| Objective: To explore the effects of estrogen on bladder function,urothelium function,and urine barrier function in the interstitial cystitis(IC/BPS)model,and explore the possible molecular mechanism of estrogen in IC/BPS on the hypoxic state of the bladder wall.Methods: Forty-two adult female SD rats were taken,and rat models with different levels of estrogen were established by bilateral ovariectomy and subcutaneous injection of estradiol benzoate,and intravesical infusion of LL-37 was performed to establish interstitial cystitis under different estrogen levels.Model.The grouping was as follows: All rats were randomly divided into three groups: the control group(Control),the bilateral ovariectomy group(OVX),and the estradiol benzoate group(EB),with 14 rats in each group.After one week of adaptive feeding,the rats in the OVX group underwent bilateral ovariectomy,and were fed normally for six weeks after operation.The EB group was injected subcutaneously every other day at 0.25mg/kg·day for six weeks.Six weeks later,14 rats in each group were randomly divided into 2 groups with7 rats in each group,and there were 6 groups in total.The grouping conditions were as follows: Control,Control+LL-37,OVX,OVX+LL-37,EB,EB+LL-37.Rats in all groups were anesthetized with pentobarbital sodium(1.5ml/kg),and the maximum bladder capacity(maximum cystometric capacity,MCC)was measured,and an epidural anesthesia catheter was inserted into the urethra.Three groups of LL-37 and EB+LL-37 were infused with antimicrobial peptide LL-37(320μM)into the bladder once a day for 45 minutes each time for three consecutive days.24 hours after the end of perfusion on the third day,the bladder capacity,pain response,and urination point counts of the rats in the LL-37 group were measured,heart blood collection and bladder samples were taken,and the serum estrogen level of the rats was determined using an ELISA kit.Carry out photographic measurement and pathological examination: HE staining,Masson staining,Alcian blue(p H=1.0)staining,mast cell staining,and immunohistochemical staining for hypoxia probe detection.Rho A,phosphorylated Rho A(Phospho-Rho A),ROCKⅠ,ROCKⅡ,HIF-1α,and VEGFR-1 were detected by Western Blot(WB).Results: Compared with Control group and EB group,estrogen deprivation(OVX group)significantly increased the MCC(P<0.001)and the length from the bladder fundus to the bladder neck(P<0.05)of female SD rats,and the urination frequency of the OVX+LL-37 group was significantly increased(P<0.05).After perfusion with LL-37,the MCC of rats in each group decreased significantly(P<0.05).Compared with Control+LL-37 group,the pain score of pelvic pain symptom OVX+LL-37 group was the highest(P<0.0001),followed by EB+LL-37 group(P<0.01).Alcian blue staining showed that the urothelial GAG layer was damaged in OVX group and EB group.Compared with the control group,bladder fibrosis and mast cell infiltration were significantly up-regulated in EB group,OVX group,EB+LL-37 group,and OVX+LL-37 group(P<0.05).In estrogen deprivation(OVX group),the expression of VEGFR-1 in rat bladder wall tissue increased,which was greater than that in Control group(P<0.001)and EB group(P<0.01),and LL-37 perfusion significantly up-regulated VEGFR-1,in Control group(P<0.0001),EB group(P<0.0001).After infusion of LL-37,HIF-1α was up-regulated in all groups.Compared with other groups,the OVX+LL-37 group significantly down-regulated the expressions of ROCKⅠ(P<0.01)and ROCKⅡ(P<0.01)in rat bladder tissue.There was no significant difference in the expressions of ROCK Ⅰ and ROCK Ⅱ among other subgroups(P>0.05).Conclusion: There is a synergistic effect between estrogen abnormality and LL-37 intervention,which up-regulates the expression of VEGFR-1 and HIF-1α,and the ROCK signaling pathway may be involved in the regulation of lower urinary tract symptoms induced by estrogen deprivation and LL-37 intervention. |
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