Effect of Acacetin on flagellin induced NLRC4 inflammasome activation in mouse bone marrow-derived macrophages

Wang Zhao-xia, Bu Juan, Zhang Xiao-ling, MAHAN Yeledan, Wu Xuan-xia, Zhou ling✉

1.Xinjiang Medical University, Urumqi, 830000, China

2.Center for Medical Research and Transformation, People's Hospital of Xinjiang Uygur Autonomous Region, Urumqi 830000, China

Keywords:Flagellin Acacetin NLRC4 activation Macrophages

ABSTRACT Objective: To investigate the effect of acacetin on flagellin induced NLRC4 inflammasome activation in mouse bone marrow-derived macrophages (BMDMs). Methods: Mouse BMDMs were divided into control group, LPS group, LPS + flagellin group and LPS + acacetin + flagellin group. All groups were added with complete medium, then primed with LPS (50 ng/mL) for 3 hrs except the control group, whereafter, LPS + flagellin group was treated with flagellin (10 μmol/L) for 0.5 hr and LPS + acacetin + flagellin group was treated with acacetin (10 μmol/L) for 0.5 hr following by flagellin (10 μmol/L) for 0.5 hr. Pro-caspase-1, pro-IL-1β in cell lysate and caspase-1, IL-1β in supernatant were detected by Western blot (WB). IL-1β, IL-18 and TNF-α in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). And the activity of LDH in supernatant was assessed by LDH test kit. Results: Compared with the control group, in LPS + flagellin group, the expression of caspase-1, IL-1β protein in supernatant were significantly increased (all P-values＜0.05), but the differences of the expression of pro-caspase-1 and pro-IL-1β protein in cell lysate were not significant. Compared with LPS + flagellin group, in LPS + acacetin+ flagellin group, the expression of caspase-1, IL-1β protein in supernatant were significantly reduced (all P-values＜0.05), while the differences of the expression of pro-caspase-1 and pro-IL-1β protein in cell lysate were not significant. ELISA showed that compared with the control group,the levels of IL-1β, IL-18, and TNF-α and the activity of LDH in supernatant of LPS + flagellin group were significantly increased (all P-values＜0.05). Compared with LPS + flagellin group, in LPS + Acacetin + flagellin group, the level of IL-1β in supernatant was significantly decreased(P＜0.05), meanwhile, the decreases of the levels IL-18, TNF-α and the activity of LDH were not significant. Conclusions: We found that Acacetin can effectively inhibit flagellin induced NLRC4 inflammasome activation and reduce cell damage in mouse BMDMs.

1. Introduction

NLRC4 inflammasome is a multiprotein complex involved in inflammation, which can be activated by pathogenassociated molecular patterns (PAMPs), or by danger-associated signals,activated cysteine-aspartic proteases-1(caspase-1),support secretion Interleukin-1β (IL-1β) and Interleukin-18 (IL-18);moreover, caspase-1 can also release the N-terminal

domain and act on the cell membrane by cutting the protein GasderminD (GSDMD), resulting in the loss of its integrity and also induce pyroptosis[1]. Many studies have proved that NLRC4 inflammasome play an important role in many diseases, such as myocardial infarction[1], ischemic stroke[4], diabetic nephropathy[5],asthma[6], colitis[7], pneumonia [8, 9]and so on. Therefore, inhibiting the activation of NLRC4 inflammasome has important clinical significance in the treatment of related diseases.

Acacetin is a kind of flavonoids, which has the effects of antitumor, anti-inflammation, antioxidation and neuroprotection, and has protective effects on inflammation-related diseases such as ischemic stroke[10, 11], pneumonia[12], arthritis[13], colitis[14] and so on. The mechanism of anti-inflammation and antioxidation is complicated.Previous studies have found that acacetin can inhibit the activation of inflammasome containing NLR family Pyrin domain protein 3(NLRP3) and reduce the release of IL-1β from mouse bone marrow macrophages[15]. NLRP3 inflammsome and NLRC4 inflammasome both belong to the NLR family, which can mediate the activation of caspase-1, promote the maturation and secretion of IL-18 and IL-1 β, and participate in the inflammatory response[16]. Can acacetin inhibit the activation of NLRC4 inflammasome? It has not been reported so far. In this study, we used mouse bone marrow-derived macrophages (BMDMs) and flagellin to induce the activation of NLRC4 inflammasome, and detected Pro-caspase-1, Pro-IL-1 β, IL-1 β, IL-18 and other related factors to explore the effects of acacetin on the activation of NLRC4 inflammasome and cell damage, so as to provide a theoretical basis for the pharmacological study of acacetin.

2. Materials and methods

2.1 Materials

The main reagent and instrument

Acacetin was obtained from Sigma (The United States); flagellin was obtained from InvivoGen (The United States); DMEM (High glucose) medium、PBS、0.25% trypsin were obtained from Excell Bio(Shanghai); RIPA lysis solution 、anti-caspase-1and anti-IL-1β were obtained from Sigma (The United States); IL-1β enzymelinked immunosorbent assay (ELISA) kit and IL-18 ELISA kit were purchased from Hangzhou Novak Biology Co., Ltd.

LDH kit Nanjing Jiancheng Company;Fluorescent inverted microscope and purchasing from Japan nikon; Protein electrophoresis apparatus, chemiluminescence imager, multifunctional enzyme mark BIO - RAD company are bought from the United States.

2.2 Methods

2.2.1 Bone marrow of mice macrophage (BMDM) separate cultivation

Three-month-old C57BL/6 mice weighing 25 ±5g were purchased from Xinjiang Medical University (license number: SCXK (new)2018-0002). The mice are free to eat and drink. The feeding environment is room temperature 21-25℃, relative humidity 50%-60%, and good ventilation. The mice were killed by cervical dislocation, the femurs on both sides of the mice were separated to the super clean platform, the muscles were separated, and the bones were exposed and cut off from both ends. The bone marrow cells were blown to the 50ml centrifuge tube with a syringe, and then centrifuged at 1500rpm for 5 minutes. Red blood cell lysate buffer was re-suspended, repeatedly blown, static 3min was placed,then DMEM medium containing 10%FBS and 1% Penicillin-Streptomycin Solution was added to mix, Centrifuge at 1500 RPM for 5 min. The DMEM medium containing granulocyte-macrophage colony-stimulating factor (10ng/ml) was resuscitated. The cells were mixed by repeated blowing, and then the cell suspension was transferred to a petri dish and cultured in a cell incubator at 37 ℃.The state of the cells was observed under the microscope every day,showing a long fusiform, indicating that the state was good, and the follow-up experiment began.

2.2.2 Cell grouping and cultureWhen cell growth to 80%,dicided into 4 groups: control group、LPS group、LPS + flagellin group and LPS + flagellin + acacetin group. Control group: BMDM was added to complete medium. LPS group: complete culture medium was added, then LPS (50ng / ml)was added for 3h. LPS group + flagellin group: complete culture medium and LPS (50 ng / ml) were added for 3 h, then flagellin (10 μ M) was added to stimulate for 0.5 h. LPS + flagellin + acacetin group: complete culture medium、LPS (50 ng/ml) were added for 3 h and Acacetin（10μmol/L）was added for 0.5h , then flagellin (10 μ M) was added to stimulate for 0.5 h.

2.2.3 Western-blot (WB) was used to detect the expression of pro-caspase-1, pro-IL-1 β, caspase-1 and IL-1β

Transfer the cell-free supernatant after centrifugation to a new centrifuge tube, add the same volume of methanol and 1/4 volume of chloroform and mixing evenly, then centrifuged at 1500rpm for 5 minutes, empty the supernatant , then add methanol and centrifuge for 5min, discard the supernatant, metal bath at 55 ℃ for 5 minutes.The collected cells were centrifuged and pour out the supernatant,mixe with the trypsin. and 500 μ L RIPA lysate (500 μL with protease inhibitor). After 40min was placed on ice, 12000rpm was centrifuged at 4 ℃ and 15min was used to collect the supernatant.BCA method kit was used to measure protein concentration, 30ug was taken for SDS-PAGE electrophoresis, and the protein on the gel was transferred to polyvinylidene fluoride (PVDF) membrane by wet transfer method. The gel was sealed with 5% skimmed milk at room temperature for 2 hours. Subsequently incubated with the anti-caspae-1 (1:1000), anti-IL-1β (1:500) and β-actin (1:1000),incubated overnight on a shaker at 4 ℃, TBST washed three times,each time 10min. Shaker incubated with diluted secondary antibody solution (1:5000) for 1 hour at room temperature ,then TBST washed again, enhanced chemiluminescence (ECL) was used to develop color and photographed with gel imaging system.

2.2.4 ELISA kit to detect the cell supernatant IL-1β, IL -18 and the content of TNF-α

According to the above steps process cells for 24 hours, collect each cell supernatant, according to the ELISA kit instruction detecting IL-1β、IL-18 and the level of TNF-α.

2.2.5 LDH Measurementcollect each cell supernatant of each group,according to the LDH kit instruction detecting。The absorbance value of wavelength 450nm was detected by enzyme labeling instrument.

2.3 Statistical Analysis

SPSS 21.0 statistical software was used for analysis. The experimental data of each group were expressed by mean ±standard deviation . Single factor analysis of variance was used for comparison among groups, and Tukey test was used for pairwise comparison between groups. The difference was statistically significant (P ＜ 0.05).

3.2 Effect of acacetin on the release of inflammatory factors from mouse bone marrow macrophages induced by flagellin

3. Results

3.1 Effect of acacetin on activation of NLRC4 inflammasome

The Western blot showed that there was no significant difference in the expression of Pro-caspase-1 and Pro-IL-1β protein in the cell lysate of each group, but the expression of caspase-1 and IL-1β in the cell supernatant of LPS+ flagellin group was significantly higher than the control group (all P ＜ 0.05). Compared with LPS+ flagellin group, the expression of caspase-1 and IL-1β in the supernatant of LPS + flagellin group decreased significantly, and the difference was statistically significant(P＜0.05).

Table1 Effect of Acacetin on expression of Pro-caspase-1、Pro-IL-1β protein in flagellin-induced macrophage lysate (n=3,±s)

Table1 Effect of Acacetin on expression of Pro-caspase-1、Pro-IL-1β protein in flagellin-induced macrophage lysate (n=3,±s)

group Pro-caspase-1 Pro-IL-1β Control group 0.685±0.015 0.820±0.050 LPS group 0.656±0.081 0.823±0.050 LPS+ flagellin group 0.694±0.090 0.864±0.016 LPS+Acacetin+flagellin group 0.666±0.057 0.873±0.052 F value 0.197 1.142 p value 0.896 0.389

Table2 Effect of Acacetin on expression of caspase-1、IL-1β protein in flagellininduced macrophage supernatant (n=3,±s)

Table2 Effect of Acacetin on expression of caspase-1、IL-1β protein in flagellininduced macrophage supernatant (n=3,±s)

Note:△Compared with control group,P＜0.05;▲Compared with LPS group,P＜0.05;◇LPS+ flagellin group,P＜0.05;

group caspase-1 IL-1β Control group 0.153±0.010 0.066±0.005 LPS group 0.174±0.009 0.073±0.004 LPS+ flagellin group 0.505±0.020△▲ 0.411±0.030△▲LPS+Acacetin+flagellin group 0.352±0.080△▲◇ 0.189±0.029△▲◇F value 47.3 174.2 p value ＜0.0001 ＜0.0001

Figure1 Effect of Acacetin on expression of Pro-caspase-1、Pro-IL-1β、caspase-1、IL-1β protein in flagellin-induced macrophage inflammatory

As shown in Table 3,compared with the control group, the expression of IL-1β, IL-18 and TNF-α in the supernatant of LPS+ flagellin group increased(P＜0.05). Compared with LPS+flagellin group, the expression of IL-1β in cell supernatant of LPS+ acacetin + flagellin group decreased, and the difference was statistically significant(P＜0.05).the expression of IL-18 and TNF-α decreased,but the difference was not statistically significant(P＞0.05).

Table3 Effect of Acacetin on expression of IL-1β、IL-18 and TNF-α protein in flagellin-induced macrophage supernatant (pg/mL, n=3,±s)

Table3 Effect of Acacetin on expression of IL-1β、IL-18 and TNF-α protein in flagellin-induced macrophage supernatant (pg/mL, n=3,±s)

Note:△Compared with control group,P＜0.05;▲Compared with LPS group,P＜0.05;◇LPS+ flagellin group,P＜0.05;

group IL-1β (pg/ml) IL-18 (pg/ml) TNF-α (pg/ml)control group 47.735±1.265 1.294±0.686 19.346±14.528 LPS group 85.443±42.909 1.942±1.452 1558.217±67.179△LPS+ flagellin group 1519.461±56.019△▲ 11.291±0.349△▲ 2978.403±58.542△▲LPS+Acacetin+flagellingroup 656.763±57.439△▲◇ 10.293±1.197△▲ 2811.843±245.629△▲F-value 6884.1 82.13 329.1 p-value ＜0.0001 ＜0.0001 ＜0.0001

3.3 Effect of acacetin on flagellin-induced injury of bone marrow macrophages in mice

As shown in Table 4,compared with the control group, The activity of LDH in LPS group increased significantly, and the activity of LDH in LPS+ flagellin group increased more significantly.Compared with LPS+ flagellin group, the LDH activity of LPS+flagellin + Acacetin group decreased significantly, and the difference was statistically significantt(P＜0.05).

Table4 Effect of Acacetin on the activity of LDH in flagellin-induced macrophage supernatant ( U/L, n=3, ±s )

Table4 Effect of Acacetin on the activity of LDH in flagellin-induced macrophage supernatant ( U/L, n=3, ±s )

Note: Compared with the control group, △P＜0.05; Compared with LPS group, ▲P＜0.05

Group LDH Activity Control 216.993±31.698 LPS 785.621±135.412△LPS+Flagellin 1 671.896±143.571△▲LPS+Acacetin+Flagellin 1 452.288±88.937△▲F 109.6 P＜0.000 1

4. Discussion

NLRC4 inflammasome are important receptors involved in immune response, which can cause inflammatory response and pyroptosis.A number of studies have shown that NLRC4 inflammatory bodies play an important role in a variety of inflammation-related diseases,so inhibiting the activation of NLRC4 inflammasome may be a potential target for the treatment of inflammation-related diseases.Ahn , H et al[17] found that acacetin can inhibit the activation of NLRC4 inflammasome by reducing the secretion of IL-18, IL-1β and caspase-1. Ahn H et al.[18] found that methylene blue could inhibit the secretion of IL-1β and caspase-1 mediated by flagellin in a dose-dependent manner through experiments in bone marrow macrophages, indicating that methylene blue can inhibit the activation of NLRC4 inflammasome. Hou, X et al.[19] found that Glaucocalyxin A can reduce the maturation and secretion of caspase-1 and IL-1β, and play an anti-inflammatory effect by inhibiting the activation of NLRC4 inflammasome.

Acacetin is a kind of flavonoids. Many studies have confirmed that acacetin has anti-inflammatory effects.For example, Wu [20] found in mice in vivo and in vitro that acacetin can protect cells from LPSinduced cell injury by reducing the release of TNF-α and IL-1β in lung tissue, inhibiting the production of reactive oxygen species and reducing lung edema. REN J[14] found that acacetin can protect ulcerative colitis by reducing the secretion of NO, IFN-γ and iNOS and down-regulating the expression of IL-1β, TNF-α and IL-6,thereby alleviating the destruction of colonic mucosal structure,ulcer and inflammatory infiltration, weight loss, hemorrhagic diarrhea and shortening of colon length. Zheng Jianjun et al.[11] was found that acacetin had a strong neuroprotective effect on rats with acute cerebral infarction by inhibiting the expression of TGF-β1,Smad3mRNA and protein, inhibit TGF-β1/Smad3 signal pathway in the animal experiments.

In our study, BMDM cells with flagellin to activate NLRC4 inflammasome, and to explore the effect of acacetin on the activation of NLRC4 inflammasome. The results showed that there was no significant difference in the expression of caspase-1 and IL-1β in the supernatant between the LPS group and the control group. In the LPS+ flagellin group, the caspase-1 and IL-1β in the supernatant were significantly increased. After add the acacetin, the expression of caspase-1 and IL-1β was significantly inhibited, and the release of inflammatory factors IL-1β and IL-18 was reduced. However,it had no significant effect on the expression of pro-caspase-1 and pro-IL-1β, indicating that acacetin could inhibit the secretion of IL-1β and had no effect on the production. Therefore, we concluded that acacetin can inhibit the activation of NLRC4 inflammasome in mouse bone marrow macrophages induced by flagellin and reduce the inflammatory response.

LDH is usually present in the cytoplasm and can be released outside the cell when the cell membrane damage or cell death.Therefore, we detected the concentration of LDH to reflect the cell damage. The results of this study showed that the concentration of LDH increased significantly after stimulating macrophages with flagellin, and decreased significantly after treatment with acacetin,indicating that acacetin can reduce cell damage.

In a word, acacetin can inhibit the activation of NLRC4 inflammasome induced by flagellin, reduce cell inflammatory response and reduce cell injury, which provides a theoretical basis for further research and clinical application of acacetin. However,the specific mechanism of the inhibitory effect of acacetin on the activation of NLRC4 inflammasome is not clear, and its specific mechanism will be further studied in the future, which is also the focus of our next step.