*Article* **Metformin Directly Binds to MMP-9 to Improve Plaque Stability**

**Xianda Chen 1,2,3,4,5,†, Shuaixing Wang 1,2,3,4,5,†, Wenli Xu 1,2,3,4,5, Mingming Zhao 1,2,3,4,5, Youyi Zhang 1,2,3,4,5 and Han Xiao 1,2,3,4,5,\***


**Abstract:** Vulnerable atherosclerotic plaque rupture is the principal mechanism that accounts for myocardial infarction and stroke. High matrix metalloproteinase-9 (MMP-9) expression and activity have been proven to lead to plaque instability. Metformin, a first-line treatment for type 2 diabetes, is beneficial to plaque vulnerability. However, the mechanism underlying its anti-atherogenic effect remains unclear. Molecular docking and surface plasmon resonance experiments showed that metformin directly interacts with MMP-9, and incubated MMP-9 overexpressing HEK293A cells with metformin (1 <sup>μ</sup>mol·L−1) significantly attenuates MMP-9's activity using zymography and MMP activity assays. Moreover, metformin treatment drives MMP-9 degradation. Next, we constructed a carotid artery atherosclerotic plaque model and administered consecutive 14-day metformin (200 mg·kg−1·d−1) treatment by intragastric gavage. Immunofluorescence staining of the right carotid common artery and serum MMP activity assay results showed that metformin treatment decreased local plaque MMP-9 protein level and circulating MMP-9 activity, respectively. Histochemical staining revealed that after metformin treatment, the collagen content in plaque was significantly preserved, and the plaque vulnerability index decreased. These findings suggested that metformin improved atherosclerotic plaque stability by directly binding to MMP-9 and driving its degradation.

**Keywords:** surface plasmon resonance; metformin; matrix metalloproteinase-9; plaque instability; atherosclerosis
