Supplementary Figure 3J–L, https://links.lww.com/CM9/C78]. The increase in PPARγ mRNA and protein expression by LEAP2 treatment was significantly reduced after knockdown of GHSR [Supplementary Figure 3M–O, https://links.lww.com/CM9/C78]. GHSR is a G-protein coupled receptor with exceptionally high constitutive activity (up to 50% of maximal activation) in triggering intracellular signals in the absence of ghrelin or its analogs,[7] suggesting an important function of constitutive activation of GHSR in MIN6 β-cells. In conclusion, LEAP2 was demonstrated to improve glucose tolerance and pancreatic islet morphology in diabetic mice in vivo, and promote glucose-stimulated insulin secretion from MIN6 β-cells in vitro. LEAP2 increased ATP content and [Ca2+]i concentrations in MIN6 cells to enhance GSIS through signaling pathways involving the GHSR-PPARγ–Gck axis [Supplementary Figure 4, https://links.lww.com/CM9/C78]. This report has demonstrated that LEAP2 is a promising hormone treating compromised glucose tolerance in HFD-induced T2D mice. Moreover, LEAP2 promotes insulin secretion, through the GHSR–PPARγ–Gck axis. LEAP2 may serve as a potential drug target to reverse damaged insulin secretion, especially for compromised GSIS in T2D. This anti-diabetic effect of LEAP2 warrants further investigation in other diabetic models and other metabolic disorders such as obesity. Funding This research was supported by grants from the National Natural Science Foundation of China (Nos.81870059, 82070068). Conflicts of interest None.

Abstract Background Idiopathic pulmonary fibrosis is a persistent disease of the lung interstitium for which there is no efficacious pharmacological therapy. Protodioscin, a steroidal saponin, possesses diverse pharmacological properties; however, its function in pulmonary fibrosis is yet to be established. Hence, in this investigation, it was attempted to figure out the anti-pulmonary fibrosis influences of protodioscin and its pharmacological properties related to oxidative stress. Methods A mouse lung fibrosis model was generated using tracheal injections of bleomycin, followed by intraperitoneal injection of different concentrations of protodioscin, and the levels of oxidative stress and fibrosis were detected in the lungs. Multiple fibroblasts were treated with TGF-β to induce their transition to myofibroblasts. It was attempted to quantify myofibroblast markers’ expression levels and reactive oxygen species levels as well as Nrf2 activation after co-incubation of TGF-β with fibroblasts and different concentrations of protodioscin. The influence of protodioscin on the expression and phosphorylation of p62, which is associated with Nrf2 activation, were detected, and p62 related genes were predicted by STRING database. The effects of Nrf2 inhibitor or silencing of the Nrf2, p62 and NBR1 genes, respectively, on the activation of Nrf2 by protodioscin were examined. The associations between p62, NBR1, and Keap1 in the activation of Nrf2 by protodioscin was demonstrated using a co-IP assay. Nrf2 inhibitor were used when protodioscin was treated in mice with pulmonary fibrosis and lung tissue fibrosis and oxidative stress levels were detected. Results In vivo, protodioscin decreased the levels of fibrosis markers and oxidative stress markers and activated Nrf2 in mice with pulmonary fibrosis, and these effects were inhibited by Nrf2 inhibitor. In vitro, protodioscin decreased the levels of myofibroblast markers and oxidative stress markers during myofibroblast transition and promoted Nrf2 downstream gene expression, with reversal of these effects after Nrf2, p62 and NBR1 genes were silenced or Nrf2 inhibitors were used, respectively. Protodioscin promoted the binding of NBR1 to p62 and Keap1, thereby reducing Keap1-Nrf2 binding. Conclusion The NBR1-p62-Nrf2 axis is targeted by protodioscin to reduce oxidative stress and inhibit pulmonary fibrosis. Graphical Abstract

The online flipped classroom (OFC) has emerged as a new teaching method in universities worldwide, which combines asynchronous and synchronous online learning. OFC differs from the traditional flipped classroom as it does not involve face-to-face interaction between teachers and students. Instead, the class meeting is conducted online, and it is focused on active and collaborative learning (e.g., discussion rather than lecturing). To evaluate the effectiveness of the Physiology OFC, we compared it with online live teaching (OLT) offered in the same school and semester. We analyzed the exam scores of the Physiology course as well as the scores for other courses offered in the same semester and after the Physiology course. We categorized the top 27% of the exam takers as high-achieving students and the bottom 27% as low-achieving students. Our analysis found no statistically significant difference between OFC and OLT in terms of overall exam scores for all students. However, high-achieving students in OFC scored higher on the total exam score and short answer questions, but the score of case study questions (CSQs) of low-achieving students was lower. Furthermore, students in OFC scored higher in Medical Immunology and courses dominated by logical thinking such as Pharmacology and Diagnostics than students in OLT. In conclusion, our findings suggest that OFC can achieve the same teaching effectiveness as OLT, with a more positive impact on high-achieving students. The positive impact extends beyond the Physiology course to other courses where logical thinking is critical. However, the lower performance of low-achieving students in CSQs highlights the need for further research to determine the reasons for their lower performance and potential strategies to improve their learning outcomes.NEW & NOTEWORTHY An online flipped classroom approach achieved the same teaching effect as online live teaching but had a more positive impact on high-achieving students. The positive impact was not only in Physiology but also in subsequent courses where logical thinking prevailed. However, for low-achieving students, the effect of online live teaching was better.