Objective

2

To discuss the possible mechanism of

Wenxin Formula

（温心方） in the treatment of coronary heart disease （CHD） from human umbilical vein endothelial cells （HUVECs） exosomes and phenotypic transformation of human aortic vascular smooth muscle cells （HAVSMCs）.

Methods

2

Thirty SD rats were given 33.75 g/（kg·d） of

Wenxin Formula

decoction by gavage for 7 consecutive days， and the blood was collected from the abdominal aorta 2 hours after the last administration to prepare drug-containing serum. HUVECs at logarithmic growth stage were divided into control group， model group，

Wenxin Formula

high-， medium- and low-dose groups. The control group was cultured with complete medium. The

Wenxin Formula

high-， medium-， and low-dose groups were cultured with 5%， 15%， and 25% of

Wenxin Formula

-containing serum for 24 hours， and then cultured with additional 50 mg/L oxidized low-density lipoprotein （ox-LDL） for 24 hours. The model group was cultured with only 50 mg/L ox-LDL for 24 hours to establish a model. Exosomes of HUVECs were obtained by high-speed centrifugation in each group， and the morphology of exosomes was observed by transmission electron microscopy. Exosome protein concentration was determined by BCA， and exosome particle size was detected. Expressions of exosome marker protein lysosome-associated membrane protein 3 （CD63） and tumor susceptibility gene 101 （TSG101） were detected by Western Blot， and miR-145 expression was detected by qRT-PCR. 50 ng exosomes of HUVECs were co-cultured with HAVSMCs in each group for 24 hours， and exosomes uptake was observed. Proliferation of HAVSMCs cells was detected by MTT assay， while cell migration ability was detected by Transwell assay， and cell apoptosis was detected by flow cytometry at 6 h， 12 h， 24 h and 48 h respectively. The protein levels of α-smooth muscle actin （α-SMA）， smooth muscle 22α protein （SM22α） and osteopontin （OPN） were determined by Western Blot.

Results

2

In this experiment， HUVECs exosomes were extracted and taken up by HAVSMCs cells successfully. Compared to those in the control group， the expressions of CD63， TSG101 and miR-145 in exosomes of HUVECs in the model group significantly decreased （

P

＜0.01）， while those in all

Wenxin Formula

groups increased than the model group， and high-dose group was superior in increasing CD36 and miR-145 than the medium- and low-dose

Wenxin Formula

group （

P

＜0.05 or

P

＜0.01）. After exosomes were co-cultured with HAVSMCs， the proliferation， migration and apoptosis rates of HAVSMCs cells in the model group significantly increased than the control group， while the expressions of α-SMA and SM22α significantly decreased， and OPN increased （

P

＜0.05 or

P

＜0.01）. The cell proliferation and apoptosis rates at 24 and 48 h and the migration number at 12， 24 and 48 h of all

Wenxin Formula

groups were significantly reduced than the model group， while the expressions of α-SMA and SM22α significantly increased， and the expression of OPN significantly decreased （

P

＜0.05 or

P

＜0.01）. Compared to those in the medium- and low-dose

Wenxin Formula

groups， the cell proliferation rate at 24 and 48 h and the migration number at 12， 24 and 48 h in the high-dose group decreased， while the expression of α-SMA and SM22α increased， and the expression of OPN was reduced （

P

＜0.05）.

Conclusion

2

Wenxin Formula

can inhibit phenotypic transformation of HAVSMCs by promoting exosomes release in HUVECs， and reduce HAVSMCs proliferation and migration， thereby playing a role in the treatment of CHD.