| Inflammation is a natural defense response to immune or non-immune types of harmful and destructive factors,which can occur in any part of the body’s tissues and organs and is a common clinical pathological process.However,if inflammation is not controlled in time,it may develop into chronic inflammation,which can lead to pathological changes in multiple organs throughout the body.Activation of nucleotide-binding oligomerization structural domain(Nod)-like receptor family pyrin structural domain 3(NLRP3)inflammasomes is closely associated with the development of several inflammatory diseases,and reactive oxygen species(ROS)act as second messengers that activate NLRP3 inflammasomes,thereby triggering a series of inflammatory responses.NIMA-related kinase 7(NEK7)is an NLRP3upstream regulator,and NEK7 is able to regulate NLRP3 signaling by binding to the LRR structural domain.Therefore,this study selected ROS/NEK7/NLRP3 pathway as an important candidate intervention target for inflammatory injury.Platycodon grandiflorus(Jacq.)A.DC.,first published in Shennong Ben Cao Jing,has the effects of opening and promoting lung qi,expectorant and relieving cough,diaphoretic and dissipating nodules,broadening the chest and draining pus.Modern research has found that Eryngium has anti-inflammatory,antibacterial,antitumor,antioxidant,hepatoprotective and immunomodulatory effects.The polysaccharides,saponins and flavonoids in Platycodon grandiflorus have certain biological activities.Platycodon grandiflorus polysaccharide(PGPS)has been identified as one of the major bioactive components and has been shown to activate a variety of immune cells and to have antiviral,antioxidant,anti-inflammatory and antibacterial and immune enhancing effects.The bioactivity of polysaccharides is inextricably linked to their structure,and the group’s preliminary research revealed that Platycodon grandiflorus total polysaccharide(PGPS_t)is a triple-helix structure with dense structure,porous and polymeric morphological characteristics,which contributes to the bioactivity of polysaccharides.At present,the mechanism of action and molecular targets of PGPS_t in alleviating the inflammatory response are less studied,and further research on them is needed.Therefore,in this study,we constructed a lipopolysaccharide(LPS)/adenosine tirphosphate(ATP)-induced cellular inflammatory injury model using porcine alveolar macrophages(3D4/21 cells)to screen the optimal effect concentration of PGPS_t on ameliorating cellular inflammatory response and observe PGPS_t inhibited ROS to reduce the inflammatory response,and further explored the effect of PGPS_t regulation of ROS/NEK7/NLRP3 inflammatory pathway in inflammatory injury,in order to reveal the inhibitory effect of PGPS_t on inflammatory injury,and to provide some theoretical basis for seeking new targets for drug therapy.Based on this,the following research was carried out in this thesis:Different concentrations of LPS were used to induce inflammatory damage in 3D4/21 cells at different times to select the appropriate concentration and time of action of LPS.CCK-8results revealed that the effect of LPS on 3D4/21 cells was dose-and time-dependent,and the inhibition rate was 50.6%at 1.0μg/m L of LPS treated cells for 4 h.Therefore,1.0μg/m L of LPS was selected to treat 3D4/21 cells for 4 h for subsequent experiments.LPS/ATP co-stimulation of 3D4/21 cells induced inflammatory injury,and changes in the expression levels of inflammation-related proteins ASC and NLRP3 and the aggregation of ASC spots were detected.The results revealed that the LPS/ATP co-stimulation group significantly increased the expression levels of NLRP3 and ASC by Western Blot.After immunofluorescence staining,it was observed that the LPS/ATP co-stimulation group significantly induced ASC to form spot aggregates close to the nucleus.The results indicated that the LPS/ATP co-stimulation group could induce a stronger inflammatory response in3D4/21 cells.To screen the optimal action concentration of PGPS_t to alleviate cellular inflammation,cells were co-treated with 100,200 and 300μg/m L PGPS_t and LPS/ATP,and the changes of expression levels of inflammation-related proteins NEK7 and NLRP3 were detected by Western Blot.The results showed that the expression levels of NEK7 and NLRP3 proteins were significantly increased in the LPS/ATP group cells;the expression levels of both NEK7 and NLRP3 proteins were decreased after 200μg/m L PGPS_t treatment.Therefore,200μg/m L PGPS_t was selected as the optimal effect concentration for subsequent experiments.To investigate whether PGPS_t inhibits NLRP3 inflammasomes activation by reducing ROS production,ROS were detected qualitatively and quantitatively in 3D4/21 cells by laser confocal technique and flow cytometry,respectively,using the ROS scavenger NAC as a positive control,RT-q PCR and ELISA were performed to detect the levels of IL-1β,IL-18m RNA and extracellular secretion,and Western Blot was performed to detect the changes of IL-1β,NEK7 and NLRP3 protein expression.The results revealed that ROS production in cells was significantly increased and the intensity of green fluorescent spots was significantly enhanced after LPS/ATP co-stimulation,while PGPS_t significantly inhibited these results,indicating that PGPS_t was able to inhibit LPS/ATP-induced intracellular ROS production.Under immunofluorescence microscopy,ASC proteins were observed to aggregate into characteristic spots,indicating that NLRP3 inflammasomes had been activated,while PGPS_ttreatment significantly reduced ASC spot formation.Meanwhile,PGPS_t significantly inhibited the expression of IL-1β,NEK7 and NLRP3 protein levels and decreased the transcription and secretion levels of inflammatory cytokine IL-1βand IL-18.These results suggest that PGPS_tinhibits the production of ROS to alleviate the cellular inflammatory response.NEK7 plays a key role in the inflammatory response centered on NLRP3 inflammasomes.To further investigate the mechanism of action of PGPS_t in alleviating inflammatory injury,the MCC950 inhibitor was used to block the NEK7-NLRP3 pathway as a positive control.Western blot was used to detect the expression of inflammation-related proteins,and the transcription level and secretion level of inflammatory cytokines were measured by RT-q PCR and ELISA.The results revealed that the PGPS_t group significantly downregulated the expression levels of IL-1β,NEK7 and NLRP3 proteins and reduced the production of inflammatory cytokines IL-1βand IL-18,indicating that PGPS_t alleviated cellular inflammatory responses by acting on the NEK7-NLRP3 pathway.To verify the role played by NEK7 in PGPS_t in alleviating cellular inflammatory damage,NEK7-si RNA was used to silence NEK7 as a positive control.The results showed that the expression of NEK7 was significantly decreased in the NEK7 silencing group,the number of NEK7 fluorescent spots was significantly reduced,and NEK7-si RNA decreased the expression of protein levels of NLRP3,ASC,NEK7,IL-1βand Caspase-1 in cells,indicating that NEK7 silencing could effectively inhibit the inflammatory response in 3D4/21cells.PGPS_t significantly inhibited the upregulation of IL-1β,NEK7,NLRP3,ASC and Caspase-1 protein expression levels induced by LPS/ATP co-stimulation,and immunofluorescence results showed that intracellular NEK7 expression was significantly blocked by PGPS_t with the same effect as the si NEK7 group.Similarly,PGPS_t significantly decreased the secretion levels of inflammatory cytokine IL-1βand IL-18,and the difference was not significant with the si NEK7 group.These results suggest that PGPS_t alleviates the cellular inflammatory response by downregulating the expression of NEK7.In summary,PGPS_t can alleviate LPS/ATP-induced inflammatory injury in 3D4/21 cells through the ROS/NEK7/NLRP3 pathway,elucidating the effect of action of PGPS_t to alleviate inflammatory responses and providing a new target for ameliorating inflammatory injury,and PGPS_t is expected to be a potential candidate for ameliorating inflammatory injury. |
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