Empagliozin Protects Atherosclerosis Progression by Modulating Lipid Proles and Sympathetic Activity

Background: Several large clinical trials have conrmed the cardioprotective role of Sodium-glucose cotransporter 2 (SGLT2) inhibitors in patients with type 2 diabetes. Here we investigated that empagliozin, as an SGLT2 inhibitor, could alleviate atherosclerosis progression. Methods: ApoE-/- mice were fed on a western diet for 12 weeks to induce atherosclerosis. On the 7 th week, a group of mice were treated with drinking water containing empagliozin (10mg/kg/day) while another group was still fed on normal water. On 12 th week, the whole aortas of each group were harvested. The Oil red O, HE and movat staining were performed for atherosclerotic lesion area and size. Mouse serum lipid proles (TC, TG, LDL-C, and HDL-C), systemic inammation level (IL-1β, IL-6 and IL-10), renin-angiotensin-aldosterone system (RAAS) and sympathetic activity (norepinephrine, and neuropeptide Y) were measured by ELISA. Results: Empagliozin reduced the atherosclerotic lesion burden in ApoE-/- mice. Besides, empagliozin decreased the body weight, lipid proles, RAAS and sympathetic activity. However, the anti-inammation effect of empagliozin was not signicantly evident. Conclusions: Empagliozin can partly prevent atherosclerosis in ApoE-/- mice, which could be attributed to its inhibition on lipid proles, and sympathetic activity.


Background
Sodium-glucose cotransporter 2 (SGLT2) is mainly distributed in the proximal tubule of the kidney and responsible for reabsorption of 80%-90% glucose load [1]. SGLT2 inhibitors can reduce glucose reabsorption of proximal tubules and increase urine glucose excretion with high selectivity and speci city, thereby reducing blood glucose levels [2]. With the loss of glucose in the urine, the body weight and blood pressure also decrease signi cantly [3].
Recent clinical studies have shown that SGLT2 inhibitors can reduce cardiovascular mortality and heart failure hospitalization rates in patients with type 2 diabetes who are at risk of atherosclerotic cardiovascular disease [4][5][6], which becomes the rst hypoglycemic agent to reduce cardiovascular adverse events independent of glycemic control [7]. These data have prompted great interest in the potential mechanisms that mediate the cardioprotective effects of SGLT2 inhibitors. Some studies revealed SGLT2 inhibitor can inhibit in ammation and improve insulin resistance [8] [9], as well as modulate the gut microbiota of type 2 diabetes mice [10]. However, the potential mechanism is still to be explored for the cardioprotective role of SGLT2 inhibitors.
In addition to glycemic control, the mechanism of cardioprotection of SGLT2 inhibitors in patients with type 2 diabetes remains unclear. Ken Lee Chin et al. systematically reviewed preclinical data on the cardioprotective effects of SGLT2 inhibitors and found that reduction of atherosclerosis was one of the underlying mechanisms [11]. Atherosclerosis is a leading cause of adverse cardiovascular events, including acute coronary syndrome (ACS) and stroke [12]. However, potential role and mechanisms of the SGLT2 inhibitors on atherosclerosis are not fully understood. Therefore, we investigated whether empagli ozin, an SGLT2 inhibitor, can inhibit the development of atherosclerosis and the possible mechanism of its vascular protection.

Atherosclerotic Lesion Analysis
After 12 weeks, the entire aorta was harvested and observed under a stereomicroscope, and xed in 4% paraformaldehyde overnight. Then the aorta was opened longitudinally and stained in Oil Red O solution for 2 hours at room temperature. Images were captured using the high-resolution camera. For plaque area analysis in aortic sinus, the upper portion of heart above the line connecting the left and right auricles and proximal aorta was xed and embedded in para n. 10-µm slides were cut and stained with hematoxylineosin or Movat (Sevicebio, Wuhan). Images were captured using the Olympus microscope. Lesion size was measured with Images J software (NIH, USA).

ELISA
Mouse serum was collected and stored at -20℃. The lipid pro le (TC, TG, LDL, and HDL), in ammatory cytokines (IL-1β, IL-6 and IL-10), RAAS mediators (renin, angiotensin II and aldosterone) and sympathetic mediators (norepinephrine and neuropeptide Y) were measured by ELISA (Jin Yibai Biological Technology Co. Ltd, Nanjing) according to the manufacturer's instructions using standard curve. The optical densities of the samples were detected using a microplate reader (BIOTEK, USA) at a wavelength of 450 nm.

Statistics
Data were shown as mean ± SEM. The 2-taild Student t test was applied for comparison between 2 groups and 1-way analysis of variance with the Bonferroni post hoc test was used for multiple comparisons. P < 0.05 was considered statistically signi cant.

1.Sglt2i attenuates atherosclerotic lesion area
To assess the therapeutic role of SGLT2i in atherosclerosis in mice, apoE-/-mice were fed a Western diet for 12 weeks (AS group), while EMPA group received empagli ozin at a dose of 10 mg/kg/day from 7th to 12th week. Macroscopically, the atherosclerotic lesion size decreased in EMPA group compared with AS group (Fig. 1A). Enface Oil Red O staining also con rmed the presence of reduced atherosclerotic lesion area in EMPA group (Fig. 1B). By HE and Movat staining analysis ( Fig. 1C and 1D), empagli ozin signi cantly reduced lesion size in aortic sinus by ~ 10%.

Sglt2i decreases lipid level in atherosclerosis
Excess lipid deposit contributed to the initiation of atherosclerosis. So we evaluated the effect of empagli ozin on lipid pro les. ELISA results showed that empagli ozin could decrease the level of triglyceride ( Fig. 2A), total cholesterol (Fig. 2B), and LDL (Fig. 2C). While HDL was not statistically different between groups (Fig. 2D).

Sglt2i minimally alleviates systemic in ammation in atherosclerosis
Chronic in ammation is also an important trigger of atherosclerosis. Therefore, we evaluated the systemic in ammation level between groups. Our results found that IL-1β (Fig. 3A) and IL-6 ( Fig. 3B) were not signi cantly decreased in the EMPA group but IL-10 (Fig. 3C). These results suggested that empagli ozin had a low anti-in ammation role.

Sglt2i inhibits Renin-Angiotensin-Aldosterone System (RAAS) and sympathetic activity
The chronic activation of Renin-Angiotensin-Aldosterone System (RAAS) was a detrimental factor in the cardiovascular diseases. Our results showed that rennin (Fig. 4A), angiotensin II (Fig. 4B) and aldosterone (Fig. 4C) were increased in AS group while they were inhibited in EMPA group other than angiotensin II. It indicated that empagli ozin could alleviate the activation of RAAS. In addition to RAAS, sympathetic activation also speeds up the progression of atherosclerosis. We found that norepinephrine (Fig. 5A) and neuropeptide Y (Fig. 5B) were partially inhibited in the EMPA group. Interestingly, empagli ozin also decreased the body weight (Fig. 5C) of AS mice to a degree.

Discussion
Our results showed that empagli ozin could mitigate the progression of atherosclerotic plaques in ApoE-/-mice. Body weight and lipid pro les of the empagli ozin-treated group were also lower than those of the untreated group. In addition, we also showed that empagli ozin signi cantly reduced expressions of norepinephrine (NE) and neuropeptide Y (NPY), as well as renin, angiotensin II, and aldosterone. These results indicate that empagli ozin alleviates the activation of sympathetic activity and RAAS, which contributes to the development of atherosclerosis. However, the anti-in ammatory effects were not signi cant in our study.
Previous studies have also demonstrated that SGLT2 inhibitors reduce the development of atherosclerotic lesions in diabetic and non-diabetic mice [13][14][15][16]. Our results were consistent with these studies. It was reported that SGLT2 inhibitors could inhibit the activation of NLRP3 in ammasome [17], reduce the secretion of vasoconstrictive eicosanoids and pro-in ammatory chemokines in the vasculature [15,18], playing an anti-in ammation role. A study showed that empagli ozin prevented the development of atherosclerosis and reduced in ammation and fat deposition in non-diabetic ApoE-/-mice [8]. Our study con rmed that inhibition of sympathetic activity and RAAS contributed to the anti-atherogenic effects of empagli ozin.
Excessive lipid deposition promotes the development of atherosclerosis. However, the effect of SGLT2 inhibitors on lipid pro les is not consistent in animal studies. Several previous studies have shown that SGLT2 inhibitors can lower lipid levels [14,19,20], while others have not [21][22][23][24][25]. Our results showed that empagli ozin could reduce the levels of triglyceride, total cholesterol and LDL, while there was no signi cant difference in HDL between the two groups. Given these ndings, further studies are warranted to fully elucidate the effects of SGLT2 inhibitors on lipid metabolism. However, the systemic in ammation level of atherosclerosis in the SGLT2 inhibitor group was not signi cantly different. Two factors can explain this difference: on the one hand, the non-diabetic ApoE-/-mice we used may not have a signi cant vascular in ammatory response induced by hyperglycemia. Nakatsu et al demonstrated that hyperglycemia rapidly induced vascular in ammatory response, which can be normalized by short-term (7 days) treatment with the SGLT2 inhibitor luseogli ozin [22]. Even though we did not detect the glucose levels in this experiment. However, previous study have con rmed no signi cant difference in glucose levels between empagli ozin-treated and untreated mice, and empagli ozin did not increase the risk of hypoglycemia in non-diabetic states [26]. On the other hand, systemic in ammation level is likely affected by the duration of treatment with SGLT2 inhibitors. The duration of our experiment was 12 weeks, and the experimental group was treated with SGLT2 inhibitors since the 7th week. Combining previous studies, we found that SGLT2 inhibitors may inhibit in ammatory mediators with a duration of at least 8 weeks [9,10,15,27]. Therefore, we speculated that short-term of empagli ozin treatment was not enough to play an anti-in ammation effect.
In recent years, some mechanisms underlying the bene cial effect of SGLT2 inhibitor on cardiovascular diseases have been proposed. The decreased toxicity of glucose to endothelial cells may be a potential mechanism in preventing diabetic ApoE-/-mice atherosclerosis [27]. And dapagli ozin could improve the differentiation of epicardial adipose tissue and perivascular adipose tissue [28]. In addition, SGLT2 inhibitor could enhance lipoprotein clearance through heparan sulfate proteoglycans (HSPG) and bile acid pathways [14], which could protect from atherosclerosis progression. In our article, we broaden our understanding of bene cial effect of SGLT2 inhibitor empagli ozin on the progression of atherosclerosis.

Conclusion
In summary, SGLT2 inhibitor empagli ozin may exert a protective role in atherosclerosis by reducing lipid levels and inhibiting sympathetic activity. Due to a relatively short-term treatment, there may be no statistical difference in some experimental results. Future long-term of empagli ozin treatment studies should be performed. Empagli ozin attenuated atherosclerotic lesion area. Representative microscopical image (A), enface Oil Red O staining (B), HE staining (C), Movat staining (D) of Sham, AS and EMPA group. *p<0.05, **p<0.01, and ***p<0.001.