Endoplasmic reticulum stress drives macrophages to produce IL-33 to favor Th2 polarization in the airways.

Interleukin (IL)-33 is a key driver of T helper 2 (Th2) cell polarization. Endoplasmic reticulum (ER) stress plays a role in the skewed T cell activation. The objective of this project is to elucidate the role of IL-33 derived from macrophages in inducing Th2 polarization in the airways. In this study, bronchoalveolar lavage fluids (BALF) were collected from patients with asthma and healthy control subjects. Macrophages were isolated from the BALF by flow cytometry cell sorting. An asthmatic mouse model was established using the ovalbumin/alum protocol. The results showed that increased IL33 gene activity and ER stress-related molecules in BALF-derived M2a macrophages was observed in asthmatic patients. Levels of IL33 gene activity in M2a cells were positively correlated with levels of asthma response in asthma patients. Sensitization exacerbated the ER stress in the airway macrophages, which increased the expression of IL-33 in macrophages of airway in sensitized mice. Conditional ablation of Il33 or Perk or Atf4 genes in macrophages prevented induction of airway allergy in mice. In conclusion, asthma airway macrophages express high levels of IL-33 and at high ER stress status. Inhibition of IL-33 or ER stress in macrophages can effectively alleviate experimental asthma.

Cortisol synergizing with endoplasmic reticulum stress induces regulatory T-cell dysfunction.

The dysfunction of regulatory T cell (Treg) is associated with the pathogenesis of many immune diseases. The regiments used to re-establish Treg's function are currently unsatisfactory and need to be improved. The purpose of this study is to elucidate the synergistic effects of cortisol and endoplasmic reticulum (ER) stress on impairing regulatory T cell functions. In this study, blood samples were collected from patients with food allergy (FA). Immune cells were purified from blood specimens by flow cytometry. A mouse model of FA was established with ovalbumin as a specific antigen. We observed that serum cortisol levels of FA patients were negatively correlated with peripheral Treg counts. Overwhelmed ER stress status was detected in Tregs of FA patients. The antigen-specific immune response induced ER stress in Tregs, which was exacerbated by concurrent cortisol exposure. ER stress mediated the effects of cortisol on impairing the immune suppressive ability of Tregs. The expression of Rnf20 was observed in Tregs upon exposure to cortisol. Rnf20 reduced the expression of Foxp3 and transforming growth factor (TGF)-ß in Tregs. Rnf20 inhibition re-established the immunosuppressive functions of Tregs obtained in patients with FA. The experimental FA in mice was attenuated by inhibition of Rnf20 in Tregs. In summary, specific immune response in synergy with cortisol to induce the expression of Rnf20 in Tregs. Rnf20 reduces the levels of Foxp3 and TGF-ß to impair the immune suppressive function. Inhibition of Rnf20 can restore the immune suppressive ability of Tregs obtained from FA patients.

Signals from the TAFA4-PTEN-PU.1 axis alleviate nasal allergy by modulating the expression of FcεRI in mast cells.

The high-affinity IgE receptor, FcεRI, plays a key role in the antigen-induced mast cell activation. Regulations for FcεRI are not yet well understood. TAFA4 is a molecule derived from neuron tissues, and has immune regulation functions. This study aims to clarify the role of TAFA4 in the regulation of FcεRI expression in mast cells. Nasal secretions were collected from patients with allergic rhinitis (AR) and healthy control (HC) subjects. TAFA4 levels of nasal secretions were evaluated by ELISA. A mouse model AR was developed using ovalbumin as the specific antigen. Negative correlation between TAFA4 and tryptase levels in nasal secretions was observed. TAFA4 could suppress the antigen-related mast cell activation. TAFA4 modulated the transcription of Fcer1g (FcεRI γ gene) in mast cells. Signals from the TAFA4-PTEN-PU.1 axis restricted FcεRI expression in mast cells. Administration of TAFA4 attenuated experimental AR. TAFA4 suppressed the expression of FcεRI in mast cells of airway tissues. TAFA4 can down regulate the expression of FcεRI in mast cells to suppress experimental AR. The data suggest that TAFA4 has translation potential to be developed as an anti-allergy therapy.

Short-Chain Fatty Acids Promote Immunotherapy by Modulating Immune Regulatory Property in B Cells.

The dysfunction of regulatory B cells (Breg) may result in immune inflammation such as allergic rhinitis (AR); the underlying mechanism is not fully understood yet. Short-chain fatty acids, such as propionic acid (PA), have immune regulatory functions. This study is aimed at testing a hypothesis that modulates PA production alleviating airway allergy through maintaining Breg functions. B cells were isolated from the blood obtained from AR patients and healthy control (HC) subjects. The stabilization of IL-10 mRNA in B cells was tested with RT-qPCR. An AR mouse model was developed to test the role of PA in stabilizing the IL-10 expression in B cells. We found that the serum PA levels were negatively correlated with the serum Th2 cytokine levels in AR patients. Serum PA levels were positively associated with peripheral CD5+ B cell frequency in AR patients; the CD5+ B cells were also IL-10+. The spontaneous IL-10 mRNA decay was observed in B cells, which was prevented by the presence of PA through activating GPR43. PA counteracted the effects of Tristetraprolin (TTP) on inducing IL-10 mRNA decay in B cells through the AKT/T-bet/granzyme B pathway. Administration of Yupinfeng San, a Chinese traditional medical formula, or indole-3-PA, induced PA production by intestinal bacteria to stabilize the IL-10 expression in B cells, which promoted the allergen specific immunotherapy, and efficiently alleviated experimental AR. In summary, the data show that CD5+ B cells produce IL-10. The serum lower PA levels are associated with the lower frequency of CD5+ B cells in AR patients. Administration with Yupinfeng San or indole-3-PA can improve Breg functions and alleviate experimental AR.

Identification of antigen-specific neutrophils in the tonsils with recurrent acute inflammation.

The pathogenesis of recurrent acute tonsillitis (Rtn) is to be further investigated. Polymorphonuclear neutrophils (PMN) often associate with the pathogenesis of acute and chronic inflammation. This study aims to identify the antigen-specific PMNs (sPMNs) isolated from the tonsillar tissues with recurrent acute inflammation. In this study, CD66b+ PMNs were isolated from surgically removed tonsils (40 tonsils were from 20 Rtn patients; 24 tonsils were from 12 tonsil tumour patients) by flow cytometry cell sorting. sPMNs were identified through immunological approaches. We found that compared with the control tonsil samples (from marginal non-tumour tissues of tonsil cancer), Rtn samples showed higher PMN frequency, higher levels of myeloperoxidase (MPO) and neutrophil elastase (NE), in which positive correlation was detected between the inflammatory scores in the Rtn tissues and PMN counts (r = .7352; p = .0002), or MPO (r = .6565, p = .0017), or NE (r = .6687, p = .0013). Upon exposure to tonsillar tissue protein extracts in the culture, a portion of Rtn PMNs was activated and released inflammatory mediators. A complex of tonsillar tissue-specific IgG and FcγRI was observed on the surface of Rtn PMNs; these PMNs could specifically recognize the Rtn tissue extracts and were designated the tonsillar antigen-specific PMNs (sPMNs). A signal transduction pathway of mitogen-activated protein kinase (MAPK)-nuclear factor of T cell activation (NFAT) was activated in sPMNs after exposure to Rtn tissue extracts. In summary, a fraction of sPMN in the Rtn tonsillar tissues was identified and characterized. The sPMNs can be activated upon exposure to tonsil-specific antigens. These sPMNs may contribute to the Rtn pathogenesis.

The low dose effects of human mammary epithelial cells induced by internal exposure to low radioactive tritiated water.

Tritium is an important radioactive waste which needs to be monitored for radiation protection. Due to long biological half-life of organically bound tritium (OBT), the adverse consequence caused by chronic exposure of tritiated water (HTO) attracts concern. In this study, fibroblast cells were exposed to 2 × 106 Bq/ml HTO to investigate the cellular behaviors. The dose relationship of survival fraction and γH2AX foci was a "U-shaped" curve. And the results of γH2AX intensity produced by ICCM, which was obtained from different doses, demonstrated bystander signal accounted for the protective effects induced by intermediate dose of 100 mGy. The comparison of temporal kinetics and spatial dynamics of DNA repair between tritium ß-rays and γ-rays showed longer time was need for the dephosphorylation of H2AX protein after HTO exposure. It indicated complex cluster DSBs induced by tritium ß-rays at the low dose impaired efficient recovery of DNA damage, which bear responsibility for the persistence of residual foci after low dose expsoure. It suggests after exposed to low dose radiation cells prefer to eliminate damage population to avoid DNA damage increasing the mutation potential.

Restoration of immune suppressor function of regulatory B cells collected from patients with allergic rhinitis with Chinese medical formula Yupingfeng San.

Immune dysregulation, such as defects in immune suppressor function, plays an important role in the pathogenesis of many immune disorders including allergic rhinitis (AR). Some Chinese traditional medical formulae have an immune regulatory function. This study aims to restore the immune suppressor function in regulatory B cells (Bregs) collected from AR patients with a Chinese medical formula, Yupingfeng San (YPFS). In this study, Bregs were isolated from blood samples collected from AR patients and healthy (HA) subjects. The capacity of Breg in suppressing effector T cell (Teff) proliferation was observed in an in vitro experiment to be used as an indicator of immune suppressor function of Breg. The effects of YPFS on promoting Bregs' immune suppressor functions were tested in a cell culture study. The results showed that the number of peripheral Breg in AR patients was not significantly different from that in HA subjects, while the immune suppressor function of AR Breg was compromised. Bcl2L12 expression was higher in AR Bregs than that in HA Bregs. A negative correlation was identified between expression of Bcl2L12 and IL-10 in AR Bregs. Exposure of AR Bregs to YPFS in the culture suppressed the expression of Bcl2L12 and improved their immune suppressor function. In conclusion, YPFS can restore the immune suppressor function of AR Bregs via inhibiting the expression of Bcl2L12. The data suggest that YPFS has the potential to be used in the improvement of immune dysfunction, such as AR.

Bcl2L12 mediates effects of protease-activated receptor-2 on the pathogenesis of Th2-dominated responses of patients with ulcerative colitis.

The immune dysregulation plays an important role in the pathogenesis of ulcerative colitis (UC). Bcl2 like protein-12 (Bcl2L12) and mast cells are involved in immune dysregulation of UC. This study aims to elucidate the role of Bcl2L12 in the contribution to the pathogenesis of T helper (Th)2-biased inflammation in UC patients. The results showed that Bcl2L12 was expressed by peripheral CD4+ T cells that was associated with Th2 polarization in UC patients. Bcl2L12 mediated the protease-activated receptor-2 (PAR2)-induced IL-4 expression in CD4+ cells. Activation of PAR2 increased expression of Bcl2L12 in CD4+ T cells. Bcl2L12 mRNA decayed spontaneously in CD4+ T cells after separated from UC patients which was prevented by activating PAR2. Bcl2L12 mediated the binding between GATA3 and the Il4 promoter in CD4+ T cells. Mice with Bcl2L12 deficiency failed to induce Th2-biased inflammation in the colon mucosa. We conclude that CD4+ T cells from UC patients expressed high levels of Bcl2L12; the latter plays an important role in the development of Th2-biased inflammation in the intestine. Bcl2L12 may be a novel therapeutic target in the treatment of Th2-biased inflammation.

Bcl2L12 Contributes to Th2-Biased Inflammation in the Intestinal Mucosa by Regulating CD4+ T Cell Activities.

The Th2-biased inflammation and immune deregulation play a critical role in the pathogenesis of ulcerative colitis (UC). Recent studies indicate that the Bcl2-like protein 12 (Bcl2L12) is associated with immune deregulation of UC. This study aims to investigate the role of Bcl2L12 in the induction of aberrant Th2-biased inflammation. In this study, peripheral blood samples were collected from patients with inflammatory bowel disease. The Th2 cell activities were analyzed by flow cytometry, real-time quantitative RT-PCR, and Western blotting. Mice with Bcl2L12-knockout CD4+ T cells were used in the experiments. The results showed that the expression of Bcl2L12 was detected in peripheral CD4+ T cells, which was significantly higher in UC patients than in healthy subjects. A positive correlation between the expression of Bcl2L12 and Th2 cytokines was detected in CD4+ T cells from UC patients. Naive CD4+ T cells with Bcl2L12 overexpression were prone to differentiate into Th2 cells. Mice with Bcl2L12 deficiency failed to induce the Th2-biased inflammation in the intestine. Bcl2L12 bound GATA3 to form a complex to enhance the binding between GATA3 and the Il4 promoter to enhance the expression of IL-4 in CD4+ T cells. CD4+ T cells with Bcl2L12 overexpression were resistant to apoptosis. In conclusion, the Bcl2L12 is a critical factor in the induction of aberrant Th2 polarization by upregulating Th2 responses and downregulating Th2 cell apoptosis. Bcl2L12 may be a novel therapeutic target in the management of the disorders with Th2-biased inflammation.

Simultaneous quantification six active compounds in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study of Pien-Tze-Huang.

Pien-Tze-Huang (PZH) is a popular traditional Chinese medicine (TCM) formula in China, but its pharmacokinetics has not been investigated yet. To better study the pharmacokinetic behaviors of PZH, an optimal ultra-performance liquid chromatography with triple quadrupole mass spectrometry (UPLC-MS/MS) method was developed for rapid quantification of six compounds (notoginsenoside R1, ginsenosides Re, Rg1, Rb1, Rd, and muscone) in rat plasma after oral administration of PZH. All analytes were extracted by protein precipitation with acetonitrile and separated on a Waters Acquity Cortecs C18 column within 3.9min, and detected by multiple-reaction monitoring in positive ion mode. This proposed method exhibited good linearity (r≥0.9932) with a lower quantification limits of 0.558-1.566ng/mL for all analytes. The intra- and inter-day precisions were within 8.24%, and the accuracy was within -10.05 to 9.87% for each analyte. The extraction recovery for each analyte ranged from 80.02 to 96.12%. This UPLC-MS/MS method was successfully applied to the pharmacokinetic study for PZH in rats.

Evaluation of toxicity studies of flavonoid fraction of Lithocarpus polystachyus Rehd in rodents.

The aim of the study was to evaluate the safety of flavonoid fraction of Lithocarpus polystachyus Rehd (Sweet Tea-F, ST-F) in mice and rats through acute and sub-chronic toxicity studies respectively. For acute toxicity study, a single dose of 5000 mg/kg of ST-F was given orally to healthy KM mice. The mice were observed mortality and toxic symptoms for 24 h, then once a day up to 14 days. In the sub-chronic toxicity study, ST-F was administered orally at doses of 0, 70, 140, 560 mg/kg/day to rats for 26 weeks. Body weight and food intake were recorded weekly. Hematological, biochemical, coagulation and organ parameters were analyzed at the end of 26 weeks administration. Vital organs were evaluated by histopathology. In the acute toxicity study, ST-F caused neither significant toxic symptoms, nor mortality in mice. In sub-chronic toxicity study, daily oral administration of ST-F at the dose of 70 mg/kg resulted in a significant increase (P < 0.05) in the relative body weight at the 10-week, and the same situation brought at the dose of 140 mg/kg/day at the 22-week. Hematological and biochemical showed significant changes (P < 0.01 or P < 0.05) in WBC, GLU, ALP, AST and serum electrolytes levels at the dose of 560 mg/kg/day. The amount of RBC decreased significantly (P < 0.05) while the content of PLT slightly increased (P < 0.05) at the dose of 140 mg/kg/day. In additional, no obvious histological changes were observed in vital organs of ST-F treated animals compared to control group. The ST-F may be exit slight side effects at the dose of 560 mg/kg/day in rats. Thus, the overall results show that the no-observed adverse effect level (NOAEL) of ST-F was considered to be 140 mg/kg for male SD rats.

Salvianolic Acid B Ameliorates Hyperglycemia and Dyslipidemia in db/db Mice through the AMPK Pathway.

BACKGROUND/AIMS: Salvianolic acid B (Sal B), a major polyphenolic compound of Salvia miltiorrhiza Bunge, has been shown to possess potential antidiabetic activities. However, the action mechanism of SalB in type 2 diabetes has not been investigated extensively. The present study was designed to investigate the effects of Sal B on diabetes-related metabolic changes in a spontaneous model of type 2 diabetes, as well as its potential molecular mechanism. METHODS: Male C57BL/KsJ-db/db mice were orally treated with Sal B (50 and 100 mg/kg) or metformin (positive drug, 300 mg/kg) for 6 weeks. RESULTS: Both doses of Sal B significantly decreased fasting blood glucose, serum insulin, triglyceride and free fatty acid levels, reduced hepatic gluconeogenic gene expression and improved insulin intolerance in db/db mice. High dose Sal B also significantly improved glucose intolerance, increased hepatic glycolytic gene expression and muscle glycogen content, and ameliorated histopathological alterations of pancreas, similar to metformin. Sal B treatment resulted in increased phosphorylated AMP-activated protein kinase (p-AMPK) protein expression in skeletal muscle and liver, increased glucose transporter 4 (GLUT4) and glycogen synthase protein expressions in skeletal muscle, and increased peroxisome proliferator-activated receptor alpha (PPARα) and phosphorylated acetyl CoA carboxylase (p-ACC) protein expressions in liver. CONCLUSION: Our data suggest that Sal B displays beneficial effects in the prevention and treatment of type 2 diabetes at least in part via modulation of the AMPK pathway.

Sweet tea leaves extract improves leptin resistance in diet-induced obese rats.

AIM OF THE STUDY: Dietary obesity is usually characterized by leptin resistance and abnormal lipid metabolism. Lithocarpus polystachyus Rehd.(Sweet Tea) leaf is a kind of Chinese folkloric medicine, and it has been widely used for obesity, diabetes, and hypertension in South China. The present study is aimed at investigating the pharmacological mechanism of the anti-hyperleptinaemia effects of Sweet Tea leaves extract in high fat diet-induced obese rats. MATERIALS AND METHODS: We induced high fat diet obesity for 14 weeks to test the corrective effects of three ST doses (75, 150 and 300 mg/kg per day) for 8 weeks. At the end of the experiment, body weight, fasting blood glucose and serum lipids, superoxide dismutase (SOD), malondialdehyde (MDA), fasting serum insulin and leptin, C-reactive protein, adiponectin and resistin levels were measured, Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) was also calculated. mRNA gene expression of PPARγ (peroxisome proliferator-activated receptor γ) and C/EBPα(CCAAT/enhancer-binding protein α) in epididymal adipose tissue of DIO control and experimental groups were evaluated. RESULTS: Sweet Tea leaves extract could significantly decrease the levels of serum lipids, attenuate body weight gain and lower circulating leptin and insulin levels, ameliorate the state of oxidative stress, raise serum adiponectin, reduce circulating CRP and resistin levels, and depress the expression of PPARγ and C/EBPα in epididymal adipose tissue of obese rats. CONCLUSION: The present findings suggest that ST can effectively attenuate the leptin resistance at least through anti-hyperlipidemic activity and thus has the therapeutic potential in treating hyperlipidemia and hyperleptinaemia related to dietary obesity.

[Effect of Nuanxin Capsule on two rat models with heart failure].

OBJECTIVE: To observe the effects of Nuanxin Capsule (NC) on the rat models of heart failure induced by abdominal aorta constriction and adriamycin. METHODS: Rats were randomly divided into the following groups: model control group, low-dose and high-dose, and digoxin group. Meanwhile, the pseudo-operation and NC groups were seperately established. After treatment for 30 days, the heart rate, systolic blood pressure (SBP), diastolic blood pressure (DBP), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximal rate of increase and decrease in left ventricular pressure (+/- dp/dt), mean peripheral blood pressure (MBP) as well as levels of serum superoxide dismustase (SOD), malondialdeh-vde (MDA), cardiac index and heart size were measured. RESULTS: SBP, LVSP, +/- dp/dt and SOD activity increased,while LVEDP,serum MDA levels decreased in high and low-dose NC groups of two models. The heart rates also decreased, but the difference was insignificant (P>0.05, compared with those of model group). Besides, the heart rate,heart size and cardiac index, as well as serum Ang II levels also decreased. The differences were significant as compared with the digoxin group (P>0.05). The high-dose NC also significatly improved MBP and SOD (P<0.05 and P<0.01). CONCLUSION: Nuanxin Capsule has good therapeutic effects on the rats models of adriamycin and abdominal aorta constriction induced heart failure.

The hypoglycemic activity of Lithocarpus polystachyus Rehd. leaves in the experimental hyperglycemic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves of Lithocarpus polystachyus Rehd. are used for the treatment of disorders such as diabetes, hypertension, and epilepsy in folk medicine of South China. The possible antidiabetic effects of the leaves were investigated in experimental type 2 and type 1 diabetic rats. MATERIALS AND METHODS: Type 2 diabetic rats received orally three different extracts of Lithocarpus polystachyus Rehd. leaves for 4 weeks (aqueous extract [ST-1], ethanol extract [ST-2], flavonoid-rich fraction [ST-3]). At the end of the experiment biochemical parameters were tested and livers and pancreases were excised for histological study. After the comparison of the pharmacological test results of the three extracts, the one which showed the best bioactivity was further studied to confirm its antidiabetes effect on both type 2 and type 1 diabetic rats. RESULTS: Compared to ST-1 and ST-2, ST-3 had better effects on regulation of blood glucose, glycosylated serum protein, cholesterol, triglyceride, malondialdehyde, superoxide dismutase and attenuation of liver injury in type 2 diabetic rats (p<0.01 or p<0.05). ST-3 administration for four weeks also significantly reduced the fasting serum insulin and C-peptide level and improved the insulin tolerance (p<0.05). In type 1 diabetic rats, ST-3 supplement for three weeks caused significant reduction in fasting blood glucose, total cholesterol, triglyceride, urea nitrogen, creatinine and liver mass, along with significantly inhibiting the decline of insulin level compared to diabetic control (p<0.05 or p<0.01). CONCLUSION: The flavonoid-rich fraction of Lithocarpus polystachyus Rehd. leaves (ST-3) had better beneficial effect than that of the ethanol or aqueous extract in experimental diabetic rats, which means that the bioactivity of the herbal leaves is probably due to the presence of flavonoids. The results also strongly suggest that the antidiabetic effect of ST-3 was possibly through multiple mechanisms of action including blood lipid and antioxidant mediation. The results indicated that the aqueous flavonoid-rich fraction of Lithocarpus polystachyus Rehd. leaves possessed significant protective activity in type 2 and type 1 diabetes.