The Three-Herb Formula Shuang-Huang-Lian stabilizes mast cells through activation of mitochondrial calcium uniporter

Abstract

Mast cells (MCs) are key effector cells of IgE-FcεRI- or MrgprX2-mediated signaling event. Shuang-Huang-Lian (SHL), a herbal formula from Chinese Pharmacopoeia, has been clinically used in type I hypersensitivity. Our previous study demonstrated that SHL exerted a non-negligible effect on MC stabilization. Herein, we sought to elucidate the molecular mechanisms of the prominent anti-allergic ability of SHL. MrgprX2- and IgE-FcεRI-mediated MC activation in vitro and in vivo models were developed by using compound 48/80 (C48/80) and shrimp tropomyosin (ST), respectively. Our data showed that SHL markedly dampened C48/80- or ST-induced MC degranulation in vitro and in vivo. Mechanistic study indicated that cytosolic Ca²⁺ (Ca²⁺_[c]) level decreased rapidly and sustainably after SHL treatment, and then returned to homeostasis when SHL was withdrawn. Moreover, SHL decreases Ca²⁺_[c] levels mainly through enhancing the mitochondrial Ca²⁺ (Ca²⁺_[m]) uptake. After genetically silencing or pharmacologic inhibiting mitochondrial calcium uniporter (MCU), the effect of SHL on the Ca²⁺_[c] level and MC degranulation was significantly weakened. Simultaneously, the activation of SHL on Ca²⁺_[m] uptake was completely lost. Collectively, by activating MCU, SHL decreases Ca²⁺_[c] level to stabilize MCs, thus exerting a remarkable anti-allergic activity, which could have considerable influences on clinical practice and research.

Introduction

Mast cells (MCs) originate from the haematopoietic progenitor cells that enter nearly all vascularized tissue, where they complete their maturation and, under some circumstances, can then migrate into epithelia^1,2,3. As tissue-resident cells, MCs are strategically situated at host-environment interfaces, such as the skin, respiratory and gastrointestinal tracts, ready to respond to immunogenic stimuli⁴, indicating that they act as key contributors of innate and adaptive immune responses^5,6. MCs are activated on IgE receptor (FcεRI) crosslinking, resulting in the release of a diverse array of preformed cytoplasmic granule-associated mediators (e.g., histamine and β-hexosaminidase, etc.), as well as newly synthesized proinflammatory lipid mediators, cytokines and chemokines^7,8. In the FcεRI-independent pathways, MCs may be activated by numerous stimuli including basic secretagogues [e.g., substance P, compound 48/80 (C48/80) and mastoparan], peptidergic drugs (e.g., icatibant), THIQ motif drugs (e.g., atracurium) and fluoroquinolone family antibiotics (e.g., ciprofloxacin). Recently research revealed that they are all ligands of MrgprX2, an orthologue of the human G-protein coupled mas-related gene receptor^9,10. But whichsoever, IgE-FcεRI- or MrgprX2-mediated MC signaling event, eventually results in the activation of protein kinase C (PKC) and the release of Ca²⁺ from the endoplasmic reticulum (ER), which in turn induces the stromal interaction molecule 1-mediated opening of the store-operated Ca²⁺ channel ORAI1 and then leads to the influx of extracellular Ca²⁺. The influx of Ca²⁺ is amplified by short transient potential Ca²⁺ channel 1. The increase in intracellular Ca²⁺ levels and the activation of PKC trigger MC degranulation^10,11.

Thus, calcium mobilization is a critical event to the activation of MCs and intracellular Ca²⁺ pools are the determining factors of MC degranulation¹². Mitochondrial Ca²⁺ (Ca²⁺_[m]) uptake is considered to buffer local or bulk cytosolic Ca²⁺ (Ca²⁺_[c]) rises¹³. But until recently, the uniporter’s veil began to be lifted. Now, it is known that the uniporter is a multi-subunit Ca²⁺ channel, with the Ca²⁺ pore formed by mitochondrial calcium uniporter (MCU) protein^14,15 and accessory proteins, including MICU1¹⁶, MICU2¹⁷, MCUb¹⁸, MCUR1¹⁹ and EMRE²⁰. Although the precise roles of these accessory proteins is far from clear, they are required either for the channel activity or for regulating MCU under various conditions. MCU, an approximate 40-kDa protein, possesses two predicted transmembrane domains, which forms (through oligomerization) a gated ion channel²¹. Mutation of a single amino acid (serine 259) resulted in a uniporter that loses the ability to be deactivated by the classical inhibitor ruthenium red. Moreover, mutations in the acidic linker domain resulted in markedly diminished calcium uptake²². The fact that mitochondria buffer the Ca²⁺_[c] rises by accumulating Ca²⁺ into their matrix raises the question whether the activating MCU may dampen MC degranulation for the treatment of allergy, anaphylaxis and asthma, etc.

Shuang-Huang-Lian (SHL), a formula containing Lonicerae Japonicae Flos, Scutellariae Radix and Fructus Forsythiae, is consistently prepared by stringent manufacturing procedure from Chinese Pharmacopoeia²³. Clinically, SHL products, generally considered as the antimicrobial agent, are delivered through different routes (e.g., oral, injectable and pulmonary routes, etc.)^23,59. The uptake is electrogenic, driven by the large voltage present across the IMM (ΔΨ m) developed by proton pum** by the respiratory chain^21,60. Balanced Ca²⁺_[m] is critical for the regulation of mitochondrial functions such as fission, fusion and ATP production⁶¹. On one hand, Ca²⁺_[m] rise is the stimulation of Ca²⁺-sensitive dehydrogenases of the Krebs cycle, tuning ATP synthesis to the increased needs of a cell; on the other hand, uncontrolled Ca²⁺_[m] overload can lead to the opening of the mitochondrial permeability transition pore with disruption of mitochondrial membrane potential (MMP)⁶². Excess Ca²⁺ entry in mitochondria has been associated with apoptosis and necrosis in many pathological states⁶³. Most recently, Vais and his colleagues found that mitochondria were protected from Ca²⁺ depletion and overload by a unique complex involving Ca²⁺ sensors on both sides of the IMM, coupled through EMRE⁵⁹. Obviously, the dynamic regulation of Ca²⁺_[m] is a highly sophisticated process. Thus, as a MC stabilizer through enhancing Ca²⁺_[m] uptake, how to strike a better balance between the effectivity and toxicity is a serious challenge. Our findings indicate that it is through activating MCU that SHL, which has been using for the allergic diseases clinically, decreases Ca²⁺_[c] level to stabilize MCs. Both the effectivity and safety (non-toxic) of SHL are compatible in vitro and in vivo, indicating that the Ca²⁺_[m] increase induced by SHL through activating MCU is sustainable to a certain degree. Of course, the pharmacological reversibility of SHL is also an essential factor. Moreover, excess Ca²⁺ entry in mitochondria (Ca²⁺_[m] overload) causes more reactive oxygen species (ROS) generation, a by-product of Krebs cycle, whose elevation is a key event that leads to further organelle depolarization and loss of MMP, thus resulting in a vicious cycle^64,65. Perhaps not by coincidence, SHL possesses scavenging effect on the excess intracellular ROS thus protecting MMP²⁵, which may also play an important role for striking the balance between effectivity and non-toxic.

It is generally recognized that MCU is a Ca²⁺-activated Ca²⁺ channel whose activation depends on the increase of Ca²⁺_[c] concentration¹⁵. But, unlike the known MCU agonist histamine¹⁵, SHL can activate MCU independent of Ca²⁺_[c] rise. Thus, we were able to observe that SHL rapidly reduced Ca²⁺_[c] levels in the resting cells (Fig. 3B) and enhanced Ca²⁺_[m] uptake in the isolated liver mitochondria (Fig. 4E), suggesting that the active constituents in SHL (e.g., quercetin, caffeic acid, ursolic acid, etc.) can rapidly enter into the cells to directly act on the mitochondria to active MCU. It is noteworthy that although five constituents reduced Ca²⁺_[c] of resting cells (Fig. 6B), their effective concentrations (10 μg/ml) on Ca²⁺_[c] was far higher than their equivalent concentrations in 2% SHL, indicating that there might be a series of active ingredients similar to these five constituents to collectively act on MCU to markedly stabilize MCs.

In summary, SHL is a potent inhibitor of MC activation through decreasing Ca²⁺_[c] level by activating MCU. By virtue of the effect on resting Ca²⁺_[c], the degree of MC activation was potently suppressed, which not only could limit allergic disease, but also might be beneficial to some non-allergic diseases involved MC activation, such as atherosclerosis⁶⁶, obesity^67,68, diabetes^67,68, chronic obstructive pulmonary disease⁶⁹, cancer⁷⁰, postoperative ileus⁷¹ and fibromyalgia⁷², etc. However, our finding, together with the fact that SHL has already been used in the clinic for decades, may offer a suitable novel target for the clinical management of aberrant MC activation in diseases.

Methods

Materials

SHL injection and its 2 intermediate fractions (ES and ELF), which were prepared according to the Chinese Pharmacopoeia²³ were from Duoduo Pharmaceutical Co., Ltd. (Jiamusi, Heilongjiang, China). C48/80, 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide, and Pluonic F-127 were purchased from Sigma-Aldrich (St Louis, MO, USA). Fluo-3 AM Ester and rhod-2/AM were from Biotium (San Francisco, CA, USA). Ketotifen and cromolyn sodium were from TCI (Tokyo, Japan) and National Institutes for Food and Drug Control (Bei**g, China), respectively. Calcium Green-5N and PerCP-eFluor 710 labeled anti-mouse FcεR1 antibody were obtained from Invitrogen (Carlsbad, CA, USA) and eBioscience (San Diego, CA, USA), respectively. The transfection reagents Entranster TM-in vivo and -H4000 were from Engreen Biosystem (Bei**g, China). Balb/c mice (male, 18–20 g) and SD rats (male, 160–180 g) were from Vital River Experimental Animal Services (Bei**g, China).

siRNA and plasmid

The MCU siRNA and their negative controls were synthesized by GenePharma Co., Ltd. (Shanghai, China). The plasmid pcDNA3.1-mito-GCaMP2 was a kind gift from Dr. **anhua Wang (Institute of Molecular Medicine, Peking University, Bei**g, China). In this plasmid, the GCaMP2 calcium indicator was ligated with a mitochondrial targeting sequence⁷³.

Cells

Rat basophilic leukemia cell line (RBL-2H3) and mouse mastocytoma cell line (P815) were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China). Human LAD2 cell line (from Michael D. Gershon, MD, Columbia University, USA) was presented as a gift from Prof. Renshan Sun (the Third Military Medical University, Chongqing, China). Peritoneal MCs were isolated from SD rats.

Isolation of ST

ST from the Metapenaeus ensis was extracted and purified by an isoelectric precipitation method as previously described⁵⁶. Protein content of the purified fraction was assayed by Bradford method⁷⁴, and the purity of the obtained ST was >98% (Fig. S4).

Production of mouse anti-ST monoclonal IgE

The preparation of antibody was similar to our previously described method⁷⁵ except for the substitution of Freund’s adjuvant for Imject Alum.

β-hexosaminidase release assay

The β-hexosaminidase release assay was performed as previously described with some modifications⁷⁶. For the measurements of IgE-induced β-hexosaminidase release, RBL-2H3 cells were seeded in the 48-well plates at 5.0 × 10⁵ cells/well and sensitized with anti-ST monoclonal IgE (25 μg/ml) at 37 °C overnight. The cells were washed by Hank’s balanced salt solution (HBSS) supplemented with 0.1% (w/v) BSA and pre-incubated with SHL in 120 μl of HBSS at 37 °C for 30 min followed by adding 20 ng/ml of ST for further 1.5 h incubation. 30 μl of the supernatant was transferred to a 96-well black flat-bottom plate accompany with 50 μl of substrate solution (0.57 mg/ml 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide in the buffer contained 133 mM sodium citrate and 133 mM NaCl, pH 4.3). The reaction proceeded at 37 °C for 1.5 h and was stopped by adding stop buffer (50 mM glycine and 5 mM EDTA-Na₂, pH 10.5; 200 μl/well). Fluorescence was determined with a fluorescence microplate reader at λ_ex 355 nm and λ_em 460 nm.

For the measurements of C48/80-induced β-hexosaminidase release, the LAD2 cells (6.0 × 10⁴ cells/well) or the peritoneal MCs (1.0 × 10⁶ cells/well) were seeded in the 96-well plates and pretreated with SHL at 37 °C for 30 min followed by adding C48/80 (10 μg/ml) for further 1.5 h incubation. β-hexosaminidase in the supernatant was determined.

C48/80-induced anaphylactic shock in mice

Balb/c mice were kept in standard laboratory conditions of temperature and humidity with a 12 h light/dark cycle. All experiments were carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Animals Ethics Committee of the Institute of Medicinal Plant Development of the Chinese Academy of Medical Sciences. The mice were given the intraperitoneal injection of C48/80 at 8 mg/kg⁷⁷. For the preventive effect, SHL or the positive controls (ketotifen or cromolyn sodium) was i.p. injected only once 30 min before C48/80 administration (designated “single treatment”). For the therapeutic effect, SHL or the positive controls was i.p. injected only once 5 min after C48/80 challenge. Mortality was monitored for 1 h after induction of anaphylactic shock.

PSA

Mice were passively sensitized intravenously (i.v.) with 40 μg/mouse of anti-ST monoclonal IgE, while the control group were given the equal volume of physiologic saline. 24 h later, the mice were challenged (i.v.) with 20 μg/mouse of ST after pretreatment with SHL or physiologic saline (i.p.) for 20 min. The rectal temperature was measured by a thermal probe (ChengDu Instrument Factory, China) for 30 min using a polygraph (RM6240, Chengdu, China).

ASA

Mice received an i.p. injection of 100 μl of Imject Alum containing 60 μg/mouse ST and were immunized again 7 days later. 2 days after the second immunization, the mice were pretreated with SHL (2.5 ml/kg or 5 ml/kg) or physiologic saline (Control group and ST model group) for 30 min and then challenged by a rapid intravenous infusion (via the lateral tail vein) of 5 μg/mouse of ST. The mice in the control group were received the same solution without ST. To monitor changes in body temperature associated with anaphylaxis, rectal temperature was measured 30 min after ST challenge.

Measurement of Ca²⁺_[c] level

Measurement of the Ca²⁺_[c] level was performed using the calcium-reactive fluorescence probe Fluo-3/AM as previously described with slight modifications⁷⁸. Briefly, the cells were resuspended (2 × 10⁶ cells/ml) and incubated for 30 min in the dark at 30 °C with Fluo-3/AM (4 μM) in the presence of 0.04% (w/v) Pluonic F-127 in HEPES buffer (10 mM HEPES, 135 mM NaCl, 1 mM Na₂HPO₄, 1 mM CaCl₂, 5 mM KCl, 0.5 mM MgCl₂, 5 mM glucose and 0.1%BSA, pH 7.4). 4 mM probenecid was added to avoid leakage of fluo-3. After removing the dye, the cells were treated with SHL or normal saline (the control group) and the fluorescent intensity was immediately determined at λ_ex 485 nm and λ_em 538 nm using a spectrofluorimeter (Thermo Electron, Washington, USA).

To assay the Ca²⁺_[c] levels in mouse peritoneal MCs, the cells in the mouse abdominal cavity were isolated and loaded with Fluo-3/AM (4 μM) at 30 °C for 30 min. MCs could be recognized and analyzed by a FACSCalibur flow cytometer after staining with PerCP-eFluor 710 labeled anti-mouse FcεR1 antibody at 25 °C for 15 min.

Measurement of Ca²⁺_[m] uptake

Measurement of Ca²⁺_[m] level in RBL-2H3 cells was performed using the mitochondrially localizing Ca²⁺-reactive fluorescence probe, rhod-2/AM, as previously described⁷⁹. To improve the discrimination between cytosolic and mitochondrially localized dye, 5 μM rhod-2/AM was reduced to the colorless, nonfluorescent dihydro-rhod-2/AM by sodium borohydride, according to the manufacturer’s protocol. RBL-2H3 cells were loaded with dihydro-rhod-2/AM (2 μM) at 37 °C for 1 h. The residual cytosolic fraction of the dye was eliminated when the cells were kept in primary culture for an additional 16 h after loading, whereas the mitochondrial dye fluorescence was maintained. The fluorescence intensity of rhod-2 was determined at λ_ex 535 nm and λ_em 590 nm.

Mouse liver mitochondria were isolated and further purified⁸⁰. Ca²⁺_[m] uptake was measured using Calcium Green-5N according to previously described¹⁴.

The recombinant adenovirus (Ad.m-GCaMP2) based on the pcDNA3.1-mito-GCaMP2 was produced by Hanbio Biotechnology Co., Ltd. (Shanghai, China). Cells were infected with Ad.m-GCaMP2. 24 h later, the fluorescence signal in the mitochondria was captured by confocal microscopy (Fluoview FV1000, Olympus, Japan) using a 100× oil objective.

MCU siRNA transfected in vivo and in vitro

The mice were injected via tail vein with MCU siRNA (siRNA-MCU1#: sense 5′-GCG CCA GGA AUA UGU UUA UTT-3′ and antisense 5′-AUA AAC AUA UUC CUG GCG CTT-3′; siRNA-MCU2#: sense 5′-CCA AAG AGA GAC CUC CUA ATT-3′ and antisense 5′-UUA GGA GGU CUC UCU UUG GTT-3′) or the negative control siRNA (sense 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and antisense 5′-ACG UGA CAC GUU CGG AGA ATT-3′) on days 1 and 4 (3 OD/mouse). Entranster TM-in vivo transfection reagent was used to deliver the siRNA according to the manufacturer′s recommendations. On day 5, the mice were sacrificed after anesthetization. Fresh liver and the peritoneal MCs were immediately isolated for the assay.

The rat MCU siRNA (siRNA-MCU1#: sense 5′-GCC AGA GAC AGA CAA UAC UTT-3′ and antisense 5′-AGU AUU GUC UGU CUC UGG CTT-3′; siRNA-MCU2#: sense 5′-GGA GAA GGU ACG GAU UGA ATT-3′ and antisense 5′-UUC AAU CCG UAC CUU CUC CTT-3′) or the negative control (sense 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and antisense 5′-ACG UGA CAC GUU CGG AGA ATT-3′) was transfected into RBL-2H3 cells using Entranster TM-H4000 as described in the manufacturer’s protocol.

Statistical analysis

Data represent the mean ± SD of at least three independent experiments. Statistical analysis was performed by one-way ANOVA. A student’s t test was used when only two groups were compared. The difference was considered to be statistically significant when P < 0.05.