| The intestinal tract of weaned piglets is immature and susceptible to external stimuli leading to dysregulation of intestinal homeostasis,functional disruption,causing reduced feed intake in piglets,and even triggering inflammatory injury,which affect the normal growth of piglets and cause severe economic losses to the pig industry.Therefore,there is an urgent need to prevent intestinal inflammatory injury and dysfunction to improve piglet growth performance and economic efficiency in the pig industry.Currently,antibiotics are widely used in clinical treatment of porcine intestinal inflammatory diseases,but long-term use or non-standard use of drugs can cause problems such as the generation of drug-resistant strains and drug residues.With the implementation of the national policy of"reducing and replacing antibodies",many herbal medicines have become potential drugs for the treatment of inflammatory injury of the intestinal tract.Recent studies have shown that Scutellaria baicalensis has many biological activities,such as anti-inflammatory,antibacterial,antioxidant and so on,which can be used as a potential drug for the treatment of intestinal inflammatory injury.Baicalin,as the main active ingredient of Scutellaria baicalensis,is widely used in the treatment of inflammatory injury,but its mechanism of alleviating intestinal inflammatory injury is unclear.Therefore,in this study,a network pharmacology approach was used to explore the targets of baicalin for alleviating intestinal inflammatory injury in pigs,and the inflammatory response was induced in IPEC-J2 cells by lipopolysaccharide(LPS)to mimic intestinal inflammatory injury in pigs,and to explore the molecular mechanism by which baicalin alleviates intestinal inflammatory injury in pigs,so as to provide a theoretical basis for ameliorating intestinal inflammatory injury.This paper will study from the following three aspects:1.To identify the potential targets of baicalin in alleviating LPS-induced intestinal inflammatory injury and its role in biological functions and signal transduction pathways.Firstly,the targets of intestinal inflammatory injury induced by lipopolysaccharide and the targets of baicalin were summarized based on network pharmacology;Secondly,the binding ability of baicalin to potential targets was verified through molecular docking and DARTS assays;Finally,GO analysis and KEGG analysis were performed on core targets to explore their roles in biological functions and signal transduction pathways.The results indicated that baicalin alleviates intestinal inflammatory injury in pigs may be related to several targets including PARP1,TLR4,and MAPK1;Molecular docking and DARTS results revealed that baicalin has the ability to stably bind with the potential core target PARP1;The results of GO analysis and KEGG analysis revealed that the potential core targets may induce inflammatory damage in the organism by mediating inflammatory injury signaling pathways to release inflammatory factors.Taken together,baicalin may alleviate intestinal inflammatory injury through PARP1 target.2.Baicalin alleviates LPS-induced inflammatory injury in IPEC-J2 cells.CCK8 assay was used to determine the effect of different concentrations of baicalin on the cell viability of IPEC-J2 cells:2×10~4cells were seeded in 96-well plates,and baicalin was added to a final concentration of 12.5,25,50,100 and 200μg/m L.IPEC-J2 cells were treated for 24 hours,and then treated with CCK8 reagent for 2hours to determine cell viability.The genes expressions of PARP1,NF-κB p65,NLRP3,Caspase1,ASC and IL-1βwere detected by RT-q PCR.The proteins expressions of PARP1,NF-κB p65,NLRP3,Caspase1,Cleaved-GSDMD,ASC,IL-18 and IL-1βwere detected by Western Blot.The experiment was divided into control group,LPS model group,and different concentrations of baicalin(25,50 and100μg/m L)+LPS group.The results showed that treatment of IPEC-J2 cells with0～100μg/m L of baicalin for 24 h with no effect on cell viability Therefore,25,50 and100μg/m L baicalin were used as concentrations for the subsequent assays.The results of genes expression showed that 10μg/m L LPS treatment significantly up-regulated the expression levels of PARP1,NF-κB p65,NLRP3,Caspase1,ASC and IL-1βin IPEC-J2 cells.Baicalin could significantly reduce the genes expression levels of inflammatory factors induced by LPS in the different concentrations(p<0.01).The results of proteins expression showed that 10μg/m L LPS treatment significantly up-regulated the expression levels of PARP1,NF-κB p65,NLRP3,Caspase1,Cleaved-GSDMD,ASC,IL-18 and IL-1βin IPEC-J2 cells.Baicalin could significantly reduce the proteins expression levels of inflammatory factors induced by LPS in the different concentrations(p<0.01).The above results indicated that 10μg/m L LPS can induce intestinal inflammatory injury in IPEC-J2 cells,and baicalin could alleviate the intestinal inflammatory injury induced by LPS,indicating that the model of intestinal inflammatory injury has been successfully established in IPEC-J2cells,and baicalin can be used as a candidate drug for treating intestinal inflammatory injury in pigs.3.Mechanism of baicalin alleviating intestinal inflammatory injury induced by LPS.The above results showed that baicalin could alleviate LPS induced intestinal inflammatory injury in pigs.Therefore,to further explore the molecular mechanism of baicalin in alleviating intestinal inflammatory injury in pig,overexpression of the core target PARP1 was carried out to mimic the effect of LPS in cells and silencing PARP1was carried out to mimic the effect of baicalin in cells.Western Blot was used to determine the protein expression levels of PARP1,NF-κB p65,NLRP3,Caspase1,Cleaved-GSDMD,ASC,IL-18 and IL-1βafter PARP1 overexpression and silencing.The overexpression experiment was divided into 5 groups:pc DNA 3.0 control group,PARP1-OE group,and different baicalin concentrations(25,50 and 100μg/m L)+PARP1-OE group.The silencing experiment was divided into 4 groups:NC control group,PARP1 si RNA group,NC control+LPS(10μg/m L)group and PARP1si RNA+LPS(10μg/m L)group.The results showed that overexpression of PARP1significantly up-regulated the protein expression levels of PARP1,NF-κB p65,NLRP3,Caspase1,Cleaved-GSDMD,ASC,IL-18 and IL-1β.Baicalin significantly reduced the expression levels of intestinal inflammatory injury-related proteins after PARP1 overexpression(p<0.01).The expression of p-NF-κB p65 in the cytoplasm and nucleus was determined by Western Blot.The experiment was divided into control group,LPS model group,and different concentrations of baicalin(25,50 and100μg/m L)+LPS group.The results showed that LPS increased phosphorylated NF-κB p65 nuclear translocation,whereas baicalin significantly decreased the phosphorylated NF-κB p65 nuclear transfer(p<0.01).The above studies suggest that baicalin negatively regulates PARP1-NF-κB/PARP1-NLRP3 signaling pathway related protein expression to alleviate LPS induced intestinal inflammatory injury in pigs.In conclusion,the induction of PARP1 protein expression by LPS causes the occurrence of intestinal inflammatory injury through two aspects.On the one hand,PARP1 mediates the transfer of NF-κB p65 from the cytoplasm to the nucleus and increases the expression of inflammatory factors IL-1βand IL-18 to induce inflammatory injury.On the other hand,PARP1 promotes NLRP3 inflammasome assembly,and activated Caspase1 cleaves pro-IL-1βand pro-IL-18 to promote the release of IL-1βand IL-18,and activated GSDMD forms perforin at the cell membrane,which promoted the release of IL-1βand IL-18 to induce inflammatory injury.Baicalin alleviates LPS-induced intestinal inflammatory injury in IPEC-J2cells by regulating the expression of PARP1 and mediating PARP1-NF-κB/PARP1-NLRP3 signaling pathway. |
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