Study Of The Effect Of Inhibition Of C-Kit And Cx43 On Bladder Contractile Function And Mechanism Of Action

Objective:A preliminary investigation of the interaction between Interstitial Cells of Cajal（ICC）and Connexin 43 in bladder contraction and its significance.Methods:Eighty male guinea pigs were randomly divided into blank control group,Glivec group,Gap27group and Glivec+Gap27 group.Four groups of guinea pigs were perfused with saline,Glivec（4μmol/L）,Gap27（26μmol/L）,and Glivec+Gap27 every morning for 2 months at 800μL each.2 months after perfusion,after successful modeling.Urodynamic examinations were performed.Bladder tissue was collected for an in vitro muscle strip test to detect muscle contraction in each group.Correlation between c-Kit and Cx43 was detected by immunofluorescence.The interaction between c-Kit and Cx43 in the bladder was further validated by qRT-PCR and Western blot.Ultrastructural changes in the muscle layer of the bladder observed by electron microscopy.Results:Urodynamic findings were bladder volume（1.70±0.06 m L）,bladder compliance（0.12±0.04 m L/cm H₂O）,residual urine（0.07±0.06 m L）,and bladder pressure（15.24±1.18 cm H₂O）in the blank control group.（0.05m L/cm H₂O）,residual urine（0.14±0.07 m L）,and bladder pressure（10.17±0.62 cm H₂O）.bladder volume（1.85±0.13m L）,bladder compliance（0.17±0.07 m L/cm H₂O）,residual urine（0.12±0.07m L）,and bladder pressure in the Gap27 group（9.89±0.69 cm H₂O）.bladder volume（2.49±0.06 m L）,bladder compliance（0.49±0.08 m L/cm H₂O）,residual urine（0.65±0.14 m L）,bladder pressure（7.21±0.41cm H₂O）in the Glivec+Gap27 group.Bladder volume,bladder compliance and residual urine were significantly increased in the Glivec group,Gap27 group and Glivec+Gap27 group compared to the blank control group（P<0.05）;bladder pressure was decreased（P<0.05）.In vitro muscle strip experiments revealed that under normal conditions bladder contraction frequency（3.07±0.05/min）and contractile tone（2.95±0.20 g）in the blank control group;bladder contraction frequency（1.6±0.06/min）and contractile tone（1.54±0.15 g）in the Glivec group;bladder contraction frequency（2.13±0.16/min）and contractile tone（1.54±0.15 g）in the Gap27 group and systolic tone（1.08±0.33 g）;bladder contraction frequency（0.93±0.16/min）and systolic tone（0.72±0.25 g）in the Glivec+Gap27 group.Bladder contraction frequency and contractile tone were reduced in the Glivec group,Gap 27 group and Glivec+Gap27 group compared to the blank control group under normal conditions（p<0.05）.Blank control bladder contraction frequency（6.0±0.14/min）and contractile tone（6.79±0.26 g）after administration of acetylcholine（ACH）;Glivec group bladder contraction frequency（2.38±0.87/min）and contractile tone（4.01±0.11 g）;Gap27group bladder contraction frequency（2.96±0.08/min）and contractile tone（2.87±0.31 g）;bladder contraction frequency（1.13±0.14/min）and contractile tone（1.82±0.09 g）in the Glivec+Gap27 group.Contraction frequency and contractile tone were lower in the Glivec group,Gap27 group and Glivec+Gap27 group compared to the normal group after the addition of ACH（P<0.05）.Immunofluorescence suggested that c-Kit was co-expressed with Cx43 on ICC cells.The level of Cx43protein expression and gene expression in guinea pig bladder tissue was lower than that in the blank control group by inhibition of c-Kit（P<0.05）;the level of c-Kit protein expression and gene expression in guinea pig bladder tissue was lower than that in the blank control group by inhibition of Cx43（P<0.05）.Electron microscopy revealed that the mitochondrial structure of bladder smooth muscle was disrupted after simultaneous inhibition of c-Kit and Cx43.Conclusion:Cx43 and c-Kit are expressed on bladder ICC and the two may be jointly involved in regulating bladder contractile function;the joint reduction of Cx43 and c-Kit may have disrupted the mitochondria of bladder smooth muscle,affecting its function and consequently bladder contractile function.