Experimental study on the protective effects of estrogen on islet transplantation mediated by the eNOS signaling pathway

Shangguan Zhaoshui; Wang Jihua; Dai Dandan; Liu Suhuan

doi:10.3969/j.issn.1672-8521.2025.04.005

Abstract：Objective:To investigate whether endothelial nitric oxide synthase(eNOS)is involved in the protective effects of estrogen(E2)on islet transplantation.Methods:(1)Optimization of application time and dose of E2:Male C57BL/6 mice were randomly divided into 4 groups:normal control group(n=12),diabetes model group(n=12),diabetes model+islet transplantation group(transplantation group,n=12),and diabetes model+islet transplantation+E2 treatment group(transplantation+E2 group,n=48).Mice in the transplantation+E2 group were given continuous E2 treatment(4.0 μg/d,subcutaneous injection,6 mice in each subgroup)for 1,3,7,and 14 days respectively to determine the optimal experimental time.In addition,mice in the transplantation+E2 group were given E2 treatment at doses of 0.2,2.0,4.0 and 20.0 μg/d respectively(for 14 consecutive days,subcutaneous injection,6 mice in each subgroup)to determine the optimal experimental dose.(2)Intervention experiment of endothelial nitric oxide synthase(eNOS)inhibitor:male C57BL/6 mice were randomly divided into 3 groups:transplantation group,transplantation+E2 group,and transplantation+E2+eNOS inhibitor Nω-nitro-L-arginine methyl ester hydrochloride group(transplantation+E2+L-NAME group),with 4 mice in each group.In the transplantation+E2 group,E2 sustained-release tablets(0.18 mg/tablet)were implanted subcutaneously in the neck,while in the transplantation+E2+L-NAME group,L-NAME inhibitor 50 mg/kg was injected intraperitoneally every day while E2 sustained-release tablets were implanted.Samples were collected after 14 days.During the experiment,the blood glucose level of mice was monitored,the glucose tolerance was detected,and the plasma insulin content was determined by enzyme-linked immunosorbent assay.The positive areas of eNOS,islet β cells and platelet endothelial cell adhesion molecule(CD31)in the grafts were analyzed by immunofluorescence staining.Results:E2 application time and dose optimization experiments showed that compared with the area under the curve of blood glucose level in the transplantation group[(553.50±37.38)dmmol/l],the area under the curve of blood glucose level in mice[(383.40±47.78)dmmol/l]was significantly reduced after 14 days of E2 treatment(t=2.848,P＜0.05),and the dose of 4.0 μg/d was the best;Using 4.0 μg/d dose of E2 for 14 days,compared with the transplantation group,the blood glucose level in the transplantation+E2 group was significantly reduced(t=3.112,P＜0.05),and the plasma insulin content and the positive area of islet β cells in the transplantation+E2 group were significantly increased(t=2.209,2.442,P＜0.05),the vascular density and eNOS positive area in the grafts were also significantly increased(t=2.131,2.205,P＞0.05).The results of L-NAME inhibitor intervention showed that compared with the transplantation+E2 group,glucose tolerance increased significantly after L-NAME intervention(t=5.871,P＜0.05),the positive area of transplated pancreatic beta cells was significantly reduced[(54.09±2.16)%in the transplantation+E2 group compared to(38.59±3.75)%in the transplantation+E2+L-NAME group,t=2.674,P＜0.05],the plasma insulin content and vascular density showed a decreasing trend(t=1.950,1.606,P＞0.05).Conclusions:E2 short-term treatment can effectively protect the survival and function of transplanted islets and promote vascular remodeling,and its mechanism may be related to eNOS signaling pathway.