Tenuigenin protects dopaminergic neurons from inflammation via suppressing NLRP3 inflammasome activation in microglia

Tenuigenin (molecular formula: C₃₀H₄₅ClO₆; average molecular weight: 537.14 kDa, chemical structure showed in Fig. 1) was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (111572-200702) with a purity of 98.7%. LPS (Escherichia coli 0111:B4, L4391), MPTP (M0896), ATP (A2385), and MSU (U2875) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse IL-1β ELISA Kits were purchased from R&D Systems. ROS-specific fluorescent probe cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Invitrogen (Thermo Fisher Scientific Inc., USA). The antibodies used in this study are the following: rabbit monoclonal anti-NLRP3 (1:1000, Cell Signaling Technology, Beverly, MA, USA), rabbit anti-caspase-1 (1:800, Santa Cruz Biotechnology, USA), goat anti-IL-1β (1:800, R&D Systems, Minneapolis, USA), mouse monoclonal anti-β-actin (1:5000, Sigma-Aldrich), mouse monoclonal anti-tyrosine hydroxylase (TH, 1:1000, Sigma-Aldrich), and rabbit polyclonal anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:500, Wako, Osaka, Japan). The reagents obtained from other sources are detailed throughout the following text.

Animals were maintained under specific pathogen-free conditions and treated according to the protocols approved by IACUC (Institutional Animal Care and Use Committee of Capital Medical University).

MPTP acute model: Male C57BL/6J mice (12-week) were randomly divided into five groups: saline, MPTP, and MPTP plus tenuigenin (low dose, 25 mg/kg), MPTP plus tenuigenin (high dose, 50 mg/kg), and tenuigenin (high dose, 50 mg/kg) alone. Mice were pre-treatment daily with tenuigenin for 10 days and then given MPTP (20 mg/kg) intraperitoneally four times at 2-h interval after tenuigenin administration. The same volume of saline was injected in the vehicle group. Two days after the last injection, behavioral assessments were performed using the open field test and rotarod test. After that, all animals were sacrificed for further study.

LPS acute model: Male C57BL/6J mice (12-week) were randomly divided into four groups: saline, LPS, and LPS plus tenuigenin (25 mg/kg) and LPS plus tenuigenin (50 mg/kg). To induce NLRP3 activation and IL-1β secretion, mice were injected intraperitoneally with LPS (20 mg/kg) alone and LPS plus tenuigenin (25 mg/kg or 50 mg/kg). After 6 h, the serum samples were collected and the IL-1β level was measured by ELISA. Animals were sacrificed, and brains were harvested.

Spontaneous locomotor activity was assessed using the open field test in a Tru Scan 2.0 system (Coulbourn Instruments, Allentown, PA, USA). Locomotor activity was assessed in automated activity chambers connected to a digital scan analyzer that transmitted the number of infrared beam breaks (activity data) to the instrument. Total movement distance (cm) was recorded across a 60-min recording period.

An accelerating rotarod was used to evaluate motor coordination and balance. Mice were placed on a rotating rod (Rota Rod Rotamex 5, Columbus Instruments, USA) at a speed of 5 rounds per minute. The speed of the rotarod accelerated to 40 rounds per minute. The latency time (sec) falling from the rod was automatically recorded. Each mouse was given three trials, and the latency times were averaged.

The dopamine (DA) contents, and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in striatum, were determined using an HPLC apparatus with an electrochemical detector (Model 5600A CoulArray Detector System ESA, MA, USA). Tissues were homogenized in 200 mM ice-cold perchloric acid and the homogenate placed in an ice bath for 60 min. The sample was then centrifuged at 15,000g for 20 min at 4 °C, and the supernatant was transferred to a clean tube. A one-half volume of the solution containing 20 mM potassium citrate, 300 mM potassium dihydrogen phosphate, and 2 mM Na₂EDTA was added and mixed thoroughly to precipitate the perchloric acid. After incubating in an ice bath for 60 min, the mixture was centrifuged at 15,000g for 20 min at 4 °C. The supernatant was filtered through a 0.22-μm filter and injected into the HPLC system. The mobile phase was 125 mM sodium citrate buffer supplemented with 20% methanol, 0.1 mM Na₂EDTA, and 0.5 mM 1-octanesulfonic acid sodium salt. The flow rate was set at 1.2 mL/min.

Brain samples were collected and postfixed in 4% PFA at 4 °C overnight. They were transferred to 15% sucrose in phosphate-buffered saline (PBS) overnight and then to 30% sucrose overnight till the brain sunk to the bottom of the tube. Coronal sections (30 μm) were cut by a freezing microtome (Leica, Germany) and stored in an antifreeze solution. Sections of substantia nigra were collected for immunohistochemistry according to a previous report [16]. Briefly, they were incubated overnight with the antibody against TH or Iba-1 overnight at 4 °C. After rinsing three times with PBS, sections were incubated with a second antibody (1:200) and AB work solution (Vector Laboratories, Burlingame, CA, USA) for 30 min at 37 °C. DAB solution was used to visualize the staining.

Images were observed, and photos were taken under a confocal microscope (Axiovert LSM510, Carl Zeiss Co., Germany). The immunostaining signals were quantitatively analyzed using the Optical Fractionator method with Microbrightfield Stereo-Investigator software (Stereo Investigator software, Microbrightfield, VT, USA). The total number of TH-IR neuron and Iba-1-IR microglia in the entire extent of SNc were counted. Briefly, the regions of SNc in the midbrain sections were outlined at low magnification (×40). For TH⁺ and Iba-1⁺ cells, the counting frame size was 50 μm × 50 μm and the sampling grid size was 100 μm × 100 μm. All stereological analyses were performed under the ×200 magnification. The sampling scheme was designed to have a coefficient of error < 10% in order to get reliable results. Each brain contained 6 serial sections at 6 intervals. For immunohistochemical staining, select one series of sections per mouse. The total numbers of immunoreactive cells in the entire extent of SNc were counted from 4 to 6 mouse brains per group.

Murine BV-2 microglia cells were maintained in DMEM/F12 (1:1) media supplemented with 10% fetal bovine serum and antibiotics at 37 °C in a humidified incubator under 5% CO₂. For inducing inflammasome activation, 1 × 10⁶ cells were plated in 6-well plate overnight and the medium were changed to opti-MEM in the following morning, and then, the cells were primed with LPS (500 ng/ml) for 3 h. After that, the cells were stimulated with 2.5 mM ATP for 30 min or 150 μg/ml monosodium urate crystals (MSU) for 4 h.

The ROS-specific fluorescent probe H2DCFDA was used to detect intracellular generation of ROS by modification. After drug treatment, BV2 cells were incubated with 10 μM DCFDA for 30 min. After washing 3 times with PBS, the intensity of fluorescence was determined by a multimode reader (Vario Skan Flash, 3001, Thermo Scientific) under an emission wavelength at 530 nm and excitation wavelength at 485 nm. The obtained values were presented as folds of the controls.

Cells were treated with different stimuli. The concentration of IL-1β in the cell culture supernatant or serum was measured by mouse IL-1β ELISA Kit according to the manufacturer’s instructions.

The cell culture supernatant was collected and centrifuged to remove dead cells, and the supernatant was transferred into new tubes. Then, 500 μL methanol and 125 μL chloroform were added to precipitate supernatant, vortex, and centrifuge 16,000g for 5 min. The upper phase was discarded without touching the protein disk, and 500 μL methanol was added for washing and centrifuged at 16,000g for 5 min. The supernatant was removed, and the pellet was dried at 37 °C for 5 min. Ultimately, 50 μL 2.5 × loading buffer was added with DTT and vortex. The samples were boiled and loaded on 15% gels.

Tissues and cells protein lysates were quantified by Bradford assays (Bio-Rad, Hercules, CA, USA). Proteins were electrophoresed through a 8–15% SDS-polyacrylamide gel and blotted to PVDF membrane. Blots were probed with the following primary antibodies: anti-NLRP3 (1:1000), anti-caspase-1 (1:500), anti-IL-1β (1:800), and anti-β-actin (1:5000). The signal was visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) according to the manufacturer’s instructions. The signals were also monitored by the Odyssey IR imaging system.

All values are expressed as the mean ± SEM and were analyzed by one-way ANOVA or Student’s t test as appropriate by using Prism 5.0 software (GraphPad Software, San Diego, CA). P < 0.05 was considered significant.

A classic systemic model is based on the administration of MPTP, with selective toxicity for dopaminergic neurons [17]. To explore the neuroprotective effects of tenuigenin in PD, we established a MPTP-induced acute model of PD in adult mice and determined whether tenuigenin improved MPTP-induced motor deficits. Experimental procedure and drug administration are shown in Fig. 2a. Open field test is a commonly used method to evaluate motor impairment. As shown in Fig. 2b, MPTP group was found to have a significant reduction in total movement distance compared to the saline group. MPTP mice pretreated with tenuigenin in both high dose (50 mg/kg) and low dose (25 mg/kg) exhibited an improvement in locomotor activity compared to MPTP group. Then, we used an accelerating rotarod test to determine motor coordination and balance. As shown in Fig. 2c, MPTP injections remarkably decreased the latency time compared with those in the saline treatment group. Moreover, high dose of tenuigenin displayed better effect on the improvement of motor behavior. These results indicate that tenuigenin administration recovered MPTP-induced motor impairment.

Furthermore, we detected the levels of DA and its metabolites, DOPAC and HVA, in the striatum by HPLC analysis. The results showed that these neurotransmitter substances in MPTP PD mice were significantly decreased by 44.4, 56.3, and 54.5%, respectively, compared with those in saline-treated mice. However, treatment of MPTP PD mice with 50 mg/kg tenuigenin increased DA level compared with untreated PD mice, while the levels of DOPAC and HVA did not significantly increase (Fig. 2d–f). Then, we calculated the ratio of (DOPAC + HVA)/DA which represented the rate of DA metabolism. As shown in Fig. 2g, high dose of tenuigenin decreased the rate of DA metabolism accelerating by MPTP. These results suggest that tenuigenin inhibited DA metabolism and elevated DA level in the striatum of MPTP PD mice.

To demonstrate whether tenuigenin protects dopaminergic neurons from MPTP damage, we detected tyrosine hydroxylase in SNc by immunohistochemistry. As shown in Fig. 3a, administration of MPTP resulted in a 38.9% loss of TH⁺ neurons in SNc compared with those in saline-treated mice. Interestingly, high dose (50 mg/kg) of tenuigenin significantly increased the number of TH⁺ neurons by 49.2% in the SNc of MPTP PD mice, while low dose (25 mg/kg) of tenuigenin increased by 20.6%. These results indicate that tenuigenin exerts a beneficial effect on dopaminergic neuronal degeneration.

Activation of microglia in both striatum and substantia nigra had been well documented to occur in the MPTP model of PD [2, 18]. Our previous studies had reported the NLRP3 inflammasome was activated in the substantia nigra of MPTP mice [11]. So we examined whether tenuigenin had an impact on the activation of NLRP3 inflammasome. As shown in Fig. 3c–g, MPTP treatment significantly evaluated the levels of inflammasome including NLRP3, caspase-1, pro-IL-1β, and IL-1β, while tenuigenin suppressed the activation of NLRP3 inflammasome in substantia nigra, especially with the high dose of tenuigenin. These date imply that tenuigenin inhibits the activation of NLRP3 inflammasome induced by MPTP in substantia nigra.

Then, we sought to determine whether tenuigenin has an effect on NLRP3 inflammasome activation in vitro. We used an immortalized murine microglial cell line, BV2, because it is an ideal alternative model system for primary microglia cultures [19]. The NLRP3 inflammasome is activated by endogenous stress-associated danger signals, such as ATP, nigericin, and MSU [9]. Since inflammasome activation is secretion of the cleaved bioactive form of IL-1β, researchers working in the inflammasome field typically “prime” the cells with LPS before performing the actual stimulation of the inflammasome with activators. Therefore, LPS-primed BV2 cells were pre-treated with tenuigenin before ATP challenge in the presence or absence of tenuigenin for 24 h. The results showed ATP induced caspase-1 activation and increased IL-1β production in BV2 microglia. The expression levels of those components of inflammasome, such as NLRP3, caspase-1, pro-IL-1β, and IL-1β, were downregulated by tenuigenin in a concentration-dependent manner (Fig. 4a–e). In particular, the inhibition of IL-1β secretion in cell supernatant also followed in a dose-dependent manner by Western blot and ELISA analysis (Fig. 4f). These data indicate that tenuigenin indeed can suppress NLRP3 inflammasome activation in microglia. To dissect the underlying molecular mechanisms, we measured the ROS production, which is believed to be a common NLRP3 activator. As shown in Fig. 4g, LPS-primed BV2 cells were pretreated with tenuigenin in different concentration before ATP challenge. High dose (8 μM) of tenuigenin remarkably decreased ATP-induced ROS production as detected by the fluorescence of H2DCFDA in BV2 microglia.

In order to find whether tenuigenin only affect ATP-induced NLRP3 inflammasome activation, we examined another NLRP3 agonists, MSU. As shown in Fig. 5a–f, tenuigenin inhibited caspase-1 cleavage and IL-1β secretion induced by MSU, coincident with the result induced by ATP. The observed inhibitory effects of tenuigenin on NLRP3 inflammasome activation were also confirmed. These results suggest that tenuigenin is a potent and broad inhibitor for NLRP3 inflammasome activation.

LPS, the principal component of the outer membrane of Gram-negative bacteria, is used to raise pro-IL-1β levels prior to the NLRP3 inflammasome stimulation. Peripheral administration of LPS alone could activate microglia in the brain and lead to inflammasome activation in vivo [20, 21]. Therefore, we detected whether tenuigenin can inhibit the activation of microglia and NLRP3 inflammasome in vivo by the treatment of LPS. Mice were injected intraperitoneally with LPS in the presence or absence of tenuigenin. After 6 h, the serum samples and brains were harvested. We performed immunohistochemistry with the classic antibody specific for Iba-1 to assess morphological microglia activation in SNc. The results showed that a large number of activated microglia with enlarged cell bodies (arrows shown in Fig. 6a) were observed in the SNc of LPS-treated mice compared with the saline group, while tenuigenin administration dramatically suppressed the activation of microglia in SNc (Fig. 6a, b). Furthermore, tenuigenin treatment significantly decreased serum IL-1β production ascending by LPS (Fig. 6c). Then, we wanted to know whether tenuigenin can prevent NLRP3 inflammasome activation induced by LPS in vivo. To address this question, we precisely isolated the tissues of ventral midbrain in each group. Western blotting analysis revealed that LPS significantly increased NLRP3, caspase-1, pro-IL-1β, and IL-1β expression, and this effect was reversed by tenuigenin treatment especially in the high-dose group (Fig. 7a–e). These results suggest that tenuigenin can inhibit the NLRP3 inflammasome activation in vivo.