Aloe emodin relieves Ang II‐induced endothelial junction dysfunction via promoting ubiquitination mediated NLRP3 inflammasome inactivation

Abstract Recent studies have revealed that aloe emodin (AE), a natural compound from the root and rhizome of Rheum palmatum L., exhibits significant pharmacologic activities. However, the pharmacologic relevance of the compound, particularly for cardiovascular disease, remains largely unknown. Here, we hypothesized that AE could improve endothelial junction dysfunction through inhibiting the activation of NOD‐like receptor family pyrin domain containing‐3 (NLRP3) inflammasome regulated by NLRP3 ubiquitination, and ultimately prevent cardiovascular disease. In vivo, we used confocal microscopy to study the expression of tight junction proteins zonula occludens‐1/2 (ZO‐1/2) and the formation of NLRP3 inflammasome in coronary arteries of hypertension. And the experimental serum was used to detect the activation of NLRP3 inflammasome by ELISA assay. We found that AE could restore the expression of the endothelial connective proteins ZO‐1/2 and decrease the release of high mobility group box1 (HMGB1), and also inhibited the formation and activation of NLRP3 inflammasome. Similarly, in vitro, our findings demonstrated that AE could restore the expression of the tight junction proteins ZO‐1/2 and decrease monolayer cell permeability that related to endothelial function after stimulation by angiotensin II (Ang II) in microvascular endothelial cells (MECs). We also demonstrated that AE could inhibit Ang II‐induced NLRP3 inflammasome formation and activation, which were regulated by NLRP3 ubiquitination in MECs, as shown by fluorescence confocal microscopy and Western blot. Together with these changes, we revealed a new protection mechanism of AE that inhibited NLRP3 inflammasome activation and decreased the release of HMGB1 by promoting NLRP3 ubiquitination. Our findings implicated that AE exhibited immense potential and specific therapeutic value in hypertension‐related cardiovascular disease in the early stage and the development of innovative drugs.

alterations in metabolism of arachidonic acid, resulting in an increase in vasoconstrictors and decrease in vasodilators, and up-regulation of endothelin. 3 Ang II plays an important role in the initiation and progression of endothelial dysfunction, including Ang II-mediated vascular tone dysfunction, 4 Ang II-and endothelium-mediated vascular inflammation, 5 Ang II-related atherosclerosis, 6 and vascular remodeling. 7 Endothelial dysfunction is a well-established fundamental cause of cardiovascular diseases and also a predictor of worse clinical outcomes. Endothelial cell barrier dysfunction results in many pathologic consequences including endothelial cell injury reflecting in tight junction protein zonula occludens-1/2 (ZO-1/2), endothelium inflammation, and impairs endothelial integrity further. 8 These interendothelial junction proteins play critical roles in protecting blood vessels and then maintain life stability. However, there are few effective drugs in clinical prevention and treatment of the endothelial injury caused by hypertension currently. As a consequence, it is imperative to find a drug that can maintain the homeostasis of endothelial tight junction proteins.
As a traditional Chinese medicine, Rheum has been widely and successfully used for many years. Aloe emodin (AE), 1,8-Dihydroxy-3-(hydroxymethyl) anthraquinone, is a major anthraquinone derived from the root and rhizome of Rheum palmatum L. Clinical studies on AE has been demonstrated that it produces beneficial effects including antioxidant, 9 anti-angiogenic, 10 anti-metastasis, 11 antiinflammatory, 12 and anti-cancer. 13 It has been proven that AE could effectively prevent cardiovascular diseases. 14 AE prevented high-fat diet-induced QT prolongation by repressing miR-1 and up-regulating its target Kir2.1. 14 AE protected against myocardial infarction via the up-regulation of miR-133, suppression of caspase-3 apoptotic signaling pathway. 15 But the protective effect of AE against endothelial dysfunction in the pathogenesis of Ang II-induced vascular complications and the underlying mechanism remains largely unknown.
It is reported that AE possessing antioxidant effect and alteration of reactive oxygen species (ROS) production may represent an important mechanism for AE mediated regulation of cellular behaviors. 15 AE has been shown to prevent high glucose-induced -cell toxicity via inhibition of ROS and down-regulation of apoptosis. 16 ROS is one of the pathways that activates NOD-like receptor family pyrin domain containing-3 ( NLRP3) inflammasome. 17 The NLRP3 inflammasome monomer is a multi-subunit protein, which consists of pattern recognition receptor NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), and inactive pro-caspase-1. Pro-caspase-1 is converted to its active form, cle-caspase-1, and subsequently cleaves its substrates such as pro-IL-1 to bioactive IL-1 . These canonical cytokine mediate inflammatory responses in many kinds of cells such as endothelial cells. 18 So it could cause or exacerbate the endothelial inflammation contributing to the development of cardiovascular diseases. NLRP3 inflammasome contributes to the increased release of high mobility group box1 (HMGB1), 19 one of the major damageassociated molecular patterns (DAMPs). The protective mechanisms of other effective drugs have been shown to prevent endothelial dysfunction related to NLRP3 inflammasome, such as Asprin 20 and puerarin. 21 It has also been reported that AE possesses anti-inflammatory effect.
Hence, we hypothesized that the protective effect of AE on cardiovascular disease may be related to NLRP3 inflammasome.
In this study, our finding raised the possibility that AE confers protection against endothelial injury induced by Ang II in endothelium and reveals the important mechanism of NLRP3 inflammasome regulated by AE for the first time. Our study showed that AE protected the expression of interendothelial junction proteins and restricted the release of HMGB1 by inhibiting the activation of NLRP3 inflammasome regulated by NLRP3 ubiquitination, and ultimately prevented cardiovascular disease. Our study implicates the clinical potential of AE for the prevention of hypertension-induced cardiovascular diseases in the early stage.

Blood pressure measurement
Blood pressure was measured using tail-cuff plethysmography (Softron Systems BP-2010 Series II Blood Pressure Analysis System) including systolic blood pressure and diastolic blood pressure. Briefly, mice were trained for a period of five consecutive days in which blood pressure was measured (30 measurements per day) but not recorded. Following this training period, blood pressure was measured for another three consecutive days, recorded, and averaged.

Immunoblotting
MECs were washed twice with ice-cold PBS and scraped in radio

Immunofluorescence microscopy analysis
To detect inflammasome activation in EOMA and the endothelium of mouse coronary arteries, we carried out immunofluorescence. Cells grown on culture dish at a density of 5 ×

ELISA assay for cytokine
After treatment, the cell supernatant and mice serum were collected and HMGB1 and IL-1 production were measured by a commercially available ELISA kit (R&D System, Minneapolis, MN, USA) according to the protocol described by the manufacturer.

Determination of trans endothelial electric resistance (TEER)
TEER was performed by using an assembly containing current-passing and voltage-measuring electrodes (EVOMZ, World Precision Instruments (WPI) is located at Sarasota, FL, USA). Cell treating is the same as steps in measuring cell permeability. MECs were also seeded on top of the transwell chamber in 24 wells (0.4 µm pore size).

Statistical analysis
All results were showed as mean ± SEM of three to eight independent experiments with each experiment including triplicate sets. One-way ANOVA analysis and post hoc tests were applied when there were more than two groups in the independent variable. The level of significance was set at a P-value of 0.05.

Aloe emodin alleviated Ang II-induced inflammasome activation and HMGB1 release in coronary arterial endothelium
Through colocalizing NLRP3 with PECAM by immunofluorescence, we found that AE inhibited the activation of NLRP3 inflammasome ( Fig. 2A, B). The activation of inflammasome reflecting in IL-1 expression of plasma in each group after stimulation and administration were shown in Fig. 2C. Similarly, we found that AE inhibited the activation of inflammation in coronary arterial endothelium. When serum HMGB1 was detected by ELISA, the release of HMGB1 was decreased after AE treatment (Fig. 2D).

Aloe emodin prevented micro-endothelial dysfunction induced by Ang II in MECs
Tight junction proteins and adhesion junction proteins play a critical role in mediating the permeability of solutes between adjacent endothelium cells. Disruption of junction proteins increases endothelial cells permeability during vascular dysfunction. As shown in Fig. 3A and B, MECs stimulated with Ang II markedly decreased the expression of tight junction proteins ZO-1, which was obviously restored by AE. The results of the endothelial permeability and TEER are consistent with the expression of ZO-1 (Fig. 3C, D), which proved that AE may have an effect of protecting the tight junction proteins for endothelium.

Aloe emodin inhibited the formation and activation of NLRP3 inflammasome and HMGB1 expression in MECs with Ang II stimulation
The NLRP3 inflammasome is best characterized as a type of inflammasome in mammalian cells that consists of a proteolytic complex formed by NLRP3, ASC, and pro-caspase-1. To biochemically assess the anti-inflammatory reduction effect of AE, we detected NLRP3 inflammasome protein expression and inflammasome formation. The immunofluorescence colocalization method was used to detect the recruitment of inflammasome ( Fig. 4A-C). Moreover, AE significantly inhibited the recruitment of NLRP3 inflammasome. To confirm the activation of NLRP3 inflammasome, we detected caspase-1 inflammasome protein expression, containing pro-caspase-1, which relates to the recruitment of NLRP3 inflammasome, and cle-caspase-1, which relates to the activation of NLRP3 inflammasomes (Fig. 4D, E). AE clearly decreased cleavage caspase-1 in Ang II-treated MECs. NLRP3 inflammasome is activated to produce a number of inflammasome products including the HMGB1 and IL-1 , which serve as a novel vascular permeability factor to mediate vascular hyper-permeability and promote endothelial cell-mediated vascular remodeling. As shown in Fig. 4F and G, we found that the release of IL-1 and HMGB1 were increased with the stimulation of Ang II and the release level was decreased after AE intervention.

Aloe emodin abolished Ang II-induced disassembly of tight junction protein via NLRP3 pathway in MECs
To prove that AE could protect the endothelial function by inhibiting

DISCUSSION
Here, we provide the evidence for a new therapeutic application of AE to protect against endothelial injury in cardiovascular diseases. To between endothelial cells. 23 Therefore, the prevention and treatment of endothelial dysfunction induced by Ang II should be taken into account. In addition, the results of this study showed that the increase in blood pressure caused by Ang II was normalized by treatment with an AT-1 receptor inhibitor losartan remarkably, and AE exerted the similar protective effect (Fig. 1A, B), demonstrating that AE is an effective treatment to hypertension-related cardiovascular diseases. Our study also explored that AE could repair the integrity of the endothelial cytomembrane under the Ang II stimulations could prevent and cure To further explore the mechanism, we focus on how AE regulated the tight junction protein ZO-1/2. Currently, it has been demonstrated that the NLRP3 inflammasome plays an important role in the endothelial cell barrier dysfunction. 25 And Ang II could cause a nonclassic inflammation response in the vascular endothelium and contribute to the inflammation response through the aggregation of intracellular ROS, which plays a critical role in NLRP3 inflammasome activation. 26 As the data showed, we demonstrated that AE alleviated the Ang II-induced vascular endothelial damage by inhibiting the activation of NLRP3 inflammasome in vivo (Fig. 2). Other studies also indicated that Ang II-induced lung fibroblast -collagen I synthesis via NLRP3 inflammasome activation. 27 25 In the present work, our data confirmed that AE decreased NLRP3 inflammasome activation and HMGB1 release in vivo. We also found that the AE has a significant effect in vitro on restoring the endothelial permeability and junction protein ZO-1/2 (Fig. 3). And we confirmed that AE could effectively inhibit the Ang II-induced NLRP3 inflammasome formation and activation (Fig. 4). The results of in vitro and in vivo experiments showed that AE could restore endothelial tight junction proteins ZO-1/2 and endothelial permeability via inhibiting the formation and activation of NLRP3 inflammasome. It means AE is effective in protecting vascular endothelium.
Straight after the shallow relationship between AE and NLRP3 inflammasome, we intended to further confirm the role of NLRP3 inflammasome on Ang II-induced endothelial dysfunction in AE group.
The reduced capacity of endothelial cells transfected with NLRP3 shRNA to release HMGB1 is consistent with recent findings that NLRP3 inflammasome activation could lead to translocation and secretion of HMGB1 by immune cells. 19 Thus, we guessed that AE may recover endothelial tight junction disruption by inhibiting endothelial NLRP3 inflammasome activation and release of HMGB1. In our experiment, we knocked down the NLRP3 gene and detected the junction protein, IL-1 , and HMGB1 (Fig. 5A). We demonstrated that AE inhibited the NLRP3 signaling pathway to alleviate endothelial dysfunction because ZO-1/2 of gNLRP3 cells has no significant change in the normal group, model group, or administration group (Fig. 5B-E). In cultured endothelial cells, Ang II induced IL-1 activation and HMGB1 production, which were blocked by NLRP3 gene silencing (Fig. 5F-I).
To sum up, AE was verified that it relieved Ang II-induced endothelial junction dysfunction, which is related to NLRP3 inflammasome signaling pathway.
At the same time, we found an interesting phenomenon that AE treatment dramatically down-regulated the expression of NLRP3 in endothelium induced by Ang II (Fig. 6A, B) forkhead-associated (FHA) and really interesting new gene (RING)like domains, facilitates NLRP3 ubiquitination. 31 These data highlight the fact that ubiquitination is a pivotal mechanism of suppressing NLRP3 inflammasome activation. Consequently, we further hypothesized that AE suppresses the NLRP3 inflammasome by increasing the ubiquitination of NLRP3, resulting in the good protection of endothelial cells. Our experiment also demonstrated that AE facilitated the ubiquitination of NLRP3 to inhibit activation of the NLRP3 inflammasome whereas the Ang II had no effect on the NLRP3 ubiquitination ( Fig. 6C). Subsequently, the expression of NLRP3 and the activated caspase-1 were reversed with the proteasome inhibitor MG-132 treatment compared with the AE treatment (Fig. 6D, E, H, I). In conclusion, AE relieves Ang II-induced NLRP3 inflammasome via promoting NLRP3 ubiquitination.

CONCLUSION
AE was explored to exert protective effects on hypertension-related cardiovascular disease. The present results reveal a new protection mechanism of AE that inhibits NLRP3 inflammasome activation and decreases the release of HMGB1 by promoting NLRP3 ubiquitination, and thus restoring the endothelial tight junction proteins and permeability. Our findings indicate that AE exhibits immense potential therapeutic value in hypertension-related cardiovascular disease and the development of an innovative drug.