Qingkailing granule alleviates pulmonary fibrosis by inhibiting PI3K/AKT and SRC/STAT3 signaling pathways.

Pulmonary fibrosis (PF) poses a significant challenge with limited treatment options and a high mortality rate of approximately 45 %. Qingkailing Granule (QKL), derived from the Angong Niuhuang Pill, shows promise in addressing pulmonary conditions. Using a comprehensive approach, combining network pharmacology analysis with experimental validation, this study explores the therapeutic effects and mechanisms of QKL against PF for the first time. In vivo, QKL reduced collagen deposition and suppressed proinflammatory cytokines in a bleomycin-induced PF mouse model. In vitro studies demonstrated QKL's efficacy in protecting cells from bleomycin-induced injury and reducing collagen accumulation and cell migration in TGF-ß1-induced pulmonary fibrosis cell models. Network pharmacology analysis revealed potential mechanisms, confirmed by western blotting, involving the modulation of PI3K/AKT and SRC/STAT3 signaling pathways. Molecular docking simulations highlighted interactions between QKL's active compounds and key proteins, showing inhibitory effects on epithelial damage and fibrosis. Collectively, these findings underscore the therapeutic potential of QKL in alleviating pulmonary inflammation and fibrosis through the downregulation of PI3K/AKT and SRC/STAT3 signaling pathways, with a pivotal role attributed to its active compounds.

Elucidating the distinctive regulatory effects and mechanisms of active compounds in Salvia miltiorrhiza Bunge via network pharmacology: Unveiling their roles in the modulation of platelet activation and thrombus formation.

Salvia miltiorrhiza Bunge. (DS), as an important traditional Chinese medicine (TCM), has a long history of usage for promoting blood circulation and removing blood stasis. Modern studies have shown that the chemical components of DS have many biological activities such as cardiovascular protection, anti-arrhythmia, anti-atherosclerosis, improvement of microcirculation, protection of myocardium, inhibition and removal of platelet aggregation. Nevertheless, the action mechanism of DS as well its active compounds on platelet activation has not been fully uncovered. This study aimed to find out the potential targets and mechanisms of DS in the modulation of platelet activation and thrombosis, using network pharmacology and biological experimental. These compounds with anti-thrombotic activity in DS, cryptotanshinone (CPT), isoeugenol (ISO) and tanshinone IIA (TSA), together with the corresponding targets being Src, Akt and RhoA are screened by network pharmacology. We confirmed that ISO, CPT and TSA dose-dependently inhibited platelet activation in vitro, mainly by inhibiting agonist-induced clot retraction, aggregation and P-selectin and ATP release. The western blot findings indicated that ISO, CPT, and TSA led to reduced levels of p-Akt and p-ERK in activated platelets. Additionally, ISO and TSA were observed to decrease p-cSrc expression while increasing RhoA expression. ISO, CPT, and TSA demonstrated a potential to restrict the advancement of carotid arterial thrombosis in vivo. We confirm that ISO, CPT and TSA are the key anti-thrombotic active compounds in DS. These active compounds exhibit unique inhibitory effects on platelet activation and thrombus formation by modulating the Akt/ERK and cSrc/RhoA signaling pathways.

Calcium/P53/Ninjurin 1 Signaling Mediates Plasma Membrane Rupture of Acinar Cells in Severe Acute Pancreatitis.

Ninjurin 1 (NINJ1) is a double-transmembrane cell-surface protein that might mediate plasma membrane rupture (PMR) and the diffusion of inflammatory factors. PMR is a characteristic of acinar cell injury in severe acute pancreatitis (SAP). However, the involvement of NINJ1 in mediating the PMR of acinar cells in SAP is currently unclear. Our study has shown that NINJ1 is expressed in acinar cells, and the expression is significantly upregulated in sodium-taurocholate-induced SAP. The knockout of NINJ1 delays PMR in acinar cells and alleviates SAP. Moreover, we observed that NINJ1 expression is mediated by Ca2+ concentration in acinar cells. Importantly, we found that Ca2+ overload drives mitochondrial stress to upregulate the P53/NINJ1 pathway, inducing PMR in acinar cells, and amlodipine, a Ca2+ channel inhibitor, can reduce the occurrence of PMR by decreasing the concentration of Ca2+. Our results demonstrate the mechanism by which NINJ1 induces PMR in SAP acinar cells and provide a potential new target for treatment of SAP.

Xanthohumol alleviates oxidative stress and impaired autophagy in experimental severe acute pancreatitis through inhibition of AKT/mTOR.

Severe acute pancreatitis (SAP) is a lethal gastrointestinal disorder, yet no specific and effective treatment is available. Its pathogenesis involves inflammatory cascade, oxidative stress, and autophagy dysfunction. Xanthohumol (Xn) displays various medicinal properties, including anti-inflammation, antioxidative, and enhancing autophagic flux. However, it is unclear whether Xn inhibits SAP. This study investigated the efficacy of Xn on sodium taurocholate (NaT)-induced SAP (NaT-SAP) in vitro and in vivo. First, Xn attenuated biochemical and histopathological responses in NaT-SAP mice. And Xn reduced NaT-induced necrosis, inflammation, oxidative stress, and autophagy impairment. The mTOR activator MHY1485 and the AKT activator SC79 partly reversed the treatment effect of Xn. Overall, this is an innovative study to identify that Xn improved pancreatic injury by enhancing autophagic flux via inhibition of AKT/mTOR. Xn is expected to become a novel SAP therapeutic agent.

NINJ1 Regulates Platelet Activation and PANoptosis in Septic Disseminated Intravascular Coagulation.

Disseminated intravascular coagulation (DIC), which is closely related to platelet activation, is a key factor leading to high mortality in sepsis. The release of contents from plasma membrane rupture after platelet death further aggravates thrombosis. Nerve injury-induced protein 1 (NINJ1) is a cell membrane protein that mediates membrane disruption, a typical marker of cell death, through oligomerization. Nevertheless, whether NINJ1 is expressed in platelets and regulates the platelet function remains unclear. The aim of this study was to evaluate the expression of NINJ1 in human and murine platelets and elucidate the role of NINJ1 in platelets and septic DIC. In this study, NINJ1 blocking peptide (NINJ126-37) was used to verify the effect of NINJ1 on platelets in vitro and in vivo. Platelet αIIbß3 and P-selectin were detected by flow cytometry. Platelet aggregation was measured by turbidimetry. Platelet adhesion, spreading and NINJ1 oligomerization were examined by immunofluorescence. Cecal perforation-induced sepsis and FeCl3-induced thrombosis models were used to evaluate the role of NINJ1 in platelet, thrombus and DIC in vivo. We found that inhibition of NINJ1 alleviates platelet activation in vitro. The oligomerization of NINJ1 is verified in membrane-broken platelets, which is regulated by the PANoptosis pathway. In vivo studies demonstrate that inhibition of NINJ1 effectively reduces platelet activation and membrane disruption, thus suppressing platelet-cascade reaction and leading to anti-thrombosis and anti-DIC in sepsis. These data demonstrate that NINJ1 is critical in platelet activation and plasma membrane disruption, and inhibition of NINJ1 effectively reduces platelet-dependent thrombosis and DIC in sepsis. This is the first study to reveal the key role of NINJ1 in platelet and its related disorders.

Berberine-mediated REDD1 down-regulation ameliorates senescence of retinal pigment epithelium by interrupting the ROS-DDR positive feedback loop.

BACKGROUND: Accumulation of age-associated senescent cells accompanied with increased reactive oxygen species (ROS) and inflammatory factors contributes to the progression of age-related macular degeneration (AMD), the main cause of blindness in the elderly. Berberine (BBR) has shown efficacy in the treatment of age-related diseases including diabetes and obesity by decreasing ROS. However, the pharmacological effect of BBR on alleviating retinal aging remains largely unknown. PURPOSE: Our study aimed to investigate the pharmacological effect of BBR as an anti-aging agent in retinal aging and its further molecular mechanisms. METHODS: D-galactose (DG)-induced ARPE-19 cell senescence and retinal aging were employed to evaluate the anti-aging effect of BBR in vivo and in vitro. The siRNA transfection, Western-Blot analyses, SA-ß-Gal assay and immunofluorescence were performed to investigate the potential mechanisms of BBR on anti-aging of RPE. RESULTS: In RPE-choroid of both natural aged and DG-induced accelerated aged mice, oxidative stress was increased along with the up-regulation of p21 expression, which was ameliorated by BBR treatment. BBR down-regulated the expression of REDD1 to decrease intracellular ROS content, attenuating DG-induced senescence in vitro and in vivo. Furthermore, p53 instead of HIF-1α was identified as the transcriptional regulator of REDD1 in DG-induced premature senescence. Importantly, NAC and BBR reversed the expression of p53 and the content of 8-OHdG, indicating that the positive feedback loop of ROS-DNA damage response (DDR) was formed, and BBR interrupted this feedback loop to alleviate DG-induced premature senescence by reducing REDD1 expression. In addition, BBR restored DG-damaged autophagy flux by up-regulating TFEB-mediated lysosomal biosynthesis by inhibiting REDD1 expression, thereby attenuating cellular senescence. CONCLUSION: BBR down-regulates REDD1 expression to interrupt the ROS-DDR positive feedback loop and restore autophagic flux, thereby reducing premature senescence of RPE. Our findings elucidate the promising effects of REDD1 on cellular senescence and the great potential of BBR as a therapeutic approach.

PD-L1 Regulates Platelet Activation and Thrombosis via Caspase-3/GSDME Pathway.

Platelets play a central role in hemostasis and thrombosis, regulating the occurrence and development of thrombotic diseases, including ischemic stroke. Programmed death ligand 1 (PD-L1) has recently been detected in platelet, while the function of PD-L1 in platelets remain elusive. Our data reveal a novel mechanism for the role of PD-L1 on platelet activation and arterial thrombosis. PD-L1 knockout does not affect platelet morphology, count, and mean volume under homeostasis and without risk of bleeding, which inhibits platelet activation by suppressing outside-in-activation of integrin by downregulating the Caspase-3/GSDME pathway. Platelet adoptive transfer experiments demonstrate that PD-L1 knockout inhibits thrombosis. And the absence of PD-L1 improves ischemic stroke severity and increases mice survival. Immunohistochemical staining of the internal structure of the thrombus proves that PD-L1 enhances the seriousness of the thrombus by inhibiting platelet activation. This work reveals a regulatory role of PD-L1 on platelet activation and thrombosis while providing novel platelet intervention strategies to prevent thrombosis.

Drug D, a Diosgenin Derive, Inhibits L-Arginine-Induced Acute Pancreatitis through Meditating GSDMD in the Endoplasmic Reticulum via the TXNIP/HIF-1α Pathway.

Acute pancreatitis (AP) is one of the most common causes of hospitalization for gastrointestinal diseases, with high morbidity and mortality. Endoplasmic reticulum stress (ERS) and Gasdermin D (GSDMD) mediate AP, but little is known about their mutual influence on AP. Diosgenin has excellent anti-inflammatory and antioxidant effects. This study investigated whether Diosgenin derivative D (Drug D) inhibits L-arginine-induced acute pancreatitis through meditating GSDMD in the endoplasmic reticulum (ER). Our studies were conducted in a mouse model of L-arginine-induced AP as well as in an in vitro model on mouse pancreatic acinar cells. The GSDMD accumulation in ER was found in this study, which caused ERS of acinar cells. GSDMD inhibitor Disulfiram (DSF) notably decreased the expression of GSDMD in ER and TXNIP/HIF-1α signaling. The molecular docking study indicated that there was a potential interaction between Drug D and GSDMD. Our results showed that Drug D significantly inhibited necrosis of acinar cells dose-dependently, and we also found that Drug D alleviated pancreatic necrosis and systemic inflammation by inhibiting the GSDMD accumulation in the ER of acinar cells via the TXNIP/HIF-1α pathway. Furthermore, the level of p-IRE1α (a marker of ERS) was also down-regulated by Drug D in a dose-dependent manner in AP. We also found that Drug D alleviated TXNIP up-regulation and oxidative stress in AP. Moreover, our results revealed that GSDMD-/- mitigated AP by inhibiting TXNIP/HIF-1α. Therefore, Drug D, which is extracted from Dioscorea zingiberensis, may inhibit L-arginine-induced AP by meditating GSDMD in the ER by the TXNIP /HIF-1α pathway.

Memory-like response in platelet attenuates platelet hyperactivation in arterial thrombosis.

Recent studies showed that in responding of pathogens stimulation, immune cells and other cells display memory-like effects. Platelets are primary effectors of hemostasis and thrombosis which also participate in immune responses. However, there is no relevant research on whether memory-like effect exists in platelets. In our study after recovery from repetitive LPS stimulus, platelets aggregation, diffusion and clot retraction exhibit a significant reduction. It proves that memory-like response could be aroused in platelets. Furthermore, in the mouse arterial thrombosis model, LPS pretreated platelets showed lower integrin activation, shorter thrombus length and longer occlusion time, indicating that the memory-like response of platelet could alleviate arterial thrombosis. Moreover, memory-like response of platelets was also found to be related to PI3K/AKT signaling pathway. The decreased mitochondrial DNA methylation reveal that platelet memory-like responses may be produced from epigenetic reprogramming. Our research proves for the first time that memory-like response in platelets protects mice from arterial thrombosis, extends the understanding of trained memory.

Tuftsin ameliorates splenic inflammatory injury by promoting neuropilin-1 in severe acute pancreatitis.

Severe acute pancreatitis (SAP)-associated spleen injury causing immune disturbances aggravates organs injuries, which contributes to higher mortality rate. However, there are no effective drugs to cure SAP-induced spleen injury. Here, we found that Tuftsin (TN) is effective for ameliorating SAP-induced pathological damage and inflammation of spleen, mainly via alleviating mitochondrial dysfunction, oxidative stress, ATP depletion and the expression of pro-inflammatory factors. We further found that TN promoted anti-inflammatory macrophage phenotype M2 via up-regulating NRP1 on macrophage in spleen during SAP. Meanwhile, EG00229 (an inhibitor of NRP1 bound to TN) weakened TN's therapeutic effect in SAP-associated spleen injury. And EG00229 also inhibited M2 macrophage, leading to increasing inflammasome formation. Additionally, EG00229 reduced the protective efficiency of TN on mitochondrial dysfunction, and inflammation injury via NRP1 in spleen caused by SAP. Similarly, siRNA-Nrp1 into macrophage also prevented TN's inhibition on apoptosis. These findings reveal that TN alleviates SAP-induced spleen injury by promoting NRP1.

STING up-regulates VEGF expression in oxidative stress-induced senescence of retinal pigment epithelium via NF-κB/HIF-1α pathway.

AIM: Aging-related dysfunction of retinal pigment epithelium (RPE) is the main pathogenic factors for pathological angiogenesis due to dysregulated vascular endothelial growth factor (VEGF) in retinal vascular diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). However, the molecular mechanism behind the up-regulation of VEGF in senescent RPE is still blurred. MATERIALS AND METHODS: As oxidative damage is the key cause of RPE dysfunction, we employed a model of oxidative stress-induced premature senescence of ARPE-19 to explore the effect of senescent RPE on VEGF. KEY FINDINGS: We reported that senescent ARPE-19 up-regulated VEGF expression under both short-term and prolonged H2O2 treatment, accompanying with increased HIF-1α, the key mediator of VEGF. STING signaling, which could be activated by oxidative stress-damaged DNA, was also observed to be increased in senescent ARPE-19 treated with H2O2. And the inhibition of STING significantly reduced HIF-1α expression to alleviate the up-regulation of VEGF. NF-κB was also shown to be involved in the regulation of VEGF in senescent ARPE-19 in response to STING signaling. Furthermore, oxidative stress impaired the lysosomal clearance of damaged DNA to enhance STING signaling, thereby up-regulating VEGF expression in senescent RPE. SIGNIFICANCE: Our data provide evidence that STING plays an important role in VEGF regulation in senescent RPE induced by oxidative stress.

Deoxyarbutin attenuates severe acute pancreatitis via the HtrA2/PGC-1α pathway.

Severe acute pancreatitis (SAP) is an inflammatory disorder of the exocrine pancreas associated with high morbidity and mortality. SAP has been proven to trigger mitochondria dysfunction in the pancreas. We found that Deoxyarbutin (dA) recovered impaired mitochondrial function. High-temperature requirement protein A2 (HtrA2), a mitochondrial serine protease upstream of PGC-1α, is charge of quality control in mitochondrial homeostasis. The molecular docking study indicated that there was a potential interaction between dA and HtrA2. However, whether the protective effect of dA against SAP is regulated by HtrA2/PGC-1α remains unknown. Our study in vitro showed that dA significantly reduced the necrosis of primary acinar cells and reactive oxygen species (ROS) accumulation, recovered mitochondrial membrane potential (ΔΨm) and ATP exhaustion, while UCF-101 (HtrA2 inhibitor), and SR-18292 (PGC-1α inhibitor) eliminated the protective effect of dA. Moreover, HtrA2 siRNA transfection efficiently blocked the protective of dA on HtrA2/PGC-1α pathway in 266-6 acinar cells. Meanwhile, dA also decreased LC3II/I ration, as well as p62, and increased Parkin expression, while UCF-101 and Bafilomycin A1 (autophagy inhibitor) reversed the protective effect of dA. Our study in vivo confirmed that dA effectively alleviated severity of SAP by reducing pancreatic edema, plasma amylase, and lipase levels and improved the HtrA2/PGC-1α pathway. Therefore, this is the first study to identify that dA inhibits pancreatic injury caused by oxidative stress, mitochondrial dysfunction, and impaired autophagy in a HtrA2/PGC-1α dependent manner.

Activation of TLR4 induces severe acute pancreatitis-associated spleen injury via ROS-disrupted mitophagy pathway.

Severe acute pancreatitis (SAP) is complicated by systemic inflammatory response syndrome and multiple organ dysfunction, the disease will eventually result in death in almost half of the case. The spleen, as the largest immune organ adjacent to the pancreas, is prone to damage in SAP, thereby aggravating the damage of other organs and increasing mortality. However, to date, the research on the mechanism and treatment of spleen injury caused by SAP is still in its infancy. Herein, we investigated the mechanism of spleen injury, and explored the application potential of tuftsin for relieving spleen damage in SAP mice. Firstly, SAP mice model was constructed via the retrograde infusion of 3.5 % sodium taurocholate into the biliopancreatic duct. Then, we proved that the up-regulation of Toll-like receptor 4 (TLR4) in spleen would lead to the accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction under SAP conditions. The splenic ROS and mitochondrial dysfunction could be improved by N-acetylcysteine (NAC) treatment or knocking out TLR4 in SAP mice. Meanwhile, we found that NAC treatment could also improve the autophagy of spleen tissue, suggesting that splenic ROS may affect impaired autophagy, causing the accumulation of damaged mitochondria, aggravating spleen damage. Furthermore, we verified the mechanism of spleen injury is caused by splenic ROS affecting PI3K/p-AKT/mTOR pathway-mediated autophagy. In addition, we detected the spleen injury caused by SAP could decrease the concentration of tuftsin in the serum of mice. Whereas, exogenous supplementation of tuftsin ameliorated the pathological damage, ROS accumulation, impaired autophagy, inflammation expression and apoptosis in damaged spleen. In summary, we verified the new mechanism of SAP-caused spleen damage that TLR4-induced ROS provoked mitophagy impairment and mitochondrial dysfunction in spleen via PI3K/p-AKT mTOR signaling, and the application potential of tuftsin in treating spleen injury, which might expand novel ideas and methods for the treatment of pancreatitis.

Vitamin B12 Attenuates Acute Pancreatitis by Suppressing Oxidative Stress and Improving Mitochondria Dysfunction via CBS/SIRT1 Pathway.

Acute pancreatitis is an inflammatory disorder of the pancreas associated with substantial morbidity and mortality, which is characterized by a rapid depletion of glutathione (GSH). Cysthionine-ß-synthase (CBS) is a key coenzyme in GSH synthesis, and its deficiency is related to a variety of clinical diseases. However, whether CBS is involved in the pathogenesis of acute pancreatitis remains unclear. First, we found that CBS was downregulated in both in vivo and in vitro AP models. The pancreatic damage and acinar cell necrosis related to CBS deficiency were significantly improved by VB 12, which stimulated clearance of reactive oxygen species (ROS) by conserving GSH. Furthermore, EX-527 (a specific inhibitor of SIRT1) exposure counteracted the protective effect of VB 12 by promoting oxidative stress and aggravating mitochondrial damage without influencing CBS, indicating that vitamin B12 regulates SIRT1 to improve pancreatical damage by activating CBS. In conclusion, we found that VB 12 protected acute pancreatitis associated with oxidative stress via CBS/SIRT1 pathway.