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The antagonism of 6-shogaol in high-glucose-activated NLRP3 inflammasome and consequent calcification of human artery smooth muscle cells

Abstract

Background

Vascular calcification is the major reason for high mortality of cardiovascular complications for diabetes. Interleukin (IL)-1β has been implicated in this pathogenesis, but its precise role and clinical evidence have not been clearly identified. Hence, this study was aimed to investigate whether high concentration of glucose (HG), which mimics the hyperglycemia environment, could initiate vascular calcification through NLRP3/IL-1β inflammasome and the underlying mechanism. Recently, 6-shogaol, a major ginger derivate, has been elucidated its pharmaceutic role for various diseases. Therefore, the aims of this study also determined 6-shogaol effect in vascular calcification of HG initiation.

Result

Human artery smooth muscle cells (HASMCs) were used in this study. Glucose concentrations at 5 and 25 mM were defined as normal and HG status, respectively. The results showed that HG could increase the NLRP3, cleaved caspase 1, and pro/mature IL-1β levels to induce the expressions of bone-related matrix proteins and subsequent HASMC calcification. This process was regulated by Akt activation and reactive oxygen species (ROS) production. Moreover, 6-shogaol could inhibit the Akt/ROS signaling and NLRP3/caspase 1/IL-1β inflammasome and hence attenuated HASMC calcification.

Conclusions

This study elucidates the detailed mechanism of HG-initiated HASMC calcification through NLRP3/caspase 1/IL-1β inflammasome and indicates a potential therapeutic role of 6-shogaol in vascular calcification complication of diabetes.

Background

Vascular calcification is highly prevalent for the patients with diabetes and chronic renal diseases and contributes to the further increased morbidity and mortality of cardiovascular complications. Although the therapeutic strategy for reducing blood glucose level has been extensively investigated, the vascular calcification development in diabetes patients have still remained [1,2,3,4,5]. Vascular calcification pathogenesis is a complex process with a phenotypic switch of vascular smooth muscle cells (VSMCs) to osteoblast- or chondrocyte-like cells, which initiates the upregulation and deposition of calcium phosphate and mineralization-related proteins, including osteopontin (OPN), osteocalcin (OCN), and alkaline phosphatase (ALP), in calcification regions and hence results in the stiffening of vessel walls [6,7,8]. Moreover, the signaling related to the bone/cartilage growth, including the bone morphogenetic proteins, Akt signaling, and runx2 transcription activity, has also been associated with the occurrence of vascular calcification complication of diabetes [8,9,10,11,12]. Currently, the precise mechanisms about how high blood glucose affects the vascular calcification pathogenesis in diabetes patients has not been completely elucidated. Therefore, more detailed investigation and understanding is still urgent and necessary for further improving the development of diabetes and its vascular calcification complications.

Vascular calcification derived from atherosclerosis and diabetes has been demonstrated to be a chronic inflammation event, which is regulated by inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Therefore, inflammasome system has recently been implicated in the pathogenesis of vascular calcification [13,14,15]. NLRP3 complex, which is composed of NLRP3 protein, adaptor protein ASC, and caspase 1, has been indicated as one of important inflammasomes because of its regulatory role in autoimmune and inflammation [16,17,18]. After receiving the stimulations, intracellular NLRP3 level upregulation and subsequent caspase 1 activation could cleave the pro-IL-1β and pro-IL-18 into mature and active IL-1β and IL-18 and then consequently elicit inflammatory responses and diseases development [16,17,18]. Accumulating data has indicated that inflammasomes, including NLRP3 complex, are the important regulatory systems for the development of chronic metabolic diseases. In this study, we further examine whether high concentration of glucose, mimics the hyperglycemia environment of diabetes patients, stimulates vascular calcification through the NLRP3 inflammasome system.

Ginger has been widely used as the flavoring agent and spice in the beverage and cooking in all over the world. Accumulating data has also demonstrated the pharmaceutic role of ginger in various diseases such as anti-cancer [19, 20], anti-arthritis [21, 22], anti-gastrointestinal disease [23, 24], anti-inflammation [25], anti-oxidation [26], and anti-atherosclerosis [27,28,29]. It has been found that the pharmaceutic function of ginger is elicited by the nature of its bioactive compounds, including gingerols and their dehydrated products, shogaols. Generally, shogaols have been suggested to be more effective than gingerols in attenuating the patients’ uncomfortable and status. And, 6-shogaol is the most dominant one [30]. However, the molecular mechanism and metabolic fate of shogaols, including 6-shogaol, in antagonizing and improving the status of these diseases have not been clearly identified. Therefore, the precise pharmaceutic effects and targets of 6-shogaol should be further elucidated and understood in order to more widely using it in the clinical application for various diseases therapy.

In this study, we investigated the role of NLRP3 inflammasome in vascular calcification in response to high glucose environment and the possible antagonized role of 6-shogaol in this process. We found that NLRP3, cleaved caspase 1, and pro/mature IL-1β proteins could be upregulated to initiate human artery SMC (HASMC) calcification under high concentration of glucose stimulation. Moreover, this NLRP3 inflammasome upregulation was resulted from the Akt activation and ROS production. Furthermore, we also demonstrated the antagonized role of 6-shogaol in NLRP3 inflammasome activation and subsequent HASMC calcification. Our findings provide new insights into the understanding of NLRP3 inflammasome-initiated HASMC calcification under high glucose stimulation and indicate a potential pharmaceutic role of 6-shogaol in cardiovascular complication of diabetes.

Results

High concentration of glucose (HG) initiates HASMC calcification

OPN, OCN, and ALP, the well-known bone matrix proteins, inductions have been recognized as the markers of vascular calcification. HASMCs were kept as the controls or were treated with 5 mM (normal concentration of glucose, NG) or 25 mM (high concentration of glucose, HG) glucose or 25 mM mannitol (M) for 3, 7, and 14 days and then the mRNA and protein expressions of OPN, OCN and ALP were examined. Cells treated with HG significantly induced the OPN, OCN, and ALP mRNA (Fig. 1a–c) and protein (Fig. 1d) expression within 3 days and persisted for 14 days in HASMCs compared to the controls and NG-/mannitol-treated cells. Moreover, we determined whether HG treatment initiates the HASMC calcification. Cells were kept as the controls or were treated with NG, mannitol, or HG for 14 days and then the HASMC calcification was examined. Cells treated with NG and mannitol had no effect on the HASMC calcification. However, cells treated with HG initiated an increase in HASMC calcification (Fig. 1e).

Fig. 1
figure1

HG initiates HASMC calcification. ad HASMCs were kept as the controls or were treated with 5 mM (normal concentration of glucose, NG) or 25 mM (high concentration of glucose, HG) glucose or 25 mM mannitol (M) for 3, 7, and 14 days and then the mRNA ac and protein d expressions of OPN, OCN and ALP were examined. e HASMCs were kept as the controls or were treated with NG, mannitol, or HG for 14 days and then the HASMC calcification was examined by ARS stain and were detected at 405 nm. Data in ac and e are mean ± SEM from three independent experiments. Results in d are representative of three independent experiments with similar results and the bar graphs are mean ± SEM from three independent experiments. *P < 0.05 vs. control cells

HG activates NLRP3 inflammasome to modulate HASMC calcification

Recently, NLRP3 inflammasome has been implicated in various diseases development. We determined whether NLRP3 inflammasome constituents, including NLRP3, caspase 1, and IL-1β, expressions could be upregulated in HASMCs in response to HG. HASMCs were kept as the controls or were treated with NG or HG for 12, 24, or 48 h and then the mRNA and protein expressions of NLRP3, caspase 1 (pro and cleaved form), and IL-1β (pro and mature form) were examined. Cells treated with HG significantly induced NLRP3 mRNA (Fig. 2a) and protein (Fig. 2b) expression within 12 h and persisted for 48 h in HASMCs compared to the controls and NG-treated cells. Moreover, HG also increased cleaved caspase 1 and pro/mature IL-1β protein expressions in HASMCs (Fig. 2b). Cells treated with NG had a partial effect on IL-1β protein expressions in HASMCs (Fig. 2b). Furthermore, cell pretreated with NLRP3 inflammasome inhibitor, MCC950, significantly inhibited OPN, OCN, and ALP mRNA expressions (Fig. 2c) and HASMC calcification (Fig. 2d) of HG induction.

Fig. 2
figure2

HG activates NLRP3 inflammasome to modulate HASMC calcification. HASMCs were kept as the controls or were treated with NG or HG for 12, 24, or 48 h or 14 days and then the mRNA a and protein b expressions of NLRP3, caspase 1 (pro and cleaved form), and IL-1β (pro and mature form), the mRNA expressions of OPN, OCN, and ALP c, and HASMC calcification d were examined. Data in a and c, d are mean ± SEM from three independent experiments. Results in b are representative of three independent experiments with similar results and the bar graphs are mean ± SEM from three independent experiments. *P < 0.05 vs. control cells. #P < 0.05 vs. DMSO/HG-treated cells

HG increases ROS level in HASMCs to trigger NLRP3 inflammasome and subsequent calcification

HASMCs were kept as the controls or were treated with NG, mannitol (M), or HG for 24 h and then the intracellular ROS level was examined. Cells treated with HG showed an increase in the ROS level in HASMCs compared to the controls and NG-/mannitol-treated cells (Fig. 3a). Cells were kept as the controls or were pretreated with DMSO or NAC, a ROS scavenger, for 1 h and then treated with HG for 2 (inflammasome protein expressions) and 14 (OPN/OCN/ALP mRNA expressions and calcification) days. HASMCs pretreated with NAC significantly downregulated HG-increased NLRP3, cleaved caspase 1, and pro/mature IL-1β protein (Fig. 3b) and OPN/OCN/ALP mRNA (Fig. 3c) expressions and HASMC calcification (Fig. 3d).

Fig. 3
figure3

HG increases ROS level in HASMCs to trigger NLRP3 inflammasome and subsequent calcification. a HASMCs were kept as the controls or were treated with NG, mannitol (M), or HG for 24 h and then the intracellular ROS level was examined. b, c HASMCs were kept as the controls or were pretreated with DMSO or NAC, a ROS scavenger, for 1 h and then treated with HG for 2 and 14 days. The NLRP3, caspase 1 (pro and cleaved form), and IL-1β (pro and mature form) protein expressions b, OPN, OCN, and ALP mRNA expressions c in HASMCs, and HASMC calcification d were examined. Data in a and c, d are mean ± SEM from three independent experiments. Results in b are representative of three independent experiments with similar results and the bar graphs are mean ± SEM from three independent experiments. *P < 0.05 vs. control cells. #P < 0.05 vs. DMSO/HG-treated cells

Akt signaling in HASMCs regulates HG-triggered ROS production, NLRP3 inflammasome and subsequent calcification

HASMCs were kept as the controls or were treated with NG, mannitol (M), or HG for 4 h and then the Akt phosphorylation was examined. Cells treated with HG significantly induced Akt phosphorylation in HASMCs compared to the control and NG-/mannitol-treated cells (Fig. 4a). Cells were kept as the controls or were pretreated with (i) DMSO or LY294008, an akt phosphorylation inhibitor, or (ii) pcDNA empty vector (EV) or dominant negative (dn)-Akt-expressed plasmid and then treated with HG for 1 (ROS production), 2 (inflammasome protein expressions) and 14 (OPN/OCN/ALP mRNA expressions and calcification) days. Akt activity inhibition in HASMCs by LY294008 or dn-Akt-expressed plasmid pretreatment significantly downregulated HG-increased NLRP3 and pro/mature IL-1β protein (Fig. 4b) and OPN/OCN/ALP mRNA (Fig. 4c) expressions, intracellular ROS production (Fig. 4c), and HASMC calcification (Fig. 4d).

Fig. 4
figure4

Akt signaling in HASMCs regulates HG-triggered ROS production, NLRP3 inflammasome and subsequent calcification. a HASMCs were kept as the controls or were treated with NG, mannitol (M), or HG for 4 h and then the Akt phosphorylation was examined. bd HASMCs were kept as the controls or were pretreated with (i) DMSO or LY294008, an akt phosphorylation inhibitor, or (ii) pcDNA empty vector (EV) or dominant negative (dn)-Akt-expressed plasmid and then treated with HG for 2 and 14 days. The NLRP3 and IL-1β (pro and mature form) protein expressions b, ROS production c, and OPN, OCN, and ALP mRNA expressions c in HASMCs, and HASMC calcification d were examined. Results in a, b are representative of three independent experiments with similar results and the bar graphs are mean ± SEM from three independent experiments. Data in c, d are mean ± SEM from three independent experiments. *P < 0.05 vs. control cells. #P < 0.05 vs. DMSO/ or EV/HG-treated cells

6-Shogaol attenuates HG-triggered Akt activation, ROS production, NLRP3 inflammasome, and calcification in HASMCs

Next, we examined whether 6-shogaol, a ginger derivate and a natural anti-inflammation factor, regulates HG effect on HASMC calcification. HASMCs were kept as the controls or were pretreated with ethanol or 6-shagaol and then treated with HG for 4 h (Akt phosphorylation) and 1 (ROS production), 2 (inflammasome protein expressions), and 14 (OPN/OCN/ALP mRNA expressions and calcification) days. It was shown that 6-shagaol significantly attenuates HG-increased Akt phosphorylation (Fig. 5a), intracellular ROS production (Fig. 5b), and NLRP3 and pro/mature IL-1β protein expressions (Fig. 5c) in HASMCs. Moreover, 6-shagaol also significantly attenuated HG-induced OPN/OCN/ALP mRNA expressions in HASMCs (Fig. 6a) and HASMC calcification (Fig. 6b).

Fig. 5
figure5

6-Shogaol attenuates HG-triggered Akt activation, ROS production, and NLRP3 inflammasome in HASMCs. a, b HASMCs were kept as the controls or were pretreated with ethanol or 6-shagaol and then treated with HG for 4, 24, and 48 h. The Akt phosphorylation, ROS production, and NLRP3/IL-1β (pro and mature form) protein expressions in HASMCs were examined. Results in a and c are representative of three independent experiments with similar results and the bar graphs are mean ± SEM from three independent experiments. Data in b are mean ± SEM from three independent experiments. *P < 0.05 vs. control cells. #P < 0.05 vs. EtOH/HG-treated cells

Fig. 6
figure6

6-shogaol attenuates HG-triggered HASMC calcification. a, b HASMCs were kept as the controls or were pretreated with ethanol or 6-shagaol and then treated with HG for 14 days. The OPN, OCN, and ALP mRNA expressions in HASMCs a and HASMC calcification b were examined. Data in a, b are mean ± SEM from three independent experiments. *P < 0.05 vs. control cells. #P < 0.05 vs. EtOH/HG-treated cells

Discussion

This study has characterized the mechanisms whereby high glucose initiates HASMC calcification development through NLRP3 inflammasome and the antagonized role of ginger derivate, 6shogaol, in this process, as summarized in Fig. 7. The systematic experiments demonstrated that (i) HG initiates an osteogenic switch of HASMCs through upregulating the expressions of osteogenic matrix proteins, i.e., OPN, OCN, and ALP and the development of calcification. (ii) This calcification-initiating development in response to HG is regulated by Akt signaling, ROS production and subsequent NLRP3/caspase 1/IL-1β inflammasome activation. (iii) 6-shogaol effectively antagonizes the HG effect on NLRP3 inflammasome activation and consequent HASMC calcification. Our findings provide new insights into the regulatory mechanism of NLRP3 inflammasome in vascular calcification development under high glucose stimulation and suggest a potential nature product, 6-shogaol, in antagonizing this process.

Fig. 7
figure7

Schematic representation of signaling pathways regulating HG-triggered vascular calcification in HASMCs and the attenuated effect of 6-shogaol

Vascular calcification accompanies the development of atherosclerosis and diabetes and is associated with the mortality risk in patients with these two diseases [31]. Both atherosclerosis and diabetes have been recognized as the chronic and low-grade inflammation events and therefore the increased cytokines in blood has been indicated as an important initiator for diseases pathogenesis. Our present results indicated that HG could initiate the HASMC calcification development through upregulating the NLRP3/caspase 1/IL-1β inflammasome. Although IL-1β is one of well-demonstrated atherosclerosis- and diabetes-stimulating cytokines, its precise role and secretion mechanism in vascular calcification complication of diabetes have not been clearly detected [31]. In contrast, accumulating data has highlighted the importance and regulation of NLRP3 inflammasome activation and subsequent IL-1β secretion in hyperglycemia. For examples, (i) it has been indicated that NLRP3-knockdown mice which decrease the secreted level of IL-1β has an improved status for insulin resistance development [32, 33]; (ii) Stienstra et al. found that the inhibition of caspase 1 activity, which is involved in NLRP3 inflammasome system and could cleave the pro-IL-1β to mature form, affects the adipocyte differentiation and insulin sensitivity [34]; (iii) Maedler et al. demonstrated that high concentration of glucose could stimulate IL-1β secretion from the β-cells and hence elicit glucotoxicity in human pancreatic islets [35]; and (iv) Zhou et al. elucidated that high concentration of glucose could also upregulate the thioredoxin-interacting protein expression (one of NLRP3 binding proteins) to increase IL-1β level [32]. Taking all of these findings together, including ours, although it still has a limitation in the clinical evidence, we strongly indicated the indispensable role of IL-1β secreted from NLRP3 inflammasome-dependent system in vascular calcification development under high glucose environment.

In addition to being a pungent flavor, the pharmaceutic role of ginger has also been demonstrated for many years. 6-shogaol is the dominant constituent in ginger to contribute to uncomfortable release and ailment alleviation. It has been suggested that the anti-oxidation and anti-inflammation properties of 6-shogaol could effectively attenuate the status of metabolic and inflammatory diseases, including the cancer, arthritis, and atherosclerosis [19,20,21,22,23,24,25,26,27,28,29]. Our results found that 6-shogaol could attenuate HG-initiated HASMC calcification development. Moreover, this attenuation is elicited through attenuating the ROS production and subsequent NLRP3 inflammasome activation in HASMCs. Our data supported the idea that ginger and 6-shogaol contribute to the anti-oxidation and anti-inflammasome activities in disease therapy. Recent diabetic mice/rats (including type I and II models) studies have shown the beneficial effects of ginger or its derivates, including 6-shogaol, on homeostatic control of blood glucose and/or insulin [36,37,38]. Moreover, the clinical trials have also found the blood glucose-lowering potential of ginger in patients with type II diabetes [39]. Although the importance of ginger/6-shogaol in diabetes has already been found, their precise mechanism in blood glucose control and even more in vascular complications development have not been elucidated clearly. Our study has provided one of antagonized mechanisms of 6-shogaol in vascular calcification under high glucose condition and further supported the possibility of its clinical application in diabetes patients.

The aberrant activation of NLRP3 inflammasome has already been implicated in the pathogenesis of many diseases, including the diabetes. Therefore, targeting the NLRP3 inflammasome has been proposed as a promised method to improve these diseased. However, the therapeutic strategies specifically antagonizing the NLRP3 inflammasome have not been effectively developed and applied in patient treatment. The current study has reported that HG condition could result in vascular calcification through inducing the bone-related matrix protein, i.e., OPN, OCN, and ALP, expressions in HASMCs, which is regulated by the Akt activation, ROS production, and subsequent NLRP3/caspase 1/IL-1β inflammasome. Also, it has been further demonstrated that 6-shogaol, a nature ginger extract, could have an antagonized effect in HASMC calcification through inhibiting HG-activated NLRP3 inflammasome.

Conclusion

The presented study provides new insights into understanding the mechanisms of NLRP3 inflammasome-regulated vascular calcification in HASMCs under hyperglycemia conditions. The results concerning the antagonized role of 6-shogaol might lead to the design and development of ginger-containing dietary therapy and/or new drugs for treating vascular calcification complication of diabetes through targeting the NLRP3 inflammasome.

Methods

Materials

Rabbit polyclonal antibody (pAb) against ALP and mouse monoclonal antibody (mAb) against OPN and OCN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit pAbs against NLRP3, caspase 1, IL-1β, pAkt, Akt and β-actin were purchased from Cell Signaling Technology (Beverly, MA). Intracellular ROS assay kit was purchased from Cell Biolabs (San Diego, CA). MCC950 (NLRP3 inflammasome inhibitor), N-acetylcysteine (NAC, ROS inhibitor), and LY294002 (PI3K/Akt inhibitor) and other chemicals were purchased from Sigma (Temecula, CA).

Cell culture

HASMCs were purchased from ATCC cell bank (Rockville, MD) and were cultured in medium (F12K, 10% FBS, and 1% antibiotics). Only 3–7 passages of HASMCs were employed for the experiments.

HASMC calcification

SMC calcification is a mineralization process. It could be examined by Alizarin Red S (ARS) stain, a tool for detecting calcium deposition [40]. HG-treated HASMCs were fixed with formaldehyde (10%) and stained with ARS reagent (40 mM). Then, the stained HASMCs were extracted with acetic acid and then were neutralized with ammonium hydroxide. The samples were centrifugated and the supernatants were collected and measured at 405 nm by colorimetric detection.

Quantitative real-time PCR

The cDNA extracted and converted from purified RNA of HASMCs was employed to determine the mRNA expression of the specific genes. The ABI StepOnePlus machine and SYBR Green kit (Applied Biosystems) were employed in the real-time PCR assay. The primers employed in the experiments included NLRP3 (positive: 5′- GATCTTCGCTGCGATCAACA-3′; negative: 5′- GGGATTCGAAACACGTGCATTA-3′), OPN (positive: 5′-GGACAGCCAGGACTCCATTG-3′; negative: 5′-TGTGGGGACAACTG GAGTGAA-3′), OCN (positive: 5′-GTGACGAGTTGGCTGACC-3′; negative: 5′-CAAGGG GAAGAGGAAAGAAGG-3′), ALP (positive: 5′-CTCCCAGTCTCATCTCCT-3′; negative: 5′-AAGACCTCAACTCCCCTGAA-3′) and GAPDH (positive: 5′-AGGTGAAGGTCGGAG TCAAC-3′; negative: 5′-CCATGTAGTTGAGGTCAATGAAGG-3′) genes. The GAPDH gene expression was indicated as the internal control.

Western blot

HASMCs (controls and HG-treated) were lysed with lysis buffer (1% NP-40/0.1% SDS/0.5% sodium deoxycholate/protease and phosphatase inhibitor cocktail). The lysates (30 µg) were separated and examined by SDS-PAGE (10% running and 4% stacking) and the indicated antibodies.

Plasmid transfection

HASMCs were cultured overnight and then transfected with the pcDNA empty vector or dominant negative Akt (dn-Akt)-expressed plasmids [41] by using the Lipofactamine 3000 Transfection Regents.

Statistical analysis

The results were shown as mean ± SEM and the statistical analysis was measured by an independent Student t-test for two groups of data and analysis of variance (ANOVA) followed by Scheffe’s test for multiple comparisons. The P value < 0.05 was shown significant. Results were assayed from at least 3 repetitions which were collected from individual experiments.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

VASMCs:

vascular artery smooth muscle cells

OPN:

osteopontin

OCN:

osteocalcin

ALP:

alkaline phosphatase

IL-1β:

interleukin-1β

pAb:

rabbit polyclonal antibody

mAb:

mouse monoclonal antibody

NAC:

N-acetylcysteine

ARS:

Alizarin Red S

dn-Akt:

dominant negative Akt

HG:

high concentration of glucose

NG:

normal concentration of glucose

References

  1. 1.

    Heath JM, Sun Y, Yuan K, Bradley WE, Litovsky S, Dell’Italia LJ, et al. Activation of AKT by O-linked N-acetylglucosamine induces vascular calcification in diabetes mellitus. Circ Res. 2014;114:1094–102.

    CAS  Article  Google Scholar 

  2. 2.

    Wang C, Xu W, An J, Liang M, Li Y, Zhang F, et al. Poly(ADP-ribose) polymerase 1 accelerates vascular calcification by upregulating Runx2. Nat Commun. 2019;10:1203.

    CAS  Article  Google Scholar 

  3. 3.

    Demer LL, Tintut Y. Vascular calcification: pathobiology of a multifaceted disease. Circulation. 2008;117:2938–48.

    Article  Google Scholar 

  4. 4.

    No author list. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet. 1998;352:837–53.

  5. 5.

    Laing SP, Swerdlow AJ, Slater SD, Burden AC, Morris A, Waugh NR, et al. Mortality from heart disease in a cohort of 23,000 patients with insulin-treated diabetes. Diabetologia. 2003;46:760–5.

    CAS  Article  Google Scholar 

  6. 6.

    Shanahan CM, Crouthamel MH, Kapustin A, Giachelli CM. Arterial calcification in chronic kidney disease: key roles for calcium and phosphate. Circ Res. 2011;109:697–711.

    CAS  Article  Google Scholar 

  7. 7.

    McCarty MF, DiNicolantonio JJ. The molecular biology and pathophysiology of vascular calcification. Postgrad Med. 2014;126:54–64.

    Article  Google Scholar 

  8. 8.

    Kapustin AN, Davies JD, Reynolds JL, McNair R, Jones GT, Sidibe A, Schurgers LJ, Skepper JN, Proudfoot D, Mayr M, Shanahan CM. Calcium regulates key components of vascular smooth muscle cell-derived matrix vesicles to enhance mineralization. Cir Res. 2011;109:e1–12.

    CAS  Article  Google Scholar 

  9. 9.

    Boström K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest. 1993;91:1800–9.

    Article  Google Scholar 

  10. 10.

    Tintut Y, Patel J, Parhami F, Demer LL. Tumor necrosis factor-alpha promotes in vitro calcification of vascular cells via the cAMP pathway. Circulation. 2000;102:2636–42.

    CAS  Article  Google Scholar 

  11. 11.

    Aikawa E, Nahrendorf M, Figueiredo JL, Swirski FK, Shtatland T, Kohler RH, Jafferer FA, Aikawa M, Weissleder R. Osteogenesis associates with inflammation in early-stage atherosclerosis evaluated by molecular imaging in vivo. Circulation. 2007;116:2841–50.

    CAS  Article  Google Scholar 

  12. 12.

    Byon CH, Javed A, Dai Q, Kappes JC, Clemens TL, Darley-Usmar VM, McDonald JM, Chen Y. Oxidative stress induces vascular calcification through modulation of the osteogenic transcription factor Runx2 by AKT signaling. J Biol Chem. 2008;283:15319–27.

    CAS  Article  Google Scholar 

  13. 13.

    Jiang D, Chen S, Sun R, Zhang X, Wang D. The NLRP3 inflammasome: role in metabolic disorders and regulation by metabolic pathways. Cancer Lett. 2018;419:8–19.

    CAS  Article  Google Scholar 

  14. 14.

    Baldrighi M, Mallat Z, Li X. NLRP3 inflammasome pathways in atherosclerosis. Atherosclerosis. 2017;267:127–38.

    CAS  Article  Google Scholar 

  15. 15.

    Wen C, Yang X, Yan Z, Zhao M, Yue X, Cheng X, et al. Nalp3 inflammasome is activated and required for vascular smooth muscle cell calcification. Int J Cardiol. 2013;168:2242–7.

    Article  Google Scholar 

  16. 16.

    Schroder K, Tschopp J. The inflammasomes. Cell. 2010;140:821–32.

    CAS  Article  Google Scholar 

  17. 17.

    He Y, Hara H, Núñez G. Mechanism and regulation of NLRP3 inflammasome activation. Trends Biochem Sci. 2016;41:1012–21.

    CAS  Article  Google Scholar 

  18. 18.

    Strowig T, Henao-Mejia J, Elinav E, Flavell R. Inflammasomes in health and disease. Nature. 2012;481:278–86.

    CAS  Article  Google Scholar 

  19. 19.

    Poltronieri J, Becceneri AB, Fuzer AM, Filho JC, Martin AC, Vieira PC, et al. [6]-Gingerol as a cancer chemopreventive agent: a review of its activity on different steps of the metastatic process. Mini Rev Med Chem. 2014;14:313–21.

    CAS  Article  Google Scholar 

  20. 20.

    Kaur IP, Deol PK, Kondepudi KK, Bishnoi M. Anticancer potential of ginger: mechanistic and pharmaceutical aspects. Curr Pharm Des. 2016;22:4160–72.

    CAS  Article  Google Scholar 

  21. 21.

    Altman RD, Marcussen KC. Effects of a ginger extract on knee pain in patients with osteoarthritis. Arthritis Rheum. 2001;44:2531–8.

    CAS  Article  Google Scholar 

  22. 22.

    Bartels EM, Folmer VN, Bliddal H, Altman RD, Juhl C, Tarp S, et al. Efficacy and safety of ginger in osteoarthritis patients: a meta-analysis of randomized placebo-controlled trials. Osteoarthritis Cartilage. 2015;23:13–21.

    CAS  Article  Google Scholar 

  23. 23.

    Prasad S, Tyagi AK. Ginger and its constituents: role in prevention and treatment of gastrointestinal cancer. Gastroenterol Res Pract. 2015;2015:142979.

    Article  Google Scholar 

  24. 24.

    Haniadka R, Saldanha E, Sunita V, Palatty PL, Fayad R, Baliga MS. A review of the gastroprotective effects of ginger (Zingiber officinale Roscoe). Food Funct. 2013;4:845–55.

    CAS  Article  Google Scholar 

  25. 25.

    Mashhadi NS, Ghiasvand R, Askari G, Hariri M, Darvishi L, Mofid MR. Anti-oxidative and anti-inflammatory effects of ginger in health and physical activity: review of current evidence. Int J Prev Med. 2013;4(Suppl 1):S36–42.

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Dugasani S, Pichika MR, Nadarajah VD, Balijepalli MK, Tandra S, Korlakunta JN. Comparative antioxidant and anti-inflammatory effects of [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol. J Ethnopharmacol. 2010;127:515–20.

    CAS  Article  Google Scholar 

  27. 27.

    Fuhrman B, Rosenblat M, Hayek T, Coleman R, Aviram M. Ginger extract consumption reduces plasma cholesterol, inhibits LDL oxidation and attenuates development of atherosclerosis in atherosclerotic, apolipoprotein E-deficient mice. J Nutr. 2000;130:1124–31.

    CAS  Article  Google Scholar 

  28. 28.

    Verma SK, Singh M, Jain P, Bordia A. Protective effect of ginger, Zingiber officinale Rosc on experimental atherosclerosis in rabbits. Indian J Exp Biol. 2004;42:736–8.

    CAS  PubMed  Google Scholar 

  29. 29.

    Wang YK, Hong YJ, Yao YH, Huang XM, Liu XB, Zhang CY, et al. 6-Shogaol protects against oxidized LDL-induced endothelial injruries by inhibiting oxidized LDL-evoked LOX-1 signaling. Evid Based Complement Alternat Med. 2013;2013:503521.

    PubMed  PubMed Central  Google Scholar 

  30. 30.

    Kou X, Wang X, Ji R, Liu L, Qiao Y, Lou Z, et al. Occurrence, biological activity and metabolism of 6-shogaol. Food Funct. 2018;9:1310–27.

    CAS  Article  Google Scholar 

  31. 31.

    Bessueille L, Magne D. Inflammation: a culprit for vascular calcification in atherosclerosis and diabetes. Cell Mol Life Sci. 2015;72:2475–89.

    CAS  Article  Google Scholar 

  32. 32.

    Zhou R, Tardivel A, Thorens B, Choi I, Tschopp J. Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat Immunol. 2010;11:136–40.

    CAS  Article  Google Scholar 

  33. 33.

    Vandanmagsar B, Youm YH, Ravussin A, Galgani JE, Stadler K, Mynatt RL, Ravussin E, Stephens JM, Dixit VD. The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance. Nat Med. 2011;17:179–88.

    CAS  Article  Google Scholar 

  34. 34.

    Stienstra R, Joosten LA, Koenen T, van Tits B, van Diepen JA, van den Berg SA, et al. The inflammasome-mediated caspase-1 activation controls adipocyte differentiation and insulin sensitivity. Cell Metab. 2010;12:593–605.

    CAS  Article  Google Scholar 

  35. 35.

    Maedler K, Sergeev P, Ris F, Oberholzer J, Joller-Jemelka HI, Spinas GA, et al. Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest. 2002;110:851–60.

    CAS  Article  Google Scholar 

  36. 36.

    Yi JK, Ryoo ZY, Ha JJ, Oh DY, Kim MO, Kim SH. Beneficial effects of 6-shogaol on hyperglycemia, islet morphology and apoptosis in some tissues of streptozotocin-induced diabetic mice. Diabetol Metab Syndr. 2019;11:15.

    Article  Google Scholar 

  37. 37.

    Li Y, Tran VH, Duke CC, Roufogalis BD. Preventive and protective properties of Zingiber officinale (ginger) in diabetes mellitus, diabetic complications, and associated lipid and other metabolic disorders: a brief review. Evid Based Complement Alternat Med. 2012;2012:516870.

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Ojewole JA. Analgesic, anti-inflammatory and hypoglycaemic effects of ethanol effect of Zingiber officinale (Roscoe) rhizomes (Zingiberaceae) in mice and rats. Phytother Res. 2006;20:764–72.

    Article  Google Scholar 

  39. 39.

    Daily JW, Yang M, Kim DS, Park S. Efficacy of ginger for treating type 2 diabetes: a systematic review and meta-analysis of randomized clinical trials. J Ethnic Foods. 2015;2:36–43.

    Article  Google Scholar 

  40. 40.

    Chen TC, Lin CT, Chien SJ, Chang SF, Chen CN. Regulation of calcification in human aortic smooth muscle cells infected with high-glucose-treated Porphyromonas gingivalis. J Cell Physiol. 2018;233:4759–69.

    CAS  Article  Google Scholar 

  41. 41.

    Yeh CC, Chang SF, Huang TY, Chang HI, Kuo HC, Wu YC, et al. Shear stress modulates macrophage-induced urokinase plasminogen activator expression in human chondrocytes. Arthritis Res Ther. 2013;15:R53.

    CAS  Article  Google Scholar 

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Acknowledgements

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Funding

This work was supported by the Kaohsiung Chang Gung Memorial Hospital (Grant Number: CMRPG8B0611, CMRPG8B0612, CMRPG8F1271 and CMRPG8G1201).

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TCC, YCL, CSS, SFC, and CNC were involved in conception and design of the study. TCC, CKY, and RZH were involved in data acquisition of data. All authors were involved in statistical analysis and interpretation of the data. TCC, SFC, and CNC wrote the manuscript. YCL, CSS, SFC, and CNC were involved in review and editing of the manuscript. All authors commented the final version. All authors had full access to all data. All authors read and approved the final manuscript.

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Correspondence to Shun-Fu Chang or Cheng-Nan Chen.

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Chen, T., Yen, C., Lu, Y. et al. The antagonism of 6-shogaol in high-glucose-activated NLRP3 inflammasome and consequent calcification of human artery smooth muscle cells. Cell Biosci 10, 5 (2020). https://doi.org/10.1186/s13578-019-0372-1

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Keywords

  • 6-Shogaol
  • Interleukin-1β
  • NLRP3 Inflammasome
  • Vascular calcification
  • Smooth muscle cells