Objective:

2

To determine whether berberine (BBR) attenuates angiotensin Ⅱ (AngⅡ)-induced endothelial dysfunction by antagonizing AT1R and modulating the phosphodiesterase 4b (PDE4b)-related signaling pathway.

Methods:

2

Primary aortic endothelial cells were isolated and cultured from control

and AngⅡ-induced hypertensive mice. Human umbilical vein endothelial cells (HUVECs)

mouse aortic endothelial cells (MAECs) and

ex vivo

mouse aortae were treated with AngⅡ (1 μmol/L)

in the presence or absence of BBR

or the PDE4b inhibitor piclamilast. Quantitative real-time PCR (qPCR)

Western blot

immunofluorescence

and microplate assays were employed to assess mRNA and protein expressions of PDE4b

AngⅡ type 1 receptor (AT1R)

endothelial nitric oxide synthase (eNOS) and protein kinase B (PKB

as known as Akt) phosphorylation

and intra- and extracellular nitric oxide (NO) levels. Vascular ring assays were conducted to evaluate the endothelium-dependent relaxation (EDR) of isolated mouse aortae. In addition

molecular docking and cellular thermal shift assays were performed to investigate the interaction between BBR and the AT1R

with comparison to valsartan.

Results:

2

AngⅡ reduced eNOS phosphorylation and NO levels in HUVECs and impaired EDR in isolated mouse aortae (

P

<

0.01)

which were reversed by BBR co-treatment (

P

<

0.05). BBR showed a binding free energy of –8.6 kCal/mol with AT1R and significantly enhanced AT1R thermal stability in 60

65

75 ℃

exhibiting similar effects as valsartan in 60

65

75 ℃ (all

P

<

0.05). AngⅡ upregulated PDE4b mRNA and protein levels in HUVECs and MAECs

while BBR reversed this effect in HUVECs (

P

<

0.05 or

P

<

0.01). AngⅡ also suppressed Akt phosphorylation in HUVECs

which was restored by BBR

like the effect of piclamilast (

P

<

0.05 or

P

<

0.01).

Conclusions:

2

BBR ameliorates AngⅡ-induced endothelial dysfunction primarily by antagonizing AT1R

suppressing PDE4b expression and restoring Akt/eNOS signaling. These findings provide mechanistic insights into BBR's endothelial-protective actions and highlight its therapeutic potential in treating hypertension-related vascular complications.