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

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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 Ca 2+ (Ca 2+ [c] ) level decreased rapidly and sustainably after SHL treatment, and then returned to homeostasis when SHL was withdrawn. Moreover, SHL decreases Ca 2+ [c] levels mainly through enhancing the mitochondrial Ca 2+ (Ca 2+ [m] ) uptake. After genetically silencing or pharmacologic inhibiting mitochondrial calcium uniporter (MCU), the effect of SHL on the Ca 2+ [c] level and MC degranulation was significantly weakened. Simultaneously, the activation of SHL on Ca 2+ [m] uptake was completely lost. Collectively, by activating MCU, SHL decreases Ca 2+ [c] level to stabilize MCs, thus exerting a remarkable anti-allergic activity, which could have considerable influences on clinical practice and research.

Shuang-Huang-Lian (SHL), a formula containing Lonicerae Japonicae Flos, Scutellariae Radix and Fructus Forsythiae, is consistently prepared by stringent manufacturing procedure from Chinese Pharmacopoeia 23 . Clinically, SHL products, generally considered as the antimicrobial agent, are delivered through different routes (e.g., oral, injectable and pulmonary routes, etc.) 23 , 24 , and widely used to treat upper respiratory tract infection, pneumonia, tonsillitis, and other respiratory diseases caused by bacterium/viruses 25 . Our previous studies indicated that SHL protected lung tissue from infections via the potential anti-inflammatory and anti-oxidative activities 25 , 26 . In addition, SHL has also been applied in the type I hypersensitivity, including bronchial asthma 27 , 28 , 29 , vernal keratitis 30 , urticaria and eczema 31 , by using the aerosol inhalation or intravenous drip. Indeed, the excellent MC stabilization effect of SHL was observed. The present study focused on the underlying molecular mechanism of SHL. Our findings reveal that, for the first time, SHL potently stabilizes MCs through decreasing Ca 2+ [c] level by activating MCU independent of Ca 2+ [c] rise, which is different from the conventional MC stabilizers (e.g., cromolyn sodium and ketotifen).

Thus, calcium mobilization is a critical event to the activation of MCs and intracellular Ca 2+ pools are the determining factors of MC degranulation 12 . Mitochondrial Ca 2+ (Ca 2+ [m] ) uptake is considered to buffer local or bulk cytosolic Ca 2+ (Ca 2+ [c] ) rises 13 . But until recently, the uniporter’s veil began to be lifted. Now, it is known that the uniporter is a multi-subunit Ca 2+ channel, with the Ca 2+ pore formed by mitochondrial calcium uniporter (MCU) protein 14 , 15 and accessory proteins, including MICU1 16 , MICU2 17 , MCUb 18 , MCUR1 19 and EMRE 20 . 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 channel 21 . 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 uptake 22 . The fact that mitochondria buffer the Ca 2+ [c] rises by accumulating Ca 2+ into their matrix raises the question whether the activating MCU may dampen MC degranulation for the treatment of allergy, anaphylaxis and asthma, etc.

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 stimuli 4 , 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 Ca 2+ from the endoplasmic reticulum (ER), which in turn induces the stromal interaction molecule 1-mediated opening of the store-operated Ca 2+ channel ORAI1 and then leads to the influx of extracellular Ca 2+ . The influx of Ca 2+ is amplified by short transient potential Ca 2+ channel 1. The increase in intracellular Ca 2+ levels and the activation of PKC trigger MC degranulation 10 , 11 .

According to the Chinese Pharmacopoeia 23 , SHL is a mixture of the extract of Scutellariae Radix (ES) and the extract of Lonicerae Japonicae Flos and Fructus Forsythiae (ELF). Thus, to identify the active constituents in SHL, we first evaluated the effects of ES and ELF at the equivalent concentrations in 2% SHL on Ca 2+ [c] in the resting RBL-2H3 cells. As shown in , ELF, rather than ES, significantly reduced Ca 2+ [c] levels compared with untreated control in a nontoxic manner (data not shown). Next, we tested the effects of 26 constituents from ELF ( Table S1 ) on the Ca 2+ [c] levels. shows that Ca 2+ [c] levels were significantly decreased in the presence of quercetin, caffeic acid, ursolic acid, D-(-)-quinic acid and methyl salicylate at 10 μg/ml in a nontoxic manner (data not shown), and the fore 3 constituents could be detected by the HPLC-UV according to the Chinese Pharmacopoeia 23 ( Fig. S3 ), suggesting that they might be the major active constituents of SHL.

In the isolated liver mitochondria from the MCU F/F and MCU −/− mice, the activation of SHL on the Ca 2+ [m] uptake was completely lost upon silencing of MCU ( ). Moreover, in the MCU −/− RBL-2H3 cells, the effect of SHL on the Ca 2+ [m] uptake and MC degranulation also disappeared ( ). It was in this MCU defective cells that we did not observe the effect of SHL in ( ), indicating that SHL did not affect NCX. Taken together, these findings reveal that SHL increases Ca 2+ [m] uptake through activating MCU to decrease Ca 2+ [c] level, thus dampens MC degranulation.

Calcium transport between the cytoplasm and the mitochondrial matrix involves the passage of Ca 2+ across both the outer and inner mitochondrial membranes (OMM and IMM). The overall permeability of the OMM for Ca 2+ is relatively high, while the IMM presents a tight barrier for Ca 2+ 40 . Early studies have revealed that MCU protein, which can form a Ca 2+ channel in lipid bilayer in the IMM, forms the basis of the primary mechanism for Ca 2+ [m] transport 14 , 15 , 41 . Moreover, our above result ( ) also showed that the effect of SHL on Ca 2+ [m] uptake can be completely blocked by ruthenium red, highly implicating that SHL enhanced Ca 2+ [m] uptake might through activating MCU. To verify whether MCU is indeed involved in the effect of SHL on Ca 2+ [m] , we silenced MCU in mice using a Entranster TM in vivo transfection reagent. The resulting mice, termed MCU −/− mice, lack MCU protein in peritoneal MCs and liver mitochondria compared with the negative control mice (MCU F/F ). The fluorescence intensity for the Ca 2+ [c] of FcεRI + cells, namely MCs, was analyzed by a flow cytometer (FACSCalibur, BD, USA). It was found that SHL potently reduced Ca 2+ [c] levels of peritoneal MCs in MCU F/F mouse with a decreased percentage of 29%, while this effect was notably weakened in the MCU −/− cells with only a decreased percentage of 3.3% ( ).

The rapid and reversible effect of SHL on Ca 2+ [c] strongly suggested an underlying non-genomic mechanism. To our knowledge, two ways are recognized to reduce Ca 2+ from the cytosol: extruding of Ca 2+ through Na + -Ca 2+ exchangers (NCX) and plasma membrane Ca 2+ -ATPase (PMCA), and (or) clearance of Ca 2+ by resequestration into the ER and mitochondria 36 . Our findings showed that under either inhibition of PMCA activity by alkaline pH 9.0 37 or suppression of sarco/endoplasmic Ca 2+ -ATPase (SERCA) by thapsigargin 38 , the reduction of SHL on Ca 2+ [c] was not affected ( ). Unexpectedly, SHL was still able to lower Ca 2+ [c] when the extracellular Na + was withdrawn 39 ( ), seemingly suggesting that SHL inhibited rather than activated NCX. But anyhow, the reduction of Ca 2+ [c] by SHL in the resting cells is independent of PMCA, NCX and SERCA. Then, we found that SHL significantly enhanced the Ca 2+ [m] uptake in a concentration dependent manner ( ). By using Calcium Green-5N, Ca 2+ [m] uptake was evaluated in the isolated mouse liver mitochondria, which is of advantage that Ca 2+ uptake phenotypes can be directly attributed to mitochondria. In agreement with the results in , SHL (≥0.06%) treatment led to a significant increase of Ca 2+ [m] uptake in response to extramitochondrial pulses of 50 μM of Ca 2+ ( ), which can be blocked by a MCU inhibitor ruthenium red 15 ( ). These results demonstrate that SHL decreases Ca 2+ [c] levels mainly through enhancing the Ca 2+ [m] uptake.

The above findings confirm that SHL dampens C48/80-MrgprX2 and IgE-FcεRI mediated MC degranulation 9 , 35 , both of which depends on the increase of Ca 2+ [c] concentration. Thus, we next investigated whether SHL could affect the Ca 2+ [c] level. As expected, ST challenge markedly elevated Ca 2+ [c] level in the sensitized RBL-2H3 cells, while pretreatment with SHL significantly reduced Ca 2+ [c] level in a concentration-dependent manner ( ), without a direct chelation (data not shown). Of note, the Ca 2+ [c] levels before ST challenge (at 0 min) had been significantly reduced in response to pretreatment with SHL compared with the control ( ), strongly suggesting that SHL decreased Ca 2+ [c] concentration before the IgE receptor cross-linking. To confirm this, we further measured the effects of SHL on the Ca 2+ [c] level in the resting RBL-2H3 cells. As shown in , the Ca 2+ [c] level decreased rapidly and sustainably after SHL treatment, and then returned to homeostasis when SHL was withdrawn. Similar effect was observed in both human (LAD2) and mouse (P815) MCs (data not shown). In contrast, cromolyn sodium and ketotifen did not affect Ca 2+ [c] in the resting cells (data not shown) at their effective concentrations on MC degranulation ( and ).

We next determined the effect of SHL on ST-induced active systemic anaphylaxis (ASA) in mice. As shown in , robust hypothermia was observed after ST challenge (ΔT ≈ −7 °C) compared with the normal control group, while pretreatment with SHL significantly attenuated the body temperature decrease (P < 0.01). Next, the effect of SHL on passive systemic anaphylaxis (PSA) was shown in and Fig. S2 , after ST challenge, the body temperature of the sensitized mice gradually decreased about 1.5 °C within 30 min, while SHL markedly prevented the body temperature decrease.

Except for C48/80-induced anaphylactoid reaction, IgE-FcεRI-mediated allergic reactions are another kind of anaphylaxis 33 . Due to the surface expression of the high-affinity FcεRI receptor for IgE and the release of chemical mediators after crosslinking 34 , the sensitized RBL-2H3 cells were used to assess the effect of SHL on the shrimp tropomyosin (ST)-induced degranulation. Our data showed that pretreatment with SHL concentration-dependently dampened IgE-FcεRI-mediated β-hexosaminidase release ( ).

Owing to the significant influence of SHL on the allergic mediator release in vitro, we next determined the effects of SHL on C48/80-induced anaphylactic shock in mice. As shown in and , intraperitoneal injection of C48/80 at 8 mg/kg caused a fatal anaphylactic shock with the mortality of 100%. In comparison, treatment of SHL either 30 min before or 5 min after C48/80 challenge under 2.5 ml/kg and 5 ml/kg dosages dramatically protected the mice against the anaphylactic shock and greatly reduced the mortality, showing the preventive and therapeutic effects of SHL on C48/80-induced anaphylactic shock in vivo.

Discussion

MCs are key effector cells that can act as potent initiators and amplifiers in allergy, immunity, and inflammation by secreting multiple mediators6,42. Our findings demonstrated that SHL markedly dampened C48/80- and IgE-mediated MC degranulation in vitro and in vivo ( and and and ), showing an impressive influence on the MC activation. Further study indicated that SHL stabilized MC via a rapid, potent and reversible effect on Ca2+[c] level of resting cells ( ), which is different from the conventional MC stabilizers (e.g., cromolyn sodium and ketotifen).

As a MC activator, C48/80 could induce a rapid release of allergic mediators and consequently lead to a systemic fatal anaphylaxis43,44. In accordance with previous studies45,46,47, intraperitoneal injection of C48/80 (8 mg/kg) induced a fatal anaphylactic shock with a mortality of 100% within 1 h. Unexpectedly but excitedly, by a single intraperitoneal treatment with 3.34 times adult oral dosage of SHL (5 ml/kg) or 600 times that of ketotifen (47 μmol/kg), the survival rate of SHL group were actually far more than that of ketotifen group ( and ).

At present, the commonly-used allergen in the IgE-FcεRI-mediated allergy research is ovalbumin (OVA) to mimic type I hypersensitivity48,49. However, our previous result showed that the sensibility of common mice response to OVA was not satisfactory, especially in the absence of an adjuvant (data not shown), which might be associated with the immune tolerance induced by a long-term consumption of eggs powder in rodents’ fodder50. As we known, seafood allergy is widely recognized as a universal health care issue51,52,53 and is one of the most common forms of food allergies54,55. Shrimp protein is a major allergen in the shellfish-induced allergy study56,57. Thus, we extracted a purified ST (Fig. S4) from the Metapenaeus ensis by isoelectric precipitation56. Satisfactorily, compared with OVA, ST dramatically elevated the total IgE level in the mouse sera, showing a more sensitive responsivity (data not shown). Therefore, ST instead of OVA was used in our study. In the IgE-FcεRI-mediated β-hexosaminidase release (in vitro) and PSA (in vivo), SHL exerted markedly anti-anaphylactic effects ( ). In ASA mice, SHL also significantly attenuated the body temperature decrease ( ). In particular, hypothermia in ASA mice was far more intense than that in PSA mice, and SHL exerted more effective protection on PSA than ASA ( ), which may be attributed to the fact that ASA is mediated not only by IgE, but also by IgG58.

Theoretically, it is feasible for a drug to stabilize MCs through buffering the Ca2+[c] rises via accumulating Ca2+[c] into mitochondrial matrix. However, difficulties lie in the experimental practices. To our knowledge, MCU mediates Ca2+ uptake into the matrix to regulate metabolism, cytoplasmic Ca2+ signaling and cell death59. The uptake is electrogenic, driven by the large voltage present across the IMM (ΔΨ m) developed by proton pumping by the respiratory chain21,60. 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 cycle64,65. 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 ( ) and enhanced Ca2+[m] uptake in the isolated liver mitochondria ( ), 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 ( ), 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, obesity67,68, diabetes67,68, 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.