Molecular Mechanism Of Panax Notoginseng Saponins(PNS)Specifically Induced Neutrophil Apoptosis In Zebrafish

Background:Sanqi(Panax notoginseng),a traditional Chinese medicine,has a history of thousands of years in Chinese traditional medicine and has been widely used in clinical practice since ancient times.It exhibits unique therapeutic effects in reducing swelling and treating inflammation,particularly in the field of surgery and trauma,earning the reputation of "surgical golden wound medicine." Currently popular traditional Chinese medicine products such as Yunnan Baiyao and Pianzaihuang are actually composed mainly of Sanqi.Although the anti-inflammatory and anti-swelling effects of Sanqi are recognized,our research team’s preliminary cell experiments have found that Sanqi can promote the chemotaxis of neutrophils to the site of inflammation and induce apoptosis of activated inflammatory neutrophils.However,the mechanism and principle of action of Sanqi remain unclear.Therefore,in this study,we plan to use live zebrafish,which share 87% similarity to human genome,as experimental animals.Taking advantage of the zebrafish’s transparent body,we aim to observe the physiological and pathological changes of neutrophils in the zebrafish body after the administration of Sanqi.By employing modern research methods,we seek to explore the mechanisms and pathways of action of the traditional Chinese medicine Sanqi.Research Objectives:The aim of this study is to achieve the following goals by using zebrafish embryos with specific fluorescent markers as an experimental animal model:1.Investigate the phenotypic changes of zebrafish neutrophils induced by Sanqi,a traditional Chinese medicine.2.Study the specific effects of Sanqi on neutrophils.3.Explore the mode of neutrophil death in zebrafish.4.Explore the molecular mechanisms or pathways through which Sanqi exerts its anti-inflammatory effects.5.Determine the optimal therapeutic concentration of Sanqi for achieving the best anti-inflammatory efficacy,with the aim of developing new medicinal applications for Sanqi.Sanqi is widely used both clinically and as a dietary supplement.It has been extensively applied in the treatment of various diseases,particularly in the field of surgical conditions such as contusions and injuries.According to traditional Chinese medicine theory,Sanqi is often used to treat inflammatory diseases,such as traumatic bleeding,fractures,chest and abdominal pain,and contusions with swelling and pain.In recent years,modern techniques have gradually revealed the chemical composition and pharmacological activities of Sanqi.Phytochemical studies have identified multiple bioactive compounds in Sanqi,including flavonoids,saponins,amino acids,and volatile oils.Among them,Panax Notoginseng Saponins(PNS)are the main bioactive components of Sanqi,which are a mixture of various saponins extracted from the root of the Sanqi plant.The pharmacological properties of PNS have been extensively investigated.Sanqi has been found to possess powerful anti-inflammatory therapeutic effects,and research on its anti-inflammatory properties primarily focuses on the various components within PNS.Moreover,direct use of PNS has been shown to effectively alleviate inflammation-related diseases.However,its mechanisms of action still require further investigation.Current evidence suggests that PNS holds promising prospects as a therapeutic agent for inflammation and related diseases.Due to the complexity of PNS components and the relatively outdated extraction processes,further research is needed to fully understand its active ingredients,potential mechanisms influencing treatment,and to identify or develop its future application scenarios while ensuring its safety.To achieve the aforementioned objectives,this study plans to utilize zebrafish embryos with specific fluorescent markers as an experimental animal model to investigate the mechanisms of action of Sanqi through observing changes in zebrafish neutrophils.Zebrafish,as a model organism,possesses transparent embryos that allow clear observation of the physiological and pathological changes of neutrophils upon the administration of Sanqi.We will employ modern research techniques to study the phenotypic changes of zebrafish neutrophils induced by Sanqi,aiming to understand its effects on cell morphology and function.Additionally,we will explore the specific effects of Sanqi on neutrophils,revealing its mechanisms targeting inflammatory reactions.Furthermore,we will investigate the molecular mechanisms or pathways through which Sanqi exerts its anti-inflammatory effects,elucidating its impact on the transmission and regulation of inflammatory signals.Finally,by testing different concentrations of Sanqi,we will determine the optimal therapeutic concentration for its anti-inflammatory efficacy,aiming to develop new clinical scenarios for the application of Sanqi as a traditional Chinese medicine.Through this study,we aim to further understand the pharmacological effects of Sanqi and provide scientific evidence for its clinical application.Additionally,in-depth research on Sanqi contributes to expanding the application scope of traditional Chinese medicine and contributes to the modernization of traditional Chinese medicine.Neutrophils are the most abundant type of granulocytes in the human body,accounting for approximately 50% to 70%.They serve as the first line of defense against various infectious pathogens,including viruses,bacteria,fungi,and protozoa.The primary functions of neutrophils are phagocytosis of pathogens and promotion of tissue repair in damaged areas.However,if neutrophils become dysregulated,the mechanisms of their infiltration and inflammatory response can cause harm to the host.Excessive activation and uncontrolled neutrophil infiltration can lead to inflammation and autoimmune diseases,such as acute lung inflammation/injury,ischemia/reperfusion injury,rheumatoid arthritis,and sepsis.In recent years,the novel coronavirus pneumonia has caused respiratory failure and death in patients due to uncontrolled inflammation leading to pulmonary inflammation and consolidation.Neutrophil apoptosis is part of the body’s homeostasis and can be triggered in senescent neutrophils.However,under specific circumstances,it can also be induced.For example,the use of serum or antibody stimulation for pathogen phagocytosis and intracellular killing can initiate a specific cellular differentiation program that ultimately leads to phagocytosis-induced cell death(PICD).Due to the anti-inflammatory properties of neutrophil apoptosis,this mechanism appears to be highly applicable in promoting tissue repair after pathogen clearance.Currently, treatment for such diseases often involves nonsteroidal anti-inflammatory drugs and anti-cytokine therapy.However,these therapies can cause damage to the innate and adaptive immune systems to some extent.Once imbalanced,inflammation may spread uncontrollably.Therefore,there is a need to develop new treatment strategies to eliminate excessive inflammatory neutrophils.Zebrafish,a small bony fish,has gradually become a focus of inflammation research over the past thirty years.Zebrafish shares a high degree of genetic similarity with humans,with over 87% of its genome being similar to those of humans.It possesses organs such as the heart,liver,pancreas,and intestines,and its immune system functions similarly to that of humans,featuring multiple conserved inflammatory signaling pathways and immune cell types.This makes zebrafish an ideal choice as a simplified model organism for studying the mechanisms of inflammation.The hematopoietic system of zebrafish is similar to that of other vertebrates and involves multiple stages of cell differentiation.The embryos and larvae of zebrafish are optically transparent,allowing for non-invasive imaging studies of the dynamic processes of inflammation.This characteristic enables real-time observation of the development and progression of inflammation.Additionally,zebrafish exhibit rapid reproduction,generating a large number of offspring,which makes high-throughput screening possible for evaluating potential anti-inflammatory compounds or gene modifications.This aids in identifying potential drug targets or guiding the development of new therapies.Therefore,zebrafish is a valuable tool for evaluating innate immune responses,and its unique characteristics make it a superior choice compared to rodent models.In animal studies,there have been several reports on the anti-inflammatory effects of PNS(Panax notoginseng saponins).However,the specific effective molecules responsible for the anti-inflammatory effects in PNS remain unclear.Moreover,the underlying mechanisms by which PNS exerts its anti-inflammatory effects have not been fully elucidated and require further research.This project aims to explore the phenotype mechanisms of specific induction of neutrophil apoptosis by PNS and conduct research related to the material basis and effects of traditional Chinese medicine.By integrating interdisciplinary approaches and modern scientific techniques,we aim to comprehensively uncover the specific molecular mechanisms underlying the anti-inflammatory properties of PNS.Materials and Methods:1.Drug treatment and animal model: Zebrafish larvae from fertilization until 72 hours post-fertilization(72 hpf),with labeled neutrophils(Tg(lyz:Ds Red2)),were exposed to PNS at concentrations of 0 mg/L,5 mg/L,10 mg/L,and 15mg/L.After 6 hours,observation and imaging of neutrophil responses to PNS were conducted under a fluorescence microscope.2.Specific evaluation of PNS on neutrophils: Using transgenic fish lines with labeled macrophages,thymic T cells,and hematopoietic stem cells,the effects of PNS on myeloid cells other than neutrophils were evaluated.3.Investigation of the mechanism of neutrophil death induced by PNS: A transgenic fish line with co-labeled macrophages and neutrophils was used to observe the process of neutrophil death induced by PNS at the single-cell level.Various inhibitors of cell death pathways,such as autophagy and necrotic apoptosis,were used to determine if they affect the PNS-induced neutrophil death process.TUNEL staining and Caspase 3 activity staining were used to determine if PNS induces neutrophil death through the apoptotic pathway.4.Exploration of signaling pathways involved in PNS-induced neutrophil apoptosis through small molecule screening: RNA-seq was used to detect transcriptomic changes induced by PNS,and genes and signaling pathways with significant changes were analyzed.Through literature research on signaling pathways enriched with significantly changed genes related to neutrophil apoptosis,a large number of small molecule drugs were used to intervene in the process of PNS-induced neutrophil apoptosis,aiming to find small molecule drugs and corresponding molecular signals that can regulate this process.5.Study on the molecular mechanism of PNS-induced neutrophil apoptosis:Antibody staining was used to explore the spatiotemporal expression of MAPK13 and protein immunoblotting was used to analyze the protein expression level of MAPK13.Finally,small molecule drugs targeting downstream proteins of MAPK13 were used for intervention to validate if they conform to the signaling pathway regulated by MAPK13.6.Exploration of the optimal therapeutic concentration of PNS for inducing inflammatory neutrophil apoptosis: Through the zebrafish tail fin inflammation model,it was found that PNS can induce the migration and apoptosis of inflammatory neutrophils at low concentrations.Further exploration of PNS-specific induction of neutrophil apoptosis was conducted using LPS to investigate the potential application prospects.Results:1.The rate of neutrophil death increases with the concentration of PNS,with higher PNS concentrations leading to faster neutrophil death.2.PNS has no significant effect on other myeloid lineage cells,such as macrophages,thymus T cells,and hematopoietic stem cells,except for neutrophils.3.PNS-induced neutrophil death is accompanied by phagocytosis by macrophages,and PNS-induced neutrophil death cannot be inhibited by autophagy or necrosis inhibitors.TUNEL and Caspase 3 activity staining show positive results during PNS-induced neutrophil death.4.PNS induces specific expression of MAPK13(p38δ)in neutrophils.The process of PNS-induced neutrophil apoptosis can also be disrupted by small molecule inhibitors targeting downstream proteins of MAPK13,such as PKD1 and PTEN.5.Low concentrations of PNS can specifically promote the migration of neutrophils to the inflamed tail region and induce apoptosis in activated inflammatory neutrophils,while having no effect on normal neutrophils.Conclusion:1.PNS can specifically induce neutrophil death2.The mode of death is achieved through apoptosis.3.PNS treatment specifically upregulates and activates the expression of p38δprotein in neutrophils,and then participates in neutrophil apoptosis by regulating downstream signaling pathways involving the activation and function of PKD1 and PTEN.4.Low concentrations of PNS(5mg/L)can rapidly and specifically induce neutrophil migration to the inflamed site and induce apoptosis in activated(inflammatory)neutrophils.A PNS concentration of 5 mg/L is able to exert the optimal anti-inflammatory effect.