[Anti-fatigue effect of Lubian on kidney Yin deficiency and kidney Yang deficiency mice and mechanism based on PI3K-Akt pathway].

This study aimed to investigate the anti-fatigue effect and mechanism of Lubian(Cervi Penis et Testis) on kidney Yin deficiency and kidney Yang deficiency mice. After one week of adaptive feeding, 88 healthy male Kunming mice were randomly divided into a blank group, a kidney Yin deficiency model group, a kidney Yin deficiency-Panacis Quinquefolii Radix(PQR) group, kidney Yin deficiency-Lubian treatment groups, a kidney Yang deficiency model group, a kidney Yang deficiency-Ginseng Radix et Rhizoma(GR) group, and kidney Yang deficiency-Lubian treatment groups, with eight mice in each group. The kidney Yin deficiency model and kidney Yang deficiency model were prepared by daily regular oral administration of dexamethasone acetate and hydrocortisone, respectively, and meanwhile, corresponding drugs were provided. The mice in the blank group received blank reagent. The treatment lasted 14 days. The exhaustive swimming time was measured 30 min after drug administration on the 14th day. On the 15th day, blood was collected from eyeballs and the serum was separated to determine the content of lactic acid(LD), blood urea nitrogen(BUN), lactate dehydrogenase(LDH), cyclic adenosine monophosphate(cAMP), and cyclic guanosine monophosphate(cGMP). The liver was dissected to determine the content of liver glycogen and the protein expression of phosphoinositide 3-kinase(PI3K) and protein kinase B(Akt). Compared with the kidney Yang deficiency model group, the kidney Yang deficiency-Lubian treatment groups showed increased body weight(P<0.05), relieved symptoms of Yang deficiency, decreased cGMP content(P<0.01), increased cAMP/cGMP(P<0.01), prolonged exhausted swimming time(P<0.01), reduced LD(P<0.01), elevated BUN content(P<0.01), increased liver glycogen content(P<0.01), and increased protein expression of PI3K and Akt in the liver(P<0.05). Compared with the kidney Yin deficiency model group, the kidney Yin deficiency-Lubian treatment groups showed increased body weight(P<0.01), relieved symptoms of Yin deficiency, increased content of cGMP(P<0.01), decreased cAMP/cGMP(P<0.01), prolonged exhausted swimming time(P<0.01), decreased LD(P<0.01), decreased BUN content(P<0.01), increased liver glycogen content(P<0.01), and increased protein expression of PI3K(P<0.05) and Akt in the liver(P<0.05). To sum up, Lubian can regulate Yin deficiency and Yang deficiency and increase glycogen synthesis by affecting the PI3K-Akt pathway, thereby exerting an anti-fatigue role.

Novel Approaches for Inhibiting the Indoor Allergen Der f 2 Excreted from House Dust Mites by Todomatsu Oil Produced from Woodland Residues.

House dust mite (HDM) is a globally ubiquitous domestic cause of allergic diseases. There is a pressing demand to discover efficient, harmless, and eco-friendly natural extracts to inhibit HDM allergens that are more likely to trigger allergies and challenging to be prevented entirely. This study, therefore, is aimed at assessing the inhibition of the allergenicity of major HDM allergen Der f 2 by todomatsu oil extracted from residues of Abies Sachalinensis. The inhibition was investigated experimentally (using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) and in silico using molecular docking. The results showed that todomatsu oil inhibits the allergenicity of Der f 2 by reducing its amount instead of the IgG binding capacity of a single protein. Moreover, the compounds in todomatsu oil bind to Der f 2 via alkyl hydrophobic interactions. Notably, most compounds interact with the hydrophobic amino acids of Der f 2, and seven substances interact with CYS27. Contrarily, the principal compounds fail to attach to the amino acids forming the IgG epitope in Der f 2. Interestingly, chemical components with the lowest relative percentages in todomatsu oil show high-affinity values on Der f 2, especially ß-maaliene (-8.0 kcal/mol). In conclusion, todomatsu oil has been proven in vitro as a potential effective public health strategy to inhibit the allergenicity of Der f 2.

Characterization of allergenicity of Platanus pollen allergen a 3 (Pla a 3) after exposure to NO2 and O3.

Pollen allergens, widely present in the atmosphere, are the main cause of seasonal respiratory diseases that affect millions of people worldwide. Although previous studies have reported that nitrogen dioxide (NO2) and ozone (O3) promote pollen allergy, the specific biological processes and underlying mechanisms remain less understood. In this study, Platanus pollen grains were exposed to gaseous pollutants (NO2 and O3). We employed environmental electron microscopy, flow cytometry, western blot assay, enzyme-linked immunoassay, ultraviolet absorption spectrometry, circular dichroism, and protein mass spectrometry to characterise the subpollen particles (SPPs) released from pollen grains. Furthermore, we determined the immunogenicity and pathogenicity induced by Platanus pollen allergen a 3 (Pla a 3). Our results demonstrated that NO2 and O3 could damage the pollen cell membranes in SPPs and increase the amount of Pla a 3 allergen released into the atmosphere. Additionally, NO2 and O3 altered the structure of Pla a3 protein through nitrification and oxidation, which not only enhanced the immunogenicity of allergens but also increased the stability of the protein. In vivo analysis using an animal model indicated that NO2 and O3 greatly aggravated pollen-induced pneumonia. Thus, our study provides guidance for the prevention of pollen allergic diseases.

Seneciphylline, a main pyrrolizidine alkaloid in Gynura japonica, induces hepatotoxicity in mice and primary hepatocytes via activating mitochondria-mediated apoptosis.

Herbal drug-induced liver injury has been reported worldwide and gained global attention. Thousands of hepatic sinusoidal obstruction syndrome (HSOS) cases have been reported after consumption of herbal medicines and preparations containing pyrrolizidine alkaloids (PAs), which are natural phytotoxins globally distributed. And herbal medicines, such as Gynura japonica, are the current leading cause of PA-induced HSOS. The present study aimed to reveal the mechanism underlying the hepatotoxicity of seneciphylline (Seph), a main PA in G. japonica. Results showed that Seph induced severe liver injury through apoptosis in mice (70 mg/kg Seph, orally) and primary mouse and human hepatocytes (5-50 µM Seph). Further research uncovered that Seph induced apoptosis by disrupting mitochondrial homeostasis, inducing mitochondrial depolarization, mitochondrial membrane potential (MMP) loss, and cytochrome c (Cyt c) release and activating c-Jun N-terminal kinase (JNK). The Seph-induced apoptosis in hepatocytes could be alleviated by Mdivi-1 (50 µM, a dynamin-related protein 1 inhibitor), as well as SP600125 (25 µM, a specific JNK inhibitor) and ZVAD-fmk (50 µM, a general caspase inhibitor). Moreover, the Seph-induced MMP loss in hepatocytes was also rescued by Mdivi-1. In conclusion, Seph induced liver toxicity via activating mitochondrial-mediated apoptosis in mice and primary hepatocytes. Our results provide further information on Seph detoxification and herbal medicines containing Seph such as G. japonica.

Studies on relationships between air pollutants and allergenicity of Humulus Scandens pollen collected from different areas of Shanghai.

Pollen pollution and allergy are becoming prominent issues in China. However, few studies on pollinosis have been reported. As an allergen in the atmosphere, allergenic Humulus scandens pollen was collected from four districts of Shanghai, including Wusong (WS), Jiading (JD), Xujiahui (XJH) and Songjiang (SJ). The mass concentrations of SO2, NO2, O3, PM10, and PM2.5 (particulate matter with air dynamic diameter less than 10 and 2.5 µm, respectively) near the four sampling sites were also recorded during Humulus scandens pollen season. The allergenicity of the Humulus scandens pollen was assessed by using of a rat model and enzyme linked immunosorbent assay (ELISA). Relationships between the allergenicity and air pollutants were correlated. Our results demonstrated that the biological viability of the pollens collected from the four districts exhibited no significant differences. ELISA and dot blotting results further demonstrated that the serum of sensitized rats exhibited much higher immune-reactive response than that of control groups. Western blotting showed that the 15 KD (1KD = 1000 dalton) proteins of Humulus pollen led to the allergic response. The allergenic intensity of Humulus pollen protein from different samples followed the pattern: WS > JD > XJ > SJ. There was a negative relationship between the allergenicity of Humulus pollens and PM10 (R = -0.99) / PM2.5 (R = -0.73), and a positive relationship with O3 (R = 0.92). These data clearly showed that PM10 and PM2.5 could enhance Humulus pollen protein release, and O3 could aggravate the allergenicity of the Humulus pollen.

The long persistence of pyrrolizidine alkaloid-derived pyrrole-protein adducts in vivo: Kinetic study following multiple exposures of a pyrrolizidine alkaloid containing extract of Gynura japonica.

Gynura japonica (also named Tusanqi in Chinese) is used as a folk herbal medicine for treating blood stasis or traumatic injury. However, hundreds of hepatic sinusoidal obstruction syndrome (HSOS) cases have been reported after consumption of preparations made from G. japonica because it contains large amounts of hepatotoxic pyrrolizidine alkaloids (PAs). To date, blood pyrrole-protein adducts (PPAs) are suggested as biomarkers for the diagnosis of PA-induced HSOS in clinics. However, the concentration of PPAs in the blood is greatly affected by several factors including the amount of PA exposure, herb intake period, and blood sampling time after the last exposure. In present study, the kinetic characters of PPAs in serum and liver as well as other potential target organs were studied systematically and comprehensively following multiple exposures of PAs in G. japonica extract (GJE). As results, PPAs content reached to a plateau both in serum and liver after the mice were treated with GJE for 2 weeks on daily basis. PPAs cleared significantly slower in liver (T1/2ke∼184.6 h, ∼7.7 days) than in serum (T1/2ke∼95.8 h, ∼4.0 days). Although more than 90 % PPAs were removed 2 weeks after the last dosing, PPAs still persisted in the liver until the end of the experiment, i.e. 8 weeks after the last dosing. The results would be of great help for understanding the importance of PPAs for PA-induced toxicity and its detoxification.

Allergenicity of recombinant Humulus japonicus pollen allergen 1 after combined exposure to ozone and nitrogen dioxide.

Ozone (O3) and nitrogen dioxide (NO2) are thought to play primary roles in aggravating air pollution-induced health problems. However, the effects of joint O3/NO2 on the allergenicity of pollen allergens are unclear. Humulus japonicus pollen allergen 1 (Hum j1) is a profilin protein that causes widespread pollinosis in eastern Asia. In order to study the effects of combined O3/NO2 on the allergenicity of Hum j1, tandem six-histidine peptide tag (His6)-fused recombinant Hum j1 (rHum j1) was expressed in a prokaryotic system and purified through His6 affinity chromatography. The purified rHum j1 was used to immunize SD rats. Rat sera with high titers of IgG and IgE antibodies against rHum j1 were used for allergenicity quantification. The rHum j1 was exposed to O3/NO2, and changes in allergenicity of the exposed rHum j1 were assayed using the immunized rat antibodies. Tandem LC-MS/LC (liquid chromatography-mass spectrometer/liquid chromatography spectrometer) chromatography and UV and circular dichroism (CD) spectroscopy were used to study the structural changes in rHum j1. Our data demonstrated that a novel disulfide bond between the sulfhydryl groups of two neighboring cysteine molecules was formed after the rHum j1 exposure to joint O3/NO2, and therefore IgE-binding affinity was increased and the allergenicity was reinforced. Our results provided clues to elucidate the mechanism behind air pollution-induced increase in pollinosis prevalence.