Fig 1.
Icariin promotes the in vivo survival of ADSCs and the ADSCs induced recovery of erectile function of DMED rats.
A) The in vivo survival of ADSCs before and after administrating icariin was assessed by Dil staining. B) and C) ICP and MAP measurements were employed in the assessment of erectile function of DMED rats. The rats were injected with saline or ADSCs followed by feeding up with saline or icariin for 4 weeks. The ICP value and the ratio of total ICP to MAP were calculated, n = 7. * p<0.01 compared with the control saline treated group, # p<0.01 compared with icariin treated group. $ p<0.01 compared with the ADSCs treated group. Scale bar: 50 μm.
Fig 2.
The treatments of icariin and ADSCs enhance the expression of vascular markers in DMED rats.
The rats were injected with saline or ADSCs followed by feeding up with saline or icariin for 4 weeks. Top panel: representative images of vWF and α-SMA-positive ADSCs (red) in corpus cavernosum of DMED rats receiving saline, ADSCs and icariin treatment. Bottom panel: Ratio of vWF and α-SMA identified in corpus cavernosum expressed as relative expression to the control group. Immunofluorescent staining analysis of cavernous tissue used vWF and α-SMA antibodies in sections of diabetic rats corpus cavernosum. DAPI staining and vessel: dash line; n = 7, scale bar: 50um, * p<0.01 compared with the control saline treated group, # p<0.01 compared with icariin treated group. $ p<0.01 compared with the ADSCs treated group.
Fig 3.
Results of histological analysis of collagen deposition.
Masson’s trichrome staining to assess the collagen deposition which indicated tissue scarring. Smooth muscles were stained in red while collagen fibers were in blue, n = 7. Scale bar: 200 μm.
Fig 4.
Characteristics of ADSCs and effects of H2O2 and icariin on the viability of ADSCs and H2O2 treated ADSCs, respectively.
A) Flowcytometry demonstrated that most ADSCs expressed CD90, CD29 but less CD45, CD34. B) Morphology of ADSCs and multipotency of ADSCs, differentiating into adipocytes stained by Oil red O and osteocytes stained by Alizarin red S (Scale bar: 100 μm). C) Cells were treated with 0, 25, 50, 100, and 200 μM H2O2 for 12 h followed by PI staining with flow cytometry to determine cell viability, n = 7. D) Cells were treated with 0, 50, 100, 150 and 200 μM icariin under 100 μM H2O2 containing medium for 12 h incubation. Cell viability was determined using PI staining, n = 7.
Fig 5.
Icariin prevents ADSCs from oxidative stress-induced cell damage and apoptosis.
(A) Cells were pretreated with 100 μM H2O2 and icariin for 12 hours, and then targeted with superoxide indicator DHE. The level of ROS was quantified by the amount of cellular ethidium formation. The produced fluorescence intensity was analyzed through a fluorescent microplate reader, n = 7. (B, C) After administration without or with H2O2 and/or icariin for 12 h, the activity of SOD and the leakage of LDH were evaluated according manufacturer’s protocols, n = 7. (D) Cells were treated under the same conditions as in (B, C) and apoptosis was evaluated by Annexin V staining, n = 7. (E-I) Cells were administrated as described in (B, C) and relative changes of p-STAT3, p-Akt, Csp3, Bcl-2, and Bax levels were measured by Western blotting. GAPDH was utilized as the loading reference. All data are expressed as mean ± SD, *p < 0.05, **p <0.01. Scale bar in A, D equals 50 μm.