| Objective: The rat model of interstitial cystitis(IC/BPS)was established by infusing protamine sulfate and lipopolysaccharide into the bladder,and The expression of TLR4/NLRP3 signaling pathway in IC models was studied to provide experimental evidence for the study of whether this signaling pathway can be used as a therapeutic target for IC/BPS.Methods: In this study,24 healthy female SD rats were randomly divided into IC/BPS group(n=12)and control group(n=12).1m L protamine sulfate +1m L lipopolysaccharide was injected into the bladder of rats in IC/BPS group to prepare an interstitial cystitis model,while 2m L normal saline was injected into the bladder of rats in control group in the same way as blank control group.Rats in both groups were injected once every 2 days.It lasts for 4 weeks.After the last bladder perfusion,urine samples of IC/BPS group and control group rats were collected using sterile plastic containers,and urine histamine levels were measured in both groups.After the statistics were completed,the rats in both groups were killed and the bladder tissues were taken out for the following experiments: 1.The pathological characteristics of the bladder tissues of rats in the two groups were observed by hematoxylin-eosin staining(HE staining).2.The infiltration degree of mast cells in the bladder tissues of the two groups was compared by toluidine blue staining(TB staining).3.The expression levels of TLR4/NLRP3 signaling pathway related proteins(TLR4,P-P65,NLRP3,Caspase-1,IL-1β)were detected by Westem blot.4.The gene expressions of TLR4,NLRP3,Caspase-1 and IL-1β were detected by q R-TPCR.5.The expression levels of TLR4/NLRP3 signaling pathway-related positive cells(TLR4,P-NF-κB,NLRP3,Caspase-1,IL-1β)were detected by immunohistochemical staining.Gap Pad software is used to plot statistics Results.SPSS 25.0 statistical analysis software was used for data processing.Results: 1.The urinary histamine level in the IC/BPS group was significantly higher than that in the control group,with a statistically significant difference(P<0.05).2.HE staining showed that the bladder mucosa epithelium of IC/BPS group rats became thinner,congested and edema,and a large number of inflammatory cells and erythrocyte infiltration could be observed in the bladder tissue,while the bladder epithelium of control group rats was intact without inflammatory cell infiltration.3.Toluidine blue staining showed that the skin on the bladder wall of IC/BPS group rats was seriously damaged,and the number of mast cells increased sharply,mainly in a degranulated state,and the number of mast cells was significantly increased compared with the control group(P<0.05).4.Westerm blotting showed that the expression levels of TLR4,P-P65,NLRP3,Caspase-1 and IL-1β related to the TLR4/NLRP3 signaling pathway in the bladder tissues of rats in IC/BPS group were significantly increased compared with the control group(P<0.05).5.The results of q RT-PCR showed that the gene expression levels of TLR4,NLRP3,Caspase-1 and IL-1β in bladder tissue of mice in IC/BPS group were significantly higher than those in control group(P<0.05).6.Immunohistochemical staining showed that The expression level of TLR4,p-NF-κB,NLRP3,Caspase-1,IL-1βin bladder tissue of rats in IC/BPS group was significantly higher than that of the control group,with a statistically significant difference(P<0.05).Conclusion: 1.The IC/BPS model of rats can be successfully constructed by injecting protamine sulfate and lipopolysaccharide into the bladder.The histamine level in the urine of IC group was significantly higher than that of the control group,and the pathological findings of IC/BPS group rats were severe skin injury on the bladder wall and a large number of inflammatory cell infiltration.2.In the IC/BPS group,the genes and proteins of TLR4/NLRP3 signaling pathway related molecules TLR4,NLRP3,Caspase-1,IL-1βwere highly expressed,and the positive cells of related molecules increased significantly.This signal pathway plays an important role in the pathogenesis of IC/BPS. |
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