Cell experiment:

Human umbilical vein endothelial cells (HUVECs) in the logarithmic growth phase are seeded into 96-well plates at a density of 1×104 cells per well, then incubated for 24 hours at 37℃, 5% CO2. After pretreatment with indicated concentration of Icariin (0, 10, 20, 40 μM) for 24 hours, the cells are incubated with or without ox-LDL (100 μg/mL) for next 24 hours. After suction of the liquid in the wells, MTT solution is added to yield a final concentration of 0.5 mg/mL, and incubation is continued for 4 h at 37℃, 5% CO2. MTT solution is removed gently and 150 μL of DMSO is added to each well for 15 min incubation. The absorbance of each sample is measrured on a microplate reader at 490 nm as cell viability[3].

Animal experiment:

Mice[2]Adult 8-week old male C57BL/6 mice are acclimated for 1-week in a temperature- and humidity-controlled facility with a standard 12-h light schedule. Mice have free access to SPF-grade rodent chow and purified drinking water. Mice are treated with Icariin (100, 200, and 400 mg/kg) for 5 days. Clofibrate (CLO, 500 mg/kg, po for 5 days) is used as a positive control, for negative controls, mice are given 2% CMC (10 mL/kg). 24 h after the last dose, livers are collected for analysis.Rats[4]Forty adult male SD rats weighing 200-290 g (12-16 weeks old) are randomly assigned to groups (n=10 per group) according to their body weight. The rats receive daily intragastric administration of Icariin at 0 (control), 50, 100, or 200 mg/kg per day for 35 consecutive days. The animals are weighed weekly, and the treatments are adjusted accordingly. At the end of the Icariin treatment period, all rats are sacrificed; blood samples are subsequently collected for further analyses of testosterone levels.

• Drug Des Devel Ther. 2022;16:2343-2363

Icariin exhibits inhibitory effects on cGMP-specific phosphodiesterase PDE5 and cAMP-specific phosphodiesterase PDE4 activities. Phosphodiesterase (PDE) is a crucial regulator of cAMP/PKA signaling. PDEs are encoded by 21 genes which can be sdivided into 11 families according to the substrate specificities and subcellular localization. PDEs are widely expressed in neurons. PDE5 has been implicated in regulating some physiological processes such as smooth muscle relaxation and neuronal survival. PDE4 has been associated with the DARPP-32 signaling pathway and dopaminergic neurotransmission [1].

In vitro: Icariin inhibited the activity of PDE5 and PDE4 in a dose- andconcentration-dependent manner. The IC50of icariin on PDE5 was 0.43 μM and the IC50 on PDE4 was 73.50 μM. Icariin showed a selective inhibitory effect on cGMP-specific PDE5 compared to cAMP-specific PDE4 [2].Icariincould also enhance the osteogenic differentiation of rat primary bone marrow stromal cells [3].

In vivo: In castrated rats, a 4-week oral administration of icariinat 1 mg/kg/day and 5 mg/kg/day improved the erectile function and increased nNOS and iNOS expression [4].Icariin also showed its effect on stimulating angiogenesis in human endothelial cells [5].

References:
Nishi A, Kuroiwa M, Miller D B, et al. Distinct roles of PDE4 and PDE10A in the regulation of cAMP/PKA signaling in the striatum[J]. The Journal of Neuroscience, 2008, 28(42): 10460-10471.
Xin Z C, Kim E K, Lin C S, et al. Effects of icariin on cGMP-specific PDE5 and cAMP-specific PDE4 activities[J]. Asian journal of andrology, 2003, 5(1): 15-18.
Chen K M, Ge B F, Ma H P, et al. Icariin, a flavonoid from the herb Epimedium enhances the osteogenic differentiation of rat primary bone marrow stromal cells[J]. Die Pharmazie-An International Journal of Pharmaceutical Sciences, 2005, 60(12): 939-942.
Liu W J, Xin Z C, Xin H, et al. Effects of icariin on erectile function and expression of nitric oxide synthase isoforms in castrated rats[J]. Asian journal of andrology, 2005, 7(4): 381-388.
Chung B H, Kim J D, Kim C K, et al. Icariin stimulates angiogenesis by activating the MEK/ERK-and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells[J]. Biochemical and biophysical research communications, 2008, 376(2): 404-408.