Tanshinone IIA inhibits exosome-induced cardiomyocyte pyroptosis through NLRP3/caspase 1 pathway

Purpose: To investigate the effect of Salvia miltiorrhiza, a traditional Chinese medicinal plant, on exosome-induced cardiomyocyte pyroptosis. Methods: Pyroptosis was induced in human AC cells using exosomes. Then, the effect of Danshen (dried roots of S. miltiorrhiza) on exosome-induced pyroptosis was determined using flow cytometry. The expressions of pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA), while protein levels of cytokines were assayed by Western blotting. Results: Tanshinone IIA (Tan IIA), the bioactive molecule in Danshen, inhibited cardiomyocyte pyroptosis by significantly reducing the expressions of proinflammatory cytokines (p < 0.001). Thus, Tan IIA reduced pyroptosis induced by cardiomyocyte-derived exosome via inhibition of the expression of NLRP3 inflammasome in human AC cells. Conclusion:


INTRODUCTION
Heart failure (HF) is elicited in myocardial death which is triggered by processes such as apoptosis, pyroptosis, necrosis and autophagy and excessive activation of the neuroendocrine system [1]. Recent research has highlighted the role of pyroptosis in progression of several cardiovascular disorders, e.g., HF [2]. Pyroptosis is a form of inflammation-induced cell death. An early step in the initiation of pyroptosis entails activation of inflammasomes. The NLRP3 inflammasome has been reported to be associated with several cardiovascular diseases [3]. In recent years, exosomes have been recognized as important extracellular factors that contribute to cardiovascular diseases, including HF which is the terminal stage of these diseases [4]. It has been reported that cardiomyocytes regulate the microenvironment via secretion or uptake of exosomes. For instance, studies have demonstrated that the viability and hypertrophy of cardiomyocytes are deeply affected by cardiac fibroblast-derived exosomes [5][6][7]. Nevertheless, the exact effect of exosomes on the progression of pyroptosis in cardiomyocytes has not been elucidated.
Salvia miltiorrhiza (Danshen) is a traditional Chinese medicinal plant. Increasing experimental evidence have revealed that Danshen exerts desirable and positive effects by preventing death and ROS accumulation in cardiomyocytes [8]. The purpose of this research was to study the influence of Tan IIA on serum exosomemediated cardiomyocyte pyroptosis, and also to elucidate the underlying mechanism.

Plasmid construction
For NLRP3 overexpression, the CDS of NLRP3 (NM_004895.4) was constructed into pCDNA3.1(+) vector using primers containing Hind III and EcoR I restriction enzyme cutting sites. The relevant primers are shown in Table 1.

Cell transfection
The AC16 cells were trypsinized and counted, and a cell suspension containing 1 × 10 6 cells per milliliter was made, 2 mL of which was inoculated in 6-well plates under the conditions of 5 % CO2 and 37 °C, followed by a 12-h incubation. Thereafter, transfection with control, vector or NLRP3 using Lipofectamine 2000 (11668-019, Invitrogen) was done.

Clinical samples
Fifteen (15) subjects were involved in the study. They comprised 9 severe burn patients and 6 healthy volunteers. Their ages ranged from 25 to 46 years, and the population had 10 males and 5 females. The inclusion and exclusion criteria for severity of burns were based on a previous report [9]. Venous blood from different subjects were collected using tubes without anticoagulant. The blood samples were centrifuged at the speed of 3500 rpm at room temperature for 8 min, to obtain sera. This study received approval from the ethical authority of our institution, and it met the criteria stipulated in the Declaration of Helsinki [9].

Extraction and identification of serum exosomes
Blood samples from severe burn patients and corresponding control were centrifuged for ½ h at 4 °C at 10,000 g, and the sera were taken up in 5 mL ultra-high speed centrifugal tubes containing PBS. The samples were then centrifuged twice for 2 h at 4 °C at 17,000 g. Then, the sediments were taken up in PBS and kept frozen at -80 °C. The exosome CD biomarkers (ab92726), CD81(ab109201) and TSG101(ab125011) were used as indexes for identification of serumderived exosomes.

PKH-67 tracer exosomes
Exosomes obtained from the serum samples of burn patients were co-cultured with AC16 cells. Endocytosis of exosomes by AC16 cells was traced using commercial kit (UR52303, Umibio). All steps used were consistent with kit instructions. Images were collected using a laserscanning microscope.

Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR)
Total RNAs were obtained with TRIzol (1596-026, Invitrogen) in line with instructions on the kit manual. Commercial cDNA Synthesis kit (Fermentas) was used for reverse-transcription of RNA to cDNA in a reaction done on ABI 7300 RT-PCR instrument (ABI-7300, Applied Biosystems). The relative level of mRNA was determined using the 2 -△△Ct method, with GAPDH as internal control. Table 2 shows the primers used.

Western blot assay
Total protein was extracted using RIPA buffer, followed by protein quantification with BSA method. Then, equal amounts of protein (25-µg portions) were resolved using SDS-PAGE, followed by transfer onto PVDF membranes.

Secretion concentrations of interleukins 18 and 1β
were measured with corresponding commercial ELISA kits.

Statistics
Data were analyzed using GraphPad Prism 7.0. Results are presented as mean ± SD. Comparison was done with one-way ANOVA. Values of p ˂ 0.05 were taken as indicative of statistically significant differences.

Serum exosomes enhanced pyroptosis of human cardiomyocytes
To investigate the influence of serum exosomes on pyroptosis, exosomes were isolated from patients with third degree burns using ultracentrifugation. First, the integrity of the exosomes was confirmed using transmission electron microscopy (TEM). The results are presented in Figure   Results that were obtained from Western blot suggested that protein levels of active Caspase-1, NLRP3 and GSDMD-N were upregulated by serum-derived exosomes in a time-dependent manner (Figure 2 D). In all, these results indicate that serum exosomes promoted pyroptosis in cardiomyocytes.

Tan IIA inhibited serum exosome-induced cardiomyocyte pyroptosis
The effects of the bioactive compounds of Danshen i.e., CTN, Tan IIA, SAA and SalB on serum exosome-mediated pyroptosis in AC16 cells were determined. In this section, the serumderived exosomes and the bioactive compounds of Danshen were used to co-culture human AC cells. As shown in Figure 3, treatment of the cells with CTN, Tan IIA, SAA and SalB led to significant reduction of pyroptosis in AC6 cells. More importantly, Tan IIA displayed the most robust inhibition of serum exosome-induced pyroptosis among all the compounds that inhibited pyroptosis.

IIA dose-dependently inhibited cardiomyocyte pyroptosis
To further test the influence of Tan IIA in pyroptosis, AC16 cells co-cultured with serum exosomes were treated with varying doses of Tan IIA, and the effects of the treatments on different factors contributing to pyroptosis were evaluated. It was found that active caspase-1 expression increased dose-dependently in Tan

Tan IIA inhibited NLRP3-induced pyroptosis of human cardiomyocytes
The regulatory effect of Tan IIA on NLPR3 inflammasome-mediated pyroptosis in human cardiomyocytes was investigated. Overexpression of NLRP3 was induced using Lentivirus, as shown in Figures 5 A and B. Tan IIA treatment significantly decreased the accumulation of IL-1β and IL-18 in oeNLRP3transfected cells (Figure 5 C). As shown in Flow cytometry was used to determine pyroptosis in oeNLRP3-transfected AC cells cocultured with Tan IIA and serum exosomes. As expected, Tan IIA effectively blocked active Caspase-1 and pyroptosis in cells treated with serum exosomes ( Figure 4F). However, treatment of cells overexpressing NLRP3 with Tan IIA significantly elevated the level of active Caspase-1 and increased pyroptosis ( Figure 4F).

DISCUSSION
Heart failure is a major health challenge in developed countries. This highlights the importance of developing new therapies that would slow down the progression of the disease. Danshen is a widely studied traditional Chinese medicine that has produced a variety of medicinal benefits. These include antiinflammation [10], anti-oxidation [10], and antithrombosis [11]. These benefits contribute to cardiovascular protection. It has been reported that Danshen contains over 200 bioactive compounds [12]. Studies have demonstrated that many of these compounds exert antiinflammatory effects, although via different mechanisms. For instance, a study has shown that salvianolic acid B produced antiinflammatory effect through suppression of TNFα-induced NF-κB activation in aortic endothelial cells [13]. Moreover, Tan IIA showed antiinflammatory function in endothelial progenitor cells [14], and cryptotanshinone exhibited antiinflammatory activity [15].
In the present study, it was revealed that Danshen inhibited serum exosome-induced pyroptosis in cardiomyocytes. Thus, it may provide protection against HF. Moreover, several bioactive components of Danshen effectively inhibited pyroptosis, with Tan IIA being the most potent anti-pyroptotic compound. Nonetheless, it was also found that the other components such as CTN, SAA and Sal B significantly blocked pyroptosis in cardiomyocytes. Future studies will determine whether these compounds have biological relevance in preventing heart disease.
The present data has shown that Tan IIA inhibited serum exosome-induced pyroptosis by blocking the expression of NLRP3 inflammasome. Elevated levels of circulating proinflammatory biomarkers have been correlated with the severity of HF [16]. This suggests that the suppressive influence of Tan IIA on expressions of proinflammatory cytokines may be be directly involved in arresting the progression of HF.
The NLRP3 inflammasome has emerged as an important factor involved in regulation of inflammation under different pathological conditions, including cardiovascular diseases [3]. It promotes cell death through pyroptosis. The loss of cardiomyocytes through pyroptosis has been shown to reduce contractile reserves, leading to HF [17]. Thus, this study has shed new light on the mechanism through which Danshen blocks pyroptosis in exosome-induced cardiomyocyte pyroptosis, thereby indicating that it may be beneficial in the cure and prevention of cardiovascular diseases. This study was performed in a cervical cancer cell line. However, it is possible that Tan IIA may play contextdependent roles in provision of health benefits for different diseases.

CONCLUSION
The present study has demonstrated that Tan IIA might be the main bioactive component of Danshen involved in suppressing pyroptosis in human cardiomyocytes. This indicates the role of Danshen as a potential agent in the treatment for HF.

DECLARATIONS
Wang, Yu Chen, Zao-Li Shen, performed the experiments. Qing Jia and Ai-Li Wang did analysis and interpretation of data. All authors read and approved the final manuscript.

Open Access
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