Effect of kaempferol on IgE-mediated anaphylaxis in C57BL/6 mice and LAD2 cells.

BACKGROUND: Immunoglobulin E (IgE)-mediated mast cell (MC) activation is crucial in multiple allergic diseases. Parkinson disease protein 7 (DJ-1) and Lyn kinase were reported as the receptor-proximal events in IgE receptor (FcεRI) signals in human MC. Kaempferol, a natural flavonol mainly derived from the rhizome of traditional Chinese herb Kaempferia galanga L. (Zingiberaceae), has been known to inhibit allergic reactions, but it was limited to the receptor-distal signals on rat basophilic leukemia cells. A thorough investigation of the inhibitory effects of kaempferol on human MC has not been done. PURPOSE: To investigate the inhibitory effects of kaempferol on IgE-mediated anaphylaxis in vivo and in human MCs, as well as the mechanism underlying its effects, especially the receptor-proximal signals. METHODS: IgE-mediated passive cutaneous anaphylaxis and systemic anaphylaxis model were applied to elucidate the antiallergic activity of kaempferol in vivo. The degranulation assay, calcium imaging, the release of cytokines and chemokines on the laboratory of allergic disease 2 (LAD2) cells were used to evaluate the antiallergic effect of kaempferol in vitro. Western blot analysis was performed to investigate the DJ-1/Lyn signaling pathway and downstream molecules. Kinase activity assay, immunofluorescence, and molecular docking were conducted to confirm the influence of kaempferol on DJ-1/Lyn molecules. RESULTS: Kaempferol dose-dependently attenuated ovalbumin/IgE-induced mice paw swelling, primary MC activation from paw skin, as well as rehabilitated the hypothermia, and reduced the serum concentrations of histamine, tumor necrosis factor-alpha, interleukin-8, and monocyte chemo-attractant protein-1. Additionally, kaempferol suppressed IgE-mediated LAD2 cell degranulation and calcium fluctuation. Remarkably, kaempferol was found to bind with DJ-1 protein, and initially prevented DJ-1 from translocating to the plasma membrane, thereby inhibited full activation of Lyn, and eventually restrained those receptor-distal signaling molecules, involved Syk, Btk, PLCγ, IP3R, PKC, MAPKs, Akt and NF-κB. CONCLUSION: Kaempferol could be used as a DJ-1 modulator for preventing MC-mediated allergic disorders through attenuating Lyn activation.

Conbercept and Ranibizumab Pretreatments in Vitrectomy with Silicone Oil Infusion for Severe Diabetic Retinopathy.

PURPOSE: We compared the efficacies of intravitreal ranibizumab (IVR) and intravitreal conbercept (IVC) as the adjuvant pretreatments for vitrectomy with silicone oil infusion for tractional retinal detachment (TRD) secondary to proliferative diabetic retinopathy. METHODS: This retrospective study comprised 74 patients (79 eyes) who underwent vitrectomy with silicone oil tamponade for diabetic TRD. They received IVC (37 eyes) or IVR (42 eyes) at standard doses 3-5 days preoperatively and were followed up for ∼6 months. Anatomic success rate, intra- and postoperative complications, and visual outcomes were compared between both groups. RESULTS: Initial (IVC vs. IVR: 97% vs. 98%) and final anatomic success rates (100% in each group) and mean visual acuity changes were not significantly different (P = 0.46). Intraoperative complications [iatrogenic retinal breaks (P = 0.58) and intraoperative bleeding (P = 0.66)], postoperative complications [fibrin formation (P = 0.51), postoperative preretinal bleeding (P = 0.88), progressing or persistent neovascular glaucoma (P = 0.63), progressive fibrovascular proliferation (P = 0.93), and recurrent retinal detachment (P = 0.93)], and surgical variables [surgical time (P = 0.53)] were similar between both groups. CONCLUSIONS: Conbercept and ranibizumab are equally effective surgical adjuvants for vitrectomy with silicone oil infusion in patients with diabetic TRD.

The anti-anaphylactoid effects of hydroxysafflor yellow A on the suppression of mast cell Ca2+ influx and degranulation.

BACKGROUND: Anaphylaxis is a type of potentially fatal hypersensitivity reaction resulting from the activation of mast cell mediators, especially histamine and lipid mediators. Non-IgE-mediated anaphylaxis can occur because of the direct activation of mast cells. Hydroxysafflor yellow A (HSYA) is the main chemical component of safflower (Carthamus tinctorius) and has been reported to have pharmacological activities. However, the anti-anaphylactoid effect of HSYA has not yet been investigated. PURPOSE: The aims of this study were to evaluate the anti-anaphylactoid activity of HSYA in vivo and to investigate the underlying mechanism in vitro. METHODS: The anti-anaphylactoid activity of HSYA was evaluated in a mouse model of hindpaw extravasation. Calcium imaging was used to assess intracellular Ca2+ mobilization. The levels of cytokines and chemokines released by stimulated mast cells were measured using enzyme immunoassay kits. Western blotting was used to explore the related molecular signaling pathways. RESULTS: HSYA markedly inhibited mast cell degranulation by suppressing the activation of intracellular Ca2+ mobilization and preventing the release of cytokines and chemokines from mast cells in a dose-dependent manner via the PKC-PLCγ-IP3R signaling pathway. CONCLUSION: In summary, HSYA has anti-anaphylactoid pharmacological activity, which makes it a potential candidate for the development of a novel agent to suppress drug-induced anaphylactoid reactions.

[Rheological properties of sinomenine in situ liquid crystal as liquid embolic agent].

Three different kinds of sinomenine in situ liquid crystal were prepared for different prescriptions, to investigate the rheological properties before and after in situ treatment and evaluate its feasibility for embolization. Rheological experiments were carried out with cone plate fixtures. Both the steady-state rheological and non-steady-state rheological properties of in-situ gels and the swelling gels were studied and compared. Steady-state rheological study results showed that all the three liquid embolic agents were non-newtonian fluid before and after in situ treatment, which would become less ropy when they were pressed with shear stress; their viscosities differed by 2-5 orders of magnitude. It had a yield value of about 10 Pa before in situ treatment and about 4 500 Pa after in situ treatment. All the six systems had thixotropy while their dynamic viscosities were not influenced by the shear rate, all less than 0.3 Pa·s before in situ treatment more than 1 Pa·s after in situ treatment, differing by an order of magnitude. The results of temperature sweeping showed a slight decrease with a steady rate in viscosity within the range of 10-50 °C, differing by 3-4 orders of magnitude. The results of unsteady rheology showed that there was no obvious linear viscoelastic region in the three kinds of agents, indicating the properties of liquid. After in situ treatment, their linear viscoelastic range γ<1% (No.3 was 5%), and their elastic modulus G' was larger than the viscous modulus G", indicating the properties of solid. Frequency scanning results showed that for the systems at low frequencies, G">G', system viscosity in a dominant position; while at high frequencies, G'>G", system elasticity in a dominant position. The results of compound viscosity test also proved that the liquid embolic agent in situ can form a cubic liquid crystal (the structure of No. 3 was destroyed after in situ treatment). The DHR-2 rheometer was used to investigate the rheological properties of in situ gels with three different prescriptions. The method is simple and the result is reliable, which can provide more theoretical reference for the in vitro evaluation and practical application of the product.

The anti-inflammation effect of Baige capsule and its principal components mixture in MCAO rats.

BACKGROUND: Baige (BG) is a compound Chinese herbal preparation, constituted of different position extracts (ethanol extracts from Pueraria lobate and SFE-CO2 extracts from Radix Angelicae dahuricae) of P. lobata and A. dahurica to treat the brain injury in patients. AIM: The goal of this study was to identify the neuroprotective properties of BG and its principal component mixture (PCM) and verify whether the material basis for BG is its PCM. METHODS: Middle cerebral artery occlusion (MCAO) was operated on male Sprague-Dawley rat for 2 h, different doses of BG or PCM or vehicle were gavaged after 3 h of MCAO. Rats were sacrificed after 30 days treatment. Blood serum inflammation factors and NGF were detected by ELISA. RESULTS: After 30 days of treatment, both BG and PCM interventions reduced the infarct volume, modified neurological severity score (mNSS) in rats, declined IL-1ß and IL-6 levels in the serum, increased NGF level in the serum and recovered the number of Nissl body in injured brain. CONCLUSIONS: Both BG and PCM exert equivalent levels of recovery effect in MCAO on rats; and PCM is the material foundation of BG. This recovery effect is associated with inflammatory inhibition and NGF production.

Harpagoside-induced anaphylactic reaction in an IgE-independent manner both in vitro and in vivo.

BACKGROUND: Harpagoside (HAR) is an active component of Scrophularia ningpoensis (SN), which has anti-inflammatory and anti-immune effects. SN is used widely in China to treat various diseases. Recently, SN has been used as a traditional Chinese medicine injection and used clinically. However, allergic responses to these injections are frequently reported. AIM: We examined whether the main component of SN, HAR, is associated with the allergic reaction to SN. METHODS: This study assessed the effects of HAR in mice and mast cell activation to characterize its anaphylactic effects and underlying mechanisms. Mice hindpaw swelling, serum allergy factor detection, enzyme-linked immunosorbent assays, and degranulation assays were performed to measure allergic mediators both in vivo and in vitro. RESULTS: The present study indicated that HAR induced paw swelling, interleukin-6, inositol triphosphate, tumor necrosis factor-α, and histamine increases in mice. Our in vitro data also showed that HAR induced ß-hexosaminidase, inositol triphosphate, and interleukin-6 release, leading to mast cell degranulation. In contrast, neither C48/80 nor HAR induced local anaphylaxis in STOCK KitW-sh/HNihrJaeBsmJNju mice. CONCLUSIONS: HAR is a potential sensitization compound in SN, and these results provide information for the safe clinical use of SN.

Saikosaponin A inhibits compound 48/80-induced pseudo-allergy via the Mrgprx2 pathway in vitro and in vivo.

Pseudo-allergic reactions-adverse, non-immunologic, anaphylaxis-like sudden onset reactions mediated through an IgE-independent pathway-are activated by various basic compounds and occur at least as frequently as IgE-mediated reactions to drugs. A large family of G protein coupled receptors (Mas-related genes; Mrgprs) is closely related to pseudo-allergies. However, few therapies can directly target pseudo-allergies and related Mrgprs. Saikosaponin A (SSA) is effective in the treatment of passive cutaneous anaphylaxis (PCA), adjuvant arthritis, and delayed hypersensitiveness. In this study, we investigated the anti-pseudo-allergy effect of SSA and its underlying mechanism. We examined the effect of SSA on both IgE-independent and IgE-dependent responses using PCA and active systemic anaphylaxis models, as well as in vitro-cultured mast cells. We also evaluated whether the anti-allergy effect is related to Mrgprs by using in vitro Mrgprx2-expressing HEK293 cells. SSA dose dependently suppressed compound 48/80 (C48/80)-induced PCA and mast cell degranulation in mice. When SSA and C48/80 were administered together through the vein, C48/80-induced systemic anaphylaxis did not occur, and C48/80-induced shock ratio decreased dose-dependently upon SSA treatment. However, SSA did not affect IgE-dependent allergy. When administered topically 24 h before antigen challenge, Evans blue leakage and paw swelling were induced in the SSA-treated group and the vehicle group. Our in vitro studies revealed that SSA reduced C48/80-induced calcium flux and suppressed degranulation in LAD2 cells. SSA could also dose-dependently inhibit C48/80-induced Mrgprx2-expressing HEK293 cell activation. As a conclusion, SSA could inhibits IgE-independent allergy, but not IgE-dependent allergy, and this effect involves the Mrgprx2 pathway. This study provided a new sight on pseudo-allergy and its therapy.

Screening anaphylactic components of MaiLuoNing injection by using rat basophilic leukemia-2H3 cell membrane chromatography coupled with HPLC-ESI-TOF-MS.

MaiLuoNing injection is a traditional Chinese medicine that used clinically since the 1950s in China. However, anaphylactic reactions, through the potentiation of mast cell degranulation, have been reported. In the present study, a rat basophilic leukemia-2H3 cell membrane chromatography coupled with high-performance liquid chromatography and electrospray ionization-ion trap-time of flight-mass spectrometry method was established for screening, analyzing, and identifying the potential anaphylactic components of MaiLuoNing injection. Harpagoside, a potential degranulator of rat basophilic leukemia-2H3 cells, was retained in rat basophilic leukemia-2H3 cell membrane chromatography. We aimed to evaluate the retained components to determine which of those were capable of inducing degranulation of basophilic leukemia cells. A ß-hexosaminidase assay revealed that harpagoside can induce rat basophilic leukemia-2H3 cell degranulation in a dose-dependent manner. BLBA/c mice also exhibit passive cutaneous anaphylaxis in response to harpagoside. These results indicate that rat basophilic leukemia-2H3 cell membrane chromatography coupled with high-performance liquid chromatography and electrospray ionization ion trap time-of-flight mass spectrometry is effective in screening for the anaphylactic components of MaiLuoNing injection.

Cloning and characterization of a phosphate transporter gene in Dunaliella salina.

The full-length cDNA of a Na(+) -dependent Pi transport gene (DsSPT1) in Dunaliella salina was cloned by 3' and 5' Rapid Amplification of cDNA Ends (RACE), with an open reading frame (ORF) encoding 716 predicted amino acids, which exhibited 60.5% identity to that of Na(+) -dependent Pi transport 1 (DvSPT1) from Dunaliella viridis. Hydrophobicity and secondary structure prediction revealed 11 conserved transmembrane domains similar to those found in DvSPT1 from D. viridis and PHO89 from Saccharomyces cerevisiae. The result of real-time quantitative PCR showed that expression level of DsSPT1 was enhanced at first and reached its peak at 90 min after salt stress; however, D. salina cells rapidly absorbed extracellular inorganic phosphorus which was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) during the first 5 min under salt stress. It suggested that D. salina on the absorption of inorganic phosphorus was regulated at DsSPTI posttranslational level.