A novel glycoprotein from earthworm extract PvE-3: Insights of their characteristics for promoting diabetic wound healing and attenuating methylglyoxal-induced cell damage.

Diabetic chronic wound is a worldwide medical burden related to overdosed methylglyoxal (MGO) synthesis, which is the major precursor of glycation of proteins and DNA and is related to the dysfunction of dermal cells thus leading to chronic refractory wounds. Previous studies proved that earthworm extract accelerates diabetic wound healing and possesses cell proliferation and antioxidative effects. However, the effects of earthworm extract on MGO-damaged fibroblasts, the inner mechanisms of MGO-induced cell damage and the functional components in earthworm extract are still poorly understood. Firstly, we evaluated the bioactivities of the earthworm extract PvE-3 on the diabetic wound model and the diabetic related cell damage model. Then the mechanisms were investigated through transcriptomics, flow cytometry and fluorescence probe. The results revealed that PvE-3 promoted diabetic wound healing and protected fibroblast function in cell-damaged conditions. Meanwhile, the high-throughput screening implied the inner mechanisms of diabetic wound healing and PvE-3 cytoprotection effect were involved in the muscle cell function, the cell cycle regulation and the mitochondrial transmembrane potential depolarization. The functional glycoprotein isolated from PvE-3 possessed EGF-like domain which had a strong binding affinity with EGFR. The findings provided references to explore the potential treatments of diabetic wound healing.

The antifibrotic effect of pheretima protein is mediated by the TGF-ß1/Smad2/3 pathway and attenuates inflammation in bleomycin-induced idiopathic pulmonary fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Pheretima is a traditional Chinese medicine that could treat various lung diseases such as asthma, pneumonia, and lung cancer effectively; however, limited studies on the use of Pheretima protein in the treatment of lung diseases have been conducted to date. AIM OF THE STUDY: The aim of this study was to explain the antipulmonary fibrosis mechanism of the Pheretima protein and elucidate its possible cell signaling pathways. MATERIAL AND METHODS: Fresh pheretima was freeze-dried to obtain the Pheretima protein. Divide C57BL/6 mice into control and bleomycin (BLM)-induced models, pirfenidone, and Pheretima protein-treatment groups. Three weeks later, they were treated with H&E and Masson's trichrome staining to assess lung injury and fibrosis. Pulmonary fibrosis was assessed using immunohistochemistry (IHC), realtime-PCR (RT-PCR), and western blotting. Inflammation was assessed using the alveolar lavage fluid. RESULTS: Pheretima protein inhibited epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition and reduced inflammation. It also reduced the levels of Smad2/3, pSmad2/3, and transforming growth factor-beta 1 (TGF-ß1). Thus, our results indicate that Pheretima protein can alleviate BLM-induced pulmonary fibrosis in a mouse model. CONCLUSION: Pheretima protein inhibits ECM, EMT, and antiinflammatory markers, which in turn ameliorates BLM-induced pulmonary fibrosis. Preliminary mechanistic studies indicated that Pheretima protein can exert its biological activity by downregulating the TGF-ß1/Smad2/3 pathway.

A rapid and simple signature peptides-based method for species authentication of three main commercial Pheretima.

Pheretima with various activities is a commonly used animal-derived traditional medicine in Asia countries. However, almost half of them are non-pharmacopoeia species in the market due to the similar morphological characteristics between medicinal and non-medicinal species. This study aims to establish an effective method based on signature peptides for species authentication of three main commercial Pheretima, including two major Pheretima species (Amynthas aspergillum, Metaphire vulgaris) and one main adulteration (Metaphire magna). Firstly, the species of 52 batches of commercial Pheretima were authenticated based on DNA barcodes. Secondly, proteomic analysis was performed for protein characterization of three main commercial Pheretima. Furthermore, their signature peptides were screened and validated using ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, a simplified sample processing method was developed. Finally, large quantities of commercial Pheretima samples were analyzed for further verifying the feasibility of the signature peptides-based method. The result showed that the established method had a great application potential for authenticity identification of commercial Pheretima. SIGNIFICANCE: The authenticity assessment of medicinal materials is a main issue in the quality control process as deceptive practices could imply severe health risks. In this study, a rapid and simple method based on signature peptides was established for species authentication of three main commercial Pheretima, which can be an effective alternative to complex DNA barcoding and difficult morphological identification, and provided a reference for improvement of Pheretima quality standards.

Authentication of three main commercial Pheretima based on amino acids analysis.

Pheretima has been used as an animal-derived traditional Chinese medicine for thousands of years in Asian countries due to its multi-activities. However, more than half of the commercial Pheretima are adulterants according to the previous research. Besides, the standards of Pheretima are still inadequate in the identification of Pheretima species. In this study, an amino acids (AAs) analytical method established based on the ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS) in multiple reaction monitoring (MRM) mode through derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was used for qualitative and quantitative analysis of the total AAs of three main commercial Pheretima (two major Pheretima species, Amynthas aspergillum, Metaphire vulgaris, and one main counterfeit, M. magna). As a result, 16 AAs were detected and quantitated in their hydrolyzed samples. Then, multivariate statistical analysis was applied to distinguish the three commercial Pheretima based on their AAs level. Finally, four AAs (Thr, Glu, Asp, and Arg) were screened as species-differential AAs, which may be used as chemical markers to distinguish the three commercial Pheretima. This study deeply described the outline of AAs in Pheretima and offered a good reference for its species authentication.

Dietary supplementation with selenium-enriched earthworm powder improves antioxidative ability and immunity of laying hens.

Selenium (Se) has been recognized as an essential dietary nutrient for decades, and organic Se sources rather than inorganic ones are increasingly advocated as Se supplements. Earthworms have been studied as a feed additive and animal protein source for many yr. The aim of this study was to evaluate the effect of Se-enriched earthworm powder (SEP) on the antioxidative ability and immunity of laying hens. A total of 120 27-wk-old laying hens were randomly divided into 4 groups (30 hens per group). Laying hens were fed diets supplemented with SEP having 0, 0.5, or 1 mg/kg of Se or with earthworm powder alone. After 5 wk of supplementation, serum from the hens was tested for nutritional components (protein, globulin, albumin, triglycerides, total cholesterol, and glucose), antioxidative properties (glutathione peroxidase, superoxide dismutase, catalase, and nitric oxide), and immune responses (lysozymes, immunoglobulin G, IL-2, and interferon gamma). We found that SEP with 1.0 mg/kg of Se upregulated the hens' total protein, albumin, glutathione peroxidase, superoxide dismutase, IgG, and IL-2 and downregulated triglycerides, total cholesterol, glucose, and nitric oxide. These results indicate that SEP improves antioxidative levels and immune function of laying hens, indicating potential benefit from use of SEP as a feed additive in the poultry industry.

Combination of c oxidase subunit I based deoxyribonucleic acid barcoding and HPLC techniques for the identification and quality evaluation of Pheretima aspergillum.

This study aimed to identify Pheretima aspergillum (Guang-Pheretima) and its adulterants using the cytochrome c oxidase subunit I based deoxyribonucleic acid barcoding technology, and further to evaluate their quality using an optimized high-performance liquid chromatography method. For deoxyribonucleic acid barcoding identification, the Kimura-2-Parameter model was used to analyze genetic distance, and phylogenetic neighbor-joining tree was constructed for species identification of 20 labeled Guang-Pheretima samples. A high-performance liquid chromatography method was developed for the simultaneous determination of seven nucleoside components for quality evaluation. Compared with the GenBank database, 10 samples were identified as real Guang-Pheretima (P. aspergillum), and the others as the adulterants-Metaphire magna. The maximum intraspecific genetic distances of c oxidase subunit I sequence for P. aspergillum were smaller than the minimum interspecific genetic distances between P. aspergillum and M. magna. Ten P. aspergillum and 10 M. magna samples were clearly clustered in the neighbor-joining tree. The contents of seven nucleosides components in P. aspergillum were significantly higher than that in its adulterant-M. magna. The incidence of adulterants for Guang-Pheretima was high (up to 50%) with an alarming quality. This study provided a powerful idea for the quality evaluation of other highly valuable plant- or animal-derived products for safety concerns to avoid misidentification.

Exploration of dipeptidyl-peptidase IV (DPP IV) inhibitors in a low-molecular mass extract of the earthworm Eisenia fetida and identification of the inhibitors as amino acids like methionine, leucine, histidine, and isoleucine.

We have reported previously that the water extract of the earthworm Eisenia fetida has inhibitory effect on human dipeptidyl-peptidase IV (DPP IV) in vitro. Here we studied to identify DPP IV inhibitors in a low-molecular mass extract (designated U3EE) under 3 kDa prepared from the water extract. U3EE showed 50 % inhibition (IC50) at the concentration of 5.3 ± 0.3 mg/mL. An inhibitory active fraction obtained by solid-phase extraction of U3EE was separated into three parts by reversed-phase HPLC. These parts were shown by GC/MS to be composed of ten (Ala, Gly, Thr, Ser, Asn, Asp, Lys, His, Orn, and cystine), two (Leu and Ile), and one (Met) amino acids, respectively. Among them, Met, Leu, and His showed strong inhibition with IC50 values of 3.4 ± 0.3, 6.1 ± 0.3 and 14.7 ± 1.2 mM, respectively; Ala, Lys, Orn, and Ile showed rather weaker inhibition than those, while the others showed no inhibition. Met, Leu, and Ile were competitive inhibitors and His was a mixed-type one. DPP IV inhibition by U3EE might be due to additive and/or synergistic effects of the inhibitory amino acids, suggesting that it could be useful as pharmaceutical and supplement for diabetes prevention.

[Study on proteins in Guangdilong by nano LC/orbitrap fusion lumos HR-MS].

In the present study,fresh Guangdilong( GD),originating from Pheretima aspergillum,was taken as the object. The total proteins from GD were firstly separated by SDS-PAGE according to their molecular weights and in-gel digestion was then performed.After that,the peptides were analyzed by nano LC/orbitrap fusion lumos high resolution mass spectrometry( nano LC/orbitrap fusion lumos HR-MS). Protein identification was implemented by comparison with Annelida. fasta database using Proteome Discoverer software.As a result,386 proteins were tentatively identified,including chain F,globin B chain,glyceraldehyde-3-phosphate dehydrogenase,fibrinolytic protein,and so on. Most of the proteins took part in cell structure and energy metabolism,and fibrinolytic protein and lombricine kinase might be related to fibrinolytic activity. Protein classification based on gene ontology was carried out using PANTHER and KEGG for metabolic pathway enrichment. The results indicated that these proteins were related to diverse signal transduction pathways,including metabolic pathways,central carbon metabolism,biosynthesis of amino acids,ribosome,glycolysis,citrate cycle( TCA cycle),and so on. This study would lay the foundation for the further research on the proteins in GD and also their functions.

Anti-Candida albicans effect of the protein-carbohydrate fraction obtained from the coelomic fluid of earthworm Dendrobaena veneta.

An antifungal active fraction (AAF) from the coelomic fluid (CF) of the earthworm Dendrobaena veneta was isolated. The aim of the study was to analyze the antifungal activity of the AAF and to carry out chemical characterization of the fraction. The active fraction showed antifungal activity against a clinical C. albicans isolate, C. albicans ATCC 10231, and C. krusei ATCC 6258. It effectively reduced the metabolic activity of C. albicans cells and influenced their morphology after 48 hours of incubation. Scanning electron microscopy (SEM) images revealed loss of integrity of the cell wall induced by the active fraction. Calcofluor White staining showed changes in the structure of the C. albicans cell wall induced by the AAF. The fungal cells died via apoptosis and necrosis after the treatment with the studied fraction. Electrophoresis under native conditions revealed the presence of two compounds in the AAF, while SDS/PAGE gel electrophoresis showed several protein and carbohydrate compounds. The active fraction was analyzed using Raman spectroscopy, MALDI TOF/TOF, and ESI LC-MS. The Raman analysis confirmed the presence of proteins and determined their secondary structure. The MALDI TOF/TOF analysis facilitated detection of four main compounds with a mass of 7694.9 m/z, 12292.3 m/z, 21628.3 m/z, and 42923.2 m/z in the analyzed fraction. The presence of carbohydrate compounds in the preparation was confirmed by nuclear magnetic resonance (NMR) and gas chromatography (GC-MS). The ATR-FTIR spectrum of the AAF exhibited high similarity to the spectrum of egg white lysozyme. The AAF showed no endotoxicity and cytotoxicity towards normal skin fibroblasts (HSF); therefore, it can be used for the treatment of skin and mucous membrane candidiasis in the future. Given its efficient and selective action, the fraction seems to be a promising preparation with antifungal activity against C. albicans.

The effect of earthworm extract on promoting skin wound healing.

Chronic nonhealing wounds pose a significant challenge to healthcare system because of its tremendous utilization of resources and time to heal. It has a well-deserved reputation for reducing the quality of life for those affected and represent a substantial economic burden to the healthcare system overall. Earthworms are used as a traditional Chinese medicine, and have been applied pharmacologically and clinically since a long time in China. However, there is paucity in data regarding its wound healing effects. Therefore, we investigated the effect of earthworm extract (EE) on skin wound healing process. The obtained data showed that EE has healing effects on local wound of mice. It decreased the wound healing time and reduced the ill-effects of inflammation as determined by macroscopic, histopathologic, hematologic, and immunohistochemistry parameters. The potential mechanism could be accelerated hydroxyproline and transforming growth factor-ß secretion-thus increasing the synthesis of collagen, promoting blood capillary, and fibroblast proliferation. It could accelerate the removal of necrotic tissue and foreign bodies by speeding up the generation of interleukin-6, white blood cells, and platelets. It thus enhances immunity, reduces the risk of infection, and promotes wound healing. All in all, the obtained data demonstrated that EE improves quality of healing and could be used as a propitious wound healing agent.

Anti-inflammatory activities of Guang-Pheretima extract in lipopolysaccharide-stimulated RAW 264.7 murine macrophages.

BACKGROUND: Guang-Pheretima, which is originated from Pheretima aspergillum, has been documented in academic Chinese herbal studies for nearly 2000 years for its prominent treating effects of various inflammatory diseases such as asthma, cough and fever. However, the anti-inflammatory activity and mechanism of Guang-Pheretima has been rarely reported. Hence, we investigated the inhibitory effect and the underlying mechanism of Guang-Pheretima aqueous extracts on inflammatory response in RAW 264.7 cells. METHOD: RAW 264.7 macrophages were pretreated with various concentrations of Guang-Pheretima decoction (GPD) or protein-free Guang-Pheretima decoction (PF-GPD) and subsequently stimulated with lipopolysaccharide (LPS) to trigger the inflammatory response. Productions of nitric oxide (NO) were determined by Griess reaction, and prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 were measured by enzyme-linked immunosorbent assays (ELISA). The protein expressions and messenger ribonucleic acid (mRNA) amounts of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were analyzed by Western Blot and Real-Time polymerase chain reaction (PCR), respectively. Finally, the translocation of nuclear factor (NF)-κB was observed by Western Blot. RESULTS: GPD of the experimental concentrations showed no anti-inflammatory activity. In contrast, PF-GPD at concentrations of 40-320 µg/mL significantly inhibited NF-κB activation and reduced the production of inflammatory mediators, such as NO, PGE2, TNF-α, as well as the related key synthases including iNOS and COX-2. Moreover, PF-GPD markedly suppressed the release of inflammatory cytokines, such as IL-1ß and IL-6. CONCLUSION: These results demonstrate the excellent anti-inflammatory properties of PF-GPD, and suggest that Guang-Pheretima may be used to treat and prevent certain inflammatory diseases.

Study of analgesic effect of earthworm extract.

Pain represents a major clinical problem and one which has exercised generations of healthcare professionals. Earthworms are used as a traditional Chinese medicine, and have been applied pharmacologically and clinically since a long time in China. However, the analgesic effects of earthworm extract (EE) are seldom studied. Hence, we evaluated the analgesic effects of EE in mice. The obtained data showed that EE increased pain threshold and exhibited peripheral but not central analgesic effects in mice; evidenced by increased inhibition ratio in acetic acid writhing test and formalin test, whereas only slight increase in inhibition ratio in hot plate test and tail immersion test. In addition, EE decreased serum norepinephrine (NE), 5-hydroxytryptamine (5-HT), and nitric oxide (NO) synthase (NOS) concentration, similar to other analgesic drugs like morphine and aspirin. In a nutshell, the obtained data have demonstrated that EE has peripheral analgesic properties and could be used as a promising analgesic drug.

Prolonging the shelf life of Lumbricus terrestris erythrocruorin for use as a novel blood substitute.

Limitations associated with the storage of red blood cells have motivated the development of novel blood substitutes that are able to withstand long-term storage at elevated temperatures. The hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an attractive blood substitute candidate, since it is resistant to oxidation and aggregation during storage. Several factors were investigated to optimize the thermal and oxidative stability of LtEc during storage, including pH, antioxidant supplements, and deoxygenation. A strategy for the reduction of fully oxidized LtEc with antioxidants was also developed. Overall, LtEc was shown to have the highest thermal stability in Ringer's Modified Lactate solution with 10 mM HEPES at pH 7.0. Deoxygenation of the LtEc was also shown to significantly reduce oxidation of the ferrous heme iron (e.g., %Fe2+ after 7 d at 37 °C = 75.7%). However, even in cases where oxidation does occur, the addition of 1.8 mM ascorbic acid (AA) was found to reduce 98.3% of the oxidized LtEc (37 µM heme). Most importantly, the oxygen transport properties of LtEc were unaffected by storage at high temperatures or oxidation followed by reduction with AA. These results show that LtEc can be stored at high temperatures (37 °C) without any significant loss of function.

Effects of Traditional Chinese Medicine, Dilong Injection, on Random Skin Flap Survival in Rats.

BACKGROUND: Dilong injection as a medicinal preparation extracted from earthworm in traditional Chinese medicine, is used to activate blood circulation and remove blood stasis. In this research, we aim to investigate its potential effect on random skin flap survival in rat models. MATERIALS AND METHODS: McFarlane flaps were established in 60 male Sprague-Dawley rats randomly divided into two groups: the control group and the Dilong injection group. Diong injection group was injected with the Diong injection (4 mL/kg) once a day for seven days, and the control group was given an equal volume of saline solution. After seven days, flaps were obtained and stained with Hematoxylin and Eosin. Histological examination was done to determine changes in histology such as thickness of granulation tissue, tissue edema, neutrophil infiltration, and the microvascular density (MVD). In addition, immunohistochemical detection was carried out to show vascular endothelial growth factor (VEGF) expression level. RESULTS: Compared with the control group, the Dilong group exhibited more fibroblastic proliferation, thinner neutrophil infiltration with less edema through histological examination. The MVD and the VEGF expression of flaps were significantly higher. The mean superoxide dismutase activity was evidently higher in the Dilong group than in the control group, while the mean MDA level was lower. CONCLUSIONS: According to the comparison made between the two groups for histological and immunohistochemical evaluation, the Dilong injection group has potential effects on the survival of random skin flaps in rat models.

[Protective effect of earthworm active ingredients against endoplasmic reticulum stress-induced acute liver injury in mice].

To study the protective effect of earthworm active ingredients(EWAs) against endoplasmic reticulum stress(ERS)-induced acute liver injury in mice. The model of liver injury was induced through intraperitoneal injection of 10%CCl4. Serum glutamic-pyruvic transaminase(ALT), glutamic-oxaloacetic transaminase(AST), superoxide dismutase(SOD) and glutathione peroxidase(GSH-PX) activity and malondialdehyde(MDA) concentration were detected by colorimetric method. Histological examination was performed through hematoxylin-eosin staining and light microscopy, and apoptosis was detected using terminal transferase dUTP nick end labeling. The expressions of ERS related proteins, including glucose regulated protein 78(GRP78), protein kinase R-like ER kinase(PERK), eukaryotic transcription initiation factor 2α(eIF2α), active transcription factor-4(ATF4) and CCAAT/enhancer binding homologous protein(CHOP), were measured by immunohistochemistry and Western blot. According to the results, compared with the model group,serological indexes in the high, middle and low doses of EWAs were significantly improved (P<0.05 or P<0.01), the extent of liver lesion was decreased and the degree of injury was significantly reduced, and that the liver index and the spleen index of mice were significantly changed(P<0.05 or P<0.01). In liver tissue, the expressions of GRP78 and CHOP were significantly decreased(P<0.05 or P<0.01). The protein expressions of GRP78, CHOP and its upstream signaling pathway PERK-eIF2-ATF4 were significantly decreased in each dose group(P<0.05 or P<0.01). In summary, EWAs has a significant protective effect on ERS-induced acute liver injury, and its mechanism may be correlated with the inhibition of oxidative stress and ERS, and down-regulation of ERS marker protein CHOP expression, andinhibition of apoptosis.

Bioactivities of crude mucus proteins from Eudrilus eugeniae (African night crawler) and Perionyx excavatus (Blue worm).

OBJECTIVE: This study aimed to investigate the enzymatic anti-oxidative, hemolytic and cytotoxic activities of crude mucus proteins from the two earthworms including Eudrilus eugeniae (African night crawler) and Perionyx excavatus (Blue worm). MATERIALS AND METHODS: The bioactivities were determined by hemolytic activity, cytotoxic activity using HepG2 human hepatocellular carcinoma cells, L-929 mouse fibroblast and TK6 human lymphoblast cell lines and antioxidant activity. RESULTS: The results indicated that the hemolytic activity of mucus proteins of P. excavatus was higher than that of E. eugeniae. The cytotoxic activity of the mucus proteins of E. eugeniae could inhibit the growth of HepG2 with the IC50 at 144.2 ± 0.18 µg/mL but showed no effect on L-929 and TK6. On the other hand, the crude proteins of P. excavatus decreased cell viability of both L-929 and HepG2 with their IC50 respectively were 6.87 ± 0.16 and 174.3 ± 0.19 µg/mL but they did not reduce the growth of TK6 cell line. The SOD-like activity of P. excavatus and E. eugeniae crude proteins were found with the IC50 at 149 µg/mL and 386.2 µg/mL, respectively. For GPx-like activity, crude proteins of P. excavatus exhibited significant (p ≤ 0.05) greater activity than those of E. eugeniae when tested at the concentration 100 µg/mL. CONCLUSIONS: This study revealed the bioactivities of crude mucus of earthworms which served as alternative natural proteins for the prophylaxis or treatment of free radical-related diseases as well as development of dietary supplements and cosmetics.

Earthworm extract attenuates silica-induced pulmonary fibrosis through Nrf2-dependent mechanisms.

Silicosis is an occupational pulmonary fibrosis caused by inhalation of silica (SiO2) and there are no ideal drugs to treat this disease. Earthworm extract (EE), a natural nutrient, has been reported to have anti-inflammatory, antioxidant, and anti-apoptosis effects. The purpose of the current study was to test the protective effects of EE against SiO2-induced pulmonary fibrosis and to explore the underlying mechanisms using both in vivo and in vitro models. We found that treatment with EE significantly reduced lung inflammation and fibrosis and improved lung structure and function in SiO2-instilled mice. Further mechanistic investigations revealed that EE administration markedly inhibited SiO2-induced oxidative stress, mitochondrial apoptotic pathway, and epithelial-mesenchymal transition in HBE and A549 cells. Furthermore, we demonstrate that Nrf2 activation partly mediates the interventional effects of EE against SiO2-induced pulmonary fibrosis. Our study has identified EE to be a potential anti-oxidative, anti-inflammatory, and anti-fibrotic drug for silicosis.

Effect of fermented earthworm cast on egg production and egg quality as well as removal of odor in feces from egg laying hens.

The objective of the present study was to determine the effect of feeding fermented earthworm casts (EEC) to layers on egg-laying performance, blood lipid profiles, cecal microflora, and fecal odor removing performance. A total of 200 Hyline Brown layer chicks at 33-week-old were used in this study. They were randomly assigned to two numerically equal groups with 100 replications per treatment for 10 weeks. All the birds were caged individually. The control group was not treated with EEC. The EEC group was treated with top dressing containing 3.5% EEC. The present study revealed that egg production and egg weight were increased after feeding diet containing EEC at the top dressing level. Haugh unit, eggshell thickness, and eggshell breaking strength of EEC group were higher than those of control group. Egg yolk was determined for fatty acid profiling. It was found that EEC group had higher ratio of unsaturated- to saturated fatty acid as compared to control group. Lower ratios of n-6 to n-3 fatty acids were found in the egg yolk of EEC group. Plasma triglyceride and total cholesterol contents were lower in the EEC group. However, high density lipoprotein-cholesterol content was higher in the EEC group as compared to that in control group. The number of cecal Lactobacillus was increased while the population of Escherichia coli and coliform bacteria decreased in the EEC group. Fecal ammonia and hydrogen sulfide contents were lower in the EEC group as compared to those in control group. Taken together, these results suggested that EEC could improve egg production and egg quality. In addition, it could remove odour from laying-hen manure.

The protective effect of the earthworm active ingredients on hepatocellular injury induced by endoplasmic reticulum stress.

The earthworm is a widely used Chinese herbal medicine. There are more than 40 prescriptions including earthworms in the "Compendium of Materia Medica". TCM theory holds that earthworms exert antispasmodic and antipyretic effects through the liver meridian to calm the liver. However, the clinical effect of earthworms on liver injury has not been clearly demonstrated. We have previously established a method to extract the active ingredients from earthworms (hereinafter referred to as EWAs) [1]. In the present study, we observed protective effect of the EWAs on tunicamycin-induced ERS (endoplasmic reticulum stress) model in human hepatic L02 cells. The results showed that the EWAs promote proliferation and reduced apoptosis of ERS model in L02 cells (P<0.01). The up-regulation of ERS-related proteins, including PERK (protein kinase RNA-like endoplasmic reticulum kinase), eIF2a (eukaryotic translation initiation factor 2a), ATF4 (activating transcription factor 4) and CHOP (CCAAT/enhancer binding protein homologous protein), in L02 cell under ERS was inhibited by treatment of the EWAs (P<0.01). In summary, our data suggest the EWAs can significant attenuate ERS-induced hepatocyte injury via PERK-eIF2a-ATF4 pathway.

Pheretima aspergillum decoction suppresses inflammation and relieves asthma in a mouse model of bronchial asthma by NF-κB inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE AND AIM OF THE STUDY: Guang-Pheretima, the live form of the earthworm Pheretima aspergillum, is a traditional Chinese medicine commonly used for the treatment of asthma, cough, stroke, epilepsy and other diseases due to its anti-inflammatory, anti-asthmatic, anti-seizure, thrombolytic and diuretic properties. Although Guang-Pheretima is effective in the relief of asthma, its pharmacological activity and the underlying molecular mechanisms are not fully understood. Hence, we investigated the effects of a Pheretima aspergillum decoction (PAD) against inflammation in a model of ovalbumin (OVA)-induced asthma in BALB/c mice, as well as the nuclear factor-κB (NF-κB) pathway involved in this process. MATERIALS AND METHODS: OVA was used to sensitize and challenge the airway of the mice, and PAD was administrated by gavage. We measured airway hyperresponsiveness (AHR) in the mice 24h following a final methacholine challenge with whole-body plethysmography. The bronchoalveolar lavage fluid (BALF), serum and pulmonary tissues were collected 48h after the last challenge. The levels of inflammatory factors and the related mRNAs were determined by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR), respectively. The number of differential inflammatory cells in the BALF was counted. Serum total and OVA-specific IgE levels were measured with ELISA. The activation of NF-κB signaling in the lung was detected by western blotting. In addition, the lung tissues were stained with hematoxylin and eosin or periodic acid Schiff stain for histopathological examination. RESULTS: PAD treatment significantly alleviated AHR in the asthmatic mice, decreased the mRNA and protein levels of IL-4, IL-5 and IL-13 and downregulated IgE. In addition, PAD treatment attenuated mucus secretion and infiltration of inflammatory cells in the lung while inhibiting the activation of NF-κB signaling. CONCLUSIONS: PAD effectively inhibited the activation of NF-κB signaling in the lungs of mice with OVA-induced asthma, and mitigated AHR and Th2 type inflammatory reactions. Therefore, PAD may serve as a drug candidate for asthma treatment.