H2S protects against diabetes-accelerated atherosclerosis by preventing the activation of NLRP3 inflammasome

Hydrogen sulfide (H2S) is an important messenger for its strong anti-inflammatory effects, which may be involved in multiple cardiovascular diseases. In our previous study, we revealed that H2S attenuates diabetesaccelerated atherosclerosis through suppressing oxidative stress. Here we report that GYY4137, a H2S donor, reduced the plaque formation of aortic roots and the levels of both intercellular cell adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) in diabetes-accelerated atherosclerotic cells and mouse models. The inflammatory factors of TNF-α, IL-1β, IL-6, and MCP1 were also significantly reduced by GYY4137. Mechanically, GYY4137 suppressed the activation of pyrin domain containing protein 3 (NLRP3) inflammasome in diabetes-accelerated atherosclerosis conditions. Upon knockdown of NLRP3, the increase of ICAM1 and VCAM1 caused by high glucose and oxLDL could be reversed, indicating that H2S protected the endothelium by inhibiting the activity of NLRP3 inflammasome. In conclusion, our study indicates that GYY4137 effectively protects against the development of diabetes-accelerated atherosclerosis by inhibiting inflammasome activation.


Introduction
Diabetes-accelerated atherosclerosis is the most common cardiovascular complication in diabetes mellitus [1] . The incidence of atherosclerosis in diabetic patients is several times higher than that of nondiabetic patients, with the onset time being early, the process being fast and the mortality rate being high [2] . At present, there is a lack of an effective treatment for atherosclerosis caused by diabetes mellitus. Therefore, it is very important to explore the pathogenesis of diabetes-accelerated atherosclerosis and find the biomolecular targets for the treatment of this disease.
Hydrogen sulfide (H 2 S) has been considered as a toxic gas [3] , but in recent years there has been growing evidence of the physiological importance of endogenous H 2 S, which is now recognized as a gaseous signaling molecule in organisms -"gasotransmitter", found after carbon monoxide (CO) and nitric oxide (NO) [4] . Recent evidence indicates that H 2 S is an important messenger that regulates the physiological and pathological functions of the body, because of its strong antioxidant and anti-inflammatory effects [5] , and thus may imply its potential protective effect on a variety of diseases, especially in the cardiovascular system [6][7] . H 2 S can stabilize atherosclerotic plaque, prevent ischemia-reperfusion injury [8] , improve endothelial dysfunction induced by hyperglycemia [9] and protect against myocardial infarction and heart failure [6] . At present, there are few reports on the role of H 2 S in diabetes-accelerated atherosclerosis, and our previous studies have pointed out that H 2 S could promote Nrf2 activation through promoting keap1 Ssulfhydration, thus protecting against diabetesaccelerated atherosclerosis [10] . In previous studies, we have revealed that the protective effects of H 2 S on diabetes-accelerated atherosclerosis were relying on the attenuation of oxidative stress and inflammation.
This study indicates that GYY4137, an H 2 S donor, can effectively inhibit the development of diabetesaccelerated atherosclerosis, which plays a protective role in vitro and in vivo. Moreover, H 2 S can inhibit the activation of pyrin domain containing protein 3 (NLRP3) inflammasome, an important inflammation signaling pathway, thus decreasing IL-1β production and eventually attenuating the pathological progress of diabetes-accelerated atherosclerosis.

Cell culture and treatment
Umbilical cords of newborn babies were derived from the Third Affiliated Hospital of Nanjing Medical University. The study was approved by the Ethics Committee of the Third Affiliated Hospital of Nanjing Medical University and was performed in compliance with the Declaration of Helsinki Principles. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured following the instruction of previous methods [11] . The HUVECs were cultured in endothelial cell medium (ECM, ScienCell, USA). HUVECs were treated with high glucose (25 mmol/L, Sigma, USA) and oxLDL (100 mg/L, Peking Union-Biology Co., Ltd., China), with or without GYY4137 (100 μmol/L), sulfide-consumed GYY4137 (SDG, a solvent for GYY4137, left corked up at atmospheric temperature for 120 hours) for 24 hours.

Small interfering RNA transfection
NLRP3 expression was silenced by using siRNA against NLRP3 which was purchased from Genepharma (China). HUVECs were transfected with siRNA oligonucleotide following the instruction of the Lipofectamine 3000 reagent (Invitrogen, USA).

RNA isolation and quantitative real-time PCR (qRT-PCR)
The whole RNA was isolated from cells or the aortic tissues with Trizol reagent (Takara, Japan). Total mRNAs were reverse-transcribed into cDNA for subsequent analyses by using the Revert Aid First Strand cDNA Synthesis kit (Vazyme, China), according to the instruction. qRT-PCR was carried out in triplicate with the resulting cDNAs using SYBR Green Premix (Takara) and ABI 7500 Real-time PCR System (ABI, Carlsbad, USA). Primers used for qRT-PCR were purchased from Thermo Fisher Scientific. Sequences of primers involved in this paper for qRT-PCR were as follows: Human TNF-α, Forward primer (

Animal treatment
Male Ldlr -/-mice (specific pathogen free, SPF), on a C57BL/6 background, were derived from Model Animal Research Center of Nanjing Medical University. Streptozotocin (STZ) [60 mg/(kg·day)] was intraperitoneally injected into Ldlr -/-mice at the age of 8 weeks for 5 days without interruption. Five days later, mice were performed with GYY4137 (a H 2 S donor) and were fed with a high-fat diet (D1-2108C) for 1 month. GYY4137 [133 μmoL/(kg·day)] or saline was intraperitoneally injected. The animals were kept under standard animal room conditions [temperature (21±1) °C; humidity 55%-60%; 12-hour light : dark cycles]. The mice were anesthetized with 3.5% isoflurane and maintained with 2.0% isoflurane during the surgery. All animal experiments were carried out in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.

Hematoxylin-eosin staining
Hematoxylin-eosin staining was conducted in the light of the instruction (Beyotime Biotechnology). Briefly, after 5 μm sections were deparaffinized and rehydrated, sections were dipped in hematoxylin solution for 10 minutes. Then, the sections were dipped in 1% acid ethanol (1% HCl in 70% ethanol) for 5 times and then washed in ultra-pure water for 20 minutes. The sections were then dipped in eosin solution for 3 minutes, and then dehydrated in graded ethyl alcohol and washed in xylene. Finally, the handled slides were then checked and photographed under a microscope (Olympus, Japan).

Masson's trichrome staining
After deparaffinization and rehydration, using graded ethyl alcohol, sections were washed in ultrapure water for 1 minute and then dipped in Weigert's iron hematoxylin working solution (Beyotime Biotechnology) for 10 minutes. Sections were then washed under warm running water for 10 minutes before being twice washed in ultra-pure water. The sections were next dipped in Biebrich scarlet-acid fuchsin solution for 10-15 minutes. Wash the sections in ultra-pure water for 2 times. The sections were then differentiated in phosphomolybdic-phosphotungstic acid solution for 10 -15 minutes while waiting for the collagen no longer turned red. Sections were then transferred directly to the aniline blue solution and tarnished for 5 -10 minutes before being washed in ultra-pure water, then washed in ultra-pure water and differentiated in 1% acetic acid solution for 2 -5 minutes. Finally, sections were washed with ultra-pure water, dehydrated in a gradient of ethanol and washed in xylene. The handled slides were then checked and photographed under a microscope (Olympus).

Statistical analysis
All data are expressed as mean±standard error of mean (mean±SEM). Gray value of the bands was analyzed by ImageJ software. Distribution of the data was first analyzed using the Kolmogorov-Smirnov test for analysis of normality. Depending on the number of experimental groups and factors to be compared, statistical analysis was performed with Student's t-test or one-way ANOVA followed by post hoc test (Bonferroni). Otherwise, Johnson transformation was applied to transform the data. P<0.05 was considered statistically significant. Statistical analysis was done with GraphPad Prism 5 (GraphPad Software Inc, San Diego, USA).

H 2 S protects against diabetes-accelerated atherosclerosis in diabetic Ldlr -/-mice
To determine the effect of H 2 S on the endothelium of diabetes-accelerated atherosclerosis mouse models, mice were injected intraperitoneally with GYY4137 or saline for 4 weeks. Meanwhile, they were kept on a high fat diet (HFD) to accelerate atherogenesis. The plaque area and collagen deposition in the aortic roots of mice's hearts were performed. As expected, treatment of diabetic Ldlr -/-mice with H 2 S significantly reduced atherosclerotic plaques and collagen deposition compared with the Ldlr -/-mice (Fig. 1A). We also found a significant decrease in the expression of intercellular cell adhesion molecule 1(ICAM1) and vascular cell adhesion molecule 1(VCAM1), both at mRNA levels ( Fig. 1B) and protein levels ( Fig. 1C). Collectively, these results revealed that exogenous H 2 S decreases atherosclerotic lesions and ICAM1 and VCAM1 expression in diabetes-accelerated atherosclerosis mouse models.

H 2 S reduces the expression of ICAM1 and VCAM1, and inflammation factors in vitro
To further investigate the protective effects of H 2 S, we established a HUVECs model treated with high glucose (25 mmol/L) and oxidized low density lipoprotein (oxLDL, 100 mg/L) in vitro, which could replicate some of the features of endothelial cells in the diabetes-accelerated atherosclerotic mouse models. We examined the expression of adhesion molecules ICAM1 and VCAM1 ( Fig. 2A and 2B) as well as inflammatory factors of tumor necrosis factorα (TNF-α), interleukine-1β (IL-1β), interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP1) (Fig. 2C) in the HUVECs of high glucose and oxLDL treatments. The increase of ICAM1 and VCAM1 induced by high glucose and oxLDL were reversed by H 2 S. The same changes were observed in inflammatory factors. These results demonstrated that H 2 S can reduce inflammation in vitro.

H 2 S decreases the activation of NLRP3 inflammasome in vivo and in vitro
The above results suggested that the inflammatory response plays a vital role in the pathological process of diabetes-accelerated atherosclerotic. Therefore, we detected the activation of caspase-1 and IL-1β, and the expression of NLRP3 (Fig. 3A) which could reflect the inflammatory infiltration in the aortic tissues of diabetic Ldlr -/-mice. Moreover, diabetic mice demonstrated an obvious increase in caspase-1 and IL-1β as well as the expression of NLRP3, compared with the non-diabetic control. In addition, the treatment of diabetic Ldlr -/-mice with GYY4137 can also reverse these changes. We next verified these in vitro with the same results (Fig. 3B). Together, these experiments indicated that H 2 S decreases the activation of NLRP3 inflammasome in diabetesaccelerated atherosclerotic cells and mouse models.

Down-regulation of NLRP3 attenuates endothelial injury in vitro
The above results suggested that H 2 S may participate in diabetes-accelerated atherosclerotic by inhibiting inflammasome. To further verify this, we knocked down the expression of NLRP3 with siRNA (Fig. 4A), and then detected the expression of adhesion molecules ICAM1 and VCAM1 (Fig. 4B). Silencing of NLRP3 significantly reduced the expression of ICAM1 and VCAM1 in high glucose and oxLDL-treated HUVECs compared with that of control. In conclusion, H 2 S can protect endothelial injury from high glucose and oxLDL treatment by inhibiting NLRP3 inflammasome.

Discussion
Diabetes-accelerated atherosclerosis is closely related to oxidative stress and inflammatory response [12] . High glucose-induced the increase of superoxide may be a key event leading to atherosclerosis [13][14][15][16] , so suppressing oxidative stress is an important measure to attenuate atherosclerosis caused by diabetes. Our previous research also reported that the anti-oxidative stress of H 2 S could effectively delay the progression of diabetesaccelerated atherosclerosis caused by high glucose and oxLDL [10] . Moreover, GYY4137 is expected to become a drug for the treatment of diabetesaccelerated atherosclerosis caused by oxidative stress with its effective release of H 2 S at a physiological concentration.
In our previous studies, we found that significant lipid plaques formed in the aortic roots of the Ldlr -/mice of high fat diet and STZ-induced diabetesaccelerated atherosclerosis, and H 2 S could effectively inhibit plaque formation. Additionally, vascular oxidation is effectively reduced by activating the Nrf2 signaling pathway [10,[17][18] . We also found that macro- HG+oxLDL Control HG+oxLDL+GYY4137
phages infiltration was evident in high fat and STZinduced diabetes-accelerated atherosclerosis mouse models, suggesting that the inflammation also plays a role in the pathological process. H 2 S treatment could significantly reduce the extent of macrophages infiltration and inhibit the inflammatory response. Based on this finding, we have now studied and demonstrated that H 2 S could not only protect against atherosclerosis in diabetes through inhibiting oxidative stress, but also prevent the progression of the disease through the attenuation of inflammatory response.
GYY4137, a H 2 S donor, could inhibit inflammation-related diseases such as peritonitis and acute ventilation by inhibiting the activation of NLRP3 inflammasome and reducing caspase-1 activation and IL-1β secretion [19][20] . GYY4137 also protects against amyloid beta-peptide induced neuronal injury via attenuating inflammatory response [7] . GYY4137 attenuates myocardial ischemia and reperfusion injury by reducing oxidative stress and apoptosis in rats [21] . In cardiovascular diseases, H 2 S regulates the inflammatory response of macrophages by inhibiting the activation of NLRP3 inflammasome, reducing caspase-1 activation and IL-1β secretion. Additionally, NLRP3 inflammasome is closely related to endothelial function [22] . However, the role of NLRP3 in diabetes-accelerated atherosclerosis has not been reported. The above reports suggest that H 2 S may reduce inflammation by reducing the activation of NLRP3 inflammasome and play a key role in the protection of diabetesaccelerated atherosclerosis.

Fig. 3 H 2 S inhibits the activity of NLRP3 inflammasome in vivo and in vitro.
A: Total protein was extracted from the aortic tissues and analyzed by Western blotting assay to determine the levels of pro-caspase-1, caspase-1, pro-IL-1β, IL-1β and NLRP3 expression ( * P<0.05, ** P<0.01, n=3). B: HUVECs were treated with high glucose and oxLDL, with or without GYY4137 (H 2 S donor) for 2 hours. The control group means without any treatment. Total proteins were extracted from cells and analyzed by SDS-PAGE to determine the level of NLRP3 ( * P<0.05, n=3).
intraperitoneally injected with STZ and kept on a HFD to induce diabetes-accelerated atherosclerosis mouse models. Moreover, endothelial cells were treated with high-glucose and oxLDL to mimic diabetesaccelerated atherosclerosis induced cell injury [23] . In the endothelial injury, we found that the expression of ICAM1 and VCAM1 was significantly increased in vivo and in vitro. At the same time, we tested the mRNA expression levels of various inflammatory factors. The expression of inflammatory factors increased in high-glucose and oxLDL-treated endothelial cells, while significantly declined after H 2 S pretreatment. Thus, H 2 S plays an important role in inhibiting inflammation of diabetes-accelerated atherosclerosis. It is worth noting that we have demonstrated that high glucose and oxLDL can induce the activation of NLRP3 inflammasome, leading to the activation of caspase-1 and the production of the proinflammatory cytokine IL-1β. H 2 S could significantly inhibit these effects. The silencing of NLRP3 significantly reduced caspase-1 activation, IL-1β production, ICAM1 and VCAM1 expression in high glucose and oxLDL-treated endothelial cells.
Taken together, our experiments demonstrated that H 2 S delayed the progression of diabetes-accelerated atherosclerosis in Ldlr -/-mice and improved high glucose and oxLDL-induced endothelial cell injury. H 2 S reduced the activation of NLRP3 inflammasome in endothelial cell injury and diabetes-accelerated atherosclerosis mouse models. H 2 S donors have therapeutic potential for cancer, erectile dysfunction, peptic ulcer disease, Parkinson's and Alzheimer's diseases, acute and chronic inflammatory diseases, atherosclerosis, arterial and pulmonary hypertension and heart failure, among other diseases [24] . Several drugs (such as statins [25] , aspirin [26] , and metformin [27] ) are also found to regulate H 2 S production, but the mechanisms and clinical significance are not fully understood. Our findings provide new evidence for the treatment of cardiovascular diseases by H 2 S donors.