1.Study of the effect of inhibition of c-Kit and Cx43 on bladder contractile function and its mechanism of action
Minghui Tan ; Ouyang Song ; Biao Qian ; Yu Hao ; Liuming Xuan ; Peng Sun ; Yongqiang Zhang ; Qinzhang Wang
Acta Universitatis Medicinalis Anhui 2022;57(12):1941-1947
Objective :
To investigation of the interaction between interstitial cells of Cajal (ICCs) and connexin 43 ( Cx43) in bladder contraction and its significance.
Methods :
Eighty male guinea pigs were randomly divided into blank control group,Glivec group,Gap27 group and Glivec + Gap27 group.Four groups of guinea pigs were per- fused with saline,Glivec,Gap 27,and Glivec + Gap 27 every morning for 2 months.Success of urodynamic testing model after 2 months.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 were observed by electron microscopy.
Results :
Urodynamics revealed increased blad- der compliance in the experimental group compared to the blank control group (P<0. 05) ; bladder compliance increased in the Glivec + Gap27 group compared to the Glivec and Gap27 groups (P<0. 01) .In vitro muscle strip experiments revealed that the frequency and tone of bladder muscle strip contractions were lower in the experimental group compared to the blank control group (P<0. 05) ,and that muscle strip contractions were weaker in the ex- perimental group after administration of acetylcholine (ACH) compared to the control group(P<0. 05) .Immuno- fluorescence showed that c-Kit was co-expressed with Cx43 on ICCs cells.qRT-PCR and Western blot suggested that the protein expression level and gene expression level of Cx43 in bladder tissues were lower after inhibition of c-Kit than in the blank control (P<0. 05) ; after inhibition of Cx43,the protein expression level and gene expres- sion level of c-Kit in bladder tissues the levels of c-Kit protein expression and gene expression in bladder tissues were lower than those in the blank control group (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 is expressed on bladder ICCs 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.
2.Effect of combined transurethral instillation of SCL and Cx43 gene lentivirus on diabetic bladder function
Liuming Xuan ; Luping Ma ; Song Ouyang ; Peng Sun ; Minghui Tan ; Yongqiang Zhang ; Qinzhang Wang
Acta Universitatis Medicinalis Anhui 2022;57(10):1546-1551
Objective :
To investigate the effect of the combination of stem cell leukemia(SCL)recombinant lentivirus and connexin 43 ( Cx43 ) recombinant lentivirus on the function of diabetic cystipathy ( DCP) in guinea pigs.
Methods :
After 90 healthy guinea pigs were fed normally for 1 week, streptozotocin(STZ)was injected intraperito⁃
neally at 200 mg/kg in a single dose, and random blood glucose was monitored weekly. After 12 weeks of normal feeding, 40 guinea pigs meeting the criteria were screened by urodynamic examination and randomly divided into 4 groups: diabetic group, SCL group, Cx43 group, and SCL + Cx43 group. In the diabetic group, 0. 2 ml of empty lentivirus without gene was instilled into the bladder through the urethra, in the SCL and Cx43 groups, 0. 2 ml of SCL lentivirus and Cx43 lentivirus were instilled using the same method, in the SCL + Cx43 lentivirus group, 0. 2 ml of each SCL and Cx43 lentivirus was instilled through the urethra. After 14 days of transfection, urodynamic examination was performed, and then the guinea pigs were executed after the examination, and the bladders were quickly removed for frozen bladder sections and fluorescent double staining.
Results :
There were no differences in urodynamic examinations between the SCL and Cx43 groups compared to the diabetic group( P > 0. 05 ) . Urodynamic examination in the SCL + Cx43 group showed improvement in detrusor pressure, abdominal pressure and bladder pressure compared to the diabetic group(P < 0. 05) . In laser confocal experiments, the number of interstitial cells of Cajal (ICC) was reduced in the diabetic group, and the spindle structure and cell protrusions were obviously destroyed and appeared in a state of cell lysis. In the SCL and Cx43 groups, there was an improvement in the spindle structure and cell protrusion of ICC⁃like cells, and there was no significant change in the number of cells. In the SCL + Cx43 group, there was an increase in the number of ICC⁃like cells, a significant improvement in the spindle structure and cell protrusion, and the formation of ICC⁃dimers.
Conclusion
Transurethral co⁃infusion of SCL and Cx43 gene recombinant lentivirus can be successfully transfected in guinea pig DCP bladder, which can restore the number and structure of damaged ICC cells and form ICC dimer structure, improve the pressure of diabetic bladder forced urinary muscle, improve the weakness of urination, ventral pressure voiding and other characteristics. It provides a new direction for the treatment of DCP.