| Objective: This study establishes a mouse interstitial cystitis(IC)model by intraperitoneal injection of cyclophosphamide,and investigates the expression of Toll-like receptor 4/p38 mitogen activated protein kinase(TLR4/p38 MAPK)signaling pathway in mouse IC model.To provide experimental evidence for studying whether the TLR4/p38 MAPK signaling pathway can be used as a therapeutic target for IC.Methods: Sixty female C57BL/6J mice were randomly divided into the IC group(n=30)and the control group(n=30).Mice in the IC group were modeled by intraperitoneal injection of cyclophosphamide(100mg/kg)on days 1,3,5,and 7,while mice in the control group were injected intraperitoneally with the same amount of saline on days 1,3,5,and 7.On the 8th day,the stereotyped behavior of the two groups of mice was observed,and the scores of the stereotyped behavior were counted.On the 9th day,the number of urination times of the two groups of mice was counted and the difference in the number of urination times between the two groups of mice was analyzed.After the statistics were completed,the mice were sacrificed,and the bladder tissue was taken for the following experiments: 1.Observing the pathological characteristics of the IC mouse model by hematoxylin-eosin staining(HE staining).2.Toluidine blue staining was used to compare the degree of mast cell infiltration in the bladder tissue of the two groups of mice.3.Detection of the contents of IL-1β,IL-6,TNF-α in the supernatant of bladder tissue homogenate of two groups of mice by enzyme-linked immunosorbent assay(ELISA).4.Detection the related proteins expression levels of TLR4/p38 MAPK pathway(TLR4,p-p38 MAPK,p38 MAPK,IL-1β,IL-6,TNF-α)by Western blot.5.Detection the m RNA expression levels of TLR4,p38 MAPK,IL-1β,IL-6,TNF-α by quantitative real-time polymerase chain reaction(q RTPCR).Graph Pad Prism 6 software was used to draw statistical results,and IBM SPSS 25.0 statistical analysis software was used for data processing.Results: 1.The stereotyped behavior scores of mice in the IC group were significantly higher than those in the control group,and the differences were statistically significant(p<0.05).2.The micturition frequency of the mice in the IC group was significantly higher than that in the control group,and the difference in urination frequency was statistically significant(p<0.05).3.HE staining showed that the bladder epithelial tissue of the IC group was obviously damaged.The local mucosa was exfoliated,some of the epithelial cells were swollen and vacuolated.And a large number of red blood cells and inflammatory cells were seen in the submucosa.4.Toluidine blue staining showed that the mast cell count in the bladder tissue of the IC group was significantly higher than that of the control group(p<0.05).5.ELISA test results showed that the contents of IL-1β,IL-6 and TNF-α in the supernatant of the bladder tissue homogenate of the mice in the IC group were significantly higher than those in the control group,and the differences were statistically significant(p<0.05).6.Western blot results showed that TLR4/p38 MAPK pathway related proteins TLR4,p-p38 MAPK,p38 MAPK,IL-1β,IL-6,and TNF-α were significantly higher in the bladder tissues of the IC group than the control group(p<0.05).7.The results of q RT-PCR showed that compared with the control group,the expressions of m RNA of TLR4,p38 MAPK,IL-1β,IL-6 and TNF-α in the bladder tissue of the IC group were significantly increased(p<0.05).Conclusions: 1.Intraperitoneal injection of cyclophosphamide can successfully build the mouse IC model.The stereotyped behavior score and urination frequency of the mice in the IC group were significantly higher than those in the control group,and a large number of red blood cells and inflammatory cells were seen in the bladder tissue of IC group.2.In the IC group,the genes and proteins of TLR4/p38 MAPK signaling pathway(TLR4,p38 MAPK,IL-1β,IL-6 and TNF-α)are highly expressed,indicating that this pathway plays an important role in the pathogenesis of IC. |
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