Study On The Mechanism Of Fushen Recipe In The Prevention And Treatment Of Peritoneal Dialysis Related Peritonitis Based On TLR4/NF-κB Pathway

Objective:To explore and analyze the inhibitory effect of Fushen Recipe on micro inflammation in uremic peritoneal dialysis rats,and its possible signaling pathway in preventing and treating peritoneal dialysis related peritonitis,To provide a reliable experimental basis for the clinical application of Fushen recipe in the prevention and treatment of peritoneal dialysis related peritonitisMethods:1.To observe the intervention effect of Fushen Recipe on micro inflammation in uremic and peritoneal dialysis ratsHealthy male SD rats were selected,and 5/6 rats were nephrectomized to make the rat model of uremia.From two weeks after operation,the experimental rats were injected intraperitoneally with 20ml(100ml·kg^-1·D^-1)dialysate（PDS）with 1.5%glucose concentration every day to make the peritoneal dialysis rat model,establish the uremia peritoneal dialysis rat model,and use low,medium and high doses of Fushen formula to treat the uremia and peritoneal dialysis rats.During the treatment,the mental state of rats which among six groups was continuously observed,and the weight of all rats was monitored every week;At the end of the 8th week of treatment,the serum creatinine and blood urea nitrogen of the rats were detected;the serum hs-CRP of the rats was detected,The levels of inflammatory factors,for such TNF-ɑand IL-6,Using H&E staining to observe the pathological morphology of rat colon tissue.2.To explore the mechanism of Fushen recipe in the prevention and treatment of peritoneal dialysis related peritonitis based on network pharmacologyAccess to TCMSP database to obtain the active compounds and target points of the traditional Chinese medicines of Fushen recipe.At the same time,the disease targets related to peritoneal dialysis related peritonitis were screened through disgenet,digsee,drugbank,GENECARD,OMIM and NCBI gene databases to obtain the intersection genes of drugs and diseases.The STRING database was allowed to make a protein-protein interactionnetwork（PPI）,and then a visualized Fushen recipe and peritoneal dialysis related peritonitis"vital core target"PPI network was constructed in Cytoscape 3.7.2 software.at the end of these steps,GO functional enrichment and KEGG pathway enrichment analysis were performed in a database called David,and the enrichment analysis results are visualized with imagegp online mapping tool.3.Effect of Fushen Recipe on the expression of key factors of TLR4/NF-κB signal pathway in uremic peritoneal dialysis ratsThe samples in this part are from the rats in the first part.The ELISA was allowed to test the levels of LPS as well as LBP in serum,Western blot was allowed to detect protein expression level of LBP,TLR4,NF-κB p65,IκBα,TNF-α,IL-1β,These key factor of TLR4/NF-κB signaling pathway in rat colonResults:1.Intervention effect of Fushen Recipe on inflammatory response in uremic peritoneal dialysis ratBiochemical experiments results indicated that the levels of serum creatinine and urea nitrogen increased in model group extraordinary increased;After receiving medical help,the Serum Creatinine and Blood urea nitrogen of among six groups of rats in the low,middle as well as high dose groups of Fushen Recipe and the medicine group of Live Combined Bifidobacterium decreased in different degrees.ELISA displayed that the substance of serum hs-CRP,Furthermore TNF-ɑas well as IL-6 were increased in the model unit;Serum hs-CRP,Furthermore TNF-ɑas well as IL-6 were reduced significantly in low,medium and high dose groups and modern medicine group of Live Combined Bifidobacterium after 8 weeks of treatment.In the sham operation unit,The pathological experiments communicated that the every layers of colonic tissue was well-defined,The villi of the colon have normal arrangement and length,the colonic mucosa,submucosa and muscular layer were arranged in an orderly manner,and inflammatory cells were rare.In the model group,the structure was abnormal in every colonic tissue,the intestinal villi were sparse and shortened,the gap was widened,There is clear infiltration of inflammatory cells in part of the colon In the low,middle as well as high dose groups of Fushen Formula and the modern medicine group of Live Combined Bifidobacterium,the Fushen formula group of low,medium as well as high dose and the medicine group of Live Combined Bifidobacterium had different degrees of damage to the intestinal villi,reduced submucosal edema,narrowed gap,and reduced infiltration of lamina propria inflammatory cells.2.To explore the mechanism of Fushen recipe in the prevention and treatment of peritoneal dialysis related peritonitis based on network pharmacologyFushen recipe mainly acts on TNF,IL-6,CASP3,IL-10 and IL-1βin peritoneal dialysis related peritonitis And other related targets,mainly involved in TNF signaling pathway,NOD like receptor signaling pathway as well as toll like receptor signaling pathway,which affect peritoneal dialysis related peritonitis mainly through TNF,IL-6,IL-1β,CXCL8,CASP8,CASP3,IL10,CCL2,etc.play a role.It is preliminarily inferred that Fushen recipe may prevent and treat peritoneal dialysis related peritonitis by restraining TLR4/NF-κB signaling pathway,and inhibiting the activities of TNF-α,IL-6,IL-1βand other related inflammatory factors.3.Effect of Fushen Recipe on the expression of key factors of TLR4/NF-κB signal pathway in uremic peritoneal dialysis ratsThe results of ELISA showed that the levels of LPS and LBP in serum of uremic peritoneal dialysis rats increased.After treatment with Fushen formula,the levels of LPS and LBP in serum of rats decreased.WB results showed that LBP,TLR4,NF-κB p65,IκBα,TNF-α,IL-1βin colon tissue of uremic peritoneal dialysis rats Increased protein expression,After treatment with Fushen formula,the LBP,TLR4,NF-κB p65,IκBα,TNF-α,IL-1βprotein content in serum and colon tissue of uremic peritoneal dialysis rats decreased,The IκBαprotein content increasedConclusion:1.Fushen recipe can improve renal function and reduce the release of serum proinflammatory factors in uremic peritoneal dialysis rats,for instance serum hs-CRP,TNF-αand IL-6 of rats.It can significantly reduce the colonic inflammation of uremic peritoneal dialysis rats,protect the intestinal barrier function of uremic peritoneal dialysis rats,and improve the micro inflammatory state of uremic peritoneal dialysis rats.2.The occurrence of peritoneal dialysis related peritonitis in uremic peritoneal dialysis rats may be related to TLR4/NF-κB pathway activation.3.The mechanism of Fushen recipe inhibiting peritoneal dialysis associated peritonitis may be through inhibiting the activation of TLR4/NF-κB signal pathway,inhibition of IκBαProtein degradation,thereby reducing TNF-α、IL-6 inflammatory factor level,prevention and treatment of peritoneal dialysis associated peritonitis.