Objective:

2

To explore the functional roles and underlying mechanisms of neferine in the context of angiotensin Ⅱ (Ang Ⅱ)-induced hypertension and vascular dysfunction.

Methods:

2

Male mice were infused with Ang Ⅱ to induce hypertension and randomly divided into treatment groups receiving neferine or a control vehicle based on baseline blood pressure using a random number table method. The hypertensive mouse model was constructed by infusing Ang Ⅱ via a micro-osmotic pump (500 ng/kg per minute)

and neferine (0.1

1

or 10 mg/kg)

valsartan (10 mg/kg)

or double distilled water was administered intragastrically once daily for 6 weeks. A non-invasive blood pressure system

ultrasound

and hematoxylin and eosin staining were performed to assess blood pressure and vascular changes. RNA sequencing and network pharmacology were employed to identify differentially expressed transcripts (DETs) and pathways. Vascular ring tension assay was used to test vascular function. A7R5 cells were incubated with neferine for 24 h and then treated with Ang Ⅱ to record the real-time Ca

2+

concentration by confocal microscope. Immunohistochemistry (IHC) and Western blot were used to evaluate vasorelaxation

calcium

and the extracellular signal-regulated kinase (ERK)1/2 pathway.

Results:

2

Neferine treatment effectively mitigated the elevation in blood pressure

pulse wave velocity

aortic thickening in the abdominal aorta of Ang Ⅱ-infused mice (

P

<

0.05). RNA sequencing and network pharmacology analysis identified 355 DETs that were significantly reversed by neferine treatment

along with 25 potential target genes

which were further enriched in multiple pathways and biological processes

such as ERK1 and ERK2 cascade regulation

calcium pathway

and vascular smooth muscle contraction. Further investigation revealed that neferine treatment enhanced vasorelaxation and reduced Ca

2+

-dependent contraction of abdominal aortic rings

independent of endothelium function (

P

<

0.05). The underlying mechanisms were mediated

at least in part

via suppression of receptor-operated channels

store-operated channels

or voltage-operated calcium channels. Neferine pre-treatment demonstrated a reduction in intracellular Ca

2+

release in Ang Ⅱ stimulated A7R5 cells. IHC staining and Western blot confirmed that neferine treatment effectively attenuated the upregulation of p-ERK1/2 both

in vivo

and

in vitro

which was similar with treatment of ERK1/2 inhibitor PD98059 (

P

<

0.05).

Conclusions:

2

Neferine remarkably alleviates Ang Ⅱ-induced elevation of blood pressure

vascular dysfunction

and pathological changes in the abdominal aorta. This beneficial effect is mediated by the modulation of multiple pathways

including calcium and ERK1/2 pathways.