AS is closely related to the incidence of coronary heart disease, hypertension and other cardiovascular and cerebrovascular diseases [11]. The process of AS begins with the dysfunction of vascular endothelial cells, which are inseparable from the activation or inhibition of autophagy [12]. There is increasing evidence that 3-MA is a widely used autophagy inhibitor [13]. However, there are few literature reports on the mechanism of 3-MA intervention in the promotion of AS autophagy inhibition.
We first observed the effect of ox-LDL on the specific effects of VSMC, which, compared to the control group, reduced systolic markers such as α-sma and SM22 and induced the synthesis of SMC markers such as OPN, thereby promoting the synthetic phenotype of vsmc and thereby inducing AS. It was reported that ox-LDL up-regulated the expression of ULK1 and Beclin1 in endothelial cells, reaching two peaks at 0.5 h and 6 h, and decreasing at 48 h [14]. Similarly, the expression of autophagy associated proteins Beclin-1 and ULK1B-II in our study was inhibited by ox-LDL, while p62 was promoted, suggesting that ox-LDL impeded autophagy in VSMC. The results of oil red O staining and transwell method showed that ox-LDL induced VSMCs migration and foaming in AS.In addition, we found that VSMCs in AS changed from a stable contractile form to an active synthesis, a process that can be simulated by the autophagy inhibitor 3-MA. In conclusion, 3-MA enhanced the autophagy inhibition of AS VSMCs and proved the correlation between 3-MA and AS.
It was also found that 3-MA inhibition of autophagy significantly reduced LPS-induced hyperpermeability of lung endothelial cells, suggesting that inhibition of autophagy could protect endothelial barrier dysfunction. 3-MA inhibits endothelial cell repair and aggravates the development of AS. Furthermore, in an in vitro experimental HUVECs model, the cholinine-derived metabolite trimethylamine n-oxide (3-MA) induced oxidative stress and activated the ROS-TXNIP-NLRP3 inflammasome signaling pathway, leading to EC inflammation and endothelial dysfunction, thereby increasing the risk of AS [15]. However, these reports did not clarify the relationship between Wnt/β-catenin and AMPK/mTOR pathway, which is supplemented by this study.
The pathological mechanism of Wnt/β-catenin and AMPK/mTOR pathways in various tumors and cardiovascular diseases has been reported in many literatures [16]. However, the literature on the pathological mechanism of AS is rarely reported. It has been reported that the tumor exosome CEMIP protein can also regulate the proliferation and migration of atherosclerotic vascular smooth muscle cells through the wnt-β-catenin signaling pathway [17]. Autophagy also plays a vital part in cardiovascular disease through several key signaling pathways, including the wnt/β-catenin/mTOR, AMPK/mTOR, IGF/EGF, p53, MAPKs, Wnt/ β-catenin, Nrf2/p62, and NF-kappaB pathways [18]. Hence, we investigated the association between proteins and genes in order to investigate the role of 3-MA in controlling autophagy in AS via the AMPK/mTOR and Wnt/β-catenin pathways. It was found that the expression of 3-MA-induced proteins (p62) was increased, while the expression of Wnt/β-catenin, p-AMPK/AMPK, Beclin-1 and ULK1, p-mTOR/mTOR was decreased. After addition of 3-MA, the expressions of p62 and other genes were enhanced, while the expressions of wnt, β-catenin, mTOR, Beclin-1 and ULK1 were down-regulated. Following treatment with the Wnt inhibitor IWP-4, there was a reversal in the expression of both the protein and gene associated with it. Therefore, our study suggest that 3-MA activates Wnt/β-catenin and AMPK/mTOR pathways to inhibit VSMCs autophagy.
Despite confirming that 3-MA regulated autophagy inhibition of VSMCs induced in AS through the Wnt/β-catenin and AMPK/mTOR pathway to promote AS, no gene regulation studies specifically involve it. The complex process of in vivo autophagy research has yet to undergo relevant in vivo experiments. Hence, removing autophagy-related genes in mice is necessary to further validate its inhibitory effect target and offer more dependable data support for AS treatment. In summary, our current data suggests that 3-MA regulates autophagy inhibition of VSMCs in AS by activating the Wnt/β-catenin and AMPK/mTOR pathway to promote AS.